CN117794523A

CN117794523A - Cancer treatment using PARP inhibitors and PLK1 inhibitors

Info

Publication number: CN117794523A
Application number: CN202280041039.0A
Authority: CN
Inventors: 玛雅·莱丁格; 马克·埃兰德
Original assignee: Cardiff Cancer Technology Co ltd
Current assignee: Cardiff Cancer Technology Co ltd
Priority date: 2021-04-09
Filing date: 2022-04-08
Publication date: 2024-03-29

Abstract

Methods, compositions, and kits are provided that include for treating cancer in a subject. The method may comprise administering to the subject a PARP inhibitor (e.g., olapari) and a PLK1 inhibitor (e.g., onvansertib) in a manner sufficient to inhibit the progression of the cancer.

Description

Cancer treatment using PARP inhibitors and PLK1 inhibitors

RELATED APPLICATIONS

U.S. provisional application No. 63/173,278 filed on day 9, 4, 2021, according to 35u.s.c. ≡119 (e); U.S. provisional application No. 63/182,674 filed on 4/30 of 2021; and U.S. provisional application No. 63/322,557, filed on 3/22, 2022, the contents of which are expressly incorporated herein by reference in their entirety.

Background

FIELD

The present application relates generally to the treatment of cancer. More specifically, combination therapies for treating cancer using PARP inhibitors in combination with polo-like kinase 1 (PLK 1) inhibitors are provided.

Description of the Related Art

Polo-like kinase 1 (PLK 1) is a well characterized member of the 5 members of the serine/threonine protein kinase family and strongly promotes cell progression through mitosis. PLK1 performs several important functions in the mitosis (M) phase of the cell cycle, including regulating centrosome maturation and spindle assembly, removing mucins from the chromosome arms, inactivating late-promoting complex/cyclin-promoting complex/cyclosome (APC/C) inhibitors, and regulating mitotic exit (mitotic exit) and cytokinesis. PLK1 plays a key role in the function of the central body and the assembly of the bipolar spindle. PLK1 also acts as a negative regulator of p53 family members, leading to ubiquitination and subsequent degradation of p53/TP53, inhibiting p73/TP 73-mediated pro-apoptotic function and phosphorylation/degradation of the cofactor bora of aurora kinase A. PLK1 is localized to the centrosome, kinetochore (kinetochores) and central spindle during different phases of mitosis. PLK1 is the major regulator of mitosis and is abnormally overexpressed in a variety of human cancers, including AML, and is associated with cell proliferation and poor prognosis.

PARP inhibitors are inhibitors of the enzyme poly (ADP-ribose) polymerase (PARP). Poly (ADP-ribose) polymerase 1 (PARP 1) is a key molecule for repairing DNA Single Strand Breaks (SSB). The induction of DNA Double Strand Breaks (DSBs) by inhibiting PARP1 functional knockout SSB repair may trigger synthetic lethality in cancer cells with homology directed DSB repair defects. Some PARP inhibitors have been approved for the treatment of certain cancer types, but patients may be resistant or develop resistance to PARP inhibitor treatment.

There is a need to find effective treatments for cancer patients, including patients resistant to PARP inhibitor treatment.

SUMMARY

Methods, compositions, and kits are provided that include use in the treatment of cancer. Some embodiments provide a method of treating cancer comprising administering a poly (ADP-ribose) polymerase (PARP) inhibitor and a Polo-like kinase 1 (PLK 1) inhibitor to a subject having cancer, thereby inhibiting cancer progression. In some embodiments, the subject has ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof. The cancer may be, for example, a homologous recombination-deficient cancer. The cancer may be, for example, a BRCA1 mutant cancer, a BRCA2 mutant cancer, or both. In some embodiments, the subject is a human. In some embodiments, the subject is resistant to or has developed resistance to PARP inhibitor treatment alone. The patient's resistance to PARP inhibitor treatment may be partially or completely absent from response to PARP inhibitor treatment alone. The PLK1 inhibitor and PARP inhibitor may be co-administered simultaneously or sequentially. In some embodiments, the PLK1 inhibitor is administered prior to the PARP inhibitor administration, and optionally, wherein the PLK1 inhibitor is administered prior to the PARP inhibitor administration on a daily basis of the PLK1 inhibitor and the PARP inhibitor to the subject. In some embodiments, the PLK1 inhibitor is administered about 30 minutes to about 5 hours prior to the PARP inhibitor on a given day. In some embodiments, the administration of the PLK1 inhibitor is oral administration, the administration of the PARP inhibitor is oral administration, or both are oral administration.

In some embodiments, the inhibition of cancer progression is greater than the combined inhibition of progression caused by PARP inhibitor alone plus PLK1 inhibitor alone. In some embodiments, the subject achieves a complete response. In some embodiments, the subject has received prior PARP inhibitor treatment. In some embodiments, the subject does not respond to treatment with PARP inhibitor alone. In some embodiments, the subject is known to be resistant to PARP inhibitor therapy. In some embodiments, the PARP inhibitor and the PLK1 inhibitor are each administered to the subject at least twice or at least five cycles within a week. In some embodiments, the PARP inhibitor, PLK1 inhibitor, or both are administered at a period of at least 7 days. The treatment cycle (e.g., each cycle of treatment) may be at least about 21 days, such as about 21 days to about 28 days. In some embodiments, the PLK1 inhibitor is administered on at least four days of the cycle. In some embodiments, the PLK1 inhibitor is not administered for at least one day of the cycle. In some embodiments, the PARP inhibitor is administered daily.

In some embodiments, the subject experiences administration of the PARP inhibitor and the PLK1 inhibitor for at least two cycles. In some embodiments, the PARP inhibitor is selective and/or specific for PARP inhibition. Non-limiting examples of PARP inhibitors include iniparib (BSI 201), tazoparib (BMN-673), olaparib (AZD-2281), AZD5305, NMS-293, ruaparib (rucaparib) (AG 014699, PF-01367338), ABT-888, uygur Li Pali (Velipparib) (ABT-888), nilaparib (nirapparib), CEP 9722, MK 4827, BGB-290 (pamiparib)), BSI-201, CEP-8983, E7016, 3-aminobenzamide, or combinations thereof; optionally the PARP inhibitor is olapari. In some embodiments, the PLK1 inhibitor is selective and/or specific for PLK 1. Non-limiting examples of PLK1 inhibitors include dihydropteridinones, pyridopyrimidines, aminopyrimidines, substituted thiazolinones, pteridine derivatives, dihydroimidazo [1,5-f ] pteridines, meta-substituted thiazolinones, benzylstyryl sulfone analogs, stilbene derivatives, or any combination thereof. In some embodiments, the PLK1 inhibitor is onvansertib, BI2536, volasertib (BI 6727), GSK461364, AZD1775, CYC140, HMN-176, HMN-214, rigossitib (ON-01910), MLN0905, TKM-080301, TAK-960, or Ro3280; and optionally the PLK1 inhibitor is onvansertib.

Onvansertib may be at 12mg/m ² -90mg/m ² Is administered to the subject. In some embodiments, the maximum concentration of onvansertib in the blood of the subject (C _max ) Is about 100nmol/L to about 1500nmol/L. In some embodiments, the area under the curve (AUC) of the plot of the concentration of onvansertib in the blood of a subject over time is from about 1000nmol/l.h to about 400000nmol/l.h. In some embodiments, the time to reach the maximum concentration of onvansertib in the subject's blood (T _max ) Is from about 1 hour to about 5 hours. In some embodiments, the elimination half-life of onvansertib in the blood of a subject (T _1/2 ) Is from about 10 hours to about 60 hours.

In some embodiments, the PARP inhibitor is olapari or NMS-293 and the PLK1 inhibitor is onvansertib. In some embodiments, the subject has received at least one prior cancer treatment. In some embodiments, the prior treatment does not include the use of PARP inhibitors, PLK inhibitors, or both. In some embodiments, the subject is in cancer remission, optionally wherein the subject in cancer remission is in Complete Remission (CR) or Partial Remission (PR). The methods disclosed herein may further comprise one or more of the following: (1) determining the cancer status of the subject, (2) determining the responsiveness of the subject to treatment with the PLK1 inhibitor, and (3) administering one or more cancer therapeutic agents or therapies for cancer. In some embodiments, the subject is a human.

