DRAY ELOISE (US)
MCHARDY STANTON F (US)
CLAIMS What is claimed is: 1. A method of treating a disorder associated with overexpression of an eyes absent (EYA) protein in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound having a structure represented by a formula: wherein R1 is selected from hydrogen and C1-C4 alkyl; wherein each of R2a, R2b, R2c, R2d, and R2e is independently selected from hydrogen, halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1- C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl; wherein R3 is selected from hydrogen and C1-C4 alkyl; wherein each of R4a and R4b is independently selected from hydrogen, halogen, and C1- C4 alkyl; wherein each of R5a, R5b, and R5c is independently selected from hydrogen, halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl; wherein each of R6a and R6b is independently selected from hydrogen and halogen; and wherein Cy1 is a C3-C8 cycloalkyl substituted with 0, 1, 2, 3, or 4 groups independently selected from halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl, provided that at least one of R6a and R6b is halogen, or a pharmaceutically acceptable salt thereof, wherein the disorder is a vascular disease, a fibrosis-related disorder, hearing loss, a cranio-facial disease, or a metabolic disease. 2. The method of claim 1, wherein the disorder is associated with overexpression of eyes absent homolog 4 (EYA4) or eyes absent homolog 2 (EYA2). 3. The method of claim 2, wherein the disorder is associated with overexpression of EYA4. 4. The method of claim 2, wherein the disorder is associated with overexpression of EYA2. 5. The method of claim 1, wherein the compound modulates expression of one or more EYA proteins. 6. The method of claim 1, wherein the compound inhibits one or more EYA proteins. 7. The method of claim 1, wherein the compound modulates expression of EYA4 or EYA2. 8. The method of claim 1, wherein the compound inhibits expression of EYA2 or EYA4. 9. The method of claim 1, wherein R6a is halogen. 10. The method of claim 9, provided that either: (a) R6b is chloro; (b) R6b is hydrogen; or (c) R6b is halogen and at least one of R5a, R5b, and R5c is not hydrogen. 11. The method of claim 1, wherein R1 is hydrogen. 12. The method of claim 1, wherein each of R2a, R2b, R2c, R2d, and R2e is hydrogen. 13. The method of claim 1, wherein R3 is hydrogen. 14. The method of claim 1, wherein each of R4a and R4b is hydrogen. 15. The method of claim 1, wherein each of R5a, R5b, and R5c is hydrogen. 16. The method of claim 1, wherein R6a is chloro. 17. The method of claim 1, wherein R6b is halogen. 18. The method of claim 17, wherein R6b is chloro. 19. The method of claim 1, wherein R6b is hydrogen. 20. The method of claim 1, wherein Cy1 is an unsubstituted C3-C8 cycloalkyl. 21. The method of claim 1, wherein Cy1 is an unsubstituted C3-C6 cycloalkyl. 22. The method of claim 1, wherein Cy1 is a cyclopentyl substituted with 0, 1, 2, 3, or 4 groups independently selected from halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1- C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. 23. The method of claim 1, wherein Cy1 is an unsubstituted cyclopentyl. 24. The method of claim 1, wherein the compound has a structure represented by a formula: or a pharmaceutically acceptable salt thereof. 25. The method of claim 1, wherein the compound has a structure represented by a formula: or a pharmaceutically acceptable salt thereof. 26. The method of claim 1, wherein the compound has a structure represented by a formula: or a pharmaceutically acceptable salt thereof. 27. The method of claim 1, wherein the compound has a structure represented by a formula: or a pharmaceutically acceptable salt thereof. 28. The method of claim 1, wherein the compound has a structure represented by a formula: or a pharmaceutically acceptable salt thereof. 29. The method of claim 1, wherein the compound is a structure selected from: or a pharmaceutically acceptable salt thereof. 30. The method of claim 1, wherein the compound is: or a pharmaceutically acceptable salt thereof. 31. The method of claim 1, wherein the subject is a mammal. 32. The method of claim 1, wherein the subject is a human. 33. The method of claim 1, wherein the subject has been diagnosed with a need for modification of the expression of one or more EYA proteins prior to the administering step. 34. The method of claim 1, wherein the subject has been diagnosed with a need for treatment of the disorder prior to the administering step. 35. The method of claim 1, further comprising identifying a subject in need of treatment of the disorder. 36. The method of claim 1, wherein the disorder is a vascular disease. 37. The method of claim 36, wherein the vascular disease is selected from atherosclerosis, peripheral artery disease, carotid artery disease, a pulmonary embolism, collagen vascular disease, and cerebrovascular disease. 38. The method of claim 1, wherein the disorder is a fibrosis-related disorder. 39. The method of claim 38, wherein the fibrosis-related disorder is selected from scleroderma, rheumatoid arthritis, Crohn’s disease, ulcerative colitis, myelofibrosis, systemic lupus erythematosus, idiopathic pulmonary fibrosis, non-alcoholic steatohepatitis (NASH), systemic sclerosis, and interstitial lung disease. 40. The method of claim 1, wherein the disorder is hearing loss. 41. The method of claim 1, wherein the disorder is a metabolic disease. 42. The method of claim 41, wherein the metabolic disease is a fatty liver disease or a neoplasia (e.g., a neoplasia of the heart or the liver). 43. The method of claim 1, wherein the disorder is a cranio-facial disease. 44. The method of claim 43, wherein the cranio-facial disease is a developmental syndrome or a heritable otitis. 45. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a compound having a structure represented by a formula: wherein R1 is selected from hydrogen and C1-C4 alkyl; wherein each of R2a, R2b, R2c, R2d, and R2e is independently selected from hydrogen, halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1- C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl; wherein R3 is selected from hydrogen and C1-C4 alkyl; wherein each of R4a and R4b is independently selected from hydrogen, halogen, and C1- C4 alkyl; wherein each of R5a, R5b, and R5c is independently selected from hydrogen, halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl; wherein each of R6a and R6b is independently selected from hydrogen and halogen; and wherein Cy1 is a C3-C8 cycloalkyl substituted with 0, 1, 2, 3, or 4 groups independently selected from halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl, provided that at least one of R6a and R6b is halogen, or a pharmaceutically acceptable salt thereof, wherein the cancer comprises a tumor that overexpresses at least one eyes absent (EYA) protein. 46. The method of claim 45, wherein the tumor overexpresses EYA4. 47. The method of claim 45, wherein the tumor overexpresses EYA2. 48. The method of claim 45, wherein R6a is halogen. 49. The method of claim 45, wherein R6b is halogen. 50. The method of claim 45, wherein each of R6a and R6b is halogen. 51. The method of claim 45, wherein the compound has a structure represented by a formula: or a pharmaceutically acceptable salt thereof. 52. The method of claim 45, wherein the compound has a structure selected from: , or a pharmaceutically acceptable salt thereof. 53. The method of claim 45, wherein the effective amount is a therapeutically effective amount. 54. The method of claim 45, wherein the effective amount is a prophylactically effective amount. 55. The method of claim 45, wherein the subject is a mammal. 56. The method of claim 45, wherein the mammal is a human. 57. The method of claim 45, wherein the subject has been diagnosed with a need for treatment of cancer prior to the administering step. 58. The method of claim 45, further comprising the step of identifying a subject in need of treatment of cancer. 59. The method of claim 45, wherein the cancer is breast cancer, cervical cancer, ovarian cancer, liver cancer, or pancreatic cancer. 60. The method of claim 45, wherein the cancer is triple-negative breast cancer (TNBC). 61. The method of claim 45, wherein the cancer is a pediatric cancer. 62. The method of claim 61, wherein the pediatric cancer is leukemia. |
or a pharmaceutically acceptable salt thereof. [00173] In various aspects, the compound has a structure represented by a formula: or a pharmaceutically acceptable salt thereof. [00174] In various aspects, the compound has a structure represented by a formula: or a pharmaceutically acceptable salt thereof. [00175] In various aspects, the compound has a structure represented by a formula:
or a pharmaceutically acceptable salt thereof. [00176] In various aspects, the compound is a structure selected from:
or a pharmaceutically acceptable salt thereof. [00177] In various aspects, the compound is: or a pharmaceutically acceptable salt thereof. a. R 1 GROUPS [00178] In one aspect, R 1 is selected from hydrogen and C1-C4 alkyl. In a further aspect, R 1 is selected from hydrogen, methyl, ethyl, n-propyl, and isopropyl. In a still further aspect, R 1 is selected from hydrogen, methyl, and ethyl. In yet a further aspect, R 1 is selected from hydrogen and ethyl. In an even further aspect, R 1 is selected from hydrogen and methyl. [00179] In various aspects, R 1 is C1-C4 alkyl. In a further aspect, R 1 is selected from methyl, ethyl, n-propyl, and isopropyl. In a still further aspect, R 1 is selected from methyl and ethyl. In yet a further aspect, R 1 is ethyl. In an even further aspect, R 1 is methyl. [00180] In various aspects, R 1 is hydrogen. b. R 2A , R 2B , R 2C , R 2D , AND R 2E GROUPS [00181] In one aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH2, ‒ OH, ‒NO 2 , methyl, ethyl, n-propyl, isopropyl, ethenyl, propenyl, ‒CCl 3 , ‒CF 3 , ‒CHCl 2 , ‒ CHF2, ‒CH2Cl, ‒CH2F, ‒CH2CH2Cl, ‒CH2CH2F, ‒CH2CH2CH2Cl, ‒CH2CH2CH2F, ‒ CH(CH 3 )CH 2 Cl, ‒CH(CH 3 )CH 2 F, ‒CH 2 CN, ‒CH 2 CH 2 CN, ‒CH 2 CH 2 CH 2 CN, ‒ CH(CH3)CH2CN, ‒CH2OH, ‒CH2CH2OH, ‒CH2CH2CH2OH, ‒CH(CH3)CH2OH, ‒OCCl3, ‒ OCF 3 , ‒OCHCl 2 , ‒OCHF 2 , ‒OCH 2 Cl, ‒OCH 2 F, ‒OCH 2 CH 2 Cl, ‒OCH 2 CH 2 F, ‒ OCH2CH2CH2Cl, ‒OCH2CH2CH2F, ‒OCH(CH3)CH2Cl, ‒OCH(CH3)CH2F, ‒OCH3, ‒ OCH 2 CH 3 , ‒OCH 2 CH 2 CH 3 , ‒OCH(CH 3 ) 2 , ‒NHCH 3 , ‒NHCH 2 CH 3 , ‒NHCH 2 CH 2 CH 3 , ‒ NHCH(CH3)2, ‒N(CH3)2, ‒N(CH3)CH2CH3, ‒N(CH2CH3)CH2CH2CH3, ‒N(CH3)CH(CH3)2, ‒CH 2 NH 2 , ‒CH 2 CH 2 NH 2 , ‒CH 2 CH 2 CH 2 NH 2 , and ‒CH(CH 3 )CH 2 NH 2 . In a still further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, ‒ CN, ‒NH 2 , ‒OH, ‒NO 2 , methyl, ethyl, ethenyl, ‒CCl 3 , ‒CF 3 , ‒CHCl 2 , ‒CHF 2 , ‒CH 2 Cl, ‒ CH2F, ‒CH2CH2Cl, ‒CH2CH2F, ‒CH2CN, ‒CH2CH2CN, ‒CH2OH, ‒CH2CH2OH, ‒OCCl3, ‒ OCF 3 , ‒OCHCl 2 , ‒OCHF 2 , ‒OCH 2 Cl, ‒OCH 2 F, ‒OCH 2 CH 2 Cl, ‒OCH 2 CH 2 F, ‒OCH 3 , ‒ OCH2CH3, ‒NHCH3, ‒NHCH2CH3, ‒N(CH3)2, ‒N(CH3)CH2CH3, ‒CH2NH2, and ‒ CH 2 CH 2 NH 2 . In yet a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH2, ‒OH, ‒NO2, methyl, ‒CCl3, ‒CF3, ‒CHCl2, ‒ CHF 2 , ‒CH 2 Cl, ‒CH 2 F, ‒CH 2 CN, ‒CH 2 OH, ‒OCCl 3 , ‒OCF 3 , ‒OCHCl 2 , ‒OCHF 2 , ‒OCH 2 Cl, ‒OCH2F, ‒OCH3, ‒NHCH3, ‒N(CH3)2, and ‒CH2NH2. [00182] In various aspects, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkyl, and C2-C4 alkenyl. In a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒ F, ‒Cl, ‒CN, ‒NH2, ‒OH, ‒NO2, methyl, ethyl, n-propyl, isopropyl, ethenyl, and propenyl. In a still further aspect, each R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH2, ‒OH, ‒NO2, methyl, ethyl, and ethenyl. In yet a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, ‒ CN, ‒NH2, ‒OH, ‒NO2, and methyl. [00183] In various aspects, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, C1-C4 alkyl, and C2-C4 alkenyl. In a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, ethenyl, and propenyl. In a still further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, methyl, ethyl, and ethenyl. In yet a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen and methyl. [00184] In various aspects, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 haloalkyl, and C1-C4 cyanoalkyl. In a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , ‒CCl 3 , ‒CF 3 , ‒CHCl 2 , ‒CHF 2 , ‒CH 2 Cl, ‒CH 2 F, ‒CH2CH2Cl, ‒CH2CH2F, ‒CH2CH2CH2Cl, ‒CH2CH2CH2F, ‒CH(CH3)CH2Cl, ‒ CH(CH 3 )CH 2 F, ‒CH 2 CN, ‒CH 2 CH 2 CN, ‒CH 2 CH 2 CH 2 CN, and ‒CH(CH 3 )CH 2 CN. In a still further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒ F, ‒Cl, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , ‒CCl 3 , ‒CF 3 , ‒CHCl 2 , ‒CHF 2 , ‒CH 2 Cl, ‒CH 2 F, ‒ CH2CH2Cl, ‒CH2CH2F, ‒CH2CN, and ‒CH2CH2CN. In yet a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , ‒CCl3, ‒CF3, ‒CHCl2, ‒CHF2, ‒CH2Cl, ‒CH2F, and ‒CH2CN. [00185] In various aspects, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, C1-C4 haloalkyl, and C1-C4 cyanoalkyl. In a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒CCl 3 , ‒CF 3 , ‒CHCl 2 , ‒ CHF2, ‒CH2Cl, ‒CH2F, ‒CH2CH2Cl, ‒CH2CH2F, ‒CH2CH2CH2Cl, ‒CH2CH2CH2F, ‒ CH(CH 3 )CH 2 Cl, ‒CH(CH 3 )CH 2 F, ‒CH 2 CN, ‒CH 2 CH 2 CN, ‒CH 2 CH 2 CH 2 CN, and ‒ CH(CH3)CH2CN. In a still further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒CCl 3 , ‒CF 3 , ‒CHCl 2 , ‒CHF 2 , ‒CH 2 Cl, ‒CH 2 F, ‒CH 2 CH 2 Cl, ‒ CH2CH2F, ‒CH2CN, and ‒CH2CH2CN. In yet a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒CCl 3 , ‒CF 3 , ‒CHCl 2 , ‒CHF 2 , ‒CH 2 Cl, ‒CH 2 F, and ‒CH2CN. [00186] In various aspects, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, and C1-C4 alkoxy. In a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH2, ‒OH, ‒NO2, ‒CH2OH, ‒CH2CH2OH, ‒ CH 2 CH 2 CH 2 OH, ‒CH(CH 3 )CH 2 OH, ‒OCCl 3 , ‒OCF 3 , ‒OCHCl 2 , ‒OCHF 2 , ‒OCH 2 Cl, ‒ OCH2F, ‒OCH2CH2Cl, ‒OCH2CH2F, ‒OCH2CH2CH2Cl, ‒OCH2CH2CH2F, ‒ OCH(CH 3 )CH 2 Cl, ‒OCH(CH 3 )CH 2 F, ‒OCH 3 , ‒OCH 2 CH 3 , ‒OCH 2 CH 2 CH 3 , and ‒ OCH(CH3)2. In a still further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , ‒CH 2 OH, ‒CH 2 CH 2 OH, ‒OCCl 3 , ‒OCF3, ‒OCHCl2, ‒OCHF2, ‒OCH2Cl, ‒OCH2F, ‒OCH2CH2Cl, ‒OCH2CH2F, ‒OCH3, and ‒ OCH2CH3. In yet a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH2, ‒OH, ‒NO2, ‒CH2OH, ‒OCCl3, ‒OCF3, ‒ OCHCl2, ‒OCHF2, ‒OCH2Cl, ‒OCH2F, and ‒OCH3. [00187] In various aspects, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, and C1-C4 alkoxy. In a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒CH2OH, ‒CH 2 CH 2 OH, ‒CH 2 CH 2 CH 2 OH, ‒CH(CH 3 )CH 2 OH, ‒OCCl 3 , ‒OCF 3 , ‒OCHCl 2 , ‒OCHF 2 , ‒ OCH2Cl, ‒OCH2F, ‒OCH2CH2Cl, ‒OCH2CH2F, ‒OCH2CH2CH2Cl, ‒OCH2CH2CH2F, ‒ OCH(CH 3 )CH 2 Cl, ‒OCH(CH 3 )CH 2 F, ‒OCH 3 , ‒OCH 2 CH 3 , ‒OCH 2 CH 2 CH 3 , and ‒ OCH(CH3)2. In a still further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒CH 2 OH, ‒CH 2 CH 2 OH, ‒OCCl 3 , ‒OCF 3 , ‒OCHCl 2 , ‒OCHF 2 , ‒ OCH2Cl, ‒OCH2F, ‒OCH2CH2Cl, ‒OCH2CH2F, ‒OCH3, and ‒OCH2CH3. In yet a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒CH 2 OH, ‒OCCl3, ‒OCF3, ‒OCHCl2, ‒OCHF2, ‒OCH2Cl, ‒OCH2F, and ‒OCH3. [00188] In various aspects, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH2, ‒OH, ‒NO2, ‒NHCH3, ‒ NHCH 2 CH 3 , ‒NHCH 2 CH 2 CH 3 , ‒NHCH(CH 3 ) 2 , ‒N(CH 3 ) 2 , ‒N(CH 3 )CH 2 CH 3 , ‒ N(CH2CH3)CH2CH2CH3, ‒N(CH3)CH(CH3)2, ‒CH2NH2, ‒CH2CH2NH2, ‒CH2CH2CH2NH2, and ‒CH(CH 3 )CH 2 NH 2 . In a still further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH2, ‒OH, ‒NO2, ‒NHCH3, ‒ NHCH 2 CH 3 , ‒N(CH 3 ) 2 , ‒N(CH 3 )CH 2 CH 3 , ‒CH 2 NH 2 , ‒CH 2 CH 2 NH 2 . In yet a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒ NH 2 , ‒OH, ‒NO 2 , ‒NHCH 3 , ‒N(CH 3 ) 2 , and ‒CH 2 NH 2 . [00189] In various aspects, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒NHCH 3 , ‒NHCH 2 CH 3 , ‒NHCH 2 CH 2 CH 3 , ‒NHCH(CH 3 ) 2 , ‒N(CH 3 ) 2 , ‒ N(CH3)CH2CH3, ‒N(CH2CH3)CH2CH2CH3, ‒N(CH3)CH(CH3)2, ‒CH2NH2, ‒CH2CH2NH2, ‒ CH 2 CH 2 CH 2 NH 2 , and ‒CH(CH 3 )CH 2 NH 2 . In a still further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒NHCH3, ‒NHCH2CH3, ‒N(CH3)2, ‒ N(CH 3 )CH 2 CH 3 , ‒CH 2 NH 2 , and ‒CH 2 CH 2 NH 2 . In yet a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒NHCH3, ‒N(CH3)2, and ‒CH2NH2. [00190] In various aspects, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, and C1-C4 alkoxy. In a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, methyl, ethyl, n-propyl, isopropyl, ‒CCl3, ‒CF3, ‒CHCl2, ‒CHF2, ‒ CH 2 Cl, ‒CH 2 F, ‒CH 2 CH 2 Cl, ‒CH 2 CH 2 F, ‒CH 2 CH 2 CH 2 Cl, ‒CH 2 CH 2 CH 2 F, ‒ CH(CH3)CH2Cl, ‒CH(CH3)CH2F, ‒OCCl3, ‒OCF3, ‒OCHCl2, ‒OCHF2, ‒OCH2Cl, ‒OCH2F, ‒OCH 2 CH 2 Cl, ‒OCH 2 CH 2 F, ‒OCH 2 CH 2 CH 2 Cl, ‒OCH 2 CH 2 CH 2 F, ‒OCH(CH 3 )CH 2 Cl, ‒ OCH(CH3)CH2F, ‒OCH3, ‒OCH2CH3, ‒OCH2CH2CH3, and ‒OCH(CH3)2. In a still further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, methyl, ethyl, ‒CCl3, ‒CF3, ‒CHCl2, ‒CHF2, ‒CH2Cl, ‒CH2F, ‒CH2CH2Cl, ‒CH2CH2F, ‒ OCCl 3 , ‒OCF 3 , ‒OCHCl 2 , ‒OCHF 2 , ‒OCH 2 Cl, ‒OCH 2 F, ‒OCH 2 CH 2 Cl, ‒OCH 2 CH 2 F, ‒ OCH3, and ‒OCH2CH3. In yet a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, methyl, ‒CCl 3 , ‒CF 3 , ‒CHCl 2 , ‒CHF 2 , ‒ CH2Cl, ‒CH2F, ‒OCCl3, ‒OCF3, ‒OCHCl2, ‒OCHF2, ‒OCH2Cl, ‒OCH2F, and ‒OCH3. [00191] In various aspects, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen and halogen. In a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, ‒Cl, and ‒Br. In a still further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, ‒F, and ‒Cl. In yet a further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen and ‒F. In an even further aspect, each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen and ‒Cl. [00192] In various aspects, each of R 2a , R 2b , R 2c , R 2d , and R 2e is hydrogen. In a further aspect, at least one of R 2a , R 2b , R 2c , R 2d , and R 2e is hydrogen. In a still further aspect, two of R 2a , R 2b , R 2c , R 2d , and R 2e is hydrogen. In yet further aspect, three of R 2a , R 2b , R 2c , R 2d , and R 2e is hydrogen. In an even further aspect, four of R 2a , R 2b , R 2c , R 2d , and R 2e is hydrogen. c. R 3 G ROUPS [00193] In one aspect, R 3 is selected from hydrogen and C1-C4 alkyl. In a further aspect, R 3 is selected from hydrogen, methyl, ethyl, n-propyl, and isopropyl. In a still further aspect, R 3 is selected from hydrogen, methyl, and ethyl. In yet a further aspect, R 3 is selected from hydrogen and ethyl. In an even further aspect, R 3 is selected from hydrogen and methyl. [00194] In various aspects, R 3 is C1-C4 alkyl. In a further aspect, R 3 is selected from methyl, ethyl, n-propyl, and isopropyl. In a still further aspect, R 3 is selected from methyl and ethyl. In yet a further aspect, R 3 is ethyl. In an even further aspect, R 3 is methyl. [00195] In various aspects, R 3 is hydrogen. d. R 4A AND R 4B GROUPS [00196] In one aspect, each of R 4a and R 4b is independently selected from hydrogen and C1-C4 alkyl. In a further aspect, each of R 4a and R 4b is independently selected from hydrogen, methyl, ethyl, n-propyl, and isopropyl. In a still further aspect, each of R 4a and R 4b is independently selected from hydrogen, methyl, and ethyl. In yet a further aspect, each of R 4a and R 4b is independently selected from hydrogen and ethyl. In an even further aspect, each of R 4a and R 4b is independently selected from hydrogen and methyl. [00197] In various aspects, each of R 4a and R 4b is independently C1-C4 alkyl. In a further aspect, each of R 4a and R 4b is independently selected from methyl, ethyl, n-propyl, and isopropyl. In a still further aspect, each of R 4a and R 4b is independently selected from methyl and ethyl. In yet a further aspect, each of R 4a and R 4b is independently ethyl. In an even further aspect, each of R 4a and R 4b is independently methyl. [00198] In various aspects, each of R 4a and R 4b is independently hydrogen. e. R 5A , R 5B , AND R 5C G ROUPS [00199] In one aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH2, ‒OH, ‒ NO 2 , methyl, ethyl, n-propyl, isopropyl, ethenyl, propenyl, ‒CCl 3 , ‒CF 3 , ‒CHCl 2 , ‒CHF 2 , ‒ CH2Cl, ‒CH2F, ‒CH2CH2Cl, ‒CH2CH2F, ‒CH2CH2CH2Cl, ‒CH2CH2CH2F, ‒ CH(CH 3 )CH 2 Cl, ‒CH(CH 3 )CH 2 F, ‒CH 2 CN, ‒CH 2 CH 2 CN, ‒CH 2 CH 2 CH 2 CN, ‒ CH(CH3)CH2CN, ‒CH2OH, ‒CH2CH2OH, ‒CH2CH2CH2OH, ‒CH(CH3)CH2OH, ‒OCCl3, ‒ OCF 3 , ‒OCHCl 2 , ‒OCHF 2 , ‒OCH 2 Cl, ‒OCH 2 F, ‒OCH 2 CH 2 Cl, ‒OCH 2 CH 2 F, ‒ OCH2CH2CH2Cl, ‒OCH2CH2CH2F, ‒OCH(CH3)CH2Cl, ‒OCH(CH3)CH2F, ‒OCH3, ‒ OCH 2 CH 3 , ‒OCH 2 CH 2 CH 3 , ‒OCH(CH 3 ) 2 , ‒NHCH 3 , ‒NHCH 2 CH 3 , ‒NHCH 2 CH 2 CH 3 , ‒ NHCH(CH3)2, ‒N(CH3)2, ‒N(CH3)CH2CH3, ‒N(CH2CH3)CH2CH2CH3, ‒N(CH3)CH(CH3)2, ‒CH 2 NH 2 , ‒CH 2 CH 2 NH 2 , ‒CH 2 CH 2 CH 2 NH 2 , and ‒CH(CH 3 )CH 2 NH 2 . In a still further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒ NH 2 , ‒OH, ‒NO 2 , methyl, ethyl, ethenyl, ‒CCl 3 , ‒CF 3 , ‒CHCl 2 , ‒CHF 2 , ‒CH 2 Cl, ‒CH 2 F, ‒ CH2CH2Cl, ‒CH2CH2F, ‒CH2CN, ‒CH2CH2CN, ‒CH2OH, ‒CH2CH2OH, ‒OCCl3, ‒OCF3, ‒ OCHCl 2 , ‒OCHF 2 , ‒OCH 2 Cl, ‒OCH 2 F, ‒OCH 2 CH 2 Cl, ‒OCH 2 CH 2 F, ‒OCH 3 , ‒OCH 2 CH 3 , ‒ NHCH3, ‒NHCH2CH3, ‒N(CH3)2, ‒N(CH3)CH2CH3, ‒CH2NH2, and ‒CH2CH2NH2. In yet a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, ‒ CN, ‒NH2, ‒OH, ‒NO2, methyl, ‒CCl3, ‒CF3, ‒CHCl2, ‒CHF2, ‒CH2Cl, ‒CH2F, ‒CH2CN, ‒ CH 2 OH, ‒OCCl 3 , ‒OCF 3 , ‒OCHCl 2 , ‒OCHF 2 , ‒OCH 2 Cl, ‒OCH 2 F, ‒OCH 3 , ‒NHCH 3 , ‒ N(CH3)2, and ‒CH2NH2. [00200] In various aspects, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkyl, and C2-C4 alkenyl. In a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒ NH2, ‒OH, ‒NO2, methyl, ethyl, n-propyl, isopropyl, ethenyl, and propenyl. In a still further aspect, each R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH 2 , ‒OH, ‒NO2, methyl, ethyl, and ethenyl. In yet a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , and methyl. [00201] In various aspects, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, C1-C4 alkyl, and C2-C4 alkenyl. In a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, ethenyl, and propenyl. In a still further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, methyl, ethyl, and ethenyl. In yet a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen and methyl. [00202] In various aspects, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 haloalkyl, and C1-C4 cyanoalkyl. In a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, ‒ CN, ‒NH 2 , ‒OH, ‒NO 2 , ‒CCl 3 , ‒CF 3 , ‒CHCl 2 , ‒CHF 2 , ‒CH 2 Cl, ‒CH 2 F, ‒CH 2 CH 2 Cl, ‒ CH2CH2F, ‒CH2CH2CH2Cl, ‒CH2CH2CH2F, ‒CH(CH3)CH2Cl, ‒CH(CH3)CH2F, ‒CH2CN, ‒ CH 2 CH 2 CN, ‒CH 2 CH 2 CH 2 CN, and ‒CH(CH 3 )CH 2 CN. In a still further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH2, ‒OH, ‒NO2, ‒ CCl 3 , ‒CF 3 , ‒CHCl 2 , ‒CHF 2 , ‒CH 2 Cl, ‒CH 2 F, ‒CH 2 CH 2 Cl, ‒CH 2 CH 2 F, ‒CH 2 CN, and ‒ CH2CH2CN. In yet a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , ‒CCl 3 , ‒CF 3 , ‒CHCl 2 , ‒CHF 2 , ‒CH 2 Cl, ‒CH 2 F, and ‒CH2CN. [00203] In various aspects, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, C1-C4 haloalkyl, and C1-C4 cyanoalkyl. In a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒CCl3, ‒CF3, ‒CHCl2, ‒CHF2, ‒CH2Cl, ‒CH2F, ‒CH2CH2Cl, ‒CH2CH2F, ‒CH2CH2CH2Cl, ‒CH2CH2CH2F, ‒CH(CH3)CH2Cl, ‒ CH(CH3)CH2F, ‒CH2CN, ‒CH2CH2CN, ‒CH2CH2CH2CN, and ‒CH(CH3)CH2CN. In a still further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒CCl3, ‒ CF 3 , ‒CHCl 2 , ‒CHF 2 , ‒CH 2 Cl, ‒CH 2 F, ‒CH 2 CH 2 Cl, ‒CH 2 CH 2 F, ‒CH 2 CN, and ‒ CH2CH2CN. In yet a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒CCl 3 , ‒CF 3 , ‒CHCl 2 , ‒CHF 2 , ‒CH 2 Cl, ‒CH 2 F, and ‒CH 2 CN. [00204] In various aspects, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, and C1-C4 alkoxy. In a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , ‒CH 2 OH, ‒CH 2 CH 2 OH, ‒CH 2 CH 2 CH 2 OH, ‒ CH(CH3)CH2OH, ‒OCCl3, ‒OCF3, ‒OCHCl2, ‒OCHF2, ‒OCH2Cl, ‒OCH2F, ‒OCH2CH2Cl, ‒OCH 2 CH 2 F, ‒OCH 2 CH 2 CH 2 Cl, ‒OCH 2 CH 2 CH 2 F, ‒OCH(CH 3 )CH 2 Cl, ‒OCH(CH 3 )CH 2 F, ‒ OCH3, ‒OCH2CH3, ‒OCH2CH2CH3, and ‒OCH(CH3)2. In a still further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , ‒ CH2OH, ‒CH2CH2OH, ‒OCCl3, ‒OCF3, ‒OCHCl2, ‒OCHF2, ‒OCH2Cl, ‒OCH2F, ‒ OCH 2 CH 2 Cl, ‒OCH 2 CH 2 F, ‒OCH 3 , and ‒OCH 2 CH 3 . In yet a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH2, ‒OH, ‒NO2, ‒ CH 2 OH, ‒OCCl 3 , ‒OCF 3 , ‒OCHCl 2 , ‒OCHF 2 , ‒OCH 2 Cl, ‒OCH 2 F, and ‒OCH 3 . [00205] In various aspects, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, and C1-C4 alkoxy. In a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒CH2OH, ‒CH2CH2OH, ‒ CH 2 CH 2 CH 2 OH, ‒CH(CH 3 )CH 2 OH, ‒OCCl 3 , ‒OCF 3 , ‒OCHCl 2 , ‒OCHF 2 , ‒OCH 2 Cl, ‒ OCH2F, ‒OCH2CH2Cl, ‒OCH2CH2F, ‒OCH2CH2CH2Cl, ‒OCH2CH2CH2F, ‒ OCH(CH 3 )CH 2 Cl, ‒OCH(CH 3 )CH 2 F, ‒OCH 3 , ‒OCH 2 CH 3 , ‒OCH 2 CH 2 CH 3 , and ‒ OCH(CH3)2. In a still further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒CH 2 OH, ‒CH 2 CH 2 OH, ‒OCCl 3 , ‒OCF 3 , ‒OCHCl 2 , ‒OCHF 2 , ‒OCH 2 Cl, ‒ OCH2F, ‒OCH2CH2Cl, ‒OCH2CH2F, ‒OCH3, and ‒OCH2CH3. In yet a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒CH 2 OH, ‒OCCl 3 , ‒OCF 3 , ‒ OCHCl2, ‒OCHF2, ‒OCH2Cl, ‒OCH2F, and ‒OCH3. [00206] In various aspects, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH2, ‒OH, ‒NO2, ‒NHCH3, ‒ NHCH2CH3, ‒NHCH2CH2CH3, ‒NHCH(CH3)2, ‒N(CH3)2, ‒N(CH3)CH2CH3, ‒ N(CH2CH3)CH2CH2CH3, ‒N(CH3)CH(CH3)2, ‒CH2NH2, ‒CH2CH2NH2, ‒CH2CH2CH2NH2, and ‒CH(CH3)CH2NH2. In a still further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH2, ‒OH, ‒NO2, ‒NHCH3, ‒NHCH2CH3, ‒ N(CH 3 ) 2 , ‒N(CH 3 )CH 2 CH 3 , ‒CH 2 NH 2 , ‒CH 2 CH 2 NH 2 . In yet a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, ‒CN, ‒NH2, ‒OH, ‒NO2, ‒ NHCH 3 , ‒N(CH 3 ) 2 , and ‒CH 2 NH 2 . [00207] In various aspects, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒NHCH3, ‒ NHCH 2 CH 3 , ‒NHCH 2 CH 2 CH 3 , ‒NHCH(CH 3 ) 2 , ‒N(CH 3 ) 2 , ‒N(CH 3 )CH 2 CH 3 , ‒ N(CH2CH3)CH2CH2CH3, ‒N(CH3)CH(CH3)2, ‒CH2NH2, ‒CH2CH2NH2, ‒CH2CH2CH2NH2, and ‒CH(CH 3 )CH 2 NH 2 . In a still further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒NHCH3, ‒NHCH2CH3, ‒N(CH3)2, ‒N(CH3)CH2CH3, ‒CH2NH2, and ‒CH 2 CH 2 NH 2 . In yet a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒NHCH3, ‒N(CH3)2, and ‒CH2NH2. [00208] In various aspects, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, halogen, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 haloalkoxy, and C1-C4 alkoxy. In a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, methyl, ethyl, n-propyl, isopropyl, ‒CCl3, ‒CF3, ‒CHCl2, ‒CHF2, ‒CH2Cl, ‒CH2F, ‒ CH 2 CH 2 Cl, ‒CH 2 CH 2 F, ‒CH 2 CH 2 CH 2 Cl, ‒CH 2 CH 2 CH 2 F, ‒CH(CH 3 )CH 2 Cl, ‒ CH(CH3)CH2F, ‒OCCl3, ‒OCF3, ‒OCHCl2, ‒OCHF2, ‒OCH2Cl, ‒OCH2F, ‒OCH2CH2Cl, ‒ OCH 2 CH 2 F, ‒OCH 2 CH 2 CH 2 Cl, ‒OCH 2 CH 2 CH 2 F, ‒OCH(CH 3 )CH 2 Cl, ‒OCH(CH 3 )CH 2 F, ‒ OCH3, ‒OCH2CH3, ‒OCH2CH2CH3, and ‒OCH(CH3)2. In a still further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, methyl, ethyl, ‒CCl 3 , ‒CF 3 , ‒ CHCl2, ‒CHF2, ‒CH2Cl, ‒CH2F, ‒CH2CH2Cl, ‒CH2CH2F, ‒OCCl3, ‒OCF3, ‒OCHCl2, ‒ OCHF 2 , ‒OCH 2 Cl, ‒OCH 2 F, ‒OCH 2 CH 2 Cl, ‒OCH 2 CH 2 F, ‒OCH 3 , and ‒OCH 2 CH 3 . In yet a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, methyl, ‒CCl 3 , ‒CF 3 , ‒CHCl 2 , ‒CHF 2 , ‒CH 2 Cl, ‒CH 2 F, ‒OCCl 3 , ‒OCF 3 , ‒OCHCl 2 , ‒ OCHF2, ‒OCH2Cl, ‒OCH2F, and ‒OCH3. [00209] In various aspects, each of R 5a , R 5b , and R 5c is independently selected from hydrogen and halogen. In a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, ‒Cl, and ‒Br. In a still further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen, ‒F, and ‒Cl. In yet a further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen and ‒F. In an even further aspect, each of R 5a , R 5b , and R 5c is independently selected from hydrogen and ‒Cl. [00210] In various aspects, each of R 5a , R 5b , and R 5c is hydrogen. In a further aspect, at least one of R 5a , R 5b , and R 5c is hydrogen. In a still further aspect, two of R 5a , R 5b , and R 5c is hydrogen. f. R 6A AND R 6B GROUPS [00211] In one aspect, R 6a is halogen and R 6b is selected from hydrogen and halogen. [00212] In one aspect, each of R 6a and R 6b is independently selected from hydrogen and halogen. In a further aspect, each of R 6a and R 6b is independently selected from hydrogen, fluoro, chloro, and bromo. In a still further aspect, each of R 6a and R 6b is independently selected from hydrogen, fluoro, and chloro. In yet a further aspect, each of R 6a and R 6b is independently selected from hydrogen and fluoro. In an even further aspect, each of R 6a and R 6b is independently selected from hydrogen and chloro. [00213] In various aspects, R 6a is halogen. In a further aspect, R 6a is selected from fluoro, chloro, and bromo. In a still further aspect, R 6a is selected from fluoro, chloro, and iodo. In yet a further aspect, R 6a is selected from fluoro, bromo, and iodo. In an even further asect, R 6a is selected from chloro, bromo, and iodo. In a still further aspect, R 6a is selected from fluoro and chloro. In yet a further asect, R 6a is selected from fluoro and bromo. In an even further aspect, R 6a is selected from fluoro and iodo. In a still further aspect, R 6a is selected from chloro and bromo. In yet a further aspect, R 6a is selected from chloro and iodo. In an even further aspect, R 6a is selected from bromo and iodo. In a still further aspect, R 6a is iodo. In yet a further aspect, R 6a is bromo. In an even further aspect, R 6a is chloro. In a still further aspect, R 6a is fluoro. [00214] In various aspects, R 6b is selected from hydrogen and halogen. In a further aspect, R 6b is selected from hydrogen, fluoro, chloro, and bromo. In a still further aspect, R 6b is selected from hydrogen, fluoro, and chloro. In yet a further aspect, R 6b is selected from hydrogen and fluoro. In an even further aspect, R 6b is selected from hydrogen and chloro. [00215] In various aspects, R 6b is halogen. In a further aspect, R 6b is selected from fluoro, chloro, and bromo. In a still further aspect, R 6b is selected from fluoro, chloro, and iodo. In yet a further aspect, R 6b is selected from fluoro, bromo, and iodo. In an even further asect, R 6b is selected from chloro, bromo, and iodo. In a still further aspect, R 6b is selected from fluoro and chloro. In yet a further asect, R 6b is selected from fluoro and bromo. In an even further aspect, R 6b is selected from fluoro and iodo. In a still further aspect, R 6b is selected from chloro and bromo. In yet a further aspect, R 6b is selected from chloro and iodo. In an even further aspect, R 6b is selected from bromo and iodo. In a still further aspect, R 6b is iodo. In yet a further aspect, R 6b is bromo. In an even further aspect, R 6b is chloro. In a still further aspect, R 6b is fluoro. [00216] In various aspects, R 6a is hydrogen. [00217] In various aspects, R 6b is hydrogen. [00218] In one aspect, each of R 6a and R 6b is independently halogen. In a further aspect, each of R 6a and R 6b is independently selected from fluoro, chloro, and bromo. In a still further aspect, each of R 6a and R 6b is independently selected from fluoro and chloro. In yet a further aspect, each of R 6a and R 6b is fluoro. In an even further aspect, each of R 6a and R 6b is chloro. g. CY 1 GROUPS [00219] In one aspect, Cy 1 is a C3-C8 cycloalkyl substituted with 0, 1, 2, 3, or 4 groups independently selected from halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1- C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In a further aspect, Cy 1 is a C3-C8 cycloalkyl substituted with 0, 1, 2, or 3 groups independently selected from halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In yet a further aspect, Cy 1 is a C3-C8 cycloalkyl substituted with 0, 1, or 2 groups independently selected from halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1- C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In an even further aspect, Cy 1 is a C3-C8 cycloalkyl substituted with 0 or 1 group selected from halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1- C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In a still further aspect, Cy 1 is a C3-C8 cycloalkyl monosubstituted with a group selected from halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In yet a further aspect, Cy 1 is an unsubstituted C3-C8 cycloalkyl. [00220] In various aspects, Cy 1 is a C3-C6 cycloalkyl substituted with 0, 1, 2, 3, or 4 groups independently selected from halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1- C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In a further aspect, Cy 1 is a C3-C6 cycloalkyl substituted with 0, 1, 2, or 3 groups independently selected from halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In yet a further aspect, Cy 1 is a C3-C6 cycloalkyl substituted with 0, 1, or 2 groups independently selected from halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1- C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In an even further aspect, Cy 1 is a C3-C6 cycloalkyl substituted with 0 or 1 group selected from halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1- C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In a still further aspect, Cy 1 is a C3-C6 cycloalkyl monosubstituted with a group selected from halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In yet a further aspect, Cy 1 is an unsubstituted C3-C6 cycloalkyl. [00221] In various aspects, Cy 1 is a cyclopentyl substituted with 0, 1, 2, 3, or 4 groups independently selected from halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In a further aspect, Cy 1 is a cyclopentyl substituted with 0, 1, 2, or 3 groups independently selected from halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1- C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In yet a further aspect, Cy 1 is a cyclopentyl substituted with 0, 1, or 2 groups independently selected from halogen, ‒CN, ‒ NH2, ‒OH, ‒NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In an even further aspect, Cy 1 is a cyclopentyl substituted with 0 or 1 group selected from halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In a still further aspect, Cy 1 is a cyclopentyl monosubstituted with a group selected from halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In yet a further aspect, Cy 1 is an unsubstituted cyclopentyl. 2. EXAMPLE COMPOUNDS [00222] In one aspect, a compound can be present as:
or a pharmaceutically acceptable salt thereof. [00223] In one aspect, a compound can be present as: , or a pharmaceutically acceptable salt thereof. [00224] In one aspect, a compound can be present as: or a pharmaceutically acceptable salt thereof. [00225] It is contemplated that one or more compounds can optionally be omitted from the disclosed invention. [00226] It is understood that the disclosed compounds can be used in connection with the disclosed methods, compositions, kits, and uses. [00227] It is understood that pharmaceutical acceptable derivatives of the disclosed compounds can be used also in connection with the disclosed methods, compositions, kits, and uses. The pharmaceutical acceptable derivatives of the compounds can include any suitable derivative, such as pharmaceutically acceptable salts as discussed below, isomers, radiolabeled analogs, tautomers, and the like. D. PHARMACEUTICAL COMPOSITIONS [00228] In one aspect, disclosed are pharmaceutical compositions comprising an effective amount of a disclosed compound and a pharmaceutically acceptable carrier. [00229] Thus, in one aspect, disclosed are pharmaceutical compositions comprising an effective amount of a compound having a structure represented by a formula:
wherein R 1 is selected from hydrogen and C1-C4 alkyl; wherein each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl; wherein R 3 is selected from hydrogen and C1-C4 alkyl; wherein each of R 4a and R 4b is independently selected from hydrogen, halogen, and C1-C4 alkyl; wherein each of R 5a , R 5b , and R 5c is independently selected from hydrogen, halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1- C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl; wherein each of R 6a and R 6b is independently selected from hydrogen and halogen; and wherein Cy 1 is a C3-C8 cycloalkyl substituted with 0, 1, 2, 3, or 4 groups independently selected from halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl, provided that at least one of R 6a and R 6b is halogen, or a pharmaceutically acceptable salt thereof., and a pharmaceutically acceptable carrier. [00230] In various aspects, the compound is a structure selected from: , Cl , or a pharmaceutically acceptable salt thereof. [00231] In various aspects, the compound is: or a pharmaceutically acceptable salt thereof. [00232] In various aspects, the compounds and compositions of the invention can be administered in pharmaceutical compositions, which are formulated according to the intended method of administration. The compounds and compositions described herein can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. For example, a pharmaceutical composition can be formulated for local or systemic administration, intravenous, topical, or oral administration. [00233] The nature of the pharmaceutical compositions for administration is dependent on the mode of administration and can readily be determined by one of ordinary skill in the art. In various aspects, the pharmaceutical composition is sterile or sterilizable. The therapeutic compositions featured in the invention can contain carriers or excipients, many of which are known to skilled artisans. Excipients that can be used include buffers (for example, citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, polypeptides (for example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, water, and glycerol. The nucleic acids, polypeptides, small molecules, and other modulatory compounds featured in the invention can be administered by any standard route of administration. For example, administration can be parenteral, intravenous, subcutaneous, or oral. A modulatory compound can be formulated in various ways, according to the corresponding route of administration. For example, liquid solutions can be made for administration by drops into the ear, for injection, or for ingestion; gels or powders can be made for ingestion or topical application. Methods for making such formulations are well known and can be found in, for example, Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, PA 1990. [00234] In various aspects, the disclosed pharmaceutical compositions comprise the disclosed compounds (including pharmaceutically acceptable salt(s) thereof) as an active ingredient, a pharmaceutically acceptable carrier, and, optionally, other therapeutic ingredients or adjuvants. The instant compositions include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. [00235] In various aspects, the pharmaceutical compositions of this invention can include a pharmaceutically acceptable carrier and a compound or a pharmaceutically acceptable salt of the compounds of the invention. The compounds of the invention, or pharmaceutically acceptable salts thereof, can also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds. [00236] The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen. [00237] In preparing the compositions for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques. [00238] A tablet containing the composition of this invention can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. [00239] The pharmaceutical compositions of the present invention comprise a compound of the invention (or pharmaceutically acceptable salts thereof) as an active ingredient, a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents or adjuvants. The instant compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. [00240] Pharmaceutical compositions of the present invention suitable for parenteral administration can be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms. [00241] Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid for easy syringability. The pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof. [00242] Pharmaceutical compositions of the present invention can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles, and the like. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations can be prepared, utilizing a compound of the invention, or pharmaceutically acceptable salts thereof, via conventional processing methods. As an example, a cream or ointment is prepared by mixing hydrophilic material and water, together with about 5 wt% to about 10 wt% of the compound, to produce a cream or ointment having a desired consistency. [00243] Pharmaceutical compositions of this invention can be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in molds. [00244] In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing a compound of the invention, and/or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form. [00245] In a further aspect, an effective amount is a therapeutically effective amount. In a still further aspect, an effective amount is a prophylactically effective amount. [00246] In a further aspect, the pharmaceutical composition is administered to a mammal. In a still further aspect, the mammal is a human. In an even further aspect, the human is a patient. [00247] In a further aspect, the pharmaceutical composition is used to treat a disorder associated with overexpression of an EYA protein. In a still further aspect the disorder is a vascular disease, a fibrosis-related disorder, hearing loss, or a metabolic disease. [00248] In a further aspect, the pharmaceutical composition is used to treat a cancer that comprises a tumor that overexpress at least one EYA protein. In a still further aspect, the cancer is breast cancer, cervical cancer, ovarian cancer, liver cancer, or pancreatic cancer. In yet a further aspect, the cancer is triple-negative breast cancer (TNBC). In an even further aspect, the cancer is a pediatric cancer (e.g., leukemia). [00249] It is understood that the disclosed compositions can be prepared from the disclosed compounds. It is also understood that the disclosed compositions can be employed in the disclosed methods of using. E. M ETHODS OF M AKING A C OMPOUND [00250] The compounds of this invention can be prepared by employing reactions as shown in the following schemes, in addition to other standard manipulations that are known in the literature, exemplified in the experimental sections or clear to one skilled in the art. For clarity, examples having a single substituent are shown where multiple substituents are allowed under the definitions disclosed herein. [00251] Preferred methods include, but are not limited to, those described below. During any of the following synthetic sequences, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This can be achieved by means of conventional protecting groups, such as those described in T. W. Greene, Protective Groups in Organic Chemistry, John Wiley & Sons, 1981; and T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Chemistry, John Wiley & Sons, 1991, which are hereby incorporated by reference. [00252] Reactions used to generate the compounds of this invention are prepared by employing reactions as shown in the following Reaction Schemes, as described and exemplified below. In certain specific examples, the disclosed compounds can be prepared by Route I, as described and exemplified below. The following examples are provided so that the invention might be more fully understood, are illustrative only, and should not be construed as limiting. 1. R OUTE I [00253] In one aspect, substituted quinazoline-2,4-diamines, or their pharmaceutically acceptable salts, can be prepared as shown below. Isolation and purification of the products is accomplished by standard procedures, which are known to a chemist of ordinary skill. S CHEME 1A. [00254] Compounds are represented in generic form, wherein X 1 and X 2 are independently halogen, and with substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below. S CHEME 1B. [00255] Referring to Scheme 1B above, condensation of an appropriate 2,4- dichloroquinazoline, e.g., 1.6 as shown above, with an appropriately substituted benzyl amine, e.g., 1.7 as shown above, in a suitable solvent such as THF, 2-methyl THF, DCE, or dioxane, at temperatures ranging from room temperature to 100 o C, produces the desired mon-substituted aminoquinazoline, e.g., 1.8 as shown above. Appropriate quinazolines and appropriate benzyl amines are commercially available or prepared by methods known to one skilled in the art. Preferred conditions for this reaction include the use of THF as the solvent at room temperature. Treatment of an appropriate aminoquinazoline, e.g., 1.8 as shown above, with an appropriate cyclic amine, e.g., 1.9 as shown above, in a suitable alcoholic solvent such as sec-butanol, butanol, pentanol, ethanol, or t-butyl alcohol, under microwave irradiation conditions (120-180 o C), produces the desired quinazoline-2,4-diamine, e.g., 1.10 as shown above. Appropriate cyclic amines are commercially available or prepared by methods known to one skilled in the art. Preferred conditions for this reaction include the use of sec-butanol as the solvent under microwave irradiation conditions at a temperature of 180 o C. As can be appreciated by one skilled in the art, the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 1.1, 1.2, 1.3, and 1.4), can be substituted in the reaction to provide substituted substituted quinazoline-2,4-diamine derivatives similar to Formula 1.5. [00256] Pharmaceutically acceptable salts of the disclosed compounds include the acid or base addition salts thereof. All salt formation reactions are typically carried out in solution. The resulting salt may precipitate out and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionization in the resulting salt may vary from completely ionized to almost non-ionized. Suitable non-toxic, acid-addition pharmaceutically acceptable salts include, but are not limited to, the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mandelates mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, salicylate, saccharate, stearate, succinate, sulfonate, stannate, tartrate, tosylate, trifluoroacetate and xinofoate salts. [00257] Suitable non-toxic, base-addition pharmaceutically acceptable salts include, but are not limited to, the aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. For a review on suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002). [00258] Also included within the scope of the present invention are all stereoisomers, geometric isomers, and tautomeric forms of the disclosed compounds, including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof. [00259] The present invention also includes all pharmaceutically acceptable isotopically-labeled analogs of the disclosed compounds, in which one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number which predominates in nature. F. METHODS OF TREATING A DISORDER ASSOCIATED WITH OVEREXPRESSION OF AN EYA PROTEIN [00260] In one aspect, disclosed are methods of treating a disorder associated with overexpression of an EYA protein in a subject in need thereof, the method comprising administering to the subject an effective amount of a disclosed compound, thereby treating the disorder. Examples of disorders for which the disclosed compounds, compositions, and methods can be useful include, but are not limited to, vascular diseases, fibrosis-related disorders, cranio-facial diseases, hearing loss, metabolic diseases, and cancer (e.g., a cancer that comprises a tumor that overexpresses at least one EYA protein). [00261] Thus, in one aspect, disclosed are methods of treating a disorder associated with overexpression of an eyes absent (EYA) protein in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound having a structure represented by a formula: wherein R 1 is selected from hydrogen and C1-C4 alkyl; wherein each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl; wherein R 3 is selected from hydrogen and C1-C4 alkyl; wherein each of R 4a and R 4b is independently selected from hydrogen, halogen, and C1-C4 alkyl; wherein each of R 5a , R 5b , and R 5c is independently selected from hydrogen, halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1- C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl; wherein each of R 6a and R 6b is independently selected from hydrogen and halogen; and wherein Cy 1 is a C3-C8 cycloalkyl substituted with 0, 1, 2, 3, or 4 groups independently selected from halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl, provided that at least one of R 6a and R 6b is halogen, or a pharmaceutically acceptable salt thereof, wherein the disorder is a vascular disease, a fibrosis-related disorder, a cranio-facial disease, hearing loss, or a metabolic disease. [00262] In various aspects, the disorder is associated with overexpression of eyes absent homolog 4 (EYA4) or eyes absent homolog 2 (EYA2). In a further aspect, the disorder is associated with overexpression of EYA4. In a still further aspect, the disorder is associated with overexpression of EYA2. [00263] In various aspects, the compound modulates expression of one or more EYA proteins. In a further aspect, the compound modulates expression of EYA4 or EYA2. In a still further aspect, the compound modulates expression of EYA4. In yet a further aspect, the compound modulates expression of EYA2. In an even further aspect, the compound modulates expression of EYA4 and EYA2 [00264] In various aspects, the compound inhibits expression of one or more EYA proteins. In a further aspect, the compound inhibits expression of EYA2 or EYA4. In a still further aspect, the compound inhibits expression of EYA4. In yet a further aspect, the compound inhibits expression of EYA2. In an even further aspect, the compound inhibits expression of EYA2 and EYA4. [00265] In various aspects, R 6a is halogen. In a still further aspect, either: (a) R 6b is chloro; (b) R 6b is hydrogen; or (c) R 6b is halogen and at least one of R 5a , R 5b , and R 5c is not hydrogen. [00266] In various aspects, R 1 is hydrogen. [00267] In various aspects, each of R 2a , R 2b , R 2c , R 2d , and R 2e is hydrogen. [00268] In various aspects, R 3 is hydrogen. [00269] In various aspects, each of R 4a and R 4b is hydrogen. [00270] In various aspects, each of R 5a , R 5b , and R 5c is hydrogen. [00271] In various aspects, R 6a is chloro. [00272] In various aspects, R 6b is halogen. In a further aspect, R 6b is chloro. [00273] In various aspects, R 6b is hydrogen. [00274] In various aspects, Cy 1 is an unsubstituted C3-C8 cycloalkyl. In a further aspect, Cy 1 is an unsubstituted C3-C6 cycloalkyl. In a still further aspect, Cy 1 is a cyclopentyl substituted with 0, 1, 2, 3, or 4 groups independently selected from halogen, ‒ CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In yet a further aspect, Cy 1 is an unsubstituted cyclopentyl. [00275] In various aspects, the compound has a structure represented by a formula: or a pharmaceutically acceptable salt thereof. [00276] In various aspects, the compound has a structure represented by a formula: or a pharmaceutically acceptable salt thereof. [00277] In various aspects, the compound has a structure represented by a formula: or a pharmaceutically acceptable salt thereof. [00278] In various aspects, the compound has a structure represented by a formula: or a pharmaceutically acceptable salt thereof. [00279] In various aspects, the compound has a structure represented by a formula: or a pharmaceutically acceptable salt thereof. [00280] In various aspects, the compound is a structure selected from:
Cl or a pharmaceutically acceptable salt thereof. [00281] In various aspects, the compound is:
or a pharmaceutically acceptable salt thereof. [00282] In a further aspect, the subject has been diagnosed with a need for treatment of modification of the expression of one or more EYA proteins prior to the administering step. In a still further aspect, the subject has been diagnosed with a need for treatment of the disorder prior to the administering step. [00283] In a further aspect, the subject is a mammal. In a still further aspect, the mammal is a human. [00284] In a further aspect, the method further comprises the step of identifying a subject in need of treatment of the disorder. [00285] In various aspects, the disorder is a vascular disease. Examples of vascular diseases include, but are not limited to dilated cardiomyopathy, cardiac hypertrophy, hypoxia-induced angiogenesis, atherosclerosis, peripheral artery disease, pericardial edema, carotid artery disease, a pulmonary embolism, collagen vascular disease, and cerebrovascular disease. [00286] In various aspects, the disorder is a fibrosis-related disorder. Examples of fibrosis-related disorders include, but are not limited to, scleroderma, pulmonary fibrosis, rheumatoid arthritis, Crohn’s disease, ulcerative colitis, myelofibrosis, systemic lupus erythematosus, idiopathic pulmonary fibrosis, non-alcoholic steatohepatitis (NASH), systemic sclerosis, and interstitial lung disease. [00287] In various aspects, the disorder is a cranio-facial disease. Examples of cranio- facial diseases include, but are not limited to, a cleft palate, mandibulofacial dysostosis, hemifacial macrosomia, plagiocephaly, Stickler syndrome, scaphocephaly, craniosynostosis, Apert syndrome, encephalocele, Saethre-Chotzen syndrome, Nager syndrome, fibrous dysplasia of bone, Down syndrome, a craniofacial cleft, and velocardiofacial syndrome. In a further aspect, the cranio-facial disease is a developmental syndrome or a heritable otitis. [00288] In various aspects, the disorder is hearing loss. [00289] In various aspects, the disorder is a metabolic disease. Examples of metabolic diseases include, but are not limited to, familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabbe disease, Maple syrip urine disease, metachromatic leukodystrophy, mitochondrial encephalopathy, lactic acidosis, stroke-like episodes (MELAS), Niemann-Pick, phenylketonuria (PKU), porphyria, Tay-Sachs disease, and Wilson’s disease. In various further aspects, the metabolic disease is a fatty liver disease or a neoplasia (e.g., neoplasia of the heart or the liver). [00290] In a further aspect, the effective amount is a therapeutically effective amount. In a still further aspect, the effective amount is a prophylactically effective amount. [00291] In a further aspect, the method further comprises administering to the subject an agent known to treat a vascular disease. Examples of agents known to treat a vascular disease include, but are not limited to, antiplatelet medicines (e.g., aspirin, clopidogrel), statins, angiotensin II receptor blockers (ARBs), and ACE inhibitors. [00292] In a further aspect, the method further comprises administering to the subject an anti-fibrotic agent. Examples of anti-fibrotic agents include, but are not limited to, nintedanib, pirfenidone, corticosteroids, azathioprine, chloroquines, and ruxolitinib. [00293] In a further aspect, the method further comprises administering to the subject an agent known to treat hearing loss. Examples of agents known to treat hearing loss include, but are not limited to, aminoglycoside antibiotics, platinum-containing chemotherapy agents, loop diuretics (e.g., furosemide), and nonsteroidal anti-inflammatory agents. [00294] In a further aspect, the method further comprises administering to the subject an agent known to treat a metabolic disease. Examples of agents known to treat a metabolic disease include, but are not limited to, ACE inhibitors, angiotensin receptor blockers, diuretics, beta blockers, statins, niacin, omega fatty acids, and insulin sensitizers (e.g., thiazolidinediones). [00295] In a further aspect, the compound and the agent are administered sequentially. In a still further aspect, the compound and the agent are administered simultaneously. [00296] In a further aspect, the compound and the agent are co-formulated. In a still further aspect, the compound and the agent are co-packaged. [00297] In a further aspect, the compound is administered as a single active agent. G. METHODS OF TREATING A CANCER THAT COMPRISES A TUMOR THAT OVEREXPRESSES AT LEAST ONE EYA PROTEIN [00298] In one aspect, disclosed are methods of treating treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a disclosed compound, wherein the cancer comprises a tumor that overexpresses at least one eyes absent (EYA) protein. [00299] Thus, in one aspect, disclosed are methods of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound having a structure represented by a formula: wherein R 1 is selected from hydrogen and C1-C4 alkyl; wherein each of R 2a , R 2b , R 2c , R 2d , and R 2e is independently selected from hydrogen, halogen, ‒CN, ‒NH 2 , ‒OH, ‒NO 2 , C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl; wherein R 3 is selected from hydrogen and C1-C4 alkyl; wherein each of R 4a and R 4b is independently selected from hydrogen, halogen, and C1-C4 alkyl; wherein each of R 5a , R 5b , and R 5c is independently selected from hydrogen, halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1- C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl; wherein each of R 6a and R 6b is independently selected from hydrogen and halogen; and wherein Cy 1 is a C3-C8 cycloalkyl substituted with 0, 1, 2, 3, or 4 groups independently selected from halogen, ‒CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl, provided that at least one of R 6a and R 6b is halogen, or a pharmaceutically acceptable salt thereof, wherein the cancer comprises a tumor that overexpresses at least one eyes absent (EYA) protein. [00300] In various aspects, the tumor overexpresses EYA4. In a further aspect, the tumor overexpresses EYA2. [00301] In various aspects, R 6a is halogen. In a still further aspect, either: (a) R 6b is chloro; (b) R 6b is hydrogen; or (c) R 6b is halogen and at least one of R 5a , R 5b , and R 5c is not hydrogen. [00302] In various aspects, R 1 is hydrogen. [00303] In various aspects, each of R 2a , R 2b , R 2c , R 2d , and R 2e is hydrogen. [00304] In various aspects, R 3 is hydrogen. [00305] In various aspects, each of R 4a and R 4b is hydrogen. [00306] In various aspects, each of R 5a , R 5b , and R 5c is hydrogen. [00307] In various aspects, R 6a is chloro. [00308] In various aspects, R 6b is halogen. In a further aspect, R 6b is chloro. [00309] In various aspects, R 6b is hydrogen. [00310] In various aspects, Cy 1 is an unsubstituted C3-C8 cycloalkyl. In a further aspect, Cy 1 is an unsubstituted C3-C6 cycloalkyl. In a still further aspect, Cy 1 is a cyclopentyl substituted with 0, 1, 2, 3, or 4 groups independently selected from halogen, ‒ CN, ‒NH2, ‒OH, ‒NO2, C1-C4 alkyl, C2-C4 alkenyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 alkylamino, (C1-C4)(C1-C4) dialkylamino, and C1-C4 aminoalkyl. In yet a further aspect, Cy 1 is an unsubstituted cyclopentyl. [00311] In various aspects, the compound has a structure represented by a formula: or a pharmaceutically acceptable salt thereof. [00312] In various aspects, the compound has a structure represented by a formula:
or a pharmaceutically acceptable salt thereof. [00313] In various aspects, the compound has a structure represented by a formula: or a pharmaceutically acceptable salt thereof. [00314] In various aspects, the compound has a structure represented by a formula: or a pharmaceutically acceptable salt thereof. [00315] In various aspects, the compound has a structure represented by a formula:
or a pharmaceutically acceptable salt thereof. [00316] In various aspects, the compound is a structure selected from:
or a pharmaceutically acceptable salt thereof. [00317] In various aspects, the compound is: or a pharmaceutically acceptable salt thereof. [00318] In a further aspect, the subject has been diagnosed with a need for treatment of cancer prior to the administering step. In a still further aspect, the subject is at risk for developing cancer prior to the administering step. [00319] In a further aspect, the subject is a mammal. In a still further aspect, the mammal is a human. [00320] In a further aspect, the method further comprises the step of identifying a subject in need of treatment of cancer. [00321] In various aspects, the cancer is selected from a sarcoma, a carcinoma, a hematological cancer, a solid tumor, breast cancer, cervical cancer, gastrointestinal cancer, colorectal cancer, brain cancer, skin cancer, prostate cancer, ovarian cancer, thyroid cancer, testicular cancer, pancreatic cancer, liver cancer, endometrial cancer, melanoma, a glioma, leukemia, lymphoma, chronic myeloproliferative disorder, myelodysplastic syndrome, myeloproliferative neoplasm, non-small cell lung carcinoma, and plasma cell neoplasm (myeloma). In a further aspect, the cancer is breast cancer, cervical cancer, ovarian cancer, liver cancer, or pancreatic cancer. In a still further aspect, the cancer is triple-negative breast cancer (TNBC). [00322] In various aspects, treating cancer comprises limiting metasis and overcoming chemotherapy resistance caused by endoreplication and genomic rearrangements. In a further aspect, the endoreplication is in response to stress. In a further aspect, the endoreplication is a result of missed mitosis initiation. [00323] In various aspects, the cancer is a pediatric cancer. In a further aspect, the pediatric cancer is Ewing sarcoma, osteosarcoma, leukemia, neuroblastoma, retinoblastoma, or melanoma. In a still further aspect, the pediatric cancer is leukemia. [00324] In a further aspect, the effective amount is a therapeutically effective amount. In a still further aspect, the effective amount is a prophylactically effective amount. [00325] In a further aspect, the method further comprises administering a chemotherapeutic agent to the subject. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., carboplatin, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmustine, busulfan, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, temozolomide, thiotepa, bendamustine, streptozocin), antimetabolite agents (e.g., gemcitabine, 5-fluorouracil, capecitabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nelarabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, methotrexate, thioguanine), antineoplastic antibiotic agents (e.g., doxorubicin, mitoxantrone, bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, valrubicin), mitotic inhibitor agents (e.g., irinotecan, topotecan, rubitecan, cabazitaxel, docetaxel, paclitaxel, etopside, vincristine, ixabepilone, vinorelbine, vinblastine, teniposide), and mTor inhibitor agents (e.g., everolimus, siroliumus, temsirolimus). [00326] In a further aspect, the compound and the agent are administered sequentially. In a still further aspect, the compound and the agent are administered simultaneously. [00327] In a further aspect, the compound and the agent are co-formulated. In a still further aspect, the compound and the agent are co-packaged. [00328] In a further aspect, the compound is administered as a single active agent. H. ADDITIONAL METHODS OF USING THE COMPOUNDS [00329] The compounds and pharmaceutical compositions of the invention are useful in treating or controlling disorders associated with overexpression of an eyes absent (EYA) protein in its wild type or mutated version (e.g., a vascular disease, a fibrosis-related disorder, hearing loss, a metabolic disease). The compounds and pharmaceutical compositions of the invention are also useful in treating or controlling cancer that comprises a tumor that overexpresses at least one eyes absent (EYA) protein. [00330] To treat or control the condition, the compounds and pharmaceutical compositions comprising the compounds are administered to a subject in need thereof, such as a vertebrate, e.g., a mammal, a fish, a bird, a reptile, or an amphibian. The subject can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. The subject is preferably a mammal, such as a human. Prior to administering the compounds or compositions, the subject can be diagnosed with a need for treatment of a disorder associated with overexpression of an eyes absent (EYA) protein (e.g., a vascular disease, a fibrosis-related disorder, hearing loss, a metabolic disease) or with a need for treatment of a cancer that comprises a tumor that overexpresses at least one eyes absent (EYA) protein. [00331] The compounds or compositions can be administered to the subject according to any method. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. A preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. A preparation can also be administered prophylactically; that is, administered for prevention of a disorder associated with overexpression of an eyes absent (EYA) protein (e.g., a vascular disease, a fibrosis-related disorder, hearing loss, a metabolic disease) or for prevention of a cancer that comprises a tumor that overexpresses at least one eyes absent (EYA) protein. [00332] The therapeutically effective amount or dosage of the compound can vary within wide limits. Such a dosage is adjusted to the individual requirements in each particular case including the specific compound(s) being administered, the route of administration, the condition being treated, as well as the patient being treated. In general, in the case of oral or parenteral administration to adult humans weighing approximately 70 Kg or more, a daily dosage of about 10 mg to about 10,000 mg, preferably from about 200 mg to about 1,000 mg, should be appropriate, although the upper limit may be exceeded. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, as a continuous infusion. Single dose compositions can contain such amounts or submultiples thereof of the compound or composition to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. 2. USE OF COMPOUNDS [00333] In one aspect, the invention relates to the use of a disclosed compound or a product of a disclosed method. In a further aspect, a use relates to the manufacture of a medicament for the treatment of a disorder associated with overexpression of an eyes absent (EYA) protein (e.g., a vascular disease, a fibrosis-related disorder, hearing loss, a metabolic disease). In a still further aspect, a use relates to the manufacture of a medicament for the treatment of a cancer that comprises a tumor that overexpresses at least one eyes absent (EYA) protein. [00334] Also provided are the uses of the disclosed compounds and products. In one aspect, the invention relates to use of at least one disclosed compound; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof. In a further aspect, the compound used is a product of a disclosed method of making. [00335] In a further aspect, the use relates to a process for preparing a pharmaceutical composition comprising a therapeutically effective amount of a disclosed compound or a product of a disclosed method of making, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, for use as a medicament. [00336] In a further aspect, the use relates to a process for preparing a pharmaceutical composition comprising a therapeutically effective amount of a disclosed compound or a product of a disclosed method of making, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, wherein a pharmaceutically acceptable carrier is intimately mixed with a therapeutically effective amount of the compound or the product of a disclosed method of making. [00337] In various aspects, the use relates to a treatment of a disorder associated with overexpression of an eyes absent (EYA) protein in a subject. In one aspect, the use is characterized in that the subject is a human. In one aspect, the use is characterized in that the disorder associated with overexpression of an eyes absent (EYA) protein is a vascular disease, a fibrosis-related disorder, hearing loss, or a metabolic disease. [00338] In various aspects, the use relates to a treatment of disorder associated with overexpression of an eyes absent (EYA) protein wherein the disorder is a vascular disease, a fibrosis-related disorder, a cranio-facial disease, hearing loss, or a metabolic disease [00339] In various aspects, the use relates to a treatment of cancer comprising a tumor having an overexpression of an eyes absent (EYA) protein. [00340] In a further aspect, the use relates to the manufacture of a medicament for the treatment of a disorder associated associated with overexpression of an eyes absent (EYA) protein in a subject. [00341] It is understood that the disclosed uses can be employed in connection with the disclosed compounds, products of disclosed methods of making, methods, compositions, and kits. In a further aspect, the invention relates to the use of a disclosed compound or a disclosed product in the manufacture of a medicament for the treatment of a disorder associated with overexpression of an eyes absent (EYA) protein in a mammal. In a further aspect, the invention relates to the use of a disclosed compound or a disclosed product in the manufacture of a medicament for the treatment of a cancer that comprises a tumor that overexpresses at least one eyes absent (EYA) protein. In a still further aspect, the cancer is breast cancer (e.g., triple-negative breast cancer), cervical cancer, ovarian cancer, liver cancer, pancreatic cancer, or a pediatric cancer (e.g., leukemia).. 3. M ANUFACTURE OF A M EDICAMENT [00342] In one aspect, the invention relates to the manufacture of a medicament for use in treating a disorder associated with overexpression of an eyes absent (EYA) protein such as, for example, a vascular disease, a fibrosis-related disorder, hearing loss, or a metabolic disease, in a subject having the condition, the method comprising combining a therapeutically effective amount of a disclosed compound or product of a disclosed method with a pharmaceutically acceptable carrier or diluent. In another aspect, the invention relates to the manufacture of a medicament for use in treating a cancer comprising a tumor that overexpresses at least one eyes absent (EYA) protein (e.g., breast cancer, triple-negative breast cancer (TNBC), cervical cancer, ovarian cancer, liver cancer, pancreatic cancer, pediatric cancer or leukemia), in a subject having the condition, the method comprising combining a therapeutically effective amount of a disclosed compound or product of a disclosed method with a pharmaceutically acceptable carrier or diluent. [00343] As regards these applications, the present method includes the administration to an animal, particularly a mammal, and more particularly a human, of a therapeutically effective amount of the compound effective in the treatment of a disorder associated with overexpression of eyes absent protein (e.g., a vascular disease, a fibrosis-related disorder, hearing loss, or a metabolic disease) or in the treatment of a cancer that comprises a tumor that overexpresss at least one eyes absent (EYA) protein. The dose administered to an animal, particularly a human, in the context of the present invention should be sufficient to affect a therapeutic response in the animal over a reasonable timeframe. One skilled in the art will recognize that dosage will depend upon a variety of factors including the condition of the animal and the body weight of the animal. [00344] The total amount of the compound of the present disclosure administered in a typical treatment is preferably between about 0.05 mg/kg and about 100 mg/kg of body weight for mice, and more preferably between 0.05 mg/kg and about 50 mg/kg of body weight for mice, and between about 100 mg/kg and about 500 mg/kg of body weight for humans, and more preferably between 200 mg/kg and about 400 mg/kg of body weight for humans per daily dose. This total amount is typically, but not necessarily, administered as a series of smaller doses over a period of about one time per day to about three times per day for about 24 months, and preferably over a period of twice per day for about 12 months. [00345] The size of the dose also will be determined by the route, timing, and frequency of administration as well as the existence, nature and extent of any adverse side effects that might accompany the administration of the compound and the desired physiological effect. It will be appreciated by one of skill in the art that various conditions or disease states, in particular chronic conditions, or disease states, may require prolonged treatment involving multiple administrations. [00346] Thus, in one aspect, the invention relates to the manufacture of a medicament comprising combining a disclosed compound or a product of a disclosed method of making, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, with a pharmaceutically acceptable carrier or diluent. I. E XAMPLES [00347] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. [00348] The Examples are provided herein to illustrate the invention, and should not be construed as limiting the invention in any way. Examples are provided herein to illustrate the invention and should not be construed as limiting the invention in any way. 1. CHEMISTRY EXPERIMENTALS [00349] All reactions were carried out in an oven-dried glassware under argon atmosphere using standard gas-tight syringe, cannula, and septa. The reaction temperatures were measured externally. Stirring was achieved with oven dried magnetic bars. All the reactions were done in anhydrous solvents (CH 2 Cl 2 , THF, MeOH) purchased from Sigma- Aldrich. All commercially purchased reagents were used without purification. The reactions were monitored by thin-layer chromatography (TLC) on a pre-coated silica gel (60 F254) glass plates from EMD Millipore and visualized using UV light (254 nm). Purification of the compounds was performed on Teledyne-ISCO Combiflash Rf 200 purification system using Redisep Rf® normal phase silica gel columns 230-400 mesh. ESI-MS spectra were recorded on a BioTof-2 time-of-flight mass spectrometer. Proton NMR spectra were recorded on a Varian Unity 400 NMR spectrometer operating at 400 MHz calibrated to the solvent peak and TMS peak. The chemical formula and Exact Mass for target compounds were determined from the (M+H) + by high resolution mass spectroscopy using an Agilent 6210 Electrospray Time of Flight. a. G ENERAL E XPERIMENTAL [00350] All operations were carried out at room or ambient temperature, that is, in the range of 18-25 o C; evaporation of solvent was carried out using a rotary evaporator under reduced pressure with a bath of up to 50 o C; reactions were monitored by thin layer chromatography (tlc) and reaction times are given for illustration only. Unless otherwise indicated all reactions were conducted in standard commercially available glassware using standard synthetic chemistry methods and setup. All air- and moisture-sensitive reactions were performed under nitrogen atmosphere with dried solvents and glassware under anhydrous conditions. Starting materials and reagents were commercial compounds of the highest purity available and were used without purification (See list of specific reagents below). Solvents used for reactions were indicated as of commercial dry or extra-dry or analytical grade. Analytical thin layer chromatography was performed on aluminum plates coated with Merck Kieselgel 60F254 and visualized by UV irradiation (254 nm) or by staining with a solution of potassium permanganate. Flash column chromatography was performed on Biotage Isolera One 2.2 using commercial columns that were pre-packed with Merck Kieselgel 60 (230– 400 mesh) silica gel. Final compounds for biological testing are all ≥95% purity as determined by HPLC-MS and 1 H NMR. 1 H NMR experiments were recorded on Agilent DD2400MHz spectrometers at ambient temperature. Samples were dissolved and prepared in deuterated solvents (CDCl3, CD3OD and DMSOd6) with residual solvents being used as the internal standard in all cases. All deuterated solvent peaks were corrected to the standard chemical shifts (CDCl3, dH = 7.26 ppm; CD3OD, dH = 3.31 ppm; DMSOd 6 , dH = 2.50 ppm). Spectra were all manually integrated after automatic baseline correction. Chemical shifts (d) are given in parts per million (ppm), and coupling constants (J) are given in Hertz (Hz). The proton spectra are reported as follows: d (multiplicity, coupling constant J, number of protons). The following abbreviations were used to explain the multiplicities: app = apparent, b = broad, d = doublet, dd = doublet of doublets, ddd = doublet of doublet of doublets, dddd = doublet of doublet of doublet of doublets, m = multiplet, s = singlet, t = triplet. All samples were analyzed on Agilent 1290 series HPLC system comprised of binary pumps, degasser and UV detector, equipped with an auto- sampler that is coupled with Agilent 6150 mass spectrometer. Purity was determined via UV detection with a bandwidth of 170nm in the range from 230-400 nm. The general LC parameters were as follows: Column - Zorbax Eclipse Plus C18, size 2.1 X 50 mm; Solvent A: 0.10 % formic acid in water, Solvent B: 0.00 % formic acid in acetonitrile; Flow rate – 0.7 mL/min; Gradient: 5 % B to 95 % B in 5 min and hold at 95 % B for 2 min; UV detector – channel 1 = 254 nm, channel 2 = 254 nm. Mass detector Agilent Jet Stream – Electron Ionization (AJS-ES). [00351] The following abbreviations are used: THF: tetrahydrofuran DCM or CH2Cl2: dichloromethane DCE: dichloroethane NaHCO 3 : sodium bicarbonate HCl: hydrogen chloride MgSO4: magnesium sulfate Na2SO4: sodium sulfate DME: dimethoxyethane n-BuLi: n-butyllithium DMF: dimethylformamide DMSO: dimethylsulfoxide Et 2 O: diethyl ether MeOH: methanol EtOAc: ethyl acetate a. R EPRESENTATIVE S YNTHESIS OF N 2 - CYCLOPENTYL -N 4 -(3,4- DICHLOROBENZYL)QUINAZOLINE-2,4-DIAMINE (AT-301; NO.38) [00352] 2-chloro-N-(3,4-dichlorobenzyl)quinazolin-4-amine (3): To a stirring solution of 2,4-dichloroquinazoline (5.0 g, 25.1 mmol) in THF (125 mL) at room temperature was added 3,4-dichlorobenzylamine (4.0 mL, 30.3 mmol). The mixture was stirred for 24 hr at room temperature, during which time a precipitate formed. The slurry was filtered and washed with hexanes. The collected filtrate was slurried with DCM/hexanes and filtered. The filtrate was washed with hexanes, collected and dried under reduce vacuum to yield 4.4 g (52% yield) of 2-chloro-N-(3,4-dichlorobenzyl)quinazolin-4-amine (3) as a white powder. [00353] N 2 -cyclopentyl-N 4 -(3,4-dichlorobenzyl)quinazoline-2,4-diamine (AT-301; 38): To a 30 mL microwave reaction vial was added 2-chloro-N-(3,4-dichlorobenzyl)quinazolin- 4-amine 3 (1.0 g, 2.95 mmol), sec-butanol (15 mL) and cyclopentylamine (251 mg, 2.95 mmol). The mixture was irradiated in an Anton Par microwave reactor for 30 minutes at 180 o C. The reaction was cooled to room temperature, during which time a precipitate formed. The solids were filtered, washed with hexanes and dried under reduced pressure to yield N 2 - cyclopentyl-N 4 -(3,4-dichlorobenzyl)quinazoline-2,4-diamine (970 mg, 85% yield) as a white powder. b. ADDITIONAL EXEMPLARY COMPOUNDS [00354] Compounds listed in Table 1 below were prepared as detailed above for Compound 4. TABLE 1.
2. TARGETING TYROSINE KINASE SUBSTRATES FOR PREVENTION AND TREAMENT O F M ETASTATIC D ISEASE a. EXAMPLE 1: EYA4, A NOVEL DNA REPAIR PROTEIN PHOSPHATASE AND A DRUGGABLE TARGET i. D EPLETION OF EYA4 INDUCES DSB S AND GROSS CHROMOSOMAL REARRANGEMENTS [00355] EYA4 depleted cells accumulate unrepaired DNA breaks: Although full knockout of EYA4 is lethal in most mouse strains shortly after birth and poorly tolerated in several lung cell lines, its expression can be reproducibly knocked down using shRNA in HeLa or U2OS cells. EYA4 was suggested to dephosphorylate residue Y142 of H2AX upon DNA damage, a modification thought to favor the subsequent phosphorylation of nearby residue S139 of H2AX (gH2AX). Not all cell lines tolerate EYA4 knock down, but HeLa cells-depleted for EYA4 are viable, and exhibit high constitutive levels of phosphorylated pY142-H2AX (FIG.1D-G), which persist after irradiation, but do not impair S139 phosphorylation nor lead to apoptosis. EYA4 depleted cells exhibit greatly elevated gH2AX protein levels and discrete foci (FIG.1A-G and FIG.2A-C) at basal level, compared to control. gH2AX activation occurred similarly in control and EYA4-depleted cells but persisted only in the latter until 20 hours post irradiation, suggesting impaired DDR. [00356] EYA4 promotes HR and limits NHEJ in normal cells: It was found that EYA4 depleted cells accumulate gH2AX foci and 53BP1 consistent with persistence of breaks and lack of DDR by HR. Importantly, EYA4 depleted cells exhibit decreased levels of pRPA following irradiation suggesting that DNA end resection might be impaired in the absence of EYA4. In support of limited DNA resection, the ssDNA that gets created following targeted DNA breaks were investigated, on stretched DNA using a novel methodology developed in the lab and in cells by BrdU incorporation and immunolabeling in non-denaturing condition, which allows specific detection of ssDNA. Less ssDNA was observed in EYA4 depleted cells than controls. Consistent with this, RAD51 is recruited to the break less efficiently in EYA4 depleted cells than in control cells. To identify if recombination-directed DSB repair might be affected a system developed by the Jasin group was used that allows direct measure of HR. EYA4 depleted cells were less proficient in repairing DSBs by HR (FIG.3A-D). Importantly, repair was restored to control levels by re-introduction of the full length EYA4, but not by any mutants that have lost Tyrosine-phosphatase activity. Like other cells impaired for HR (e.g., those with mutated BRCA1 or BRCA2) cells depleted for EYA4 were found sensitive to the PARPi Olaparib in an MTT assay. Overall, preliminary data indicate that EYA4 is involved in HR-mediated DDR, and that its depletion favors NHEJ (FIG.3E-3H), a premise validated by using reporter assays. [00357] Elevated NHEJ in EYA4 depleted cells promotes chromosome fusion: Fluorescence in situ hybridization (FISH) using telomeric and centromeric probes followed by cytogenetic analysis were used to quantitate chromosomal abnormalities. An increase in chromosome fusions (fused chromosomes with multiple centromeres) were identified, EYA4 depleted cells. The significant increase in chromosome fusions in response to EYA4 depletion implicates DNA repair by the engagement of NHEJ pathways. [00358] EYA4 forms dynamic foci: The 3D organization of the human genome is complex, with long-range chromatin loops occurring between promoters and enhancers to initiate transcription (topologically associating domains or TAD). Similarly, loops called “insulated neighborhoods” can organize and isolate enhancers outside of the loop to prevent their association with gene promoters. Recently, many groups have provided strong evidence that DSB can occur within the loops due to topological constraints to cause genomic instability, and that proteins including 53BP1 organize within and around loops, to form foci in which DSB repair occurs. Dynamic movement or sliding of DNA within loops favors recruitment of DNA repair proteins, DNA homology search in HR, and repair. Compartmentalization of regions in the nucleus is not restricted to the chromatin, and many DNA repair proteins have been described to undergo “phase separation” in the nucleus, forming structures larger than classical DNA repair foci, and this state is believed to reflect changes in the structure, interactions, and function of the phase separated protein. [00359] Interestingly, when a GFP-tagged EYA4 construct was expressed in cells the formation of dense chromatin regions were observed in S-G2 phase, which resemble TAD, and EYA4 forms large foci at these locations. In G1, both GFP-EYA4 and endogenous EYA4 form small, discrete foci that do not colocalize with gH2AX without stress. Interestingly, in S and G2-M, a significant proportion of EYA4 foci (20 and 40%, respectively) colocalize with spontaneous gH2AX foci. This proportion reaches 55% when cells are irradiated, consistent with a possible role of EYA4 at the DSB. ii. EYA4 DEPHOSPHORYLATES DDR PROTEINS [00360] Histone variant H2AX is a substrate of EYA4. EYA4 can be expressed as a GFP fusion in 293FT cells in suspension, and purify it near homogeneity using an anti-GFP nanobody, and previously described chromatography conditions. Based on the observation that cells depleted for EYA4 accumulate phosphorylated H2AX, the inventors wanted to verify whether EYA4 dephosphorylate H2AX, on residue S139 (gH2AX) and/or Y142 (pH2AX). Purified EYA was therefore incubated with peptides containing either pS139 or pY142 and released phosphate was quantified by a colorimetric method using malachite green-molybdate (FIG.2C). Under conditions optimal for EYA4 tyrosine phosphatase activity (acidic, with Mg 2+ ), a peptide containing pY142 was dephosphorylated efficiently in 20 minutes, while dephosphorylation of pS139 was minimal. Under basic pH and in the absence of a metal ion, conditions in whichEYA4 Y domain is not functional, pY142 was not dephosphorylated, demonstrating the specificity of the reactions. Based on the published model, these data indicate that EYA4 dephosphorylates Y142 to promote activation of H2AX as gH2AX, initiating DNA damage repair signaling and preventing apoptosis. Interestingly, the phosphatase activity of EYA4 is greatly enhanced by addition of ssDNA in the reaction. [00361] EYA4 dephosphorylates ATM/ATR targets. To obtain further insights on the functional roles of EYA4 in genomic instability the inventor searched for EYA4 targets. ATM and ATR kinases are the master transducers of the DNA damage response, and they phosphorylate a plethora of DNA repair proteins following DSB, including the effector kinases Chk1 and Chk2, which amplify the signal by activating other DNA damage repair proteins. PTM Scan Technology from Cell Signaling Technology and mass spectrometry (performed at the Proteomic Resource Center, CST, and validated at the mass spec core facility, UQ, Australia) were used to investigate whether EYA4 phosphatase activity could reverse some of the ATM/ATR activating phosphorylation events. A total of 216 peptides were found significantly hyper-phosphorylated in EYA4 deficient or mutant cells when compared to control. Enrichment analysis of the peptides showed that a third of the possible EYA4 targets identified are proteins in the RNA-DNA metabolism pathways (37% combined of transcription, RNA binding, DNA repair, and cell cycle). Depletion of EYA4 leads to elevated levels of phosphorylated DNA repair proteins (8.8% of all peptides), indicating that EYA4 could regulate major DNA repair pathways. Phospho-peptides were found for RAD50, NBS1, 53BP1 and Fanconi Anemia pathway proteins FANC-A and FANC-I, among others. Residues found were for the most part not described before, but the inventor has shown that EYA4 target residues have biological significance. For example, hyper-phosphorylation of 53BP1 promotes NHEJ and chromosome fusion, and phosphorylations on FANC-I and RAD50 change their interaction with known protein partners. It is contemplated that EYA4 controls the activity of key DNA repair proteins in several pathways. [00362] EYA4 dephosphorylates 53BP1 and RAD51. Of the identified DDR substrates of EYA4, the inventor elected to focus on 53BP1, because it was a top hit with seven residues in 53BP1 found differentially phosphorylated in EYA4 depleted cells, with hyperphosphorylation being observed on four sites (S523, S1068, S1368, and T1370). Importantly, the phenotype induced by EYA4 depletion resembles that of 53BP1 mutations (70) and it was asked if this could be explained at least in part by 53BP1 hyperphosphorylation. The 53BP1 residues in question are novel and thus specific, commercial, antibodies are unavailable. However, several key residues in 53BP1, e.g., S25, S29, T543, and S1618, are known targets of ATM or ATR, and the phosphorylation state of these residues controls the functionality of 53BP1. Immunofluorescence was used to monitor two known phosphorylation events in 53BP1: activating ATM-mediated phosphorylation of T543 upon DNA damage and an inactivating phosphorylation of S1618 catalyzed by PLK1 during mitosis to prevent recruitment of 53BP1. It was observed that residue T543 is constitutively phosphorylated, while S1618 is unchanged or slightly decreased by EYA4 depletion. Since phosphorylation of T543 is in response to DNA damage, it is likely that 53BP1 becomes hyperactivated in EYA4 depleted cells. Using the colorimetric assay described earlier, 53BP1 residues were found hyperphosphorylated in cells: S1067, S1068, S1368 and T1370 are indeed targets of EYA4. Out of all targets tested 53BP1 is overall the preferred substrate of EYA4. [00363] These results demonstrate that the atypical protein phosphatase EYA4 is a novel DNA repair gene, that not only dephosphorylates H2AX on Y142 (as its homologs EYA1/3), but also contributes to DNA damage repair by targeting RAD51 on Y315. The dephosphorylation of RAD51, promotes a more stable and active presynaptic filament, consequently favoring strand exchange, showing a direct implication of EYA4 in the RAD51-dependent homologous recombination repair. b. E XAMPLE 2: EYA4 TYROSINE PHOSPHATASE CONTROLS RAD51 FUNCTION AT THE DOUBLE STRAND BREAK AND PROMOTES REPAIR BY H OMOLOGOUS RECOMBINATION . [00364] Cross talk between various double strand break (DSB) repair pathways and their synchronization with the cell cycle ensures the most efficient repair possible, and limits genome rearrangements. Post translational modifications of histones and DNA damage repair (DDR) proteins influence pathway choice, timing and efficacy of the DSB repair. Phosphorylation events are known contributors of DDR activation, and many serine/threonine protein phosphatases play a role in DNA damage repair. It was found that the protein phosphatase EYA4, and in particular its tyrosine phosphatase function, is essential to promote Homologous Recombination at DSBs. EYA4 binds to DNA and the chromatin, and forms dynamic foci in response to DNA damage. It can dephosphorylate histone variant H2AX to promote repair of DSBs and dephosphorylates RAD51. Over-expression of EYA4 in cells leads to overly stable RAD51, and phosphomutants that mimic dephosphorylated RAD51 in vitro are forming longer and more stable filaments onto ssDNA than WT RAD51, with enhanced strand exchange activity. iii. R ESULTS [00365] EYA4 deficient cells accumulate DNA damage. Complete knock out of EYA4 is lethal in most mice strains shortly after birth (Depreux et al., The Journal of clinical investigation 2008, 118(2):651-8), and poorly tolerated in several lung cell lines (Wilson et al., Oncogene 2014, 33(36):4464-73) and other cell lines that were tested. However, shRNAs were able to significantly decrease EYA4 expression and protein levels in HeLa cells (FIG. 10B), U2OS cells, MDA-MB-231, and establish mutants of the catalytic domains (see FIG. 10A). [00366] Upon knocking down EYA4, HeLa cells readily acquired nuclear defects indicative of genomic instability (FIG.10C). Cells depleted for EYA4 showed aneuploidy, DNA bridges, and accumulation of micronuclei. To investigate whether this genomic instability could result from defective DNA damage repair the level of H2AX in controls and cells depleted for EYA4 by any of three shRNAs was determined. More than 50% of EYA4 depleted cells exhibited 10 or more γH2AX discrete foci and an overall 8-fold increase of γH2AX foci in normal growth conditions (5% CO2, 37 °C) without any exogenous stress, compared to cells expressing a non-targeting shRNA (FIG.10D). While accumulation of γH2AX is a reliable marker for double stranded DNA breaks, one could not readily exclude that H2AX S139 could be a target of EYA4 phosphatase activity and remain phosphorylated regardless of the DNA repair status, in the absence of EYA4. To test whether EYA4 depleted cells accumulate unrepaired DNA breaks or whether they are proficient for repair but deficient for dephosphorylating H2AX, DSBs were introduced by irradiation, and the stimulation and resolution of γH2AX foci were followed. A time course of γH2AX foci by immunofluorescence (FIG.11A) and the protein levels followed by western blotting (FIG. 11B) both confirmed that γH2AX is constitutively elevated in cells depleted for EYA4, but still increases following irradiation. Interestingly, a portion of DSBs is repaired in the absence of EYA4, as γH2AX is resolved overtime and return to its initial, elevated levels, 2-4 hours post irradiation (FIG.11A and FIG.11B). [00367] Histone variant H2AX pY142 but not pS139, is a substrate of the EYA4 phosphatase activity. PP4 and WIP1 are the known phosphatase for γH2AX (Chowdhury et al., Mol Cell.2008, 31(1):33-46) and EYA4 is unlikely to dephosphorylate S139, but EYA1- 3 proteins have been found directly or indirectly to dephosphorylate residue Y142 of H2AX upon DNA damage (Hegde et al., Crit Rev Biochem Mol Biol.2020, 55(4):372-85; Wilson et al., Oncogene 2014, 33(36):4464-73), and by similarity, EYA4 was suggested to possess the same activity (Wilson et al., Oncogene 2014, 33(36):4464-73). pY142-H2AX is a low abundance modification (Hatimy et al., International Journal of Mass Spectrometry 2016, 391:139-45) but it is thought essential for the promotion of DNA damage repair over apoptosis (Cook et al., Nature 2009, 458(7238):591-6; Solier and Pommier, Cell Cycle 2009, 8(12):1853-9). To investigate whether EYA4 dephosphorylates H2AX, EYA4 was expressed and purified as a GFP fusion using previously described chromatography conditions (FIG. 11C). Purified EYA was incubated with phosphorylated H2AX peptides (FIG.11A-E) containing either pS139 or pY142 at 37 °C for 60 min. Released phosphate was quantified by a colorimetric method using malachite green-molybdate (FIG.11D). Both threonine and tyrosine phosphatase domains of EYA4 have been described as functional in vitro (Okabe et al., Nature 2009, 460:520-4). Under conditions optimal for EYA4 tyrosine phosphatase activity (acidic, with Mg 2+ ), the synthetic pY142 peptide was dephosphorylated 5-fold more efficiently than pS139 peptides (FIG.11D). Using the serine threonine-optimized conditions, pS139 was still minimally dephosphorylated and pY142 was not dephosphorylated, indicating that the experimental conditions are specific for each of the activity tested. EYA4 is not the phosphatase for γH2AX but its efficient dephosphorylation of H2AX on pY142 could promote DNA damage repair signaling following DNA damage. Surprisingly, it was found that the phosphatase activity of EYA4 is greatly stimulated by addition of single- stranded DNA (ssDNA) in the reaction (FIG.11D). In vitro findings were verified in cell, using a specific antibody directed against pY142 of H2AX, for immunofluorescence and in Western blots. Higher levels of pY142 were detected in EYA4 depleted cells compared to controls (FIG.11E). As previously reported in the literature, Y142 phosphorylation level decreases in response to DNA damage (Cook et al., Nature 2009, 458(7238):591-6), but an accumulation of pY142-H2AX was observed in EYA4 depleted cells, at 2 hours following irradiation (FIG.11E). This suggests that EYA4 could target H2AX pY142 for dephosphorylation, but also contributes to its accumulation, possibly by targeting upstream kinases. [00368] EYA4 is a DNA binding protein and interact with DNA, histones and nucleosomes. The unexpected stimulation of the reaction by DNA raised the possibility that EYA4 could interact directly with DNA. To investigate this purified EYA4, GFP full length or m-cherry-tagged N-terminus (aa 1-365, FIG.12A-F) were used. Electromobility shift assay (EMSA) were performed using purified EYA4 and synthetic substrates to mimic ssDNA or dsDNA. EYA4 was found to bind to DNA and is extremely avid for ssDNA (FIG. 12B and FIG.12C). The N-terminus of EYA4 contains the DNA binding activity, and exhibits the same substrate specificity as the full-length, namely preferring ssDNA over dsDNA in the short (FIG.12B) and long system (FIG.12C). Using a long fragment encompassing the entire tyrosine domain in the C-terminus part of the protein (aa-aa), EMSAs were repeated and observe that this region possesses no or little, DNA binding activity. In the long system, both ssDNA and the dsDNA are closed-circular forms and no DNA end is available. Super-shifts observed with increasing concentration of EYA4 indicates that it accumulates on the phage DNA and does not bind solely to the DNA ends. As dephosphorylation of H2AX and DNA binding activity makes EYA4 a good candidate for a role in DNA repair, the EYA4 protein network in response to DNA damage was investigated. Immunoprecipitation followed by mass spectrometry was performed in HeLa cells expressing endogenous levels of EYA4 and subjected to either no irradiation or 10 Gy irradiation. EYA4 was found strongly associated with chromatin and bound to histones and associated proteins in the absence of IR. Interestingly, binding to histones is weakened or lost following irradiation (FIG.12D), suggesting that EYA4 could be an early responder to DNA damage and is displaced following dephosphorylation of H2AX, possibly to allow chromatin accessibility by repair machineries. Since EYA4 binds DNA, direct binding to histones in vitro was verified. Using the GFP-EYA4 protein immobilized on GFP-nanobody beads (Schellenberg et al., Protein Sci.2018;27(6):1083-92) and purified histones, in vitro pull- downs were performed that identified extremely robust interactions between EYA4 and H2A or H2B (FIG.12E). Despite its preference for ssDNA, EYA4 also binds strongly to dsDNA nucleosomes (FIG.12E). [00369] EYA4 is part of DNA damage repair foci. To gain a better understanding of EYA4 cellular behavior, GFP-tagged version of EYA4 were followed in cells over time. EYA4 forms spontaneous foci in cells. Untreated cells possess an average of 20, small discrete EYA4 foci, that appear randomly distributed in the nucleus (FIG.12D) and fuse into bigger structures in G2. When using an antibody directed against the endogenous EYA4 in WT cells, it was observed that irradiation promptly stimulates EYA4 foci formation, and these colocalize with γH2AX (FIG.13A). To further interrogate the colocalization of EYA4 with other DNA repair proteins, but also use a different system to confirm observations are not due to the irradiation or the antibody the DIvA system (DSB inducible via AsiSI (24)) generously provided by the Legube lab was transfected with GFP- EYA4 and probed for the GFP tag during immunolocalization. In DivA cells untreated, a low basal level of γH2AX foci was observed, and low or absent RAD51 foci were observed, as expected (FIG.13B, top panel). Cells expressing highest levels of EYA4 were also found to exhibit RAD51 foci, even in the absence of γH2AX (FIG.13B, top panel). This suggest that EYA4 could co-localize with non-repair RAD51 foci, and/or that RAD1 accumulates in the presence of EYA4. Following the addition of tamoxifen and subsequent translocation of AsiSI, DSBs were introduced as shown by increased γH2AX, and EYA4 formed robust foci that colocalize almost systematically with RAD51 foci (FIG.13B, bottom panel) and strongly with γH2AX. [00370] In the absence of EYA4, homologous recombination efficiency is compromised. Accumulation of unrepaired DSBs in EYA4-depleted cells and the colocalization of EYA4 with γH2AX and RAD51 indicate that EYA4 might be a hitherto unsuspected player of the DNA damage repair response. To test this hypothesis, the repair of DSBs in the absence of EYA4, after no or 4Gy irradiation was investigated. Ionizing- radiation induced foci formation by γH2AX, pRPA, and RAD51 were followed. EYA4 depleted cells accumulate γH2AX foci and replicative pRPA (phosphorylated at residue S33) but fail to form pRPA (S4/S8) foci that are surrogate markers of end resection at the break (FIG.14A). This suggests that the signaling of the break is perturbed in the absence of EYA4 and processing of the break might not be fully proficient. It was verified using a previously published protocol, where incorporation of BrdU is measured in native conditions (O'Sullivan et al., J Vis Exp.2021(170)). The lack of a denaturing step allows to measure exclusively ssDNA, as a readout for resection efficiency. Using this technique evidenced that cells depleted for EYA4 exhibit diminished resection compared to controls, in response to IR. Consistent with this, RAD51 is recruited less efficiently to the break in EYA4 depleted cells, and later than in control cells (FIG.14C). Lack of RAD51 foci indicate that recombination- directed DSB repair might be affected, and to ascertain this, an in vitro system developed by the Jasin group (Pierce et al., Genes & Development.1999, 13(20):2633-38; Nakanishi et al., Methods in Molecular Biology.2011, 745:283-91) was used, which allows direct measurement of HR. EYA4 depleted cells were found less proficient in repairing DSBs by HR (FIG.14D). Repair was restored to control levels by re-introduction of the full length EYA4. The EYA4 depleted cells were complemented with EYA4 constructs mutated in the catalytic domain (FIG.14E). Doing so evidenced that the serine threonine phosphatase domain of EYA4 is essential for its role in HR. A specific inhibitor of EYA4’s tyrosine phosphatase activity was recently identified. To try to confirm data observed with catalytic mutants, cells were subjected to increasing concentrations (0-500nM) of a tyrosine- phosphatase inhibitor CIDD-0149689 (FIG.14F). In the DR-GFP system, the tyrosine inhibition decreases HR efficiency. Interestingly, the same inhibitor was found to have only a moderate effect on single strand annealing (SA), as tested by the SA-GFP system developed and generously provided by the Stark lab. Like HR, SA is a recombination-based repair. However, it relies on RAD52 and not RAD51, and the data herein indicate that EYA4 might promote specifically RAD51-dependent repair. In accord with a HR repair defect, cells depleted for EYA4 were found sensitive to the PARP inhibitor Olaparib in an MTT assay (FIG.14G). [00371] RAD51 Y315 is a target of EYA4. RAD51 phosphorylation levels on residues Y54 and Y315 could influence its function (Popova et al., FEBS Letters.2009, 583(12):1867- 72; Subramanyam et al., PNAS.2016, 113(41):E6045-E54). The tyrosine kinase c-ABL, or its oncogenic fusion protein BCR/ABL, phosphorylates RAD51 in a sequential manner, on residue Y315 followed by Y54 (Popova et al., FEBS Letters.2009, 583(12):1867-72). This sequential phosphorylation is important for RAD51 recruitment to DSB sites and its strand exchange activity (Subramanyam et al., PNAS.2016, 113(41):E6045-E54). Persistent phosphorylation of Y315, and consequently double-phosphorylated status of RAD51, is known to have an inhibitory effect on HR, due to a defect in nucleofilament formation (Alligand et al., Biochimie.2017, 139(114-24)). Using a specific antibody recognizing pY315, it was observed that cells depleted for EYA4 exhibit elevated foci of pY315RAD51, compared to controls (FIG.15A). Interestingly, control cells exhibit some pRAD51 in the absence of stress, which decrease in response to IR (FIG.15B). However, in cells depleted for EYA4, pY315 remains elevated (FIG.15B). Using the malachite green in vitro phosphatase assay, Y315, but not Y54, was identified to be a substrate for EYA4 (FIG.15C). Interestingly, the dephosphorylation of RAD51 by EYA4 was again found stimulated by the addition of ssDNA suggesting a common activation mechanism for its phosphatase function. Biochemical characterization of Y54 and Y315 have been performed independently; however, the combined actions of these two residues has not yet been investigated. The double mutant RAD51 (Y54F, Y315F) that cannot be phosphorylated, as well as the double phospho-mimetic RAD51 (Y54E, Y315E) , which mimics a phosphorylated tyrosine but cannot be dephosphorylated (FIG.15D) were purified. Based on cell-based data and the literature available on these RAD51 tyrosine residues, it was decided to investigate the ability of RAD51 (Y54F, Y315F) to polymerize onto ssDNA and form nucleoprotein filaments. Surprisingly, it was observed by electron microscopy (FIG.15E) that this mutant is much more proficient at polymerizing than the WT protein. It can form filaments across ~10 lengths of ssDNA (83dT) in the mutant (FIG.15E), while the WT RAD51 coats ssDNA as individual filaments, without any tethering activity (FIG.15E). This increased activity is not linked to a change in ATPase activity as tested by malachite green. Unexpectedly, it had previously been observed in cells that the overexpression of EYA4-WT in cells led to an increase of RAD51 staining in otherwise untreated cells (see FIG.13A and FIG.13B). Since EYA4 overexpression leads to accumulation of RAD51 in vivo, it was wondered whether RAD51 (Y54F, Y315F) might be more stable than RAD51 WT or RAD51 (Y54E, Y315E) . To answer this query, pulse chase experiments were conducted with cycloheximide in cells, followed by western blot analysis. It was found that the expression of plasmid based RAD51 leads to a more stable protein overall compared to the endogenous protein. When comparing cells transfected with RAD51 WT, RAD51 (Y54F, Y315F) , or RAD51 (Y54E, Y315E) , it was observed that the phosphomutant of RAD51 has a slightly increased half-life compared to WT and the phospho-mimetic. The combination of this increase in stability coupled to an improved polymerization activity of RAD51 (Y54F, Y315F) could explain RAD51 strong staining in cells overexpressing EYA4. [00372] Taken together, the data demonstrate that EYA4 plays a role in promoting DNA repair by homologous recombination, through the dephosphorylation and subsequent stabilization of RAD51. c. M ATERIAL AND METHODS [00373] Cell culture and maintenance, transfections and stable cell lines establishment. Cells obtained from ATCC, U2OS-DiVA gifted by Gaelle Legube, and U2OS/DR-GFP gifted by Jeremy Stark were maintained in cell-adhesion treated vessels at 37˚C in 5% CO2 incubators. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco ® ) supplemented with 10% fetal bovine serum (FBS) and passaged at 80% confluence or less. DiVA were supplemented with Glutamax (Gibco). For virus production 1.2*10 6 HEK 293FT cells were reverse transfected using Lipofectamine ® 2000 Reagent (Invitrogen ™ ) with MISSION ® TRC2 pLKO.5-Puro (Sigma-Aldrich ® ) empty vector or MISSION ® TRC2 pLKO.5-Puro (Sigma-Aldrich ® ) EYA4 shRNA constructs (shRNA1, TRCN0000244430; shRNA2, TRCN0000218273; shRNA3, TRCN0000244429) and Lenti- vpak plasmids from OriGene to create lentivirus particles. Viruses were harvested at 48 and 72 hours post transfection and filtered through a 0.45 µm filter, then used to infect cell lines with polybrene (4 µg/mL) in a 6 cm dish. Stable cell lines were selected after 48 hours using 1-2 µg/mL of puromycin. For complementation, stable cells expressing shRNA1 were transfected with the pcDNA ™ 3.1/nV5-DEST construct coding for EYA4-resistant (full length, FL) and selected with 500 µg/mL geneticin. For DSB induction, DiVA cells were treated for 4h with 300nM hydroxytamoxifen (4OHT) added directly to the culture medium. The origin of all cells was confirmed by short tandem repeat (STR) analysis. All cells were tested negative for Mycoplasma. [00374] RT-PCR and Western blot analyses. RNA was isolated by phenol chloroform extraction (TRIzol ® , Invitrogen ™ ) followed by nucleic acid precipitation. The GoScript ™ Reverse Transcription System (Promega) was used to generate first-strand cDNA. Real-Time quantitative PCR (RT-qPCR) was performed using TaqMan ® probes for human EYA4 (Invitrogen ™ , Hs01012406_mH, Cat. # 4351372) and human 18S (Invitrogen ™ , Hs99999901_s1, Cat. # 4331182) to amplify 70 bp and 187 bp fragments, respectively.18S was used as an endogenous control and used for normalization. [00375] Western blot analysis was conducted according to the standard procedures ((31)). Briefly, cells were lysed on ice in RIPA buffer (10 mM Tris HCl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% (v/v) Triton X-100, 0.1% (w/v) sodium deoxycholate, 0.1% (v/v) SDS, 140 mM NaCl) supplemented with 1 mM PMSF, 1 mM Na 3 VO 4 , 1 mM NaF, 4X protease inhibitor cocktail (1 μg/mL each), 1 mM benzamidine and 0.025 U/μL benzonase, sonicated 2 minutes (40%) in an ultrasonic water bath (Sonics Vibra-Cell VCX400). The primary antibodies were: anti-EYA4 (Abcam ab93865, 1:3,000), anti-γH2AX (S139) (Millipore 05-636, 1:3,000), anti-RAD51 (H-92, Santa Cruz sc8349, 1:5,000), α-pRAD51 (Y315) (Abcam ab61111, 1:3,000), α-β-Tubulin (9F3, Cell Signaling #2128, 1:2,000) and anti- ^-Actin antibody (C-4, Santa Cruz sc47778, 1:10,000). [00376] Phosphatase and ATPase assays. To measure phosphatase activity, increasing concentrations of EYA4 (0-200 nM) were incubated in the presence of 400 nM potential substrates (peptides sourced from Genscript, see list and sequence in figures) in 50 μL of reaction buffer A (50 µM MES pH 6, 2 mM MgCl2, 50 µM DTT) for 1h at 37 ºC. To measure RAD51 phosphatase activity, RAD51 WT or mutant protein (0-100 nM) was incubated at 37 ºC for 5 minutes with 0.25 mM ATP with or without ssDNA poly(dT)83 (300 nM nucleotides) in 20 μl of buffer B (25 mM Tris-HCl, pH 7.2, 5 mM KCl, 0.2 mM MgCl2, 0.1 mM DTT, and 1 μg/ml BSA). At the end of the first incubation, the reaction mix containing malachite green was added to phosphatase or ATPase reactions, and the mixture was further incubated for 30 minutes at 37 ºC. For both experiments, plates were read at 590 nm in a plate reader. The quantity of phosphatase released was inferred from the standard curve prepared as per manufacturer recommendation (Sigma). [00377] Indirect immunofluorescence. Stable cell lines expressing shRNA or control plasmid were grown on coverslips for 24 hours and treated with 4 Gy ^-rays. Cell nuclei were pre-extracted with nuclear extraction buffer (NEB; 10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA (pH 8.0), 0.5% (v/v) Triton X-100) for 2 minutes at room temperature and fixed with 4% (v/v) paraformaldehyde (PFA) for 10 minutes at 4 °C. Nuclei were blocked in PBS with 5% (w/v) BSA and 0.3% (v/v) Triton X- 100 for 2 hours at room temperature or overnight at 4˚C, immunoblotted with a primary antibody (1:500 dilution in PBS with 1% (w/v) BSA and 0.3% (v/v) Triton X-100) for 2 hours at room temperature, followed by secondary antibody (2 ^g/mL in PBS with 1% (w/v) BSA and 0.3% (v/v) Triton X-100) for 2 hours at room temperature. DNA was stained with ProLong ® Gold/Diamond Antifade Mountant with DAPI (Invitrogen ™ ). The slides were viewed at 120× magnification in an Olympus FV3000 confocal microscope. The primary antibodies were: anti-γH2AX (S139) (Millipore 05-636), anti-pRPA (S4/S8) (Bethyl A300- 245A), α-RAD51 (H-92, Santa Cruz sc8349) and α-pRAD51 (Y315) (Abcam ab61111). The secondary antibodies were: anti-Mouse (Abcam ab150103, Alexa Fluor ® 647), anti-Rabbit (Abcam ab150081, Alexa Fluor ® 488), anti-Mouse (Abcam ab150117, Alexa Fluor ® 488) and anti-Rabbit (Santa Cruz sc362292, CFL-647). The number of cells with nuclear foci was quantified using CellProfiler. When indicated, cells were irradiated (4 and 10 Gy) in regular growth medium and a Gammacell ® 40 Exactor (radiation source: caesium 137 ) unit. [00378] Analyses of chromosomal aberrations. HeLa control and knock down cells were synchronized in G2/M phase with demecolcine (300 ng/mL) for 4 hours, detached from the culture vessel, washed in PBS, and allowed to swell in 75 mM KCl at 37˚C for 40-60 minutes. Cells were fixed with methanol:acetic acid (3:1), dropped onto glass coverslips and dried by evaporation in a humidity chamber. Metaphase spreads were stained with ProLong ® Gold/Diamond Antifade Mountant with DAPI (Invitrogen ™ ).30 metaphases were imaged at 100× magnification on an Olympus FV3000 confocal. For sister chromatid exchange, cells were incubated with BrdU for two cell cycle, before metaphase spreads were prepared. This ensured differential labeling of the sister chromatids. [00379] Electro Mobility Shift Assay (EMSA). Electro Mobility Shift Assay was performed with primers (short system) or Phi-X DNA (long system) as previously described (31, 32). Increasing concentrations of EYA4 GFP- full length or mCherry- 1-365 was incubated with fixed amounts of DNA in 10 μL of reaction buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl 2 , 1 mM dithiothreitol, 100 μg/mL BSA and 2.5 mM ATP) at 37 ºC for 20 min. The reaction mixtures were resolved in 8% polyacrylamide gels in TBE buffer (100 mM Tris-borate (pH 8.3), 2 mM EDTA), for the short system, followed by imaging at 488 or 647 nm wavelength and quantification using QuantumStudio (BioRad). The long system EMSA were resolved on a 1% Agarose gel, stained with ethidium bromide, destained in water, and imaged under UV excitation on a BioRad imager then quantified. [00380] DNA substrates. Cy5-labeled oligo P1 (31) from IDT (TTATATCCTTTACTTTGAATTCTATGTTTAACCTTTTACTTATTTTGTATTAGCCG GATCCTTATTTCAATTATGTTCAT) was used as the ssDNA substrate. dsDNA was generated by annealing Cy5-labeled oligo P1 with its exact complement P2 ATGAACATAATTGAAATAAGGATCCGGCTAATACAAAATAAGTAAAAGGTTAAA CATAGAATTCAAAGTAAAGGATATAA. The annealed product was purified by SDS- PAGE then concentrated and quantified. For the long system assays, substrates were sourced from NEB. The ssDNA is the ^X174 virion DNA and the dsDNA its replicative form ^X174 RF I DNA. [00381] Electron Microscopy. The reaction was carried out at 37 ºC in buffer containing 2 mM ATP, 2.5 mM MgCl2, 50 mM KCl, and no BSA. To assemble nucleoprotein filaments, RAD51 (2.4 μM) was incubated with an 83mer Oligonucleotide polydT (7.2 μM nucleotides) for 5 min and then the reaction mixtures were diluted 8-fold with the same buffer, and a 4 μl aliquot was applied to 400-mesh grids coated with carbon film and which had previously been glow-discharged in air. After staining for 30s with 4% uranyl acetate, the samples were examined in a JEOL JSM-6610LV electron microscope equipped with a tungsten filament at 100 keV. Digital images were captured with a charge- coupled device camera at a nominal magnification of X 63,000. 3. RAD51 DEPHOSPHORYLATION BY THE EYA4 TYROSINE PHOSPHATASE CONTROLS ITS FILAMENT FORMATION AND ITS ACTIVITY IN HOMOLOGOUS RECOMBINATION [00382] The observation that EYA4 is hypermethylated and possibly overexpressed in triple-negative breast cancer samples prompted the study of its cellular role beyond organogenesis. EYA4 was inactivated or overexpressed in a variety of cell lines and the resulting phenotypes were investigated. In addition, the DNA repair efficiency was evaluated and the identity of the EYA4 targets and other interacting partners were sought. [00383] Novel ssDNA-binding activity of EYA4 was uncovered, which greatly stimulated its phosphatase activity. In addition to binding ssDNA, EYA4 is chromatin bound through direct interaction with histones. It was found that upon DNA damage, EYA4 dephosphorylates H2AX on residue pY142, as previously suggested by homology. Futher it was found that EYA4 is also phosphorylated in response to irradiation, which causes it to detach from the chromatin, likely to facilitate access to DSBs by the DNA repair machinery. In addition, it was found that EYA4 targets RAD51 on residue Y315, a tyrosine which influences RAD51 polymerization and presynaptic filament formation. In addition, it was found that while cells depleted for EYA4 are sensitive to genotoxic stress, deficient for HR, and exhibit chromatin compaction defects, overexpression of EYA4 drives the accumulation of hyper-active and stable dephosphorylated RAD51 protein, which forms longer presynaptic filaments in vitro. Conversely it was found that phosphorylation of RAD51 reduces its DNA binding activity. Taken together, these findings indicate that RAD51 phosphorylation status changes the nature of the filament and controls the recombination function of RAD51. Thus, it was determined that EYA4 is a key player of DDR. Therefore, overexpression of EYA4 in tumors could yield accumulation of RAD51, which is linked with hyperrecombination and drug resistance phenotypes, even in the absence of copy number variation. These discoveries suggest that the EYA proteins, and, in particular, the EYA4 protein is a druggable target that could be used in the treatments of disorders associated with overexpression of EYA proteins including, but not limited to cancer treatments to limit metastasis and combat drug resistance caused by elevated RAD51 levels or secondary mutations in HR genes that restore HR (H. L. Klein (2008) DNA Repair (Amst) 7, 686-693; Y. Feng et al. (2021) Cancer Cell Int 21, 249). a. M ATERIALS AND M ETHODS i. C ELL CULTURE AND MAINTENANCE , TRANSFECTIONS , AND STABLE CELL LINES ESTABLISHMENT [00384] Cell lines were obtained from ATCC, U2OS-DiVA gifted by Gaelle Legube, and U2OS/DR-GFP gifted by Jeremy Stark were maintained in cell-adhesion treated vessels at 37 °C in 5% CO 2 incubators. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco®) supplemented with 10% fetal bovine serum (FBS) and passaged at 80% confluence or less. DiVA were supplemented with Glutamax (Gibco). For virus production 1.2*106 HEK 293FT cells were reverse-transfected using Lipofectamine® 2000 Reagent (Invitrogen™) with MISSION® TRC2 pLKO.5-Puro (Sigma-Aldrich®) empty vector or MISSION® TRC2 pLKO.5-Puro (Sigma-Aldrich®) EYA4 shRNA constructs (shRNA1, TRCN0000244430; shRNA2, TRCN0000218273; shRNA3, TRCN0000244429) and Lenti- vpak plasmids from OriGene to create lentivirus particles. Viruses were harvested at 48 and 72 hours post transfection and filtered through a 0.45 µm filter, then used to infect cell lines with polybrene (4 µg/mL) in a 6 cm dish. Stable cell lines were selected after 48 hours using 1-2 µg/mL of puromycin. For complementation, stable cells expressing shRNA1 were transfected with the pcDNA™3.1/nV5-DEST construct coding for EYA4-resistant (full length, FL) and selected with 500 µg/mL geneticin. For DSB induction, DiVA cells were treated for 4h with 300nM hydroxytamoxifen (4OHT) added directly to the culture medium. The origin of all cells was confirmed by short tandem repeat (STR) analysis. All cells were regularly tested for Mycoplasma and cell of origin was validated by STR profiling. ii. RT-QPCR AND WESTERN BLOT ANALYSES. [00385] Total RNA was isolated by phenol chloroform extraction (TRIzol, Invitrogen) followed by nucleic acid precipitation. The GoScript Reverse Transcription System (Promega) was used to generate first-strand cDNA. Real-Time quantitative PCR (RT-qPCR) was performed using TaqMan probes for human EYA4 (Invitrogen, Hs01012406_mH) and human 18S (Invitrogen, Hs99999901_s1) to amplify 70 bp and 187 bp fragments, respectively. The relative expression of EYA4 was determined using 2 - ^ ^Ct method with 18S as an endogenous control for normalization. Western blot analysis was conducted according to standard procedures (C. Wiese et al., Mol Cell 28, 482-490 (2007)). Briefly, cells were lysed on ice in RIPA buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) supplemented with cOmplete Mini EDTA-free protease inhibitor cocktail (Roche), 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, 1 mM benzamidine and 0.025 U/μL benzonase, sonicated 2 minutes (40%) in an ultrasonic water bath. The primary antibodies were: EYA4 (Abcam), γH2AX (S139) (Millipore), β-Tubulin (9F3, CST) and ^-Actin (C-4, Santa Cruz). iii. INDIRECT IMMUNOFLUORESCENCE [00386] Indirect immunofluorescence was performed as described elsewhere (B. de la Peña Avalos, E. Dray. Journal of Visualized Experiments 160, e61447 (2020)). Stable cell lines expressing control or shRNA plasmid were grown on coverslips for 24 hrs and treated with 4 Gy ^-rays. Cell nuclei were pre-extracted with nuclear extraction buffer (NEB; 10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA (pH 8.0), 0.5% Triton X-100) for 2 minutes at RT and fixed with 4% paraformaldehyde (PFA) for 10 minutes at 4 °C. Nuclei were blocked in PBS with 5% BSA and 0.3 % Triton X-100 for 2 hrs at RT, immunoblotted with a primary antibody (1:500 dilution in PBS with 1% BSA and 0.3% Triton X-100) for 2 hours at RT, followed by secondary antibody (2 ^g/mL in PBS with 1% BSA and 0.3% Triton X-100) for 2 hours at RT. DNA was counterstained with DAPI. Slides were viewed on an Olympus FV3000 confocal microscope. Primary antibodies were: γH2AX (S139) (Millipore), pH2AX (Y142) (Abcam), EYA4 (Abcam), pRPA (S4/S8) (Bethyl), pRPA (S33) (Bethyl), RAD51 (H-92, Santa Cruz) and α-pRAD51 (Y315) (Abcam). Secondary antibodies were: ^-Mouse (Abcam, Alexa Fluor 647), ^-Rabbit (Abcam, Alexa Fluor 488), ^-Mouse (Abcam ab, Alexa Fluor 488) and ^-Rabbit (Santa Cruz, CFL-647). The number of nuclear foci and their colocalization was quantified using CellProfiler. When indicated, cells were irradiated (4 and 10 Gy) in regular growth medium and a Gammacell® 40 Exactor (radiation source: caesium 137 ) unit. iv. PROTEINS EXPRESSION AND PURIFICATION [00387] EYA4 full-length, cloned in pEGFP-C1 was expressed in mammalian cells using the expi293 expression system (ThermoFisher).250 mL of culture was infected then grown for four days, and cell pellets were resuspended in lysis buffer (50 mM Tris-HCl (pH 8.9), 150 mM NaCl, 10% sucrose, 10% glycerol, 0.5 mM EDTA, 1 mM TCEP, 1 mM PMSF, 0.5% Igepal and protease inhibitors), sonicated 20 times for 15 seconds (50%). and the lysate was clarified by centrifugation (20,000 ^ g, 30 minutes). The supernatant was diluted in 50 mM Tris-HCl (pH 8.9), 10% glycerol, and loaded onto a 7.4 mL Source 30 Q column equilibrated in buffer A (lysis + 75 mM NaCl). The protein was fractionated in 4 mL fractions using a linear gradient to 100% of buffer B (buffer A + 1 M NaCl). Fractions containing the peak EYA4 were pooled and incubated with 1 mL of agarose resin anti-GFP for 2 hrs at 4 °C. The resin was collected, washed with 20 CV of buffer B, followed by 10 CV of buffer C (50 mM Tris-HCl (pH 8), 150 mM NaCl, 10% glycerol). GFP-EYA4 was either left on beads for interaction studies or eluted by cleavage of the GFP-tag with TEV protease (4 °C 2h). EYA4 was collected the next day by flow in buffer C. TEV was removed by incubating elution on Ni-NTA2+ resin for 1 hr. Flow through and washes (buffer C) were then pooled, buffer exchanged in storage buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 30% glycerol) and concentrated before storage. v. PHOSPHATASE ASSAYS [00388] To measure phosphatase activity, increasing concentrations of EYA4 (0-200 nM, alone or pre-incubated with ssDNA) were incubated in the presence of 400 nM potential substrates (peptides sourced from Genscript, see list and sequence in figures) in 50 μL of reaction buffer A (50 µM MES pH 6, 2 mM MgCl 2 , 50 µM DTT) for 1h at 37 ºC. At the end of the first incubation, the reaction mix containing malachite green was added to phosphatase or ATPase reactions, and the mixture was further incubated for 30 minutes at 37 ºC. Plates were read at 620 nm in a plate reader. The quantity of phosphatase released was inferred from the standard curve prepared as per manufacturer recommendation (Sigma). vi. ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA) [00389] Electrophoretic Mobility Shift Assay was performed with primers (short system, IDT, see DNA substrates (below)) or Phi-X DNA (long system, sourced from NEB) as previously described (C. Wiese et al., Mol Cell 28, 482-490 (2007), M. H. Dunlop et al., J Biol Chem 286, 37328-37334 (2011)). Increasing concentrations of purified protein was incubated with fixed amounts of DNA in 10 μL of reaction buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM MgCl 2 , 1 mM dithiothreitol, 100 μg/mL BSA and 2.5 mM ATP) at 37 ºC for 20 min. The reaction mixtures were resolved in 8% polyacrylamide gels in TBE buffer (100 mM Tris-borate (pH 8.3), 2 mM EDTA), for the short system, followed by imaging at 488 or 647 nm wavelength and quantification using QuantumStudio (BioRad). The long system EMSA were resolved on a 1% Agarose gel, stained with ethidium bromide, destained in water, and imaged under UV excitation on a BioRad imager then quantified. vii. MICROSCALE THERMOPHORESIS (MST) [00390] A total of 12 concentrations of ligand proteins (e.g., EYA (1-365)) were serially diluted by 1:1 using MST buffer from 5 uM to 2.4 nM, 10 ul of each dose of ligand protein was mixed with 10 ul of cy5-labeled dsDNA/ssDNA supernatant. The mixture of EYA and DNA was loaded into premium capillaries for MST assay with parameters set up at auto-detected excitation power and medium MST power. Binding affinity was analyzed by MO. Affinity Analysis software (version 2.1.3, NanoTemper Technologies) using the signal from an MST-on time of 20 s. Three individual experiments were merged to generate standard deviation. viii. ELECTRON MICROSCOPY [00391] The reaction was carried out at 37 ºC in buffer containing 2 mM ATP, 2.5 mM MgCl2, 50 mM KCl, and no BSA. To assemble nucleoprotein filaments, RAD51 (2.4 μM) was incubated with an 83mer Oligonucleotide polydT (7.2 μM nucleotides) for 5 min and then the reaction mixtures were diluted 8-fold with the same buffer, and a 4 μl aliquot was applied to 400-mesh grids coated with carbon film and which had previously been glow- discharged in air. After staining for 30s with 4% uranyl acetate, the samples were examined in a JEOL JSM-6610LV electron microscope equipped with a tungsten filament at 100 keV. Digital images were captured with a charge-coupled device camera at a nominal magnification of X 63,000. ix. IMMUNOPRECIPITATION (IP) AND MASS SPECTROMETRY. [00392] 1.2x106 HeLa cells were reverse transfected with 1 μg of DNA (empty vector or pEGFPC1-EYA4). Media was changed at 24 hrs, and 48 hrs post transfection cells were irradiated (or not) with 10 Gy, incubated for an additional 1 hr, and proteins were extracted by autolyze on ice, using 1 mL of extraction buffer for each condition. Resuspended cells were sonicated, spun down at 14,000 rpm at 4 °C for 10 minutes. Cell extracts were rocked with 200 μL of resin (IgG anti-mouse or nanobody anti-GFP, immobilized on beads) for 2 hrs at 4 °C. Beads were washed 4 times in 50 mM Tris-HCl (pH 8.0), 750 mM NaCl, 10% glycerol, and proteins bound were eluted in Laemmli buffer, loaded on gel, digested, and subjected to analysis by mass spectrometry (UTHSCSA Core facility). Data were visualized and analyzed using Scaffold. x. AFFINITY PULL-DOWN [00393] Affinity pull-downs were performed as described elsewhere (E. Dray et al., Proc Natl Acad Sci U S A 108, 3560-3565 (2011), E. Dray et al., Nat Struct Mol Biol 17, 1255-1259 (2010)).2 μg (2.5 μL) of GFP-tagged EYA4 fusion protein immobilized on beads was incubated with 2 μg of purified proteins (H2A, H2B) in 10 μL of reaction buffer (25 mM Tris-HCl (pH 7.5), 120 mM KCl, 1 mM ^-mercaptoethanol) for 30 minutes at 4 °C. Unbound proteins were collected as supernatant (S). Beads were washed (wash W) three times with 100 μL of the same buffer and proteins complexed with EYA4 were eluted (E) in 20 μL of Laemmli buffer.10 μL of the supernatant (S), first wash (W), and eluation (E) were analyzed by SDS-PAGE. xi. CANCER CELL LINE ENCYCLOPEDIA (CCLE) ANALYSES [00394] Protein-level expression data from cell lines in the CCLE dataset with available EYA4 protein levels (n=90) were downloaded (D. P. Nusinow et al., Cell 180, 387- 402 e316 (2020)). Chromosome instability (CIN70) and homologous recombination deficiency (HRD) scores in corresponding cell lines were determined as previously described (L. Carter, et al., Nat Genet 38, 1043-1048 (2006), G. Peng et al., Nat Commun 5, 3361 (2014)). xii. CHROMOSOMAL BREAK IN VITRO REPORTER SYSTEM AND FLOW CYTOMETRY [00395] The HEK293-puro-DR-GFP cell line has been described elsewhere (P. Caron et al., Cell Rep 13, 1598-1609 (2015), J. O'Sullivan, et al., J Vis Exp 10.3791/62553 (2021)). Cells were reversed co-transfected with 3 µg I-SceI expression plasmid (pCβASce; (54)) and 500 ng MISSION TRC2 pLKO.5-Puro empty vector or shRNA constructs, as indicated. Transfected cells were kept in regular growth medium and analyzed by flow cytometry after 72 hrs to measure the percentage of cells expressing GFP (E. Dray et al., Proc Natl Acad Sci U S A 108, 3560-3565 (2011)). For complementation, cells were co-transfected with shRNA1 and pcDNA3.1 Myc/His EYA4 or containing a mutant version of EYA4. xiii. CELL CYTOTOXICITY ASSAY (MTT) [00396] HeLa cells (empty vector or knocked down for EYA4) were seeded in 96-well plates. After 24 hrs, increasing concentrations of PARP inhibitor (olaparib) were added. Cell cytotoxicity was measured following manufacturer’s protocol (Abcam ab211091). Briefly, 50 µL serum-free media and 50 µL MTT reagent was added to each well and incubated at 37 °C for 3 hrs. MTT media was replaced with 150 µL of MTT solvent and incubated with agitation for 15 min. Absorbance was measured at 590 nm. xiv. ANTI-GFP NANOBODY AGAROSE PRODUCTION [00397] Plasmid encoding the sequence of the anti-GFP nanobody was obtained and the nanobody was purified (M. J. Schellenberg, et. al., Protein Sci 27, 1083-1092 (2018)). The protein was expressed in E. coli BL21 (DE3) strain with an induction with 0.1 mM of IPTG for 16 hrs at 16 °C. Cells were harvested and resuspended in lysis buffer (50 mM Tris- HCl (pH 8.0), 1 M NaCl, 1 mM TCEP, protease inhibitors: aprotinin, chymostatin, leupeptin, and pepstatin A at 3 μg/ml each). The resuspended cells were sonicated with 5 cycles of 15 seconds with 30 seconds cooling periods (50% maximal output). Subsequently, lysed cells were clarified by centrifugation at 100,000 ^ g for 45 minutes at 4 °C, the supernatant was collected and incubated with 5 mL of Ni2+-NTA beads at 4 °C with agitation for 2 hrs. Beads were collected, washed with 200 mL of 50 mM Tris-HCl (pH 8.0), 1 M NaCl, and the protein eluted in 10 ^ 5 mL fractions in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 300 mM imidazole. Fractions containing the nanobody, as identified by SDS-PAGE gel, were pooled, diluted in 50 mM Tris-HCl (pH 8) and passed over a 5 mL HiTrap Q fast flow column (Cytiva). The flow-through containing the unbound nanobody was concentrated to 5 mL and resolved onto a Hi load 16/200 Superdex 200 preparative column run in 0.1 M NaHCO3 (pH 8.4) with 0.5 M NaCl. Fractions containing the purified nanobody were pooled together and used for the coupling reaction. Purified anti-GFP nanobody was coupled to cyanogen bromide-activated agarose (Sigma-Aldrich) following the manufacturer’s indications. After coupling, blocking and washing, the beads were resuspended in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 50% glycerol and kept at 4 °C until further use. xv. PROTEIN EXPRESSION AND PURIFICATION [00398] Full length m-cherry EYA4 or the N-terminus domain of EYA4. Full length m-cherry EYA4 or the N-terminus domain of EYA4 (residues 1-365), was sub-cloned as a Strep-mCherry-EYA4-His construct in pFastBac1. Plasmids were used to make viral particles by first establishing a bacmid in DH10Bac (Invitrogen) then infecting Sf9 cells.200,000 particles /ml were used to infect 400 ml of Sf9 cells (1.2 x 10 6 cells/ml), grown 72h at 27 ºC, and harvested by centrifugation (5 minutes 1000 x g). Pellets were resuspended in lysis buffer (50 mM CAPS (pH 10.9), 100 mM NaCl, 20% sucrose, 10% glycerol, 0.5 mM EDTA, 1 mM TCEP, 1 mM PMSF, Igepal and protease inhibitors), sonicated 15 times for 20 seconds (50%). Lysed cells were clarified by centrifugation at 40,000 ^ g for 40 minutes at 4 °C. Supernatant was incubated with Ni-NTA 2+ resin for 3 hr with agitation. The resin was washed with 10CV Buffer B, 10CV Buffer C + 10 mM Imidazole, then eluted in buffer C+ 200 mM Imidazole. Elutions were then pooled, loaded onto a Q column, and eluted using a salt gradient 0-100% as described for the GFP-WT protein. Fractions containing EYA4 were pooled, dialyzed against buffer A to lower the conductivity, and loaded onto a monoQ column. The protein was eluted using a 15-60% gradient of buffer C. Fractions containing EYA4 were pooled, buffer exchanged in storage buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 30% glycerol) and concentrated before storage at -80 ºC. [00399] The C-terminus domain of EYA4 (residues 358-639). The C-terminus domain of EYA4 (residues 358-639), was sub-cloned as a GST-EYA4 (358-639) construct. The protein was expressed in E. coli C43 (DE3) strain with an induction with 0.1 mM of IPTG for 16 hrs at 16 °C. Cells were harvested and resuspended in lysis buffer (50 mM Tris- HCl (pH 8.0), 150 mM NaCl, 20% sucrose, 10% glycerol, 0.5 mM EDTA, 1 mM TCEP, 1 mM PMSF and protease inhibitors), sonicated 10 times for 10 seconds. IGEPAL CA 630 was added (1% v/v), then lysed cells were clarified by centrifugation at 100,000 ^ g for 45 minutes at 4 °C. Clarified supernatant was then incubated with 5 mL affinity resin (Glutathione sepharose, Cytiva) for 90 min under agitation at 4 ^C. The resin was washed with 20 CV of buffer B, followed by 10 CV of buffer C. The protein was then eluted in 5 x 5 mL fractions in elution buffer D (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 25 mM glutathione, 10% glycerol). Fractions with EYA4 (358-639), as identified on SDS-Page, were pooled (approx.25 mL), diluted with dilution buffer (50 mM Tris-HCl (pH 8), 10% glycerol) and loaded onto a Fast Flow Q column. The protein was fractionated in 3 mL fractions using a linear gradient to 100% of buffer B (50 mM Tris-HCl (pH 8.0), 1 M NaCl, 10% glycerol). Fractions containing the peak EYA4 were pooled and resolved onto a Superdex200 column run in Tris-HCl (pH 8.0) with 300 mM NaCl. Fractions containing EYA4 (358-639) were pooled together and concentrated before storage. [00400] RAD51 WT and mutants cloned in pET11. RAD51 WT and mutants cloned in pET11 were expressed in BLR DE3 pLyS E. coli strain, and purified following previously described procedures (C. Wiese et al., Mol Cell 28, 482-490 (2007)). For GFP-RAD51 proteins, RAD51WT was a kind gift from Roland Kanaar (Erasmus); Y54F, Y315F and Y54D, Y315D were sub-cloned from pEt11d into pEGFP-C1 and all constructs were expressed in 293 suspension cells. Pellets from 250ml suspension were resuspended in RAD51 lysis buffer (T. T. Paull, Curr Opin Genet Dev 71, 55-62 (2021).) containing complete Roche anti-proteases, homogenized in a Dounce homogenizer, sonicated 10 times at 50% for 20 seconds, and spun at 40,000xg for 30 minutes at 4 ºC. The clarified soluble proteins extract was loaded onto a 30ml Fast Flow Q column and eluted with a 0-100% gradient of KCl in T buffer. Fractions eluted from 38-mM salt were combined, dialyzed against T buffer, and loaded onto a 5ml macrohepatite column. Fractions containing RAD51 were pooled and incubated with 1ml of GFP-nanobody agarose beads as described above. After 2h incubation with agitation at 4 ºC, 400 uL of beads were collected, washed in T+1000, T+150, then resuspended in storage buffer (T+300mM NaCl, 30% glycerol), aliquoted in 10 uL aliquots, and frozen at -20 ºC to be used for pulldowns. The 600 uL beads left over were eluted by adding 200 mM Glycine pH2 in T+ 150mM NaCl, to the resin and collected in 10 uL NaOH to neutralize the pH. Elutions were pooled, pH was adjusted to 9, diluted 1:1 in T buffer to lower the sample conductivity, and loaded onto a monoQ column. Fractions containing RAD51 were combined, concentrated, and frozen. xvi. DNA SUBSTRATES [00401] Cy5-labeled oligo P1 (T. T. Paull Curr Opin Genet Dev 71, 55-62 (2021)). from IDT (TTATATCCTTTACTTTGAATTCTAT- GTTTAACCTTTTACTTATTTTGTATTAGCCGGATCCTTATTTCAATTATGTTCAT; SEQ ID NO:1) was used as the ssDNA substrate. dsDNA was generated by annealing Cy5- labeled oligo P1 with its exact complement P2 ATGAACATAATTGAAATAAGGATCCGGCTAATACA-AAATAAGTAAAAG GTTAAACATAGAATTCAAAGTAAAGGATATAA (SEQ ID NO:2). The annealed product was purified by SDS-PAGE then concentrated and quantified. For the long system assays, substrates were sourced from NEB. The ssDNA is the ^X174 virion DNA and the dsDNA its replicative form ΦX174 RF I DNA. xvii. ATPASE ASSAY [00402] To measure ATPase activity, previously published protocols were followed (R. Prakash et al., Genes Dev 23, 67-79 (2009)). xviii. MASS PHOTOMETRY [00403] To investigate the oligomerization vs polymerization states of RAD51 species, samples were diluted to mass photometry analysis on (Reyfen). Proteins were diluted in EMSA reaction buffer containing ATP and MgCl 2 to 50 nM and the contrast was recorded over 60 seconds. For DNA binding analysis, DNA was added to the buffer + protein sample, allowed to equilibrate for 60 seconds, then recorded for 60 seconds. Covalbumin (75 kDa), Aldolase (158 kDa) and Ferritin (440 kDa) were used to establish the mass calibration. [00404] Alternatiely, samples were diluted to mass photometry analysis on (Reyfen). Proteins were diluted in Buffer A or reaction buffer. Covalbumin (75 kDa), Aldolase (158 kDa) and Ferritin (440 kDa) were used to establish the mass calibration. xix. EYA4-DEFICIENT CELLS ACCUMULATE DNA DAMAGE [00405] Complete knockout of EYA4 is lethal in most mouse strains shortly after birth (F. F. Depreux et al., The Journal of Clinical Investigation 118, 651-658 (2008)), and poorly tolerated in several lung cell lines (I. M. Wilson et al., Oncogene 33, 4464-4473 (2014)) and other cell lines. However, using short hairpin RNAs (shRNAs) a significant decrease EYA4 expression and protein levels in HeLa cells is observed (FIG.16B), HEK-293T cells, and MDA-MB-231 cells, and mutants of the EYA4 catalytic domains were established (see FIG. 16A for details). [00406] Referring to FIG.16A, a representative depiction illustrating that two domains of EYA4 that possess phosphatase activity are well conserved among vertebrates is shown. Mutations used in this study are indicated by stars. FIG.16B shows representative data illustrating that incomplete knock down can be achieved by shRNA, and leads to decrease of both proteins. [00407] Upon knocking down EYA4, HeLa cells readily acquired nuclear defects indicative of genomic instability (FIG.16C). Cells depleted for EYA4 showed aneuploidy, DNA bridges, and accumulation of micronuclei. This is consistent with various mitotic defects, particularly in metaphase, previously observed upon EYA4 knockdown in Mitocheck screens (Table 2)(Y. Cai et al., Nature 561, 411-415 (2018)). To further assess this phenotype in a broader panel of cell lines, 90 cell lines were identified in the Cancer Cell Line Encyclopedia (CCLE) dataset with available EYA4 protein-level data (D. P. Nusinow et al., Cell 180, 387-402 e316 (2020)). The CIN70 signature was used to evaluate the degree of genomic instability (S. L. Carter, et al., Nat Genet 38, 1043-1048 (2006)). Cell lines with low EYA4 protein levels showed significantly higher protein expression levels of CIN70 signature proteins, a surrogate for the degree of genomic instability (Y. Cai et al., Nature 561, 411-415 (2018)), when compared to cell lines with high EYA4 protein levels (p = 0.0028, t test) (FIG.16D). [00408] Referring to FIG.16C, representative data illustrating that cells depleted for EYA4 present with large numbers of nuclear aberrations (bridges, micronuclei, lost chromosomes; quantification N>300) is shown. FIG.16D representative data illustrating that EYA4 depletion leads to elevated γH2AX foci (i) microscopy and (ii) quantification is shown. TABLE 2.
[00409] The level of γH2AX was investigated in control cells and cells depleted for EYA4 by any of three shRNAs. When compared to cells expressing a non-targeting shRNA, more than 50% of EYA4-depleted cells exhibited an overall 8-fold increase of γH2AX foci in normal growth conditions (5% CO2, 37 °C) without any exogenous stress and contained 10 or more discrete γH2AX foci (FIG.16E). Accumulation of γH2AX is a reliable marker for DSBs, however there is the possibility that H2AX S139 could be a target of EYA4 phosphatase activity and thus remain phosphorylated regardless of the DNA repair status, even in the absence of EYA4. A test for whether EYA4-depleted cells accumulate unrepaired DNA breaks or whether they are proficient for repair but deficient for dephosphorylating H2AX was created by causing DSBs with irradiation, then followed the stimulation and resolution of γH2AX foci. A time course of γH2AX foci by immunofluorescence (FIG.17A) and measurement of protein levels by Western blotting (FIG.17B) both show that γH2AX is constitutively elevated in cells depleted for EYA4, but still increases following irradiation. Interestingly, some DSBs are repaired in EYA4-depleted cells because γH2AX is resolved over time and returns to its initial, elevated levels, 2-4 hours post irradiation (FIG.17A and FIG.17B). These observations demonstrate that EYA4 depleted cells exhibit accumulation of DNA damage but repair, at least partially, some irradiation-induced DSBs. [00410] Referring to FIG.16E, representative data illustrating the CIN70: chromosomal instability score based on expression levels of 70 proteins, showing CIN linked to EYA4 expression is shown. 90 cell lines from CCLE split in two equal-size groups: EYA4 low and EYA4 high with median EYA4 protein expression level used as cut-off. Means +/- SEM are shown. P value: p=0.0028, t test. FIG.17A, representative data illustrating the kinetics of γH2AX foci formation over time, in cells proficient and deficient for EYA4, and after irradiation is shown. Specifically, in (i) representative images are shown and in (ii) foci are quantified and plotted +/- standard deviation. FIG.17B shows representative data illustrating γH2AX protein levels followed by Western blot after irradiation (10Gy). b. HISTONE VARIANT H2AX PY142 BUT NOT PS139, IS A SUBSTRATE OF THE EYA4 PHOSPHATASE ACTIVITY [00411] PP4 and WIP1 are the known phosphatases for γH2AX (D. Chowdhury et al., Mol Cell 31, 33-46 (2008) and H. Cha et al., Cancer Res 70, 4112-4122 (2010)) and EYA4 is unlikely to dephosphorylate S139, but EYA1-3 proteins have been found directly or indirectly to dephosphorylate residue Y142 of H2AX upon DNA damage (R. S. Hegde, et al., Crit Rev Biochem Mol Biol 55, 372-385 (2020), I. M. Wilson et al., Oncogene 33, 4464-4473 (2014)), and by similarity, EYA4 was suggested to possess the same activity (I. M. Wilson, et al., Oncogene 33, 4464-4473 (2014)). The two residues are in close proximity (FIG.17C) and while pY142-H2AX is a low abundance modification (A. A. Hatimy, et al., International Journal of Mass Spectrometry 391, 139-145 (2016).), it is thought to be essential for the promotion of DNA damage repair over apoptosis (P. J. Cook et al., Nature 458, 591-596 (2009), S. Solier, et al., Cell Cycle 8, 1853-1859 (2009)). The dephosphorylation of H2AX by EYA4 was investigated, EYA4 was expressed and purified as a GFP-fusion protein using previously described chromatography conditions (FIG.17D). Purified EYA4 was incubated with phosphorylated H2AX peptides (FIG.17E, and Table 3) containing either pS139 or pY142 at 37 °C for 60 min. Released phosphate was quantified by a colorimetric method using malachite green-molybdate (FIG.17E). Both threonine and tyrosine phosphatase domains of EYA4 have been described as functional in vitro (Y. Okabe, et al., Nature 460, 520-524 (2009)). Under conditions optimal for EYA4 tyrosine phosphatase activity (acidic, with Mg 2+ ), the synthetic pY142 peptide was dephosphorylated 5-fold more efficiently than pS139 peptides (FIG.17E). Using the serine threonine- optimized conditions (described in supplemental methods), pS139 was still minimally dephosphorylated and pY142 was not dephosphorylated, indicating that the experimental conditions are specific for each of the activities tested. EYA4 is not the phosphatase for γH2AX but its efficient dephosphorylation of H2AX on pY142 could promote DNA damage repair signaling following DNA damage. The results show that the phosphatase activity of EYA4 is greatly stimulated by addition of single-stranded DNA in the reaction (+/- DNA, FIG.17E). Verification of the results was obtained of the in vitro findings in cells, using a specific antibody directed against pY142 of H2AX, for immunofluorescence and in Western blots. Higher levels of pY142 were detected in EYA4 depleted cells compared to controls (FIG.17F and FIG.22A). Y142 phosphorylation level decreases in response to DNA damage (P. J. Cook et al., Nature 458, 591-596 (2009)), but also an accumulation of pY142-H2AX in EYA4 depleted cells, at 2 hours following irradiation (FIG.17F and FIG.22A). Without wishing to be bound by theory, this suggests that EYA4 could target H2AX pY142 for dephosphorylation, but also contributes to its accumulation, possibly by targeting upstream kinases. [00412] Referring to FIG.17C representative data illustrating that residues S139 and Y142 are on the extreme C-terminal position of the histone tail, H2AX (PDB P16104) is depicted in darker shade on a nucleosome structure is shown. FIG.17D shows representative data illustrating a protein purification scheme (left) used to purify GFP-EYA4. SDS PAGE and Coomassie stain (right) shows the purified protein. FIG.17E shows representative data illustrating a schematic of the malachite green colorimetric assay (left) used to investigate possible substrates of EYA4 (right) such as residues pS139 and pY142 in H2AX. FIG.17F shows representative data illustrating the Foci formation in EYA4 depleted cells using an antibody specific of phospho-tyrosine 142. TABLE 3. c. EYA4 IS A DNA BINDING PROTEIN AND INTERACTS WITH DNA, HISTONES AND NUCLEOSOMES [00413] The unexpected stimulation of phosphatases reaction by DNA raised the possibility that EYA4 could interact directly with DNA. Purified EYA4, GFP full length (FIG.17D), N-terminally m-cherry-tagged EYA4 (aa 1-365, FIG.18A and FIG.23A), and GST-C-terminal EYA4 (aa 358-639; FIG.23A) was used to explore this possibility. Electromobility shift assay (EMSA) using purified N-EYA4 (1-365) and synthetic substrates to mimic ssDNA or dsDNA was performed. It was found that EYA4 binds to both ssDNA and dsDNA (FIG.18B and FIG.23B) and in a competition, identified substrate specificity for ssDNA. The N-terminus of EYA4 contains the DNA binding activity and exhibits the same substrate specificity as full-length EYA4, namely preferring ssDNA over dsDNA using a “short system” where substrates are purified oligonucleotides (FIG.18B) or a “long system”, which utilizes intact plasmids with no DNA end available (FIG.18B). The C- terminus EYA4, encompassing the tyrosine domain (358-639) does not possess DNA binding activity (FIG.23D). Super-shifts observed with increasing concentration of EYA4 in both the short and long systems indicate that it accumulates on the phage DNA and does not bind solely to DNA ends. Dephosphorylation of H2AX and DNA binding activity makes EYA4 a good candidate for a role in DNA repair. Immunoprecipitation was performed followed by mass spectrometry in HeLa cells expressing endogenous levels of EYA4 and subjected to either no irradiation or 10 Gy irradiation. Among other interactions, EYA4 was found strongly associated with chromatin, and bound to histones and associated proteins in the absence of IR. Binding to histones is weakened or lost following irradiation (FIG.18D), suggesting that EYA4 could be an early responder to DNA damage and is displaced following dephosphorylation of H2AX, possibly to allow chromatin accessibility by repair machineries. Looking at EYA4 sequence, ATM-ATR consensus site SQTQ in position 209- 212 was identified. Mutations were introduced to create a phosphomimetic EYA4 double mutant: S209D, T211D. The DNA binding experiments were repeated, and observed a near complete abolishment of the DNA binding activity in the double mutant (FIG.18E and TABLE 4). This partially explains the observation that EYA4 relocates from the chromatin following DNA damage, likely upon phosphorylation by ATM. Since EYA4 binds to DNA, it was to be determined if the histones binding observed in IP is direct or through DNA. Using the GFP-EYA4 protein immobilized on GFP-nanobody beads (M. J. Schellenberg, et al., Protein Sci 27, 1083-1092 (2018)) and purified histones, in vitro pull-downs were performed, and extremely robust interactions between EYA4 and H2A or H2B were identified (FIG.18F). Despite its preference for ssDNA, EYA4 also binds strongly to dsDNA nucleosomes (FIG.18F). [00414] Referring to FIG.18A representative data illustrating FL WT, or FL Mutated (** indicate S209, T211residues), N-terminal 1-365 or C-terminal construct were purified for testing DNA binding activity is shown. FIG.18B shows representative data illustrating increasing concentrations (0-300nM) of EYA41-365 were incubated with ssDNA, dsDNA or a mixture of both. Electromobility shift assay was visualized by tracking the Cy5-DNA on gel under UV (Biorad imager). The percentage of bound DNA was quantified and plotted. Error bars = mean ± SEM of n>3. FIG.18C shows representative data illustrating MST experiment shows binding of EYA4-N to ssDNA binding (KD 303 nM) and dsDNA (KD 732 nM). FIG.18D shows representative data illustrating IP-MS workflow and a subset of peptides pulled-down with GFP-EYA4 in HeLa cells non-irradiated (0 Gy) or 1 hr after irradiation (10Gy). FIG.18E shows representative data illustrating a mutant of EYA4 that mimics phosphorylation on residues S209 and T211 does not bind DNA as shown by MST (KD > 2 ^M). FIG.18F shows in vitro pull-down with purified histones H2A and H2B(left) or nucleosomes (NEB kit). FIG.23A shows M-cherry constructs (FL WT, FL (S209D, S211D), and 1-365) or a GST(359-639) construct were expressed and purified to test DNA binding properties of EYA4. FIG.23B shows EYA4 full length WT was found to bind ssDNA using phage DNA. FIG.23C shows that using the 1-365 construct, a comparable ssDNA binding activity was observed, and at concentration>500nM 1-365 also binds to dsDNA. FIG.23D shows conversely, the 358-639 fragment does not possess significant DNA binding activity, for ssDNA nor dsDNA. TABLE 4. d. EYA4 IS PART OF DNA DAMAGE REPAIR FOCI [00415] To gain a better understanding of EYA4 cellular behavior, the GFP-tagged version of EYA4 in cells was followed over time. EYA4 forms spontaneous foci in cells. Untreated cells possess an average of 20, small discrete EYA4 foci, that appear randomly distributed in the nucleus (FIG.18D) and fuse into bigger structures in G2 phase. When using an antibody directed against the endogenous EYA4 in WT cells, it was found that irradiation promptly stimulates EYA4 foci formation, and these colocalize with γH2AX (FIG.19A). Further interrogation of the colocalization of EYA4 with other DNA repair proteins, but also the utilization of a different system to confirm that the observations are not due to the irradiation or the antibody was performed using the DIvA system (DSB inducible via AsiSI (P. Caron et al., Cell Rep 13, 1598-1609 (2015)), provided by the Legube lab, transfected with GFP-EYA4, probing for the GFP tag during immunolocalization. In untreated DIvA cells, a low basal level of γH2AX foci, and low or absent RAD51 foci were observed (FIG.19B, top panel). Cells expressing highest levels of EYA4 were also found to exhibit RAD51 foci, even in the absence of γH2AX (FIG.19B, top panel). Without wishing to be bound by theory, this suggest that EYA4 co-localizes with non-repair RAD51 foci, and that RAD51 accumulates in the presence of EYA4. Following the addition of tamoxifen and subsequent translocation of AsiSI, DSBs were introduced as shown by increased γH2AX, and EYA4 formed robust foci, a large proportion of which colocalize with RAD51 foci (54%, FIG.19B, bottom panel) and with γH2AX (25%). [00416] Referring to FIG.19A shows representative data illustrating EYA4 (2nd column) and γH2AX (3rd column) foci were observed by indirect immune-fluorescence on samples fixed after no irradiation (0Gy) or 4 Gy irradiation, at the indicated time points. Foci were quantified and plotted. FIG.19B shows representative data illustrating GFP-EYA4 transfected DIVA cells, with no DSB (-4OHT) or after induction of DSB by hydroxytamoxifen (+4OHT) were imaged and co-localization of EYA4, RAD51, and γH2AX at the break was quantified and plotted. e. IN THE ABSENCE OF EYA4, HOMOLOGOUS RECOMBINATION EFFICIENCY IS COMPROMISED [00417] Accumulation of unrepaired DSBs in EYA4-depleted cells and the colocalization of EYA4 with γH2AX and RAD51 indicate that EYA4 might be a hitherto unidentified player of the DNA damage repair response. The repair of DSBs in the absence of EYA4, after no or 4Gy irradiation was investigated. Ionizing radiation-induced foci formation by γH2AX, pRPA, and RAD51 was followed. EYA4 depleted cells were found to accumulate γH2AX foci and replicative pRPA (phosphorylated at residue S33; FIG.24A) but failed to form pRPA (S4/S8) foci that are surrogate markers of end resection at the break (FIG.19C and FIG.24B). This suggests that the signaling of the break is perturbed in the absence of EYA4 and processing of the break might not be fully proficient, which was verified using a previously published protocol, where incorporation of BrdU is measured in native conditions (J. O'Sullivan, et al., J Vis Exp 10.3791/62553 (2021)). The lack of a denaturing step allows to measure exclusively ssDNA, as a readout for resection efficiency. Use of this technique provided evidenced that cells depleted for EYA4 exhibit diminished resection compared to control cells, in response to IR (FIG.19D). Consistent with this, RAD51 is recruited less efficiently to the break in EYA4 depleted cells, and later than in control cells (FIG.19E and FIG.25A). Using cells stably expressing the Fucci system (R. Tropee et al., Breast Cancer Res Treat 185, 601-614 (2021)), in which clover-geminin and mKO-cdt1 label cells in green when in S-G2-M and in red when in G1 respectively, it was verified that EYA4 depleted cells present no significant defect in cell cycle that would explain decreased RAD51 foci formation (FIG.25B). [00418] Referring to FIG.19C representative data illustrating γH2AX and pRPA foci (S4/S8) were observed overtime, after no (0 Gy) or 4 Gy irradiation, in controls (EV) or cells depleted for EYA4 (shRNA3) is shown. Foci were imaged (i) and quantified (ii). FIG.19D shows representative data illustrating BrdU incorporation in control cells or silenced for EYA4 after irradiation allows indirect measurement of resection. FIG.19E shows representative data illustrating that the same experiment as in FIG.19C was performed to follow γH2AX and RAD51 IR-induced foci. FIG.24A shows the accumulation of RPA phosphorylated at S33 under normal and stress conditions are observed in the absence of EYA4. Representative images of foci formation are shown (i; scale bar 10 μm) and quantified (ii; mean ± SD; n ≥ 100; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001). Induction is plotted (iii). FIG.24B shows the time-course of pRPA (S4/S8) following radiation (4Gy) induced DSB formation at T = 0 (untreated) and T = 1, 2 and 4 hours (4 Gy) are shown for three hairpins that deplete EYA4 (shRNA1-3) and a control (EV) Scale bar 10 μm. Nuclear foci were quantified and plotted as mean ± SD; n ≥ 300; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. FIG.25A shows the time-course of RAD51 foci formation following radiation (4Gy) at t = 0 (untreated), t = 1, 2 and 4 hours post irradiation. Scale bar 10 μm. Nuclear foci were quantified and plotted as mean ± SD; n ≥ 300; ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. FIG.25B shows that nuclear RAD51 foci in HeLa Fucci cells were observed without irradiation, or 2h post 10 Gy-irradiation. Images were acquired by high throughput content imager (Operetta) and analyzed using the GE analysis software. [00419] Lack of RAD51 foci indicate that recombination-directed DSB repair might be affected. To ascertain this, the use of the in vitro system developed by the Jasin group (A. J. Pierce, et al., Genes & Development 13, 2633-2638 (1999), K. Nakanishi, et al., Methods in Molecular Biology 745, 283-291 (2011)) was made, which allows direct measurement of HR. EYA4 depleted cells were found less proficient in repairing DSBs by HR (FIG.20A). Repair was restored to control levels by re-introduction of the full-length EYA4. The EYA4 depleted cells were systematically complemented with various EYA4 constructs containing one or more mutations in the catalytic domain (FIG.16A). Doing so evidenced that the tyrosine phosphatase domain of EYA4 is essential for HR (FIG.20B). [00420] Referring to FIG.20A shows representative data illustrating that the DR-GFP system (i) was used in cells and the percentage of GFP-positive cells was quantified by flow cytometry on >100,000 cells to estimate HR efficiency. shRNA used are indicated. FIG.20B shows representative data illustrating where mutations were introduced in EYA4 to inactivate phosphatase activity (i). These constructs were used to transfect DR-GFP cells and quantify their individual contribution to HR (ii). [00421] To assess whether reduced EYA4 protein levels might cause HR deficiency more broadly, previously defined HR deficiency scores (G. Peng et al., Nat Commun 5, 3361 (2014)) were determined in 87 CCLE cell lines (Y. Cai et al., Nature 561, 411-415 (2018)). This revealed that cell lines with low EYA4 protein levels show significantly higher HR deficiency compared to cell lines with high EYA4 protein levels (p=0.0167, t test) (FIG. 20C). In accordance with a HR repair defect, cells depleted for EYA4 were also found sensitive to the PARP inhibitor Olaparib in an MTT assay (FIG.20D). [00422] FIG.20C shows representative data illustrating 87 cell lines from CCLE. Split in two equal-size groups: EYA4 low and EYA4 high with median EYA4 protein expression level used as cut-off. Means +/- SEM are shown. P value: p=0.0028, t test. HRD score: homologous recombination deficiency score, as defined by Peng et al (G. Peng et al., Nat Commun 5, 3361 (2014)). FIG.20D shows representative data illustrating a MTT assay was used to measure survival in response to the PARP inhibitor olaparib. For all panels * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. f. RAD51 Y315 IS A TARGET OF EYA4 [00423] RAD51 phosphorylation levels on residues Y54 and Y315 could influence its function (D. Chowdhury et al., Mol Cell 31, 33-46 (2008), H. Cha et al., Cancer Res 70, 4112-4122 (2010)). The tyrosine kinase c-ABL, or its oncogenic fusion protein BCR/ABL, phosphorylates RAD51 in a sequential manner, on residue Y315 followed by Y54 (M. Popova et al., FEBS Letters 583, 1867-1872 (2009)). This sequential phosphorylation is important for RAD51 recruitment to DSB sites and its strand exchange activity (S. Subramanyam, et al., PNAS 113, E6045-E6054 (2016)). Persistent phosphorylation of Y315, and consequently double-phosphorylated status of RAD51, is known to have an inhibitory effect on HR, due to a defect in nucleofilament formation (B. Alligand, et al., Biochimie 139 (2017)). Using a specific antibody recognizing pY315, it was observed that cells depleted for EYA4 exhibit elevated foci of pY315RAD51, compared to control cells (FIG.21A). Interestingly, control cells exhibit some pRAD51 in the absence of stress, which decrease in response to IR (FIG.21B). However, in cells depleted for EYA4, pY315 remains elevated (FIG.21B and FIG.26A). Using the malachite green in vitro phosphatase assay, Y315 was identified, but not Y54, to be a substrate for EYA4 (FIG.21C). Interestingly, the dephosphorylation of RAD51 by EYA4 was again found stimulated by the addition of ssDNA suggesting a common activation mechanism for its phosphatase function. Biochemical characterization of Y54 and Y315 have been performed independently ((M. Popova et al., FEBS Letters 583, 1867-1872 (2009), S. Subramanyam, et al., PNAS 113, E6045-E6054 (2016), B. Alligand, et al., Biochimie 139 (2017)) and very elegant studies from the Spies and Fleury laboratories evidences the role of these residues in RAD51 recombinase activity. However, there are instances where dual phosphorylations can have a very distinct effect than single PTM, as previously exemplified by S139, Y142, in H2AX (J. A. Brown, et al., FEBS Open Bio 2, 313-317 (2012)). Therefore, the combined action of these two residues was investigated. Hence, the double-mutant RAD51 (Y54F, Y315F) that cannot be phosphorylated was purified, as well as the double phospho-mimetic RAD51(Y54D, Y315D), which mimics a phosphorylated tyrosine that cannot be dephosphorylated (FIG.21D). Based on the cell-based data and the literature available on these RAD51 tyrosine residues, the ability of RAD51(Y54F, Y315F) to polymerize onto ssDNA and form nucleoprotein filaments was investigated. Surprisingly, this mutant was observed by electron microscopy and negative staining (FIG.21E) to be much more proficient at polymerizing than the WT protein (FIG.21E). It can form filaments across ~10 lengths of ssDNA (83dT) in the mutant (FIG.21E), while WT RAD51 coats ssDNA as individual filaments, without any tethering activity (FIG.21E). In light of these surprising data, a T309A mutant that is less efficient in HR (P. B. Narayanaswamy, et al., Cell Death Dis 7, e2383 (2016)) but does not affect filament formation was purified and used. This mutant did not exhibit significant differences when compared to WT RAD51. The RAD51 (Y54D, Y315D) phosphomimetic formed globular structures that are reminiscent of the hexameric ring. Minimum variations in the ATPase activity were observed between RAD51 WT and mutants (FIG.26B), that cannot fully explain the phenotype observed. DNA binding experiments by microscale thermophoresis (MST) were conducted (FIG.21F). Surprisingly, it was found that Y54F, Y315F binds to ssDNA with an affinity comparable to the WT RAD51. However, it is much more avid for dsDNA. In this experiment, the hill coefficient was very high at 7.07 (Table 5 and FIG.26C), which is consistent with cooperative binding, phase separation and/or condensation. These data indicate a different DNA binding modality for the Y54F, Y315F mutant, possibly by aggregation onto the DNA. Even more surprisingly, the phospho-mimetic Y54D, Y315D was found to bind neither ssDNA nor dsDNA (FIG.26F) with a good affinity. While this was consistent with the EM observations, it could not rule out that this phospho-mimetic can bind to nucleic acid. Indeed, in two independent protein preparations (no added DNA and no ATP nor MgCl2 in the storage buffer), rare, long filament structures were observed (FIG.26C). Whether RAD51 (Y54D, Y315D) co-purifies with specific sequences of DNA, RNA, or forms spontaneous filamentous polymers on its own remains to be elucidated. Mass Photometry analysis (Reyfen) confirmed that the WT RAD51 forms hexameric rings in solution at the expected ~200kDa mark, the Y54D, Y315D and Y54F, Y315F readings indicated possibly different configurations (FIG.21G). When introducing GFP-fused WT; Y54F, Y315F or Y54D, Y315D constructs in cells either expressing EYA4 (EV) or not (shRNA3), it was observed that the WT forms structures as previously described for GFP-RAD51 (M. Reuter et al., J Cell Biol 207, 599-613 (2014)) in the EV cells. However, the Y54D, Y315D is found non- nuclear, consistent with its inability to bind to DNA (FIG.21H). In cells transfected with shRNA3, both the WT and the phospho-mutant constructs form condensates that are consistent with the MST data, namely that dephosphorylated RAD51 is prone to phase separation on DNA. It had been previously observed that the overexpression of EYA4-WT in cells led to an increase of RAD51 staining in otherwise untreated cells (see FIG.19B). Since EYA4 overexpression leads to accumulation of RAD51 in vivo (FIG.19) and dephosphorylates RAD51, the question remained whether RAD51 (Y54F, Y315F) might be more stable than RAD51 WT or RAD51 (Y54D, Y315D). Cells expressing GFP-RAD51 WT were subjected to photo bleaching experiments. Analysis of fluorescence intensity overtime after photodamage showed that RAD51 WT mobility is diminished in cells depleted for EYA4 when compared to control cells expressing EYA4 (FIG.26D). [00424] Referring to FIG.21A shows representative data illustrating phosphorylation at residue Y315 can be observed in cells using a specific antibody. FIG.21B shows representative data illustrating the pY315 levels in cell respond to irradiation. FIG.21C shows representative data illustrating EYA4 dephosphorylates residue pY315 in a malachite green assay and the activity is stimulated by DNA. FIG.21D shows representative data illustrating RAD51 WT, phospho-mutant Y54F, Y315F and phospho-mimic Y54D, Y315D were purified near homogeneity. FIG.21E shows representative data illustrating representative electron microscopy micrographs of the RAD51 proteins on DNA. FIG.21F shows representative data illustrating microscale thermophoresis measurements of RAD51 proteins on ssDNA or dsDNA and the affinity constants. FIG.21G shows representative data illustrating mass photometer measurement of apparent sizes for RAD51 polymers in suspension. FIG.21H shows representative data illustrating that introduction of RAD51 proteins as GFP-constructs in HeLa cells allows live imaging and the tracking of RAD51 structures formation and their subcellular localization. FIG.26A shows pY315RAD51 form dynamic foci that decrease in response to irradiation. Top panel (0Gy) is depicted in the main figures, bottom panel shows response to irradiation (2h post 4Gy). FIG.26B shows ATPase activity was measured by thin layer chromatography in the presence of 32P labelled ATP and hydrolyzed ATP was quantified over time and plotted. FIG.26C shows a fraction of the RAD51 Y54D, Y315D mutant protein preparation forms long pre-synaptic filament-like structures without added nucleic acid. FIG.26D shows WT-GFP-RAD51 transfected in control or EYA4 depleted (shRNA3) cells shows variation of mobility over time following laser (405) micro ablation. GFP intensity of the track was measured using Olympus CellSens and relative intensity to t0 (prior laser) is plotted. TABLE 5. [00425] Without wishing to be bound by theory, the combination of this increase in stability coupled to a high avidity for dsDNA and a slightly decreased ATPase activity of RAD51 (Y54F, Y315F) could explain RAD51 strong staining in cells overexpressing EYA4. [00426] Taken together, the data demonstrate that EYA4 promotes DNA repair by homologous recombination. It is phosphorylated upon DNA damage, dephosphorylates histone H2AX to promote repair over apoptosis, and contributes to HR repair through the dephosphorylation and subsequent stabilization of RAD51. g. THE TIMING OF REPAIR EVENTS IS AFFECTED IN EYA4 DEPLETED CELLS [00427] EYA4 depleted cells exhibit elevated levels of pY412-H2A as shown by western blotting and immunofluorescence. When cells were subjected to irradiation (10Gy for western blots or 4Gy for foci formation), minimal variations were observed over time by western blotting (FIG.22A). Without wishing to be bound by theory, this could be explained by the low abundance of the protein modification, which has been described previously as rare in proteomics studies, and the difficulty to protect phospho-tyrosines during protein extraction. In cells, the accumulation of H2AX-pY142 was more evident and elevated pY142 was observed in the cells depleted for EYA4 when compared to the controls. In addition, pY142 remains elevated at 4h post irradiation in EYA4 depleted cells and decrease in the controls from 2h. At 20h post irradiation, the shRNA treated cells only exhibit large clusters of pY142 stain (FIG.22B). h. EYA4 MAPPING OF THE DNA BINDING DOMAIN [00428] Various constructs were used to investigate the DNA binding activity of EYA4. EYA4 is a very difficult protein to purify, as it is largely disorganized from residues 1 to 320. EYA4 is prone to aggregation and phase separation, and for these studies, it could not be expressed or purified from E. coli despite trying many constructs. Using new generation fluorescent tags that are super folder and can help solubilize proteins in vivo, it was possible to express and purify GFP or m-cherry (FIG.23A) in human cell suspensions and Sf9 insect cells, respectively. Full length EYA4, as well as the N-terminal (1-365) and the C-terminal (358-639) halves of the protein, were expressed and purified (FIG.23A). EMSA was conducted using these three constructs and it was observed that EYA4 full length binds ssDNA with a high affinity, but in the range of concentration used (10-500 nM; FIG.23B) it exhibits little activity in dsDNA binding. Using the two halves of EYA4, it was verified that the N-terminus contains the full activity (FIG.23C) while the C-terminus portion does not contain any DNA binding activity. As 1-365 is easier to purify and concentrate, and, thus, more suited for experiments such as MST than the full length WT EYA4, subsequent DNA binding activity investigations were performed using 1-365. Interestingly, the full length EYA4 containing the SQTQ mutations S209D, S211D is extremely well expressed, can be purified in large amounts, and can be easily concentrated. i. DISCUSSION [00429] Phosphorylations are frequent post-translational modifications on proteins and they play key roles in a wide range of essential cellular functions. This includes regulation of DNA damage repair pathways, as kinases trigger cascades of protein activation and orchestrate their timely recruitment to DNA breaks and adducts. Most phosphorylation events in DNA repair that are well characterized and now utilized as readout of repair progression and efficiency, involve serine and threonine residues. Here, it is described that tyrosine residues on H2AX and RAD51, which have been described before as phosphorylated by the c-Abl kinase, are favored substrates of the protein phosphatase EYA4. Like the kinase DNA- PK, the activity of EYA4 is greatly stimulated by DNA binding. A fraction of EYA4 is nuclear, and it locates at the chromatin in dividing cells, through its N-terminal domain, which is shown here to directly interact with histones H2A and H2B, as well as with DNA. Following DNA damage, EYA4 dephosphorylates pY142 on H2A, detaches from the chromatin likely after being phosphorylated by ATM, and dephosphorylates RAD51 to promote HR. [00430] In recent years, it has become clear that RAD51 plays an important role in DNA metabolism, beyond vegetative double strand break repair and its function is not always mediated by BRCA2 alone (F. Prado, Genes (Basel) 12 (2021); M. J. Cabello-Lobato et al., Cell Rep 36, 109440 (2021); M. Tarsounas, D. Davies, S. C. West, Oncogene 22, 1115-1123 (2003)) or even BRCA-dependent. Here, it is shown that dephosphorylated RAD51 is more stable than its phosphorylated version, in cells and in vitro, and more proficient to nucleate onto ssDNA. It exhibits DNA binding modalities that diverge strongly from the WT protein, and was found to accumulate rapidly onto dsDNA. Whether this promotes duplex capture for homologous recombination or targets different DNA structures warrants further studies, including a biophysical characterization of the nucleoprotein filaments it can assemble. RAD51 is often upregulated in p53 deficient cancers. It is known to be overexpressed in breast, cervical, ovarian, pancreatic and other cancers (T. Takenaka et al., CInt J Cancer 121, 895-900 (2007); H. Maacke et al., Int J Cancer 88, 907-913 (2000)), where it causes drug resistance (H. L. Klein, DNA Repair (Amst) 7, 686-693 (2008); Y. Feng, et. al., Cancer Cell Int 21, 249 (2021); M. M. Hoppe et al., EMBO Mol Med 13, e13366 (2021)). Lack of phosphorylation by cAbl or excessive dephosphorylation of RAD51 by EYA4 could both lead to its accumulation and pathogenicity. The exact mechanism by which EYA4 undergoes post-translational modifications in response to DNA damage and how it regulates its interaction with other DNA repair proteins remains to be investigated. However, overall, these data start explaining the link between EYA4 and carcinogenesis. Decreased EYA4 levels in breast cancer samples might help identify HR deficiency, while overexpressed or hyperactive tyrosine phosphatases could predict RAD51 stabilization, accumulation, and resulting drug resistance. Targeting EYA4 could be of interest for the future development of novel cancer therapies, especially these aimed at decreasing uncontrolled DNA damage repair in drug resistant tumors. 4. EYA4 INHIBITION AFFORD BROAD SPECTRUM UTILITY IN CANCER TREATMENT [00431] As detailed herein above, the atypical protein phosphatase EYA4 plays a central role in regulating and coordinating DNA damage repair pathways. By promoting faithful double strand break repair through dephosphorylation of RAD51 and H2AX, and limiting non-homologous end joining, by dephosphorylating 53BP1 EYA4 is a protector of genomic stability and integrity. It limits gross chromosome rearrangements, chromosome fusions, and steers cells toward repair pathways rather than apoptosis following DNA damage. [00432] DNA repair is tightly regulated and accumulation of DNA repair proteins is almost always as deleterious as their depletion or mutation. Increase in either homologous recombination or DNA end joining can lead to illegitimate recombination, break point fusions, and severe genomic rearrangements. Overexpression of 53BP1 and RAD51 for instance, are linked to aggressive cancers and drug resistance. [00433] EYA4 is often found overexpressed in breast cancer and in esophageal cancer, and its accumulation can destabilize the genome by promoting homologous recombination even in G1 phase of the cell cycle, when it should be avoided at all cost and end joining is to be the dominant repair mechanism. EYA4 could thus be an ideal target for drug development, and its inactivation could be used to treat the many tumors overexpressing it. Here, the synthesis and characterization of a novel class of molecules that inhibit specifically and efficiently the tyrosine phosphatase activity of EYA4 is described. It is found that blocking the tyrosine phosphatase activity of EYA4 recapitulates major phenotypes observed in cells depleted for EYA4. Chemical blockade of the tyrosine dephosphorylation activity results in the accumulation of phosphorylated RAD51, which is less stable than its dephosphorylated counterpart and less prone to hyper-recombination. It is also found that DNA damage repair function by homologous recombination is lessened by increasing doses of the inhibitor, and higher doses of EYA4 inhibitor kills cancer cells of various subtypes by promoting apoptosis. Taken together, these results evidence that EYA4 could be efficiently targeted in a broad range of cancers. These include tumors that present excessive DNA repair capabilities acquired by secondary mutations in the course of chemotherapy, as well as tumors overexpressing RAD51 and other DDR proteins. In addition, tumor growth and metastasis could be tamed or even fully suppressed by EYA4 inhibition, in cancers known to overexpress EYA4 such as esophageal and triple negative breast cancers. a. MATERIALS AND METHODS i. CHEMICAL SYNTHESIS [00434] All operations were carried out at room or ambient temperature, that is, in the range of 18-25 oC; evaporation of solvent was carried out using a rotary evaporator under reduced pressure with a bath of up to 50 oC; reactions were monitored by thin layer chromatography (tlc) and reaction times are given for illustration only. Unless otherwise indicated all reactions were conducted in standard commercially available glassware using standard synthetic chemistry methods and setup. All air- and moisture-sensitive reactions were performed under nitrogen atmosphere with dried solvents and glassware under anhydrous conditions. Starting materials and reagents were commercial compounds of the highest purity available and were used without purification (See list of specific reagents below). Solvents used for reactions were indicated as of commercial dry or extra-dry or analytical grade. Analytical thin layer chromatography was performed on aluminum plates coated with Merck Kieselgel 60F254 and visualized by UV irradiation (254 nm) or by staining with a solution of potassium permanganate. Flash column chromatography was performed on Biotage Isolera One 2.2 using commercial columns that were pre-packed with Merck Kieselgel 60 (230– 400 mesh) silica gel. Final compounds for biological testing are all ≥95% purity as determined by HPLC-MS and 1H NMR. 1H NMR experiments were recorded on Agilent DD2400MHz spectrometers at ambient temperature. Samples were dissolved and prepared in deuterated solvents (CDCl3, CD3OD and DMSOd6) with residual solvents being used as the internal standard in all cases. All deuterated solvent peaks were corrected to the standard chemical shifts (CDCl3, dH = 7.26 ppm; CD3OD, dH = 3.31 ppm; DMSOd6, dH = 2.50 ppm). Spectra were all manually integrated after automatic baseline correction. Chemical shifts (d) are given in parts per million (ppm), and coupling constants (J) are given in Hertz (Hz). The proton spectra are reported as follows: d (multiplicity, coupling constant J, number of protons). The following abbreviations were used to explain the multiplicities: app = apparent, b = broad, d = doublet, dd = doublet of doublets, ddd = doublet of doublet of doublets, dddd = doublet of doublet of doublet of doublets, m = multiplet, s = singlet, t = triplet. All samples were analyzed on Agilent 1290 series HPLC system comprised of binary pumps, degasser and UV detector, equipped with an auto- sampler that is coupled with Agilent 6150 mass spectrometer. Purity was determined via UV detection with a bandwidth of 170 nm in the range from 230-400 nm. The general LC parameters were as follows: Column - Zorbax Eclipse Plus C18, size 2.1 X 50 mm; Solvent A: 0.10 % formic acid in water, Solvent B: 0.00 % formic acid in acetonitrile; Flow rate – 0.7 mL/min; Gradient: 5 % B to 95 % B in 5 min and hold at 95 % B for 2 min; UV detector – channel 1 = 254 nm, channel 2 = 254 nm. Mass detector Agilent Jet Stream – Electron Ionization (AJS-ES). ii. FUCCI ANALYSIS BY LIVE IMAGING [00435] Experimental set up: Red and Green U2932 cells were synchronized using a double thymidine block prior to the start of this experiment. After release from the last thymidine block, the cells were allowed to rest in fresh media for 5 hours to prevent synchronization related cell death. Following this rest period, approximately 10-50 cells were added to a 96 well plate that was pretreated with Poly-L-Ornithine (increases cell adhesion to plate, keeping cells in frame for the experiment). The cells were then treated with alisertib, Aurkin A, EYA4, or combination therapy with a final volume of 1% DMSO (in 100uL well- RPMI 1640 with 10% FBS). Each drug condition had a 3x replicate. Plates were analyzed via an Incucyte SX5 plate reader, scanning at 20x in both red and green fluorescence every 30 minutes. [00436] Analysis: Cell by cell analysis was preformed via the Incucyte software. Red and Green fluorescent gating was preformed based on the control image time course. The average fluorescent (colorless G0, red G1, green S-G2-M, yellow G1/S transition) cell counts for each experimental group (each data point is an average of 3x replicates). The number of red, green, and yellow fluorescent cells was added together to determine the total cell fluorescent count for each experimental group. Each color was then divided by the total fluorescent cell count to get a percentage fluorescence (raw data with formals found in the excel spread sheets attached). This was the Red and Green conditions were then graphed in Prism (data file with graphs attached). Colorless cells (late M/G0/senescent) population was excluded from the analysis. b. SYNTHESIS OF MOLECULES AND DESCRIPTION OF THE LIBRARIES [00437] Libraries of compounds were synthesized using the procedures detailed elsewhere herein. For compounds 3 and 4 used in this study, the specific steps were followed: 2-chloro-N-(3,4-dichlorobenzyl)quinazolin-4-amine (3): To a stirring solution of 2,4- dichloroquinazoline 1 (5.0 g, 25.1 mmol) in THF (125 mL) at room temperature was added 3,4-dichlorobenzylamine 2 (4.0 mL, 30.3 mmol). The mixture was stirred for 24 hr at room temperature, during which time a precipitate formed. The slurry was filtered and washed with hexanes. The collected filtrate was slurried with DCM/hexanes and filtered. The filtrate was washed with hexanes, collected and dried under reduce vacuum to yield 4.4 g (52% yield) of 2-chloro-N-(3,4-dichlorobenzyl) quinazolin-4-amine (3) as a white powder. [00438] For the N2-cyclopentyl-N4-(3,4-dichlorobenzyl) quinazoline-2,4-diamine (4, CIDD-0149689), synthesis was performed using the following procedure: To a 30 mL microwave reaction vial was added 2-chloro-N-(3,4-dichlorobenzyl)quinazolin-4-amine 3 (1.0 g, 2.95 mmol), sec-butanol (15 mL) and cyclopentylamine (251 mg, 2.95 mmol). The mixture was irradiated in an Anton Par microwave reactor for 30 minutes at 180 oC. The reaction was cooled to room temperature, during which time a precipitate formed. The solids were filtered, washed with hexanes and dried under reduced pressure to yield N2- cyclopentyl-N4-(3,4-dichlorobenzyl) quinazoline-2,4-diamine (970 mg, 85% yield) as a white powder. c. DISCUSSION [00439] EYA1-3 proteins have been described to dephosphorylate residue Y142 on the histone variant H2AX and the same activity was recently confirmed in EYA4. Using the synthetic peptide KATQASQE{pTyr} as substrate and a commercial colorimetric malachite green assay (Sigma) that allows to quantify ATP released in the buffer by a phosphatase, EYA4’s tyrosine phosphatase activity was assayed in the presence of putative inhibitors. [00440] In a malachite green assay, the tyrosine but not serine/threonine phosphatase activity was inhibited by CIDD molecule - 149689. For this assay, full length EYA4 purified near homogeneity as previously described was incubated with increasing amounts of inhibitor, then mixed with either peptide KATQASQE{pTyr} from H2AX or with peptide from RAD51. In both cases, the phosphatase activity was significantly reduced. Interestingly, when a large polypeptide encompassing the first 365 residues of EYA4 was incubated, and which contains the serine/threonine phosphatase domain but not the tyrosine domain, with phosphatase inhibitors in the presence of a 53BP1- derived peptide, the phosphatase activity was not inhibited. [00441] Both the serine/threonine and the tyrosine phosphatase domains contain similar catalytic residues, mostly acidic but the tyrosine inhibitor was found specific. [00442] Referring to FIG.28A and FIG.28B, EYA4 tyrosine phosphatase inhibitor recapitulates the HR deficient phenotype but does not affect other DNA damage repair pathways. [00443] Referring to FIG.29A-C, an exemplary compound efficiently inhibits tyrosine phosphatase in the micromolar concentration range, and efficiently suppress cell growth in cells expressing high levels of EYA4. [00444] Referring to FIG.30A, FIG.30B, and FIG.31A-D, EYA4i is synthetic lethal with kinase inhibitors in breast and leukemia cell lines. 5. EYA4 DRIVES BREAST CANCER PROGRESSION AND METASTASIS THROUGH ITS NOVEL ROLE IN REPLICATION STRESS AVOIDANCE [00445] The Eyes Absent family (EYA1-4) is a unique group of dual-functioning protein phosphatases, which have been shown to promote cell proliferation, invasion, migration, and survival in a variety of cancers (Kong D, et al. (2019) American Journal of Translational Research 11(4): 2328-38; Xu H, J et al. (2019) Frontiers in Oncology 9(26); Zhu J, et al. (2021) World Neurosurgery 149: e1174-e9). Members of the EYA family possess N-terminal transcriptional co-activation and threonine phosphatase activity, and C- terminal tyrosine phosphatase activity (Tootle TL, et al. (2003) Nature 426: 299-302; Okabe Y, et al. (2009) Nature 460: 520-4; Rebay I. (2016) Molecular and Cellular Biology 36(5): 668-77). The highly conserved C-terminal domain, also known as the EYA domain (ED), contains a haloacid dehalogenase (HAD) signature sequence, making them the only known HAD-family tyrosine phosphatases (FIG.38A) (Tootle TL, et al. (2003) Nature 426: 299- 302). As the founding members of a new class of non-thiol-based protein tyrosine phosphatases, EYAs have a unique active site, using aspartic acid rather than cysteine as the nucleophile, making these atypical phosphatases attractive targets for specific inhibition with small molecules. However, the biological functions and cellular targets of these dual- phosphatases remain largely unknown, particularly for EYA4. [00446] Defects in EYA4 have been linked to different developmental disorders including hearing loss (Morín et al. (2020) Scientific Reports 6213) and cardiomyopathy (Ahamadmehrabi et al. (2021) Human Genetics 140: 957-67). EYA4 has also been associated with cancer in various organs. In malignant peripheral nerve sheath tumors (MPNST) EYA4 is over-expressed (Miller et al. (2010) Oncogene 29(3): 368-79), whilst it is down-regulated in esophageal adenocarcinoma ( Zou et al. (2005) Cancer Epidemiology, Biomarkers and Prevention 14(4): 830-4; Luo et al. (2018) Cancer Science 109(6): 1811-24), hepatocellular carcinoma (Hou et al. (2014) Annals of Surgical Oncology 21(12): 3891-9), lung cancer (Wilson et al. (2014) Oncogene 33(36): 4464-73) and colorectal cancer (Kim et al. (2015) Molecular Carcinogenesis 54(12): 1748-57), where the EYA4 gene promoter has been found to be hypermethylated. Consistent with this, EYA4 was identified as a potential novel breast cancer gene (Stirzaker (2015) et al. Nature Communications 6: 5899). Specifically, the observation that EYA4 is hypermethylated in the first intron-exon junction particularly in triple-negative breast cancer patients when compared to matched normal samples prompted the study of its role in carcinogenesis and its cellular functions. EYA4 was inactivated or overexpressed in a variety of cell lines and investigated the resulting phenotypes, including cell cycle progression and DNA replication efficiency. [00447] Over-expression of EYA4 was shown to increase proliferation and migration in breast cancer cells, features that are linked with aggressive breast cancer in vivo. The function of EYA4 in promoting breast cancer growth and metastasis is also supported by in vivo xenograft studies showing that silencing of EYA4 expression in MDA-MB-231 cells leads to reduced cancer burden and distant metastasis. The serine/threonine phosphatase activity of EYA4 was found to be essential for breast cancer progression and metastasis, but not its tyrosine phosphatase. In cells, EYA4 depletion was revealed to promote endoreplication and consequently polyploidy, a phenomenon that can occur in response to stress (Lang L, et al. Science Direct.2020;54:85-92; Matsuda M, et al. Plant Cell Reports. 2018;37:913-21) and can cause drug resistance (Shu Z, et al. Trends in Cell Biology. 2018;28(6):465-74). The absence of EYA4 leads to spontaneous replication stress characterized by activation of key cell cycle checkpoints (pChk1 and pChk2), sensitivity to hydroxyurea, and accumulation of endogenous DNA damage, as indicated by increased γH2AX levels. Upon induction of replication stress by hydroxyurea in EYA4-depleted cells, enhanced levels of unresolved DNA breaks are observed. Without wishing to be bound by theory, EYA4 plays a crucial role in the repair of replication-associated DNA damage. [00448] Taken together, these data indicate that EYA4 is a novel oncogene in breast cancer and could play a role in cell cycle maintenance. Without wishing to be bound by theory, this makes EYA4 an attractive, druggable target in cancer treatments, especially in triple-negative breast cancer, to limit metastasis and overcome chemotherapy resistance. a. MATERIALS AND METHODS i. PLASMIDS [00449] MISSION TRC2 pLKO.5-Puro empty vector (EV) or EYA4 shRNA constructs (shRNA1, TRCN0000244430; shRNA2, TRCN0000218273; shRNA3, TRCN0000244429) were obtained from Sigma-Aldrich. pcDNA3.1-nV5 EYA4 full length (FL) and pDEST26-His EYA4 FL were cloned and sent for sequencing. pcDNA3.1-Myc-His EYA4 mutant (3YF281 and pY dead) were obtained from General Biosystems. ii. Cell culture and maintenance, transfections, and stable cell line establishment [00450] HeLa, MDA-MB-231 and MCF-7 cells were sourced from ATCC. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% CO2 incubators and passaged at 80% confluence or less. MCF-7 cells were supplemented with 10 ^g/mL insulin and 1 mM sodium pyruvate. 1.2 ^106 HEK 293T cells were reverse-transfected using Lipofectamine 2000 reagent (Invitrogen) with pLKO.5 empty vector or EYA4 shRNA constructs and Lenti-vpak plasmids from OriGene to create lentivirus particles. Viruses were harvested at 48 and 72 h post- transfection, filtered through a 0.45 ^m filter, and used to infect HeLa or MDA-MB-231 cells with 4 ^g/mL polybrene. Stable cell lines were selected using 1-2 ^g/mL of puromycin. For complementation, stable HeLa or MDA-MB-231 cells expressing shRNA1 were transfected with pcDNA3.1 Myc/His containing a mutant version of EYA4 in the S/T domain (Y281F, Y284F, Y285F; referred to as 3YF281) or pY dead (D375N, D377N, T548A, E606Q, E607Q, E608Q)) and selected with 500 ^g/mL geneticin. MCF-7 cells were transfected with pcDNA3.1-nV5 EYA4 FL or pDEST26-His EYA4 FL and selected with 500 ^g/mL geneticin. MDA-MB-231/Luc and MCF-7/Luc cells stably expressing firefly luciferase were established as described above. HeLa cells were transduced with FUCCI (red/green) plasmids (Sakaue-Sawano A, et al. (2008) Cell 132(3): 487-98) and FACS sorted to select homogenous positive cell populations. The origin of all cells was confirmed by short-tandem repeat (STR) profiling and tested regularly for Mycoplasma contamination. iii. RNA EXTRACTION AND QUANTITATIVE REVERSE TRANSCRIPTION PCR (QRT-PCR). [00451] Total RNA was isolated from transfected or transduced cells by phenol- chloroform extraction (TRIzol; Invitrogen) followed by nucleic acid precipitation. The GoScript Reverse Transcription System (Promega) was used to generate first-strand cDNA. Quantitative PCR was performed using TaqMan probes spanning across exons for human EYA4 (Invitrogen Hs01012406_mH) and human 18S (Invitrogen Hs99999901_s1) to amplify 70 bp and 187 bp fragments, respectively. The relative expression of EYA4 was determined using the 2- ^ ^Ct method with 18S as an endogenous control for normalization. iv. IMMUNOBLOTTING [00452] Immunoblotting analysis was conducted according to standard procedures (C. Wiese et al., (2007) Mol Cell 28: 482-490). Cells were collected and lysed in RIPA buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) supplemented with cOmplete Mini EDTA-free protease inhibitor cocktail (Roche), 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, 1 mM benzamidine and 0.025 U/μL benzonase, followed by sonication for 2 min (40%) in an ultrasonic water bath (Sonics Vibra-Cell VCX400). Proteins were resolved in 4-20% Mini- Protean TGX gels (Bio-Rad) and transferred to Immobilon-P PVDF membranes (Merck). Membranes were then blocked with either 5% skim milk or bovine serum albumin (BSA) in TBS-T. Blots were incubated with primary antibody at either 4 °C overnight or room temperature (RT) for 2 h, washed, then incubated with secondary HRP-conjugated antibodies for 1 h at RT. Bands were visualized using the Clarity Western ECL substrate (Bio-Rad). Primary antibodies: EYA4 (Abcam ab93865), cyclin E1 (HE12; Cell Signaling #4129), CDK2 (78B2; Cell Signaling #2546), p21WAF1/CIP1 (12D1; Cell Signaling #2947), p27KIP1 (D69C12; Cell Signaling # 3686), cyclin A (B-8; Santa Cruz sc-271682), pChk1 (S345) (133D3; Cell Signaling # 2348), pChk2 (T68) (Cell Signaling # 2661), γH2AX (S139) (Millipore 05-636), PCNA (PC10; Santa Cruz sc-56), GAPDH (14C10; Cell Signaling #2118) and β-Actin (C4; Santa Cruz sc-47778). v. SUBCUTANEOUS TUMOR XENOGRAFTS IN IMMUNODEFICIENT MICE [00453] For subcutaneous injections, MCF-7/Luciferase wild type (WT), pcDNA3.1- nV5 EYA4 FL and pDEST26-His EYA4 FL cells (1.0 x 106) were resuspended in 100 ^L of 0.9% (w/v) NaCl and injected in the left mammary fat pad (MFP) of 24 non-obese diabetic/severe combined immunodeficiency gamma (NSG, NOD scid gamma) female mice (6 weeks of age; 8 mice per cell line). A 17β-estradiol pellet (1.7 mg/pellet, 60-days release, Innovative Research of America) was implanted close to the neck using a precision trochar, 24 h prior to MFP injections. Weekly, all mice were weighed, tumor growth was measured by using a caliper and detected in vivo by bioluminescent imaging. For in vivo imaging, mice were first injected with D-luciferin (150 mg/kg, 10 min prior to imaging), anesthetized with 3% isoflurane and then imaged in an IVIS spectrum imaging system (Caliper, Newton, USA). Images were analyzed with Living Image software (Caliper, Newton, USA). Bioluminescent flux (photons/sec/sr/cm2) was determined for the tumors. Tumor volume was calculated according to the following formula: (length ^ width2)/2. MCF-7/Luciferase mice were sacrificed before tumors reached 10 mm (8 weeks post-injection). Harvested tumor tissues were placed in liquid nitrogen and then frozen at -80 °C or fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained. Antibodies used: anti-Estrogen Receptor (SP1; Roche 790-4324; CC164 min), anti-Ki-67 (30-9; Roche 790-4286; CC164 min), anti- γH2AX (pS319; Abcam ab2893; CC164 min; 1:600). vi. MOUSE TAIL-VEIN ASSAY [00454] MDA-MB-231/Luciferase WT, EYA4 shRNA1 and EYA4 shRNA2 cells (1.0 x 106 cells/100 ^L 0.9% (w/v) NaCl) were injected in the lateral tail-vein of 9 female NOD scid gamma mice (6 weeks of age; 3 mice per cell line). For complementation, MDA-MB- 231/Luciferase WT, EYA4 shRNA1 and pcDNA3.1-Myc-His EYA4 mutant (3YF281 and pY dead) cells (1.0 x 106 cells/100 ^L 0.9% (w/v) NaCl) were injected in the lateral tail-vein of 22 female NSG mice (7 weeks of age; 7 WT mice and 5 mice per cell line). Mice were detected every week for metastatic foci by bioluminescent imaging as described above. MDA-MB-231/Luciferase mice were monitored and culled 4-5 weeks post-injection. Bioluminescent flux (photons/s/cm2/sr) was determined. Organs in which metastatic foci were observed were harvested and fixed in 4% PFA, followed by 70% EtOH, then embedded in paraffin, sectioned, and stained. Antibodies used as described above. vii. CELL PROLIFERATION ASSAY. [00455] Cells were seeded in a 96-well plate at 2.0 x 103 cells/well. Phase contrast images of cells were acquired every 2 h using an IncuCyte Zoom (Essen BioScience) live imaging system. Proliferation was measured as a percentage of confluency. viii. IN VITRO MIGRATION ASSAY [00456] Cells were cultured in a 96-well plate for 24 h to achieve 100% confluency. An IncuCyte Woundmaker was used to make a scratch in the cell monolayer. Cells were then incubated in serum-free media and automatically imaged every 2 h using an IncuCyte Zoom (Essen BioScience) live imaging system. The scratch gap width and confluence were measured at each time point and compared between groups. ix. APOPTOSIS [00457] HeLa cells were seeded in a 96-well plate (100 cells/well). After 24 h, annexin V (red) reagent was added according to manufacturer’s protocol (IncuCyte). Images (phase contrast/orange) were acquired every 2 h using an IncuCyte SX5 (Sartorius) live-cell imaging system. Apoptosis was measured as total integrated intensity (OCU ^ ^m2/image). x. DOUBLE THYMIDINE BLOCK AND CELL CYCLE PROGRESSION (FLOW CYTOMETRY) [00458] HeLa cells were synchronized in early S-phase by a double thymidine block. Briefly, cells were blocked with 2 mM thymidine for 18 h, released for 9 h, and blocked again with 2 mM thymidine for 17 h. After the second block, cells (asynchronized and synchronized) were released and collected according to time points, then fixed in ice-cold 70% ethanol at -20 °C for at least 24 h. DNA was stained with 38 mM trisodium citrate, 100 ^g/mL RNase A and 150 ^g/mL propidium iodide (PI) for 1 h at RT. A DNA control PI (trout erythrocytes) was used as an internal control to normalize the cell cycle. Data were collected using a CytoFLEX Flow Cytometer (Beckman Coulter) and cell cycle profiles were analyzed with FlowJo to determine the percentage of cells in G1, S and G2/M.10,000 events were collected, and aggregated cells were gated out. xi. FUCCI [00459] HeLa FUCCI cells stably transfected with empty vector or EYA4 shRNAs were seeded in a 96-well plate (100 cells/well). Phase contrast and green/orange images were acquired every 2 h to monitor cell cycle progression using an IncuCyte SX5 (Sartorius) live- cell imaging system. Images were analyzed using cell-by-cell analysis software and population subsets were classified based on green and red fluorescence. G1 phase (red), G1-S transition (green + red), S/G2/M phase (green) and M-G1 transition (non-fluorescent) (19). xii. INDIRECT IMMUNOFLUORESCENCE [00460] Indirect immunofluorescence was performed as described elsewhere (B. de la Peña Avalos, E. Dray. Journal of Visualized Experiments 160, e61447 (2020)). Cells were grown on coverslips for 24 h and treated with 4 Gy ^-irradiation (Gammacell40 Exactor unit) or 4 mM hydroxyurea. Cell nuclei were pre-extracted with nuclear extraction buffer (NEB; 10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA (pH 8.0), 0.5% Triton X-100) for 2 min at RT then fixed with 4% paraformaldehyde (PFA) for 10 min at 4 °C. Nuclei were blocked in 5% BSA and 0.3% Triton X-100 in PBS, immunoblotted with a primary antibody (1:500 in dilution buffer; 1% BSA and 0.3% Triton X-100 in PBS), followed by secondary antibody (2 ^g/mL in dilution buffer). DNA was counterstained with DAPI. Slides were viewed on an Olympus FV3000 confocal microscope. Primary antibodies: CENP-F (H-260; Santa Cruz sc-22791), γH2AX (S139) (Millipore 05-636). Secondary antibodies: ^-Rabbit (Abcam ab150081, Alexa Fluor 488), ^-Mouse (Abcam ab150103, Alexa Fluor 647). Nuclear foci quantification was performed using CellProfiler. xiii. MTT CELL CYTOTOXICITY ASSAY [00461] For genotoxic stress, cells were seeded into 96-well plates (200 cells/well). Twenty-four hours after seeding, increasing concentrations of ATR inhibitor (AZ20) or hydroxyurea were added to the culture (24 h pulse). Cell cytotoxicity was measured after 96 h following manufacturer’s protocol (Abcam ab211091). Briefly, 50 µL serum-free media (no phenol red) and 50 µL MTT reagent was added to each well and incubated at 37 °C for 3 h. MTT media was replaced with 150 µL of MTT solvent and incubated with agitation for 15 min. Absorbance was measured at 590 nm. The cell viability was calculated using the following equation: ^^ ^^ ODtreated and ODcontrol represented the absorbance of sampled and control, respectively. xiv. EDU INCORPORATION [00462] HeLa control and EYA4 knockdown cells (4.0 x 104 cells/well) were seeded in 12-well plates with coverslips for 24 h.5-ethynyl-2’-deoxyuridine (EdU) incorporation was performed according to manufacturer’s protocol (Base Click). Briefly, cells were treated with 4 mM hydroxyurea for 2 hours, released for 10 minutes, then labeled with 10 ^M of EdU for 30 min at 37 °C, then fixed with 4% PFA for 10 min at 4 °C, followed by permeabilization with 0.3% Triton X-100 in PBS for 20 min at RT. Reaction cocktail with 6-FAM azide was added to fixed cells and incubated for 30 min at RT. DNA was counterstained with DAPI. Slides were viewed on an Olympus FV3000 confocal microscope. EdU-stained cells were quantified using CellProfiler. xv. DNA FIBER ASSAY [00463] Exponentially growing HeLa cells (3.0 x 105) were labeled with a 5-iodo-2’- deoxyuridine (IdU; 50 ^M) pulse for 30 min. After labeling, cells were harvested, embedded in agarose and DNA was prepared then combed onto silanized coverslips using the FiberComb Molecular Combing System (Genomic Vision). Following combing, coverslips were baked for 2 h at 65 °C. Combed DNA fibers were denatured with 0.5 M NaOH + 1 M NaCl for 8 min at RT, neutralized with PBS (3 times, 3 min), then dehydrated in ethanol (70%-90%-100%, 3 min each), and air-dried. Combed DNA was blocked with BlockAid blocking solution (Invitrogen B10710) for 15 min at RT, followed by immunostaining with mouse ^-BrdU (to detect IdU; BD Biosciences 347580) for 1 h at 37 °C, washed with PBS-T, and probed with secondary antibody ( ^-mouse Cy3, SIGMA C2181) for 45 min at 37 °C. Single-stranded DNA was counterstained with ^-ssDNA mouse antibody (DSHB University of Iowa) for 2 h at 37 °C, followed by ^-mouse BV480 (Jackson ImmunoResearch 115–685- 166) for 45 min at 37 ^C. Coverslips were washed in PBS, subjected to a graded ethanol series, air-dried, and then mounted with 25 ^L of Vectashield mounting medium (Vector Laboratories). DNA fiber images were acquired on an Olympus FV3000 confocal microscope. Track lengths were measured with ImageJ. To calculate replication fork speed, the following equation was used to convert fork length from ^m to kb/min: length ^m ^ 2/labeling time in min = fork speed kb/min (conversion factor of 2 kb/ ^m specific for DNA combing method). xvi. STATISTICAL ANALYSIS [00464] The statistical analyses were conducted using GraphPad Prism 9 and a p < 0.05 was considered statistically significant. Student’s t-test was used to test for significant differences between groups, considering a normal distribution. Unpaired two-tailed tests were applied to all data if not specified. Samples sizes were chosen according to previously published methods where significant biological conclusions were reported. b. EYA4 IS A NOVEL BREAST CANCER GENE [00465] EYA4 was investigated as to whether it is expressed in specific breast cancer subtypes using real-time quantitative PCR and immunoblotting in several breast cancer cell lines (FIG.38B and FIG.38C). The expression of EYA4 varied greatly across cell lines, however, the triple-negative breast cancer cell line MDA-MB-231 showed the highest endogenous expression of EYA4. In most mouse strains, knockout of EYA4 is lethal shortly after birth (F. F. Depreux et al., (2008) The Journal of Clinical Investigation 118: 651-658) and is toxic in several lung cancer cell lines (I. M. Wilson et al., (2014) Oncogene 33: 4464- 4473) and other cell lines that were tested. Using short-hairpin RNAs (shRNAs), EYA4 expression could be significantly decreased in MDA-MB-231 cells (FIG.38D) or in HeLa cells (FIG.34A). The most efficient hairpin, shRNA3, induces cell death in MDA-MB-231, indicating that EYA4 is essential in these cells. In parallel, EYA4 was over-expressed using two different vectors (FIG.38E) in the ER+/PR+ breast cancer cell line, MCF-7, which expresses low or no detectable endogenous EYA4 (FIG.38B and FIG.38C and Cancer Cell Line Encyclopedia, https://sites.broadinstitute.org/ccle). The effects of EYA4 deregulation on primary cancer growth and metastasis were assessed in vivo using luciferase-expressing cell lines. A human tumor xenograft model was established using NOD scid gamma mice. MCF-7/Luc wild type (WT) and EYA4 over-expressing cells were injected subcutaneously into the left mammary fat pad (MFP) of female mice supplemented with 17 ^-estradiol and monitored by caliper measurement and in vivo imaging for 8 weeks. Following an intraperitoneal injection with D-luciferin (150 mg/kg), the firefly luciferase enzyme catalyzes this substrate, which results in light photons that are captured by a charge-coupled device (CCD) camera mounted within an IVIS® Spectrum Imaging System (Carceles-Cordon M, et al. Bio-Protocol. 2016;6(6)). As shown in FIG. 32A and FIG. 32B, the bioluminescence intensity (BLI) signal measurement confirmed tumor engraftment for all mice. Primary tumors show a significant increase in volume when EYA4 is over-expressed. BLI signal correlated with caliper measurements as observed in FIG.32C, and with tumor volume and weight (FIG. 32E and FIG. 32F) once surgically removed postmortem (FIG. 32D). EYA4 over-expression leads to a more aggressive breast cancer, as observed by immunohistochemistry (IHC) staining (FIG.32G). These results correspond with previous reports that in MPNST, EYA4 is dramatically upregulated in cells and primary tumors, and its depletion leads to reduced cell adhesion and migration in vitro and has an inhibitory effect in tumorigenesis in vivo (Miller SJ, et al. Oncogene.2010;29(3):368-79). [00466] Estrogen receptor alpha (ER- ^) co-stain was used to validate human cells. Interestingly, cells expressing high levels of EYA4 also showed high expression of ER- ^, the proliferation-related antigen Ki-67, and γH2AX, a marker of DNA damage (FIG. 32G and FIG. 32H). ER- ^ has a well-established role in supporting estrogen-dependent breast tumor growth through its association with aberrant proliferation (up-regulating Ki-67), which can result in the accumulation of random DNA mutations (marked by γH2AX), and when highly expressed it is associated with poor prognosis in breast cancer (Thomas C, et al. Nature Reviews. 2011;11:597-608, Liao X-H, et al. FEBS Journal. 2014;281:927-42), which can explain the aggressive breast cancer subtype observed when EYA4 is over-expressed. [00467] Since breast cancer subtypes are associated with unique patterns of metastatic spread, the metastatic capacity was assessed utilizing MDA-MB-231 stably expressing firefly luciferase. MDA-MB-231/Luciferase WT cells and cells in which EYA4 was stably knocked down (shRNA1 and shRNA2) were injected into the tail vein and monitored by in vivo imaging over 5 weeks. While WT and EYA4-depleted cells colonized the lungs as expected following systemic injection, a decrease in BLI signal was observed in mice injected with EYA4-depleted cells compared to the control (FIG.33A). This was directly linked to a lesser number and a decrease in the area of metastatic foci observed in livers as revealed by histological analyses (FIG.33B-E). Importantly, these IHC analyses also showed significant areas of central necrosis with inflammatory cells and blood vessel congestion (left panel) and scant fibrosis (right panel) was observed in the control group but not in the EYA4 knockdown mice (FIG.33F). This particular observation could be due to the role that EYA4 plays in innate immune system regulation by enhancing the expression of IFN- ^ and CXCL10, in response to DNA stimulation (Y. Okabe, et al., Nature 460, 520-524 (2009)). In cancer cells, the cGAS-STING pathway is constitutively activated, inducing chronic IFN- ^ expression, triggered by the accumulation of DNA damage due to replication fork collapse or reactive oxygen species (ROS) that leads to the presence of DNA in the cytoplasm (Cheon H, et al. Trends in Cancer.2023;9(1):83-92). Altogether, the data suggest that EYA4 is a driver of breast cancer and that decreasing its expression reduces tumor and metastatic burdens. c. THE S/T PHOSPHATASE DOMAIN OF EYA4 CONTRIBUTES TO BREAST CANCER DEVELOPMENT [00468] EYA4 possesses both serine/threonine (S/T) and tyrosine (Y) phosphatase activities (FIG.38A) (Rebay I. Molecular and Cellular Biology.2016;36(5):668—77). To investigate the relevance of these activities on tumor growth, MDA-MB-231/Luc cells expressing either EYA4 mutated in the S/T domain (Y281F, Y284F, Y285F; henceforth, 3YF281) or the pY dead combination mutant (D375N, D377N, T548A, E606Q, E607Q, E608Q) were injected into the tail-vein and monitored by in vivo imaging for 4 weeks using the luciferase reporter. The phosphatase mutants (3YF281 and pY dead) caused even more significant outcomes that EYA4 depletion, especially the 3YF281 mutant. Both EYA4 phosphatase mutants did not complement EYA4 depletion with shRNA1, as observed by both BLI signal (FIG.33G) and by metastatic foci observed in livers (FIG.33H-K). However, the serine/threonine phosphatase activity of EYA4 (3YF281) is the one that shows more significant outcomes, as observed not only by decreased tumor burden to lungs (FIG.39C- E), but also by a lesser number of metastatic foci to the liver, with an average of 2 foci for 3YF281, compared to 6 for EYA4 shRNA2 and 7 for pY dead (FIG.33J). In addition, as observed by IHC staining, when a metastatic site is observed (marked by H&E) in mouse injected with 3YF281 cells, there is no stain by Ki-67 or γH2AX (FIG.39F). For γH2AX, only a background level (mouse cells stained), can be observed. Notably, all mice injected with 3YF281 cells showed liver enlargement and hyperplasia (FIG.33H and FIG.33I), which could be driven by an increased hepatocyte number, prompting further investigation. Without wishing to be bound by theory, these data suggest that the serine/threonine phosphatase activity of EYA4 is essential for breast cancer progression and metastasis. d. EYA4 PROMOTES CELL PROLIFERATION AND MIGRATION [00469] Without wishing to be bound by theory, one simple explanation for variations in primary tumor sizes is the accumulation of larger cells (Qiu J, et al. (2022) Cancer Management and Research 14: 2235-41; Zhou X, et al. (2022) Frontiers in Cell and Developmental Biology 10) or increased proliferation rates. Uncontrolled and unlimited cell proliferation is a hallmark of cancer (Hanahan D. (2022) Cancer Discovery 12(1): 31-46) and another member of the Eyes Absent family, EYA2, has been shown to increase cell proliferation in lung cancer (Li Z, et al. (2017) Oncotarget 8(67): 110837-48). Stable knockdowns were generated in HeLa cells, using three independent short-hairpin RNAs, and a significant decrease in EYA4 protein levels was achieved (FIG.34A). Growth rates were followed by live-cell imaging. In both, HeLa (FIG.34B) and MDA-MB-231 (FIG.40A) cells, depletion of EYA4 led to lower proliferation rates compared to control. On the contrary, the over-expression of EYA4 in MCF-7 leads to higher proliferation rates when compared to control (FIG.40C), suggesting that EYA4 promotes cell proliferation. In addition, the effect of EYA4 on cell migration was investigated by comparing the number of control, EYA4 knocked down and EYA4 over-expressing cells at the scratch wound at different time points by live-cell imaging. HeLa (FIG.34C) and MDA-MB-231 (FIG.40B) cells depleted for EYA4, exhibited significantly lower migratory capacity relative to cells expressing the empty vector (EV) control, whilst EYA4 over-expression in MCF-7 (FIG. 40D) primes the migration capacity of cells, indicating that EYA4 plays a role in driving cell migration. EYA4 phosphatase mutants, specially 3YF281, display a phenotype comparable, or even more dramatic, than EYA4 depleted cells when tested for proliferation and migration capacities in HeLa cells (FIG.40E-G), showing a significant decrease for both. However, the same phenotype was not observed in MDA-MD-231 cells (FIG.34H and FIG.34J), suggesting that the role in cell migration might be cell line dependent. As the apparent slower proliferation being caused by cell death cannot exclude, HeLa control and EYA4 knockdown cells were followed after the addition of the apoptosis marker, annexin V. Compared to HeLa control cells, EYA4 shRNA3 showed a slight increase in apoptosis in normal growth conditions (FIG.34D and FIG.40K), which could explain, at least partially, the slower proliferation rate observed for shRNA3. The increase in apoptosis observed in HeLa EYA4 shRNA3 cells reflects the fact that this hairpin could not be used in MDA-MB-231 cells, as severe knockdown of EYA4 is incompatible with cell viability. e. EYA4 PERTURBS CELL CYCLE PROGRESSION [00470] Cell cycle is tightly regulated via checkpoints that are activated by DNA damage, low nutrient content, or other endogenous and external stresses. Aberrant cell cycle progression tends to result in genome instability and contributes to cancer progression. To determine how EYA4 might affect cell cycle progression, flow cytometry was used to profile asynchronous populations of either control or EYA4-depleted cells (FIG.35A). A slight increase (2-3%) in S-phase was observed when EYA4 is silenced and a significant increase (8%) in the G2/M population for shRNA1 (FIG.35A), when compared to empty vector control, which, without wishing to be bound by theory, suggested a delay in cell cycle progression upon EYA4 depletion. However, shRNA3 does not show a significant increase in G2/M, which could be explained due to its characteristic phenotype (enlarged, flat and multinucleated cells, FIG.40L), and this subpopulation could have been gated out by flow cytometry (raw data in FIG.41). The FUCCI system (Sakaue-Sawano A, et al. (2008) Cell 132(3): 487-98) was used and live-cell imaging (FIG.35B and FIG.42A) to overcome these technical issues and profile single cells. A subtly different behavior was observed for EYA4 shRNA3, especially when it comes to cells in S-G2-M (FIG.35B). This correlates with cells depleted for EYA4 (especially with shRNA3) undergoing endoreplication (FIG.35C). Endoreplication refers to a cell cycle variant that only consists of the G and S phases, during which cells replicate their DNA content without dividing, thus giving rise to polyploid cells (Shu Z, et al. (2018) Trends in Cell Biology 28(6): 465-74; Lee HO, et al. (2009) Genes & Development 23: 2461-77). The result is either a cell that maintains separate nuclei and remains multinucleated, due to a process called endomitosis, or a cell with an enlarged-single nucleus containing all the DNA, derived from a process called endocycling (Shu Z, et al. (2018) Trends in Cell Biology 28(6): 465-74). As described above, shRNA3 cells tend to be enlarged and multinucleated, which is characteristic of endomitosis, a major form of endoreplication in which mitosis is initiated but not completed (green/non-fluorescent/green; white arrowhead; FIG.35C). The endoreplication and consequent polyploidy observed, which can occur in response to stress, is a phenomenon that has been linked to cancer progression and chemotherapy resistance (Tagal V, et al. (2021) Cancer Research 81(2): 400- 13). f. EYA4 INDUCES CELL CYCLE ARREST [00471] The most common change leading from a mitotic to an endoreplication cycle is a switch in activation/inactivation of cyclins and cyclin-dependent kinases (CDKs), key regulators of cell cycle progression (Gandarillas A, et al. (2018) Cell Death & Differentiation 25: 471-6). To investigate if EYA4 expression impacts individual phases of the cell cycle, cells were arrested in early S-phase with a double thymidine block (FIG.35D) and assessed for cell cycle progression. Propidium iodide (PI) staining of the DNA and flow cytometry in HeLa cells showed that EYA4 decrease (shRNA1) leads to a delay in S-phase restart compared to control (FIG.35D). Upon release, 74.9% of control cells entered G2/M after 6 h, compared to 49.43% of EYA4-depleted cells (raw data can be found in FIG.41). EYA4- depleted cells resumed/finished S with a 2 h delay, and 78.3% of depleted cells entered G2/M 8 h post-release, showing that EYA4 depletion extends S-phase and delays cell division. Without wishing to be bound by theory, the most logical explanations for such observations are defects in DNA replication and aberrant checkpoint signals. Since EYA4 depletion appears to halt the cell cycle in the transition between S-phase and G2, the activation of several proteins involved in G1 checkpoint (G1/S transition) and G2 checkpoint were evaluated (schematic in FIG.42C). The G1/S transition was examined to assess if the cells can initiate DNA replication. For this, the expression of cyclin E1, its partner CDK2, and its corresponding CDK inhibitors, p21 WAF1/CIP1 and p27 KIP1 (FIG.35E) were determined. After double thymidine block (G1/S transition), synchronized EYA4 depleted cells appeared to accumulate p21 WAF1/CIP1 and p27 KIP1 , especially EYA4 depleted with shRNA3. However, CDK2 does not seem to be affected by the CDK inhibitors, since the level of expression appears to be similar between control and EYA4 silenced cells. Cyclin E1 levels increase sharply in late G1, where it interacts and activates CDK2 allowing G1/S transition, then decrease in S-phase (Mazumder S, et al. (2004) Current Cancer Drug Targets 4(1): 65-75), as observed in control cells, but not in EYA4-depleted cells. This correlates with the accumulation of cells in G1-S at 6 h observed in cells depleted for EYA4 (FIG.35D). Cyclin E1/CDK2 is an important part of the G1 checkpoint and deregulation in the G1/S transition may impair normal DNA replication, causing replication stress and DNA damage (Fagundes R, and Teixeira LK. (2021) Frontiers in Cell and Developmental Biology 9: 774845). Nevertheless, EYA4 silenced cells appear to be able to overcome the G1 checkpoint and initiate DNA replication with little or no delay. Upon release from the thymidine block, EYA4-depleted cells, especially shRNA1, exhibited a notable delay in S-phase compared to the EV control (FIG.35D). This was confirmed by the accumulation of cyclin A (highly expressed in S-phase, decreasing in G2) for up to 10 hours post-release (FIG.35E). Altogether, these data indicate that in the absence of EYA4, S-phase and its subsequent transition into G2 become prolonged. Without wishing to be bound by theory, these effects could stem from faulty DNA replication and/or the accumulation of DNA damage during S- phase. g. SPONTANEOUS REPLICATION STRESS IS OBSERVED IN THE ABSENCE OF EYA4 [00472] Since EYA4-depleted cells transition through G1/S and enter DNA replication, but S-phase appears to be longer and the S-G2 transition halted, the level of expression of pChk1 (S345) and pChk2 (T68) by immunoblotting was evaluated, to assess if the cells have accumulated spontaneous damaged DNA. To do so, cells were arrested in early S-phase with a double thymidine block. Checkpoint kinase 1 (Chk1) is a key player of DNA- damage-activated checkpoint response that acts downstream of ATR (Ataxia Telangiectasia and Rad3 related) kinase, in response to the formation of single-stranded DNA due to DNA damage of blocked replication forks (FIG.36A). It is activated by all known forms of DNA damage, particularly triggering the intra-S- and G2/M-phase checkpoints (Qiu Z, et al. (2018) Radiotherapy and Oncology 126(3): 450-64). Chk2 is a stable protein expressed throughout the cell cycle. In response to DNA double-strand breaks, Chk2 becomes rapidly phosphorylated at threonine 68 by ATM (Ataxia Telangiectasia Mutated) (FIG.36A). The kinase activity of Chk2 depends on the severity of DNA damage (Ward IM, et al. (2001) Journal of Biological Chemistry 276: 47755-8). Under normal conditions, EYA4-depleted cells accumulated pChk1 (S345) up to 8 h after release (FIG.36A), but not the control, implying that replication fork stalling occurs in the absence of EYA4, and its resolution becomes delayed. Additionally, pChk2 (T68) is highly expressed in the absence of EYA4 (FIG.36A), which, without wishing to be bound by theory, suggests the accumulation of double-stranded breaks (DSBs) that might be a consequence of replication fork collapse. Spontaneous accumulation of DNA damage was confirmed by evaluating the expression of the phosphorylated histone variant H2AX (S319, γH2AX) in early S-phase. Accumulation of γH2AX was observed in EYA4-depleted cells (FIG.36A), but not in the control, indicating the presence of replication stress, which probably triggers the phosphorylation of H2AX on S139 by ATR (FIG.36A). In accordance with these results, cells depleted for EYA4 were also found sensitive to AZ20, an ATR inhibitor (FIG.36B). Since longer S-phase and halted cell cycle observed in the absence of EYA4 might be due to accumulation of replication stress, the assessment as to if the cells are able to progress throughout the cell cycle upon DNA damage induction was sought. The formation of CENP-F foci was followed after 4 Gy ^-irradiation, to identify S-phase and G2/M. CENP-F gradually accumulates during the cell cycle until it reaches peak levels in G2/M phases (weakly positive in S-phase), where it first associates with kinetochores in late G2 (Liao H, et al. (1995) Journal of Cell Biology 130(3): 507-18). Control cells accumulate in S-G2/M after ^-irradiation, indicating that the cell cycle is halted (1 h after 4 Gy) but they progress once DNA damage is resolved. Nevertheless, in the absence of EYA4, accumulation of CENP-F was observed up to 4 h after irradiation, indicating that the cells are taking longer to resolve DNA damage (FIG.36C). h. EYA4 CONTRIBUTES TO HU RESISTANCE [00473] To address the potential role of EYA4 in the cellular response to replication stress, the effects of knocking down EYA4 were examined on the sensitivity to hydroxyurea (HU), which causes replication stress by depleting the intracellular pool of deoxynucleotides (Musialek MW, and Rybaczek D. (2021) Genes (Basel) 12(7): 1096). In accordance with the accumulation of replication stress and checkpoint activation, cells depleted for EYA4 were found to be sensitive to hydroxyurea in an MTT assay (FIG.37A). In order to monitor DNA synthesis, cells were treated with 4 mM hydroxyurea for 2 h and then measured 5-ethynyl-2’- deoxyuridine (EdU) incorporation after the removal of HU. Under these conditions, silencing EYA4 resulted in a slightly increased rate of EdU incorporation (FIG.37B), indicating that EYA4 might be involved in maintaining replication fork stability since EYA4-depleted cells appear to overcome the HU blockage and resume synthesis. i. EYA4 DEPLETION RESULTS IN INCREASED AND UNRESOLVED LEVELS OF HU-INDUCED DSBS [00474] Replication fork collapse resulting from chronic HU exposure generates double-stranded breaks (Musialek MW, and Rybaczek D. (2021) Genes (Basel) 12(7): 1096), which are rapidly marked by γH2AX. To examine the possible role of EYA4 in the repair of HU-induced DSBs, HeLa cells were incubated with 4 mM HU for 2 h and then allowed to recover for 2 h in the absence of the drug. Even though EYA4-depleted cells have high levels of endogenous DNA damage, an increase in HU-induced DSBs was observed in the absence of EYA4 (FIG.37C). Next, HeLa cells were incubated with 4 mM HU for 16 h and then released for 18 h, to assess for unresolved DNA damage in the absence of EYA4. Although residual γH2AX foci were present in HeLa control cells after recovery from HU exposure, ~10 % more cells with > 10 γH2AX foci per cell (FIG.37D) were observed in the absence of EYA4, implying that these cells have a diminished ability to resolve HU-induced DNA damage. Without wishing to be bound by theory, these results together suggest that EYA4 contributes to replication-associated DNA damage repair. j. EYA4 IMPACTS REPLICATION FORK SPEED [00475] To investigate the functional role of EYA4 in DNA replication, single- molecule analysis of replicated DNA fibers were utilized to test if the increased DSBs in EYA4-deficient cells affected replication fork (RF) progression and speed. It was found that the replication tracts are much shorter in EYA4 deficient cells compared to control cells, under normal conditions (FIG.37E), demonstrating that genome-wide RF progression is strongly impaired by EYA4 depletion. Interestingly, the fork slowing observed was even more dramatic in the 3YF281 mutant cells, but not in the pY dead cells, showing that the serine/threonine phosphatase activity of EYA4 is essential for replication fork progression. k. DISCUSSION [00476] EYA protein phosphatases have been associated with cancer pathologies, and they exhibit characteristics of oncogenic and tumor-suppressive activities depending on the tissue of origin. Because EYA are protein phosphatases, without wishing to be bound by theory, it is expected that lack of phosphorylation would impact a variety of cellular pathways depending on the protein substrates expressed and targeted in specific tissues. In this study, it was attempted to gain a better understanding of the cellular processes impacted by EYA4 deregulation in cancer, and specifically understand the possible role of EYA4 in breast carcinogenesis. [00477] As previously reported the EYA4 gene is hypermethylated in the first intron- exon junction (Stirzaker C, et al. (2015) Nature Communications 6: 5899), and possibly over- expressed in triple-negative breast cancer patients, which correlates with publicly available TCGA dataset that shows amplification as the most common alteration in breast cancer patients. In this study provided herein, HeLa and breast cancer cell lines were used to investigate the proliferation rates of cells knocked down or over-expressing EYA4, ex vivo and as xenografts in small animals. While EYA4 is often not expressed in normal breast, it was found that MDA-MB-231 over-express EYA4, and are depending on its expression for survival. Over-expression of EYA4 was shown to drive the growth of ER + primary tumors, and promote metastasis to distant organs such as lungs and livers. In triple-negative breast cancer xenografts, the knockdown of EYA4 was able to efficiently limit the spread of metastasis and the overall cancer burden. These two xenograft studies suggest that EYA4 therapeutic targeting is an interesting avenue that should be pursued for anti-breast cancer drug development. Over-expressing EYA4 in cancer could be used to predict patient outcomes and drug response. [00478] EYA4 level is inversely correlated with ER status, with high expression largely found in triple-negative breast cancer, while ER + tumors and cell lines express little or no EYA4. This warrants further investigation to fully understand the connection between Eyes Absent phosphatases and the hormonal status of cancerous tissues. In breast cancer, it is well-established that estrogen is a major driver of breast tumor growth through its role in cell proliferation, as well as an effective therapeutic target. It has been proposed that in MCF-7 cells, ER- ^ induces cell proliferation by regulating the cell cycle by stimulating the expression of PCNA and Ki-67 and suppressing of p53/p21 transcription (Liao X-H, et al. (2014) FEBS Journal 281: 927-42). The data provided herein shows that EYA4-depleted cells exhibit slower division rates as measured by live imaging. Further investigation by live imaging and the FUCCI system demonstrated that cells silenced for EYA4 undergo slower DNA synthesis, halting cell cycle progression, and undergoing endoreplication as a result of missed mitosis initiation. The data provided herein confirm previous observations in glioma where EYA4 over-expression promotes cell proliferation by directly suppressing the expression of p27 KIP1 , suggesting p27 KIP1 as a transcriptional target of EYA4 (Li Z, et al. (2018) Cellular Physiology and Biochemistry 49: 1856-69). [00479] In EYA4-depleted cells, cell cycle arrest and DNA damage response (DDR) activation were observed. EYA4, and specifically the serine/threonine domain of EYA4, was shown to play an important novel role in replication fork progression. Several human diseases have been associated with defects in replication stress signaling, including Bloom syndrome (Shastri VM, et al. (2021) Nucleic Acids Research 49(15): 8699-713), Fanconi anemia (Michl J, et al. 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It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
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