WO2024186523A2

WO2024186523A2 - Glucocorticoid receptor antagonists for cancer treatment

Info

Publication number: WO2024186523A2
Application number: PCT/US2024/017560
Authority: WO
Inventors: Jinsong Liu; Anil K. Sood; Xudong Zhang; Jun Yao; Xiaoran Li; Na NIU; Yan Liu
Original assignee: University of Texas System; University of Texas at Austin
Current assignee: University of Texas System; University of Texas at Austin
Priority date: 2023-03-07
Filing date: 2024-02-27
Publication date: 2024-09-12
Anticipated expiration: 2025-09-07
Also published as: WO2024186523A3

Abstract

Provided herein are methods of treating poly(ADP-ribose) polymerase inhibitor (PARPi)-naïve cancer with a combination therapy such as a PARPi and a glucocorticoid receptor antagonist, as well as methods of treating PARPi-resistant cancer with glucocorticoid receptor antagonist compounds. The methods include identifying subjects suitable for cancer treatment with a glucocorticoid receptor antagonist or with a PARPi-glucocorticoid receptor antagonist combination therapy.

Description

GLUCOCORTICOID RECEPTOR ANTAGONISTS FOR CANCER TREATMENT BACKGROUND OF THE INVENTION

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority under U.S.C. §119(e) to U.S. Provisional Application Serial No. 63/450,645, filed on March 7, 2023, the entire contents of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to poly(ADP-ribose) polymerase inhibitor (PARPi) resistance in cancer and more specifically to methods of treating cancer with a PARPi as well as methods of treating PARPi-resistant cancer.

BACKGROUND INFORMATION

[0003] Approximately 50% of high-grade serous carcinomas (HGSCs) exhibit defective homologous recombination (HR) DNA repair caused by genetic or epigenetic alterations of HR pathway genes, most commonly in the tumor-suppressor genes BRCA1 and BRCA2. HR- deficient tumors with BRCA1/2 alterations are susceptible to PARPi, which selectively kill BRCAl/2-deficient cells while sparing BRCAl/2-proficient normal cells. Currently, 3 PARPi — olaparib, rucaparib, and niraparib — have been granted regulatory approval as maintenance therapy for platinum-sensitive recurrent ovarian cancer based on substantial improvements in progression-free survival in 3 randomized phase III trials.

[0004] PARP inhibitors offer significant clinical benefits in ovarian cancer patients with or without deficiencies in the homologous recombination DNA repair pathway. However, most patients with advanced cancer eventually acquire resistance to PARPi, and the mechanisms by which tumors escape PARPi therapy are still not fully understood. Despite the remarkable clinical benefit of PARPi, acquired drug resistance is observed in most patients with advanced HGSC and other cancers. Multiple potential mechanisms of PARPi resistance have been proposed in preclinical models, including overexpression of drug- efflux transporter genes, decreased PARP trapping, stabilization of stalled replication forks, or reactivation of HR. However, most of these mechanisms have only been studied in vitro, and it is unclear whether they apply to treated tumors in patients. Moreover, up to 50% of patients with HR-proficient HGSC do not substantially benefit from PARPi treatment, highlighting the importance of identifying alternative clinically relevant mechanisms that potentiate the therapeutic effect of PARPi and the mechanisms of acquired resistance in patients regardless of HR status. [0005] Emerging evidence suggests that moderate, clinically relevant doses of anticancer drugs can trigger senescence of cancer cells in addition to apoptosis in solid tumors and tumor- derived cell lines. These senescent cells contain either a highly enlarged nucleus or multiple nuclei, often accompanied by markedly increased cell size and genomic content and are now referred to as polyploid giant cancer cells (PGCCs). Although PGCCs were often overlooked or mispresented as "dead cells" in the past owing to their inability to execute mitosis, they are now known to generate therapy-resistant daughter cells via nuclear budding or bursting, forming transient cell-in-cell structures called fecundity cells. PGCCs recapitulate properties of the blastomere-stage embryonic program of dedifferentiation. PGCCs can also contribute to therapy resistance in various types of solid tumors, including ovarian, prostate, colon, and breast cancers and melanoma. However, whether PGCCs account for resistance to PARPi in HGSC remains unknown.

SUMMARY OF THE INVENTION

[0006] The present invention is based on the seminal discovery that the use of antiglucocorticoids, including drugs or agents that reduce glucocorticoid activity in the body, e.g., glucocorticoid receptor (GR) antagonist compounds, are effective for treating PARPi-resistant cancer and such compounds increase the efficacy of PARPi treatment for PARPi-naive cancer. The present disclosure demonstrates in an illustrative example, that mifepristone, an FDA- approved drug, blocks the growth of ovarian cancer cells in multiple ovarian and breast cell lines, organoids, and patient-derived xenograft models for ovarian cancer by blocking the growth of PARPi-resistant cancer. The disclosure provides for the use of a anti -glucocorticoids, such as a glucocorticoid receptor (GR) antagonist, either alone or in combination with a PARPi, to treat cancer.

[0007] In one embodiment, the present invention provides a method of treating cancer in a subject which includes administering a therapeutically effective amount of a poly(ADP -ribose) polymerase inhibitor (PARPi) and a glucocorticoid receptor (GR) antagonist to a subject who has not previously received PARPi treatment, thereby treating the cancer.

[0008] In one aspect, the PARPi is olaparib, niraparib, rucaparib, talazoparib, or a combination thereof. In a particular aspect, the PARPi is olaparib. In one aspect, the therapeutically effective amount of the PARPi is between about 5 and 250 mg/kg/day. In another aspect, the therapeutically effective amount of the PARPi is about 50 mg/kg/day. [0009] In one aspect, the glucocorticoid receptor (GR) antagonist is relacorilant, ketoconazole, aminoglutethimide, metyrapone, mifepristone, or a combination thereof. In a particular aspect, the glucocorticoid receptor (GR) antagonist is mifepristone. In one aspect, the therapeutically effective amount of the glucocorticoid receptor (GR) antagonist is between about 2.5 and 125 mg/kg/day. In another aspect, the therapeutically effective amount of the glucocorticoid receptor (GR) antagonist is about 25 mg/kg/day. In some aspects, the therapeutically effective amount of the PARPi is about 1 to 10 times the therapeutically effective amount of the glucocorticoid receptor antagonist.

[0010] In one aspect, the cancer is breast cancer, ovarian cancer, carcinoma, or adenocarcinoma. In a further aspect, the subject has not previously received PARPi treatment within the previous 15 days, within the previous 20 days, within the previous 25 days, within the previous 30 days, within the previous 40 days, within the previous 50 days, within the previous 60 days, within the previous 70 days, within the previous 80 days, within the previous 90 days, within the previous 100 days, or within the previous year. In another aspect, the subject does not exhibit elevated levels of polyploid giant cancer cells (PGCCs) or endoreplicating cancer cells.

[0011] In another embodiment, the present invention provides a method of treating cancer in a subject, wherein the subject has previously received poly(ADP-ribose) polymerase inhibitor (PARPi) treatment including: ceasing the PARPi treatment; and, at least ten days after cessation of the PARPi treatment, administering a therapeutically effective amounts of a poly(ADP- ribose) polymerase inhibitor (PARPi) and a glucocorticoid receptor (GR) antagonist to the subject in combination, thereby treating the cancer.

[0012] In another embodiment, the present invention provides a method of treating cancer in a subject which includes administering a therapeutically effective amount of a glucocorticoid receptor (GR) antagonist to a subject who has an elevated polyploid giant cancer cell (PGCC) level, an elevated endoreplicating cancer cell level, or a combination thereof, thereby treating the cancer.

[0013] In one aspect, the glucocorticoid receptor (GR) antagonist is relacorilant, ketoconazole, aminoglutethimide, metyrapone, mifepristone, or a combination thereof. In another aspect, the glucocorticoid receptor (GR) antagonist is mifepristone. In one aspect, the therapeutically effective amount of the glucocorticoid receptor (GR) antagonist is between about 5 and 250 mg/kg/day. In another aspect, the therapeutically effective amount of the glucocorticoid receptor (GR) antagonist is about 50 mg/kg/day.

[0014] In one aspect, the elevated PGCC level, the elevated endoreplicating cancer cell level, or the combination thereof is a level of at least about 3%, at least about 5%, at least about 7.5%, at least about 10%, at least about 12.5%, at least about 15%, at least about 17.5%, at least about 20%, at least about 22.5%, at least about 25%, at least about 27.5%, or at least about 30% among cells from a biological sample from the subject. In one aspect, the biological sample includes saliva, blood, plasma, serum, vaginal fluid, interstitial fluid, ocular fluid, seat, mucus, glandular secretion, nipple aspirate, seme, spinal fluid, emphatic fluid, sputum, pus, huffy coat, tumor tissue, circulating tumor cells, skin tissue, lung tissue, kidney tissue, bone marrow, pancreatic tissue, liver tissue, muscle tissue, gall bladder tissue, colon tissue, brain tissue, prostate tissue, esophageal tissue, thyroid tissue, a solid-tumor biopsy, a liquid tumor biopsy, or a combination thereof.

[0015] In one aspect, the cancer is breast cancer, ovarian cancer, carcinoma, adenocarcinoma. In another aspect, the subject received PARPi treatment within the previous day, within the previous 2 days, within the previous 3 days, within the previous 4 days, within the previous 5 days, within the previous 6 days, within the previous 7 days, within the previous 10 days, within the previous 15 days, within the previous 20 days, within the previous 25 days, within the previous 30 days, within the previous 40 days, within the previous 50 days, within the previous 60 days, within the previous 70 days, within the previous 80 days, within the previous 90 days, within the previous 100 days, or within the previous year. In another aspect, the PARPi is olaparib.

[0016] In yet another embodiment, the present invention provides a method of identifying a subject with cancer as a candidate for treatment with a poly(ADP-ribose) polymerase inhibitor (PARPi) and a glucocorticoid receptor (GR) antagonist including: measuring a level of polyploid giant cancer cells (PGCCs), endoreplicating cancer cells, or a combination thereof in a biological sample from the subject; and classifying the subject as having a cancer cell PGCC level, an endoreplicating cancer cell level, or a combination thereof of at most 5% as a likely responder to the PARPi and the glucocorticoid receptor antagonist, thereby identifying the subject as suitable for treatment with the PARPi and the glucocorticoid receptor antagonist, or classifying the subject as having a cancer cell PGCC level, an endoreplicating cancer cell level, or a combination thereof of at most 5% as a likely non-responder to the PARPi and the glucocorticoid receptor antagonist, thereby identifying the subject as unsuitable for treatment with the PARPi and the glucocorticoid receptor antagonist; thereby identifying the subject with cancer as a candidate for treatment with the PARPi and the glucocorticoid receptor antagonist. [0017] In one aspect, the PARPi is olaparib, niraparib, rucaparib, talazoparib, or a combination thereof. In a further aspect, the PARPi is olaparib. In one aspect, the treatment with the PARPi includes administration of between about 5 and 250 mg/kg/day. In a further aspect, the treatment with the PARPi includes administration of about 50 mg/kg/day.

[0018] In one aspect, the glucocorticoid receptor antagonist is relacorilant, metyrapone, ketoconazole, aminoglutethimide, mifepristone, or a combination thereof. In a further aspect, the glucocorticoid receptor is mifepristone. In one aspect, the treatment with the glucocorticoid receptor includes administration of between about 2.5 and 125 mg/kg/day. In a further aspect, the treatment with the glucocorticoid receptor includes administration of about 25 mg/kg/day. [0019] In some aspects, the cancer is breast cancer, ovarian cancer, carcinoma, or adenocarcinoma. In some aspects, the method includes measuring a level of PGCCs in the biological sample. In some aspects, the biological sample includes saliva, blood, plasma, serum, vaginal fluid, interstitial fluid, ocular fluid, seat, mucus, glandular secretion, nipple aspirate, seme, spinal fluid, emphatic fluid, sputum, pus, huffy coat, tumor tissue, circulating tumor cells, skin tissue, lung tissue, kidney tissue, bone marrow, pancreatic tissue, liver tissue, muscle tissue, gall bladder tissue, colon tissue, brain tissue, prostate tissue, esophageal tissue, thyroid tissue, a solid-tumor biopsy, a liquid tumor biopsy, or a combination thereof.

[0020] In another embodiment, the present invention provides a method of identifying a subject with cancer as a candidate for treatment with a glucocorticoid receptor (GR) antagonist including: measuring a level of polyploid giant cancer cells (PGCCs), endoreplicating cancer cells, or a combination thereof in a biological sample from the subject; and classifying the subject as having a cancer cell PGCC level, an endoreplicating cancer cell level, or a combination thereof of at least 5% as a likely responder to the glucocorticoid receptor antagonist, thereby identifying the subject as suitable for treatment with the glucocorticoid receptor antagonist; or classifying the subject as having a cancer cell PGCC level, an endoreplicating cancer cell level, or a combination thereof of at most 5% as a non-likely responder to the glucocorticoid receptor antagonist, thereby identifying the subject as unsuitable for treatment with the glucocorticoid receptor antagonist; thereby identifying the subject with cancer as a candidate for treatment with the glucocorticoid receptor antagonist. [0021] In one aspect, the glucocorticoid receptor (GR) antagonist is relacorilant, metyrapone, ketoconazole, aminoglutethimide, mifepristone, or a combination thereof. In a particular aspect, the glucocorticoid receptor antagonist is mifepristone. In one aspect, the treatment with the glucocorticoid receptor antagonist includes administration of between about 5 and 250 mg/kg/day. In a particular aspect, the treatment with the glucocorticoid receptor antagonist includes administration of about 50 mg/kg/day.

[0022] In one aspect, the cancer is breast cancer, ovarian cancer, carcinoma, adenocarcinoma, or a combination thereof. In a further aspect, the method includes measuring a level of PGCCs in the biological sample. In an additional aspect, the biological sample includes saliva, blood, plasma, serum, vaginal fluid, interstitial fluid, ocular fluid, seat, mucus, glandular secretion, nipple aspirate, seme, spinal fluid, emphatic fluid, sputum, pus, huffy coat, tumor tissue, circulating tumor cells, skin tissue, lung tissue, kidney tissue, bone marrow, pancreatic tissue, liver tissue, muscle tissue, gall bladder tissue, colon tissue, brain tissue, prostate tissue, esophageal tissue, thyroid tissue, a solid-tumor biopsy, a liquid tumor biopsy, or a combination thereof.

[0023] In another embodiment, the present application provides a method of treating cancer in a subject that includes administering a therapeutically effective amount of a poly(ADP- ribose) polymerase inhibitor (PARPi) and an anti-glucocorticoid to a subject, thereby treating the cancer. In some aspects, the subject has not previously received PARPi treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0025] FIGS. 1A-1H are a series of graphs and images illustrating the establishment of olaparib-resistant PDX models of BR(’A''‘' ^[ and BRCAl''^{A [} ovarian HGSC. FIGS. 1A-1C illustrate tumor growth curves of HGSC xenografts. Mice were treated with vehicle or olaparib (50 mg/kg/day, 5 days/week) for 60 days. Data are shown as mean ± SD. FIG. 1A illustrates data for BRCA^W1 PDX-2445: vehicle (n = 3), olaparib (n = 10). FIG. IB illustrates data for BRCA'* PDX-2428: vehicle (n = 3), olaparib (n = 8). FIG. 1C illustrates data for BRCA /'" ¹ PDX-2462: vehicle (n = 3), olaparib (n = 7). FIGS. 1D-1F illustrate validation of acquired o lap arib resistance in xenograft tumors. Olaparib-treated PDX tumors were harvested, retransplanted into different mice, and expanded. PDX-bearing mice were then treated with vehicle or olaparib (50 mg/kg/day, 5 days/week). FIG. ID illustrates data for PDX- 2445: vehicle (n = 3), olaparib (n = 5). FIG. IE illustrates data for PDX-2428: vehicle (n = 3), olaparib (n = 6). FIG. IF illustrates data for PDX-2462: vehicle (n = 3), olaparib (n = 3). Data are shown as mean ± SD (two-way ANOVA). FIG. 1G illustrates data for Representative H&E- stained sections of PDX-2428 and PDX-2462 xenografts. The vehicle-treated tumors mostly consist of relatively uniform tumor cells. In contrast, the tumors with acquired olaparib resistance exhibit enriched polyploid giant cancer cells (PGCCs) in the forms of multinucleated giant cells (black arrows) or mononucleated giant cells (arrows). Scale bars, 50 pm. FIG. 1H illustrates PI-FACS quantification of polyploidy in PDX- 2428 and PDX-2462 xenografts. Cells with DNA content > 4C were defined as PGCCs. Data are shown as mean ± SD. The exact P values are shown on the graph (Welchs /-test).

