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Title:
ACRIDINONE COMPOSITIONS FOR TREATING HEAD AND NECK CANCERS AND METHODS OF PROGNOSING HEAD AND NECK CANCERS
Document Type and Number:
WIPO Patent Application WO/2016/179702
Kind Code:
A1
Abstract:
The invention relates to compositions useful in the treatment of a head and neck cancer, the composition comprising a therapeutically effective amount of an acridinone compound having the formula (I): or a pharmaceutically acceptable salt thereof, wherein: - R1 and R2, which may be the same or different, are hydrogen, substituted or unsubstituted lower alkyl, -OR5, -OCOR6, or CF3, wherein R5 is hydrogen or substituted or unsubstituted lower alkyl, and R6 is substituted or unsubstituted lower alkyl; - R3 is amino, alkylamino, -R7-NH2 or -R8-OH, wherein R7 and R8 are substituted or unsubstituted lower alkyl; and - and R4 is H or substituted or unsubstituted lower alkyl; and a pharmaceutically acceptable carrier, excipient, or vehicle. The composition optionally includes at least one chemotherapeutic agent, such as a platinum anti-cancer drug. Also provided is a method of prognosing a head and neck cancer, in particular oral squamous cell carcinoma, in a subject, the method comprising measuring the expression levels of the biomarkers PLK-1 and/or Syk.

Inventors:
RALHAN RANJU (CA)
FU GUODONG (CA)
Application Number:
PCT/CA2016/050541
Publication Date:
November 17, 2016
Filing Date:
May 11, 2016
Export Citation:
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Assignee:
RALHAN RANJU (CA)
FU GUODONG (CA)
International Classes:
A61K31/473; A61K31/194; A61P35/00; C07D219/14
Foreign References:
US20050232969A12005-10-20
Other References:
P. SINGH ET AL.: "Search for MDR Modulators: Design, Syntheses and Evaluations of N-substituted Acridones for Interactions with p-Glycoprotein and Mg2+'';", BIOORG. & MED. CHEM., vol. 17, 2009, pages 2423 - 2427., XP025981939
R. KNECHT ET AL.: "Prognostic Significance of Polo-like Kinase (PLK) Expression in Squamous Cell Carcinomas of the Head and Neck'';", CANCER RESEARCH, vol. 59, no. 12, 1999, pages 2794 - 2797, XP055331083
Y.-B. FENG ET AL.: "Overexpression of PLK1 is associated with poor survival by inhibiting apoptosis via enhancement of survivin level in esophageal squamous cell carcinoma'';", INT. J. CANCER, vol. 124, no. 3, 2009, pages 578 - 588, XP055002858
Z.-M. DU ET AL.: "Clinical significance of elevated spleen tyrosine kinase expression in nasopharyngeal carcinoma'';", HEAD & NECK, vol. 32, no. 10, 2012, pages 1456 - 1464, XP055331084
Attorney, Agent or Firm:
CHARI, Santosh K. et al. (Cassels & Graydon LLP199 Bay Street, Suite 4000,Commerce Court Wes, Toronto Ontario M5L 1A9, CA)
Download PDF:
Claims:
WHAT IS CLAIMED IS:

1. A pharmaceutical composition for treatment of a head and neck cancer, the composition comprising a therapeutically effective amount of an acridinone compound having the formula (I):

R2 R3 or a pharmaceutically acceptable salt thereof, wherein:

- R1 and R2, which may be the same or different, are hydrogen, substituted or unsubstituted lower alkyl, -OR5, -OCOR6, or CF3, wherein R5 is hydrogen or substituted or unsubstituted lower alkyl, and R6 is substituted or unsubstituted lower alkyl;

- R3 is amino, alkylamino, -R7-NH2 or -R8-OH, wherein R7 and R8 are substituted or unsubstituted lower alkyl; and

- and R4 is H or substituted or unsubstituted lower alkyl;

and a pharmaceutically acceptable carrier, excipient, or vehicle.

2. The composition of claim 1 , wherein the acridinone compound is 10-(3-aminopropyl)- 3,4-dimethyl-9(10H)-acridinone, or a pharmaceutically acceptable salt thereof.

3. The composition of claims 1 or 2, wherein the acridinone compound is 10-(3- aminopropyl)-3,4-dimethyl-9(10H)-acridinone maleate.

4. The composition of any one of claims 1 to 3 further comprising a chemotherapeutic agent.

5. The composition of claim 2, wherein the chemotherapeutic agent is a platinum anticancer drug.

6. The composition of claim 4 or 5, wherein the chemotherapeutic agent is cisplatin, carboplatin, oxaliplatin, [cis-Pt(diaminocyclohexane)CI2], nedaplatin, stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin, enloplatin, iproplatin, lobaplatin, ormaplatin, spiroplatin, or zeniplatin.

7. Use of the composition according to any one of claims 1 to 6 in the treatment of a head and neck cancer.

8. Use of the composition according to any one of claims 1 to 4 in the

chemosensitization of a head and neck cancer cells to a chemotherapeutic agent.

9. Use of the composition of any one of claims 1 to 6 in the preparation of a

medicament for the treatment of a head and neck cancer.

10. A kit form of a composition as claimed in any one of claims to 1 to 6.

1 1. A kit comprising a first container containing the acridinone compound according to any one of claims 1 to 3 and second container containing the chemotherapeutic agent according to any one of claims 4 to 6.

12. A method of treating a head and neck cancer in a subject comprising administering to the subject the composition of any one of claims 1 to 6.

13. A method of delivering an effective amount of an acridinone compound as defined in any one of claims 1 to 3 to a subject with head and neck cancer, wherein the effective amount is sufficient to chemosensitize the head and neck cancer cells to the

chemotherapeutic agent of any one of claims 4 to 6, comprising combining the acridinone compound with the chemotherapeutic agent and administering the combination to the subject.

14. A method of treating a head and neck cancer in a subject comprising administering to the subject the composition of any one of claims 4 to 6, wherein the acridinone compound and the chemotherapeutic agent are administered to the subject sequentially.

15. A method of treating a head and neck cancer in a subject comprising administering to the subject effective amounts of an acridinone compound as defined in any one of claims 1 to 3 and monitoring the efficacy of the acridinone compound using PLK-1 and/or Syk as biomarkers of drug response.

16. A method prognosing head and neck cancer in a subject comprising:

- measuring the expression of PLK-1 and/or Syk in a biological sample from the subject;

- comparing the amount of expression of the PLK-1 and/or Syk with a control value corresponding to an expression amount in non-cancerous tissue; and,

- prognosing the head and neck cancer when the measured expression amount is greater than the control value.

17. The method of claim 16, wherein the head and neck cancer is oral cancer.

18. The method of claim 16, wherein the head and neck cancer is oral squamous cell carcinoma.

Description:
ACRIDINONE COMPOSITIONS FOR TREATING HEAD AND NECK CANCERS AND METHODS OF PROGNOSING HEAD AND NECK CANCERS

CROSS REFERENCE TO PRIOR APPLICATION

The present application claims priority under the Paris Convention to US Application number 62/159,551 , filed on May 1 1 , 2015. The entire contents of such prior application is incorporated herein by reference. FIELD OF THE INVENTION The invention relates generally to compositions and methods comprising an acridinone compound for treatment of head and neck cancer. The invention also relates to biomarkers for prognosing head and neck cancers. BACKGROUND OF THE INVENTION Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide (1 ). HNSCC patients diagnosed at stages I and II have five-year survival rates of 70% - 90% (2-4), while those diagnosed in stages III and IV have 50 % survival, limited treatment options and poor prognosis (5). Current treatments including primary surgery and/or a combination of chemo- and radio- therapy for oral squamous cell carcinoma (OSCC) patients are traumatic, disfiguring and drastically compromise their quality of life (6,7). Chemotherapy (CT) using cisplatin/carboplatin, methotrexate or taxanes as single agents or in combination are given in recurrent or metastatic HNSCC; dose-limiting toxicities restrict their clinical utility (3,8-1 1). Monotargeted therapies including inhibitors of EGFR, STAT3, NFKB and mammalian target of rapamycin (mTOR) have shown limited efficacy (12- 15). There exists urgent need for development of new drugs for oral cancer. Small bioactive molecules are being explored as inhibitors of novel kinases as molecular therapeutic targets including Spleen tyrosine kinase (Syk) and Polo-like kinase 1 (PLK1). Syk is a cytoplasmic tyrosine kinase with critical roles in B cell development, initiation of inflammatory responses, and acts as a pro-survival factor in cancers of both hematopoietic and epithelial origins (16, 17). The expression of Syk is increased in HNSCC and lymph node metastases and promotes migration of HNSCC cells (18). Syk inhibitors are likely to be effective in inhibiting cell migration and metastasis in HNSCC, making this kinase an attractive therapeutic HNSCC target. PLK1 is an essential mitotic kinase that

phosphorylates Ser/Thr residues in proteins and has pleiotropic roles in regulation of cell division, including effects on G2/M transition, centrosome maturation, mitotic spindle formation, chromosome segregation, and cytokinesis (19). PLK1 is involved in checkpoint recovery and resuming cell cycle progression after DNA damage-induced cell cycle arrest (20); it is overexpressed in several human cancers (21 ,22), although such overexpression has not been previously noted in oral cancers; and is a potential target for antitumor therapy with increasing efforts being made for developing small-molecule PLK inhibitors. SUMMARY OF THE INVENTION The invention relates to compositions and methods for treatment of head and neck cancers. In an aspect, the invention relates to compositions and methods for inhibiting, reducing or suppressing the growth or survival of head and neck cancer cells. In an aspect, the invention provides a pharmaceutical composition comprising an effective amount of an acridinone compound for use in treating head and neck cancers. In an aspect a pharmaceutical composition is provided for inhibiting, reducing or suppressing the growth or survival of a head and neck cancer cell, in particular an oral cancer cell, comprising an effective amount of an acridinone compound. In another aspect, a

pharmaceutical composition of the invention is administered to a head and neck cancer cell, in particular an oral cancer cell, capable of malignant transformation, thereby suppressing or preventing transformation. A pharmaceutical composition of the invention may optionally comprise a pharmaceutically acceptable carrier, excipient, or vehicle. In an embodiment, the invention provides a pharmaceutical composition for the treatment of head and neck cancer characterized in that it comprises an effective amount of an acridinone compound together with a pharmaceutically acceptable carrier, excipient or vehicle. A composition of the invention may comprise one or more additional component or it may be administered in combination with another treatment, in particular a component or procedure for treating or preventing head and neck cancer, including but not limited to a chemotherapeutic agent, a non-steroidal anti-inflammatory drug, vaccines with autologous tumor cells, vaccines against tumor-associated antigens, monoclonal antibodies against tumor antigens, gene therapy including gene correction, virus-directed enzyme prodrug treatment, or a treatment such as radiation therapy or surgery. In an aspect, a pharmaceutical composition of the invention comprises

therapeutically effective amounts of an acridinone compound and a chemotherapeutic agent, in particular a platinum anti-cancer drug. In a particular aspect the invention provides a pharmaceutical composition comprising therapeutically effective amounts of an acridinone compound and a platinum anti-cancer drug that provide beneficial effects relative to an acridinone compound alone following treatment. In other particular aspects the beneficial effects comprise reduced toxicity and/or increased antineoplastic activity. In an aspect, the invention provides a pharmaceutical formulation comprising an acridinone compound and a platinum anti-cancer drug formulated in a pharmaceutically acceptable excipient and suitable for use in humans to treat a head and neck cancer. The invention also relates to an anti- cancer pharmaceutical composition for prophylaxis or treatment of a head and neck cancer comprising an acridinone compound and a platinum anti-cancer drug. A pharmaceutical composition containing an acridinone compound, and optionally a chemotherapeutic agent, in particular a platinum anti-cancer drug, is useful especially as a preventive drug, a therapeutic drug or as a cell proliferation inhibitor of head and neck cancer. In an aspect, the invention relates to a two component pharmaceutical composition for treating a head and neck cancer in a subject comprising one or more daily doses of an acridinone compound together with a pharmaceutically acceptable carrier, in combination with one or more daily doses of a platinum anti-cancer drug together with a pharmaceutically acceptable carrier. The amount of platinum anti-cancer drug used in combination with an acridinone compound may be substantially less than in methods or preparations where the platinum anti-cancer drug is used alone. The invention also contemplates a pharmaceutical composition in separate containers and intended for simultaneous or sequential administration to prevent or treat a head and neck cancer comprising an acridinone compound and a chemotherapeutic agent, in particular a platinum anti-cancer drug, both optionally together with pharmaceutically acceptable carriers, excipients, or vehicles. The invention provides a cancer therapeutic formulation with reduced toxicity including an effective dose of an acridinone compound and an effective dose of a chemotherapeutic agent, in particular a platinum anti-cancer drug. The invention further contemplates a conjugate comprising an acridinone compound interacting with or linked to a chemotherapeutic agent, in particular a platinum anti-cancer drug. The invention further contemplates a single combination dosage unit comprising an acridinone compound and a chemotherapeutic agent, in particular a platinum anti-cancer drug. The invention also contemplates methods for preparing compositions of the invention that result in compositions to prevent or treat a head and neck cancer. In an aspect of the invention, a method is provided for preparing a pharmaceutical composition of an acridinone compound comprising preparing a composition comprising the acridinone compound and a pharmaceutically acceptable carrier, excipient, or vehicle. In another aspect of the invention, a method is provided for preparing a pharmaceutical composition of an acridinone compound and a chemotherapeutic agent, in particular a platinum anti-cancer drug, adapted to provide beneficial effects, following treatment, comprising preparing a composition comprising the acridinone compound, chemotherapeutic agent, and a pharmaceutically acceptable carrier, excipient, or vehicle. In another aspect of the invention, a method is provided for preparing a stable pharmaceutical composition of an acridinone compound comprising mixing an acridinone compound, a chemotherapeutic agent, in particular a platinum anti-cancer drug, and a pharmaceutically acceptable carrier, excipient, or vehicle effective to physically stabilize the acridinone compound. The invention provides a method for inhibiting, reducing or suppressing the growth or survival of head and neck cancer cells, in particular oral cancer cells, comprising contacting the cells with an effective amount of an acridinone compound, or composition of the invention. In another aspect, the invention provides a method of suppressing or preventing malignant transformation of head and neck cancer cells, in particular oral cancer cells, comprising contacting the head and neck cancer cells, in particular oral cancer cells, with an effective amount of an acridinone compound, or composition of the invention. In this embodiment, an acridinone compound, or composition is administered to head and neck cancer cells, in particular oral cancer cells, capable of malignant transformation, thereby suppressing or preventing transformation. The invention also provides a method of inducing apoptosis in transformed head and neck cancer cells, in particular oral cancer cells, comprising contacting the cells with an acridinone compound, or composition of the invention. In this embodiment, an acridinone compound, or composition of the invention is administered to transformed head and neck cancer cells, in particular oral cancer cells, thereby promoting apoptosis. The invention provides methods for treating a head and neck cancer in a patient in need of such treatment comprising administering to the patient an effective amount of an acridinone compound, or composition of the invention. In an aspect, the invention provides a method of treating a subject diagnosed with head and neck cancer, comprising delivering to the subject in need thereof, an acridinone compound, or composition of the invention. In a further aspect, the invention provides a method of treating a susceptible head and neck cancer in a mammal, comprising: administering to the mammal effective amounts of an acridinone compound, or composition of the invention. In a still further aspect, the invention relates to a method of inhibiting a head and neck cancer in a patient at risk for developing a head and neck cancer comprising administering to the patient an acridinone compound, or composition of the invention. In an aspect, the invention provides for the use of one or more daily doses for treating a head and neck cancer each dose comprising an effective amount of an acridinone compound, or composition of the invention. The invention provides a method for treating a head and neck cancer in a subject comprising administering to the subject effective amounts of an acridinone compound and monitoring the response to, or efficacy of the acridinone compound using PLK-1 and/or Syk as biomarkers of drug response. The invention provides a method of prophylactically treating a subject at risk of developing a head and neck cancer comprising the steps of: a) identifying a subject at risk of developing a head and neck cancer; and b) administering to the subject an amount of an acridinone compound, or composition of the invention effective to inhibit or delay onset of the head and neck cancer. A method of the invention may comprise one or more additional component or treatment, in particular a component or procedure for treating or preventing head and neck cancer. Thus, the invention provides a combination treatment for head and neck cancer comprising administering to a subject in need thereof an effective amount of an acridinone compound, and one or more additional component or treatment. In an aspect, the invention provides methods for treating head and neck cancer in a patient in need of such treatment comprising administering to the patient an effective amount of an acridinone compound, and one or more other component or treatment chosen or selected from the group consisting of a chemotherapeutic agent, a non-steroidal anti-inflammatory drug, immunotherapies such as vaccination with autologous tumor cells, vaccination against tumor-associated antigens, monoclonal antibodies against tumor antigens, gene therapy including gene correction, virus-directed enzyme prodrug treatment, matrix metalloproteinase inhibitors, radiation therapy or surgery. In an aspect, the additional component or treatment is a

chemotherapeutic agent, in particular a platinum anti-cancer drug. Similarly, the invention provides a use of the acridinone compounds described herein in the treatment of a cancer, such as a head and neck cancer in a patient. Such use may include the use of a chemotherapeutic agent, in particular a platinum anti-cancer drug, as described herein. The invention relates to a combination treatment for preventing or treating a head and neck cancer in a subject comprising administering to the subject a therapeutically effective amount of at least one acridinone compound and at least one platinum anti-cancer drug, or a composition, conjugate or dosage unit of the invention. In an aspect, the invention provides a method for the prevention and/or intervention of a head and neck cancer in a subject comprising administration of at least one acridinone compound and at least one platinum anti-cancer drug, or a dosage unit, composition or conjugate of the invention. In an aspect, the invention provides a method for the prevention and/or intervention of a head and neck cancer in a subject comprising administration of at least one acridinone compound and at least one platinum anti-cancer drug to a subject in need thereof to provide therapeutic effects. In another aspect, the invention provides a method for the prevention and/or intervention of a head and neck cancer in a subject comprising co-administering at least one acridinone compound and at least one chemotherapeutic agent, in particular a platinum anti- cancer drug, to a subject in need thereof. In an aspect the invention provides a method of substantially reducing dosage levels of a platinum anti-cancer drug for administration to a subject with a head and neck cancer which comprises administering a combination of at least one acridinone compound with the platinum anti-cancer drug. In a particular aspect the invention provides a method of substantially reducing dosage levels of a platinum anti-cancer drug for administration to a subject with head and neck cancer, with comprises administering a combination of one or more daily doses of at least one acridinone compound with one or more daily doses of the platinum anti-cancer drug. In an aspect the invention provides a method of delivering an effective dose of a platinum anti-cancer drug to a subject with head and neck cancer that reduces the toxic side effects of the platinum anti-cancer drug that includes the step of combining a platinum anti- cancer drug with an acridinone compound and administering the combination to the subject. The invention provides a method of increasing response to a chemotherapeutic agent for treating head and neck cancer, in a subject who is a poor responder to the chemotherapeutic agent, which comprises administering to the subject a therapeutically effective amount of a composition of the invention. In an aspect the invention provides a method of increasing response to a platinum anti-cancer drug in a subject with head and neck cancer who is a poor responder to the platinum anti-cancer drug, which comprises administering an acridinone compound in combination with the platinum anti-cancer drug. In a particular aspect the invention provides a method of increasing response to a platinum anti-cancer drug in a subject with head and neck cancer who is a poor responder to the platinum anti-cancer drug, which comprises administering a combination of one or more daily doses of at least one platinum anti-cancer drug in combination with one or more daily doses of at least one acridinone compound. The invention provides a method of delivering an effective amount of an acridinone compound to a subject with head and neck cancer that increases the sensitivity of head and neck cancer cells to chemotherapy (i.e. chemosensitizes), comprising combining the acridinone compound with a chemotherapeutic agent and administering the combination to the subject. In an aspect, the invention provides a method of delivering an effective amount of an acridinone compound to a subject with head and neck cancer that increases the sensitivity of head and neck cancer cells to platinum anti-cancer drug, comprising combining the acridinone compound with the platinum anti-cancer drug and administering the combination to the subject. In an embodiment, the invention provides a method of promoting treatment of patients having a head and neck cancer comprising packaging, labelling and/or marketing an acridinone compound alone or in combination with a platinum anti-cancer drug to be used in treating a patient having a head and neck cancer. In an aspect, the invention provides a method of promoting treatment of patients having head and neck cancer comprising packaging, labelling and/or marketing an acridinone compound to be used in conjoint therapy with a platinum anti-cancer drug for treating a patient having head and neck cancer. The invention contemplates the use of a composition comprising an acridinone compound for preventing or treating a head and neck cancer or in the preparation of one or more medicament for preventing or treating a head and neck cancer. In another aspect, the invention relates to the use of a composition comprising an acridinone compound for the preparation of a medicament which has a protracted profile of action in treating head and neck cancer. The invention also contemplates the use of a composition comprising a combination of at least one acridinone compound and at least one additional treatment, in particular a chemotherapeutic agent, more particularly a platinum anti-cancer drug, for the preparation of one or more medicament for preventing or treating head and neck cancer. The invention further contemplates use of an acridinone compound in combination with at least one additional treatment, in particular a chemotherapeutic agent, more particularly a platinum anti-cancer drug, for the manufacture of a medicament for the treatment of head and neck cancer. Still further the invention provides use of an acridinone compound for the

manufacture of a medicament for the treatment of head and neck cancer used in

combination with at least one additional treatment, in particular a chemotherapeutic agent, more particularly a platinum anti-cancer drug. In an aspect, the invention relates to the use of synergistically effective amounts of at least one acridinone compound and at least one additional treatment, in particular a chemotherapeutic agent, more particularly a platinum anti-cancer drug, for the preparation of a medicament for preventing or treating head and neck cancer. In another aspect, the invention relates to the use of an acridinone compound and at least one additional treatment, in particular a chemotherapeutic agent, more particularly a platinum anti-cancer drug, for the preparation of a medicament which has a protracted profile of action. The invention additionally provides uses of a pharmaceutical composition and a conjugate of the invention in the preparation of medicaments for the prevention and/or treatment of head and neck cancer. The invention further contemplates use of a composition comprising an acridinone compound in combination with a chemotherapeutic agent for the treatment of a head and neck cancer or for the manufacture of a medicament for the treatment of a head and neck cancer. The invention further provides for the use of one or more daily doses of a composition of the invention in combination with one or more daily doses of a

chemotherapeutic agent for treating a subject suffering from a head and neck cancer who is a poor responder to the chemotherapeutic agent. In an aspect, the invention relates to the use of additive or synergistically effective amounts of a composition comprising an acridinone compound, and a chemotherapeutic agent for the preparation of a medicament for preventing or treating a head and neck cancer. In an aspect of the invention, the chemotherapeutic agent is a platinum anti-cancer drug. In an aspect, the invention relates to the use of an acridinone compound and a platinum anti-cancer drug, or a composition, or conjugate of the invention for the prevention and/or treatment of head and neck cancer in a subject. In a particular aspect the invention provides for the use of one or more daily doses of a platinum anti-cancer drug for treating head and neck cancer, each dose comprising a therapeutically effective amount of an acridinone compound for inducing or augmenting antineoplastic activity of the platinum anti- cancer drug. The invention further relates to the use of one or more daily doses of an acridinone compound in combination with one or more daily doses of a chemotherapeutic agent, in particular a platinum anti-cancer drug, for treating head and neck cancer in a subject wherein the amount of chemotherapeutic agent, in particular platinum anti-cancer drug, is substantially reduced as compared with the use of the chemotherapeutic agent, in particular platinum anti-cancer drug, on its own. The invention further provides for the use of one or more daily doses of an acridinone compound in combination with one or more daily doses of a chemotherapeutic agent, in particular a platinum anti-cancer drug, for treating a subject who is a poor responder to the chemotherapeutic agent, in particular platinum anti-cancer drug. Since the present invention relates to a method of treatment and/or prevention involving compositions comprising an acridinone compound, the invention also provides a kit comprising a composition of the invention in kit form. In embodiments of the methods, conjugates, uses, kits and compositions of the invention, the acridinone compound is 10-(3-aminopropyl)-3,4-dimethyl-9(10H)-acridinone, or a pharmaceutically acceptable salt thereof. In aspects of the invention, a platinum anti-cancer drug used in the methods, compositions, uses, kits and conjugates of the invention is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, [cis-Pt(diaminocyclohexane)CI 2 ], nedaplatin, stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin, enloplatin, iproplatin, lobaplatin, ormaplatin, spiroplatin, and zeniplatin. In particular embodiments of the methods, conjugates, uses, kits and compositions of the invention, the acridinone compound is 10-(3-Aminopropyl)-3,4-dimethyl-9(10H)- acridinone maleate (referred to herein as "EM maleate" or "EMR") and the platinum anti- cancer drug is carboplatin. In particular embodiments of the methods, conjugates, uses, kits and compositions of the invention, the acridinone compound is 10-(3-Aminopropyl)-3,4- dimethyl-9(10H)-acridinone maleate and the platinum anti-cancer drug is cisplatin. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. ER maleate inhibited cell proliferation, survival, spheroid formation and colony formation in OSCC cells. HNSCC cell lines treated with different doses of ER maleate (0, 0.5, 1 , 2 μΜ) show a significant dose-dependent decrease in cell viability in each cell line. "Indicates p value less than 0.05. (A) Primary quantitative high throughput screening with B- scores analysis along with Z prime and Z factor of small molecule Kinase Inhibitor library. The line at B score of 0 (shown in red) represents the mean score and the lines above and below the B score of 0 (shown in yellow) show standard deviations. (B) The chemical structure of ER maleate. (C) Secondary screening was performed to select only the dose responsive hits. ER maleate showed cytotoxic effect using three doses (4 μΜ ("high dose"), 1.33 μΜ ("medium dose") and 0.44 μΜ ("low dose")) in SCC4, Cal33, HSC2 and MDA1986 cells from second round validation of these 48 inhibitors. ER maleate inhibited cell proliferation in a dose dependent manner (0-5 μΜ) in SCC4 cells (D) and Cal33 cells (E) by MTT assay. A similar inhibition of cell proliferation was observed in FADU (F), SCC25 (G), and SCC9 (H) cells on treatment with ER maleate. Cisplatin (0-10 μΜ) inhibited cell proliferation (I) and ER maleate (0-2 μΜ) enhanced Carboplatin (0-50 μΜ) inhibited cell proliferation (J) in SCC4 cells by MTT assay. (K) Spheroid formation. ER maleate incubation with SCC4 cells for 9 days decreased the cell density and size of spheroids in a dose dependent manner (0-2 μΜ) and failed to form the spheroid at 5 μΜ (lower panel).