Disclosed herein includes a method of sensitizing cancer cells to PARP inhibitors. In some embodiments, the method comprises: contacting the cancer cells with a composition comprising a Polo-like kinase 1 (PLK 1) inhibitor, thereby sensitizing the cancer cells to a PARP inhibitor.

In some embodiments, the PLK1 inhibitor is onvansertib, the PARP inhibitor is olapari, or the PLK1 inhibitor is onvansertib and the PARP inhibitor is olapari. In some embodiments, contacting the cancer cells with the composition occurs in vitro, ex vivo, and/or in vivo. In some embodiments, contacting the cancer cells with the composition is in a subject, and optionally, wherein the subject is not responsive to, or is known to be resistant to, the PARP inhibitor. In some embodiments, the subject has prior treatment with a PARP inhibitor. In some embodiments, the subject is a mammal, e.g., a human.

In some embodiments, the method comprises determining sensitization of the cancer cells to the PARP inhibitor after contact with the composition. In some embodiments, the method comprises contacting the cancer cell with a PARP inhibitor, e.g., contacting the cancer cell with the PARP inhibitor occurs in the subject. In some embodiments, the method comprises determining the response of the subject to the PARP inhibitor. In some embodiments, contacting the cancer cell with the PARP inhibitor is simultaneous with contacting the cancer cell with the composition, or after contacting the cancer cell with the composition.

Also disclosed herein is a kit comprising a Polo-like kinase 1 (PLK 1) inhibitor; and providing a manual for instructions for co-administering the PLK1 inhibitor and the poly (ADP-ribose) polymerase (PARP) inhibitor to the subject to treat the cancer. In some embodiments, the subject has ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof. The cancer may be, for example, a Homologous Recombination (HR) deficient cancer. In some embodiments, the cancer is BRCA1 mutant cancer, BRCA2 mutant cancer, or both.

The instructions may, for example, comprise instructions for co-administering the PLK1 inhibitor and the PARP inhibitor simultaneously, or instructions for sequential co-administration of the PLK1 inhibitor and the PARP inhibitor. In some embodiments, the instructions comprise (1) instructions for orally administering the PLK1 inhibitor, (2) instructions for orally administering the PARP inhibitor, or both.

In some embodiments, the instructions comprise instructions that the subject has received prior PARP inhibitor treatment. In some embodiments, the instructions comprise instructions that the subject does not respond to treatment with PARP inhibitor alone. In some embodiments, the instructions comprise instructions for a subject known to be resistant to PARP inhibitor therapy. In some embodiments, the instructions comprise instructions for administering each of the PARP inhibitor and the PLK1 inhibitor to the subject at least twice or at least five cycles within a week.

In some embodiments, the instructions comprise instructions for administering the PARP inhibitor, the PLK1 inhibitor, or both, at a period of at least 7 days. In some embodiments, each treatment cycle is at least about 21 days, e.g., each treatment cycle is about 21 days to about 28 days. In some embodiments, the instructions comprise instructions for administering the PLK1 inhibitor on at least four days of the cycle. In some embodiments, the instructions comprise instructions for not administering the PLK1 inhibitor for at least one day of the cycle. In some embodiments, the instructions comprise instructions for daily administration of the PARP inhibitor. In some embodiments, the instructions comprise instructions for administering the PARP inhibitor and the PLK1 inhibitor for at least two cycles.

In some embodiments, the PARP inhibitor is selective and/or specific for PARP1 inhibition and/or PARP2 inhibition. Non-limiting examples of PARP inhibitors include iniparib (BSI 201), tazopanib (BMN-673), AZD5305, olaparib (AZD-2281), rupa-ril (AG 014699, PF-01367338), ABT-888, vitamin Li Pali (ABT-888), nilaparib, CEP 9722, MK 4827, BGB-290 (pamipril), BSI-201, CEP-8983, E7016, 3-aminobenzamide, NMS-293, or combinations thereof. In some embodiments, the PARP inhibitor is Olaparib or NMS-293.

In some embodiments, the PLK1 inhibitor is selective and/or specific for PLK 1. Non-limiting examples of PLK1 inhibitors include dihydropteridinones, pyridopyrimidines, aminopyrimidines, substituted thiazolinones, pteridine derivatives, dihydroimidazo [1,5-f ] pteridines, meta-substituted thiazolinones, benzylstyryl sulfone analogs, stilbene derivatives, or any combination thereof. In some embodiments, the PLK1 inhibitor is onvansertib, BI2536, volasertib (BI 6727), GSK461364, AZD1775, CYC140, HMN-176, HMN-214, rigossitib (ON-01910), MLN0905, TKM-080301, TAK-960, or Ro3280; and optionally wherein the PLK1 inhibitor is onvansertib.

In some embodiments, the instructions include instructions for use at 12mg/m ² -90 mg/m ² Instructions for administering onvansertib. In some embodiments, the PARP inhibitor is olapari or NMS-293 and the PLK1 inhibitor is onvansertib. In some embodiments, the instructions comprise instructions that the subject has received at least one prior treatment for cancer, and optionally, wherein the prior treatment does not include the use of a PARP inhibitor, a PLK inhibitor, or both. In some embodiments, the instructions comprise instructions that the subject is in cancer remission, and optionally, wherein the subject in cancer remission is in Complete Remission (CR) or Partial Remission (PR). In some embodiments, the kit further comprises a PARP inhibitor.

Brief Description of Drawings

Fig. 1 is a non-limiting schematic diagram showing the combination of onvansertib and PARP inhibitors in the treatment of cancer.

Figure 2 shows a non-limiting dosing schedule used in example 1.

Fig. 3A is a Kaplan Meier curve showing the survival probability of BRCA1 mutant High Grade Severe Ovarian Cancer (HGSOC) patient-derived xenograft (PDX) model #hoc22 treated with control, olapari, onvansertib, or a combination of onvansertib and olapari. Fig. 3B is a Kaplan Meier curve showing the survival probability of ovarian BRCA1 mutant HGSOC PDX model #hoc266 treated with control, olapari, onvansertib or a combination of onvansertib and olapari.

Fig. 4A-4B are graphs showing tumor volume changes in BRCA 1-Wild Type (WT) HGSOC PDX model #hoc124 treated with control, olapari, onvansertib, or a combination of onvansertib and olapari.

Fig. 5 is a table showing molecular and pharmacological characteristics of three PDX models (MNHOC 22, MNHOC266, and MNHOC316 DDPs) used in exemplary embodiments.

Fig. 6A is a graph showing the change in body weight in mice in MNHOC22 model treated with control, olapari, onvansertib, or a combination of onvansertib and olapari. Fig. 6B is a Kaplan Meier curve showing the probability of survival in an MNHOC22 model treated with control, olapari, onvansertib, or a combination of onvansertib and olapari. Fig. 6C is a graph showing the change in body weight in mice in MNHOC66 model treated with control, olapari, onvansertib, or a combination of onvansertib and olapari. Fig. 6D is a Kaplan Meier curve showing the probability of survival in MNHOC66 model treated with control, olapari, onvansertib, or a combination of onvansertib and olapari. Fig. 6E is a graph showing changes in mouse body weight in MNHOC316DDP model treated with control, olapari, onvansertib, or a combination of onvansertib and olapari. Fig. 6F is a Kaplan Meier curve showing survival probability in an MNHOC316DDP model treated with control, olapari, onvansertib, or a combination of onvansertib and olapari.

Fig. 7 is a table showing median survival time and lifetime Increase (ILS) for three PDXs (MNHOC 22, MNHOC266, and MNHOC316 DDP) treated with control, olaparib, onvansertib, or a combination of onvansertib and olaparib.

Fig. 8 provides a graph showing evaluation of Ki67 positivity (panel a), mitosis (panel B), apoptosis (panel C) and RAD51 foci formation (panel D) in PDX MNHOC22 and MNHOC266 treated with controls, olapari, onvansertib or a combination of onvansertib and olapari.

FIG. 9 shows the levels of pSer10-H3 and pSer139- γH2AX in tumors of PDX MNOCC 22 and MNOCC 266 treated with control, olaparib, onvansertib or a combination of onvansertib and Olaparib.