[0026] FIGS. 2A-2F are a series of graphs, timelines, and images which illustrate that olaparib induces the formation of PGCCs. FIG. 2A is representative phase-contrast microscopy images of Hey HGSC cells exposed to the indicated concentrations of olaparib or vehicle (0.1% DMSO) for 7 days. The higher concentrations of olaparib (>100 pM) caused cell death; however, the sublethal concentration of olaparib (50 pM) led to formation of PGCCs, characterized by enlarged cytoplasm and nuclei. Scale bars, 100 pm. FIG. 2B is a schematic illustration of induction of PGCCs and PGCC-derived daughter cells. Hey cells were treated with 50 pM olaparib for 7 days. Cells were then allowed to recover in drug-free culture medium for up to 10 days to generate daughter cells. R0 refers to the day on which olaparib was withdrawn. FIGS. 2C-2D illustrate PI-FACS quantification of polyploidy in Hey cells exposed to 50 pM olaparib at the indicated times. Each data point corresponds to one biological replicate, and data are shown as mean ± SD. The exact P values are shown on the graph (oneway ANOVA). FIG. 2E illustrates representative phase-contrast microscopy images showing the morphological changes in Hey cells exposed to olaparib at the indicated times. Freshly seeded Hey cells are slender. Once exposed to 50 pM of olaparib, cells gradually became flattened with enlarged cytoplasm and nuclei. PGCCs proliferated and produced daughter cells during recovery (R) days 3-10. Scale bars, 100 pm. FIG. 2F illustrate PI-FACS quantification of polyploidy (left) and PGCCs as a percentage of total tumor cells (right) in human ovarian and cancer cell lines. OVCA-432 PGCCs were induced by exposure to 400 pM olaparib for 72 h, then allowed to recover for another 72 h. SKOV3 and MCF-7 PGCCs were induced by exposure to 50 pM olaparib for 7 days. Each data point corresponds to one biological replicate, and data are shown as mean ± SD. The exact P values are shown on the graph (Welchs /-test).

[0027] FIGS. 3A-3E are a series of graphs and images which illustrate that PGCCs exhibit multiple characteristics of senescent cells. FIG. 3A is representative images (left) and quantification (right) of |3-galactosidase (P-gal) staining of control Hey cells and Hey PGCCs. The P-gal-positive cells exhibit dark blue staining in the cytoplasm. Scale bars, 50 pm. Four randomly selected fields per group (10x objective lenses) were used for quantification analysis. FIG. 3B is immunofluorescence images of y-H2AX foci and p21 expression (left) and quantification (right) of y-H2AX foci number in control Hey cells and Hey PGCCs at the indicated times. Both y-H2AX foci and p21 expression were highly elevated in the nuclei of PGCCs on treatment day 7 and gradually decreased during the recovery period in the PGCC-derived daughter cells (white arrows). Scale bars, 50 pm. Filamentous actin (F-actin) was visualized by phalloidin staining. At least 50 cells per group were counted and used for quantification. FIG. 3C illustrate that Hey PGCCs were induced by exposure of Hey cells to 50 pM olaparib for 7 days and allowed to recover for 3 or 10 days. Supernatants were collected and analyzed for IL- 1 p and IL-6 secretion by ELISA. Each data point corresponds to one biological replicate. FIG. 3D is phase-contrast images (left) and quantification (right) of P-gal staining of control OVCA-432, SKOV3, and MCF-7 cells and their corresponding PGCCs. Scale bars, 50 pm. Four randomly selected fields per group (10x objective lenses) were used for quantification analysis. FIG. 3E illustrate immunofluorescence images (left) ofy-H2AX foci, p 16^{I 4a}. and p21 expression and quantification (right) of y-H2AX foci in the indicated cell lines and their corresponding PGCCs. Scale bars, 50 pm. At least 50 cells per group were counted and used for quantification (27 cells for SKOV3 PGCCs). Data are shown as mean ± SD in FIGS. 3A- 3E. The exact P values are shown on the graph (Welchs /-test).

[0028] FIGS. 4A-4D are images which indicate that olaparib induces the formation of PGCCs through endoreplication. FIGS. 4A-4C illustrate time-lapse monitoring of Hey cells labeled with FUCCI. FIG. 4D illustrates time-lapse monitoring of Hey cells labeled with or histone H2B-mCherry and EGFP-a-tubulin. The FUCCI system labels nuclei in red at the G1 phase, yellow at the Gl/S transition phase, and green at the S/G2/M phases. FIG. 4A illustrates that the typical mitotic cell cycle consists of interphase (Gl, S, G2) and mitosis (M). FIG. 4B is images which track cell cycle changes in a Hey cell exposed to 50 pM olaparib. A diploid Hey cell gradually becomes a mononucleated PGCC after undergoing multiple cycles of endoreplication without cell division under olaparib treatment. FIG. 4C depicts a Hey PGCC re-entering the mitosis cycle to generate daughter cells with strong self-renewal capacity. FIG. 4D depicts a mononucleated PGCC generates a multinucleated PGCC via restitutional multipolar endomitosis. Arrows in FIGS. 4A-4D indicate micronuclei.

[0029] FIGS. 5A-5E are a series of graphs which illustrate that mifepristone inhibits olaparib- mediated PGCC formation by promoting apoptosis. FIG. 5A illustrates apoptosis data for Hey cells and Hey PGCCs were treated with vehicle (0.1% DMSO) or olaparib at the indicated concentrations for 7 days, stained with PI, and analyzed with FACS. Representative percentages of dead cells are shown on the left, and statistical analysis results are shown on the right. FIG. 5B illustrates quantitative analysis of olaparib sensitivity in Hey and Hey PGCC daughter cells. Hey cells and Hey PGCC-derived daughter (Dau) cells (derived from a single PGCC or pooled PGCCs) were exposed to the indicated increasing concentrations of olaparib for 7 days and assayed by PI staining. Data represent mean ± SD from two independent experiments. FIG. 5C illustrates cell viability data for Hey cells treated with the indicated concentrations of mifepristone (MF) alone, olaparib alone, or a combination of both drugs for 7 days. Cell viability was determined by PI-FACS. Percentages of dead cells are shown on the left, and statistical analysis results are shown on the right. FIG. 5D illustrates apoptosis data for Hey cells treated with the indicated concentrations of MF or olaparib alone or with a combination of both drugs for 3 or 7 days. Apoptotic cells were identified by Annexin-V-PI staining. Q2 and Q3 represent the late apoptotic cells and early apoptotic cells, respectively. Statistical analysis results are shown on the right. FIG. 5E illustrates cell viability data for Hey cells exposed to 50 pM olaparib with or without MF for 7 days to induce PGCC formation. Polyploidy was measured by PI-FACS analysis, and the percentage of PGCCs is shown. Data are shown as mean ± SD in FIGS. 5A-5E. Each data point corresponds to one biological replicate in FIGS. 5A, 5C, 5D and 5E. The exact P values are shown on the graph (Welchs /-test).

[0030] FIGS. 6A-6D are a flowchart and series of graphs and images which illustrate that olaparib enhances polyploidy in patient-derived ovarian cancer organoids. FIG. 6A is a flowchart of procedures for establishing organoids from patient-derived xenografts. MACS, magnetic-activated cell sorting. FIG. 6B is phase-contrast images of 3 HGSC organoids (left) together with H&E and immunohistochemical staining (right) in organoids and corresponding parental tumors. Tumors and organoids showed robust expression of p53, PAX8 (a marker of serous subtype), and WT1 in the nucleus. Scale bars, 100 pm (right panel). FIG. 6C illustrates PI-FACS analysis (left) and quantification (right) of polyploidy in human ovarian cancer-derived organoids. Organoids (Org) were exposed to vehicle (DMSO) or olaparib at the indicated concentrations for 7 days, then collected and dissociated into single cells. Polyploidy was determined by PI-FACS analysis. FIG. 6D illustrates PI-FACS analysis (left) and quantification (right) of polyploidy in Org-2414. Org-2414 was exposed to the indicated concentrations of mifepristone (MF), 50 pM olaparib, or a combination of both drugs for 7 days. Data are shown as mean ± SD in FIGS. 6C-6D. Each data point corresponds to one biological replicate. The exact P values are shown on the graph (Welchs /-test).

[0031] FIGS. 7A-7C are a series of graphs which illustrate that mifepristone mitigates tumor growth in ovarian HGSC PDX models. FIG. 7A illustrates tumor volume data for ovarian HGSC PDX-3008 xenografts BRCA^ olaparib-naive tumors) treated with olaparib (n = 6), mifepristone (n = 7). vehicle (n = 5), or olaparib in combination with mifepristone (n = 7) for 60 days. The left panel shows the mean tumor volume in the xenograft-bearing mice at the indicated times; the right panel shows the tumor mass after 60 days of treatment. Mifepristone monotherapy and mifepristone/olaparib treatment significantly suppressed tumor growth compared with vehicle (two-way ANOVA, ****/> < 0.0001). Tumors treated with mifepristone alone (Welch’s /-test, ***p = 0.0002) and those treated with mifepristone/olaparib (****p < 0.0001) had significantly smaller masses at harvest than did vehicle-treated tumors. Error bars indicate SD. FIG. 7B illustrates tumor volume data for olaparib-resistant BRCA^ ovarian HGSC PDX-2445 xenografts following harvested, reimplantation, and expansion. Tumor-bearing mice were then treated with olaparib (n = 7), mifepristone (n = 5), vehicle (n = 9), or mifepristone/olaparib (n = 8) for 60 days. The left panel shows the mean tumor volume in the xenograft-bearing mice at the indicated times; the right panel shows the tumor mass after 60 days of treatment. Both mifepristone monotherapy (two-way ANOVA, ****P < 0.0001) and mifepristone/olaparib combination therapy (****? < 0.0001) significantly suppressed tumor growth compared with vehicle treatment. Tumors treated with mifepristone monotherapy (Welchs /-test, **P = 0.0061) and mifepristone/olaparib combination therapy (*P = 0.0257) had significantly smaller masses at harvest than did vehicle-treated tumors. Error bars indicate SD. FIG. 7C illustrates olaparib-resistant BRCA^ ovarian HGSC PDX-2428 xenografts treated with olaparib (n = 8), mifepristone (n = 8), vehicle (n = 8), or mifepristone/olaparib (n = 7) for 53 days. The left panel shows the mean tumor volume in xenograft-bearing mice at the indicated times; the right panel shows the tumor mass after 53 days of treatment. Both mifepristone monotherapy (two-way ANOVA, ****P < 0.0001) and mifepristone/olaparib combination treatment (**** < 0.0001) significantly suppressed tumor growth compared with vehicle treatment. Tumors treated with mifepristone monotherapy (Welchs /-test, ****P < 0.0001) and mifepristone/olaparib combination therapy (*** = 0.0006) had significantly smaller masses at harvest than did vehicle- treated tumors. Error bars indicate SD.

[0032] FIG. 8 is a schematic model on how mifepristone potentiates olaparib-induced therapeutic response and blocks acquired resistance to PARPi in ovarian cancer. Continuous exposure to olaparib results in unrepaired DNA damage, causing the activation of the aberrant endoreplication cell cycle. Cells that undergo endoreplication develop into senescent PGCCs, which give rise to daughter cells via a variety of modes of depolyploidization. The daughter cells acquired resistance via the life cycle of PGCCs and escaped senescence, eventually leading to tumor recurrence. The combined use of mifepristone and olaparib could block the development of PGCCs, thereby suppressing tumor growth. In tumors with acquired resistance, mifepristone can directly attenuate tumor growth, possibly by inducing differentiation toward benign lineages.

[0033] FIG. 9 is images of high-grade serous carcinoma (HGSC) cancer cells. The top and middle rows show representative H&E-stained sections of BRCA^ and BRCA l''ⁿ HGSD PDXs showing that PGCC formation was induced in response to olaparib treatment. The bottom row shows representative Ki-67 staining in sections of BRCAl''^['' ^[ HGSC PDXs showing that PGCCs can proliferate, as indicated by the robust expression of Ki-67. Black arrows indicate PGCCs. Scale bars, 50 pm.

[0034] FIGS. 10A-10C are a series of graphs which illustrate data concerning PGCC induction by PARP inhibitors in various cancer cell lines. FIG. 10A illustrates dose-response curves for olaparib and niraparib for indicated cell lines. Cells were treated with olaparib or niraparib for 5 days and assayed with CCK-8 Kit. Each data point corresponds to 4 biological replicates. Error bars indicate SD. FIG. 10B illustrates PI-FACS analysis (top three rows) and quantification (bottom row) of polyploidy in OVCAR8, OBCAR5, and PEO-1 cell lines. Cells were treated with DMSO or olaparib at the indicated concentration for 7 days. Each data point corresponds to 2 biological replicates. Data are shown as mean ± SD. The exact P values are shown on their graph (Welch’s /-test). FIG. IOC illustrates PI-FACS analysis (top three rows) and quantification (bottom row) of polyploidy in Hey, MCF-7, OVCA432, SKOV3, 0VCAR8, 0VCAR5, and PEO-1 cell lines. Cells were treated with DMSO or niraparib at the indicated concentration for 7 days. Each data point corresponds to 2 biological replicates. Data are shown as mean ± SD. The exact P values are shown on their graph (Welch’s /-test).

[0035] FIGS. 11A-10C are a series of heatmaps and images which illustrate transcriptome signatures of PGCCs at different stages of PGCC induction by olaparib. FIG. 11A is heatmaps of differentially expressed genes in DMSO-treated human cancer cell lines or organoids versus PGCCs. NC-D3: DMSO treatment, day 3; PGCC D3, D7, R7 (cell lines only), R14 (organoids only): olaparib-induced PGCCs on indicated treatment days (D) or recovery days (R). Shading denotes upregulated and downregulated genes. The most remarkable gene expression changes were observed on day 7 of olaparib incubation. FIG. 11B illustrates gene set enrichment analysis (GSEA) of representative pathways enriched in PGCCs versus DMSO-treated cell lines or organoids. FIG. 11C is a series of images of Western blotting of lysates from DMSO-treated Hey cells or Hey PGCCs was performed at the indicated times with the indicated antibodies. The values under the lane are normalized protein expression (the gray value of the target protein divided by the gray value of the actin of the corresponding sample) analyzed with Image J 1.52 software. Data are representative of 2 independent experiments.

