WO2017201573A1

WO2017201573A1 - A method of classification and treatment

Info

Publication number: WO2017201573A1
Application number: PCT/AU2017/050482
Authority: WO
Inventors: Richard Pearson; Ross Duncan HANNAN; Elaine SANIJ; Karen Elizabeth SHEPPARD; Katherine Margaret HANNAN; Keefe Thomas CHAN; Nadine HEIN; Jaclyn Elizabeth QUIN; Gretchen POORTINGA
Original assignee: Peter Maccallum Cancer Institute
Priority date: 2016-05-23
Filing date: 2017-05-23
Publication date: 2017-11-30

Abstract

The present disclosure relates generally to methods of treatment and the classification of subjects that will respond to such methods of treatment. In particular, the present disclosure provides a means to classify subjects who will respond to RNA Polymerase I (Pol I) inhibitor therapy and the treatment of subjects with a Pol I inhibitor or a treatment regimen comprising a Pol I inhibitor.

Description

A METHOD OF CLASSIFICATION AND TREATMENT FIELD OF THE INVENTION

[0001] The present invention relates generally to methods of treatment and the classification of subjects that will respond to such methods of treatment. In particular, the present invention provides a means to classify subjects who will respond to RNA Polymerase I (Pol I) inhibitor therapy and the treatment of subjects with a Pol I inhibitor or a treatment regimen comprising a Pol I inhibitor.

BACKGROUND OF THE INVENTION

[0002] Ovarian cancer is associated with the highest mortality rate of all gynecologic malignancies. This high mortality rate is commonly attributed to diagnosis of advanced stage disease. Current treatment for ovarian cancer involves cytoreductive surgery combined with platinum-based chemotherapy. In most cases, this treatment regimen is initially successful, with approximately 80% of patients responding to first-line treatment. However, the majority of patients relapse within 12 months and currently there are no curative treatments for recurrent disease. Accordingly, there is a need to identify new therapeutic strategies to improve the outcomes for patients diagnosed with this disease.

[0003] Targeted therapeutics directly interfere with the biological events that drive tumorigenesis. Unlike traditional chemotherapeutics, which exert their effect on rapidly proliferating cells, targeted therapeutics are designed to target the molecular characteristics that distinguish malignant cell populations from normal cells. Accordingly, inherent to the development of such targeted therapeutics is the identification of molecular defects or characteristics that are unique to tumour cells.

[0004] MYC is a potent oncogene whose deregulated expression plays a significant role in the development of ovarian cancer. The abnormal expression and activity of MYC has been shown to regulate cell growth, proliferation and survival. Furthermore, MYC has also been demonstrated to transcriptionally upregulate components involved in ribosome biogenesis, including the direct binding to ribosomal RNA (rRNA) genes, which hyper-activates the synthesis of rRNA to drive cell growth (Poortinga et al. 2015, Oncogene, 34:403-412). Inhibition of rRNA synthesis using a specific Pol I inhibitor is associated with significant therapeutic benefit in the treatment of MYC driven hematologic tumors (Bywater et al. 2012, Cancer Cell, 22: 51-65). Treatment with a Pol I inhibitor has also been demonstrated to induce autophagy and senescence among solid tumour cell lines (Drygin et al. 2011, Cancer Research, 71(4): 1418-1430). Additionally, studies with human solid tumors grown in murine xenograft models have shown that oral administration of Pol I inhibitor results in favorable pharmacokinetics and anti-tumor efficacy without changes in body weight or overt toxicity (Drygin et al. 2011, Cancer Research, 71(4): 1418-1430). However, many tumors do not respond to treatment, and of those that do respond, a number of subjects will eventually relapse and develop acquired resistance to Pol I inhibitor therapy.

[0005] Accordingly, there is an ongoing need to develop methods for the effective classification of subjects who will respond to Pol I inhibitor therapy to ensure that patients are best suited for certain treatments or treatment regimens.

SUMMARY OF THE INVENTION

[0006] The present inventors have identified a gene expression signature that classifies ovarian cancer subjects as sensitive to RNA Polymerase I (Pol Γ) inhibitor therapy. In particular, the present invention provides a BRCA1 mutation gene signature that is predictive of sensitivity to Pol I inhibitor therapy. The present invention further provides methods and kits useful for obtaining and utilizing gene expression information for the genes identified herein, to classify ovarian cancer subjects as sensitive to Pol I inhibitor therapy and thereafter, to treat subjects with Pol I inhibitor or a treatment regimen comprising a Pol I inhibitor.

[0007] Accordingly, in one aspect, the present invention provides a method of classifying a subject with ovarian cancer comprising:

analysing the gene expression of a panel of genes according to Table 1 below by Gene Set Enrichment Analysis (GSEA); and

classifying the subject as sensitive to RNA Polymerase I (Pol I) inhibitor therapy, wherein the Enrichment Score (ES) reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 10 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy. Table 1: Gene signature for the prediction of sensitivity to CX-5461

[0008] Beneficially, the classification of subjects as sensitive to Pol I inhibitor therapy allows for the stratification of patients that will benefit from treatment with a Pol I inhibitor or a treatment regimen comprising a Pol I inhibitor. Patient stratification results in the avoidance of overtreatment or under treatment of subjects with ovarian cancer and also minimizes the potential adverse side effects associated with the administration of targeted therapeutics.

[0009] In a second aspect, the present invention provides a method for treating a subject with ovarian cancer comprising:

classifying the subject according to the method of the present invention;

selecting RNA Polymerase I (Pol I) inhibitor therapy for a subject classified as sensitive to Pol I inhibitor therapy, wherein the Enrichment Score (ES) reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 10 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy; and

treating the subject classified as sensitive to Pol I inhibitor therapy with an effective amount of a Pol I inhibitor.

[0010] In a third aspect, the present invention provides a method for treating a subject with ovarian cancer comprising:

classifying the subject according to the method of the present invention;

selecting a treatment regimen comprising a RNA polymerase I (Pol I) inhibitor for a subject classified as sensitive to RNA Polymerase I (Pol I) inhibitor therapy, wherein the Enrichment Score (ES) reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 10 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy; and treating the subject classified as sensitive to Pol I inhibitor therapy with a treatment regimen comprising an effective amount of a Pol I inhibitor.

[0011] In a fourth aspect, the present invention provides a kit for classifying a subject with ovarian cancer comprising detection agents capable of detecting the expression products of the panel of genes according to the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a graphical representation of in vitro sensitivity of ovarian cancer cell lines (x-axis) to CX-5461 GI50 (nM; y-axis).