Fig. 10 is a graph showing inhibition of tumor growth in prostate BRCA 2-mutant 22RV1 xenograft models treated with control, olapari, BI2536 or a combination of BI2536 and olapari.

Fig. 11 depicts two pie charts (circles) showing the cancer types of cell lines tested in two-dimensional cell cultures (upper panels) and three-dimensional cell cultures (lower panels).

Fig. 12A-12B depict the onvansertib and olapari synergy scores in cells cultured in two-dimensional cell cultures (fig. 12A) and three-dimensional cell cultures (fig. 12B).

Detailed description of the preferred embodiments

In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like numerals generally identify like components unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein and make part of this disclosure.

All patents, published patent applications, other publications, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety for the relevant art.

Definition of the definition

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. See, e.g., singleton et al, dictionary of Microbiology and Molecular Biology, 2 nd edition, j.wiley & Sons (New York, NY 1994); sambrook et al Molecular Cloning, ALaboratory Manual, cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For the purposes of this disclosure, the following terms are defined below.

As used herein, "subject" refers to an animal that is the subject of treatment, observation, or experiment. "animals" include cold and warm-blooded vertebrates and invertebrates such as fish, crustaceans, reptiles and in particular mammals. "mammal" includes, but is not limited to, mice; a rat; a rabbit; guinea pigs; a dog; a cat; sheep; a goat; cattle; a horse; primates such as monkeys, chimpanzees, and apes and in particular humans.

As used herein, "patient" refers to a subject who is treated by a medical professional, such as a doctor (i.e., symptomatic or orthopedic medical doctor) or veterinary doctor, in an attempt to cure or at least ameliorate the effects of a particular disease or disorder, or to first prevent the occurrence of a disease or disorder. In some embodiments, the patient is a human or an animal. In some embodiments, the patient is a mammal.

As used herein, "administration" or "administering" refers to a method of administering a dose of a pharmaceutically active ingredient to a vertebrate.

As used herein, "dose" refers to a combined amount (combined amountof) of active ingredients (e.g., PLK1 inhibitors (e.g., onvansertib) or PARP inhibitors (e.g., olapari)).

As used herein, "unit dose" refers to the amount of therapeutic agent administered to a patient in a single dose.

As used herein, the term "daily dose" or "daily dose" refers to the total amount of a pharmaceutical composition or therapeutic agent that will be administered within 24 hours.

As used herein, the term "delivery" refers to methods, formulations, techniques and systems for delivering a pharmaceutical composition or therapeutic agent into a patient as needed to safely achieve its desired therapeutic effect. In some embodiments, an effective amount of the composition or agent is formulated for delivery into the blood stream of a patient.

As used herein, the term "formulated" or "formulation" refers to a process of combining different chemicals comprising one or more pharmaceutically active ingredients to produce a dosage form. In some embodiments, two or more pharmaceutically active ingredients may be co-formulated into a single dosage form or a combined dosage unit, or formulated separately and subsequently combined into a combined dosage unit. Sustained release formulations are formulations designed to release the therapeutic agent slowly in the body over an extended period of time, while immediate release formulations are formulations designed to release the therapeutic agent rapidly in the body over a shortened period of time.

As used herein, the term "pharmaceutically acceptable" indicates that the indicated material does not have properties that would lead to a reasonably cautious medical practitioner avoiding administration of the material to a patient in view of the disease or condition to be treated and the corresponding route of administration. For example, such materials are often required to be substantially sterile.

As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, which involves carrying or transporting any supplement or composition or component thereof from one organ or part of the body to another organ or part of the body, or delivering an agent to diseased tissue or tissue adjacent to diseased tissue. A carrier or excipient may be used to produce the composition. The carrier or excipient may be selected to facilitate administration of the drug or prodrug. Examples of carriers include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or various types of starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents. Examples of physiologically compatible solvents include water for injection (WFI), saline solution, and sterile solutions of dextrose.

As used herein, the term "pharmaceutically acceptable salt" refers to any acid or base addition salt whose counter ion is non-toxic to the patient at the pharmaceutical dosage of the salt. Many pharmaceutically acceptable salts are well known in the pharmaceutical arts. If pharmaceutically acceptable salts of the compounds of the present disclosure are used in these compositions, these salts are preferably derived from inorganic or organic acids and bases. Such acid salts include the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate (e.g., hydrochloride and hydrobromide), sulfate, phosphate, nitrate, sulfamate, malonate, salicylate, methylene-bis-b-hydroxynaphthalene formate, gentisate, hydroxyethylsulfonate, di-p-toluoyl tartrate, ethanesulfonate (ethane sulfonate), cyclohexanetate, quinite, etc. Pharmaceutically acceptable base addition salts include, but are not limited to, those derived from alkali metal bases or alkaline earth metal bases or conventional organic bases (such as triethylamine, pyridine, piperidine, morpholine, N-methylmorpholine), ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts of organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine salts, and salts of amino acids such as arginine, lysine, and the like.

As used herein, the term "hydrate" refers to a complex formed by the combination of water molecules and molecules or ions of a solute. As used herein, the term "solvate" refers to a complex formed by a combination of a solvent molecule and a molecule or ion of a solute. The solvent may be an organic compound, an inorganic compound, or a mixture of both. Solvates are intended to include hydrates, hemihydrates, channel hydrates, and the like. Some examples of solvents include, but are not limited to, methanol, N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, and water.

As used herein, "therapeutically effective amount" or "pharmaceutically effective amount" refers to the amount of a therapeutic agent that has a therapeutic effect. The dosage of the pharmaceutically active ingredient useful in the treatment is a therapeutically effective amount when administered alone or in combination with one or more additional therapeutic agents. Thus, as used herein, a therapeutically effective amount refers to an amount of a therapeutic agent that produces a desired therapeutic effect as judged by clinical trial results and/or model animal studies. The therapeutically effective amount will vary depending on the compound, disease, disorder or condition and its severity, the age, weight, etc., of the mammal to be treated. The dosage may conveniently be administered, for example, as a divided dosage up to four times daily or in sustained release form.

As used herein, the term "treatment" or "treatment" refers to administration of a therapeutic agent or pharmaceutical composition to a subject for prophylactic and/or therapeutic purposes. The term "prophylactic treatment" refers to treating a subject that has not exhibited symptoms of a disease or condition, but is susceptible to or otherwise at risk of a particular disease or condition, whereby treatment reduces the likelihood that the patient will develop the disease or condition. The term "therapeutic treatment (therapeutic treatment)" refers to the administration of a treatment to a subject already suffering from a disease or condition. As used herein, a "therapeutic effect" reduces one or more symptoms of a disease or disorder to some extent. For example, the effect of a treatment may be observed by a reduction in subjective discomfort conveyed by the subject (e.g., reduced discomfort recorded in a self-administered patient questionnaire).

As used herein, the terms "prevention", "prevention" and grammatical variations thereof refer to the prophylactic treatment of a subclinical disease state of a subject (e.g., a mammal (including a human)) to reduce the probability of occurrence of the clinical disease state. The method may partially or completely delay or exclude the onset or recurrence of one or more of the disorder or condition and/or its concomitant symptoms, or prevent or reduce the risk of the subject obtaining or reacquiring the disorder or condition. The subject is selected for prophylactic therapy based on factors known to increase the risk of developing a clinical disease state as compared to the general population. "prevention" therapy can be divided into (a) primary prevention and (b) secondary prevention. Primary prevention is defined as treatment of a subject who has not yet presented with a clinical disease state, while secondary prevention is defined as prevention of a second occurrence of the same or similar clinical disease state.

As used herein, each of the terms "partial response" and "partial remission" refers to an improvement in the cancerous state as measured by, for example, tumor size and/or cancer marker levels in response to treatment. In some embodiments, "partial response" means that the tumor or blood markers indicative of the tumor is reduced in size or level by about 50% in response to treatment. The treatment may be any treatment for cancer including, but not limited to, chemotherapy, radiation therapy, hormonal therapy, surgery, cell or bone marrow transplantation, and immunotherapy. The size of the tumor can be detected by clinical or radiological means. Markers indicative of tumors can be detected by means well known to the skilled artisan, e.g., ELISA or other antibody-based tests.

As used herein, each of the terms "complete response" or "complete remission" means that the cancerous state as measured by, for example, tumor size and/or cancer marker levels has disappeared after treatment, including, but not limited to, chemotherapy, radiation therapy, hormonal therapy, surgery, cell or bone marrow transplantation, and immunotherapy. The presence of a tumor can be detected by clinical or radiological means. Markers indicative of tumors can be detected by means well known to the skilled artisan, e.g., ELISA or other antibody-based tests. However, a "complete response" does not necessarily indicate that the cancer has been cured, as there is a possibility of recurrence after a complete response.

Cancer of the human body

The methods, compositions and kits disclosed herein can be used to treat cancer. In some embodiments, a method for treating cancer comprises administering to a subject (e.g., patient) in need thereof a PARP inhibitor (e.g., olapari or NMS-293) or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof, and a Polo-like kinase 1 (PLK 1) inhibitor (e.g., onvansertib) or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof.

The methods, compositions, and kits disclosed herein can be used for various types of cancers, including, but not limited to, melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC)), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies. Additionally, the diseases or conditions provided herein include refractory or recurrent malignant tumors, the growth of which can be inhibited using the methods and compositions disclosed herein. In some embodiments, the cancer is an epithelial cancer, squamous cell carcinoma, adenocarcinoma, sarcoma, endometrial cancer, breast cancer, ovarian cancer, cervical cancer, fallopian tube cancer, primary peritoneal cancer, colon cancer, colorectal cancer, anogenital area squamous cell carcinoma, melanoma, renal cell carcinoma, lung cancer, non-small cell lung cancer, lung squamous cell carcinoma, gastric cancer, bladder cancer, gallbladder cancer, liver cancer, thyroid cancer, laryngeal cancer, salivary gland cancer, esophageal cancer, head and neck cancer, glioblastoma, glioma, head and neck squamous cell carcinoma, prostate cancer, pancreatic cancer, mesothelioma, sarcoma, hematologic cancer, leukemia, lymphoma, neuroma, or a combination thereof. In some embodiments, the cancer is epithelial cancer, squamous cell cancer (e.g., cervical canal, eyelid, conjunctiva, vagina, lung, oral cavity, skin, bladder, tongue, larynx, and esophagus), and adenocarcinoma (e.g., prostate, small intestine, endometrium, cervical canal, large intestine, lung, pancreas, esophagus, rectum, uterus, stomach, breast, and ovary). In some embodiments, the cancer is a sarcoma (e.g., myogenic sarcoma), leukemia, neuroma, melanoma, and lymphoma.

The cancer may be a solid tumor, a liquid tumor, or a combination thereof. In some embodiments, the cancer is a solid tumor including, but not limited to, melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, gall bladder cancer, laryngeal cancer, liver cancer, thyroid cancer, gastric cancer, salivary gland cancer, prostate cancer, pancreatic cancer, merkel cell carcinoma, brain and central nervous system cancer, and any combination thereof. In some embodiments, the cancer is a liquid tumor. In some embodiments, the cancer is a hematologic cancer. Non-limiting examples of hematologic cancers include diffuse large B-cell lymphoma ("DLBCL"), hodgkin lymphoma ("HL"), non-hodgkin lymphoma ("NHL"), follicular lymphoma ("FL"), acute myelogenous leukemia ("AML"), and multiple myeloma ("MM").

The cancer may be, for example, ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof. The cancer may be pancreatic ductal carcinoma, pancreatic adenocarcinoma, ovarian serous adenocarcinoma, breast ductal carcinoma, high-grade serous ovarian adenocarcinoma, or a combination thereof. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is prostate cancer. The cancer may be BRCA1 mutant cancer, BRCA2 mutant cancer, or both. In some embodiments, the cancer is BRCA2 mutant prostate cancer. In some embodiments, the cancer is BRCA1 mutant ovarian cancer. The cancer may be BRCA wild-type cancer having a wild-type BRCA1 and/or BRCA2 sequence, such as BRCA1 wild-type ovarian cancer and/or BRCA2 wild-type ovarian cancer. In some embodiments, the cancer is BRCA1 wild-type prostate cancer.

In some embodiments, the cancer is sensitive to PARP inhibitor treatment. In some embodiments, the cancer is characterized by a defect in DNA repair. The cancer may be a Homologous Recombination (HR) deficient cancer with impaired HR-mediated DNA repair function. In some embodiments, a subject with cancer has one or more pathogenic variants of one or more genes involved in HR-mediated DNA repair mechanisms, including, but not limited to BRCA1, BRCA2, 53BP1, ATM, ATR, ATRIP, BARD1, BLM, BRIP1, DMC1, MRE11A, NBN, PALB2, RAD50, RAD51B, RAD C, RAD51D, RIF1, RMI2, RPA1, TOP3A, TOPBP1, XRCC2, and XRCC3.

In some embodiments, the cancer is BRCA 1-deficient and/or BRCA 2-deficient. In some embodiments, HRDetect scores for cancers or tumors may be calculated to detect BRACA1/BRCA2 deficient tumors. HRDetect is a classifier based on whole genome sequencing designed to predict BRCA1 and BRCA2 defects based on six mutant features. Details on the HRDetect method are in Nat med.2017 by Davies H et al; 23:517-25, the contents of which are incorporated herein by reference in their entirety. In some embodiments, an HRDetect score equal to or greater than about 0.7 indicates an HR defect, while less than about 0.7 indicates HR potency (HR proficiency). In some embodiments, the cancer has an HRDetect score equal to or greater than 0.7. In some embodiments, the cancer has an HRDetect score of less than 0.7.

In some embodiments, the cancer is PARP inhibitor resistant cancer or has developed resistance to PARP inhibitors. The cancer may be PARP inhibitor resistant (e.g., olapari resistant) breast cancer, PARP inhibitor resistant (e.g., olapari resistant) ovarian cancer, PARP inhibitor resistant (e.g., olapari resistant) pancreatic cancer, or PARP inhibitor resistant (e.g., olapari resistant) prostate cancer. In some embodiments, combined inhibition of PLK1 and PARP can be effective in treating PARP inhibitor resistant cancers by significantly reducing tumor size, increasing cancer survival and extending the duration of cancer survival compared to single agent treatment (PARP inhibitor or PLK1 inhibitor).

In some embodiments, RAD51 focus assays are performed to distinguish between PARP inhibitor-sensitive cancers and PARP inhibitor-resistant cancers. RAD51 refers to a homologous recombinant DNA repair protein that forms a focal point of a nucleus after DNA damage, and can be used as an index of the repair function of the homologous recombinant DNA. RAD51 foci can be quantified using immunofluorescence-based methods in formalin-fixed paraffin-embedded tumor samples treated with vehicle or PARP inhibitors. A low RAD51 score may be associated with PARP inhibitor (e.g., olapari or NMS-293) sensitivity, while a high RAD51 score may be associated with PARP inhibitor (e.g., olapari or NMS-293) resistance. Details regarding RAD51 focus determination can be found, for example, in guffnti F et al, br J Cancer,2022Jan;126 120-128, the contents of which are incorporated herein by reference. In some embodiments, the cancer has a RAD51 focus level equal to or greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%. In some embodiments, the cancer has a RAD51 focus level of less than 30%, 25%, 20%, 15%, 10%, 5%, 2%, or 1%.

PARP inhibitors and PLK inhibitors

The methods, compositions, and kits disclosed herein can be used to treat cancer, such as ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof. In some embodiments, a method for treating cancer comprises administering to a subject (e.g., patient) in need thereof a PARP inhibitor (e.g., olapari or NMS-293) or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof, and a PLK1 inhibitor (e.g., onvansertib) or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof. The method may comprise administering a pharmaceutically effective amount of a PARP inhibitor and a pharmaceutically effective amount of a PLK1 inhibitor.

PARP inhibitors

Poly (ADP-ribose) polymerase (PARP) plays a key role in DNA damage response and affects many DDR pathways, including BER, HR, NER, NHEJ and MMR, directly or indirectly. PARP inhibitors are inhibitors of PARP, developed for a variety of indications, including the treatment of cancer. PARP is critical for repair of single-stranded DNA breaks (SSBs). Failure to repair SSBs by PARP inhibition results in double-stranded DNA breaks (DSBs). In cells with a functionally Homologous Recombination (HR) pathway, DSBs are repaired. In cells with dysfunctional HR pathways, such as BRCA1 and/or BRCA2 mutant cells, the lesions are not sufficiently repaired, resulting in cell death.