[0036] FIGS. 12A-12F is a series of time-lapse tracking images of PGCC division in Hey cells. FIG. 12A is images of Hey cells labeled with FUCCI, showing a mononucleated PGCC dividing via multipolar mitosis and giving rise to daughter cells continuously undergoing endocycling. FIGS. 12B-12F are images of chromosomes labeled with histone H2B-mCherry and microtubules with EGFP-a-tubulin in FIGS 12B-12F. FIG. 12B is an image showing typical bipolar mitosis process in a Hey cell. During mitosis, chromosomes are condensed, then align in the center of the dividing cell at 4:00 (h:mm). The chromosomes are separated into the nuclei of 2 daughter cells (5:30), and cytokinesis is completed. FIG. 12C is an image of an example of a PGCC giving rise to daughter cells via bipolar mitosis. FIG. 12D is images of PGCCs dividing via multipolar mitosis. Three daughter cells were formed after tripolar mitosis (2:45), and 2 fuse shortly thereafter, forming 1 binucleated cell (16:00). The white arrow at 2:45 indicates the midbody, and the arrows indicate micronuclei. FIG. 12E is an image of PGCC division by multipolar mitosis. Three daughter cells were formed, and each daughter cell contains fragmented chromosomes. Arrows indicate micronuclei. FIG. 12F is an image of PGCC division by acytokinetic mitosis. Arrows indicate micronuclei.

[0037] FIGS. 13A-13C is a series of plots which indicate that PGCC-derived daughter cells exhibit high resistance to olaparib. FIG. 13A illustrates Hey cells and Hey PGCC-derived daughter (Dau) cells (derived from a single PGCC or pooled PGCCs) were exposed to the indicated concentrations of olaparib for 7 days, and cell death was assayed by PI staining and FACS. At least 10,000 events were recorded per sample, and the percentages of dead cells are shown. FIG. 13B illustrates growth curves of Hey cells and Hey PGCC daughter cells. Cells were seeded on a 96-well plate at 2000 cells/well. Cell proliferation was determined with a CCK- 8 kit every other day. FIG. 13C illustrates dose-response curves for Hey cells and Hey PGCC daughter cells. Cells were treated with carboplatin or paclitaxel for 5 days and assayed with a CCK-8 kit. In FIGS. 13B-13C, each data point corresponds to 4 biological replicates. Error bars indicate SD. The exact P values are shown on each graph (two-way ANOVA).

[0038] FIG. 14 is a series of plots and graphs which illustrate the effect of mifepristone/olaparib combination treatment on apoptosis. OVCA432, MCF-7, and SKOV3 cells were exposed to DMSO (control), mifepristone (MF, 25 pM), olaparib at the indicated concentrations, or the combination of olaparib and mifepristone for 3 (for MCF-7) or 7 days (for OVCA 432 and SKOV3). Apoptosis was assayed by Annexin V-FITC and PI staining. At least 10,000 events were recorded per sample. Statistical analysis results are shown on the right. Data are shown as mean ± SD, and each data point corresponds to one biological replicate. The exact P values are shown on the graph (Welchs /-test).

[0039] FIGS. 15A-15D are a series of plots and images which illustrate the effect of olaparib on the proportion of PGCCs in HGSC organoids. FIG. 15A illustrates dose-response curves for Org-2414, Org-2445, and Org-3008 organoids treated with olaparib. Each data point corresponds to 4 biological replicates. Error bars indicate SD. FIG. 15B is phase-contrast images (top) and quantification (bottom) of P-galactosidase (P-gal) staining of organoid-2414. Organoid-2414 was treated with vehicle (0.1% DMSO) or 50 pM olaparib for 7 days, recovered in drug-free medium for another 7 days, and then stained for 13-gal. Scale bars, 50 pm. Four randomly selected fields per group (10x objective lenses) were used for quantification. FIG. 15C is immunofluorescence images of y-H2AX foci, p!6INK4a expression, and p21 expression (top) and quantification of y- H2AX foci (bottom) in organoid-2414. Organoid-2414 was dissociated into single cells and seeded on a 12-well cell culture plate. After attaching to the plate, cells were treated with 0.1% DMSO or 50 j M olaparib for 7 days, and immunofluorescence assays were conducted. Scale bars, 50 jun. At least 50 cells per group were counted and used for y-H2AX foci quantification. Data are shown as mean ± SD. The exact P values are shown on the graph (Welchs /-test). FIG. 15D illustrates apoptosis data for Org-2414, Org-2445, and Org-3008 exposed to DMSO (control), mifepristone (MF, 25 pM), olaparib at the indicated concentrations, or the combination of olaparib and mifepristone for 7 days. Apoptosis was assayed by Annexin V-FITC and PI staining. At least 10,000 events were recorded per sample. Statistical analysis results are shown on the right. Data are shown as mean ± SD. Each data point corresponds to one biological replicate in FIGS. 15B and 15D. The exact P values are shown on the graph (Welchs /-test). [0040] FIGS. 16A-16C are a series of graphs and images which illustrate that mifepristone monotherapy and mifepristone/olaparib combination therapy both suppress tumor growth in ovarian HGSC PDXs with acquired olaparib resistance. Representative H&E-stained sections and immunohistochemistry (left) and quantification (right) of Ki-67 expression are shown in FIG. 16A for PDX-3008, FIG. 16B for PDX-2445, and FIG. 16C for PDX-2428 tumors from the indicated treatment groups. Scale bars, 200 pm. Data are shown as mean ± SD. Each data point corresponds to one independent sample. The exact P values are shown on the graph (Welch’s /-test).

DETAILED DESCRIPTION OF THE INVENTION

[0041] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0042] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0043] As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. [0044] As used herein, the term “about” in association with a numerical value is meant to include any additional numerical value reasonably close to the numerical value indicated. For example, and based on the context, the value can vary up or down by 5-10%. For example, for a value of about 100, means 90 to 110 (or any value between 90 and 110).

[0045] As used herein and in the claims, the terms “comprising,” “containing,” and “including” are inclusive, open-ended and do not exclude additional unrecited elements, compositional components or method steps. Accordingly, the terms “comprising” and “including” encompass the comparably more restrictive terms “consisting of’ and “consisting essentially of.”

[0046] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0048] The present invention is based on the seminal discovery that glucocorticoid receptor antagonist compounds increase the efficacy of PARPi treatment against PARPi-naive cancer. While PARPi treatment can induce DNA damage and inhibit mitosis in some cancers, many advanced-stage cancers develop replication-competent, PARPi-resistant PGCCs, rendering PARPi monotherapies ineffective for treatment. The inclusion of a glucocorticoid receptor antagonist with the PARPi can synergistically block endoreplication and survival of the PGCCs, increasing the anti cancer efficacy above that of either drug administered alone.

[0049] While use of glucocorticoid receptor antagonists is disclosed as an illustrative example, it should be understood that the present disclosure also includes other ways to reduce glucocorticoid activity. For example, the disclosure includes the use of anti-glucocorticoids including drugs that reduce glucocorticoid activity in the body. By way of example, such drugs or agents include: 1) Direct glucocorticoid receptor antagonists, such as mifepristone; 2) Synthesis inhibitors such as metyrapone, ketoconazole, and aminoglutethimide; and 3) anabolic steroids that prevent cortisol from binding to the glucocorticoid receptor. [0050] The examples cover several different preclinical ovarian cancer models, including multiple ovarian cell lines with or without p53 mutation and one breast cancer cell line, HGSC organoids, and patient-derived xenograft (PDX) models, to investigate the role of PGCCs in the therapeutic response as well as PARPi resistance in ovarian cancer. The phenotype and division of PGCCs into daughter cells were monitored to evaluate the therapeutic effect of PARPi and overcoming PARPi resistance by eliminating PGCCs.

[0051] As disclosed herein, treatment with the PARPi olaparib induces the formation of polyploid giant cancer cells (PGCCs) in ovarian and breast cancer cell lines (Hey, SKOV3, MCF-7), three human high-grade serous ovarian cancer-derived organoids, and high-grade serous ovarian cancer patient-derived xenografts. Live-cell fluorescence time-lapse tracking of ovarian cancer cells labeled with FUCCI (fluorescent ubiquitination cell-cycle indicator) or histone H2B-mCherry/a-tubulin-EGFP revealed that PGCCs primarily developed from the endoreplication of diploid cancer cells after exposure to sublethal concentrations of olaparib. PGCCs exhibited markers of senescent cells; however, they were able to escape senescence to generate mitotically competent daughter cells via budding, multipolar mitosis, and acytokinetic mitosis following olaparib withdrawal. PGCCs and derived daughter cells conferred resistance to olaparib-induced cytotoxicity, which could be blocked by mifepristone. Whole transcriptome analysis by RNA sequencing of ovarian cancer cell lines and ovarian cancer- derived organoids showed activation of a senescence-associated secretory phenotype with upregulated cytokines and chemokines and downregulation of MYC signaling in PGCCs. PARPi/glucocorticoid receptor antagonist (e.g., mifepristone/olaparib) combination therapy significantly mitigated tumor growth in patient-derived xenograft models with acquired resistance to a glucocorticoid receptor antagonist (e.g., olaparib). Thus, targeting of PGCCs overcomes PARPi resistance in recurrent ovarian cancer.

[0052] Mifepristone/olaparib combination therapy significantly reduced tumor growth in PDX models without previous exposure to olaparib, while mifepristone alone decreased tumor growth in PDX models with acquired resistance to olaparib. Thus, targeting PGCCs increases the therapeutic effect of PARPi and overcoming P ARPi-induced resistance.

[0053] Leveraging this discovery, one embodiment of the present invention provides a method of treating cancer in a subject which includes administering a therapeutically effective amount of a poly(ADP -ribose) polymerase inhibitor (PARPi) and a glucocorticoid receptor antagonist to a subject who has not previously received PARPi treatment, thereby treating the cancer.

[0054] As used herein, the term “subject” refers to any individual or patient to which the disclosed methods are performed, to whom the disclosed compositions are administered, or from whom a biological material (e.g., a tissue sample, a cell, or a biofluid) is obtained. Generally, the subject is human, although as will be appreciated by those in the art, the subject may be a non-human animal. Thus, other animals, including vertebrate such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, chickens, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.

[0055] The term "treatment" is used interchangeably herein with the term "therapeutic method" or “therapy” and refers to 1) therapeutic treatments or measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic conditions or disorder (e.g., idiopathic pulmonary fibrosis), and/or 2) prophylactic/ preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder (i.e., those needing preventive measures).

[0056] The terms “administration of’ and or “administering” should be understood to mean providing a pharmaceutical composition in a therapeutically effective amount to the subject in need of treatment. Administration routes can be enteral, topical or parenteral. As such, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal, oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, and nebulization. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration. In some aspects, administering includes intravenous injection, intrapulmonary administration, intratracheal administration, intrabronchial administration, intranasal administration, nebulization, powder inhalation, intrapulmonary injection, intraperitoneal, intrathecal, or pulmonary artery infusion. In some cases, the administering includes intravenous or intrapulmonary administration.

[0057] The methods of the present invention utilize a range of PARP inhibitors. As used herein, the terms “PARP inhibitor” and “PARPi” refer to compounds which reduce the enzymatic activity of poly(ADP-ribose) polymerase (PARP). PARP is an abundant family of proteins that generate polymeric adenosine diphosphate ribose (PAR) in the presence of singlestrand DNA breaks. Examples of PARP proteins include PARP1, PARP2, PARP3, PARP6, PARP7, PARP8, PARP9, PARP 10, PARP11, PARP 12, PARP 14, PARP 15, PARP 16, VP ARP, Tankyrase-1, and Tankyrase-2. PARP activity modulates a number of pathways, including DNA repair, metabolism, and inflammatory gene expression. As disclosed herein, PARP inhibition induces the formation of polyploid giant cancer cells (PGCCs) in numerous cancer cell lines. Many advanced-stage cancers develop replication-competent, PARPi-resistant PGCCs, often rendering PARPi monotherapies ineffective for treatment.

[0058] As non-limiting examples, a PARPi can have an ICso of at most about 50 pM, at most about 25 pM, at most about 10 pM, at most about 5 pM, at most about 2 pM, at most about 1 pM, at most about 500 nM, at most about 250 nM, at most about 100 nM, at most about 50 nM for a PARP (e.g., human PARPI). A PARPi can be a small molecule (e.g., olaparib), a nucleic acid, an oligopeptide, a polypeptide, or a combination thereof. The PARPi can reduce the enzymatic activity of a particular PARP enzyme, such as PARPI, PARP2, PARP3, PARP6, PARP7, PARP8, PARP9, PARP 10, PARPI 1, PARP 12, PARP 14, PARP 15, PARP 16, VP ARP, Tankyrase-1, Tankyrase-2, or an alternatively spliced or processed analogue thereof. The PARPi can also reduce the enzymatic activity of multiple PARP enzymes, such as PARP 1-4.

[0059] As PARP inhibitors can exhibit disparate partitioning and PARP -inhibitory activity (e.g., olaparib typically exhibits greater inhibition of PARPI and PARP2 than PARP3 or PARP 4), the method utilizes a single PARP inhibitor or a combination of PARP inhibitors for cancer treatment. As nonlimiting examples, the PARP inhibitor can be olaparib, niraparib, rucaparib, talazoparib, veliparib, iniparib, AZD 2461, CEP 9722, E7016, INO-1001, LT-673, MP-124, NMS-P118, XAV93, or a combination thereof. In one particular aspect, the PARPi is olaparib, niraparib, rucaparib, talazoparib, or a combination thereof. In a particular aspect, the PARPi is olaparib. As demonstrated herein, olaparib not only exhibits high efficacy against numerous cancers such as breast and ovarian cancer, but can exhibit enhanced activity against these cancers when paired with a glucocorticoid receptor antagonist such as mifepristone.

[0060] As used herein, the term "effective amount" of an active agent refers an amount that is non-toxic to a subject but is an amount of the active agent that is sufficient to provide a desired effect (e.g., an amount effective to inhibit DNA repair in cancer cells in a subject). This amount may vary from subject to subject, depending on the species, age, and physical condition of the subject, the severity of the disease that is being treated, the particular conjugate, or more specifically, the particular active agent used, its mode of administration, and the like. Therefore, it is difficult to generalize an exact "effective amount," yet, a suitable effective amount may be determined by one of ordinary skill in the art.

[0061] In one aspect, the therapeutically effective amount of the PARPi is an amount sufficient to inhibit DNA repair in cancer cells from the subject. Certain cancers are highly responsive to low PARPi doses. In particular, BRCA1 or 2 mutant breast and ovarian cancers, which can lack BRCA^1/2-mediated repair of homologous recombination, can exhibit heightened reliance on PARP activity for DNA damage repair, and thus can exhibit heightened responsiveness to PARPi treatment. In one aspect, the therapeutically effective amount of the PARPi is between about 5 and 250 mg/kg/day. In some aspects, the therapeutically effective amount of the PARPi can be between about 5 and 25 mg/kg/day, between about 10 and 50 mg/kg/day, between about 25 and 75 mg/kg/day, between about 40 and 100 mg/kg/day, between about 60 and 150 mg/kg/day, or between about 100 and 250 mg/kg/day. In some aspects, the PARPi is administered at a dose of less than about 5 mg/kg/day, for example between about 1 and 5 mg/kg/day, between about 0.2 and 2 mg/kg/day, or between about 0.05 and 0.5 mg/kg/day. In some aspects, the therapeutically effective dose of the PARPi is greater than 250 mg/kg/day, for example between about 250 mg/kg/day and 1 g/kg/day. In some aspects, the therapeutically effective amount of the PARPi is about 1 mg/kg/day, about 5 mg/kg/day, about 10 mg/kg/day, about 15 mg/kg/day, about 20 mg/kg/day, about 25 mg/kg/day, about 30 mg/kg/day, about 40 mg/kg/day, about 50 mg/kg/day, about 60 mg/kg/day, about 70 mg/kg/day, about 80 mg/kg/day, about 90 mg/kg/day, about 100 mg/kg/day, about 110 mg/kg/day, about 120 mg/kg/day, about 130 mg/kg/day, about 140 mg/kg/day, about 150 mg/kg/day, about 160 mg/kg/day, about 170 mg/kg/day, about 180 mg/kg/day, about 190 mg/kg/day, about 200 mg/kg/day, about 210 mg/kg/day, about 220 mg/kg/day, about 230 mg/kg/day, or about 240 mg/kg/day, about 250 mg/kg/day. In some aspects, the therapeutically effective amount of the PARPi is about 50 mg/kg/day (e.g., 45 to 55 mg/kg/day or 47.5 to 52.5 mg/kg/day, two 22.5-27.5 mg/kg doses per day, one 45 to 55 mg/kg dose per day, one 225 to 275 mg/kg dose every five days, or one 450 to 550 mg/kg dose every ten days).

[0062] In one aspect, the glucocorticoid receptor antagonist is relacorilant, metyrapone, ketoconazole, aminoglutethimide, mifepristone, or a combination thereof. As used herein, the term “glucocorticoid receptor antagonist” can be used interchangeably with “antiglucocorticoid” and “glucocorticoid blocker” can refer to species which antagonizes (e.g., blocks or allosterically inhibits) a glucocorticoid receptor, diminishes binding between a glucocorticoid receptor and an agonist, or suppresses a downstream response from glucocorticoid receptor activation (e.g., transactivation). As non-limiting examples, a glucocorticoid receptor antagonist can have an IC50 of at most about 50 pM, at most about 25 pM, at most about 10 pM, at most about 5 pM, at most about 2 pM, at most about 1 pM, at most about 500 nM, at most about 250 nM, at most about 100 nM, or at most about 50 nM for a glucocorticoid receptor (e.g., a type 1 or type 2 glucocorticoid receptor). In particular aspects of the disclosed methods, the glucocorticoid receptor antagonist is mifepristone.

[0063] In one aspect, the therapeutically effective amount of the glucocorticoid receptor antagonist is between about 2.5 and 125 mg/kg/day. For example, the therapeutically effective amount of the glucocorticoid receptor antagonist can be between about 2.5 and 12.5 mg/kg/day, between about 5 and 25 mg/kg/day, between about 10 and 50 mg/kg/day, between about 25 and 75 mg/kg/day, between about 40 and 100 mg/kg/day, or between about 50 and 125 mg/kg/day. In some aspects, the therapeutically effective amount of the glucocorticoid receptor antagonist is about 0.5 mg/kg/day, about 1 mg/kg/day, about 5 mg/kg/day, about 10 mg/kg/day, about 15 mg/kg/day, about 20 mg/kg/day, about 25 mg/kg/day, about 30 mg/kg/day, about 40 mg/kg/day, about 50 mg/kg/day, about 60 mg/kg/day, about 70 mg/kg/day, about 80 mg/kg/day, about 90 mg/kg/day, about 100 mg/kg/day, about 110 mg/kg/day, or about 125 mg/kg/day. In a particular aspect, the therapeutically effective amount of the glucocorticoid receptor antagonist is about 25 mg/kg/day.

[0064] As it was discovered herein that an excess of PARPi relative to glucocorticoid receptor antagonist is efficacious for treating cancer (e.g., as outlined in EXAMPLE 7), the therapeutically effective amount of the PARPi can be greater than the therapeutically effective amount of the glucocorticoid receptor antagonist. In some aspects, the therapeutically effective amount of the PARPi is about 1 to 25 times, about 1.25 to 25 times, about 1.5 to 25 times, about 2 to 25 times, about 5 to 25 times, about 10 to 25 times, about 1 to 10 times, about 1.25 to 10 times, about 1.5 to 10 times, about 2 to 10 times, about 5 to 10 times, about 1 to 5 times, about 1.25 to 5 times, about 1.5 to 5 times, about 2 to 5 times, about 1 to 2 times, about 1.25 to 2 times, or about 1.5 to 2 times the therapeutically effective amount of the glucocorticoid receptor antagonist. In some aspects, the therapeutically effective amount of the PARPi is about 1 to 10 times the therapeutically effective amount of the glucocorticoid receptor antagonist.

[0065] The method targets a wide range of cancers. In some aspects, the method targets a cancer with a heightened responsiveness to PARPi-treatment, for example a cancer with deficient DNA repair activity. In particular aspects, the cancer includes a mutation in a BRCA gene. In some aspects, the cancer is breast cancer, ovarian cancer, carcinoma, or adenocarcinoma. In some aspects, the cancer is carcinoma or adenocarcinoma. In some aspects, the cancer is breast cancer or ovarian cancer. In some aspects, the cancer is breast carcinoma, breast adenocarcinoma, ovarian carcinoma, or ovarian adenocarcinoma. In some aspects, the cancer is serous ovarian cancer. In a particular aspect, the cancer is resistant to a PARPi. For example, in some aspects, the cancer is resistant to a PARPi coadministered with a glucocorticoid receptor antagonist. In some aspects, the cancer is resistant to olaparib.

[0066] While it is demonstrated herein that cancers can generate PARPi-resistance over the course of treatment, it is further shown that PARPi-resistance can diminish following cessation of the PARPi treatment. In particular, the prevalence of PARPi-resistant PGCCs and PARPi- resistant PGCC-derived daughter cells can return to baseline following cessation of the PARPi treatment, in some cases enabling efficacious PARPi-glucocorticoid receptor antagonist combination therapy. In some aspects, the subject has not previously received PARPi treatment with the previous 15 days, within the previous 20 days, within the previous 25 days, within the previous 30 days, within the previous 40 days, within the previous 50 days, within the previous 60 days, within the previous 70 days, within the previous 80 days, within the previous 90 days, within the previous 100 days, or within the previous year. In another aspect, the subject does not exhibit elevated levels of polyploid giant cancer cells (PGCCs) or endoreplicating cancer cells (e.g., the prevalence of PGCCs or endoreplicating cells does not diminish between biopsies collected from the subject over 1 week, 2 weeks, 3 weeks, or 4 weeks). In some cases, the subject does not exhibit elevated levels of PGCCs with increased SA-|3-gal activity. In some cases, the subject does not exhibit elevated levels of p21 positive PGCCs. In some cases, the subject does not exhibit elevated levels of PGCCs with increased y-H2AX foci. In some cases, the subject does not exhibit elevated levels of PGCCs with increased secretion of SASP factors.

[0067] In one embodiment, the present invention provides a method of treating cancer in a subject, wherein the subject has previously received poly(ADP-ribose) polymerase inhibitor (PARPi) treatment including: ceasing the PARPi treatment; and, at least ten days after cessation of the PARPi treatment, administering a therapeutically effective amounts of a poly(ADP- ribose) polymerase inhibitor (PARPi) and an glucocorticoid receptor antagonist to the subject in combination, thereby treating the cancer. In some aspects, the therapeutically effective amounts of the PARPi and the glucocorticoid receptor antagonist are administered at least 15 days, at least 20 days, at least 25 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, or at least 100 days after cessation of the PARPi treatment.

[0068] It is disclosed herein that PARPi-treatment promotes PGCC development by inducing cancer cells to shift from mitosis to endoreplication, and that such PGCCs exhibit heightened resistances to PARPi and apoptosis. However, it also is also disclosed that such cancers are often responsive to glucocorticoid receptor antagonist treatment, and that such treatments can exhibit greater efficacy when not paired with a PARPi.

[0069] Leveraging this discovery, the present disclosure provides a method of treating cancer in a subject including administering a therapeutically effective amount of a glucocorticoid receptor antagonist to a subject who has an elevated polyploid giant cancer cell (PGCC) level, an elevated endoreplicating cancer cell level, or a combination thereof, thereby treating the cancer. In some aspects, the method does not include administering a PARPi.

[0070] In one aspect, the glucocorticoid receptor antagonist is relacorilant, metyrapone, ketoconazole, aminoglutethimide, mifepristone, or a combination thereof. In another aspect, the glucocorticoid receptor antagonist is mifepristone. In one aspect, the therapeutically effective amount of the glucocorticoid receptor antagonist is between about 5 and 250 mg/kg/day. In some aspects, the therapeutically effective amount of the glucocorticoid receptor antagonist is between about 5 and 25 mg/kg/day, between about 10 and 50 mg/kg/day, between about 25 and 75 mg/kg/day, between about 40 and 100 mg/kg/day, between about 60 and 150 mg/kg/day, or between about 100 and 250 mg/kg/day. In some aspects, the glucocorticoid receptor antagonist is administered at a dose of less than about 5 mg/kg/day, for example between about 1 and 5 mg/kg/day, between about 0.2 and 2 mg/kg/day, or between about 0.05 and 0.5 mg/kg/day. In some aspects, the therapeutically effective dose of the glucocorticoid receptor antagonist is greater than 250 mg/kg/day, for example between about 250 mg/kg/day and 1 g/kg/day. In some aspects, the therapeutically effective amount of the glucocorticoid receptor antagonist is about 1 mg/kg/day, about 5 mg/kg/day, about 10 mg/kg/day, about 15 mg/kg/day, about 20 mg/kg/day, about 25 mg/kg/day, about 30 mg/kg/day, about 40 mg/kg/day, about 50 mg/kg/day, about 60 mg/kg/day, about 70 mg/kg/day, about 80 mg/kg/day, about 90 mg/kg/day, about 100 mg/kg/day, about 110 mg/kg/day, about 120 mg/kg/day, about 130 mg/kg/day, about 140 mg/kg/day, about 150 mg/kg/day, about 160 mg/kg/day, about 170 mg/kg/day, about 180 mg/kg/day, about 190 mg/kg/day, about 200 mg/kg/day, about 210 mg/kg/day, about 220 mg/kg/day, about 230 mg/kg/day, or about 240 mg/kg/day, about 250 mg/kg/day. In some aspects, the therapeutically effective amount of the glucocorticoid receptor antagonist is about 50 mg/kg/day.

[0071] In one aspect, the elevated PGCC level, the elevated endoreplicating cancer cell level, or the combination thereof is at a level of at least about 3%, at least about 5%, at least about 7.5%, at least about 10%, at least about 12.5%, at least about 15%, at least about 17.5%, at least about 20%, at least about 22.5%, at least about 25%, at least about 27.5%, or at least about 30% among cells from a biopsy sample from the subject. In one aspect, the subject has an elevated PGCC level, of at least about 3%, at least about 5%, at least about 7.5%, at least about 10%, at least about 12.5%, at least about 15%, at least about 17.5%, at least about 20%, at least about 22.5%, at least about 25%, at least about 27.5%, or at least about 30% among cells from a biological sample from the subject.

[0072] In some aspects, the biological sample includes saliva, blood, plasma, serum, vaginal fluid, interstitial fluid, ocular fluid, seat, mucus, glandular secretion, nipple aspirate, seme, spinal fluid, emphatic fluid, sputum, pus, huffy coat, tumor tissue, circulating tumor cells, skin tissue, lung tissue, kidney tissue, bone marrow, pancreatic tissue, liver tissue, muscle tissue, gall bladder tissue, colon tissue, brain tissue, prostate tissue, esophageal tissue, thyroid tissue, a solid-tumor biopsy, a liquid tumor biopsy, or a combination thereof.

[0073] In some aspects, the biological sample is a tumor biopsy sample. The tumor biopsy sample can be isolated from the subject through any known technique, including surgery or fine needle aspirate. As non-limiting examples, the biopsy can be a core biopsy, an incisional biopsy, an excisional biopsy, an endoscopic biopsy, a liquid biopsy, a shave biopsy, a percutaneous biopsy, or a fine needle aspiration. In a particular aspect, the tumor biopsy sample is a solid-tumor biopsy sample.

[0074] In some aspects, the cancer is breast cancer, ovarian cancer, carcinoma, or adenocarcinoma. In some aspects, the cancer is carcinoma or adenocarcinoma. In some aspects, the cancer is breast cancer or ovarian cancer. In some aspects, the cancer is breast carcinoma, breast adenocarcinoma, ovarian carcinoma, or ovarian adenocarcinoma. In some aspects, the cancer is serous ovarian cancer. In some aspects, the cancer includes a mutation in a BRCA gene.

[0075] In another aspect, the subject received PARPi treatment within the previous day, within the previous 2 days, within the previous 3 days, within the previous 4 days, within the previous 5 days, within the previous 6 days, within the previous 7 days, within the previous 10 days, within the previous 15 days, within the previous 20 days, within the previous 25 days, within the previous 30 days, within the previous 40 days, within the previous 50 days, within the previous 60 days, within the previous 70 days, within the previous 80 days, within the previous 90 days, within the previous 100 days, or within the previous year. In another aspect, the PARPi is olaparib.

[0076] In one embodiment, the present invention provides a method of identifying a subject with cancer as a candidate for treatment with a poly(ADP-ribose) polymerase inhibitor (PARPi) and an glucocorticoid receptor antagonist including: measuring an abundance of polyploid giant cancer cells (PGCCs), endoreplicating cancer cells, or a combination thereof in a biological sample from the subject; and classifying the subject as having a cancer cell PGCC abundance, an endorepli eating cancer cell abundance, or a combination thereof of at most 5% as a likely responder to the PARPi and the glucocorticoid receptor antagonist, thereby identifying the subject as suitable for treatment with the PARPi and the glucocorticoid receptor antagonist, or classifying the subject as having a cancer cell PGCC abundance, an endoreplicating cancer cell abundance, or a combination thereof of at most 5% as a likely nonresponder to the PARPi and the glucocorticoid receptor antagonist, thereby identifying the subject as unsuitable for treatment with the PARPi and the glucocorticoid receptor antagonist; thereby identifying the subject with cancer as a candidate for treatment with the PARPi and the glucocorticoid receptor antagonist.

[0077] In one aspect, the PARPi is olaparib, niraparib, rucaparib, talazoparib, or a combination thereof. In a particular aspect, the PARPi is olaparib. In one aspect, the treatment with the PARPi includes administration of between about 5 and 25 mg/kg/day, between about 10 and 50 mg/kg/day, between about 25 and 75 mg/kg/day, between about 40 and 100 mg/kg/day, between about 60 and 150 mg/kg/day, or between about 100 and 250 mg/kg/day. In one aspect, the treatment with the PARPi includes administration of between about 5 and 250 mg/kg/day. In some aspects, the treatment with the PARPi includes administration of about 1 mg/kg/day, about 5 mg/kg/day, about 10 mg/kg/day, about 15 mg/kg/day, about 20 mg/kg/day, about 25 mg/kg/day, about 30 mg/kg/day, about 40 mg/kg/day, about 50 mg/kg/day, about 60 mg/kg/day, about 70 mg/kg/day, about 80 mg/kg/day, about 90 mg/kg/day, about 100 mg/kg/day, about 110 mg/kg/day, about 120 mg/kg/day, about 130 mg/kg/day, about 140 mg/kg/day, about 150 mg/kg/day, about 160 mg/kg/day, about 170 mg/kg/day, about 180 mg/kg/day, about 190 mg/kg/day, about 200 mg/kg/day, about 210 mg/kg/day, about 220 mg/kg/day, about 230 mg/kg/day, or about 240 mg/kg/day, about 250 mg/kg/day. In a particular aspect, the treatment with the PARPi includes administration of about 50 mg/kg/day.

[0078] In one aspect, the glucocorticoid receptor antagonist is relacorilant, ketoconazole, aminoglutethimide, metyrapone, mifepristone, or a combination thereof. In a particular aspect, the glucocorticoid receptor antagonist is mifepristone. In some aspects, treatment with the glucocorticoid receptor antagonist includes administration of between about 2.5 and 12.5 mg/kg/day, between about 5 and 25 mg/kg/day, between about 10 and 50 mg/kg/day, between about 25 and 75 mg/kg/day, between about 40 and 100 mg/kg/day, or between about 50 and 125 mg/kg/day. In a particular aspect, the treatment with the glucocorticoid receptor antagonist includes administration of between about 2.5 and 125 mg/kg/day. In some aspects, treatment with the glucocorticoid receptor antagonist includes administration of about 0.5 mg/kg/day, about 1 mg/kg/day, about 5 mg/kg/day, about 10 mg/kg/day, about 15 mg/kg/day, about 20 mg/kg/day, about 25 mg/kg/day, about 30 mg/kg/day, about 40 mg/kg/day, about 50 mg/kg/day, about 60 mg/kg/day, about 70 mg/kg/day, about 80 mg/kg/day, about 90 mg/kg/day, about 100 mg/kg/day, about 110 mg/kg/day, or about 125 mg/kg/day. In one aspect, treatment with the glucocorticoid receptor antagonist includes administration of about 25 mg/kg/day.

[0079] In some aspects, the cancer is breast cancer, ovarian cancer, carcinoma, or adenocarcinoma. In some aspects, the cancer is carcinoma or adenocarcinoma. In some aspects, the cancer is breast cancer or ovarian cancer. In some aspects, the cancer is breast carcinoma, breast adenocarcinoma, ovarian carcinoma, or ovarian adenocarcinoma. In some aspects, the cancer is serous ovarian cancer. In some aspects, the cancer includes a mutation in a BRCA gene.

[0080] In some aspects, the biological sample includes saliva, blood, plasma, serum, vaginal fluid, interstitial fluid, ocular fluid, seat, mucus, glandular secretion, nipple aspirate, seme, spinal fluid, emphatic fluid, sputum, pus, huffy coat, tumor tissue, circulating tumor cells, skin tissue, lung tissue, kidney tissue, bone marrow, pancreatic tissue, liver tissue, muscle tissue, gall bladder tissue, colon tissue, brain tissue, prostate tissue, esophageal tissue, thyroid tissue, a solid-tumor biopsy, a liquid tumor biopsy, or a combination thereof. In some aspects, the biological sample is a tumor biopsy sample. In particular aspects, the tumor biopsy sample is a solid-tumor biopsy sample.

[0081] In one embodiment, the present invention provides a method of identifying a subject with cancer as a candidate for treatment with an glucocorticoid receptor antagonist including: measuring an abundance of polyploid giant cancer cells (PGCCs), endorepli eating cancer cells, or a combination thereof in a biological sample from the subject; and classifying the subject as having a cancer cell PGCC abundance, an endoreplicating cancer cell abundance, or a combination thereof of at least 5% as a likely responder to the glucocorticoid receptor antagonist, thereby identifying the subject as suitable for treatment with the glucocorticoid receptor antagonist; or classifying the subject as having a cancer cell PGCC abundance, an endoreplicating cancer cell abundance, or a combination thereof of at most 5% as a non-likely responder to the glucocorticoid receptor antagonist, thereby identifying the subject as unsuitable for treatment with the glucocorticoid receptor antagonist; thereby identifying the subject with cancer as a candidate for treatment with the glucocorticoid receptor antagonist.

[0082] In some aspects, the glucocorticoid receptor antagonist is relacorilant, metyrapone, ketoconazole, aminoglutethimide, mifepristone, or a combination thereof. In some aspects, the glucocorticoid receptor antagonist is mifepristone. In some aspects, treatment with the glucocorticoid receptor antagonist includes administration of between about 2.5 and 12.5 mg/kg/day, between about 5 and 25 mg/kg/day, between about 10 and 50 mg/kg/day, between about 25 and 75 mg/kg/day, between about 40 and 100 mg/kg/day, or between about 50 and 125 mg/kg/day. In some aspects, treatment with the glucocorticoid receptor antagonist includes administration of about 25 mg/kg/day, between about 5 and 25 mg/kg/day, between about 10 and 50 mg/kg/day, between about 25 and 75 mg/kg/day, between about 40 and 100 mg/kg/day, between about 60 and 150 mg/kg/day, or between about 100 and 250 mg/kg/day. In one aspect, the treatment with the glucocorticoid receptor antagonist includes administration of between about 5 and 250 mg/kg/day. In some aspects, the treatment with the glucocorticoid receptor antagonist includes administration of about 1 mg/kg/day, about 5 mg/kg/day, about 10 mg/kg/day, about 15 mg/kg/day, about 20 mg/kg/day, about 25 mg/kg/day, about 30 mg/kg/day, about 40 mg/kg/day, about 50 mg/kg/day, about 60 mg/kg/day, about 70 mg/kg/day, about 80 mg/kg/day, about 90 mg/kg/day, about 100 mg/kg/day, about 110 mg/kg/day, about 120 mg/kg/day, about 130 mg/kg/day, about 140 mg/kg/day, about 150 mg/kg/day, about 160 mg/kg/day, about 170 mg/kg/day, about 180 mg/kg/day, about 190 mg/kg/day, about 200 mg/kg/day, about 210 mg/kg/day, about 220 mg/kg/day, about 230 mg/kg/day, or about 240 mg/kg/day, about 250 mg/kg/day. In a particular aspect, the treatment with the glucocorticoid receptor antagonist includes administration of about 50 mg/kg/day.

[0083] In some aspects, the biological sample includes saliva, blood, plasma, serum, vaginal fluid, interstitial fluid, ocular fluid, seat, mucus, glandular secretion, nipple aspirate, seme, spinal fluid, emphatic fluid, sputum, pus, huffy coat, tumor tissue, circulating tumor cells, skin tissue, lung tissue, kidney tissue, bone marrow, pancreatic tissue, liver tissue, muscle tissue, gall bladder tissue, colon tissue, brain tissue, prostate tissue, esophageal tissue, thyroid tissue, a solid-tumor biopsy, a liquid tumor biopsy, or a combination thereof. In some aspects, the biological sample is a tumor biopsy sample. In particular aspects, the tumor biopsy sample is a solid-tumor biopsy sample.

[0084] In a further embodiment, the present invention provides a method of treating cancer in a subject by administering a therapeutically effective amount of olaparib and mifepristone to the subject, thereby treating the cancer. In some aspects, the subject has not previously received PARPi treatment for the cancer. In some aspects, the subject has not previously received olaparib treatment for the cancer.

[0085] Presented below are examples discussing cancer treatment with PARP inhibitors and glucocorticoid receptor antagonists, as well as the mechanisms for PARP inhibitor resistance in PARP inhibitor-treated cancers, contemplated for the discussed applications. The following examples are provided to further illustrate the embodiments of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLES

EXAMPLE 1

Study Design

[0086] Several preclinical ovarian cancer models were included in the below-outlined studies, including cell lines, organoids, and PDXs models, to investigate the essential role of PGCCs in PARPi resistance in ovarian cancer. A panel of markers of senescent cells were used to identify PGCCs in the cell lines and organoids. Time-lapse photography was used to monitor how PGCCs evolved. The in vivo efficacy of mifepristone was evaluated in mouse PDX models.

Cell lines and organoids

[0087] The human ovarian cancer cell lines Hey, SKOV3, OVCA-432, OVCAR8, OVCAR5, and PEO-1, and the human breast cancer cell line MCF-7 were obtained from laboratory stocks. The p53 status of the cell lines was as follows: WT p53: Hey (Watson et al., Mol. Car cinog. 2010; 49: 13-24) and MCF-7 (Troester et al., BMC Cancer, 2006; 6:276); p53-null: SKOV3 (Yaginuma and Westphal, Cancer Res., 1992; 52:4196-4199); mutant p53: OVCA (Muenyi et al., toxicol. Sci., 2012; 727:139-149), OVCAR8, OVCAR5 (Feudis etal., Br. J. Cancer, 1997; 76:474-479), and PEO-1 (Cooke etal., Oncogene, 2010; 29: 4905-4913). Hey, OVCA-432 and MCF-7 cells were cultured in Eagle’s minimum essential medium (30-2003, American Type Culture Collection (ATCC)) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. SKOV3 cells were grown in modified McCoy’s 5A media (16600082, Gibco) with 10% FBS and 1% penicillin/streptomycin. OVCAR5 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (10-013-CV, Coming) supplemented with 10% FBS and 1% penicillin/streptomycin. OVCAR8 and PEO-1 cells were cultured in RPMI- 1640 medium (10-040-CV, Coming) with 10% FBS and 1% penicillin/streptomycin. Organoids (2414, 2445, 3008) were established from xenografts derived from patients with ovarian HGSC. MD Anderson Characterized Cell Line Core facility confirmed the authenticity of the cell lines and organoids via short tandem repeat sequence analysis.

Human Specimens

[0088] HGSC tissues were obtained from patients undergoing resection of ovarian tumors at The University of Texas MD Anderson Cancer Center. The Institutional Review Board at MD Anderson approved the use of these samples. In addition, all patients gave informed consent to their tissue’s use for scientific research. The clinicopathological and genomic characterization data of the patients participating in the study are summarized in TABLE 1.

TABLE 1: The clinicopathological and genomic characterization data of patients

Ovarian Cancer PDX Tissue Processing

[0089] Under sterile conditions, xenografts were removed from tumor-bearing mice and transferred onto a 60 mm petri dish. The necrotic tissue was removed with a scalpel and forceps, and well-formed tumor tissue was rinsed with ice-cold phosphate-buffered saline (PBS) at least 3 times. The tumor tissue was then minced and transferred to a 15 mL conical tube containing 10 mL prewarmed basal medium (Advanced DMEM/F12 with 1 x Glutamax, 10 mM HEPES, [Gibco], and 100 pg/mL Primocin antimicrobial agent [Invivogen]) supplemented with 0.6-2.4 U/mL dispase II (17105041, Gibco) and 10 pM Rock inhibitor (Y-27632, Selleckchem). The tube was incubated in a 37 °C water bath for 15 min to dissociate the tissue, and the cell slurry was manually agitated every 5 min. The digested cell suspension was sheared using a 5 mL serological pipette and transferred onto a cell strainer (100 pm mesh) placed on top of a 50 mL conical tube. Next, 2% fetal calf serum (FCS) was added to the strained cell suspension, and the mixture was centrifugated at 300 relative centrifugal force (ref) for 5 min. The cell pellet was then dissociated with 1 mL of TrypLE Express enzyme (Invitrogen) containing 10 pM Y- 27632 at room temperature for 5 min. The dissociated cell clusters were sheared into a singlecell suspension by using a Pl 000 pipette with a P20 tip without a filter, resuspended with 10 mL of basal medium supplemented with 5% FBS and 10 pM Y-27632, and passed through a 40 pm cell strainer. Human tumor cells were enriched by using a mouse cell depletion kit (130- 104-694, Miltenyi Biotec) according to the manufacturer's instructions.

Organoid Culture

[0090] The purified human tumor cell pellet was resuspended in a small volume of ice-cold growth-factor-reduced Matrigel (354230, Coming). Up to four 50 pL drops of the Matrigel- cell suspension were plated into a prewarmed 6-well cell suspension culture plate (M9062, Greiner) at a density of about 15,000 to 20,000 cells per drop. The Matrigel was solidified for 15 min at 37 °C, and 3 mL prewarmed organoid culture medium (TABLE 2) was added to each well. In addition, 10 pM Y-27632 was added upon plating to supplement the culture medium for 3 days. Organoids were cultured at 37 °C in an atmosphere of 5% CO2 in a humidified incubator. The medium was changed every 3 to 4 days, and organoids were passaged at a ratio of 1:2 to 1:4 every 2 weeks. For passaging, 1 mg/mL of dispase II was added to the culture medium and incubated at 37 °C for 1 h. The Matrigel was then mechanically disrupted, and organoids were transferred into a 15 mL conical tube and centrifuged at 300 ref for 5 min. Subsequently, organoids were dissociated by resuspension in 1 mL of TrypLE Express enzyme containing 10 pM Y-27632. Organoids were then incubated at room temperature for 3 min and mechanically sheared into small cell clusters with a Pl 000 pipette connected to a P20 tip without a filter. Organoid fragments were then washed with 3 mL basal medium, spun down, and reseeded as described above. Organoids were frozen in 90% FBS and 10% dimethyl sulfoxide (DMSO) to make stocks and stored in liquid nitrogen.

TABLE 2: Ovarian cancer PDX-derived organoid medium recipe

Drug Response Assay

[0091] Cell viability of cancer cell lines was determined with a Cell Counting Kit-8 (CCK-

8, CK04-13, Dojindo). Cells were seeded on 96-well plates (3595, Coming) at a density of 2000 cells/well in quadruplicate and incubated overnight. The cells were then exposed to olaparib (LC Laboratories), niraparib (LC Laboratories), carboplatin (Sigma), or paclitaxel (Sigma) at from 5 to 7 different concentrations for 5 days. After drug exposure, CCK-8 solution was added to each well, and the plates were incubated for 2 h at 37 °C. The absorbance was measured at 450 nm using a microplate reader (BMG Labtech CLARIOstar). Cell viability of organoids was assayed with CellTiter-Glo® 3D kit (G9683, Promega). Organoids were collected 3 days after passaging and strained with a 100 pm cell strainer (431752, Coming) to remove large organoids. Organoids were then plated in a 4 pL organoid culture media and Matrigel mix (v/v 1:3) in a 96-well white plate (655083, Greiner Bio-One) at a density of 100 to 200 organoids/well in quadruplicate. At 24 h after plating, organoids were exposed to olaparib at 6 to 7 different concentrations for 5 days. Cell viability was determined according to the manufacturer’s instructions, and results were normalized to vehicle controls. Data were analyzed using the GraphPad Prism 8.0.2 software, and the values of IC50 were calculated by applying nonlinear regression (curve fit) and the equation [Inhibitors] versus responsevariable slope (four parameters).

Cell Viability, Cell Cycle, Apoptosis, and Immunofluorescence Assays

[0092] Target cells were plated in a 6-well culture plate at 1 * 10⁵ cells/well. The next day, the cells were treated with olaparib, mifepristone (Sigma-Aldrich), or both for the times indicated in the figure legends. Cell survival was determined by measuring propidium iodide (Pl)-stained cells by flow cytometry as described previously. The DNA content of tumor cells was detected by PI staining and flow cytometry as described previously. For apoptosis assays, cells were incubated with the drugs for the times indicated in the figure legends and evaluated with a fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit (556547, BD Biosciences). All flow cytometry experiments were conducted on a FACSCalibur flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (Tree Star, Inc.). For immunofluorescence, cells or organoids were seeded onto a coverslip, treated as described in the figure legends, fixed, and stained with primary antibodies (TABLE 3) overnight at 4 °C. Samples were then incubated with a secondary antibody labeled with FITC (Invitrogen) at room temperature for 1 h. After F-actin staining with phalloidin, samples were mounted with Vectashield mounting medium containing 4°,6-diamidino-2-phenylin-dole (DAPI, Vector Laboratories). Images were acquired with an Axio Imager A2 microscope.

TABLE 3: Antibodies

7?A 4 Sequencing and Data Analysis

[0093] RNA samples were retrieved from cell lines and organoids that had been treated with olaparib or vehicle (Table 4). Total RNA was extracted using RNeasy Mini Kits (Qiagen). The RNA quality was determined by the RNA integrity number (RIN) value with an Agilent 2100 Bioanalyzer. Only specimens with RIN values ? 7.0 and a 28S/18S ratio ? 1.0 were used in this study. RNA library preparation and transcriptome resequencing (20M reads/sample, DNA-Seq 100 paired ends) were performed by BGI on an Illumina HiSeq 1000 sequencing system. Differentially expressed genes (DEGs) between sample pairs (PGCC vs. control) were obtained by identifying genes with more than 2-fold changes in Transcripts Per Kilobase of exon model per Million mapped reads (TPM), with more highly expressed genes having TPM more than 0.5. DEG was collected only if it showed changes in a scenario shown in FIG. 10: changed in PGCCs on day 3 only, changed in PGCCs on day 3/7, changed in PGCCs on day 3/7 and recovery day 7, changed in PGCCs on day 7 and recovery day 7, changed in PGCCs on day 7 only, and changed on recovery day 7 only.

TABLE 4: Sample information for RNA-seq

Western Blotting

[0094] Cells were lysed in radioimmunoprecipitation assay (RIP A) buffer (89900, Thermo Fisher Scientific) supplemented with a protease and phosphatase inhibitor cocktail (78442, Thermo Fisher Scientific) for 30 min on ice, followed by centrifugation at 15,000 g for 15 min at 4 °C. Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a poly vinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% bovine serum albumin (BSA) and probed with antibodies against GATA4 (1:4000, abl24265, Abeam), p21(l:1000, 2947S, Cell Signaling Technology), or betaactin (1:4000, Al 978, Sigma- Aldrich). Detection was performed with a chemiluminescent substrate (32132, Thermo Fisher Scientific) followed by exposure to a ChemiDoc MP Imaging System (Bio-Rad).

Histology and Immunohistochemistry [0095] Tissue and organoids were processed for paraffin sectioning, and H&E staining was performed on 5 pm paraffin sections using standard protocols. In brief, organoids were harvested with dispase II, washed in PBS, fixed in 4% paraformaldehyde at 4 °C overnight, and centrifuged at 300 ref for 5 min. The cell pellet was resuspended in 30 pL of HistoGel specimen processing gel (HG-4000-012, Thermo Fisher) before processing and embedding. For immunohistochemical staining, 5 pm paraffin sections were deparaffinized in xylene, rehydrated with a graded series of ethanol, and treated with a heat retrieval solution (RV1000M, Biocare Medical) in a digital electric pressure cooker (Decloaking Chamber, Biocare Medical). Slides were incubated with Ki-67 antibody (1:200 dilution, ab!6667, Abeam) diluted in Da Vinci Green diluent (PD900M, Biocare Medical) overnight at 4 °C. Slides were then incubated with a Polink-2 HRP Plus Rabbit DAB Detection System (D39-18, GBI Labs) according to the manufacturer’s instructions. Immunohistochemical staining of p53, PAX-8, and WT1 was performed by the Research Histology Core Laboratory at MD Anderson. Images were obtained with an Axio Imager A2 microscope (Carl Zeiss).

Plasmids, Lentiviral Manipulation, and Time-Lapse Cell Cycle Imaging

[0096] For production of lentiviral particles, human embryonic kidney -293T (HEK293T) cells were transfected using fuGENE transfection reagent (Promega) with a mixture of the following plasmids: 10 pg of a lentiviral plasmid (TABLE 5), 5 pg of the packaging plasmid psPAX2 (12260, Addgene), and 2.5 pg of the pMD2.G envelope-expressing plasmid (12259, Addgene). The supernatant containing the lentiviral particles was collected 48 and 72 hours after transfection, pooled, filtered (0.45 pm), and concentrated with PEG-it Virus Precipitation Solution (LV810A-1, SBI). Hey cells were subsequently infected with the lentiviral vectors pLenti6-H2B-mCherry and L304-EGFP -Tubulin- WT to visualize the chromosomes and cytoskeleton, respectively. In addition, the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system was employed to record cell cycle changes.

TABLE 5: Vectors

[0097] To label cells with FUCCI, Hey cells were infected with the lenti viral vectors mK02- hCdtl(30/120) and mAG-hGeminin (1/110). For time-lapse imaging, fluorescently labeled Hey cells or Hey PGCCs were plated on a glass-bottomed 6-well plate (P06G-1.0-20-F, MatTek) at a density of 1 x io³ cells per well. The following day, cells were maintained in the imaging medium (Table S4.b) and imaged with a Lionheart FX automated microscope (BioTek) in a humidified chamber kept at 37 °C with 5% CO2. Cells were imaged every 15 min using a 10x objective lens for up to 1 week. The imaging program includes: 1) stabilize incubation temperature to 37°C. 2) laser autofocus. 3) phase-contrast image acquisition. 4) fluorescence image acquisition. 5) move to the next beacon. Loop steps 2-5. The raw images were imported into Fiji ImageJ software (RRID: SCR_002285) (version 1.52p) to generate image stacks with pseudocolor rendering procedures. The time-lapse videos were further edited in Adobe Premiere Pro (RRID: SCR 021315) and coded in H.264 format.

TABLE 6: Time-lapse imaging medium recipe

Enzyme-Linked Immunosorbent Assay

[0098] Supernatants were collected from control Hey cells, Hey PGCCs, or Hey PGCC- derived daughter cells. ELISA was performed according to the instructions included with commercial kits (IL-1 [3: #DLB50; IL-6: #D6050, R&D Systems). The data were normalized to the number of cells in each sample and presented as pg/mL protein per 10⁶ cells.

Senescence-Associated fi-Galactosidase (SA-fi-gal) Staining

[0099] Cells were stained for SA-P-gal activity at pH 6.0 as described previously. For quantification, cells were counterstained with DAPI to determine the total cell number. Four randomly selected fields (10x objective lenses) were photographed, and the number of cells blue with x-gal was divided by the total cell number.

Xenograft Studies

[0100] 6- to 8-week-old athymic nude (nu/nu) female mice were purchased from Envigo/Harlan Labs. All mouse experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) at MD Anderson Cancer Center (protocol number: 00001249-RN03). To establish the PDX model, fresh ovarian cancer tumor tissue obtained from debulking surgeries conducted at MD Anderson Cancer Center was sectioned into chunks (4 x 4 x 4 mm) and engrafted in both flanks of the mice subcutaneously. Once the tumor volume reached approximately 700 to 1000 mm³, tumors were harvested, expanded, and banked for future use. To develop PDXs of tumors with acquired olaparib resistance, cryopreserved tumor tissue was thawed, washed twice with PBS, and engrafted subcutaneously to one flank of each nude mouse. Tumor length (L) and width (W) were measured with calipers. Tumor volume (V = [L* W2]/2) was calculated as previously described. Once tumor volume reached around 200 mm3, mice were randomly assigned to treatment with vehicle (10% 2-hydroxy-propyl-beta- cyclodextrin/PBS) or olaparib (50 mg/mL solubilized in DMSO and diluted to 5 mg/mL with vehicle, 50 mg/kg/day, intraperitoneally (i.p.), 5 days/week) for at least 8 weeks until tumors grew back. The recurring tumors were expanded in different mice and treated according to the same regimen. Acquired resistance to olaparib was defined as a comparable growth rate in olaparib-treated recurring tumors and vehicle-treated recurring tumors. To investigate the effect of mifepristone on tumor growth, olaparib-naive tumors or tumors with acquired olaparib resistance were transplanted subcutaneously into nude mice. Once tumor volume reached 150 to 200 mm3, mice were randomized to the following treatment arms: vehicle (10% 2-hydroxy-propyl-beta- cyclodextrin/PBS), olaparib (50 mg/kg/day, i.p., 5 days/week), mifepristone (50 mg/mL solubilized in DMSO and diluted to 5/3 mg/mL with vehicle, 25 mg/kg/day, i.p., 5 days/week), and olaparib/mifepristone (olaparib 50 mg/kg/day + mifepristone 25 mg/kg/day, i.p., 5 days/week). Tumor volume and body weight were monitored twice weekly. Animals were euthanized by inhalation of CO2 followed by cervical dislocation after 6 to 8 weeks of treatment. Tumors were collected and snap-frozen for protein analysis and immunohistochemistry.

Statistical Analyses

[0101] Statistical methods are described in the respective figure legends. All statistical analyses were performed with GraphPad Prism 8.0.2 software. Data from multiple experiments are presented as mean ± SD. Statistical differences were determined by unpaired two-tailed Welch’s /-test or two-way analysis of variance (ANOVA) P < 0.05 was considered statistically significant.

EXAMPLE 2

PGCCs ACCUMULATE IN PARPi-RESISTANT HGSC MODELS WITH ACQUIRED RESISTANCE TO OLAPARIB

[0102] Patient derived xenograft (PDX) models recapitulate the original tumor’s heterogeneity and preserve its 3-dimensional histologic and architectural characteristics of cancer tissue. To establish an olaparib-resistant PDX model, HGSC PDX tumors (2 BRCA1/2 wild type

and 1 BRCA1 mutant, RCA l '' ^{n [} ) were transplanted subcutaneously into the left flanks of female nude mice. When the tumor volume reached about 200 mm³, mice were given olaparib (50 mg/kg) or vehicle intraperitoneally daily for at least 8 weeks.

[0103] The response of BRCA '' ¹ and BR( i'^{i[ l} tumors to olaparib displayed great intratumor and intertumor heterogeneity. Among the BRCA'' ^[ PDX-2445 xenografts treated with olaparib, 7 of 10 xenografts were innately resistant, 1 was sensitive, and 2 gradually developed resistance (FIG. 1A). Similarly, 4 of the 8 BRCA^ PDX-2428 xenografts were innately resistant to olaparib, 3 were sensitive, and 1 was initially sensitive but acquired olaparib resistance after 6 weeks of treatment (FIG. IB). In contrast, all of the BR( l'^M PDX-2462 xenografts were initially sensitive to olaparib, and 3 of 7 developed resistance over time (FIG. 1C).

[0104] To evaluate whether the xenografts that acquired olaparib resistance were bona fide resistant tumors, they were harvested, retransplanted, and expanded in different mice, and the mice were treated with olaparib or vehicle as above. As expected, these xenografts displayed resistance to olaparib, and their tumor growth was similar to that of vehicle-treated xenografts regardless of their BRCA status (FIGS. 1D-1F). Histologically, the olaparib-sensitive tumors were mainly composed of residual tumor cells on a background of fibrotic stromal cells. PGCCs constituted one of the major components of the residual tumor cells, especially in the germline BRCAl^¹ tumors (FIG. 9). PGCCs were also highly enriched in both the BRCA''' ^[ PDX-2428 and the BRCA 1'' ^V PDX- 2462 tumors with acquired olaparib resistance (FIG. 1G). Quantitative analysis using flow cytometry shows that there was approximately two-fold enrichment of PGCCs in tumors with acquired resistance as compared with vehicle-treated controls (FIG. 1H). Thus, the PDX experiments showed that PGCCs survived the antitumor effect of olaparib and were accumulated in HGSC tumors with acquired olaparib resistance.

EXAMPLE 3

A SUBLETHAL CONCENTRAION OF OLAPARIB LEADS TO THE DEVELOPMENT OF PGCCs IN OVARIAN AND BREAST CANCER CELL LINES [0105] To investigate the mechanism underlying the increased proportion of polyploid giant cancer cells (PGCCs) in tumors with acquired olaparib resistance, Hey ovarian cancer cells were treated with various concentrations of olaparib for 1 week. High concentrations of olaparib (>100 pM) induced massive cell death. However, lower concentrations of olaparib (25 and 50 pM) resulted in the enlargement of the cytoplasm and nucleus (FIG. 2A). Therefore, 50 pM olaparib was selected as the optimal concentration for inducing Hey PGCCs for the following experiments. Generally, Hey PGCCs were induced by treating Hey cells with 50 pM olaparib for 7 days. Olaparib was then withdrawn from culture on day 7, and PGCCs were allowed to recover in drug-free culture medium and to generate daughter cells (FIG. 2B).

[0106] To determine how PGCCs change over time, Hey cells were exposed to 50 pM olaparib for 7 days and allowed to recover for another 10 days. The percentage of PGCCs (defined by DNA content > 4C) in untreated Hey cells was only 1.05%, but it markedly increased, to 34.4%, after 7 days of treatment. During recovery, the proportion of PGCCs in Hey cells decreased over time (FIGS. 2C, 2D)

[0107] The morphological characteristics of PGCCs observed under a light microscope closely corresponded with flow cytometry analyses of these cells (FIG. 2E). Freshly seeded Hey cells mainly consisted of diploid cells (Day 0). By Day 3 of exposure to olaparib, the cells became flat, and the cytoplasm gradually enlarged. At Day 7, olaparib exposure had induced the enlargement of the nuclei and cytoplasm of Hey cells. Once olaparib was withdrawn from the culture, PGCCs proliferated and produced progeny cells (recovery [R] Days 3-10).

[0108] Whether olaparib could facilitate the formation of PGCCs in 5 other ovarian cancer cell lines and 1 breast cancer cell line as compared with the Hey cell line was examined. First, olaparib and niraparib sensitivities were determined in these cell lines (FIG. 10A). Then, olaparib concentrations were optimized to induce PGCCs in different cell lines. Olaparib induced a remarkable increase in the proportion of PGCCs in all cell lines tested (FIG. 2F, FIG. 10B, and TABLE 8). Finally, whether PARPi could also induce PGCCs was also tested. Indeed, niraparib induced PGCCs at an even lower concentration in all cell lines tested (FIG. 10C). These results suggest that the emergence of PGCCs may be a general biologic response to PARPi treatment.

TABLE 8: Percentage of PGCC induced by PARPi in different cell lines

EXAMPLE 4

PGCCs EXHIBIT HALLMARKS OF CELLULAR SENSECENCE

[0109] The enlarged size and flattened morphology of PGCCs suggest that these cells may be senescent. The senescence phenotype is often characterized by the induction of y-H2A histone family member X (y-H2AX) nuclear foci (a marker of DNA damage), cell cycle arrest regulated by the cyclin-dependent kinase inhibitors pl6iNK4aand p21, an increase of senescence- associated p-gal actosidase (SA-p-gal) activity, and enhanced expression of cytokines (e.g., interleukin- 1 (IL-1), IL-6, and IL-8).

[0110] A panel of markers that are commonly used for senescence detection were evaluated. Hey cells were first subjected to acidic -gal staining. The untreated Hey cells were barely stained with p-gal. However, Hey PGCCs exhibited remarkable blue-green positive staining in the cytoplasm (FIG. 3 A). The PGCC progeny cells at RIO expressed less p-gal than did the parental PGCCs. The expression of y-H2AX foci and p21 in Hey cells was then assessed by immunofluorescence staining. The untreated Hey cells showed minimal staining for y- H2AX and p21. Conversely, y-H2AX foci and p21 expression were highly enhanced in the nuclei of PGCCs and dropped to an undetectable level in the progeny cells (FIG. 3B). Another senescence marker, p 16^{I K4a}. was not detected in any of the samples. Finally, levels of IL-ip and IL-6, 2 components of the senescence- associated secretory phenotype (SASP) were analyzed in the culture medium of Hey cells by the enzyme-linked immunosorbent assay (ELISA). The secretion of IL-ip and IL-6 was significantly higher in PGCCs that had recovered in olaparib-free medium for 3 days than in control Hey cells or Hey PGCCs that had not been allowed to recover (FIG. 3C).

[0111] It was next evaluated whether the PGCCs induced by olaparib in other cancer cell lines were also senescent. PGCCs derived from OVCA-432 ovarian cancer and MCF-7 breast cancer cell lines were strongly stained with p-gal, but p-gal staining in SKOV3 ovarian cancer PGCCs was almost negligible (FIG. 3D). Furthermore, the expression of senescence marker proteins varied among the cell lines. For example, PGCCs derived from OVCA-432 or MCF- 7 cells expressed abundant y-H2AX foci and p21 protein. OVCA-432 PGCCs were also positively stained with p!6iNK4a (FIG. 3E). However, SKOV3 PGCCs were only positively stained for y-H2AX foci. These results suggest that PGCCs do not necessarily express all markers involving senescence, depending on their genetic background.

[0112] To identify the potential biomarkers of PGCCs, RNA sequencing analysis of whole transcriptomes was performed in the above-used cell lines and 3 ovarian cancer organoids treated with olaparib (FIG. 11A and TABLE 4). Gene Set Enrichment Analysis indicated that multiple pathways were enriched in the PGCCs and PGCC-derived daughter cells. The most prominent enriched pathways involved cytokines and chemokines associated with the SASP phenotype such as increased tumor necrosis factor alpha (TNFa) signaling and cytokine activity. Enrichment of other gene sets, including methylation and MYC signaling (FIG. 11B), demonstrated the downregulation of major cell proliferation-related pathways in PGCCs. The protein level changes of core senescence-determining genes such as GATA binding protein 4 (GATA4) and p21 from the PGCCs to subsequent cell division were also evaluated. The expression of GATA4 and p21 were upregulated in PGCCs (day 7) and gradually down- regulated in PGCC-derived daughter cells. Together, these data demonstrated that PGCCs display several major hallmarks of cellular senescence.

EXAMPLE 5

PGCCs ESCAPE FROM SENESCENCE AND GENERATE MITOTIC COMPETENT DAUGHTER CELLS

[0113] Senescent cells are traditionally considered to be nondividing cells because they lack the ability to undergo mitosis. One of the major unsolved questions about PGCCs is how they survive PARPi-induced therapeutic stress and whether they can escape senescence and resume proliferation. To answer these questions, Hey cells were labeled with the fluorescent ubiquitination- based cell cycle indicator (FUCCI) system and tracked cell cycle changes with time-lapse photography. The mitotic cell cycle consists of 2 main stages: interphase (G1 phase, S phase, G2 phase) and M phase (mitosis and cytokinesis). The FUCCI system labels nuclei in red at the G1 phase, yellow at the Gl/S transition phase, and green at the S/G2/M phases. Consistent with the earlier finding with paclitaxel-induced PGCCs, it was found that untreated Hey cells divided via the canonical mitotic cell cycle and produced 2 identical daughter cells once they completed cytokinesis (FIG. 12A). However, olaparib (50 pM) prevented Hey cells from undergoing mitosis. Instead, in olaparib-treated Hey cells, the cell cycle consisted of alternating S and G phases without cell division. Eventually, the cells became flattened and their nuclei enlarged due to the accumulation of genomic DNA (FIG. 12b) This process is termed endoreplication or endocycling. B

[0114] It was next investigated whether PGCCs can exit the endoreplication cycle and reenter mitosis when cultured in the drug-free recovery medium. Tripolar mitosis occurred in some cases; FIG. 12A shows that a PGCC can generate 3 daughter cells, 2 of which re-fused. All daughter cells then continued endocycling. PGCCs also gave rise to daughter cells via bipolar mitosis. In the example shown in FIG. 12C, the first cell division occurred at 1 :30, and the resulting daughter cells underwent the second and third rounds of cell division at 36:30 and 64:45, respectively. To visualize the dynamic changes of chromosomes and the spindle movement in PGCCs during the division process, Hey cell chromosomes were labeled with histone H2B-mCherry and microtubules with enhanced green fluorescent protein (EGFP)-a- tubulin. Untreated Hey cells divided via canonical bipolar mitosis (FIG. 12B). By contrast, olaparib-induced Hey PGCCs exhibited diverse modes of division. In some cases, PGCCs produced 2 separate daughter cells via bipolar mitosis (FIG. 12C). In other cases, PGCCs underwent tripolar mitosis. As shown in FIG. 12D, a mononucleated PGCC gave rise to 3 daughter cells connected at the midbody, 2 of which re-fused and formed a binucleated PGCC. In another case, a PGCC divided into 3 multinucleated daughter cells via tripolar mitosis (FIG. 12E). In addition, during division, PGCCs can undergo restitution multipolar endomitosis (RMEM), resulting in a massively fragmented multinucleated PGCC (FIGS. 12D and 12F) with multiple micronuclei (arrows in FIGS. 4D and 12F).

[0115] These results collectively indicated that a sublethal concentration of olaparib resulted in a switch from the mitotic cell cycle to various modes of endoreplication with defective nuclear division or cytokinesis. PGCCs then divided in various ways to produce mononucleated or multinucleated daughter cells with massively altered genomes via creating genomic chaos, a subset of daughter cells can survive and acquire therapeutic resistance.

EXAMPLE 6

PGCCs AND DAUGHTER CELLS ARE RESISTANT TO CELL DEATH TRIGGERED BY OLAPARIB

[0116] To test whether PGCCs and PGCC-derived daughter cells confer olaparib resistance, olaparib sensitivity was compared between control Hey cells, Hey PGCCs, and PGCC- derived daughter cells. As shown in FIG. 5A, Hey and Hey PGCCs (pretreated with 50 pM olaparib for 1 week) were exposed to concentrations of olaparib ranging from 25 to 400 pM for 7 days. As also shown in FIG. 5A, olaparib induced the death of control Hey cells in a dose-dependent manner. In contrast, Hey PGCCs showed extreme resistance to olaparib, especially to high concentrations (>100 pM). To test whether PGCC-derived progeny cells confer drug resistance, the Hey PGCCs were subcloned and established 4 daughter cell lines derived from a single PGCC. Compared with control Hey cells, all the tested daughter cell lines exhibited varying degrees of resistance to olaparib-induced cell death (FIG. 5B and FIG. 13A). To determine whether the varying sensitivity of PGCC subclones is related to the proliferation of the clones, the proliferative capacity of these daughter cell clones were compared and determined to exhibit no differences (FIG. 13B). The sensitivity of the PGCC subclones to common chemotherapy drugs such as carboplatin and paclitaxel was also assessed (FIG. 13C). Although the dose response of PGCC subclones to carboplatin was consistent with that of Hey cells, they were more resistant to paclitaxel, especially PGCC subclone #10. These data demonstrate that daughter cells derived from PGCCs maintain the PGCCs’ acquired resistance to olaparib.

EXAMPLE 7

PGCCs EXHIBIT SENSITIVITY TO THE COMBINATION OF OLAPARIB AND MIFEPRISTONE

[0117] PGCCs dedifferentiate from mature somatic cells by recapitulating a blastomerestage cleavage program that augments the nucleus and gives rise to new embryonic life. Because they mimic early embryonic development, it was hypothesized herein that PGCCs may represent “somatic cell pregnancy”. Therefore, it was reasoned that certain contraceptive drugs may be able to block the life cycle of PGCCs to block development of somatic blastomeres. One candidate drug is mifepristone, which has been used as an emergency contraceptive drug for decades. Mifepristone can also inhibit repopulation of ovarian cancer cells after cisplatin/paclitaxel combination therapy and delay the growth of ovarian carcinoma xenografts. Therefore, it was hypothesized that mifepristone might decrease tumor recurrence by inhibiting olaparib-induced PGCC formation.

[0118] To test this hypothesis, the cytotoxic effect of mifepristone on Hey cells was analyzed. A lower concentration (25 pM) of mifepristone had minimal effect on the viability of Hey cells, and olaparib alone resulted in only 16.0% cell death. However, the combination of mifepristone and olaparib led to 87.8% cell death (FIG. 5C). The synergy between mifepristone and olaparib disappeared when a higher concentration of mifepristone (50 pM) was used, probably because it alone was sufficient to kill most cells. Analysis of the apoptotic response of cells to the compounds showed that treatment with mifepristone (25 pM) in combination with olaparib for 3 days accelerated the production of early apoptotic cells (42.5%, Q3) compared to cells treated with mifepristone (1.1%) or olaparib alone (14.8%) (FIG. 5D). The combination treatment also led to the emergence of more late apoptotic cells (59.9%, Q2) than did treatment with mifepristone (8.3%) or olaparib (19.0%) alone for 7 days. Cell cycle analysis further revealed that mifepristone alone did not significantly reduce the proportion of preexisting PGCCs among Hey cells. However, the combined use of mifepristone and olaparib dramatically inhibited the olaparib-mediated development of PGCCs in a concentration-dependent manner (FIG. 5E). The above findings were further validated in OVCA-432, MCF-7, and SK0V3 cells. In these cells, too, the combination of mifepristone and olaparib resulted in more early and late apoptotic cells than did mifepristone or olaparib alone (FIG. 14). Taken together, these data demonstrate that the mifepristone/olaparib combination blocked olaparib-induced PGCC development by promoting apoptosis.

EXAMPLE 8

OLAPARIB ENHANCES THE FREQUENCY OF PGCCs IN HGSC-DERIVED ORGANOIDS

[0119] Organoids can faithfully maintain the heterogeneity and the histomorphological characteristics of the parental tumor and can better predict drug response than cell lines. Several organoids from HGSC PDXs were established to develop ex vivo platforms of organoid models (FIG. 6A and TABLE 7). Hematoxylin and eosin (H&E) staining of these HGSC PDX-derived organoids revealed that the organoids harbored multiple histologic and characteristics of their parental tumors, such as the presence of histologic architectures, papillary (HGSC-2414, HGSC- 2445) or solid (HGSC-3008) patterns, and nuclear and cellular atypia (FIG. 6B). The expression of HGSC protein biomarkers between organoids and parental tumors was also compared. HGSC parental tumors were characterized by intense nuclear staining of paired box gene 8 (PAX8), a marker of the serous subtype and Wilms tumor 1 (WT1), as well as either positive nuclear staining or completely absent expression of tumor protein p53 (TP53). As shown in FIG. 6F, the organoids retained the TP53, PAX8, and WT1 expression status of their corresponding parental tumors.

[0120] It was next investigated whether olaparib induces the development of PGCCs in organoids. Olaparib treatment remarkably increased the number of PGCCs in organoid (Org)- 3008 and Org- 2445 when used at a sublethal concentration (FIG. 6C and FIG. 15A). Similarly, exposure to 50 pM olaparib induced the highest proportion of PGCCs in Org-2414. PGCCs induced by olaparib in the organoids exhibited the phenotypes of senescent cells (FIGS. 15B, 15C). y-H2AX foci, pl6^INK4a, and p21 were highly expressed in Org-2414 PGCCs (FIG. 15C). Consistent with our observations in Hey cells, mifepristone alone did not change the percentage of PGCCs in Org-2414. However, the proportion of PGCCs in this organoid shrank from 28.4% with olaparib monotherapy to 7.94% with combined mifepristone and olaparib (50 pM each) (FIG. 6D). A combination of mifepristone and olaparib enhanced apoptosis in Org-2414, Org-2445, and Org-3008 (FIG. 15D). These data suggest that mifepristone blockage of olaparib-induced PGCC formation can be applied to HGSC organoid models.

TABLE 7: Xenograft-derived organoids lines

EXAMPLE 9

MIFEPRISTONE SUPPRESSES TUMOR GROWTH IN OLAPARIB-NAIVE AND OLAPARIB-RESISTANT HGSC PDX MODELS

[0121] To assess whether blockage of PGCC development could suppress tumor growth, the in vivo efficacy of mifepristone in mouse PDX models was measured. The effects of mifepristone monotherapy and a mifepristone/olaparib combination using an olaparib-naive PDX model (i.e., one without prior exposure to olaparib) were compared. Mifepristone monotherapy induced mild tumor growth inhibition in BRCA^ PDX-3008 compared to vehicle treatment, with increased tumor necrosis and a reduction in the number of proliferating cells as shown by Ki-67 staining (FIG. 7A and FIG. 16A). By contrast, the combination of mifepristone with olaparib was more effective at reducing tumor growth than either single agent. Furthermore, both mifepristone monotherapy and the mifepristone/olaparib combination significantly decreased the tumor mass compared to vehicle-treated tumors (P = 0.0002 and P < 0.0001, respectively).

[0122] Next, the role of mifepristone in the acquired olaparib resistance models PDX- 2445 and PDX-2428 was assessed (FIG. 1). The olaparib-resistant tumors were expanded in mice, which were then treated with vehicle, olaparib only, mifepristone only, or olaparib and mifepristone. The olaparib-treated tumors had a similar growth pattern to that of vehicle-treated tumors, demonstrating that they were bona fide olaparib-resistant tumors. Moreover, mifepristone monotherapy and the mifepristone/olaparib combination therapy significantly inhibited tumor growth compared to vehicle treatment (FIG. 7B). Interestingly, mifepristone monotherapy showed significantly better suppression of tumor growth than did mifepristone/olaparib combination therapy (P < 0.0001). Mifepristone monotherapy and the mifepristone/olaparib combination also led to massive tumor necrosis and reduced cell proliferation, as indicated by Ki-67 staining (FIG. 8B). Similar results were obtained in PDX-2428 (FIG. 7C and FIG. 16C). In addition, in a subset of tumors that responded to mifepristone, there are a marked increase in fibrosis and macrophages (FIG. 16B), suggesting that mifepristone may attenuate the tumor growth in patient tumors with acquired resistance by targeting embryonic properties of PGCCs toward benign lineages. Inclusion of olaparib may in fact interfere with the differentiation ability of mifepristone in PDX models that have already acquired resistance. Taken together, our data suggest that combination therapy with olaparib/mifepristone may be a better choice for olaparib-naive tumors, while mifepristone monotherapy may be more effective for treating olaparib-resistant tumors

EXAMPLE 10

DISCUSSION

[0123] The inevitable tumor recurrence in both patients with BRCA mutations and those with WT BRCA following olaparib therapy suggests that the underlying mechanism of PARPi resistance may be independent of the BRCA status of the tumor. Multiple mechanisms of resistance to PARPi have been described, including upregulation of drug efflux via overexpression of ATP-binding cassette, restoration of homologous recombination or wild type BRCA sequence, target specific resistance, or restoration of stalled replication fork protection. While these mechanisms remain important ones to understand how resistance occurs to PARPi in different subtypes of tumor; however, it is unlikely that they represent all of mechanisms that confer the resistance, which prompted a search for additional new mechanisms and to look into PGCCs for a generalized resistance mechanism for whole genome reprogramming in response to catastrophic stress.

[0124] To address this issue, PDX models with acquired olaparib resistance from both BRCAl*^ and BRCA^ HGSC patient tumors were developed. It was found that PGCCs were more common in the olaparib-treated PDXs, suggesting that PGCCs, not BRCA status, are associated with acquired resistance to olaparib in patients with relapsed ovarian cancer. This result is consistent with increased ploidy in PARPi resistant ovarian cancer. To understand how PGCCs are associated with PARPi resistance and tumor recurrence, the effect of olaparib on the development of PGCCs was evaluated in 6 human ovarian cancer cell lines and 1 breast cancer cell line. Long-term (1-week) exposure to sublethal concentrations of olaparib markedly increased the proportion of PGCCs in these cancer cell lines.

[0125] Using time-lapse photography of FUCCI-labeled Hey cells, it was demonstrated herein that olaparib led to a transition of the cell cycle from mitosis to endoreplication, a common cell cycle variant during which cells increase their genomic DNA content without dividing. The endoreplication cycle eventually resulted in the development of PGCCs (mostly mononucleated PGCCs). Here, it was found that PGCCs divided into daughter cells through diverse variants of mitosis, such as multipolar mitosis and RMEM. These aberrant mitoses generated multiple micronuclei and inevitably increased chromosomal instability, genomic chaos, and cancer macroevolution, which is known to contribute to chemotherapy resistance. Thus, PGCCs are not “dead,” as has been assumed in the past, but rather undergo a variety of aberrant cell cycles that may underlie their ability to promote cancer recurrence and therapy resistance via whole genomic duplication-mediated reprogramming. Consistent with early observations on paclitaxel-induced PGCCs, the presently disclosed data demonstrate that olaparib-induced PGCCs exhibit most of the phenotypes of senescent cells. Most of the Hey-derived PGCCs showed enhanced SA-P-gal activity and p21 expression, greater numbers of y-H2AX foci, and increased secretion of SASP factors, whereas the daughter cells of PGCCs minimally expressed P-gal, p21, and y-H2AX. This finding suggested that PGCC progeny cells can escape senescence to facilitate recurrence, consistent with earlier observations.