[0013] Figure 2 shows the ovarian cancer cell lines that are sensitive to CX-5461 undergo cell death and ovarian cancer cell lines that are resistant to CX-5461 undergo senescence. (A) A graphical representation of in vitro cell death in ovarian cancer cell lines that are sensitive to CX-5461 (x-axis) according to PI staining (% Cells; y-axis). (B) A photographic representation of cellular senescence according to β- galactosidase staining (blue). D API-stained nuclei are shown in grey.

[0014] Figure 3 shows the mutations and protein components of the PI3K/mTOR and RAS/ERK pathways. (A) A graphical representation of cell lines with activating mutations in PIK3A. (B) A graphical representation of a heat map showing expression of PTEN proteins and phosphorylated proteins in exponentially growing cells. The levels of PTEN protein, P-PRAS, P-rpS6 and P-ERKl/2 were quantitated from immunoblots. Numbers and colors represent percent of human ovarian surface epithelial (HOSE) cell expression and are the average of at least two biological repeats.

[0015] Figure 4 shows that administration of CX-5461 and PF502 is effective for the treatment of established ovarian tumours. (A) A graphical representation of tumour volume (mm³; y-axis) against time (day; x-axis) for mice treated with CX-5461 (red line) or vehicle (black line). (B) A graphical representation of tumour volume (mm³; y-axis) against time (day; x-axis) for mice treated with PF502 (red line) or vehicle (black line).

[0016] Figure 5 shows a heat map of gene expression for gene signature that is predictive of sensitivity to CX-5461. A graphical representation of gene expression for sensitive (orange) and resistant (grey) ovarian cancer cell lines, wherein high expression is shown in red and low expression is shown in blue.

[0017] Figure 6 is a graphical representation of a BRCA1 gene expression signature that is predictive of sensitivity to CX-5461.

[0018] Figure 7 shows that administration of a combination of CX-5461 and PF502 synergizes to improve survival in vivo. A graphical representation of survival (%; y-axis) against time (days post-transplant; x-axis) of C57B1/6 mice transplanted with EuMyc B- cell lymphoma cells treated with CX-5461 (black line); everlolimus (yellow line); a combination of CX-5461 and everlolimus (green line) and vehicle (blue line).

[0019] Figure 8 shows that administration of a combination of CX-5461 and DDR inhibitors synergizes to improve in vitro cell survival. (A) A graphical representation of the synergistic effects of siRNA knockdown of HR genes (y-axis) in combination with CX-5461 in resistant ovarian cancer cell lines according to combination index (y-axis). (B) A graphical representation of cell viability (% of untreated control; y-axis) against concentration of BMN673 (logioM; y-axis). (C) A graphical representation of synergistic effect of CX-5461 (pink line) and PARP (blue line) inhibition alone and in combination (light blue line) according to confluence (%; y-axis) against time (hours; x-axis). (D) A graphical representation of cell death (% cells PI +ve; y-axis) in cells treated with vehicle, CX-5461 and BMN673, either alone or in combination.

[0020] Figure 9 shows that administration of a combination of CX-5461 and DDR inhibitors is effective for the treatment of established ovarian tumors in vivo. (A) A graphical representation of time (day; x-axis) against tumour volume (mm³; y-axis). (B) A graphical representation of time (day; x-axis) against tumour volume ratio (y-axis).

[0021] Figure 10 shows that administration of a combination of CX-5461 and HDAC inhibitor cooperates to improve in vitro cell survival in OVCAR4 ovarian cancer cells.

(A) A graphical representation of the cooperative effect of BML-281 and CX-5461 in combination (yellow line) compared with BML-210 inhibition alone (blue line) according to cell number (y-axis) against dose (-logio(concentration); x-axis). (B) A graphical representation of the cooperative effect of Vorinostat and CX-541 in combination (yellow line) compared with Vorinistat inhibition alone (blue line) according to cell number (y- axis) against dose (-log₁₀(concentration); x-axis).

[0022] Figure 11 shows that administration of CX-5461 attenuates DNA damage response. (A) A photographic representation of BJ-T cells treated with either vehicle or CX-5461 and exposed to 250 j/m² UV irradiation. (B) A graphical representation of comet tails (extent tail moment; y-axis) normalized to vehicle control.

[0023] Figure 12 shows that treatment with CX-5461 activates CHK1 and CHK2. A photographic representation of a Western Blot analysis of sensitive and resistant ovarian cancer cell lines treated with CX-5461 or vehicle.

[0024] Figure 13 shows that treatment with CX-5461 upregulates pro-inflammatory cytokines in resistant ovarian cancer cell lines. A graphical representation of gene expression (fold change in mRNA expression; y-axis) and time (days; x-axis) in the resistant OV90 cell line.

DETAILED DESCRIPTION

[0025] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers by not the exclusion of any other element or integer or group of elements or integers.

[0026] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgement or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0027] It is to be understood that unless otherwise indicated, the subject invention is not limited to specific manufacturing methods, formulation components, dosage regimens, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. [0028] Unless otherwise indicated the biochemical, cell culture and immunological techniques utilised in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0029] All publications mentioned in this specification are herein incorporated by reference in their entirety.

[0030] It must be noted that, as used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a single agent, as well as two or more agents; reference to "the disclosure" includes single and multiple aspects taught by the disclosure; and so forth. Aspects taught and enabled herein are encompassed by the term "invention". All aspects are enabled within the width of the present invention.

Ovarian cancer

[0031] Ovarian cancer is associated with the highest mortality rate of all gynecologic malignancies. This high mortality rate is commonly attributed to diagnosis of advanced stage disease. Current treatment for ovarian cancer involves cytoreductive surgery combined with platinum-based chemotherapy. In most cases, this treatment regimen is initially successful, with approximately 80% of patients responding to first-line treatment. However, the majority of patients relapse within 12 months and currently there are no curative treatments for recurrent disease. [0032] Ovarian cancer is largely asymptomatic, which often results in the detection of the tumour once the disease has already progressed to an advanced stage. The presence of ovarian cancer is typically confirmed by microscopic analysis of ovarian tissue obtained by surgical biopsy. Other tests that can be performed to confirm the presence of ovarian cancer include, but are not limited to, physical examination, blood tests, ultrasound and other imaging tests.

[0033] Histologically, there are four major subtypes of ovarian cancer: (i) serous; (ii) mucinous; (iii) endometrioid; and (iv) clear cell. The most predominant subtype of ovarian cancer is high-grade serous ovarian cancer, which accounts for -70% of deaths related to ovarian cancer.

[0034] Ovarian cancer may also be classified according to molecular subtypes. For example, gene expression profiling has identified four common molecular subtypes of high-grade serous ovarian cancer: (i) immunoreactive; (ii) differentiated; (iii) proliferative; and (iv) mesenchymal.

[0035] Throughout this specification, the term "ovarian cancer" refers to all histological and molecular subtypes of ovarian cancer.

[0036] In an embodiment, the ovarian cancer contemplated by the present invention is epithelial ovarian cancer. In another embodiment, the ovarian cancer is high-grade serous ovarian cancer.

[0037] If cancer cells are found in a tissue sample, an assessment is usually undertaken to determine the stage, or extent, of the disease, with respect to size and spread of the ovarian cancer. The FIGO system is often employed for this purpose, where stage I, Π, ΠΙ or IV is assigned to the ovarian cancer. Stage I is the earliest stage of ovarian cancer and limited to in situ tumours with no detectable cancer cells in the peritoneal cavity or metastases at distant sites. Stage I tumours are classified into six distinct sub-stages (IA, IB, IC, IC1, IC2 and IC3), which are defined according to the detection of cancer cells on one or both of the ovaries and the presence of positive ascites. Stage Π tumours are characterised by pelvic extension of the tumour or primary peritoneal tumours, which involve one or both ovaries. Stage Π tumours are classified into two distinct sub-stages, stage ΠΑ have detectable tumours on the uterus or fallopian tubes; and stage IIB have detectable tumours elsewhere in the pelvis. Stage ΙΠ tumours are classified into seven distinct sub-stages, stage ΙΠΑ tumours have metastases present in regional lymph nodes or extrapelvic regions; stage ΙΠΑ1 tumours have metastases limited to regional lymph nodes, stage ΙΠΑ1(ί) have metastases less than 10 mm in diameter and stage IIIAl(ii) have metastases greater than 10 mm in diameter; stage ΠΙΑ2 have microscopic metastases in the peritoneum; stage ΙΠΒ have metastases present in the peritoneum less than 2 cm in diameter, regardless of regional lymph node status or at distance sites; and stage niC have metastases present in the peritoneum greater than 2 cm in diameter, regardless of regional lymph node status or at distance sites. Finally, Stage IV tumours represent the most advanced stage of disease and are characterised by metastases present in regional lymph nodes and distant sites; stage IVA have pleural effusion containing cancer cells and stage IVB have metastasis to distance sites.

Treatment of ovarian cancer

[0038] The therapeutic regimen for the treatment of ovarian cancer can be determined by a person skilled in the art and will typically depend on factors including, but not limited to, the age, weight, menopausal status and general health of the subject in addition to the type, size, stage, molecular characteristics of the ovarian cancer. Another determinative factor may be the stage of the ovarian cancer. Accordingly, for a subject identified as having a more advanced stage of ovarian cancer, for example, stage ΠΙ or IV disease, a more aggressive therapeutic regimen may be prescribed as compared, for example, for a subject that has a less advanced stage of ovarian cancer.

[0039] Reference to the terms "treat", "treatment" and "treating" refer to any and all uses, which remedy a condition or symptom, or otherwise prevent, hinder, retard, abrogate or reverse the progression of a condition or disease or other undesirable symptoms in any way whatsoever. Thus, the terms "treating" and the like are to be considered in their broadest possible context. For example, treatment does not necessarily imply that a subject is treated until total recovery or cure. In conditions that display or are characterised by multiple symptoms, the treatment need not necessarily remedy, prevent, hinder, retard, abrogate or reverse all of said symptoms, but may remedy, hinder, retard, abrogate or reverse one or more of said symptoms. In the context of ovarian cancer, the agents, uses, methods and protocols of the present disclosure that involve treatment may prevent, reduce, ameliorate or otherwise delay the progression of ovarian cancer following resection.

[0040] The term "inhibiting" and variations thereof, such as "inhibition" and "inhibits", as used herein, do not necessarily imply the complete inhibition of the specified event, activity or function. Rather, the inhibition may be to an extent, and/or for a time, sufficient to produce the desired effect. Inhibition may be retardation, reduction, abrogation or otherwise hindrance of an event, activity or function. Such inhibition may be in magnitude and/or be temporal in nature.

[0041] Suitable therapeutic regimens would be known to persons skilled in the art, in the context of ovarian cancer, most subjects will have surgery to remove the cancerous tissue, followed by a course of chemotherapy.

[0042] Reference to "surgery" includes cytoreductive surgery or debulking. For example, initial cytoreductive surgery by laparotomy is performed to debulk the tumour mass. This generally includes a total hysterectomy, bilateral salpingo-oophorectomy, omentectomy and the removal of any visible cancer within the abdomen.

[0043] Reference to "chemotherapy" means any agent that is administered to inhibit the growth of cancer cells or induce cancer cell death. Chemotherapeutic treatments for ovarian cancer include, but are not limited to, actinomycin-D, bleomycin, carboplatin, cyclophosphamide, cisplatin, doxorubicin hydrochloride, etoposide, gemcitabine hydrochloride, topotecan hydrochloride, paclitaxel, vinblastine sulfate and vincristine sulfate.

[0044] Chemotherapeutic agents may be administered as single agents or in combination with one or more agents. For example, carboplatin and paclitaxel; gemcitabine and cisplatin; bleomycin, etoposide and cisplatin (BEP); vincristine sulfate, actinomycin-D and cyclophsopamide (VAC) and vinblastine sulfate, ifosfamide and cisplatin (VelP) are commonly used combinations of chemotherapeutic agents that are used in the treatment of ovarian cancer.

[0045] Ovarian cancer may also be treated with targeted therapies. [0046] Reference to "targeted therapy" includes any targeted therapeutic agent that is specifically designed to interfere with molecular alterations that are specific to cancer cells. Targeted therapeutic agents may include, but are not limited to, monoclonal antibodies and small molecule inhibitors. For example, everolimus is a small molecule inhibitor or the PDK/mTORCl pathway, which inhibits mTORCl function, disrupting the activity of P70 S6 kinase and the phosphorylation of its substrates including ribosomal protein S6; KU- 60019 and AZD6738 are small molecule inhibitors of ATM and ATR, respectively; LY2606368 is a small molecule inhibitor of CHKl/2; and BMN673 is a small molecule inhibitor of the nuclear enzyme poly(ADP-ribose) polymerase (PARP), which inhibits PARP-mediated repair of single strand DNA breaks.

[0047] Other targeted therapies have been developed to specifically target immune cells in the subject that promote the growth and survival of cancer cells or activate the subject's immune system to target cancer cells. Such "immunotherapies" include monoclonal antibodies that improve the activation and function of tumour specific T-cells; antibodies targeting cell surface proteins such as neoantigens, glycoproteins and receptor tyrosine kinases; cytokine therapies that modulate immune responses such as interferons and interleukins; and passive immunization therapies such as adoptive T-cell therapy. For example, anti-CTLA-4, anti-PD-1 and anti-PDL have all demonstrated efficacy for the treatment of tumours that are otherwise resistant to traditional chemotherapy.

[0048] In an embodiment, the therapeutic regimen for the treatment of ovarian cancer comprises surgery and the administration of chemotherapy, either alone or in combination with targeted therapy.

[0049] In another embodiment, the therapeutic regimen comprises a combination of two or more treatment modalities (for example, 2, 3 or more, 4 or more, 5 or more, 6 or more). Treatment modalities will typically be selected with a view to treating ovarian cancer and preventing ovarian cancer recurrence.

Subject

[0050] The terms "subject", "individual" and "patient" are used interchangeably herein to refer to any subject to which the present disclosure may be applicable, particularly a vertebrate subject, and even more particularly a mammalian subject. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordate including primates, rodents (for example, mice, rates, guinea pigs), lagomorphs (for example, rabbits, hares), bovines (for example, cattle), ovines (for example, sheep), caprines (for example, goats), porcines (for example, pigs), equines (for example, horses), canines (for example, dogs), felines (for example, cats), avians (for example, chickens, turkeys, duck, geese, companion birds such as canaries, budgerigars, etc.), marine mammals (for example, dolphins, whales), reptiles (for example, snakes, frogs, lizards, etc.), and fish. In some embodiments, the subject is a primate (for example, a human, ape, monkey, chimpanzee). In a preferred embodiment, the subject is a human.

RNA Polymerase I inhibitor for the treatment of ovarian cancer

[0051] In an aspect, the present invention provides a method for treating a subject with ovarian cancer comprising classifying a subject as sensitive to RNA polymerase I (Pol I) inhibitor therapy; selecting Pol I inhibitor therapy for a subject classified as sensitive to Pol I inhibitor therapy; and treating the subject classified as sensitive to Pol I inhibitor therapy with an effective amount of a Pol I inhibitor.

[0052] Reference to the term "RNA polymerase I inhibitor" or "Pol I inhibitor" means a compound or ligand, or a pharmaceutically acceptable salt thereof, which inhibits RNA polymerase I from transcribing the ribosomal RNA genes (rDNA) in the cell. In an embodiment, the Pol I inhibitor of the present invention is selected from the group consisting of CX-5461, BMH 21 and 9-hydroxyellipiticine. In a further embodiment, the Pol I inhibitor is CX-5461.

[0053] An "effective amount" is intended to mean that the amount of Pol I inhibitor when administered to a subject, in particular a human subject, in need of such treatment, is sufficient to treat ovarian cancer.

[0054] The Pol I inhibitor contemplated by the present invention may be prepared in a manner known in the art and are those suitable for enteral, such as oral or rectal, and parental administration to mammals (warm-blooded animals), particularly humans, comprising a therapeutically effective amount of Pol I inhibitor alone, or in combination with one or more pharmaceutically acceptable carriers or diluents, especially suitable for enteral or parenteral application.

[00SS] Suitable carriers, diluents or excipients include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that compositions of the invention may also include other supplementary physiologically active agents.

[0056] The carrier must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the subject. Compositions include that suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parental (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

[0057] In another aspect, the present invention provides a method for treating a subject with ovarian cancer comprising classifying a subject as sensitive or resistant to RNA polymerase I (Pol I) inhibitor therapy; selecting Pol I inhibitor therapy for a subject classified as sensitive to Pol I inhibitor therapy; and treating the subject classified as sensitive to Pol I inhibitor therapy with treatment regimen comprising an effective amount of a Pol I inhibitor.

[0058] In an embodiment, the treatment regimen comprises a Pol I inhibitor in combination with any one or more of the group consisting of chemotherapy and targeted therapy. In another embodiment, the targeted therapy is selected from the group consisting of any one or more of PDK/mTORCl pathway inhibitors, DNA damage repair (DDR) pathway inhibitors, epigenetic and/or transcription modifier inhibitors and immunotherapies. [0059] Reference to "PI3K/mTORCl pathway inhibitors" means any compound or ligand, or a pharmaceutically acceptable salt thereof, which inhibits the PDK/mTORCl pathway in a cell. In an embodiment, the PDK/mTORCl inhibitor is an allosteric inhibitor. In another embodiment, the PDK/mTORCl inhibitor is a catalytic inhibitor.

[0060] Examples of PDK/mTORCl pathway inhibitors contemplated by the present invention include AR245408, AZD6482, BAY80-6946, BEZ235, BGT226, BKM120, BYL719, taselisib, GDC-0941, GDC-0980, GSK2126458, GSK2636771, LY3023414, MLN1117, rigosertib, PF-05212384, PX-866, SAR245409, VS-5584, XL147, XL765, GDC-0032, AZD5363, everolimus and PF-04691502.

[0061] Reference to "DNA damage repair pathway inhibitors" or "DDR pathway inhibitors" means any compound or ligand, or a pharmaceutically acceptable salt thereof, which inhibits the DDR pathway in a cell. In an embodiment, the DDR pathway inhibitor is an allosteric inhibitor. In another embodiment, the DDR pathway inhibitor is a catalytic inhibitor.

[0062] Examples of DDR pathway inhibitors contemplated by the present invention include mirin, telomelysin, KU-55933, KU-60019, CP466722, schisandrin B, NU-6027, VE-821, SAR-020106, VRX0466617, NU7026, NU7441, KU-0060648, SCR7, L67, B02, A03, A10, UCN-01, SCH900776, LY2603618, XL844, AZD7762, PF-00477736, CC-115, TRC102, lucanthone, rucaparib, olaparib, veliparib, INO-1001, MK4827, CEP-9722, E7016, AZD6738, LY2606368 and BMN673.

[0063] Reference to "epigenetic and/or transcription modifier inhibitors" means any compound or ligand, or a pharmaceutically acceptable salt thereof, which inhibits epigenetic and/or transcription modifiers in a cell. In an embodiment, the epigenetic and/or transcription modifier inhibitor is an allosteric inhibitor. In another embodiment, the epigenetic and/or transcription modifier inhibitor is a catalytic inhibitor.

[0064] Examples of epigenetic and/or transcription modifier inhibitors contemplated by the present invention include BML-281, vorinostat, panobinostat, dinaciclib, I-BET151 and JQl. [0065] Reference to "immunotherapies" means any compound or ligand, or a pharmaceutically acceptable salt thereof, which activates or inhibits tumour associated immune cells.

[0066] Examples of immunotherapies contemplated by the present invention include monoclonal antibodies that improve the activation and function of tumour-specific T cells, such as anti-CTLA-4 antibodies, ipilimumab, tremelimumab, anti-PD-1 antibodies, nivolumab, pembrolizumab, anti-PD-Ll antibodies, BMS-936559, MEDI4736, MPDL33280A and MSB0010718C.

[0067] Selection of the appropriate treatment regimen will be dependent upon the molecular and pathological characteristics of the subject's ovarian cancer. For example, treatment regimens comprising a Pol I inhibitor and a PDK/mTORCl pathway inhibitor will be effective where MYC is overexpressed and/or amplified in the subject's ovarian cancer.

[0068] Reference to the terms "overexpression" or "overexpressed" is used to describe gene expression whereby the gene is expressed at a higher level than normal.

[0069] Reference to the terms "amplification" or "amplified" is used to describe gene amplification whereby there is an increase in the number of copies of a gene.

[0070] Methods for the detection of genomic characteristics such as amplification and overexpression are well known in the art.

[0071] Alternatively, treatment regimens comprising a Pol I inhibitor and chemotherapy and/or DDR pathway inhibitors will be effective where the subject's ovarian cancer has homologous recombination deficiency.

[0072] Reference to the terms "homologous recombination deficiency" or HR deficiency" means any defect in DNA repair via homologous recombination due to genetic and epigenetic alterations of HR pathway genes that results in the inability to repair double- stranded DNA breaks.

[0073] HR deficiency is generally characterised by BRCAl/2 inactivation by mutation or epigenetic silencing. Subjects with HR deficiency may also be identified by applying the homologous recombination deficiency (HRD) test, wherein a high HRD score (over 42) correlates with BRCAl/2 mutation and HRD (Telli et al. 2015, Abstract P3-07-12, San Antonio Breast Cancer Symposium, presented December 10, 2015). The HRD test combines loss of heterozygosity, telomeric allelic imbalance and large-scale transition scores. All three scores are then correlated with BRCAl/2 defects to derive the HRD score.

[0074] Furthermore, treatment regimens comprising a Pol I inhibitor and immunotherapies will be effective where the subject's ovarian cancer exhibits an immune gene expression signature.

[0075] Reference to an "immune gene expression signature" means a signature of gene expression that reflects an increased infiltration of intra-tumour CD3+ T cells as described by Tothill et al. 2008, Clincal Cancer Research, 14(16): 5198-5208, which is incorporated by reference.

Method of classification

[0076] The present invention is predicated of the inventors' finding that sensitivity to a specific Pol I inhibitor can be predicated using a BRCA1 mutation gene expression signature that is predictive of sensitivity to Pol I inhibitor therapy.

[0077] Accordingly, in an aspect, the present invention provides a method of classifying a subject with ovarian cancer comprising analysing the gene expression of a panel of genes according to Table 1 by Gene Set Enrichment Analysis (GSEA); and classifying the subject as sensitive to Pol I inhibitor therapy or resistant to Pol I inhibitor therapy, wherein the Enrichment Score (ES) reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 10 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy.

[0078] Sequences for the panel of genes according to Table 1 are publically available on the RefSeq: NCBI Reference Sequence Database via the National Centre for Biotechnology Information, U.S. National Library of Medicine website, www.ncbi.nlm.nih.aov/reseq/. RefSeq accession numbers for each of the genes is provided in Table 2. Table 2: Genes predictive of sensitivity to CX-S461 including RefSeq accession numbers

[0079] In an embodiment, the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 20 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy. In another embodiment, the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 30 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy. In another embodiment, the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 40 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy. In another embodiment, the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for all of the genes in the panel is indicative of sensitivity to Pol I inhibitor therapy.

[0080] Methods for measuring gene expression are well known in the art, which include low-throughput methods that analyse single genes and high-throughput methods that analyse the whole genome or large cohorts of genes simultaneously (exome and arrays). For example, such methods include northern blots, polymerase chain reaction (qPCR), microarrays, RNA sequencing (RNA-seq), targeted RNA sequencing, and NanoString gene expression analyses.

[0081] Each of these technologies produces a value, or a set of values for each of the products measured. A measured value for any gene expression product is defined as a measured biomarker, as measured by the processing instrument or device. Examples of a measured biomarker include a protein concentration for a specified protein, the transcript count for a single transcript in the case of RNA sequencing, the expression value for an exon or transcript in the case of microarrays, an m/z value in the case of mass spectrometry or a fluorescence value in the case of flow cytometry. Measured biomarkers can be understood as 'raw data', as measured by the instrument. Multi-biomarker assays will measure a number of biomarkers in parallel, reporting a collection of measured biomarkers.

[0082] Reference to the term "biomarker" means a gene that is differentially expressed in subjects that are sensitive to Pol I inhibitor therapy when compared to subjects that are resistant to Pol I inhibitor therapy.

[0083] Gene Set Enrichment Analysis (GSEA) provides a knowledge-based approach for interpreting genome-wide expression profiles. This method for analysing gene expression data is set out by Subramanian et al. 2005, PNAS, 102(43): 15545-15550, which is incorporated in its entirety by reference.

[0084] Reference to the term "Enrichment Score" or "ES" means the measured value calculated by GSEA that reflects the degree to which a set (5) is overrepresented at the extremes (top or bottom) of an entire ranked list of genes assessed by genome wide mRNA expression analysis. The score is calculated by walking down the list L, increasing a running-sum statistic when a gene in S is encountered and decreasing it when a gene in S is not encountered. The magnitude of the increment depends on the correlation of the gene with the phenotype. The ES is the maximum deviation from zero encountered in the random walk; it corresponds to a weighted Komogorov-Smirnov-like statistic.

[0085] Reference to the term "core enrichment" means high scoring gene sets that contribute to the ES. Gene sets comprising a core enrichment value of "yes" represent the list of genes that contribute most to the enrichment result.

[0086] In an embodiment, a subject will be classified as sensitive to Pol I inhibitor therapy, wherein the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 10 genes in the panel of genes according to Table 1 is indicative of sensitivity to Pol I inhibitor therapy. In another embodiment, a subject will be classified as sensitive to Pol I inhibitor therapy, wherein the ES reflecting the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment representing the list of genes contributing most to the enrichment result for at least 20 genes in the panel of genes according to Table 1 is indicative of sensitivity to Pol I inhibitor therapy. In another embodiment, a subject will be classified as sensitive to Pol I inhibitor therapy, wherein the ES reflecting the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment representing the list of genes contributing most to the enrichment result for at least 30 genes in the panel of genes according to Table 1 is indicative of sensitivity to Pol I inhibitor therapy. In another embodiment, a subject will be classified as sensitive to Pol I inhibitor therapy, wherein the ES reflecting the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment representing the list of genes contributing most to the enrichment result for at least 40 genes in the panel of genes according to Table 1 is indicative of sensitivity to Pol I inhibitor therapy. In another embodiment, a subject will be classified as sensitive to Pol I inhibitor therapy, wherein the ES reflecting the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment representing the list of genes contributing most to the enrichment result for all of the genes in the panel of genes according to Table 1 is indicative of sensitivity to Pol I inhibitor therapy. Kits

[0087] The present invention contemplates a kit for classifying a subject with ovarian cancer comprising detection agents capable of detecting the expression products of the panel of genes according to Table 1. Examples of suitable detection agents include, but are not limited to, primers specific to an mRNA encoding the panel of genes according to Table 1, probes specific to an mRNA encoding the panel of genes according to Table 1, adapters specific to an mRNA encoding the panel of genes according to Table 1, and the like. The skilled person would be familiar with standard mRNA detection agents as described in the art.

[0088] All essential materials and reagents required for detecting and quantifying expression of the panel of genes according to Table 1 may be assembled together in a kit. The kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, blotting membranes, microtitre plates, dilution buffers and the like. For example, an mRNA detection kit may include (i) primers or probes that specifically hybridize to each of the panel of genes according to Table 1. Also included may be enzymes suitable for amplifying mRNA, including various polymerases and buffers to provide the necessary reaction mixture for amplification. The kit may also feature various devices (for example, one or more) and reagents (for example, one or more) for performing any one of the assays described herein; and/or printed instructions for using the kit to quantify gene expression.

[0089] In another aspect, the present invention also contemplates a kit for the treatment of ovarian cancer in a subject comprising pharmaceutical-grade Pol I inhibitor.

[0090] All essential materials and reagents required for treating ovarian cancer in a subject may be assembled together in a kit. The kits may optionally include appropriate therapeutic agents to be administered in combination with a Pol I inhibitor, including, but not limited to the group consisting of chemotherapy and/or targeted therapy.

[0091] The present invention also teaches a commercial package comprising a kit for the classification of a subject with ovarian cancer or a kit for the treatment of ovarian cancer according to the present invention, together with instructions for use. [0092] In an embodiment, the commercial package comprises mRNA detection agents, together with instructions for use for the classification of a subject with ovarian cancer; or pharmaceutical-grade Pol I inhibitor, together with instructions for use for the treatment of ovarian cancer.

[0093] Those skilled in the art will appreciate that the invention described is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

EXAMPLES

[0094] Aspects of certain embodiments of the present invention are further described by reference to the following non-limiting Examples.

Protocols

Cell lines

[0095] Individuality of ovarian cell lines listed in Table 3 was routinely confirmed by polymerase chain reaction (PCR)-based short tandem repeat (STR) analysis using six STR loci.

Table 3: Ovarian cancer cell lines, their corresponding histotype

Therapeutics

[0096] CX-5461 was provided by Cylene Pharmaceuticals (now fully owned by Senhwa Biosciences) or synthesised in-house. PF-04691502 was provided by Pfizer Oncology. Everolimus (SI 120), BMN673 and AZD7762 were from Selleckchem. Cell proliferation assay

[0097] Cells were drug treated for 72 h, and cell number assessed using the Incucyte imaging system or the sulforhodamine B assay. Cells were less than 90% confluent in control wells at the end of incubation. GI50 was determined using GraphPad Prism. GI50 values did not follow a Gaussian distribution and the geometric mean (-300 nM) was used to define cells as resistant or sensitive.

[0098] To assess drug synergy, dose response curves were generated for both single agents and their combinations. A mutually exclusive combination index (CI) was determined using CalcuSyn where: CI < 1 = synergy; CI > 1 = antagonism; CI = 1 = additive. The combination ratio was fixed and based on the GI50 for each drug, wherein the highest and lowest combination ratio was eight times and 1/8^Λ the GI50, respectively.

Cell death assay

[0099] Cell death was determined using propidium iodide (PI) staining followed by flow cytometry (LSRII) and data analysed using FCS Express (De Novo Software).

Cell count assay

[0100] OVCAR4 cells were plated in 384-well plates and treated with increasing dose of HDAC inhibitor BML-281 or vorinostat in the absence or presence of CX-5461 (GI25: 50 nM).

[0101] After 120 h incubation, cell counts were performed using DAPI staining followed by high-content image acquisition (Cellomics ArrayScan) and analysis using HCS Studio Cell Analysis Software (Cellomics).

Immunoblotting

[0102] Cells were lysed with RIPA buffer, subjected to SDS-PAGE, immunoblotted and protein bands visualised and quantified using ImageQuant (GE Healthcare).

Gene expression

[0103] Cells were harvested at 50-80% confluency. RNA was extracted using the RNeasy kit (Qiagen). In vitro transcribed and biotin labelled cRNA was fragmented and hybridised to Affymetrix LOST expression arrays, as per the manufacture's instructions (accession number GSE43765). Differential gene expression was determined using the Limma R package after RMA normalisation and background correction.

Gene mutational analysis

[0104] Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen). PCR primers and annealing temperatures are in Table 3. Cycle sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit and analysed on a 3130 Genetic Analyzer (Applied Biosystems).

Human ovarian cancer xenograft assays

[0105] Female Balb/c nude mice were injected subcutaneously with 5 x 10⁶ cells in 0.05 mL of 50% Matrigel. When tumours reached -100 mm³ mice were randomised into groups of 10 and daily oral gavaged for 28 consecutive days with (i) CX-5461 every three days at 30 to 40 mg/kg in 25 nmol/L NattPC^ (pH 4.5); (ii) everolimus was given daily at 5 mg/kg in DMSO (5%) and 1% methylcellulose (95%); and/or (iii) BMN673 was given daily at 0.33 mg/kg.

Gene Set Enrichment Analysis (GSEA)

[0106] For GSEA 1000 iterations were performed using the default weighted enrichment statistic and a signal-to-noise metric to rank genes based on their differential expression across sensitive and resistant cell lines.

Statistical analysis

[0107] Student's t-test or one-way analysis of variance followed by Tukey's Multiple Comparison Test was performed using GraphPad PRISM. To calculate the correlation between two variables, a two-tailed Spearman correlation test was performed. Chi-squared tests were used to assess associations between mutation status and sensitivity. Cell number/dose response curves were generated from non-linear fits using R.

[0108] Differences of p < 0.05 were considered significant. All data are expressed as mean ± standard error of mean. CX-5461 inhibits ovarian cancer cell proliferation

[0109] Evaluation of CX-5461 over a panel of 29 ovarian cancer cell lines covering all major histological subtypes (Table 2) showed differential growth inhibition. The CX-5461 concentration that inhibited proliferation by 50% (GI50) ranged from 12 to 5170 nM (Figure 1; Table 4).

Table 4: GIS0 values for ovarian cancer cell lines

[0110] In sensitive cell lines, CX-5461 caused cell cycle arrest and death, while resistant cell lines underwent p53-independent cell cycle arrest and exhibited markers of senescence (Figure 2). Sensitivity to CX-5461 did not correlate with histological subtypes or mutations in TP '53, suggesting that treatment with CX-5461 may be efficacious for the treatment of high-grade serous ovarian cancer, which is ubiquitously characterized by the presence of TP53 mutations. However, sensitivity to CX-5461 did correlate with PI3K pathway activation, as defined by the presence of activating mutations in PI3KCA and loss of PTEN (Figure 3).

[0111] To confirm the efficacy of CX-5461 in inhibiting ovarian cancer cell proliferation, we tested the anti-tumour effect of CX-5461 in an in vivo xenograft model. Accordingly, treatment of the ES2 xenograft model with CX-5461 significantly inhibited tumour growth (Figure 4).

[0112] These data indicate that at least a subset of ovarian cancer is inherently reliant on oncogene-induced increases in ribosome biogenesis.

Generation of a predictive gene expression signature of CX-5461

[0113] To identify molecular pathways that confer sensitivity and/or resistance to CX- 5461, we examined the difference in gene expression between 10 CX-5461 sensitive and 11 resistant ovarian cancer cell lines using GSEA. Using GSEA, a predictive gene signature of sensitivity to CX-5461 was generated, which is characterized by a BRCA1 mutation signature (Figures 5 and 6). Accordingly, sensitivity to CX-5461 may be further characterized by deficiencies in the homologous recombination (HR) repair pathway, with particularly compromised DNA damage repair.

Efficacy of CX-5461 combination therapeutic regimens

[0114] Given the prevalence of hyper activation of the PDK/RAS pathways, MYC amplification and homologous recombination deficiency, we hypothesised that combining CX-5461 with other agents that induce DNA damage, inhibit PDK/RAS signalling and target immune activation against ovarian tumours may increase the efficacy of CX-5461 treatment. CX-5461 and PI3K/RAS pathway inhibitors

[0115] We have previously shown that the combined inhibition of the PDK/mTORCl/2 and the RAS/MEK/ERK signalling pathways is a potentially effective new approach in the treatment of ovarian cancer (Sheppard et al. 2013, European Journal of Cancer, 49(18): 3936-3944). Therefore, given that sensitivity to CX-5461 correlates with PDK pathway activation, we investigated whether dual inhibition of Pol I, PDK/mTORCl/2 and/or the RAS/MEK/ERK signalling pathways resulted in greater inhibition of proliferation and/or cell death compared to single agent treatment.

[0116] Inhibiting PDK/mTORCl/2 signalling using PF-04691502 significantly inhibits ES2 tumour growth in vivo. Furthermore, we have also shown that the co-ordinated targeting of Pol I and PDK/AKT/mTORCl -dependent ribosome biogenesis and protein translation provided a remarkable improvement in treating MYC-driven B-cell lymphoma (Figure 7), using a combination of CX-5461 and everolimus (Devlin et al. 2016, Cancer Discovery, 6(1): 1-12). Considering that the PDK pathway is frequently deregulated in all ovarian cancer subtypes and MYC is amplified frequently in high-grade serous ovarian cancer, we predict that more effective targeting of ribosome biogenesis may be achieved by combining CX-5461 with PDK/AKT/mTORCl inhibitors, such as everolimus.

CX-5461 andDNA damage repair pathway inhibitors

[0117] We investigated whether dual inhibition of Pol I and DNA damage repair (DDR) pathway activity resulted in greater inhibition of proliferation and/or cell death when compared to single agent treatment. Accordingly, we combined BMN673, which inhibits the repair of single strand breaks by PARP, with CX-5461 to treat both resistant and sensitive cell lines. This combination approach resulted in a synergistic reduction in cell proliferation and cell death (mutually non-exclusive CI < 1) in both sensitive and resistant cell lines (Figure 8). To confirm that the combination of CX-5461 and BMN673 was relevant in vivo, we tested anti-tumour effects in xenografts. Accordingly, ES2 tumours demonstrated improved efficacy of the combination of CX-5461 and BMN673 when compared to single agent treatment (Figure 9).

[0118] Consistent with this outcome, pre-treatment of minimally immortalised human fibroblasts with CX-5461 prior to UV irradiation leads to an increase in UV-induced DNA damage, suggesting that CX-S461 treatment increases sensitivity to DNA damaging agents (Figure 11). This also suggests that CX-5461 may cooperate with current chemotherapies {i.e. platinum-based chemotherapies) that are used to treat ovarian cancer.

[0119] An alternative approach to targeting DDR pathways is provided by inhibiting Ataxia telangiectasia mutated (AIM) and Ataxia telangiectasia and Rad3 related (ATR) kinase signalling. ATM and ATR recognise DNA damage and promote repair, while also activating cell cycle arrest by the checkpoint kinases CHK1 and CHK2. Inhibition of CHKl/2 in combination with CX-5461 has been demonstrated to allow cells to bypass the G2 checkpoint leading to mitotic catastrophe. We have also shown that CX-5461 activates ATM and ATR signalling in both sensitive and resistant ovarian cancer cell lines, as indicated by the phosphorylation of CHK1 at the T68 phosphorylation site and CHK2 at the S345 phosphorylation site (Figure 12). Therefore, the combination of CX-5461 and CHKl/2 inhibitors (such as AZD7762) is predicted to lead to a synergistic effect in both sensitive and resistant ovarian cancer cell lines.

CX-5461 and immunotherapies

[0120] Previous studies have identified a correlation between immune gene expression signatures in high-grade serous ovarian cancer, characterised by increased infiltration of intra-tumour CD3+ T cells (Tothill et al. 2008 Clinical Cancer Research, 14(16): 5198- 5208). Immunotherapies that improve activation and function of tumour specific T cells, including anti-CTLA-4, anti-PD-1 and anti-PD-L monoclonal antibodies, have entered into clinical trials for ovarian cancer patients. We have shown that CX-5461 upregulates the expression of PD-L1 in an ovarian cancer cell line (Figure 13). Upregulation of PD-L1 can promote tumour progression by inhibiting tumour associated T cells. Therefore, combining immunotherapies, such as anti-PD-1, is predicted to improve the efficacy of CX-5461 against ovarian cancer.

CX-5461 and epigenetic/transcription modifier inhibitors

[0121] Hi stone deacetylase (HDAC) inhibitors have been shown to exert anti-tumour effects through hyperacetylation of histones and demethylation of genomic DNA resulting in the reactivation of genes that inhibit proliferation. Previous studies have demonstrated that HDAC inhibition synergizes with conventional chemotherapies to induce potent cytotoxic effects in ovarian cancer models (see, for example, Budman et al. 2011 Investigational New Drugs, 29: 1224-1229; Cooper et al. 2007 Gynecologic Oncology, 104: 596-601; and Qiu et al. 2013 Future Oncology, 9: 255-269).

[0122] We investigated whether dual inhibition of Pol I and HDAC enzyme activity resulted in greater inhibition of proliferation and/or cell death when compared to single agent treatment. Accordingly, we combined vorinostat, which inhibits class I, Π and IV HDAC enzymes, or BML-281, which is a selecting HDAC3 and HDAC6 inhibitor, with CX-5461 to treat the OVCAR4 cell line. This combination approach resulted in a cooperative reduction in cell number using both the vorinostat and BML-281 (Figure 10).

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Claims

1. A method of classifying a subject with ovarian cancer comprising:

a. analysing the gene expression of a panel of genes according to Table 1 below by Gene Set Enrichment Analysis (GSEA); and

b. classifying the subject as sensitive to RNA Polymerase I (Pol I) inhibitor therapy, wherein the Enrichment Score (ES) reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 10 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy.

Table 1

2. The method according to claim 1, wherein the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 20 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy.

3. The method according to claim 1, wherein the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 30 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy.

4. The method according to claim 1, wherein the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 40 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy.

5. The method according to claim 1, wherein the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for all genes in the panel is indicative of sensitivity to Pol I inhibitor therapy.

6. A method for treating a subject with ovarian cancer comprising:

a. classifying the subj ect according to the method of claim 1 ; b. selecting RNA Polymerase I (Pol I) inhibitor therapy for a subject classified as sensitive to Pol I inhibitor therapy, wherein the Enrichment Score (ES) reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 10 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy; and

c. treating the subject classified as sensitive to Pol I inhibitor therapy with an effective amount of a Pol I inhibitor.

7. The method according to claim 6, wherein the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 20 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy.

8. The method according to claim 6, wherein the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 30 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy.

9. The method according to claim 6, wherein the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 40 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy.

10. The method according to claim 6, wherein the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for every gene in the panel is indicative of sensitivity to Pol I inhibitor therapy.

11. The method according to any one of claims 6 to 10, wherein the Pol I inhibitor therapy is selected from the group consisting of CX-5461, BMH 21, and 9- hydroxyellipiticine.

12. The method according to claim 11, wherein the Pol I inhibitor therapy is CX-5461.

13. A method for treating a subject with ovarian cancer comprising:

a. classifying the subj ect according to the method of claim 1 ;

b. selecting a treatment regimen comprising a RNA Polymerase I (Pol I) inhibitor for a subject classified as sensitive to Pol I inhibitor therapy, wherein the Enrichment Score (ES) reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 10 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy; and c. treating the subject classified as sensitive to Pol I inhibitor therapy with a treatment regimen comprising an effective amount of a Pol I inhibitor.

14. The method according to claim 13, wherein the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 20 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy.

15. The method according to claim 13, wherein the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 30 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy.

16. The method according to claim 13, wherein the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for at least 40 genes in the panel is indicative of sensitivity to Pol I inhibitor therapy.

17. The method according to claim 13, wherein the ES reflects the highest degree to which a gene set is overrepresented and a "yes" value for core enrichment for every gene in the panel is indicative of sensitivity to Pol I inhibitor therapy.

18. The method according to any one of claims 13 to 17, wherein the treatment regimen comprises a Pol I inhibitor in combination with any one or more of the group consisting of PDK/mTORCl pathway inhibitors, chemotherapy, DNA damage repair (DDR) pathway inhibitors, epigenetic/ transcription modifier inhibitors and immunotherapies.

19. The method according to claim 18, wherein the treatment regimen comprises a Pol I inhibitor in combination with PDK/mTORCl pathway inhibitors where MYC is overexpressed and/or amplified in the subject's ovarian cancer.

20. The method according to claim 18, wherein the treatment regimen comprises a Pol I inhibitor in combination with chemotherapy and/or DDR pathway inhibitors where the subject's ovarian cancer has a homologous recombination deficiency.

21. The method according to claim 20, wherein homologous recombination deficiency is determined according to the homologous recombination deficiency (HRD) test.

22. The method according to claim 18, wherein the treatment regimen comprises a Pol I inhibitor in combination with immunotherapies where the subject's ovarian cancer has an immune gene expression signature.

23. A kit for classifying a subject with ovarian cancer comprising detection agents capable of detecting the expression products of the panel of genes according to claim 1.