Representative SCC4 cell spheroid is shown from each group (upper panel). Data are represented as mean +SEM relative to the control from three independent experiments. (L) Colony formation assay. SCC4 cells were treated with ER maleate (0.5 μΜ -1 μΜ), carboplatin (25 μΜ) or in combination of ER maleate (0.5 μΜ -1 μΜ) and carboplatin (25 μΜ) for 12 days. Colonies formed were stained and counted. Histogram analysis showed significant reduction in colony forming ability in ER maleate treated cells which were further reduced in combination treatment of ER maleate and carboplatin (lower panel).

Representative stained colonies were shown from each group (upper panel). Data are represented as mean +SEM relative to the control from three independent experiments. Treatment groups denoted by different letters represent a significant difference at p<0.05 (ANOVA followed by Fisher's LSD test). Figure 2. ER maleate inhibited cell invasion and migration potential, and modulated the expression of TIMP-MMPs in OSCC cells. (A) ER maleate significantly inhibited invasive capability of SCC4 cells in a dose dependent manner (0 - 2μΜ) after 24 h incubation by transwell invasion assay. Bar graphs show the decrease in invaded cell number with ER maleate treatment in a dose dependent manner. (B) ER maleate significantly suppressed cell migration to the wound area in SCC4 cells in comparison with vehicle control cells in 24 h by wound healing assays. Histogram analysis showing significantly low number of cells in wound of ER maleate treated cells. (C) ER maleate treatment decreased the expression of MMP-1 , MMP-10, MMP-12 and MMP-13, while TIMP-2 expression increased with no significant change in TIMP-1 at the mRNA level in SCC4 cells analyzed by illumine mRNA profiles. The bar graph data were presented as mean ± SEM; compared to the

corresponding control, * p < 0.05. Figure 3. ER maleate induced cell apoptosis in OSCC cells. (A) ER maleate (2 μΜ) showed significant increase in apoptosis in SCC4 by the Annexin-V and 7-ADD double staining assay. ER maleate induced early apoptosis in 1 1.16%, 10.41% and 7.21% cells, and late apoptosis in 1 1.08%, 44.21%, and 74.58% SCC4 cells at 24 h, 48 h and 72 h, respectively. (B) ER maleate also induced similar apoptotic effects in Cal 33 cells by the Annexin-V and 7-ADD double staining assay. Furthermore, carboplatin (CBP) treatment induced apoptotic cell population and this induction was further enhanced by combining with ER maleate in SCC4 cells (A) as well as in Cal33 cells (B). (C, D) ER maleate increased the expression of cleaved PARP, caspase 9 and caspase 3 in a dose dependent manner (0-2 μΜ), while the control group cells showed a single band of PARP, full length caspase 9 and caspase 3 in SCC4 (C) and Cal 33 cells (D) by western blot analysis. (E, F) ER maleate induced phosphorylated Bad level by western blot analysis (E) and Bad mRNA expression by illumine mRNA profiles (F) in SCC4 cells treated with ER maleate for 24 h. (G) The bar graph data were presented as mean ± SEM; Compared to the corresponding control, * p < 0.05. Figure 4. ER maleate arrested cell in G2 phase and induced polyploid population. (A) FACS analysis of SCC4 cells. ER maleate treatment decreased the diploid fraction (76.09%), whereas it increased polyploid population (23.91 %) in a dose dependent manner. For the diploid cells, cell population in G2 phase was increased from 22.38% to 50.98% and in G 1 phase decreased from 46.70% to 19.14% in a dose dependent manner (0 - 2 μΜ) after ER maleate treatment of SCC4 cells for 48 h through Modfit analysis. For the polyploid cell population, most cells (97.49%) accumulate in S phase. (B) FACS analysis of Cal33 cells. ER maleate decreased the diploid population (44.72%) and increased the polyploid population (55.28%) in Cal 33 cells. In Cal33 cells, diploid fraction was increased in S phase of cell cycle from 29.81 % to 39.14% and in polyploid cells, S phase increased (34.51 %) by ER maleate treatment (0-2 μΜ) for 48 h. G2 cell population was decreased from 10.47% to 5.18% in diploid population but increased from 0 to 6.66% in polyploid fraction. (C)

ImageStream FACS of SCC4 cells. Imagestream and Ideas program analysis showed ER maleate induced the tetraploid/anueploid (polyploid) cell population in SCC4 cells at 24 h and 48 h; (D) Imagestream nuclear morphology of SCC4. Cell nuclei stained with PI and run on Amnis Imagestream MKII reveal a significant increase in tetraploid/anueploid (polyploid) cell opulation in SCC4 cells; (E) ImageStream FACS of Cal33 cells. Imagestream and Ideas program analysis showed ER maleate induced the tetraploid/anueploid (polyploid) cell population in Cal33 cells; (F) Imagestream nuclear morphology of Cal33. Cell nuclei stained with PI and run on Amnis Imagestream MKII reveal a significant increase in

tetraploid/anueploid (polyploid) cell population in Cal33 cells. Figure 5. ER maleate inhibits PLK1 , Syk and PI3K/Akt signaling in OSCC cells. OSCC were treated with ER maleate (0-2 μΜ), or carboplatin (CBP, 25 μΜ) for 24 h, cell lysates were prepared for western blot analysis or total RNA prepared for real-time PCR quantification. The expression of PLK1 , Syk and EGFR kinase was decreased in a dose dependent manner in SCC4 (A) and Cal33 cells (B). A similar decreased expression was also observed in SCC9, SCC25 and FADU cells on treatment with ER maleate. Both phosphorylation level of Akt and the expression of total Akt were suppressed with ER maleate treatment in SCC4 (A) and Cal33 cells (B). Cell cycle regulatory molecule, Cyclin D1 , was also decreased at protein level by ER maleate in SCC4 (A) and Cal33 cells (B). GAPDH served as a loading control. Consistent with its change at protein level, illumina mRNA profiles showed Syk gene expression was suppressed at mRNA level by ER maleate in SCC4 and Cal33 cells (C). ER maleate down-regulated the gene expression of CHEK2 (D), PLK1 (E) at mRNA level, but not PLK4 expression (F). The bar graph data were presented as mean ± SEM; compared to the corresponding control, * p < 0.05. SCC4 and Cal33 cells were treated with ER maleate (2 μΜ) for a short period (5-120 min). ER maleate treatment reduced the levels of pAkt473 and pAkt308 in SCC4 (G) and Cal33 cells (H). Similarly, phosphorylated mTOR (pmTOR) was also decreased whereas pS6 increased in both cells (G, H). On contrary, the activity of phosphorylated ERK (pERK) was induced by ER maleate in both cells (G, H). β-actin served as a loading control. Figure 6. ER maleate anticancer potential in tumor xenograft mice model and its expression in human clinical tissues. (A) ER maleate inhibits growth of tumor xenografts in mice. Cal33 cells were injected in the right flank of 6 weeks old immunocompromised mice (NOD/SCID/crl). ER maleate treatment was started after 3 weeks when tumor xenografts volume was about 250 mm3 with doses ranging from 0.1 -3.0 mg/kg mice body weight for 10 weeks. Analysis of xenograft tumors from mice treated with ER maleate showed a dose dependent suppression of tumor growth within initial 6 weeks with an efficacious pharmacodynamic effect of complete inhibition of tumor growth at 1 mg/kg and 3mg/kg by the 10th week. From the 7th week, the group of mice pre-treated with 0.1 mg/kg ER maleate was treated with carboplatin at 75mg/kg and the group with ER maleate at 0.3mg/kg within first 6 weeks received a combination treatment of ER maleate (1 mg/kg) and carboplatin (75mg/kg) shown in the grey box. The combination treatment with ER maleate and carboplatin inhibited tumor growth in vivo from the 8th week; in comparison, inhibition of tumor growth by carboplatin alone was lesser than in combination with ER maleate. (B) Effect of ER maleate treatment on body weight of mice. Weekly measurements of mice body weight after Cal33 cell injection among different groups. (C) Hematoxylin and eosin stained liver, kidney and heart tissue sections. Histology of liver (I), kidney (II) and heart (III) tissues obtained at conclusion of the in vivo study. Hematoxylin-eosin (H&E) staining showed normal histology with no obvious signs of oncocytic necrosis or fibrosis observed in tissue sections (a-e) from different treatment groups. Original magnification is 400x. (D) Immunohistochemical analysis of Cyclin D1 , Syk and PLK1 in tumor xenografts in immunocompromised mice. Panel I shows H&E stained tumor tissue sections in untreated control mice (a) and the treatment groups (b-e). Panels II, III and IV show nuclear Cyclin D1 , Syk and PLK1 expression in untreated control mice (a); reduced Cyclin D1 , Syk and PLK1 expression in CBP (75mg/kg) treated tumors (b); combination of CBP (75mg/kg) and ER maleate (1 mg/kg) shows further reduction in Cyclin D1 , Syk and PLK1 (c); ER maleate treatment at 1 mg/kg and 3mg/kg show reduced Cyclin D1 , Syk and PLK1 expression in comparison with untreated controls (d and e), respectively. Original magnification is x400. (E) IHC analysis of Syk in OSCC. IHC studies showed no detectable expression of Syk in normal oral mucosa. In OSCC, both nuclear (N) and cytoplasmic (C) expression of Syk were increased. (F) IHC analysis of PLK1 in OSCC. IHC analysis showed no detectable expression of PLK1 in normal oral mucosa. In OSCC, both nuclear (N) and cytoplasmic (C) expression of PLK1 were increased. Original magnification 400x. (G) Kaplan-Meier survival analysis of Syk. OSCC patients showing cytoplasmic Syk overexpression (Syk positive, Cytoscore >4.56) followed up over a period of up to 140 months showed significant reduction in mean disease free survival (DFS) (DFS = 43 months) as compared to patients who did not show cytoplasmic Syk positivity (Syk negative, Cytoscore <3) (DFS = 89 months; p = 0.001). (H) Kaplan-Meier survival analysis of PLK1 . Survival analysis over a period of up to 100 months showed significant reduction in mean DFS in OSCC patients overexpressing nuclear PLK1 (PLK1 positive, Nucscore >3.5) (DFS = 58.7 months) as compared to patients who did not show nuclear PLK1 positivity (PLK1 negative, Nucscore <3.5) (DFS = 89.8 months; p = 0.004). Figure 7. Patient-derived OSCC cells treated with different doses of ER maleate (0, 0.25, 0.5, 1 , 2 μΜ) shows a significant dose-dependent decrease in cell viability in each cell line. Seven patient-derived oral cancer cells were obtained from tissues and successfully grown in culture. ER maleate treatment reduced cell viability in a dose-dependent manner in all patient cells with 60-80% decrease in cell viability with treatment of the highest dose. Figure 8. Expression of Plk1 and Syk in HNSCC cell lines treated with different doses of ER maleate for 48 hours. All cell lines show a dose-dependent decrease in Plk1 and a significant decrease in Syk at the highest dose of ER maleate treatment (2 μΜ). The effect of ER maleate treatment on head and neck cancer cell lines SCC4, SCC9, SCC25, Cal33, and FADU was determined on Plk1 and Syk expression. Western blotting revealed that ER maleate reduced the expression of Plk1 in all cell lines in a dose-dependent manner and significantly reduced Syk expression at the highest-dose of ER maleate (2uM) in all cell lines with the exception of SCC9 which had a baseline level of Syk that was too low for detection. Figure 9. Knockdown of Plkl and Syk resulted in cell death. FADU cells showed a 37.6% and 27.7% decrease in cell viability and Cal33 cells showed a 36.8 and 34.42% decrease in cell viability when treated with two different Plk1 siRNA oligomers. Knockdown of Plk1 using two siRNA oligos which target different regions of Plk1 mRNA resulted in a significant decrease in cell viability of FADU (37.6% and 27.7%) and Cal33 (36.87% and 34.41 %) cells after 48 hours. Figure 10. Western blots display knockdown of Plk1 expression using to siRNA oligomers, which in turn leads to decreased expression of p-PTEN, pAkt and Akt. Western blot confirmed the knockdown of Plk1 at the protein level and revealed that knockdown of Plk1 expression also led to the decrease in P-PTEN and P-Akt, suggesting Plk1 is an upstream modulator of the PI3K/Akt cell proliferation pathway. Figure 11. FADU cells showed a 18.5% and 31.5% decrease in cell viability with treatment with two different Syk siRNA oligomers. Cal33 cells showed a decrease in cell viability that was not significant with treatment with two Syk siRNA oligomers. Knockdown of Syk using two siRNA oligos which target different regions of Syk mRNA resulted in a significant decrease in cell viability only in FADU cells (18.5%, 31 .5%) after 48 hours. Figure 12. Western blots display the knockdown of Syk expression in SCC4, Fadu and Cal33 cells using two different Syk siRNA oligomers. Western blot confirmed the knockdown of Syk at the protein level. Figure 13. (A) Overexpression of Plk1 plasmid in FADU cells followed by treatment with ER maleate at different doses let to an approximately 20% rescue in cell viability at all doses of treatment. (B) Western blot confirms the overexpression of Plk1 in transfected cells compared to control. Figure 14. (A) Overexpression of Syk plasmid in FADU cells followed by treatment with ER maleate at different doses did not result in rescue of cell viability at all doses of treatment. (B) Western blot confirms the overexpression of Syk in transfected cells compared to control. Figure 15: Time-dependent AUC for Example 2. Figure 16: Kaplan-Meier survival curve using median cut-off value of 12. Figure 17: Recursive partitioning for biomarker risk score to identify the best potential cut-off. Figure 18: Kaplan-Meier survival curves and median survival times for Example 2. DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about." Further, it is to be understood that "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing "a compound" includes a mixture of two or more compounds. The term "about" means plus or minus 0.1 to 50%, 5-50%, or 10-40%, preferably 10-20%, more preferably 10% or 15%, of the number to which reference is being made. The terms "administering" and "administration" refer to the process by which a therapeutically effective amount of compounds or a composition contemplated herein are delivered to a subject for prevention and/or treatment purposes. Compositions are administered in accordance with good medical practices taking into account the subject's clinical condition, the site and method of administration, dosage, patient age, sex, body weight, and other factors known to physicians. The terms "subject" and "patient" are used interchangeably herein and refer to an animal including a warm-blooded animal such as a mammal, which is afflicted with or suspected of having or being pre-disposed to a head and neck cancer. Typical subjects for treatment include persons susceptible to, suffering from or that have suffered a head and neck cancer. Preferably, the terms refer to a human. The terms also include domestic animals bred for food, sport, or as pets, including horses, cows, sheep, poultry, fish, pigs, cats, dogs, and zoo animals. The methods herein for use on subjects and patients contemplate prophylactic as well as curative use. The term "pharmaceutically acceptable carrier, excipient, or vehicle" refers to a medium which does not interfere with the effectiveness or activity of an active ingredient and which is not toxic to the hosts to which it is administered. A carrier, excipient, or vehicle includes diluents, binders, adhesives, lubricants, disintegrates, bulking agents, wetting or emulsifying agents, pH buffering agents, and miscellaneous materials such as absorbants that may be needed in order to prepare a particular composition. The use of such media and agents for an active substance is well known in the art. In certain aspects of the invention, a carrier, excipient, or vehicle is selected to stabilize an acridinone compound and/or a platinum anti-cancer drug. Suitable pharmaceutical carriers, excipients, and vehicles are described in the standard text, Remington: The Science and Practice of Pharmacy,

[(formerly called Remington's Pharmaceutical Sciences), Editor: University of the Sciences in Philadelphia (USIP), 21 st Edition, May, 2005]. As used herein, the term "treat" or "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a head and neck cancer. Treatment may be administered to a subject who does not exhibit signs of a head and neck cancer and/or exhibits only early signs of a head and neck cancer for the purpose of decreasing the risk of developing pathology associated with the condition. Thus, depending on the state of the subject, the term in some aspects of the invention may refer to preventing a condition, and includes preventing the onset, or preventing the symptoms associated with a condition. The term also includes maintaining the condition and/or symptom such that the condition and/or symptom do not progress in severity. The term also refers to reducing the severity of the condition or symptoms associated with the condition prior to affliction with the condition. Such prevention or reduction of the severity of the condition prior to affliction refers to administration of a therapy to a subject that is not at the time of administration afflicted with the condition. Preventing also includes preventing the recurrence of a condition, or of one or more symptoms associated with such condition. The terms

"treatment" and "therapeutically" refer to the act of treating, as "treating" is defined above. The purpose of intervention is to combat the condition and includes the administration of therapy to prevent or delay the onset of the symptoms or complications, or alleviate the symptoms or complications, or eliminate the condition. For example, a treatment may be used to decrease the risk of a head and neck cancer or metastasis of a head and neck cancer A "beneficial effect" refers to an effect of a combination of an acridinone compound and a platinum anti-cancer drug, or dosage unit, composition or conjugate thereof that is greater than the effect of an acridinone compound alone. A beneficial effect includes favorable pharmacological and/or therapeutic effects, and improved pharmacokinetic properties and biological activity. A beneficial effect may be an additive effect or synergistic effect. In aspects of the invention, beneficial effects include but are not limited to reduced toxic effects and/or increased efficacy of an agent. In a particular aspect of the invention, the beneficial effect is a "sustained beneficial effect" where the beneficial effect is sustained for a prolonged period of time after termination of treatment. The period of time a beneficial effect is sustained may correlate with the duration and timing of the treatment. A subject may be treated continuously for about 1 to 3 days, 1 to 5 days, 1 to 7 days, 2 to 7 days, 1 to 2 weeks, 1 to 4 weeks, and 1 to 6 weeks, 2 to 16 weeks, 2 weeks to 6 months or periodically. The beneficial effect may be a statistically significant effect in terms of statistical analysis of an effect of the two agents versus the effects of an acridinone compound alone or platinum anti-cancer drug alone. "Statistically significant" effects or levels with two agents compared with each compound alone, in particular platinum anti-cancer agent alone, may represent effects or levels that are higher or lower than a standard. In embodiments of the invention, the difference may be 1 .5, 2, 3, 4, or 5 times higher or lower compared with the effect or levels obtained with each compound alone. An "additive effect" of an acridinone compound and a platinum anti-cancer drug refers to an effect that is equal to the sum of the effects of the two individual agents. A "synergistic effect" of an acridinone compound and a platinum anti-cancer drug refers to an effect that is greater than the additive effect which results from the sum of the effects of the two individual agents. "Combination treatment", "combination therapy", and "in combination" are used interchangeably herein and mean that the active ingredients are administered concurrently to a patient being treated. When administered in combination each component may be administered at the same time, or sequentially in any order at different points in time. Each component may be administered separately, but sufficiently close in time to provide a desired effect, in particular a beneficial, additive, or synergistic effect. The first agent may be administered in a regimen which additionally comprises treatment with the second agent. In certain embodiments, the term refers to administration of an acridinone compound and a platinum anti-cancer drug to a patient daily, within one week, one month, 6 months, or one year, including separate administration of two medicaments each containing one of the agents as well as simultaneous administration whether or not the two agents are combined in one formulation or whether they are two separate formulations. "Effective amount" or "therapeutically effective amount" relates to the amount or dose of active agents (i.e., acridinone compound or platinum anti-cancer drug), compositions or conjugates of the invention that will lead to one or more desired therapeutic effect or beneficial effect, in particular one or more sustained beneficial effects. A "therapeutically effective amount" can provide a dosage which is sufficient in order for a treatment of a subject to be effective compared with no treatment or a platinum anti-cancer drug alone. "Synergistically effective amount" relates to the amount of dose of active agents (i.e., acridinone compound and platinum anti-cancer drug), compositions or conjugates of the invention that will provide a synergistic effect, in particular a synergistic beneficial effect. A "subtherapeutic dose" refers to a dose of an active pharmaceutical agent that is functionally insufficient to elicit an intended pharmacological effect by itself, or that is quantitatively less than the established therapeutic dose for that agent. An established therapeutic dose may be published in a reference, for example, the Physicians' Desk Reference, 62nd Ed., 2008, Thomson Healthcare or Brunton, et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, 1 1th edition, 2006, McGraw-Hill Professional. In some aspects, a subtherapeutic dose is about 1%, 2%, 3%, 5%, 10%, 12%, 15%, 20%, or 25% of the amount of the agent conventionally administered. A subtherapeutic dose may also be an amount that provides blood levels of the agent which are lower, either systemically or locally, than that obtained when a conventional dose for the agent is administered. Thus, a subtherapeutic dose may be the result of administration of a lower than conventional dose of the agent or by a route or dosing regimen different from a conventional dosage or administration regimen. The terms "linked" or "interacting" refer to any physical association between molecules. The terms more particularly refer to a stable association between two molecules due to, for example, electrostatic, hydrophobic, ionic, hydrogen-bond interactions, or covalent interactions. Certain interacting or associated molecules interact only after one or more of them have been activated. "Head and neck cancer" refers to any neoplastic disorder, including cellular disorder, in the head and/or neck. A head and neck cancer can originate from head and neck cells, or it may originate from other organs that metastasize to the head and neck. The head and neck cancer may not be treatable by conventional chemotherapy and/or radiotherapy. Head and neck cancers typically begin in the squamous cells that line the moist, mucosal surfaces inside the head and neck (for example, inside the mouth, the nose, and the throat) or in the salivary glands. Squamous cell cancers are often referred to as head and neck squamous cell carcinoma (HNSCC). Head and neck cancers include, without limitation, cancers which begin in the oral cavity, pharynx, larynx, paranasal sinuses, nasal cavity and the salivary glands. Oral squamous cell carcinoma (OSCC) includes cancers in the oral cavity including the lips, the front two-thirds of the tongue, the gums, the lining inside the cheeks and lips, the floor (bottom) of the mouth under the tongue, the hard palate (bony top of the mouth), and the small area of the gum behind the wisdom teeth. In particular, head and neck cancers also include, without limitation, hypopharyngeal cancer, nasopharynx cancer, and oropharyngeal cancer in which malignant cells form in the tissues of the hypopharynx, nasopharynx, and oropharynx, respectively; laryngeal cancer in which malignant cells form in the tissues of the larynx; metastatic squamous neck cancer with occult primary treatment in which squamous cell cancer spreads to lymph nodes in the neck and it is not known where the cancer originates; paranasal sinus and nasal cavity cancer in which malignant cells form in the tissues of the paranasal sinuses and nasal cavity; and salivary gland cancer in which malignant cells form in the tissues of the salivary glands. The term "acridinone compound" refers to a compound having the following formula I:

R2 R3 or a pharmaceutically acceptable salt thereof, wherein:

- R1 and R2, which may be the same or different, are hydrogen, substituted or unsubstituted lower alkyl, -OR5, -OCOR6, or CF3, wherein R5 is hydrogen or substituted or unsubstituted lower alkyl, and R6 is substituted or unsubstituted lower alkyl;

- R3 is amino, alkylamino, -R7-NH2 or -R8-OH, wherein R7 and R8 are substituted or unsubstituted lower alkyl; and

- and R4 is H or substituted or unsubstituted lower alkyl. In one aspect, the acridinone is provided in the form of a pharmaceutical composition in combination with a pharmaceutically acceptable carrier, excipient, or vehicle.

In one aspect, R1 and R2 are lower alkyl, and R3 is aminoalkyl. In another aspect, R1 and R2 are lower alkyl, and R3 is -NR9 wherein R9 is lower alkyl which may optionally be substituted. In another aspect, R1 and R2 are the same lower alkyl and R3 is aminomethyl, aminopropyl or aminobutyl. In a particular aspect, R1 and R2 are methyl or ethyl and R3 is aminopropyl. In other particular aspects of the invention, the acridinone compound is 10-(3- aminopropyl)-3,4-dimethyl-9(10H)-acridinone, or a pharmaceutically acceptable salt thereof. More particularly, the acridinone compound is 10-(3-aminopropyl)-3,4-dimethyl-9(10H)- acridinone maleate (referred to herein as "ER maleate" or "ERM"). "Lower alkyl" refers to a straight or branched saturated hydrocarbon chain having 1 , 2, 3, 4, 5, or 6 carbon atoms, which may optionally be substituted. The term is exemplified by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, t-butyl, n-pentyl, n-hexyl, and the like. The term "amino" as used herein refers to groups of the form -NR6 wherein R6 is hydrogen or lower alkyl groups which may optionally be substituted. This term is exemplified by groups such as aminomethyl, aminoethyl, aminopropyl, aminobutyl and aminopentyl. "Pharmaceutically acceptable salt(s)," means a salt that is pharmaceutically acceptable and has the desired pharmacological properties, in particular it refers to those salts which are suitable for use in contact with the tissues of a subject or patient without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are described for example, in S. M. Berge, et al., J. Pharmaceutical Sciences, 1977, 66:1. Suitable salts include salts that may be formed where acidic protons in the compounds are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals, e.g. sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g. ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Suitable salts also include acid addition salts formed with inorganic acids (e.g. hydrochloride and hydrobromic acids) and organic acids (e.g. acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic acid and benezenesulfonic acid). In aspects of the invention, the pharmaceutically acceptable salts are formed with organic acids including acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid. In particular aspects of the invention, the pharmaceutically acceptable salts are formed with maleic acid, hydroxymaleic acid or methylmaleic acid, preferably maleic acid. Acridinone compounds may be evaluated for effectiveness in the treatment of head and neck cancer by a variety of methods, including, for example, evaluating the killing effects of an acridinone compound in head and neck cancer cell lines.

A "platinum anti-cancer drug" or "platinum cancer drug" refers to a class of compounds that comprise platinum complexes which react in vivo, binding to and causing crosslinking of DNA triggering apoptosis. Examples of platinum anti-cancer drugs for use in accordance with the invention include cisplatin, carboplatin, oxaliplatin, [cis- Pt(diaminocyclohexane)CI 2 ], nedaplatin, stratoplatin, paraplatin, platinol, cycloplatin, dexormaplatin, enloplatin, iproplatin, lobaplatin, ormaplatin, spiroplatin, and zeniplatin.

phenanthriplatin, pyriplatin. Of these, cisplatin and carboplatin are generally preferred. Compositions and Methods of Administration The invention relates to therapeutic and related uses of acridinone compounds to treat subjects suffering from a head and neck cancer. For administration to patients, the acridinone compound or its salt is provided, in one aspect of the invention, in

pharmaceutically acceptable form, e.g., as a preparation that is sterile-filtered, and substantially pyrogen-free. Desirably, the acridinone compound meets standards set by the various national bodies which regulate quality of pharmaceutical products. For therapeutic use, the chosen acridinone compound is formulated with a carrier, excipient or vehicle that is pharmaceutically acceptable and is appropriate for administering the compound to the subject by the chosen route of administration so as to deliver the compound to the subject. For use as injectables, the compounds may be administered in a vehicle such as distilled water or, more desirably, in saline, phosphate buffered saline or dextrose solution. As an alternative to injectable formulations, an acridinone compound may be formulated for administration to patients and delivered to the patient by other routes. Oral dosage forms, such as tablets, capsules and the like, can be formulated in accordance with standard pharmaceutical practice. By way of example, for oral administration in the form of a capsule or tablet, the active components can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, methyl cellulose, magnesium stearate, glucose, calcium, sulfate, dicalcium phosphate, mannitol, sorbital, and the like. For oral administration in a liquid form, the agents may be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Suitable binders (e.g. gelatin, starch, corn sweeteners, natural sugars including glucose, natural and synthetic gums, and waxes), lubricants (e.g. sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride), disintegrating agents (e.g. starch, methyl cellulose, agar, bentonite, and xanthan gum), flavoring agents, and coloring agents may also be combined in the compositions or components thereof. Formulations for parenteral administration of a composition of the invention may include aqueous solutions, syrups, aqueous or oil suspensions and emulsions with edible oil such as cottonseed oil, coconut oil or peanut oil. Dispersing or suspending agents that can be used for aqueous suspensions include synthetic or natural gums, such as tragacanth, alginate, acacia, dextran, sodium carboxymethylcellulose, gelatin, methylcellulose, and polyvinylpyrrolidone. Compositions for parenteral administration may include sterile aqueous or non- aqueous solvents, such as water, isotonic saline, isotonic glucose solution, buffer solution, or other solvents conveniently used for parenteral administration of therapeutically active agents. A composition intended for parenteral administration may also include conventional additives such as stabilizers, buffers, or preservatives, e.g. methylhydroxybenzoate or similar additives. Compositions, conjugates, and dosage units may be sterilized by, for example, filtration through a bacteria retaining filter, addition of sterilizing agents to the composition, irradiation of the composition, or heating the composition. Alternatively, the compositions, conjugates, and dosage units of the present invention may be provided as sterile solid preparations e.g. lyophilized powder, which are readily dissolved in sterile solvent immediately prior to use. After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labelled for treatment of an indicated condition. For administration of a composition of the invention, such labelling would include amount, frequency, and method of administration. In some aspects, an acridinone compound may be administered by a targeted drug delivery system. Preferably, the delivery systems may be employed for targeting drug delivery to the head and neck. Such drug delivery systems include covalent linkage compositions, polymer coated compositions, compositions embedded in matrices, time- released compositions, redox-sensitive polymer compositions, bioadhesive compositions, micropartical coating compositions, and osmotic delivery compositions. Those skilled in the art will appreciate the use of such compositions for the purposes of targeting delivery of the compounds of the present invention, or a salt or solvate thereof, to a specific site of the subject being treated. An acridinone compound may also be formulated as a slow release implantation device for extended and sustained administration of the compounds. Examples of such sustained release formulations include composites of bio-compatible polymers, such as poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like. The structure, selection and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including, A. Domb et al., Polymers for Advanced Technologies 3:279-292 (1992). Additional guidance in selecting and using polymers in pharmaceutical formulations can be found in the text by M. Chasin and R. Langer (eds.), "Biodegradable Polymers as Drug Delivery Systems", Vol. 45 of "Drugs and the Pharmaceutical Sciences", M. Dekker, New York, 1990. Liposomes may also be used to provide for the sustained release of an acridonine compound . Details concerning how to use and make liposomal formulations of drugs of interest can be found in, among other places, U.S. Pat. Nos. 4,921 ,706; 5,008,050; 4,921 ,706; 4,927,637; 4,452,747; 4,016,100;

4,31 1 ,712; 4,370,349; 4,372,949; 4,529,561 ; 5,009,956; 4,725,442; 4,737,323; and

4,920,016. An acridinone compound or compositions disclosed herein can be administered by any means that produce contact of the active agent(s) with the agent's sites of action in the body of a subject or patient to produce a desired effect. The compounds and compositions disclosed herein may be administered by conventional methods including without limitation orally, intranasally, by inhalation, intraperitoneally, subcutaneously, intramuscularly, transdermally, sublingually or intravenously. For combination treatments (e.g., acridinone compound and chemotherapeutic agent such as a platinum anti-cancer drug), the active ingredients can be administered simultaneously or sequentially and in any order at different points in time, to provide the desired effects. A acridinone compound or composition of the invention can be formulated for sustained release, for delivery locally or systemically. It lies within the capability of a skilled physician or veterinarian to select a form and route of administration that optimizes the effects of the compounds, compositions and combinations, and treatments of the present invention. The therapeutic dosing and regimen most appropriate for patient treatment will of course vary with the disease or disease stage to be treated, and according to the patient's weight and other parameters. The dosage sizes and dosing regimen most appropriate for human use may be guided by the results herein presented, and can be confirmed in properly designed clinical trials. An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject, including the size of the patient, the age of the patient, the general condition of the patient, the particular disease being treated, the severity of the disease, the presence of other drugs in the patient, the in vivo activity of the acridinone compound and the like. The trial dosages would be chosen after consideration of the results of animal studies and the clinical literature. It will be appreciated by the person of ordinary skill in the art that information such as binding constants and Ki derived from in vitro binding competition assays may also be used in calculating dosages.

Kits

The invention provides kits comprising or consisting essentially of an acridinone compounds and optionally a chemotherapeutic agent, in particular a platinum anti-cancer drug, or compositions described herein. The kit is a package that houses a container which contains agents (an acridinone compound and optionally a platinum anti-cancer drug), a composition or conjugate of the invention, and also houses instructions for administering the agents, conjugate or composition to a subject. In an aspect of the invention, a pharmaceutical pack or kit is provided comprising one or more containers filled with one or more of the ingredients of a composition of the invention. As there may be advantages to mixing a component of a composition of the invention and a pharmaceutically acceptable carrier, excipient or vehicle near the time of use, the invention encompasses kits in which components of the compositions are packaged separately. For example, the kit can contain an agent in a powdered or other dry form in, for example, a sterile vial or ampule and, in a separate container within the kit, a carrier, excipient, or vehicle, or a component of a carrier, excipient, or vehicle (in liquid or dry form). In an aspect, the kit can contain a component (e.g. an acridinone compound) in a dry form, typically as a powder, often in a lyophilized form in, for example, a sterile vial or ampule and, in a separate container within the kit, a carrier, excipient, or vehicle, or a component of a carrier, excipient, or vehicle. Alternatively, the kit may contain a component in the form of a concentrated solution that is diluted prior to administration. Any of the components described herein, any of the carriers, excipients or vehicles described herein, and any combination of components and carriers, excipients or vehicles can be included in a kit. A kit can include two or more of the agents e.g., an acridinone compound and a chemotherapeutic agent, in particular a platinum anti-cancer drug (in any sufficiently stable form). The components can be combined with a carrier, excipient, or vehicle or packaged separately. For example, a kit can contain an acridinone compound, and, in a separate container, a platinum anti-cancer drug. Optionally, a kit may also contain instructions for preparation or use (e.g. , written instructions printed on the outer container or on a leaflet placed therein) and one or more devices to aid the preparation of the solution and/or its administration to a patient [e.g. , one or a plurality of syringes, needles, filters, tape, tubing (e.g., tubing to facilitate intravenous administration) alcohol swabs and/or Band-Aids®]. Compositions that are more

concentrated than those administered to a subject can be prepared. Accordingly, such compositions can be included in the kits of the invention with, optionally, suitable materials (e.g. , water, saline, or other physiologically acceptable solutions) for dilution. Instructions included with the kit can include, where appropriate, instructions for dilution. In other embodiments, the kits of the invention can include pre-mixed compositions and instructions for solubilizing any precipitate that may have formed during shipping or storage. Kits containing solutions of one or more acridinone compound and a chemotherapeutic agent, in particular a platinum anti-cancer drug, and one or more carriers, excipients or vehicles may also contain any of the materials mentioned above (e.g., any device to aid in preparing the composition for administration or in the administration per se). The instructions in these kits may describe suitable indications (e.g. , a description of patients amenable to treatment) and instructions for administering the solution to a patient. The invention will be described in greater detail by way of a specific example. The following example is offered for illustrative purposes, and is not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results. Example 1 - ER Maleate in the Treatment of Head and Neck Cancer Head and neck squamous cell carcinoma (HNSCC) patients diagnosed in late stages have limited chemotherapeutic options, underscoring the great need for development of new anticancer agents. Using quantitative high-throughput screening, we identified ER maleate as a promising potential cytotoxic agent for oral cancer. ER maleate induced cell apoptosis, inhibited cell proliferation, colony formation and cell migration and invasion in oral cancer cells. Imagestream cell cycle analysis revealed ER maleate treatment of oral cancer cells resulted in cell cycle arrest in G2/M phase, blocked cell division and increased polyploid cell population, unraveling deregulation of cell division and cell death. Mechanistically, ER maleate decreased expression of polo-like kinase 1 (PLK1) and spleen tyrosine kinase (Syk), induced cleavage of poly ADP ribose polymerase (PARP), caspase 9 and caspase 3. Notably, ER maleate increased chemosensitivity to carboplatin by enhancing apoptosis and inhibiting proliferation of oral cancer cells. Importantly, ER maleate treatment significantly suppressed tumor growth and increased antitumor activity of carboplatin in tumor xenografts, underscoring its chemosensitization effects in vivo. ER maleate treated tumor xenografts showed reduced PLK1 and Syk expression and clinical investigations revealed

overexpression of PLK1 and Syk in oral SCC patients. Taken together, both in vitro and in vivo findings provide a strong rationale for pre-clinical efficacy of ER maleate as a novel anti- cancer therapeutic candidate and chemosensitizer of platinum drug for head and neck cancer management by inhibiting PLK1 and Syk expression. The following materials and methods were used in the study described in this Example. Cell Culture Human OSCC cell line, SCC4 was purchased from the American Type Culture Collection (ATCC, Manassas, VA), MDA1986 was a kind gift from MD Anderson Cancer Centre (Texas, USA) and HSC2 (JCRB0622) was obtained from Health Science Research Resources Bank, Japan (HSRRB). Cal33 is a HNSCC cell line established from a moderately differentiated squamous cell carcinoma of the tongue of a 69-year old male (23). All these cell lines were characterized using short tandem repeat polymorphism analysis and used within 10 passages. Cells were grown in monolayer cultures in Dulbecco's modified Eagles medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (Sigma, MO) as described earlier (24,25). Small molecule inhibitor libraries screening using High throughput assays Primary screening using six chemical libraries (5170 compounds) was performed at the Simple Modular Assay and Robotic Technology (SMART) Facility of the Lunenfeld Tanenbaum Research Institute (LTRI), Mount Sinai Hospital, Toronto, Canada. Cell cycle flow cytometry and Imagstream analysis Cell cycle analysis of SCC4 and Cal33 was performed using Gallios Flow Cytometer (Beckman Coulter, CA, USA) and cell cycle modeling was carried out with ModFit LT software (Verity Software House, ME) (25). In addition to the cell cycle analysis based on DNA content, image analysis features can be used to distinguish mitotic events from G2 cells based on nuclear morphology. Amnis' imaging flow cytometry combining high speed multispectral image acquisition and automated image analysis was run on ImageStreamX Mark II High resolution microscope and analyzed with IDEAS® Software (Amnis, EMD Millipore, WA). Cell viability assay and Annexin V assay Cell viability and proliferation were determined by MTT assay (Sigma, MO, USA). Apoptosis effect of ER maleate treated control in SCC4 and Cal33 cells was evaluated using Annexin V and AAD7 double staining as described earlier (25) using Gallios Flow Cytometer (Beckman Coulter, CA, USA). Colony formation assay and Hanging-drop culture for spheroid formation. The anchorage dependent clonogenicity assay was performed using SCC4 cells. Hanging-drop culture was performed using a method described previously (27). Briefly, SCC4 cells were incubated with different doses of ER maleate (0-5 μΜ) in suspension culture medium. 15 μΙ droplets, each containing 10,000 cells, were plated on the inner surface of a petri dish cover of a 10 cm petri dish. Approximately 20 droplets from each group were plated onto one cover. The covers were then inverted and placed on a dish containing 15 ml PBS. After 9 days of culture, spheroids were photographed and their size was measured using the ImageJ software, Cell invasion and migration assay The cell invasion assay was performed in a 24-well transwell plate (Costar™ Transwell™, Corning INC, NY) with 8 μΜ polyethylene terephthalate membrane filters separating the lower and upper culture chambers. Cell migration was performed using wound healing assay as described earlier (28). Western blot assay and mRNA profiling SCC4 and Cal33 cells were treated with ER maleate (0.5 μΜ - 2 μΜ). After 24 h - 48 h, protein lysates were prepared in RIPA lysis buffer for western blot assay described previously (29). Total RNA were extracted with Trizol (Invitrogen, CA, USA), two hundred nanograms of total RNA from each samples were used for lllumina mRNA profiling by following the protocol from Princess Margaret Genomics Centre (Toronto). RNAs were labelled using lllumina TotalPrep-96 RNA Amplification kit Lot # 1312037 (Ambion, CA, USA) as per amplification protocol. 750 ng of cRNA generated from each samples were hybridized onto one Human HT-12 V4 Beadchip by incubation at 58°C, with rotation speed 5 for 18 hours. The BeadChip was washed and stained as per lllumina protocol and scanned on the iScan (lllumina, CA, USA). The data files were quantified in GenomeStudio Version 201 1.1 (lllumina, CA, USA). All samples passed lllumina sample dependent and independent QC Metrics. Data was imported in GeneSpring v12.6.1 for analysis. During import, the data was normalized using a standard (for lllumina arrays) quantile normalization followed by a "per probe" median centred normalization. In vivo Mouse Xenograft Model A mouse xenograft model was developed by subcutaneously injecting 6 x 10 6 Cal33 cells in PBS in six weeks old immunocompromised NOD/SCID/Crl mice in the right flank for tumor development, according to a protocol approved by the Animal Ethics Committee of Mount Sinai Hospital in accordance with the Toronto Centre of Phenogenomics (TCP) guidelines. Mice with palpable tumors (250mm 3 ) were randomly assigned into 5 groups (4 mice/group), Group 1 (PBS control), Group 2 (ER maleate 0.1 mg/kg b. wt.); Group 3 (ER maleate 0.3mg/kg); Group 4 (ER maleate 1.0mg/kg) and Group 5 (ER maleate 2.0 mg/kg). The mice were injected with ER maleate intraperitoneally (i.p.) weekly for 6 weeks. During this period tumor volume and body weight were monitored weekly. Tumor volumes were measured using vernier callipers and calculated according to the formula: (the shortest diameter) 2 χ (the longest diameter) χ 0.5. At the end of the experiment (or earlier if tumors exceeded 20% body mass), mice were sacrificed. At the end of the study, blood was collected from saphenous vein of mice in each group for isolating serum and toxicology studies before euthanization as per TCP guidelines. Following euthanasia, tumors and organs were harvested and stored in 10% buffered formalin for preparation of formalin fixed paraffin embedded tissue sections for histological assessment using H & E staining or IHC analysis. Immunohistochemistry analysis Serial FFPE tissue sections (4 μηι thickness) of OSCCs and normal oral tissues from patients, or tumor xenografts from ER maleate treated and vehicle control group mice xenografts were deparaffinized in xylene, hydrated through graded alcohol series, antigen was retrieved by microwave treatment, endogenous peroxidase activity was blocked and non-specific binding was blocked using normal horse serum (10%) as described earlier (30). The sections were incubated with anti-Syk (1 :500 dilution) or anti-PLK1 antibodies (1 : 100) (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. Slides were incubated with biotinylated secondary antibody for 20 min, followed by VECTASTAIN Elite ABC reagent (Vector labs, Burlingame, CA) using diaminobenzidine as the chromogen. Slides were washed with Tris-buffered saline (TBS, 0.1 M, pH = 7.4), 3-5 times after every step. Sections were counterstained with Mayer's hematoxylin. In the negative control tissue sections, the primary antibody was replaced by isotypespecific non-immune mouse/rabbit IgG. The sections were evaluated by light microscopic examination. Immunohistochemically stained clinical cases were evaluated and scored as positive if epithelial cells showed immunopositivity in the nucleus and cytoplasms as described earlier (30). The slides were also scanned using Nanozoomer 2.0 (Hamamatsu Photonics K. K., Hamamatsu City, Japan). Visiopharm Integrator System software Ver. 4.6.3.857 (Visiopharm, Hoersholm, Denmark) was used for image quantitation. The list of antibodies used in this application is given in Table 1. Statistical analysis For statistical analysis data presented as mean + SEM were compared using the Student t-test or with one-way ANOVA when three or more groups were present using GraphPad Prism 6.0 and clinical data were analyzed by SPSS 22. Two-tailed Student t-test was applied for two-group comparison. A p-value < 0.05 was considered as statistically significant. Kaplan-Meier survival analysis was used to determine the prognostic value of Syk and PLK1 as markers for OSCC patients. The results of the study are set out below. ER maleate is a small molecule inhibitor identified in OSCC In search of novel anti-cancer agents, six chemical libraries (Prestwick, Spectrum, Kinase Inhibitors, Cell Signaling, Tocris and NIH) containing 5170 small molecule inhibitors were screened to identify anti-proliferative agents for OSCC cells (Personal

communications). Among 48 candidates identified, ER maleate emerged as a promising inhibitor in the kinase inhibitor library screen [Z score range 0.58- 0.79; coefficient of variation < 7%] (Figure 1 A, B). Second round verification of ER maleate using cytotoxicity assays with three-fold dilutions (4, 1.3 and 0.44 μΜ) in four OSCC cell lines (SCC4, Cal33, MDA1986 and HSC2) confirmed its cytotoxic potential (Figure 1 C). ER maleate inhibited cell proliferation, survival, spheroid formation and colony formation To determine the anti-proliferative effects of ER maleate in OSCC, cell based assays were performed by incubating SCC4 and Cal33 cells with ER maleate (0-5μΜ). A dose dependent decrease in cell viability with 50% inhibitory concentration (IC50) of 2 μΜ at 48 h and cell death in 95.54 % SCC4 cells at 5 μΜ (Figure 1 D ) and 90.10 % in Cal33 (Figure 1 E), was observed by MTT assay. An in vitro 3D culture model was used to examine the effect of ER maleate on oral spheroid formation. Incubation of SCC4 cells with ER maleate for 9 days decreased the cell density and size of spheroids in a dose dependent manner (0-2 μΜ) and failed to form the spheroids at 5 μΜ (Figure 1 H). Colony forming assay revealed ER maleate significantly suppressed the proliferation potential of OSCC cells in long term cultures (12 days) at doses of 0.5-1 μΜ (Figure 1 1). ER maleate inhibited cell invasion and migration potential in OSCC cells Transwell matrigel invasion assays showed ER maleate significantly inhibited invasive capability of SCC4 cells in a dose dependent manner (0 - 2μΜ) within 24h (Figure 2A). Similarly, wound healing assays revealed ER maleate significantly suppressed cell migration to the wound area in SCC4 cells in comparison with control cells in 24 h (Figure 2B), suggesting ER maleate inhibited cell migration. Matrix metalloproteinases (MMP) MMP1 , MMP10, MMP12 and MMP13 expression was decreased at mRNA level, while tissue inhibitor of metalloproteinase2 (TIMP2) expression increased with no significant change in TIMP1 (Figure 2C), suggesting inhibitory effect of ER maleate on invasion and migration potential in OSCC cells was mediated by modulation of expression of MMPs and TIMP2. ER maleate induced cell apoptosis ER maleate (2 μΜ) showed significant increase in apoptosis in SCC4 and Cal33 cells by Annexin-V and 7-ADD double staining assay (Figure 3A). ER maleate induced early apoptosis in 1 1.16%, 10.41 %, and 7.21 %, and late apoptosis in 1 1 .08%, 44.21 %, and 74.58% of SCC4 cells at 24 h, 48 h and 72 h respectively (Figure 3A) and similar apoptotic effect was observed in Cal33 cells (Figure 3B). ER maleate increased expression of cleaved PARP, caspase9 and caspase3 in a dose dependent manner (0-2 μΜ), while untreated control cells showed single band of these full length proteins confirming ER maleate induced apoptosis in SCC4 and Cal33 cells (Figure 3C, D). ER maleate induced phosphorylation of Bad, a pro-apoptotic protein, in SCC4 and Cal33 cells (Figure 3E, F) as well as Bad expression at the mRNA level in SCC4 and Cal33 cells treated with ER maleate for 24 h (Figure 3G) confirming apoptotic cell death. ER maleate blocked cell division and induced cell polyploid population To further characterize ER maleate induced anti-proliferative effects on cell cycle, flow cytometry (FACS) using propidium iodide (PI) staining was performed. Modfit analysis showed ER maleate decreased diploid cell fraction and increased polyploid population in a dose dependent manner (Figure 4A, Table 2). For diploid cells, cell population was increased in G2 phase (50.98%) and decreased in G1 phase (19.14%) in a dose dependent manner (0 - 2 μΜ) on ER maleate treatment of SCC4 cells for 48 h (Figure 4A). For polyploid cell population, most cells (97.49%) accumulated in S phase but did not continue the cell cycling. Similarly, ER maleate decreased diploid fraction and increased polyploid population in Cal33 cells (Figure 4B, Table 3). In both diploid and polyploid Cal33 cells, S phase fraction was also increased (Figure 4B). Imagestream analysis showed increase in cell size and number of polyploid cells with multiple nuclei, including tetraploid and anueploid cells in both SCC4 (Figure 4C, D) and Cal 33 cells (Figure 4E, F), providing image analysis based evidence that DNA synthesis and replication in oral cancer cells continued but cell division was inhibited, which eventually resulted in cell death. These observations consistently support ER maleate inhibited cell proliferation (Figure 1 D, E) and induced apoptosis in SCC4 and Cal33 cells (Figure 3A, B, C, and D). ER maleate inhibited PLK1 , Syk, and PI3K/Akt signaling To unravel potential molecular targets and the related signaling pathways involved in ER maleate action, western blot analysis was performed to evaluate its effect on expression of PLK1 , Syk, EGFR and PI3K/Akt and their downstream targets. The expression of PLK1 , Syk and EGFR was decreased in a dose dependent manner with ER maleate (0-2 μΜ) incubation of SCC4 and Cal33 cells for 24 h (Figure 5A, B). Furthermore, both the phosphorylation level of Akt (pAkt473) and expression of total Akt were suppressed, whereas pERK was induced, by ER maleate in SCC4 and Cal33 cells. Cell cycle regulator, Cyclin D1 was also decreased by ER maleate (Figure 5A, B). Consistent with its change at the protein level, Syk gene expression was suppressed at mRNA level by ER maleate in both cells (Figure 5C). ER maleate down-regulated CHEK2 mRNA expression (Figure 5D), which emerges as a tumor suppressor gene, as well as PLK1 mRNA level (Figure 5E), but not PLK4 expression (Figure 5F). ER maleate treatment reduced the levels of pAkt473 and pAkt308 in a time dependent manner in SCC4 and Cal33 cells respectively (Figure 5G, H). Phosphorylated mTOR (pmTOR) and ERK (pERK) were transiently induced then decreased by ER maleate in both cells (Figure 5G, H). ER maleate is a chemosensitizer of platinum drugs Cisplatin or carboplatin inhibited cell proliferation in SCC4 cells (Figure 1 F, G).

Notably, ER maleate in combination with carboplatin further reduced SCC4 cell viability and colony formation compared to treatment with ER maleate or carboplatin alone (Figure 1 G, I). Carboplatin further enhanced the ER maleate induced cell apoptosis (Figure 3A, B), and cleaved PARP, caspase9 and caspase3 in SCC4 and Cal33 cells (Figure 3C, D).

Importantly, ER maleate inhibited PLK1 , Syk and EGFR expression were further decreased on treatment with combination of ER maleate and carboplatin in both SCC4 and Cal33 cells (Figure 5A, B), suggesting PLK1 , Syk and EGFR are targets in carboplatin

chemosensitization by ER maleate. Downregulated pAkt and total Akt levels were further reduced while activated ERK activity was further induced by combination of ER maleate and carboplatin. The expression of downstream target Cyclin D1 , down-regulated by ER maleate, was further decreased by combination with carboplatin in SCC4 and Cal33 cells (Figure 5A, B). Together, ER maleate in combination with carboplatin further inhibited cell proliferation and induced cell death, suggesting its potential as chemosensitizer of platinum drugs in oral cancer cells. In vivo characterization of anticancer potential of ER maleate To characterize the anticancer potential of ER maleate in an oral cancer animal model and determine its tolerability and pharmacokinetic properties, an in vivo efficacy study was first performed by injecting Cal33 cells in immunocompromised mice (NOD/SCID/crl), and 3 weeks later followed by ER maleate treatment with doses ranging 0.1 -3.0 mg/kg mice body weight for 10 weeks. The tolerated dose for once-weekly administration of ER maleate was determined by the maximum dose that did not cause loss in body weight or other tolerability features (signs of illness, abnormal behavior including poor grooming, hunched posture, diarrhea, urine stains, and dehydration) in accordance with the Institutional humane endpoint guidelines. Doses within 0.1 - 3.0 mg/kg range were well tolerated in an oral tumor xenograft model. Analysis of xenograft tumors from mice treated with ER maleate (1 mg/kg and 3mg/kg) showed an efficacious pharmacodynamic effect of complete inhibition of tumor growth by the 10th week without any significant body weight loss (Figure 6A, B). After 6 weeks of ER maleate treatment, mice groups administered with a low dose of ER maleate (0.1 mg/kg and 0.3mg/kg) continued to show increases in tumor volume, so treatment was changed to examine the combined effects of ER maleate and carboplatin. From the 7th week, the group of mice pre-treated with 0.1 mg/kg ER maleate was treated with carboplatin at 75mg/kg, and the group receiving 0.3mg/kg ER maleate for the first 6 weeks received a combination of ER maleate (1 mg/kg) and carboplatin (75mg/kg). Notably, ER maleate and carboplatin combination inhibited tumor growth in vivo from the 8th week; carboplatin alone inhibited tumor growth marginally compared to the control group (Figure 6A). ER maleate at 0.1 -3.0mg/kg, carboplatin at 75mg/kg or their combination did not show any apparent toxicity to normal tissues by microscopic examination of hematoxylin-eosin (H&E) stained liver, kidney and heart tissues (Figure 6C), biochemistry and hematology analysis (Table 4, 5). Importantly, the findings showed the chemosensitizing effect of ER maleate on carboplatin in vivo supporting the in vitro data. Immunohistochemical (IHC) analysis of Cyclin D1 , Syk and PLK1 expression in tumor xenografts and image analysis based quantitation of

immunostained tissue sections using Visiopharm software showed 54%, 46% and 33% nuclear positivity in the ER maleate treated group (1 mg/kg), 21%, 31% and 15% in ER maleate treated group (3mg/kg), 5%, 21 % and 2.6% in group receiving a combination of carboplatin and ER maleate, 41 %, 35% and 19% in carboplatin treatment alone, and 82%, 92% and 77% in control untreated group respectively (Figure 6D). These in vivo studies provided evidence for downregulation of Cyclin D1 , Syk and PLK1 in response to ER maleate treatment in oral tumor xenografts in support of PLK1 and Syk being molecular targets of ER maleate in OSCC. The expression of Syk and PLK1 is increased in oral cancer patients To assess the clinical significance of Syk and PLK1 in oral cancer, nuclear (N) and cytoplasmic (C) expression levels of Syk and PLK1 protein were compared in normal oral mucosa and OSCCs. IHC studies revealed no detectable expression of Syk in normal oral mucosa (n = 16), while both nuclear (N) and cytoplasmic (C) Syk expression were observed in OSCC (n=32) (Figure 6E, Table 6). Kaplan-Meier survival analysis of follow up of these OSCC patients over a period of up to 140 months showed significant reduction in mean disease free survival (DFS) in patients overexpressing cytoplasmic Syk (DFS=43 months) as compared to patients who did not show cytoplasmic Syk positivity (DFS = 89 months; p = 0.001) (Figure 6G). Similarly, the nuclear overexpression of PLK1 was observed in OSCC (n=30) as compared to normal oral mucosa (n=16) (Figure 6F). Kaplan-Meier survival analysis also showed a significant reduction in mean DFS in patients with overexpression of the nuclear PLK1 (DFS=58.7 months) as compared to patients with lower expression of the nuclear PLK1 (DFS=89.8 months, p = 0.004) (Figure 6H, Table 7). The in vitro and in vivo studies demonstrated that ER maleate downregulated the expression of Syk and PLK1 , and Syk and PLK1 are useful as biomarkers for assessing the efficacy of ER maleate in patients. ER maleate inhibited cell proliferation and cell survival in oral cancer cells derived from patients with oral cancer. Seven patient-derived oral cancer cells were obtained from tissues and successfully grown in culture. ER maleate treatment reduced cell viability in a dose-dependent manner in all patient cells with 60-80% decrease in cell viability with treatment of the highest dose (Figure 7). ER maleate treatment reduced PLK1 and Syk expression in head and neck cancer cell lines. The effect of ER maleate treatment on head and neck cancer cell lines SCC4, SCC9, SCC25, Cal33, and FADU was determined on Plk1 and Syk expression. Western blotting revealed that ER maleate reduced the expression of Plk1 in all cell lines in a dose- dependent manner and significantly reduced Syk expression at the highest-dose of ER maleate (2uM) in all cell lines with the exception of SCC9 which had a baseline level of Syk that was too low for detection (Figure 8). Knockdown of Plk1 and Syk resulted in cell death. Knockdown of Plk1 using two siRNA oligos which target different regions of Plk1 mRNA resulted in a significant decrease in cell viability of FADU (37.6% and 27.7%) and Cal33 (36.87% and 34.41 %) cells after 48 hours (Figure 9). Western blot confirmed the knockdown of Plk1 at the protein level and revealed that knockdown of Plk1 expression also led to the decrease in P-PTEN and P-Akt, suggesting Plk1 is an upstream modulator of the PI3K/Akt cell proliferation pathway (Figure 10).

Knockdown of Syk using two siRNA oligos which target different regions of Syk mRNA resulted in a significant decrease in cell viability only in FADU cells (18.5%, 31.5%) after 48 hours (Figure 1 1). Western blot confirmed the knockdown of Syk at the protein level (Figure 12). Overexpression of Plk1 resulted in rescue of cell viability. FADU cells showed high transfection efficiency; Plk1 and Syk plasmids were transfected into FADU cells. Through selection of transfected cells with G418 antibiotic over a 1 -week period, stable Plk1 and Syk transfectants were successfully generated in FADU cells. After treating both the FADU cells with control vector and those overexpressing Plk1 , it was observed that overexpressing Plk1 in these cells rescued ~20-30% cell viability at all of the doses tested (Figure 13). However, the cells overexpressing Syk did not show any rescue in cell viability with ER maleate treatment when compared with the control cells (Figure 14) suggesting that Plk1 is more likely a downstream target of ER maleate compared to Syk. Discussion of results ER maleate was identified as a potent anti-proliferative agent for OSCC. The in vitro studies demonstrated novel effects of ER maleate treatment in oral cancer cells including G2/M arrest, blockade of cell division, aberrant mitosis and cells arrested as large polyploid cells. At the molecular level, downregulation of PLK1 and CHEK2 was observed in ER maleate treated cells that may account for deregulation of cell division. PLK1 is

overexpressed during cancer development and plays a critical role in cell division as a major cell cycle regulator controlling entry into mitosis and regulating the spindle checkpoint (21 ,22). ER maleate inhibited PLK1 expression in SCC4 and Cal33 cells, suggesting PLK1 inhibition might serve as a novel molecular target for ER maleate to mediate cell anti- proliferative effect, cell cycle arrest in G2 phase, increase in polyploidy/aneuploidy with an increase in cell size. As a consequence, the mitotic cell cycle checkpoint is affected and cell division is inhibited. Faulty chromosomal alignment and distribution may lead to cell tetraploidy/aneuploidy. Downregulation of PLK1 expression thus provides a potential mechanism for mitotic arrest and subsequent apoptosis in response to ER maleate in oral cancer cells. In support of these findings, shRNA mediated decreased cellular PLK1 levels resulted in increase of cells in G2/M phase, apoptosis and antiproliferative effects (31). Inhibition of PLK1 by siRNA in cancer cells in vitro has been shown to result in mitotic arrest and subsequent apoptosis demonstrating specific killing of cancer cells, while the normal cells survived (32). Interestingly, PLK1 is associated with PI3K and MAPK pathways, both of which were shown to be affected by ER maleate in OSCC. Hence, it is likely that PLK1 actively contributes to ER maleate driven oral cancer cell death. Importantly, ER maleate was shown to downregulate CHEK2 in OSCC and this finding is important as CHEK2 is a critical regulator of G2/M cell cycle checkpoint (33). ER maleate mediated decrease in CHEK2 expression may lead to interruption of its interaction with CHEK1 and inhibit their roles in maintenance of G2/M checkpoint, cell mitosis and cell cycle progression in OSCC cells thereby accounting for defective mitosis and polyploidy observed in OSCC cells. The findings of these studies provide more advanced knowledge on biochemical basis, targets, preclinical data and rationale supporting development of ER maleate for OSCC therapy. The studies showed that in addition to targeting Syk, PLK1 and CHEK2, ER maleate also modulates PI3K/Akt/mTOR, and MAPK signaling which are important pathways in OSCC. ER maleate induced cleavage of PARP suggested induction of apoptosis was mediated by the PARP pathway (34). Activation of cleaved caspase9 and caspase3 suggested involvement of the intrinsic mitochondrial pathway of apoptosis in ER maleate treated OSCC cells (35). ER maleate induced phosphorylation of pro-apoptotic BAD (pBAD1 12) and its altered mRNA level, further supported the apoptotic effect of ER maleate. To further gain insight into signaling of ER maleate induced cell apoptosis and death in OSCC cells, its effects on PI3K/Akt/mTOR pathway were investigated. PI3K/Akt/mTOR signaling regulates cellular proliferation, growth, survival, metabolism, motility and is often deregulated in several cancers including OSCC (36). Activated PI3K/Akt signaling also controls cell growth and proliferation via mTOR associated protein S6. the results revealed decrease in pAkt (s473 and s308) levels with no change in total Akt supporting our data on ER maleate induced apoptosis in OSCC cells. Akt pathway promotes cell survival and proliferation by inhibiting the pro-apoptotic activity of Bad (pBad-S1 12) and hence activation of caspase9. ER maleate modulated mTOR phosphorylation and its downstream molecule S6 phosphorylation (pS6) in SCC4 and Cal 33 cells, suggesting PI3K/Akt/mTOR pathway plays an important role in mediating ER maleate induced cell death. Extracellular MMPs play pivotal roles in oral cancer progression by promoting motility, invasion/metastasis of OSCC. Increased mRNA expression of MMP-9 is correlated with invasive behavior in UMSCC1 OSCC cells (37). Several studies have demonstrated correlation between elevated TIMP1 levels and diminished MMP9 activity and invasiveness and migration (38). However, no significant change of MMP9 and TIMP1 was observed in ER maleate treated SCC4 cells. However, ER maleate inhibition resulted in decreases in other MMPs, MMP-1 , -10, -12 and -13. TIMPs are natural inhibitors of MMPs. A parallel increase in TIMP2 expression on ER maleate treatment was observed, suggesting TIMP2 may have a role in regulation of ER maleate mediated regulation of MMPs. In support of these findings, upregulation of MMP-1 , -10, -12 and -13 has also been shown in oral tongue SCC HSC-2 and Ca9-22 cells by genome-wide transcriptomic profiling (39). Several studies have highlighted the dual functions of TIMP2 in regulating MMPs processing and inhibition of the active enzyme (40), and taken together with the findings of these studies, it may be concluded that TIMP2-MMPs contribute to ER maleate inhibited invasion and migration in SCC4 cells. In support of the in vitro data demonstrating efficiency of ER maleate as a novel drug for OSCC, in vivo mouse xenograft studies revealed markedly reduced tumor growth without any noticeable reduction in weight of ER maleate treated and untreated control animal groups. Moreover, the clinical chemistry profiles, hematology and organ function tests of ER maleate treated mice also did not show any apparent normal tissue toxicity. IHC analysis of ER maleate treated tumor tissue sections showed decreases in PLK1 , Syk and Cyclin D1 suggesting that the in vitro findings were reproduced in vivo as well. Taken together, these preclinical findings suggest that ER maleate has therapeutic effect on oral cancer in vivo. Chemoresistance is one of the main causes for treatment failure in advanced cancers, where local therapies are insufficient. Carboplatin disrupts microtubule dynamics and is known to invoke the mitotic checkpoint. Combinatorial studies using ER maleate and carboplatin provide the first evidence for synergistic activities in SCC4 and Cal33 cells in vitro as well as in suppressing tumor growth in a mice model in vivo. The combination with ER maleate induced apoptosis based on cleavage of PARP, caspases 3 and 9. ER maleate inhibition of PLK1 and CHEK2 has also been demonstrated to modulate the mitotic checkpoint. Thus the in vitro and in vivo studies underscore the chemosensitization potential of ER maleate in OSCC. The in vitro experimental findings demonstrate downregulation of Syk expression in response to ER maleate and in vivo treatment of oral tumor xenografts with ER maleate showed tumor regression and decrease in Syk and PLK1 expression suggesting Syk and PLK1 as plausible targets for ER maleate in oral cancer. Importantly, the clinical studies showed Syk and PLK1 overexpression in OSCC patients and its association with reduced disease free survival. Notably, IHC analysis of Syk and PLK1 overexpression in clinical samples and their association with reduced disease free survival of OSCC patients taken together with reduction in Syk and PLK1 expression in ER maleate treated oral xenografts as well as patient cells in vitro suggest their novel potential as intermediary biomarkers in oral cancer patients treated with ER maleate. In conclusion, the study provides in vitro and in vivo evidence for ER maleate being an anticancer agent for OSCC and chemosensitizer for platinum drugs, and Syk and PLK1 are its molecular targets. ER maleate mediated downregulation of Syk, PLK1 and CHEK2 may account for aberrant mitosis, cell arrested as large polyploid cells, blockade of cell division and cell death. The clinical data showing Syk and PLK1 overexpression in OSCC and its association with poor prognosis suggest ER maleate may be used for intervention in patients with Syk and PLK1 overexpressing OSCC to reduce the risk of disease recurrence, using Syk and PLK1 as intermediary biomarkers. Example 2: Syk and Plk1 as Prognostic biomarkers for Head and Neck Cancer As discussed above, Syk and Plk1 have been found by the present inventors to have unique expression characteristics in oral cancers. As such, their use as prognostic markers in head and neck cancers was studied. Statistical analysis All statistical analyses were carried out using R version 3.2.1 (http://www.r- project.org/). Univariate and multivariable Cox regression analyses were used to assess the prognostic value of biomarker's sub-cellular localizations alone and adjusted for clinical parameters (histology grade, perineural involvement, tumor size, and nodal status). The response was the time-to-event of recurrence, while the predictors are ordinal biomarker scores. Two-sided p values were calculated and p < 0.05 was considered to be significant. Harrell's c-statistic was used to summarize the overall discriminatory value of individual biomarkers as well. Stepwise variable selection was further used to acquire a panel of biomarkers' sub-cellular localizations from which a signature score was derived. A signature score is the linear combination of biomarker expressions using regression estimates as weights. Improvements by biomarkers signature score upon clinical parameters were assessed using nested multivariable Cox regression analyses and time-dependent Area Under the Curve (AUC) plots. This was done using a baseline model with clinical parameters alone, and an extended baseline model with both clinical parameters and biomarkers signature score (41) All cox proportional hazard models were fitted using rms package in R (41). Cox proportional hazards assumption was ensured via chi-squared test for goodness of fit on Schoenfeld residuals (42). Time-dependent AUC plots were done using riskset ROC R package (43). A median risk score value derived from test set was used to classify subjects into high and low-risk groups of recurrence and further verified in the validation set. Kaplan- Meier survival curves were used to assess survival time of participants in the high- and low- risk groups. Differences in survival times were assessed using log-rank test and median survival times. Analysis of Syk and PLk1 as Prognostic biomarkers for OSCC Immunohistochemical analysis of sub-cellular expression of Syk and PLK1 was carried out in 58 normal oral tissues and 101 OSCCs, correlated with clinic-pathological parameters and clinical outcome over 14 years to develop a risk model for prediction of recurrence free survival. The analyses for variations in expression levels of Syk and PLK1 in normal oral tissues and in OSCCs and the correlations of protein expressions with patients' demographic characteristics (age and gender) as well as clinical and pathological parameters (tumor site, histopathological grade (HP Grade), tumor (T) stage, nodal status and clinical stage) are are summarized in Table 8. The univariable Cox-regression analysis for nuclear, cytoplasmic and total expression of Syk and PLk1 is given in Table 9. Sensitivity analysis of biomarkers' prognostic value adjusted for age, gender, tumor size, and nodal status is given in Table 10. Individual prognostic value of the clinical factors is given in Table 1 1. Development of Biomarker risk score Full and final models were used to develop the Biomarker risk score (Table 12). Biomarkers signature score was calculated as a linear combination of nuclear PLK1 and cytoplasmic Syk, with regression estimates as weights (Score = 23.85 χ [nuclear PLK1] + 1.0 x [cytoplasmic Syk]) (Table 12). Biomarkers signature score was associated with time of recurrence [HR = 1.07 (95% CI = 1.02, 1.13); p = 0.0055], and achieved a discriminatory c- statistic value of 0.59 (Table 13). The biomarkers signature score was also found to hold a prognostic value adjusted for those clinical parameters, and does improve upon them. The reference baseline model achieved a discriminatory c-statistic of 0.6227. Adding the clinical parameters only marginally improved the discriminatory value to 0.6887(Table 14), suggesting a clinical value of these biomarkers signature score. Overall, the prognostic value of this biomarkers signature score adds improvements to the classical clinical parameters for assessing prognosis of OSCC patients. The time-dependent AUC plot of the baseline and improved baseline models confirmed that biomarkers together with clinical parameters (age, gender, histopathological grade, nodal status, tumor stage, and clinical stage) hold better overall discriminatory ability throughout time compared to the use of clinical parameters alone (Figure 15). A cut-off was derived as the median risk score to stratify subjects into high and low risk groups of recurrence (Score = 12.00). Kaplan-Meier survival analyses show that the high and low risk groups have significantly different survival times (log-rank test: p = 0.0164, Figure 16). The high risk group had a median survival time of 19.5 months. The low risk group in comparison did not reach a survival probability less than 50%. Recursive partitioning for biomarker risk score was also carried out to identify the best potential cut-off value of 14.37 (Figure 17). Kaplan-Meier survival analyses show that the high and low risk groups (based on cut-off 14.37) have significantly different survival times (log-rank test: p = 0.0005, Figure 18). The high risk group had a median survival time of 64 months. The low risk group in comparison did not reach a survival probability less than 50%. The present invention is not to be limited in scope by the specific embodiments described herein, since such embodiments are intended as but single illustrations of one aspect of the invention and any functionally equivalent embodiments are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. All publications, patents and patent applications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. The citation of any reference herein is not an admission that such reference is available as prior art to the instant invention.

Table 1 : List of antibodies

Table 2: ER maleate (ERM) effect on cell cycle in SCC4 cells using FACS and Modfit analysis. Dip Diploid G 1 phase; Dip G 2 : Diploid G 2 phase; Dip S: Diploid S phase; Anu G^ Polyploid G 1 phase; Anu G 2 : Polyploid G 2 phase; Anu S: Polyploid S phase

Table 3: ER maleate (ERM) effect on cell cycle in Cal33 cells using FACS and Modfit analysis. Dip G^ Diploid G 1 phase; Dip G 2 : Diploid G 2 phase; Dip S: Diploid S phase; Anu G^ Polyploid G 1 phase; Anu G 2 : Polyploid G 2 phase; Anu S: Polyploid S phase

Table 4 Clinical chemistry profiles among different mice groups with ER maleate or carboplatin treatment.

Table 5: Hematology test among different mice groups with ER maleate or carboplatin treatment.

Table 6: Clinicopathological characteristics of OSCC patients and Syk expression

oral tissues analyzed

Table 7: Clinicopathological characteristics of OSCC patients and PLK1 expression

*No detectable Syk expression was observed in 16 normal oral tissues analyzed Table 8: Immunohistochemical analysis of Syk and PLKl in normal oral tissues and OSCCs

Table 9: Univariable Cox-regression analysis:

Table 10: Sensitivity analysis of biomarkers' prognostic value adjusted for age, gender, tumor size, and nodal status

Table 11 : Individual prognostic value of clinical factors

Table 12: Development of biomarker risk score - Full and Final Model:

Full Model

AIC: 467.64

C-statistic: 0.6421

HR [95% CI] P-value

Nuclear Plk1 1.21 [0.95, 1.53] 0.1264

Cytoplasmic Plk1 1.06 [0.86, 1.31] 0.5956

Nuclear Syk 0.95 [0.78, 1.14] 0.5681

Cytoplasmic Syk 1.15 [0.94, 1.41] 0.1684

Final Model

AIC: 464.33

c-statistic: 0.6347

HR [95% CI] P-value

Nuclear Plk1 1.27 [1.05, 1.54] 0.0140 *

Cytoplasmic Syk 1.10 [0.96, 1.26] 0.1562 ' Table 13: Prognostic value of biomarker risk score Biomarker Risk Score:

Score = 23.85 X [Nuclear Plk1] + 1.0 X [Cytoplasmic Syk] Table 14: Baseline Model and Improved Model

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