[0126] The expression of senescence markers in PGCCs varied in different cell types. For instance, SA-P-gal activity was elevated in the PGCCs of all but one cell line, which only exhibited increased y-H2AX nuclear foci. These differences could be atributable to the different genetic backgrounds of the parental cell lines. To better model tumor’s therapy response, organoids were developed from PDXs from patients with ovarian HGSC. A relatively simple medium modified from the media used in previous reports was formulated to foster the HGSC PDX-derived organoids. These HGSC organoids recapitulated the histomorphological characteristics of the parental tumor and were able to proliferate in vitro over the long term (> 40 passages). The organoid-derived PGCCs induced by olaparib also exhibited the phenotypes of senescent cells. These data provide further evidence that sustained DNA damage results in a senescent cell-like phenotype in PGCCs and that this conserved mechanism allows these cells to resist apoptosis.

[0127] Mutation in the TP53 gene was found in 96% of HGSC. This study used both p53 wild type and multiple p53 mutant ovarian cancer cell lines. A high frequency of PGCCs in all cell lines regardless of p53 mutations was observed. These data support our early conclusion that PGCCs represent a general genomic response to high level stresses via an evolutionarily conserved polyploidization program. Although mutation or loss of p53 can sensitize the cancer cell to polyploidization due to defective cell checkpoints, however, the mutation in p53 per se is not required. Depending on the types of stressors, the level of polyploidization program could respond at the different levels by varying speed or number of genomic copy to generate newly programmed daughter cells for resistance.

[0128] PGCCs can confer acquired therapeutic resistance and dormancy by derepressing an embryonic program that is suppressed during human growth and development. PGCCs may therefore represent “somatic blastomeres” in tumor initiation, resistance, dormancy, and metastasis. The different life cycle stages of PGCCs may offer vulnerabilities for potential therapeutic intervention. The data disclosed herein provide additional proof of principle for this hypothesis.

[0129] By using the glucocorticoid receptor antagonist, antiprogestin, and contraceptive drug mifepristone to block the initiation of PGCCs by olaparib, it was found that mifepristone can block the formation and survival of PGCCs induced by olaparib. Mifepristone synergistically acts with olaparib to promote apoptosis of cells that are undergoing endoreplication, resulting in the inability to form PGCCs. This mechanism suggests that mifepristone may be more effective in killing newly formed PGCCs induced by therapeutic stress than preexisting PGCCs in patient tumors. Mifepristone can directly decrease tumor growth with acquired resistance to PARPi. One possibility is that mifepristone could also promote the differentiation of PGCCs and daughter cells toward differentiation into benign lineages, as early studies demonstrated that PGCCs acquired blastomere- like sternness and are prone to adipose or fibrous cells. Indeed, massive fibrosis was observed in mifepristone-treated tumors (FIG. 16B). However, such observation needs to be validated in additional studies and an increased number of PDX models. In addition, mifepristone is also known to block the function of the glucocorticoid receptor, and it remains to be determined if dysfunctional glucocorticoid receptors play any role in resistance formation and tumor recurrence following PARPi treatment. This mechanistic understanding may explain our finding that the combined use of olaparib and mifepristone achieved the most potent tumor inhibition in olaparib-naive PDXs, while mifepristone monotherapy showed better tumor inhibition effect in PDXs with acquired olaparib resistance. This difference suggests that olaparib partially interferes with the tumor inhibition effect of mifepristone if the patient’s tumor has already acquired PARPi-induced resistance. This finding has important clinical implications: addition of mifepristone to a PARPi could potentiate therapeutic effects of PARPi for patients who have not been previously exposed to PARPi, and mifepristone alone can block tumor growth in patients who have been already treated with PAPRi and have acquired resistance.

[0130] A model (FIG. 8) was formulated based on the above data to elucidate the mechanism of acquired resistance to olaparib in ovarian HGSC. In tumors that have not been exposed to olaparib, the administration of olaparib induces DNA damage, shuts down mitosis, and leads to activation of the life cycle of PGCCs via de-repressing a pre-embryonic program that is suppressed in cancer growth and progression to generate somatic blastomeres, which leads to whole genomic reorganization at both genomic and epigenetic levels and produces genomically reprogrammed and mitotically competent progeny cells with acquired resistance, causing tumor recurrence. Mifepristone promotes apoptosis of cells undergoing endoreplication, thereby blocking the formation of PGCCs, preventing the development of somatic cell pregnancy, and suppressing tumor growth. The inclusion of mifepristone with olaparib can synergistically block the endoreplication and survival of PGCCs, increasing the antitumor efficacy compared to either drug alone. In addition, mifepristone can directly block tumor growth with acquired resistance to PARPi, as these resistant tumors have acquired high level of embryonic sternness and prone to differentiation toward benign lineages. Thus, targeting the life cycle of PGCCs represents a promising approach to potentiate the therapeutic effect of PARPi and to overcome acquired resistance in ovarian cancer.

[0131] Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What Is Claimed Is:

1. A method of treating cancer in a subj ect comprising: administering a therapeutically effective amount of a poly(ADP-ribose) polymerase inhibitor (PARPi) and a glucocorticoid receptor antagonist to a subject who has not previously received PARPi treatment, thereby treating the cancer.

2. The method of claim 1, wherein the PARPi is olaparib, niraparib, rucaparib, talazoparib, or a combination thereof.

3. The method of claim 2, wherein the PARPi is olaparib.

4. The method of claim 1, wherein the therapeutically effective amount of the PARPi is between about 5 and 250 mg/kg/day.

5. The method of claim 4, wherein the therapeutically effective amount of the PARPi is about 50 mg/kg/day.

6. The method of claim 1, wherein the glucocorticoid receptor antagonist is relacorilant, metyrapone, ketoconazole, aminoglutethimide, mifepristone, or a combination thereof.

7. The method of claim 6, wherein the glucocorticoid receptor antagonist is mifepristone.

8. The method of claim 1, wherein the therapeutically effective amount of the glucocorticoid receptor antagonist is between about 2.5 and 125 mg/kg/day.

9. The method of claim 8, wherein the therapeutically effective amount of the glucocorticoid receptor antagonist is about 25 mg/kg/day.

10. The method of claim 1, wherein the therapeutically effective amount of the PARPi is about 1 to 10 times the therapeutically effective amount of the glucocorticoid receptor antagonist.

11. The method of claim 1, wherein the cancer is breast cancer, ovarian cancer, carcinoma, or adenocarcinoma.

12. The method of claim 1, wherein the subject has not previously received PARPi treatment within the previous 15 days, within the previous 20 days, within the previous 25 days, within the previous 30 days, within the previous 40 days, within the previous 50 days, within the previous 60 days, within the previous 70 days, within the previous 80 days, within the previous 90 days, within the previous 100 days, or within the previous year.

13. The method of claim 1, wherein the subject does not exhibit elevated levels of polyploid giant cancer cells (PGCCs) or endoreplicating cancer cells.

14. A method of treating cancer in a subject, wherein the subject has previously received poly(ADP-ribose) polymerase inhibitor (PARPi) treatment comprising: ceasing the PARPi treatment; and at least ten days after cessation of the PARPi treatment, administering a therapeutically effective amounts of a poly(ADP-ribose) polymerase inhibitor (PARPi) and a glucocorticoid receptor antagonist to the subject in combination, thereby treating the cancer.

15. A method of treating cancer in a subject comprising: administering a therapeutically effective amount of a glucocorticoid receptor antagonist to a subject who has an elevated polyploid giant cancer cell (PGCC) level, an elevated endoreplicating cancer cell level, or a combination thereof, thereby treating the cancer.

16. The method of claim 15, wherein the glucocorticoid receptor antagonist is relacorilant, metyrapone, ketoconazole, aminoglutethimide, mifepristone, or a combination thereof.

17. The method of claim 16, wherein the glucocorticoid receptor antagonist is mifepristone.

18. The method of claim 15, wherein the therapeutically effective amount of the glucocorticoid receptor antagonist is between about 5 and 250 mg/kg/day.

19. The method of claim 18, wherein the therapeutically effective amount of the glucocorticoid receptor antagonist is about 50 mg/kg/day.

20. The method of claim 15, wherein the elevated PGCC level, the elevated endoreplicating cancer cell level, or the combination thereof is a level of at least about 3%, at least about 5%, at least about 7.5%, at least about 10%, at least about 12.5%, at least about 15%, at least about 17.5%, at least about 20%, at least about 22.5%, at least about 25%, at least about 27.5%, or at least about 30% among cells from a biological sample from the subject.

21. The method of claim 20, wherein the biological sample comprises saliva, blood, plasma, serum, vaginal fluid, interstitial fluid, ocular fluid, seat, mucus, glandular secretion, nipple aspirate, seme, spinal fluid, emphatic fluid, sputum, pus, huffy coat, tumor tissue, circulating tumor cells, skin tissue, lung tissue, kidney tissue, bone marrow, pancreatic tissue, liver tissue, muscle tissue, gall bladder tissue, colon tissue, brain tissue, prostate tissue, esophageal tissue, thyroid tissue, a solid-tumor biopsy, a liquid tumor biopsy, or a combination thereof.

22. The method of claim 15, wherein the cancer is breast cancer, ovarian cancer, carcinoma, adenocarcinoma.

23. The method of claim 15, wherein the subject received PARPi treatment within the previous day, within the previous 2 days, within the previous 3 days, within the previous 4 days, within the previous 5 days, within the previous 6 days, within the previous 7 days, within the previous 10 days, within the previous 15 days, within the previous 20 days, within the previous 25 days, within the previous 30 days, within the previous 40 days, within the previous 50 days, within the previous 60 days, within the previous 70 days, within the previous 80 days, within the previous 90 days, within the previous 100 days, or within the previous year.

24. The method of claim 23, wherein the PARPi is olaparib.

25. A method of identifying a subject with cancer as a candidate for treatment with a poly(ADP-ribose) polymerase inhibitor (PARPi) and a glucocorticoid receptor antagonist comprising: a) measuring an level of polyploid giant cancer cells (PGCCs), endoreplicating cancer cells, or a combination thereof in a biological sample from the subject; and i) classifying the subject as having a cancer cell PGCC level, an endoreplicating cancer cell level, or a combination thereof of at most 5% as a likely responder to the PARPi and the glucocorticoid receptor antagonist, thereby identifying the subject as suitable for treatment with the PARPi and the glucocorticoid receptor antagonist, or ii) classifying the subject as having a cancer cell PGCC level, an endoreplicating cancer cell level, or a combination thereof of at most 5% as a likely non-responder to the PARPi and the glucocorticoid receptor antagonist, thereby identifying the subject as unsuitable for treatment with the PARPi and the glucocorticoid receptor antagonist; thereby identifying the subject with cancer as a candidate for treatment with the PARPi and the glucocorticoid receptor antagonist.

26. The method of claim 25, wherein the PARPi is olaparib, niraparib, rucaparib, talazoparib, or a combination thereof.

27. The method of claim 26, wherein the PARPi is olaparib.

28. The method of claim 25, wherein the treatment with the PARPi comprises administration of between about 5 and 250 mg/kg/day.

29. The method of claim 28, wherein the treatment with the PARPi comprises administration of about 50 mg/kg/day.

30. The method of claim 25, wherein the glucocorticoid receptor antagonist is relacorilant, metyrapone, ketoconazole, aminoglutethimide, mifepristone, or a combination thereof.

31. The method of claim 30, wherein the glucocorticoid receptor antagonist is mifepristone.

32. The method of claim 25, wherein the treatment with the glucocorticoid receptor antagonist comprises administration of between about 2.5 and 125 mg/kg/day.

33. The method of claim 32, wherein the treatment with the glucocorticoid receptor antagonist comprises administration of about 25 mg/kg/day.

34. The method of claim 25, wherein the treatment with the PARPi and the glucocorticoid receptor antagonist comprises administration of 1 to 10 times the amount of the PARPi as compared with the amount of the glucocorticoid receptor antagonist.

35. The method of claim 25, wherein the cancer is breast cancer, ovarian cancer, carcinoma, or adenocarcinoma.

36. The method of claim 25, wherein the method comprises measuring a level of PGCCs in the biological sample.

37. The method of claim 25, wherein the biological sample comprises saliva, blood, plasma, serum, vaginal fluid, interstitial fluid, ocular fluid, seat, mucus, glandular secretion, nipple aspirate, seme, spinal fluid, emphatic fluid, sputum, pus, huffy coat, tumor tissue, circulating tumor cells, skin tissue, lung tissue, kidney tissue, bone marrow, pancreatic tissue, liver tissue, muscle tissue, gall bladder tissue, colon tissue, brain tissue, prostate tissue, esophageal tissue, thyroid tissue, a solid-tumor biopsy, a liquid tumor biopsy, or a combination thereof.

38. A method of identifying a subject with cancer as a candidate for treatment with a glucocorticoid receptor antagonist comprising: a) measuring a level of polyploid giant cancer cells (PGCCs), endoreplicating cancer cells, or a combination thereof in a biological sample from the subject; and i) classifying the subject as having a cancer cell PGCC level, an endoreplicating cancer cell level, or a combination thereof of at least 5% as a likely responder to the glucocorticoid receptor antagonist, thereby identifying the subject as suitable for treatment with the glucocorticoid receptor antagonist; or ii) classifying the subject as having a cancer cell PGCC level, an endoreplicating cancer cell level, or a combination thereof of at most 5% as a non-likely responder to the glucocorticoid receptor antagonist, thereby identifying the subject as unsuitable for treatment with the glucocorticoid receptor antagonist; thereby identifying the subject with cancer as a candidate for treatment with the glucocorticoid receptor antagonist.

39. The method of claim 38, wherein the glucocorticoid receptor antagonist is relacorilant, metyrapone, ketoconazole, aminoglutethimide, mifepristone, or a combination thereof.

40. The method of claim 38, wherein the glucocorticoid receptor antagonist is mifepristone.

41. The method of claim 38, wherein the treatment with the glucocorticoid receptor antagonist comprises administration of between about 5 and 250 mg/kg/day.

42. The method of claim 38, wherein the treatment with the glucocorticoid receptor antagonist comprises administration of about 50 mg/kg/day.

43. The method of claim 38, wherein the cancer is breast cancer, ovarian cancer, carcinoma, adenocarcinoma, or a combination thereof.

44. The method of claim 38, wherein the method comprises measuring a level of PGCCs in the biological sample.

45. The method of claim 38, wherein the biological sample comprises saliva, blood, plasma, serum, vaginal fluid, interstitial fluid, ocular fluid, seat, mucus, glandular secretion, nipple aspirate, seme, spinal fluid, emphatic fluid, sputum, pus, huffy coat, tumor tissue, circulating tumor cells, skin tissue, lung tissue, kidney tissue, bone marrow, pancreatic tissue, liver tissue, muscle tissue, gall bladder tissue, colon tissue, brain tissue, prostate tissue, esophageal tissue, thyroid tissue, a solid-tumor biopsy, a liquid tumor biopsy, or a combination thereof.

46. A method of treating cancer in a subject comprising: administering a therapeutically effective amount of a poly(ADP -ribose) polymerase inhibitor (PARPi) and an anti -glucocorticoid to a subject who has not previously received PARPi treatment, thereby treating the cancer.