Several forms of cancer are more dependent on PARP than conventional cells, which makes PARP an attractive target for cancer therapy. In addition to its use in cancer therapy, PARP inhibitors are also considered potential treatments for acute life threatening diseases such as stroke and myocardial infarction, as well as long term neurodegenerative diseases. DNA is damaged thousands of times during each cell cycle and the damage must be repaired. BRCA1, BRCA2 and PALB2 are proteins important for repair of double-stranded DNA breaks by error-free homologous recombination repair or HRR pathway. When a mutation occurs in the gene of any one of the proteins, such a change may lead to an error in DNA repair, which may ultimately cause breast cancer. When subjected to sufficient damage at a time, the altered gene may cause cell death. PARP1 is a protein important for repair of single strand breaks (a "nick" in DNA). If such a nick is not repaired until the DNA is replicated (which must be before the cell divides), the replication itself may cause the formation of a double strand break. Drugs that inhibit PARP1 cause multiple double strand breaks to form in this way, and in tumors with BRCA1, BRCA2 or PALB2 mutations, these double strand breaks cannot be repaired effectively, leading to cell death. Normal cells that do not replicate their DNA as often as cancer cells, and normal cells that lack any mutated BRCA1 or BRCA2, still have a homologous repair process, which allows them to survive PARP inhibition. Some cancer cells lacking the tumor suppressor PTEN may be sensitive to PARP inhibitors because of down-regulation of Rad51 (a key homologous recombination component), although other data suggest that PTEN may not regulate Rad51. In some embodiments, the PARP inhibitor is a PARP inhibitor that is effective against one or more PTEN deficient tumors (e.g., some invasive prostate cancers). Cancer cells that are hypoxic (e.g., in fast growing tumors) are sensitive to PARP inhibitors. PARP inhibitors were initially thought to act primarily by blocking PARP enzyme activity, thereby preventing repair of DNA damage and ultimately causing cell death. PARP inhibitors have additional modes of action: PARP proteins are localized at DNA damage sites, which are associated with their anti-tumor activity. Captured PARP protein-DNA complexes are highly toxic to cells because they block DNA replication. The PARP protein family in humans includes PARP1 and PARP2, which are DNA binding and repair proteins. When activated by DNA damage, these proteins recruit other proteins to accomplish the actual work of repairing DNA. Under normal conditions, PARP1 and PARP2 are released from DNA after the repair process begins. But when they bind to PARP inhibitors, PARP1 and PARP2 become trapped on the DNA. It was shown that the captured PARP-DNA complex was more toxic to cells than the unrepaired single-stranded DNA breaks accumulated in the absence of PARP activity, indicating that the PARP inhibitor acts as a PARP poison. As described herein, there are two classes of PARP inhibitors: (1) A catalytic inhibitor which is mainly used for inhibiting the activity of PARP enzyme and does not trap PARP protein on DNA, and (2) a dual inhibitor which both blocks the activity of PARP enzyme and acts as a PARP poison. Non-limiting examples of PARP inhibitors include: inibarib (BSI 201) (e.g., for breast cancer and squamous cell lung cancer); olapari (AZD-2281) (e.g., for breast, ovarian and colorectal cancers); rukapali (AG 014699, PF-01367338, e.g., for metastatic breast and ovarian cancer); dimension Li Pali (ABT-888) (e.g., for metastatic melanoma and breast cancer); CEP 9722 (e.g., for non-small cell lung cancer (NSCLC)); MK 4827 inhibiting both PARP1 and PARP 2; BMN-673 (e.g., for advanced hematologic malignancies and for advanced or recurrent solid tumors); and 3-aminobenzamide. The PARP inhibitor may be a PARP1 inhibitor or a PARP2 inhibitor. In some embodiments, PARP inhibitors may inhibit PARP1 and PARP2. The PARP inhibitor may be a selective inhibitor of PARP1, PARP2 or both.

Methods and compositions disclosed herein for treating cancer in combination with one or more PLK1 inhibitors may comprise one or more PARP inhibitors (including, but not limited to, olapari, tazopari (BMN-673), AZD5305, ruaparil, vitamin Li Pali, nilaparib, CEP 9722, MK 4827, BGB-290 (pamipril), ABT-888, AG014699, BSI-201, CEP-8983, E7016, NMS-P293, and 3-aminobenzamide). PARP inhibitors are known to exhibit synthetic lethality, for example, in tumors with BRCA1/2 mutations. Olaparib has been approved by the FDA for treatment of ovarian cancer patients with BRCA1 or BRCA2 mutations. In addition to olapari, other FDA approved PARP inhibitors for ovarian cancer include nilaparib and rupa. Talazolepal has recently been approved for the treatment of breast cancer with germline BRCA mutations and is in phase III trials of hematologic malignancies and solid tumors, and efficacy in SCLC, ovarian cancer, breast cancer and prostate cancer has been reported. Valipali was in phase III trials of advanced ovarian cancer, TNBC and NSCLC. Not all PARP inhibitors are dependent on BRCA mutant status, and nilaparil has been approved for maintenance therapy of recurrent platinum-sensitive ovarian cancer, fallopian tube cancer, or primary peritoneal cancer, independent of BRCA status. NMS-P293 is described, for example, in the following: abstract 4843 NMS-P293, a PARP-1selective inhibitor with no trapping activity and high CNS penetration,possesses potent in vivo efficacy and represents a novel therapeutic option for brain localized metastases and glioblastoma,Proceedings:AACR Annual Meeting 2018; april 14-18,2018; chicago, IL.

Some PARP inhibitors have been approved for use in BRCA1/2 mutant ovarian, breast, prostate and pancreatic cancer patients. Although the initial response to PARP inhibitors is high, patients eventually develop resistance. Resistance mechanisms to PARP inhibitors include recovery of Homologous Recombination (HR).

The PARP inhibitor may be, for example, iniparib (BSI 201), tazopanib (BMN-673), AZD5305, olaparib (AZD-2281), rupa line (AG 014699, PF-01367338), ABT-888, vitamin Li Pali (ABT-888), nilaparib, CEP 9722, MK 4827, BGB-290 (pamiproli), BSI-201, CEP-8983, E7016, 3-aminobenzamide, or a combination thereof. In some embodiments, the PARP inhibitor is olapari.

PLK1 inhibitors

Polo-like kinases (PLKs) are a family of five highly conserved serine/threonine protein kinases. PLK1 is the primary regulator of mitosis and involves several steps of the cell cycle including mitotic entry, centrosome maturation, bipolar spindle formation, chromosome segregation and cytokinesis. PLK1 has been shown to be overexpressed in solid tumors and hematological malignancies, including AML. PLK1 inhibition induces G2-M phase arrest and subsequent apoptosis in cancer cells and has emerged as a promising targeted therapy. Several PLK inhibitors have been studied in clinical trials. In a randomized phase II study on AML patients not treated but not suitable for induction therapy, the pan PLK inhibitor volasertib (BI 6727) intravenously administered in combination with LDAC showed a significant increase in OS when compared to LDAC alone. Subsequent randomized phase III studies did not find benefit from the combination and described an increased risk of severe infection. PLK1 promotes HR during double-stranded DNA break (DSB) repair. PLK1 phosphorylates Rad51 and BRCA1, promoting their recruitment to DSB sites and thereby effecting HR-mediated DNA repair.

The onvansertib (also known as "compound of formula (I)" in PCM-075, NMS-1286937, NMS-937, U.S. Pat. No. 8,927,530; IUPAC name 1- (2-hydroxyethyl) -8- { [5- (4-methylpiperazin-1-yl) -2- (trifluoromethoxy) phenyl ] amino } -4, 5-dihydro-1H-pyrazolo [4,3-H ] quinazoline-3-carboxamide) is a selective ATP-competitive PLK1 inhibitor. Biochemical assays showed that onvansertib has high specificity for PLK1 in the 296 kinase group (including other PLK members). In models of both solid and hematological malignancies, onvansertib has strong antitumor activity in vitro and in vivo. onvansertib was the first PLK 1-specific ATP-competitive inhibitor administered by the oral route to enter clinical trials with demonstrated antitumor activity in different preclinical models. The onvansertib inhibits cell proliferation in AML cell lines and tumor growth in AML xenograft models at nanomolar concentrations. The onvansertib also significantly increases the antitumor activity of cytarabine in a disseminated AML model.

onvansertib shows high potency in proliferation assays, with low nanomolar activity against a large number of cell lines from both solid as well as hematological tumors. Following oral administration at well-tolerated doses in mice, onvansertib effectively causes mitotic cell cycle arrest and subsequent apoptosis in cancer cell lines and inhibits xenograft tumor growth, with a well-defined PLK 1-related mechanism of action. Furthermore, onvansertib shows activity in combination therapies with approved cytotoxic drugs (such as irinotecan), where there is enhanced tumor regression in HT29 human colon adenocarcinoma xenografts compared to each dose alone, and prolonged survival of animals in a disseminated model of AML in combination therapies with cytarabine. The advansertib has favorable pharmacological parameters and good oral bioavailability in rodent and non-rodent species, as well as demonstrated antitumor activity in different non-clinical models using multiple dosing regimens, which may provide a high degree of flexibility in dosing schedules, warranting investigation in a clinical setting. onvansertib has several advantages over volasertib (BI 6727, another PLK1 inhibitor), including a higher degree of potency and specificity for PLK1 isozymes and a higher degree of oral bioavailability.

Phase I, first time in humans, dose escalation studies in patients with advanced/metastatic solid tumors established neutropenia and thrombocytopenia as the major dose limiting toxicities. These hematological toxicities are expected based on the mechanism of action of the drug and are reversible, recovery occurring within 3 weeks. The half-life of onvansertib was determined to be between 20 hours and 30 hours. The oral bioavailability of onvansertib coupled with its short half-life provides an opportunity for convenient, controlled and flexible dosing schedules, potentially minimizing toxicity and improving the therapeutic window. Pharmacodynamics and biomarker studies have been performed, including baseline genomic profiling (baseline genomic profiling), continuous monitoring of mutant allele fractions (mutant allele fractions) in plasma, and extent of PLK1 inhibition in circulating blasts, to identify biomarkers associated with clinical response, and are described in WO 2021/146322, the contents of which are incorporated herein by reference in their entirety.

As disclosed herein, combination therapies of PARP inhibitors and PLK1 inhibitors (including onvansertib) can result in significantly enhanced efficacy against cancer (e.g., breast cancer, ovarian cancer, colorectal cancer, prostate cancer, head and neck cancer, non-small cell lung cancer, intrahepatic cholangiocarcinoma, gastric cancer, urothelial cancer, small cell lung cancer, endometrial cancer, cervical cancer, rhabdomyosarcoma, cholangiocarcinoma, or a combination thereof), resulting in tumor regression and cancer survival (cancer survivin). Tumor regression and cancer survival/duration by combination can surprisingly be synergistic (i.e., beyond additive, better than cumulative anti-tumor efficacy caused by PARP inhibitors and PLK1 inhibitors alone). The PLK1 inhibitor may be onvansertib. Provided herein include methods, compositions, and kits for treating cancer in a subject (e.g., a human patient suffering from cancer). The method comprises administering to the patient a PARP inhibitor and a PLK1 inhibitor in a manner sufficient to inhibit or reduce progression of the cancer. For example, the PARP inhibitor and PLK1 inhibitor may be administered simultaneously, separately or sequentially to a subject suffering from cancer. Surprisingly, the tumor regression and cancer survival/duration by combination exceeds additive, i.e. superior to the cumulative anti-tumor efficacy caused by PARP inhibitors and PLK1 inhibitors alone. Provided herein include methods, compositions, and kits for treating cancer in a subject (e.g., a human patient suffering from cancer). The method comprises administering to the patient a PARP inhibitor and a PLK1 inhibitor in a manner sufficient to inhibit progression of the cancer. For example, the PARP inhibitor and PLK1 inhibitor may be administered simultaneously, separately or sequentially to a subject suffering from cancer.

In some embodiments, the inhibition or reduction of cancer progression is not merely additive, but rather enhanced or synergistic (i.e., inhibition is greater than the combined inhibition of progression caused by PARP inhibitor alone plus PLK1 inhibitor alone). In different embodiments, the enhanced or synergistic efficacy or inhibition of any combination of PARP inhibitors and PLK1 inhibitors of the present disclosure may be different. In some embodiments, the enhanced or synergistic efficacy or inhibition of any combination of a PARP inhibitor and a PLK1 inhibitor of the present disclosure is greater than, at least less than, at most greater than, or at most greater than the inhibition of progression by the PARP inhibitor alone plus the PLK1 inhibitor alone: 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or a number or range between any two of these values.

The molar ratio of PLK1 inhibitor (e.g., onvansertib) to PARP inhibitor (e.g., olapari or NMS-293) may be, for example, a number or range between about 1:200, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:1, 10:1, 20:1, 30:1, 40:1, 50:1, 100:1, 1000:1, 2000:1, or 5000:1 or any two of these values. In some embodiments, the enhanced or synergistic efficacy or inhibition of cancer progression caused by a combination of a PARP inhibitor and a PLK1 inhibitor (e.g., onvansertib) is greater than, about less than, at least about less than, at most less than, or at most about less than the combined inhibition of progression caused by a PARP inhibitor alone plus a PLK1 inhibitor alone (e.g., onvansertib). 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300% or a number or range between any two of these values. For example, a combination of PARP inhibitor and PLK1 inhibitor may result in 50%, 60%, 70%, 80%, 90% or more inhibition of cancer progression (50%, 40%, 30%, 20%, 10% or less of cancer cell viability), while under the same conditions, a combination of PARP inhibitor alone plus PLK1 inhibitor alone may result in 10%, 20%, 25%, 30% or less inhibition of cancer progression (90%, 80%, 75%, 70% or more of cancer cell viability). Thus, the enhanced or synergistic efficacy or inhibition of cancer progression caused by a combination of PARP inhibitor and PLK1 inhibitor is, for example, 50%, 60%, 70%, 80%, 90%, 100% or more greater than the combined inhibition of progression caused by PARP inhibitor alone plus PLK1 inhibitor alone. In some embodiments, the PARP inhibitor is olaparib and the PLK1 inhibitor is onvansertib.

The methods described herein using a combination of PARP inhibitors and PLK1 inhibitors are expected to be effective against a variety of cancers, such as head and neck cancer, non-small cell lung cancer, intrahepatic cholangiocarcinoma, gastric cancer, urothelial cancer, small cell lung cancer, breast cancer, endometrial cancer, cervical cancer, rhabdomyosarcoma, cholangiocarcinoma, liver cancer, ovarian cancer, prostate cancer, colorectal cancer, pancreatic cancer, prostate cancer, or combinations thereof.

As described herein, the patient may achieve a complete response or a partial response following treatment with PARP inhibitors and PLK1 inhibitors. In some embodiments, the patient achieves a complete response. In some embodiments, the patient achieves a partial response. In some embodiments, the patient is not responsive to treatment with only one or more PARP inhibitors. In some embodiments, the patient does not respond to treatment with PARP inhibitor alone.

PARP inhibitors and PLK1 inhibitors may be administered to a patient in any manner that is considered effective in treating cancer. PARP inhibitors may be administered with PLK1 inhibitors or separately with PLK1 inhibitors. When administered alone, the PARP inhibitor may be administered before or after the PLK1 inhibitor, or in different administration cycles.

The PLK1 inhibitor and PARP inhibitor may be co-administered (i.e., administered simultaneously) or sequentially. In some embodiments, it may be advantageous to administer a PLK1 inhibitor (e.g., onvansertib) to a subject prior to administration of a PARP inhibitor (e.g., olapari or NMS-293) to the subject, e.g., on one or more days or daily of the administration days of the PLK1 inhibitor and the PAPR inhibitor to the subject, such that the PLK1 inhibitor may sensitize cells (e.g., cancer cells) to the PARP inhibitor (e.g., by injury to HR) to achieve effective treatment. The time interval between administration of the PLK1 inhibitor and PARP inhibitor may be, for example, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, any value between any two of these values, or any value between 30 minutes and 12 hours. In some embodiments, both the PLK1 inhibitor (e.g., onvansertib) and the PARP inhibitor (e.g., olapari) are administered to the subject on the following, or at least about the following, days in one cycle (e.g., in each cycle during the combination treatment): 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, and optionally, on each day of administration of both, PLK1 inhibitor is administered to the subject prior to PARP inhibitor, e.g., PLK1 inhibitor is administered 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, a range between any two of these values, or any value between 30 minutes and 12 hours prior to PARP inhibitor administration.

The PARP inhibitor and PLK1 inhibitor may each be administered on any schedule, for example, daily or once a week or more; once, twice, three times, four times, five times, six times or seven times per week (daily); for one week or more; etc. In some embodiments, the PARP inhibitor and the PLK1 inhibitor are each administered to the patient at a period of at least two times a week. In other embodiments, the PARP inhibitor and the PLK1 inhibitor are each administered to the patient at least five cycles over a week. In further embodiments, the patient experiences administration for at least two cycles. The patient may undergo one cycle or more than one cycle of administration, for example, two cycles, three cycles, four cycles, five cycles, or more. The administration of two adjacent cycles may be continuous, i.e. without interruption between the last day of the first cycle and the first day of the second cycle. In some embodiments, the administration of two adjacent cycles has an interruption therebetween, i.e., the interval between the last day of the first cycle and the first day of the second cycle. The interruption (i.e., interval) may be or at least be: one day, two days, three days, five days, seven days, ten days, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or numbers or ranges between any two of these values. In some embodiments, the patient experiences administration for three or four cycles, wherein each cycle comprises at least five times a week (e.g., 5 days a week). Each cycle of the multicycle administration may have the same or different dosing schedule. For example, one cycle of a multicycle administration may be five consecutive days of daily administration of PLK1 inhibitor and PARP inhibitor and two days of the week discontinued for four weeks, and one or more other cycles of the same multicycle administration may be 28 consecutive days of daily administration of PLK1 inhibitor and PARP inhibitor over a four week period.

PARP inhibitors may be administered to a patient at any suitable dose, for example, at about, at least, or at most the following doses: 0.1mg/kg, 1mg/kg, 5mg/kg, 10mg/kg, 20mg/kg, 30mg/kg, 40mg/kg, 50mg/kg, 60mg/kg, 70mg/kg, 80mg/kg, 90mg/kg, 100mg/kg, 200mg/kg, 300mg/kg, 400mg/kg, 500mg/kg, 600mg/kg, 700mg/kg, 800mg/kg, 900mg/kg, 1000mg/kg, 1500mg/kg, 2000mg/kg or numbers between any two of these values. The weight-based dosage unit (mg/kg) may be converted to another unit (e.g., mg/m) using a conversion table such as a Body Surface Area (BSA) conversion table ² ) As will be appreciated by those skilled in the art. In some embodiments, the PARP inhibitor is olapari or NMS-293, which is administered at a dose of about below, at least below, or at most below: 1mg/kg, 5mg/kg, 10mg/kg, 20mg/kg, 30mg/kg, 40mg/kg, 50mg/kg, 60mg/kg, 70mg/kg, 80mg/kg, 90mg/kg, 100mg/kg, 200mg/kg, 300mg/kg, 400mg/kg, 500mg/kg, 600mg/kg, 700mg/kg, 800mg/kg, 900mg/kg, 1000mg/kg or numbers between any two of these values.

PARP inhibitors may be administered to a patient once daily, twice daily or three times daily. In some embodiments, the PARP inhibitor is administered in a period of 7-56 days of daily administration. In some embodiments, the PARP inhibitor is administered in a period of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 35, 42, 49 or 56 days. In some embodiments, the PARP inhibitor is administered on 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 32 days, 35 days, 42 days, 49 days, or 56 days in one cycle. In some embodiments, the PARP inhibitor is administered on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, day 28, day 29, day 30, day 31, day 32, day 33, day 34, day 35, day 36, day 37, day 38, day 39, day 40, day 41, day 42, day 43, day 44, day 45, day 46, day 47, day 48, day 49, day 50, day 51, day 52, day 53, day 54, and/or day 56. In some embodiments, the PARP inhibitor is not administered on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, day 28, day 29, day 31, day 32, day 33, day 34, day 35, day 36, day 37, day 38, day 39, day 40, day 41, day 42, day 43, day 44, day 45, day 46, day 47, day 48, day 49, day 50, day 51, day 52, day 53, day 54, day 55 and/or day 56. For example, olapari or NMS-293 may be administered at a period of 5, 6, 7, 8, 9 or 10 days. The olapari may be administered daily on each day of the administration cycle or on the selected day. In some embodiments, the olapari is administered in a 7 day cycle, wherein the daily administration is for 5 days (e.g., days 1-5) and the administration is not for 2 days (e.g., days 6-7).

Any PARP inhibitor now known or later discovered may be used in these methods, including PARP inhibitors that are selective for PARP (e.g., PARP1, PARP2, or both), as well as PARP inhibitors that also inhibit other protein activities. Non-limiting examples of PARP inhibitors include Iniparib (BSI 201), tazopanib (BMN-673), AZD5305, olaparib (AZD-2281), rupa line (AG 014699, PF-01367338), ABT-888, vitamin Li Pali (ABT-888), nilaparib, CEP 9722, MK 4827, BGB-290 (pamippa), BSI-201, CEP-8983, E7016, 3-aminobenzamide, or combinations thereof. In some embodiments, the PARP inhibitor is 2X 121, ABT-767, AZD 2461, BGB-290, BGP 15, CEP 8983, CEP 9722, DR 2313, E7016, E7449, fluxazopali (SHR 3162), IMP 4297, INO1001, JPI 289, JPI 547, monoclonal antibody B3-LysPE40 conjugate, MP 124, nilaparil (ZEJULA) (MK-4827), NMS-P293, NOV-140101, NU 1025, NU 1064, NU 1076, NU1085, olaparil (AZD 2281), 0N02231, pa Mi Pali, PD 128763, R503, R554, rubrapali (RUCA) (AG-014699, PF-01367338), SBP 101, SC 34, simipari, taxazopali (BMN-673), AZD Li Pali, AZT-5, UK-5, olaparil (AZK-6), or a combination thereof. In some embodiments, the PARP inhibitor is olapari.

In some embodiments, the PARP inhibitor is NMS-P293.NMS-P293 is a non-capturing, potent and selective PARP-1 inhibitor. NMS-P293 has excellent preclinical characteristics, including: high in vitro cross-species metabolic stability, lack of cytochrome and drug transporter interactions, low protein binding and excellent pharmacokinetic profile, low clearance and almost complete oral bioavailability in both rodents and non-rodents; the permeability of the brain barrier is higher than that of the competitor, and opens up opportunities for treating brain tumor and brain metastasis; excellent tumor distribution and prolonged pharmacodynamic effects; complete regression of BRCA mutated tumor models and cure mice with high single agent antitumor efficacy; synergistic efficacy and tolerability in combination with Temozolomide (TMZ) -resistant MGMT hypomethylated GBM in Glioblastoma (GBM) tumor models, including TMZ.

Similarly, any PLK1 inhibitor now known or later discovered may be used in these methods, including PLK1 inhibitors that are selective for PLK1, as well as PLK1 inhibitors that also inhibit other protein activities. In some embodiments, the PLK1 inhibitor is a dihydropteridinone, pyridopyrimidine, aminopyrimidine, substituted thiazolinone, pteridine derivative, dihydroimidazo [1,5-f ] pteridine, meta-substituted thiazolinone, benzylstyryl sulfone analog, stilbene derivative, or a combination thereof. In some of these embodiments, the PLK1 inhibitor is onvansertib, BI2536, volasertib (BI 6727), GSK461364, AZD1775, CYC140, HMN-176, HMN-214, rigossitib (ON-01910), MLN0905, TKM-080301, TAK-960 or Ro3280.

In some embodiments, the PLK1 inhibitor is onvansertib. In these embodiments, the onvansertib is administered to the patient in any suitable dosage, e.g., less than 12mg/m ² Is less than or equal to 24mg/m ² Or greater than 24mg/m ² Is a dose of (a). In some embodiments, the onvansertib is administered to the patient daily. In further embodiments, the onvansertib is administered in a period of 3-10 days of daily administration of the onvansertib, wherein no onvansertib is administered for 2-16 days. In some embodiments, the onvansertib is administered to the patient at least five times a week. The patient may undergo two, three or four cycles of administration. In some embodiments, the patient experiences four cycles of administration with at least five daily cycles of administration of onvansertib, wherein no onvansertib is administered for 1-2 days.

In some embodiments, the PLK1 inhibitor alone or in combination with the PARP inhibitor is administered to a patient who undergoes a drug holiday after undergoing one or more administration cycles. Drug holidays as used herein refers to the period of time when the patient ceases to take PLK1 inhibitors and/or PARP inhibitors. The drug holiday may be several days to several months. In some embodiments, the drug holiday may be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or any value or range between any two of these values.

As will be appreciated by those of skill in the art, the amount of co-administration of the PARP inhibitor and the PLK1 inhibitor, as well as the time of co-administration, may depend on the type (species, sex, age, weight, etc.) and condition of the subject being treated, as well as the severity of the disease or condition being treated. PARP inhibitors and PLK1 inhibitors may be formulated as a single pharmaceutical composition or as two separate pharmaceutical compositions. The active ingredient may also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, in hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.

The methods, compositions, kits, and systems disclosed herein can be applied to different types of subjects. For example, the subject may be a subject receiving a cancer treatment, a subject in remission of cancer, a subject who has received one or more cancer treatments, or a subject suspected of having cancer. The subject may have stage I cancer, stage II cancer, stage III cancer, and/or stage IV cancer. The cancer may be ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof. The cancer may be a BRCA mutant cancer, such as a BRCA1 mutant cancer, a BRAC2 mutant cancer, or a BRAC1 and BRAC2 mutant cancer. The cancer may be a BRCA wild-type cancer (e.g., without BRCA mutations), such as a BRCA1 wild-type cancer or a BRCA2 wild-type cancer. The method may further comprise administering an additional therapeutic intervention to the subject. Additional therapeutic interventions may include therapeutic interventions other than administration of PLK1 inhibitors and PARP inhibitors, antibodies, adoptive T cell therapies, chimeric Antigen Receptor (CAR) T cell therapies, antibody-drug conjugates, cytokine therapies, cancer vaccines, checkpoint inhibitors, radiation therapies, surgery, chemotherapeutic agents, or any combination thereof. The therapeutic intervention may be administered at any time of treatment, such as when the subject has early cancer, and wherein the therapeutic intervention is more effective than if the therapeutic intervention were administered to the subject at a later time.

Without being bound by any particular theory, it is believed that PLK1 inhibitors (e.g., onvansertib) may sensitize cells (e.g., cancer cells) to PARP inhibitor treatment (e.g., damage through HR) to achieve effective cancer treatment.

Dose and pharmacokinetics

Treatment of the present disclosure may include administration of a PLK1 inhibitor (e.g., onvansertib) for a desired duration in one or more treatment cycles, and administration of a PARP inhibitor.

Treatment may, for example, include daily administration of a PARP inhibitor (e.g., olapari) at or about below: a number or range between 0.01mg, 0.05mg, 0.1mg, 0.15mg, 0.2mg, 0.25mg, 0.3mg, 0.35mg, 0.4mg, 0.45mg, 0.5mg, 0.55mg, 0.6mg, 0.65mg, 0.7mg, 0.75mg, 0.8mg, 0.85mg, 0.9mg, 0.95mg, 1mg, 5mg, 10mg, 20mg, 50mg, 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1000mg, 1100mg, 1200mg, or any two of these values. In some embodiments, the daily dose of PARP inhibitor (e.g., olapari) may be adjusted (e.g., increased or decreased in range) during the treatment of the subject. Daily administration of PARP inhibitors may be in different amounts on different days or between different periods. For example, treatment may include daily administration of a PARP inhibitor (e.g., olapari) at 0.1mg to 20mg during cycle 1, 0.25mg to 50mg during cycle 2, 0.5mg to 100mg during cycle 3, 1mg to 200mg during week 4, and 2mg to 400mg at and after week 5. For example, treatment may include daily administration of 300mg on day 1, 450mg on day 2, 600mg on day 3, 750mg on and after day 4, or 600mg of a PARP inhibitor (e.g., olapari). In some embodiments, the PARP inhibitor (e.g., olapari) is administered orally to the subject twice daily (two 150mg tablets each), with or without food, at a total daily dose of 600mg. In some embodiments, the PARP inhibitor (e.g., olapari) is orally administered to the subject twice daily (100 mg tablet and 150mg tablet each time), with or without food, at a total daily dose of 500mg. In some embodiments, the PARP inhibitor (e.g., olapari) is administered orally to the subject twice daily (two tablets of 100mg each), with or without food, at a total daily dose of 400mg.

Maximum concentration of PARP inhibitor (e.g., olapari or NMS-293) in the blood of a subject (C) when the PARP inhibitor is administered alone or in combination with the PLK1 inhibitor _max ) The (during and/or after treatment) may be about 0.1pg/mL (picogram/mL) to about 10 μg/mL (microgram/mL). For example, when PARP inhibitors are administered alone or in combination with PLK1 inhibitorsIn the case, the PARP inhibitor (e.g., olapari) C in the blood of the subject _max The following may be or about: 0.1. Mu.g/mL, 0.2. Mu.g/mL, 0.3. Mu.g/mL, 0.4. Mu.g/mL, 0.5. Mu.g/mL, 0.6. Mu.g/mL, 0.7. Mu.g/mL, 0.8. Mu.g/mL, 0.9. Mu.g/mL, 1. Mu.g/mL, 1.1. Mu.g/mL, 1.2. Mu.g/mL, 1.3. Mu.g/mL, 1.4. Mu.g/mL, 1.5. Mu.g/mL, 1.6. Mu.g/mL, 1.7. Mu.g/mL 1.8. Mu.g/mL, 1.9. Mu.g/mL, 2. Mu.g/mL, 2.1. Mu.g/mL, 2.2. Mu.g/mL, 2.3. Mu.g/mL, 2.4. Mu.g/mL, 2.5. Mu.g/mL, 2.6. Mu.g/mL, 2.7. Mu.g/mL, 2.8. Mu.g/mL, 2.9. Mu.g/mL, 3. Mu.g/mL, 3.1. Mu.g/mL, 3.2. Mu.g/mL, 3.3. Mu.g/mL 3.4. Mu.g/mL, 3.5. Mu.g/mL, 3.6. Mu.g/mL, 3.7. Mu.g/mL, 3.8. Mu.g/mL, 3.9. Mu.g/mL, 4. Mu.g/mL, 4.1. Mu.g/mL, 4.2. Mu.g/mL, 4.3. Mu.g/mL, 4.4. Mu.g/mL, 4.5. Mu.g/mL, 4.6. Mu.g/mL, 4.7. Mu.g/mL, 4.8. Mu.g/mL, 4.9. Mu.g/mL, and 5.1. Mu.g/mL, 5.2. Mu.g/mL, 5.3. Mu.g/mL, 5.4. Mu.g/mL, 5.5. Mu.g/mL, 5.6. Mu.g/mL, 5.7. Mu.g/mL, 5.8. Mu.g/mL, 5.9. Mu.g/mL, 6. Mu.g/mL, 6.1. Mu.g/mL, 6.2. Mu.g/mL, 6.3. Mu.g/mL, 6.4. Mu.g/mL, 6.5. Mu.g/mL, and, 6.6. Mu.g/mL, 6.7. Mu.g/mL, 6.8. Mu.g/mL, 6.9. Mu.g/mL, 7.1. Mu.g/mL, 7.2. Mu.g/mL, 7.3. Mu.g/mL, 7.4. Mu.g/mL, 7.5. Mu.g/mL, 7.6. Mu.g/mL, 7.7. Mu.g/mL, 7.8. Mu.g/mL, 7.9. Mu.g/mL, 8.1. Mu.g/mL, 8.2. