NEOPLASIA

FIELD OF INVENTION

[0001] The present invention provides, inter alia, methods for identifying a target for treating a neoplasia and for identifying a candidate agent that may be useful for treating a neoplasia.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims benefit to U.S. provisional application serial no. 61/790941 filed March 15, 2013, the entire contents of which are incorporated by reference.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] This application contains references to amino acids and/or nucleic acid sequences that have been filed concurrently herewith as sequence listing text file "0327656pct.txt", file size of 27.5 KB, created on March 13, 2014. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1 .52(e)(5).

BACKGROUND OF THE INVENTION

[0004] Target identification and validation is often employed by pharmaceutical and biotechnology companies. This approach provides a gateway to drug discovery and potentially identifies drugs with improved efficacy but fewer undesirable side effects. Significant resources, therefore, have been invested in acquiring and developing technologies for identifying and validating new drug targets.

[0005] In cancer biology, target identification and validation has traditionally been founded on demonstrating that target perturbations lead to functional impairment in cultured cancer cells in vitro. Large scale gene deletion studies in cancer cell lines have been used to assign target relevance on the basis of altered in vitro characteristics, such as rate of proliferation and/or apoptosis and/or autophagy. Using this screening paradigm, gene perturbations that do not impact in vitro cellular characteristics are discarded and not regarded as useful targets for cancer therapy. Thus far, a screening paradigm that seeks out targets that have no impact on in vitro growth characteristics but do alter tumor growth in a cell autonomous manner has not been used.

[0006] Accordingly, there is a need for additional methods of identifying targets for treating a neoplasia and for identifying a candidate agent that may be useful for treating a neoplasia. The present invention is directed to meeting this and other needs.

SUMMARY OF THE INVENTION

[0007] One embodiment of the present invention is a method for identifying a target for treating a neoplasia. This method comprises:

(a) generating a second neoplastic cell line having a single gene knockdown from a first neoplastic cell line;

(b) growing cells from the first and second neoplastic cell lines in vitro and in vivo; (c) determining whether there is a difference with respect to a cancer marker between the first and second neoplastic cell lines grown in vitro;

(d) determining whether there is a difference with respect to the same cancer marker in step (c) between the first and second neoplastic cell lines grown in vivo, wherein

(i) if there is no difference in step (c) between the first and second neoplastic cell lines grown in vitro with respect to the cancer marker and

(ii) there is a difference in step (d) between the first and second neoplastic cell lines grown in vivo with respect to the cancer marker, then the gene that is knocked down in the second cancer cell line is a target for treating a neoplasia.

[0008] An additional embodiment of the present invention is a target for treating a neoplasia identified according to any method disclosed herein.

[0009] Another embodiment of the present invention is a method for identifying a candidate agent that may be useful for treating a neoplasia. This method comprises:

(a) contacting neoplastic cells obtained from the same neoplastic cell line that are growing in vivo and in vitro with the candidate agent; and

(b) comparing the growth rate of the neoplastic cells grown in vivo in the presence and in the absence of the candidate agent and comparing the growth rate of the neoplastic cells grown in vitro in the presence and in the absence of the candidate agent, wherein a reduced in vivo growth rate of the neoplastic cell in the presence of the candidate agent compared to the neoplastic cell in the absence of the candidate agent and an unaffected in vitro growth rate of the neoplastic cell in the presence or absence of the candidate agent indicate that the candidate agent may be useful for treating neoplasia.

[0010] Another embodiment of the present invention is a candidate agent useful for treating a neoplasia identified according to any method disclosed herein.

[0011] Yet another embodiment of the present invention is a kit for identifying a candidate gene useful for targeting in the treatment of neoplasia. This kit comprises:

(a) a neoplastic cell;

(b) a nucleic acid for inhibiting a candidate gene or a product of the gene in the neoplastic cell; and

(c) instructions for the use of the neoplastic cell and the nucleic acid.

[0012] An additional embodiment of the present invention is a method for identifying a second target for treating a neoplasia. This method comprises:

(a) generating a second neoplastic cell line having a first target gene knockdown from a first neoplastic cell line;

(b) growing cells from the first and the second neoplastic cell line in vitro and in vivo;

(c) determining whether there are differences with respect to an expression level of a gene between the first and the second neoplastic cell lines grown in vitro and between the first and the second neoplastic cell lines grown in vivo; wherein

if the expression levels of a gene are different between the first and the second neoplastic cell lines grown in vitro and in vivo, then the gene whose expression level changed both in vitro and in vivo is a second target for treating a neoplasia.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0014] Figure 1 shows the in vivo anti-tumor effects of two independent shRNAs targeting BMPR2.

[0015] Figure 2 shows antitumor activity of various BMPR2 shRNAs across three independent experiments.

[0016] Figure 3 shows the growth of individual tumors in various groups of HRLN female nu/nu mice. Figure 3A shows individual tumor growth in groups 1 -6. Figure 3B shows individual tumor growth in groups 7-12. Figure 3C shows individual tumor growth in groups 13-18. Figure 3D shows individual tumor growth in groups 19-22.

[0017] Figure 4 shows tumor volume distributions in various groups of mice (groups 1 -22) on day 17.

[0018] Figure 5 shows median tumor growth in various groups of mice (groups 1 -22) over time. [0019] Figure 6 shows mean tumor growth in various groups of mice (groups 1 -22).

[0020] Figure 7 shows percent body weight change of various groups of mice (groups 1 -22) in comparison to the body weight of the mice on day 1 of the study.

[0021] Figure 8 shows that BMPR2 knockdown in vitro does not alter cell proliferation or survival. A2058 cells were transfected with various synthetic siRNA duplexes as shown in Figures 8A and 8B. 2-4 days after transfection, cells were assessed for proliferation and apoptosis using colorimetric or lumogenic detection assays (MTS for proliferation, Cell- CaspaseGlo for apoptosis). Cells treated with BMPR2 siRNA duplexes did not exhibit changes in either in vitro proliferation (Figure 8C) or apoptosis (Figure 8D) over the 2-4 day period. siRNAs introduced against the cell- essential polo-like kinase (PLK1 ) gene, however, prevented normal proliferation and induced elevated apoptosis over the same time frame.

[0022] Figure 9 shows that loss of BMPR2 regulates gene expression in a largely context-specific fashion. Whole-genome microarray gene expression profiling was conducted on stable RNAi cell line clones, as well as matched samples harvested from xenograft tissue isolated after 28 days in vivo, or when the tumor volume of the experimental cohorts had reached 2000 mm3, whichever occurred first. Several genes exhibit changes consistently in cell line clones with targeted BMPR2 constructs (Figure 9A). A majority of these genes, however, appear to not change consistently in paired xenograft trials (Figure 9B). Meanwhile, NOX4 expression changed in both in vitro and in vivo samples, and therefore may represent a BMPR2-dependent target that mediates the in vivo specific lethality observed in BMPR2 shRNAi clones (Figures 9A and 9B). Likewise, numerous gene expression changes in BMPR2 RNAi clones appear to be in vivo specific. The expression levels of TYRP1 and LXN, however, change both in vivo and in vitro in BMPR2 RNAi clones (Figures 9C and 9D). Thus, TYRP1 and LXN may be genes whose expression either correlates or mediates the BMPR2-dependent effects on xenograft growth.

DETAILED DESCRIPTION OF THE INVENTION

[0023] One embodiment of the present invention is a method for identifying a target for treating a neoplasia. This method comprises:

(a) generating a second neoplastic cell line having a single gene knockdown from a first neoplastic cell line;

(b) growing cells from the first and second neoplastic cell lines in vitro and in vivo;

(c) determining whether there is a difference with respect to a cancer marker between the first and second neoplastic cell lines grown in vitro;

(d) determining whether there is a difference with respect to the same cancer marker in step (c) between the first and second neoplastic cell lines grown in vivo, wherein

(i) if there is no difference in step (c) between the first and second neoplastic cell lines grown in vitro with respect to the cancer marker and (ii) there is a difference in step (d) between the first and second neoplastic cell lines grown in vivo with respect to the cancer marker, then the gene that is knocked down in the second cancer cell line is a target for treating a neoplasia.

[0024] As used herein, a "neoplasia" or a "neoplasm" means an abnormal growth of body tissue. Neoplasia include cancers, which are malignant, and noncancerous growth/tumors, which are benign.

[0025] As used herein, a "neoplastic cell line" means cancer/tumor cell lines, for example those which are readily available, through, e.g., the ATCC, and may be immortal (e.g., may be propagated indefinitely in vitro).

[0026] As used herein, a "target" for treating a neoplasia means a protein, a gene, or other molecule(s) produced by a cell, that are linked to the onset or the progression of neoplasia. Such a target may be a gene or protein on which the drugs act to treat neoplasia.

[0027] As used herein, to "knockdown" a gene means to reduce the expression of the gene or the expression and/or the activity of the gene product. Genes may be knocked down either through genetic modification or by treatment with a reagent, such as, e.g., by contacting the cell with a biologic or a chemical. The biologic may be a nucleic acid, a protein, and combinations thereof. Preferably, the nucleic acid comprises an shRNA. The chemical may be a small molecule inhibitor of the gene product, for example.

[0028] As used herein, a "cancer marker" means a characteristic that is affected by neoplastic cells. Cancer markers may be growth rate, invasiveness of the neoplasia, the metastatic capability of the neoplasia, the resistance of the neoplasia to conventional chemotherapeutic agents, or a combination of the above.

[0029] For example, the cancer marker may be growth rate of the first and second neoplastic cell lines. Preferably, the growth rate of the second neoplastic cell line in vivo, as represented by the size of the tumor, is reduced in comparison to the first cell line. Growth rates may be assayed using any conventional methods known in the art, including those disclosed herein.

[0030] The cancer marker may also be altered invasiveness of the neoplasia or metastatic capability of the neoplasia. As used herein, "invasiveness" refers to the capacity of a tumor or cancer to spread beyond the layer of tissue in which it developed and to grow into surrounding, healthy tissues. "Metastatic capability" refers to the ability of a tumor or a cancer to spread to a secondary location in the body. Invasiveness of the neoplasia or metastatic capability of the neoplasia rates may be assayed using any conventional methods known in the art, including by for example, histological analysis of the tissues.

[0031] As used herein, a "conventional chemotherapeutic agent" is a drug that is used to treat cancer or tumor. Chemotherapeutic agents may be DNA damaging agents, antimetabolites, anti-microtubule agents, or antibiotic agents. DNA damaging agents include alkylating agents, intercalating agents, and enzyme inhibitors of DNA replication. Non-limiting examples of DNA alkylating agents include cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, carmustine, lomustine, streptozocin, busulfan, temozolomide, cisplatin, carboplatin, oxaliplatin, a pharmaceutically acceptable salt thereof, a prodrug thereof, and combinations thereof. Preferably, the DNA alkylating agent is temozolomide, a prodrug thereof, or a pharmaceutically acceptable salt thereof. Non-limiting examples of intercalating agents include doxorubicin, daunorubicin, idarubicin, and mitoxantrone. Non-limiting examples of enzyme inhibitors of DNA replication include irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, and teniposide. Antimetabolites include folate antagonists such as methotrexate and premetrexed, purine antagonists such as 6-mercaptopurine, dacarbazine, and fludarabine, and pyrimidine antagonists such as 5- fluorouracil, arabinosylcytosine, capecitabine, gemcitabine, and decitabine. Anti-microtubule agents include without limitation vinca alkaloids, paclitaxel (Taxol®), docetaxel (Taxotere®), and ixabepilone (Ixempra®). Antibiotic agents include without limitation actinomycin, anthracyclines, valrubicinepirubicin, bleomycin, plicamycin, and mitomycin. The resistance of the neoplasia to conventional chemotherapeutic agents may be assayed using any conventional method, for example, by measuring the growth rate of the cells after the application of conventional chemotherapeutic agents.

[0032] In one aspect of this embodiment, the single gene is knocked down by contacting the first neoplastic cell with a biologic or a chemical. As used herein, a "biologic" means a substance which is derived from or produced by a living organism or synthesized to mimic an in Vo-derived agent or a derivative or product thereof. A biologic may be, for example, a nucleic acid, a polypeptide, or a polysaccharide. Preferably, the biologic is a nucleic acid, a protein, or a combination thereof. More preferably, the nucleic acid comprises an shRNA. [0033] As used herein, a "chemical" means a substance that has a definite chemical composition and characteristic properties and that is not a biologic. Non-limiting examples of chemicals include small organic compounds and small inorganic compounds.

[0034] In yet another aspect of this embodiment, the first neoplastic cell line is A101 D, A172, A204, A2058, A253, A2780, A3-KAW, A375, A388, A4- Fuk, A427, A431 , A498, A549, A673, A704, ABC-1 , ACHN, ACN, AGS, ALL- PO, AM-38, AML-193, AN3-CA, ARH-77, ATN-1 , AU565, AsPC-1 , BALL-1 , BB30-HNC, BB49-HNC, BB65-RCC, BC-1 , BC-3, BCPAP, BE-13, BEN, BFTC-905, BFTC-909, BHT-101 , BHY, BL-41 , BL-70, BOKU, BONNA-12, BPH-1 , BT-20, BT-474, BT-549, BV-173, Becker, BxPC-3, C-33-A, C-4-II, C2BBe1 , C32, C3A, C8166, CA46, CADO-ES1 , CAKI-1 , CAL-120, CAL-12T, CAL-148, CAL-27, CAL-33, CAL-39, CAL-51 , CAL-54, CAL-62, CAL-72, CAL- 85-1 , CAMA-1 , CAPAN-1 , CAS-1 , CCF-STTG1 , CCRF-CEM, CESS, CFPAC- 1 , CGTH-W-1 , CHL-1 , CHP-126, CHP-134, CHP-212, CMK, CML-T1 , COLO- 205, COLO-320-HSR, COLO-668, COLO-678, COLO-679, COLO-680N, COLO-684, COLO-741 , COLO-792, COLO-800, COLO-824, COLO-829, COR-L105, COR-L23, COR-L279, COR-L88, COR-L96CAR, CP50-MEL-B, CP66-MEL, CP67-MEL, CPC-N, CRO-AP2, CRO-AP5, CTB-1 , CTV-1 , CW-2, Ca-Ski, Ca9-22, CaR-1 , Calu-1 , Calu-3, Calu-6, Caov-3, Caov-4, Capan-2, ChaGo-K-1 , CoCM-1 , D-245MG, D-247MG, D-263MG, D-283MED, D- 336MG, D-384MED, D-392MG, D-397MG, D-423MG, D-458MED, D-502MG, D-538MG, D-542MG, D-556MED, D-566MG, DB, DBTRG-05MG, DEL, DG- 75, DJM-1 , DK-MG, DMS-1 14, DMS-153, DMS-273, DMS-53, DMS-79, DOHH-2, DOK, DSH1 , DU-145, DU-4475, DV-90, Daoy, Daudi, Detroit562, DoTc2-4510, EB-3, EB2, EC-GI-10, ECC10, ECC12, ECC4, EFE-184, EFM- 19, EFO-21, EFO-27, EGI-1, EHEB, EKVX, EM-2, EPLC-272H, ES1, ES3, ES4, ES5, ES6, ES7, ES8, ESS-1, ETK-1, EVSA-T, EW-1, EW-11, EW-12, EW-13, EW-16, EW-18, EW-22, EW-24, EW-3, EW-7, EoL-1-cell, FADU, FTC-133, G-361, G-401, G-402, GA-10-Clone-4, GAK, GAMG, GB-1, GCIY, GCT, GDM-1, GI-1, GI-ME-N, GMS-10, GOTO, GP5d, GR-ST, GT3TKB, H- EMC-SS, H4, H9, HA7-RCC, HAL-01, HC-1, HCC1143, HCC1187, HCC1395, HCC1419, HCC1569, HCC1599, HCC1806, HCC1937, HCC1954, HCC2157, HCC2218, HCC2998, HCC38, HCC70, HCE-4, HCE-T, HCT-116, HCT-15, HD-MY-Z, HDLM-2, HEC-1, HEL, HGC-27, HH, HL-60, HLE, HMV-II, HN, HO-1-N-1, HOP-62, HOP-92, HOS, HPAF-II, HSC-2, HSC-3, HSC-4, HT, HT- 1080, HT-1197, HT-1376, HT-144, HT-29, HT-3, HT55, HTC-C3, HUH-6- clone5, HUTU-80, HeLaSF, Hs-578-T, HuCCTI, HuH-7, HuO-3N1, HuO9, HuP-T3, HuP-T4, IA-LM, IGR-1, IGROV-1, IM-9, IMR-5, IPC-298, IST-MEL1, IST-MES1, IST-SL1, IST-SL2, ITO-II, J-RT3-T3-5, J82, JAR, JEG-3, JVM-2, JVM-3, JiyoyeP-2003, K-562, K052, K5, KALS-1, KARPAS-299, KARPAS- 422, KARPAS-45, KASUMI-1, KATOIII, KE-37, KG-1, KGN, KINGS-1, KLE, KM-H2, KM12, KMOE-2, KMS-12-PE, KNS-42, KNS-62, KNS-81-FD, KOSC- 2, KP-4, KP-N-RT-BM-1, KP-N-S19S, KP-N-YN, KP-N-YS, KS-1, KU-19-19, KU812, KURAMOCHI, KY821, KYSE-140, KYSE-150, KYSE-180, KYSE-270, KYSE-410, KYSE-450, KYSE-510, KYSE-520, KYSE-70, L-363, L-428, L- 540, LAMA-84, LAN-6, LB1047-RCC, LB2241-RCC, LB2518-MEL, LB373- MEL-D, LB647-SCLC, LB771-HNC, LB831-BLC, LB996-RCC, LC-1F, LC-2- ad, LC4-1, LCLC-103H, LCLC-97TM1, LK-2, LN-405, LNCaP-Clone-FGC, LOUCY, LOXIMVI, LP-1, LS-1034, LS-123, LS-174T, LS-411N, LS-513, LU- 134-A, LU-135, LU-139, LU-165, LU-65, LU-99A, LXF-289, LoVo, M059J, M14, MC-1010, MC-CAR, MC-IXC, MC1 16, MCF7, MDA-MB-134-VI, MDA- MB-157, MDA-MB-175-VII, MDA-MB-231 , MDA-MB-361 , MDA-MB-415, MDA-MB-435, MDA-MB-453, MDA-MB-468, ME-180, MEG-01 , MEL-HO, MEL-JUSO, MES-SA, MFE-280, MFE-296, MFH-ino, MFM-223, MG-63, MHH-CALL-2, MHH-CALL-4, MHH-ES-1 , MHH-NB-1 1 , MHH-PREB-1 , MIA- PaCa-2, MJ, MKN1 , MKN28, MKN45, MKN7, ML-2, MLMA, MMAC-SF, MN- 60, MOLT-13, MOLT-16, MOLT-4, MONO-MAC-6, MPP-89, MRK-nu-1 , MS-1 , MSTO-21 1 H, MUTZ-1 , MV-4-1 1 , MZ1 -PC, MZ2-MEL, MZ7-mel, Malme-3M, Mewo, Mo-T, NALM-1 , NALM-6, NB1 , NB10, NB12, NB13, NB14, NB17, NB5, NB6, NB69, NB7, NBsusSR, NCCIT, NCI-ADR-RES, NCI-H1048, NCI-H1092, NCI-H1 105, NCI-H1 155, NCI-H1 173, NCI-H1 184, NCI-H128, NCI-H1284, NCI-H1299, NCI-H1304, NCI-H1355, NCI-H1395, NCI-H1417, NCI-H1436, NCI-H1437, NCI-H146, NCI-H1522, NCI-H1563, NCI-H157, NCI-H1573, NCI- H1581 , NCI-H1618, NCI-H1623, NCI-H1648, NCI-H1650, NCI-H1651 , NCI- H1666, NCI-H1693, NCI-H1694, NCI-H1703, NCI-H1734, NCI-H1755, NCI- H1770, NCI-H1792, NCI-H1793, NCI-H1838, NCI-H187, NCI-H1882, NCI- H1926, NCI-H1930, NCI-H1963, NCI-H1975, NCI-H1993, NCI-H2009, NCI- H2029, NCI-H2030, NCI-H2052, NCI-H2081 , NCI-H2087, NCI-H209, NCI- H2107, NCI-H2122, NCI-H2126, NCI-H2141 , NCI-H2170, NCI-H2171 , NCI- H2196, NCI-H2227, NCI-H2228, NCI-H226, NCI-H2291 , NCI-H23, NCI- H2330, NCI-H2342, NCI-H2347, NCI-H2405, NCI-H2452, NCI-H250, NCI- H28, NCI-H292, NCI-H295, NCI-H322M, NCI-H345, NCI-H358, NCI-H378, NCI-H441 , NCI-H446, NCI-H460, NCI-H508, NCI-H510A, NCI-H520, NCI- H522, NCI-H524, NCI-H526, NCI-H596, NCI-H630, NCI-H64, NCI-H650, NCI- H661, NCI-H69, NCI-H711, NCI-H716, NCI-H719, NCI-H720, NCI-H727, NCI- H747, NCI-H748, NCI-H774, NCI-H810, NCI-H82, NCI-H835, NCI-H838, NCI- H889, NCI-N417, NCI-N87, NCI-SNU-1, NCI-SNU-16, NCI-SNU-5, NEC8, NH-12, NH-6, NKM-1 , NMC-G1 , NOMO-1, NOS-1, NTERA-S-cl-DI , NUGC-3, NY, no-10, no-11, OAW-28, OAW-42, OC-314, OCI-AML2, OCUB-M, OE19, OE33, OMC-1 , ONS-76, OPM-2, OS-RC-2, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, P12-ICHIKAWA, P30-OHK, P31-FUJ, PA-1, PANC-03-27, PANC- 08-13, PANC-10-05, PC-14, PC-3, PF-382, PFSK-1, PLC-PRF-5, PSN1, QIMR-WIL, RCC10RGB, RCM-1, RD, REH, RERF-LC-FM, RERF-LC-MS, RF-48, RH-1, RH-18, RKO, RL, RL95-2, RMG-I, RO82-W-1, RPMI-2650, RPMI-6666, RPMI-7951, RPMI-8226, RPMI-8402, RPMI-8866, RS4-11, RT- 112, RT4, RTSG, RVH-421, RXF393, Raji, Ramos-2G6-4C10, S-117, SAS, SBC-1, SBC-5, SCC-15, SCC-25, SCC-3, SCC-4, SCC-9, SCCH-26, SCH, SCLC-21H, SF126, SF268, SF295, SF539, SH-4, SHP-77, SIG-M5, SIMA, SJRH30, SJSA-1, SK-CO-1, SK-HEP-1, SK-LMS-1, SK-LU-1, SK-MEL-1, SK- MEL-2, SK-MEL-24, SK-MEL-28, SK-MEL-3, SK-MEL-30, SK-MEL-5, SK- MES-1, SK-MG-1, SK-MM-2, SK-N-AS, SK-N-DZ, SK-N-FI, SK-NEP-1, SK- OV-3, SK-PN-DW, SK-UT-1, SKG-llla, SKM-1, SN12C, SNB19, SNB75, SNG-M, SNU-387, SNU-423, SNU-449, SNU-475, SNU-C1, SNU-C2B, SR, ST486, SU-DHL-1, SUP-B8, SUP-T1, SW1088, SW1116, SW13, SW1417, SW1463, SW1573, SW1710, SW1783, SW1990, SW48, SW620, SW626, SW684, SW756, SW780, SW837, SW872, SW900, SW948, SW954, SW962, SW982, Saos-2, SiHa, T-24, T47D, T84, T98G, TALL-1, TC-YIK, TCCSUP, TE-1, TE-10, TE-11, TE-12, TE-15, TE-161-T, TE-206-T, TE-441-T, TE-5, TE- 6, TE-8, TE-9, TGBC11TKB, TGBC1TKB, TGBC24TKB, TGW, THP-1, TI-73, TK10, TT, TUR, TYK-nu, U-1 18-MG, U-2-OS, U-266, U-698-M, U-87-MG, U031 , U251 , UACC-257, UACC-62, UACC-812, UACC-893, UM-UC-3, UMC- 1 1 , VA-ES-BJ, VM-CUB-1 , VMRC-MELG, VMRC-RCZ, WERI-Rb-1 , WM-1 15, WSU-NHL, YAPC, YH-13, YKG-1 , YT, ZR-75-30, 22RV1 , 23132-87, 380, 5637, 639-V, 647-V, 697, 769-P, 786-0, 8-MG-BA, 8305C, or 8505C. Preferably, the first neoplastic cell line is a melanoma cell line.

[0035] In an additional aspect of this invention, the first neoplastic cell line is obtained or derived from a human.

[0036] In another aspect of this invention, the first neoplastic cell line is obtained from a one or more of the following neoplasms: adrenocortical carcinoma, anal cancer, bladder cancer, bone cancer, brain tumor, breast cancer, carcinoid tumor, carcinoma, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing family of tumors, extracranial germ cell tumor, eye cancer, gallbladder cancer, gastric cancer, germ cell tumor, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, kidney cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, malignant mesothelioma, Merkel cell carcinoma, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pituitary cancer, plasma cell neoplasm, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell cancer, transitional cell cancer of the renal pelvis and ureter, salivary gland cancer, Sezary syndrome, skin cancer (such as cutaneous t-cell lymphoma, Kaposi's sarcoma, and melanoma), small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, Wilms' tumor, and combinations thereof. Preferably, the first neoplastic cell line is obtained from a melanoma.

[0037] An additional embodiment of the present invention is a target for treating a neoplasia identified according to any method disclosed herein.

[0038] Another embodiment of the present invention is a method for identifying a candidate agent that may be useful for treating a neoplasia. This method comprises:

(a) contacting neoplastic cells obtained from the same neoplastic cell line that are growing in vivo and in vitro with the candidate agent; and

(b) comparing the growth rate of the neoplastic cells grown in vivo in the presence and in the absence of the candidate agent and comparing the growth rate of the neoplastic cells grown in vitro in the presence and in the absence of the candidate agent,

wherein a reduced in vivo growth rate of the neoplastic cell in the presence of the candidate agent compared to the neoplastic cell in the absence of the candidate agent and an unaffected in vitro growth rate of the neoplastic cell in the presence or absence of the candidate agent indicate that the candidate agent may be useful for treating neoplasia.

[0039] Suitable neoplastic cell lines are as disclosed herein.

[0040] In one aspect of this embodiment, the candidate agent is a biologic or a chemical. Suitable biologies and chemicals are as disclosed herein. Preferably, the biologic is a nucleic acid, which comprises an shRNA. [0041] Another embodiment of the present invention is a candidate agent useful for treating a neoplasia identified according to any method disclosed herein.

[0042] Yet another embodiment of the present invention is a kit for identifying a candidate gene useful for targeting in the treatment of neoplasia. This kit comprises:

(a) a neoplastic cell;

(b) a nucleic acid for knocking down a candidate gene in the neoplastic cell; and

(c) instructions for the use of the neoplastic cell and the nucleic acid.

[0043] In one aspect of this embodiment, the nucleic acid comprises an shRNA.

[0044] In another aspect of this embodiment, the kit further comprises a container for growing the neoplastic cell in vitro. Such containers are known in the art and include a tissue culture container.

[0045] An additional embodiment of the present invention is a method for identifying a second target for treating a neoplasia. This method comprises:

(a) generating a second neoplastic cell line having a first target gene knockdown from a first neoplastic cell line;

(b) growing cells from the first and the second neoplastic cell line in vitro and in vivo;

(c) determining whether there are differences with respect to an expression level of a gene between the first and the second neoplastic cell lines grown in vitro and between the first and the second neoplastic cell lines grown in vivo; wherein

if the expression levels of a gene are different between the first and the second neoplastic cell lines grown in vitro and in vivo, then the gene whose expression level changed both in vitro and in vivo is a second target for treating a neoplasia.

[0046] Numerous assays for measuring an expression level of a gene are known in the art. In general, these assays include methods involving hybridization analysis of polynucleotides, methods involving amplification of polynucleotides, and sequencing-based methods.

[0047] Hybridization analysis methods include northern blotting and in situ hybridization (See, e.g., Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNAse protection assays (Hod, Biotechniques 13:852- 854 (1992)). Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Methods using microarrays, which are commercially available from e.g., Affymetrix (Santa Clara, CA) and lllumina Inc. (San Diego, CA), also depend on hybridization principals to analyze the expression of genes. (See, e.g., Freeman, W.M., D.J. Robertson, and K.E. Vrana. 2000. Fundamentals of DNA hybridization arrays for gene expression analysis. (BioTechniques 29:1042-1055) As used herein, "microarrays" means small, solid supports onto which different nucleic acid sequences are immobilized, or attached, at fixed locations.

[0048] Amplification methods include reverse transcription polymerase chain reaction (RT-PCR) (Weis et al., Trends in Genetics 8:263-264 (1992)), such as TAQMAN™ assay (Roche Molecular Systems, Inc.). (See, e.g., Holland et al., Proc. Natl. Acad. Sci., U.S.A. (1991 ) 88:7276-7280; U.S. Pat. Nos. 5,538,848, 5,723,591 , and 5,876,930). RT-PCR methods include real time quantitative PCR (RT-qPCR), which measures PCR product accumulation through a dual-labeled fluorogenic probe. See, e.g., Held et al., Genome Research 6:986-994 (1996); VanGuilder HD, Vrana KE, Freeman WM (2008). "Twenty-five years of quantitative PCR for gene expression analysis". Biotechniques 44 (5): 619-626.)

[0049] Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and high throughput sequencing, such as gene expression analysis by massively parallel signature sequencing (MPSS) (See, e.g., Ding and Cantor, Proc. Nat'l Acad Sci 100:3059-3064 (2003); Brenner, Sydney; Johnson, Maria; Bridgham, John; Golda, George; Lloyd, David H.; Johnson, Davida; Luo, Shujun; McCurdy, Sarah et al. (2000). "Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays". Nature Biotechnology 18 (6): 630-4.)

[0050] In one aspect of this embodiment, the expression level is analyzed using a technique selected from reverse-transcription polymerase chain reaction (RT-PCR), microarrays, high-throughput sequencing, serial analysis of gene expression (SAGE).

[0051] In another aspect of this embodiment, the first target gene is knocked down by contacting the first neoplastic cell with a biologic or a chemical. Preferably, the biologic is selected from the group consisting of a nucleic acid, such as one that comprises an shRNA or an siRNA, a protein, and combinations thereof.

[0052] Suitable cell lines are as set forth above.

Additional Definitions

[0053] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0054] For recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitly contemplated.

Nucleic Acid

[0055] "Nucleic acid" or "oligonucleotide" or "polynucleotide" used herein mean at least two nucleotides covalently linked together. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.

[0056] Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequences. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be synthesized as a single stranded molecule or expressed in a cell (in vitro or in vivo) using a synthetic gene. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.

[0057] The nucleic acid may also be a RNA such as a mRNA, tRNA, short hairpin RNA (shRNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), transcriptional gene silencing RNA (ptgsRNA), Piwi-interacting RNA, pri-miRNA, pre-miRNA, micro-RNA (miRNA), or anti-miRNA, as described, e.g., in U.S. Patent Application Nos. 1 1/429,720, 1 1/384,049, 1 1/418,870, and 1 1/429,720 and Published International Application Nos. WO 2005/1 16250 and WO 2006/126040.

[0058] siRNA gene-targeting may be carried out by transient siRNA transfer into cells, achieved by such classic methods as lipid-mediated transfection (such as encapsulation in liposome, complexing with cationic lipids, cholesterol, and/or condensing polymers, electroporation, or microinjection). siRNA gene-targeting may also be carried out by administration of siRNA conjugated with antibodies or siRNA complexed with a fusion protein comprising a cell-penetrating peptide conjugated to a double- stranded (ds) RNA-binding domain (DRBD) that binds to the siRNA (see, e.g., U.S. Patent Application Publication No. 2009/0093026).

[0059] An shRNA molecule has two sequence regions that are reversely complementary to one another and can form a double strand with one another in an intramolecular manner. shRNA gene-targeting may be carried out by using a vector introduced into cells, such as viral vectors (lentiviral vectors, adenoviral vectors, or adeno-associated viral vectors for example).

[0060] The nucleic acid may also be an aptamer, an intramer, or a spiegelmer. The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target. Aptamers are derived from an in vitro evolutionary process {e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), disclosed in U.S. Pat. No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries. Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules. The nucleotide components of an aptamer may have modified sugar groups {e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH ₂ ), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood. Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system. Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker (Brody, E. N. and L. Gold (2000) J. Biotechnol. 74:5-13).

[0061] The term "intramer" refers to an aptamer which is expressed in vivo. For example, a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).

[0062] The term "spiegelmer" refers to an aptamer which includes L- DNA, L-RNA, or other left-handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.

[0063] A nucleic acid will generally contain phosphodiester bonds, although nucleic acid analogs may be included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those disclosed in U.S. Pat. Nos. 5,235,033 and 5,034,506. Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included within the definition of nucleic acid. The modified nucleotide analog may be located for example at the 5'-end and/or the 3'-end of the nucleic acid molecule. Representative examples of nucleotide analogs may be selected from sugar- or backbone-modified ribonucleotides. It should be noted, however, that also nucleobase-modified ribonucleotides, i.e. ribonucleotides, containing a non-naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridines or cytidines modified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosines and guanosines modified at the 8-position, e.g. 8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g. N6-methyl adenosine are suitable. The 2'-OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH ₂ , NHR, NR ₂ or CN, wherein R is C1 -C6 alkyl, alkenyl or alkynyl and halo is F, CI, Br or I. Modified nucleotides also include nucleotides conjugated with cholesterol through, e.g., a hydroxyprolinol linkage as disclosed in Krutzfeldt et al., Nature (Oct. 30, 2005), Soutschek et al., Nature 432:173-178 (2004), and U.S. Patent Application Publication No. 20050107325. Modified nucleotides and nucleic acids may also include locked nucleic acids (LNA), as disclosed in U.S. Patent Application Publication No. 200201 15080. Additional modified nucleotides and nucleic acids are disclosed in U.S. Patent Application Publication No. 20050182005. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments, to enhance diffusion across cell membranes, or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs may be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.

Peptide, Polypeptide, Protein

[0064] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein. In the present invention, these terms mean a linked sequence of amino acids, which may be natural, synthetic, or a modification, or combination of natural and synthetic. The term includes antibodies, antibody mimetics, domain antibodies, lipocalins, targeted proteases, and polypeptide mimetics. The term also includes vaccines containing a peptide or peptide fragment intended to raise antibodies against the peptide or peptide fragment.

[0065] "Antibody" as used herein includes an antibody of classes IgG, IgM, IgA, IgD, or IgE, or fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single chain antibodies, diabodies, bispecific antibodies, and bifunctional antibodies. The antibody may be a monoclonal antibody, polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom. The antibody may also be a chimeric antibody. The antibody may be derivatized by the attachment of one or more chemical, peptide, or polypeptide moieties known in the art. The antibody may be conjugated with a chemical moiety. The antibody may be a human or humanized antibody. These and other antibodies are disclosed in U.S. Published Patent Application No. 20070065447.

[0066] Other antibody-like molecules are also within the scope of the present invention. Such antibody-like molecules include, e.g., receptor traps (such as entanercept), antibody mimetics (such as adnectins, fibronectin based "addressable" therapeutic binding molecules from, e.g., Compound Therapeutics, Inc.), domain antibodies (the smallest functional fragment of a naturally occurring single-domain antibody (such as, e.g., nanobodies; see, e.g., Cortez-Retamozo et al., Cancer Res. 2004 Apr 15;64(8):2853-7)).

[0067] Suitable antibody mimetics generally can be used as surrogates for the antibodies and antibody fragments described herein. Such antibody mimetics may be associated with advantageous properties {e.g., they may be water soluble, resistant to proteolysis, and/or be nonimmunogenic). For example, peptides comprising a synthetic beta-loop structure that mimics the second complementarity-determining region (CDR) of monoclonal antibodies have been proposed and generated. See, e.g., Saragovi et al., Science. Aug. 16, 1991 ;253(5021 ):792-5. Peptide antibody mimetics also have been generated by use of peptide mapping to determine "active" antigen recognition residues, molecular modeling, and a molecular dynamics trajectory analysis, so as to design a peptide mimic containing antigen contact residues from multiple CDRs. See, e.g., Cassett et al., Biochem Biophys Res Commun. Jul. 18, 2003;307(1 ):198-205. Additional discussion of related principles, methods, etc., that may be applicable in the context of this invention are provided in, e.g., Fassina, Immunomethods. October 1994;5(2):121 -9.

[0068] As used herein, "peptide" includes targeted proteases, which are capable of, e.g., substrate-targeted inhibition of post-translational modification such as disclosed in, e.g., U.S. Patent Application Publication No. 20060275823.

[0069] In the present invention, "peptide" further includes anticalins. Anticalins can be screened for agents that decrease the number of cancer stem cells. Anticalins are ligand-binding proteins that have been constructed based on a lipocalin scaffold (Weiss, G. A. and H. B. Lowman (2000) Chem. Biol. 7:R177-R184; Skerra, A. (2001 ) J. Biotechnol. 74:257-275). The protein architecture of lipocalins can include a beta-barrel having eight antiparallel beta-strands, which supports four loops at its open end. These loops form the natural ligand-binding site of the lipocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities. The amino acid substitutions can be made using methods known in the art, and can include conservative substitutions {e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity. [0070] In general, a polypeptide mimetic ("peptidomimetic") is a molecule that mimics the biological activity of a polypeptide, but that is not peptidic in chemical nature. While, in certain embodiments, a peptidomimetic is a molecule that contains no peptide bonds (that is, amide bonds between amino acids), the term peptidomimetic may include molecules that are not completely peptidic in character, such as pseudo-peptides, semi-peptides, and peptoids. Examples of some peptidomimetics by the broader definition (e.g., where part of a polypeptide is replaced by a structure lacking peptide bonds) are described below. Whether completely or partially non-peptide in character, peptidomimetics according to this invention may provide a spatial arrangement of reactive chemical moieties that closely resembles the three- dimensional arrangement of active groups in a polypeptide. As a result of this similar active-site geometry, the peptidomimetic may exhibit biological effects that are similar to the biological activity of a polypeptide.

[0071] There are several potential advantages for using a mimetic of a given polypeptide rather than the polypeptide itself. For example, polypeptides may exhibit two undesirable attributes, i.e., poor bioavailability and short duration of action. Peptidomimetics are often small enough to be both orally active and to have a long duration of action. There are also problems associated with stability, storage and immunoreactivity for polypeptides that may be reduced with peptidomimetics.

[0072] Polypeptides having a desired biological activity can be used in the development of peptidomimetics with similar biological activities. Techniques of developing peptidomimetics from polypeptides are known. Peptide bonds can be replaced by non-peptide bonds that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original polypeptide. Further modifications can also be made by replacing chemical groups of the amino acids with other chemical groups of similar structure, shape or reactivity. The development of peptidomimetics can be aided by determining the tertiary structure of the original polypeptide, either free or bound to a ligand, by NMR spectroscopy, crystallography and/or computer-aided molecular modeling. These techniques aid in the development of novel compositions of higher potency and/or greater bioavailability and/or greater stability than the original polypeptide (Dean (1994), BioEssays, 16: 683-687; Cohen and Shatzmiller (1993), J. Mol. Graph., 1 1 : 166-173; Wiley and Rich (1993), Med. Res. Rev., 13: 327-384; Moore (1994), Trends Pharmacol. Sci., 15: 124-129; Hruby (1993), Biopolymers, 33: 1073-1082; Bugg et al. (1993), Sci. Am., 269: 92-98.

Polysaccharides

[0073] The term "polysaccharides" means polymeric carbohydrate structures, formed of repeating units (either mono- or di-saccharides) joined together by glycosidic bonds. The units of mono- or di-saccharides may be the same or different. Non-limiting examples of polysaccharides include starch, glycogen, cellulose, and chitin.

Small organic or inorganic molecules

[0074] The phrase "small organic" or "small inorganic" molecule includes any chemical or other moiety, other than polysaccharides, polypeptides, and nucleic acids, that can act to affect biological processes. Small molecules can include any number of therapeutic agents presently known and used, or can be synthesized in a library of such molecules for the purpose of screening for biological function(s). Small molecules are distinguished from macromolecules by size. The small molecules of this invention usually have a molecular weight less than about 5,000 daltons (Da), preferably less than about 2,500 Da, more preferably less than 1 ,000 Da, most preferably less than about 500 Da.

[0075] As used herein, the term "organic compound" refers to any carbon-based compound other than biologies such as nucleic acids, polypeptides, and polysaccharides. In addition to carbon, organic compounds may contain calcium, chlorine, fluorine, copper, hydrogen, iron, potassium, nitrogen, oxygen, sulfur and other elements. An organic compound may be in an aromatic or aliphatic form. Non-limiting examples of organic compounds include acetones, alcohols, anilines, carbohydrates, mono-saccharides, di- saccharides, amino acids, nucleosides, nucleotides, lipids, retinoids, steroids, proteoglycans, ketones, aldehydes, saturated, unsaturated and polyunsaturated fats, oils and waxes, alkenes, esters, ethers, thiols, sulfides, cyclic compounds, heterocyclic compounds, imidizoles, and phenols. An organic compound as used herein also includes nitrated organic compounds and halogenated {e.g., chlorinated) organic compounds. Collections of small molecules, and small molecules identified according to the invention are characterized by techniques such as accelerator mass spectrometry (AMS; see Turteltaub et al., Curr Pharm Des 2000 6:991 -1007, Bioanalytical applications of accelerator mass spectrometry for pharmaceutical research; and Enjalbal et al., Mass Spectrom Rev 2000 19:139-61 , Mass spectrometry in combinatorial chemistry.) [0076] Preferred small molecules are relatively easier and less expensively manufactured, formulated or otherwise prepared. Preferred small molecules are stable under a variety of storage conditions. Preferred small molecules may be placed in tight association with macromolecules to form molecules that are biologically active and that have improved pharmaceutical properties. Improved pharmaceutical properties include changes in circulation time, distribution, metabolism, modification, excretion, secretion, elimination, and stability that are favorable to the desired biological activity. Improved pharmaceutical properties include changes in the toxicological and efficacy characteristics of the chemical entity.

[0077] The following examples are provided to further illustrate the methods of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way.

EXAMPLES

Example 1

[0078] shRNAs were purchased from Sigma (St. Louis, MO). A2058 parental cell line was obtained from ATCC (Manassas, VA). [0079] Cell lines were generated through viral mediated transduction of cell lines using vesicular stomatitis virus G glycoprotein (VSVG) pseudotyped lentiviral particles containing shuttle inserts that encode short hairpin RNA interference sequences designed to reduce the mRNA expression of human genes. RNAi 'libraries' designed by The RNAi Consortium containing hairpin sequences were cloned in the pLKO.1 vector. Plasmid DNA containing a desired hairpin was mixed with a series of 'packaging' plasmids which express gene products for viral particle encapsidation of the RNAi vector. These plasmids were introduced into HEK293-T helper cell lines via liposomal transfection. Virus containing media supernatants were isolated at 72 and 96 hours post-transfection.

[0080] Virus particles were then used to transduce a cell line of interest, or the A2058 cell line, with a desired RNAi insert. Cells in standard culture conditions were exposed to viral supernatant, typically at multiplicities of infection ranging from 10 to 100 viral particles per cell. 72-96 hours postinfection, cells were provided with selective growth media containing puromycin (typically 1 -5 g/mL). The pLKO.1 vector contains a puromycin resistance cassette and thus allows for isolation of RNAi insert-containing cells via positive selection in puromycin containing media. Polyclonal cell lines, containing cell clones with varying numbers of viral insertions with the RNAi cassette, were expanded and used in further in vitro and in vivo experiments.

Cell line characterization

[0081] Cell lines were typically assessed for in vitro doubling time over 96 hours using standard tissue culture methods such as hemacytometer- based counting. Cell lines of interest which show differences in growth in vivo were occasionally re-assessed for levels of knockdown of the targeted gene, or by microarray-based gene expression analysis. BMPR2 clones were verified for levels of mRNA knockdown using 'real-time', quantitative RT-PCR methods.

In vivo Growth Data

[0082] For candidate screening, groups of mice, or xenograft lines, are assessed for differences in growth across the duration experiment. Most often, 'end-point' assessments are made at the latest time-point where the majority of xenograft lines still have subjects with implanted tumors.

[0083] Specifically, HRLN female nu/nu mice were injected with 1x107 A2058 tumor cells in 50% Matrigel subcutaneously in flank. The injection volume was 0.2 mL/mouse. Age of the mice at the start of the experiment was 8 to 12 weeks. Body weight was measured biweekly, starting on day 4, until the end of the experiment. Tumor size was also measured biweekly, starting on day 4, until the end of the experiment. Animals are to be monitored individually. The endpoint of the experiment is a tumor volume of 2000 mm ³ or 17 days, whichever comes first. Responders can be followed longer. When the endpoint is reached, the animals are to be euthanized.

[0084] Xenograft measures are typically aggregated in a 'carry-forward' analysis: for subjects missing at a given time point due to sacrifice, the largest tumor measurement from the nearest earlier assessment is use to represent the subject at that later day. With group estimates across the all xenograft lines, a standard one-way ANOVA analysis, with a post-hoc Dunnett multiple testing comparison, is used to identify lines which show growth different from either the RNAi negative control line expressing a 'scrambled' shRNA that does not target a human gene, or the 'parental', untransduced A2058 line. Significance is assessed at p values less than 0.05. Drags and Treatments

[0085] Tumor growth for A2058 lines generated by lentiviral transduction were monitored when implanted in nude mice in accordance with the procedure set forth above.

[0086] In one experimental setup, the following dosing solutions were prepared:

Table 1 Various clones and their corresponding target genes

A2058 Clone 95 GSK3A (T39766)

A2058 Clone 96 HKDC1 (T78723)

A2058 Clone 97 ITPK1 (T37698)

A2058 Clone 98 LIMK1 (T00826)

A2058 Clone 99 MKNK2 (T06098)

A2058-BVD None (control)

[0087] Dosing volume was 1 x 10 ⁷ cells in 0.20 mL of 50% Matrigel in PBS buffer.

[0088] The protocol design is shown in Table 2 below.

Table 2 Protocol design

[0089] The response summary of the above experiment is shown in Table 3 below. No regression of the tumor was seen in any of the mice. Further data are shown in Figures 3-7.

Table 3 Response summary

[0090] In sum, the inventors have taken a novel approach based on an alternative assumption: that a gene perturbation may become essential/important when the cancer cells are in the in vivo environment, and not necessarily in the in vitro culture environment. To test this hypothesis, the inventors used shRNAs targeting kinases in the melanoma cell line A2058, to generate cell lines with single gene knockdowns. Resulting knockdown lines were expanded and the growth properties confirmed as similar to parental A2058 cells. Cell lines were then implanted (1 ,000,000 cells in 50% Matrigel) into 7 week old nu/nu mice and tumor growth monitored biweekly using calipers. A cell line that exhibited a reduced rate of tumor growth in vivo but had normal growth in vitro was scored as a positive hit and a potential target for cancer drug discovery.

[0091] One such target is bone morphogenetic protein receptor type II (BMPR2; aka PPH 1 , BMPR3, BRK-3, T-ALKI), a gene product previously not described as important in melanoma growth. Knock down of BMPR2 using two independent shRNAs did not substantially impact A2058 doubling time in vitro but significantly reduced tumor growth in vivo (Figure 1 ). The two shRNA nucleotide sequences targeting BMPR2 and demonstrating antitumor activity are listed in Table 4 below.

Table 4 BMPR2 shRNA sequences tested for antitumor activity

[0092] The above results were verified by three independent sets of experiments, as set forth in Table 5 below.

Table 5 Summary of antitumor activity in three independent experiments

Example 2

[0093] Secondary assay validated lack of BMPR2 effects in vitro. Transient RNAi knockdown experiments were performed. Cell lines

[0094] Unique clones from A2058 cell lines transduced with stable RNA interference constructs (pLKO/RNAi Consortium) were harvested by trypsin dissociation, washed with PBS, pelleted and stored frozen at -80°C. [0095] A2058 cells were transiently transfected with synthetic RNAi duplexes (from Thermo Fisher Scientific, Inc., Waltham, MA and Life Technologies Inc., Carlsbad, CA) using Lipofectamine 2000 liposomal transfection reagent. After 96 hours, cells were harvested by dissociation and stored as pellets at -80°C. QPCR Expression Analysis

[0096] Total RNA was prepared from cell line and tumor xenograft samples as described above. cDNA was prepared using Quantitect reverse transcription kits (Qiagen). cDNA were analyzed for gene expression using Taqman gene expression assays for human BMPR2 (Hs00176148_m1 ) on an ABI7900HT thermocycler under standard cycling conditions.

In-vitro Cell Viability and Apoptosis

[0097] A2058 cells were transiently transfected with synthetic RNAi duplexes (Dharmacon/Lifetech) using Lipofectamine 2000 liposomal transfection reagent. At multiple time points, cells were collected and analyzed for growth and apoptosis. Proliferation was measured using Cell- Titer-Glo luminescent detection kits (Promega); apoptosis was assessed with Cell-Caspase-Glo cleaved caspase 3/7 luminescent detection kits. All experiments were conducted using N=4 experimental replicates, and statistical comparisons were made with Excel.

[0098] The results are shown in Figure 8. BMPR2 knockdown cells showed no changes in proliferation or apoptosis over a period of 96 hours after transfection with RNAi. Thus, these data further confirm the lack of in vitro effects observed in the shRNAi clones used for xenograft studies.

Example 3

[0099] Gene expression analysis highlights in vivo versus in vitro consequences of BMPR2 loss. shRNAi clones were analyzed for global gene expression changes by microarray. Many genes were expressed at notably different levels compared to the non-silencing control samples. Importantly, genes affected by BMPR2 loss in-vitro were often unchanged or changed in different fashion when paired samples were compared in the xenograft experiments. The converse was also true.

Cell and Tissue samples

[0100] Cell lines were prepared, and transfection was performed as set forth in the previous example.

[0101] Tumor fragments were excised from xenograft specimens after 28 days growth, or when cohorts reached 2000 mm ³ on average. Tissue samples were snap-frozen and pulverized in liquid nitrogen before storage at - 80°C.

Microarrav analysis

[0102] RNA isolated by treating samples with Trizol reagent (Lifetech), followed by standard separation techniques. Isolated RNA was further purified using Qiagen (Germantown, MD) RNeasy column kits, and assessed for quality using Agilent (Santa Clara, CA) Bioanalyzer kits, both under manufacturer recommendations. Suitable RNA was directly labeled with Agilent cDNA kits, and hybridized to Human GE 4x44K v2 microarrays. Array detection and processing were performed using manufacturer recommendations. Statistical and comparative expression analysis were calculated in Excel. All experiments were conducted using N=4 experimental replicates, which were labeled separately and then pooled before hybridization.

[0103] Several genes exhibit changes consistently in cell line clones with targeted BMPR2 constructs. A majority of these genes, however, appear to not change consistently in paired xenograft trials. Meanwhile, NADPH Oxidase 4 (NOX4) expression changed in all samples, and therefore may represent a BMPR2-dependent target that mediates the in-vivo specific lethality observed in BMPR2 shRNAi clones. NOX4 regulates growth and transcription in melanoma cells and contributes to transformation of melanoma cells by regulating cell cycle progression (Brar et al., 2002; Yamaura et al., 2009). NOX4 is also linked to inflammation-induced renal cell carcinoma metastasis. (Fitzgerald et al., 2012) Furthermore, NOX4 is overexpressed in the majority of breast cancer cell lines, primary breast tumors, and ovarian tumors (Graham, 2010). Additionally, NOX4 is prominently expressed in various neuroepithelial tumors, such as astrocytomas and glioblastomas, and specific knockdown of Nox4 expression by RNA interference results in cell-growth inhibition and enhances induction of apoptosis by chemotherapeutic agents, such as cisplatin, in cultured glioma cell lines. (Shono et al., 2008). Thus, NOX4 may be used as a target for treating neoplasia.

[0104] Likewise, numerous gene expression changes in BMPR2 RNAi clones appear to be in vivo specific. The expression levels of Tyrosinase- related protein 1 (TYRP1 ) and Latexin (LXN), however, were changed both in vivo and in vitro. Thus, TYRP1 and LXN may be genes whose expression either correlates or mediates the BMPR2-dependent effects on xenograft growth.

[0105] TYRP1 mRNA was detected in 15 of the 43 melanoma patients (34.9%), and TYRP1 mRNA in 16 of the 43 (37.2%) (Jin et al., 2003). TYRP1 mRNA expression level, at least in skin metastases, is a prognostic marker for melanoma (Journ et al., 201 1 ). (Xiao et al., 2012) TYRP1 is a mediator of melanoma immunogenicity, and TYRP1 protein or peptide derivatives may be used in various cancer vaccinations. (Guevara-Patifio et al., 2006) LXN is one of the three genes that are densely methylated in greater than 95% of uncultured melanoma tumor samples (Muthusamy et al., 2006). It is a tumor suppressor gene that is involved in the regulation of proliferation, apoptosis, self-renewal, and subsequently pool size, of normal hematopoietic stem cells (HSCs) (Li et al., 201 1 ; Liang et al., 2007; Liu et al., 2012). Lxn inhibits human carboxypeptidase A4 (hCPA4), whose expression is induced by histone deacetylase inhibitors in prostate cancer cells, and is associated with cancer progression (Garcia-Castellanos et al., 2005). Accordingly, TYRP1 and LXN may also be used as targets for cancer.

[0106] Lastly, it is notable that expression of apparent BMPR2- dependent genes can change drastically in the cellular or xenograft settings. This again suggests how critical mediators of cancerous growth and survival may only be relevant in particular experimental contexts.

Documents

Brar, Sukhdev S., Thomas P. Kennedy, Anne B. Sturrock, Thomas P. Huecksteadt, Mark T. Quinn, A. Richard Whorton, and John R. Hoidal, An NAD(P)H oxidase regulates growth and transcription in melanoma cells Am J Physiol Cell Physiol 282:(6) C1212-C1224 (2002).

Fitzgerald JP, Nayak B, Shanmugasundaram K, Friedrichs W, Sudarshan S, et al. Nox4 Mediates Renal Cell Carcinoma Cell Invasion through Hypoxia- Induced Interleukin 6- and 8- Production. PLoS ONE 7(1 ): e30712 (2012). Garcia-Castellanos, R., Bonet-Figueredo, R., Pallares, I., Ventura, S., Aviles, F.X., Vendrell, J., Gomis-Riitha, F.X. Detailed molecular comparison between the inhibition mode of A/B-type carboxypeptidases in the zymogen state and by the endogenous inhibitor latexin. Cell. Mol. Life Sci. 62(17):1996-2014 (2005)

Graham KA, Kulawiec M, Owens KM, Li X, Desouki MM, Chandra D, Singh KK., "NADPH oxidase 4 is an oncoprotein localized to mitochondria." Cancer Biol Ther. Aug 1 ;10(3):223-31 (2010).

Guevara-Patino, J. A. et al. Optimization of a self antigen for presentation of multiple epitopes in cancer immunity J. Clin. Invest. 1 16, 1382-1390 (2006). Jin, H.Y., Yamashita, T., Minamitsuji, Y., Omori, F., Jimbow, K. Detection of tyrosinase and tyrosinase-related protein 1 sequences from peripheral blood of melanoma patients using reverse transcription-polymerase chain reaction. J. Dermatol. Sci. 33(3):169-76 (2003)

Journe, F., Boufker, H. I., Van Kempen, L., Galibert, M. -., Wiedig, M., Sales, F. et al.. TYRP1 mRNA expression in melanoma metastases correlates with clinical outcome. British Journal of Cancer, 105(1 1 ), 1726-1732 (201 1 ). Li Y, Basang Z, Ding H, Lu Z, Ning T, et al. Latexin expression is downregulated in human gastric carcinomas and exhibits tumor suppressor potential. BMC cancer 1 1 : 121 (201 1 ).

Liang, Ying, et al. "The quantitative trait gene latexin influences the size of the hematopoietic stem cell population in mice." Nature genetics 39.2: 178-188 (2007).

Liu, Y., Howard, D., Rector, K., Swiderski, C, Brandon, J., Schook, L., et al. Latexin Is Down-Regulated in Hematopoietic Malignancies and Restoration of Expression Inhibits Lymphoma Growth. PloS one, 7(9), e44979 (2012).

Muthusamy, V., Duraisamy, S., Bradbury, CM., Hobbs, C, Curley, D ., Nelson, B., Bosenberg, M., Epigenetic silencing of novel tumor suppressors in malignant melanoma. Cancer Res. 66(23):1 1 187-93 (2006).

Shono, Tadahisa, Nobuhiko Yokoyama, Toshio Uesaka, Junya Kuroda, Ryu Takeya, Tomoko Yamasaki, Toshiyuki Amano, Masahiro Mizoguchi, Satoshi O Suzuki, Hiroaki Niiro, Kyoko Miyamoto, Koichi Akashi, Toru Iwaki, Hideki Sumimoto, Tomio Sasaki, Enhanced expression of NADPH oxidase Nox4 in human gliomas and its roles in cell proliferation and survival. Int J Cancer, 123 787-92 (2008)

Yamaura M, Mitsushita J, Furuta S, Kiniwa Y, Ashida A, Goto Y, Shang WH, Kubodera M, Kato M, Takata M, Saida T, Kamata T. NADPH oxidase 4 contributes to transformation phenotype of melanoma cells by regulating G2- M cell cycle progression. Cancer Res. Mar 15;69(6):2647-54 (2009).

[0107] All documents cited in this application are hereby incorporated by reference as if recited in full herein. [0108] Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the invention.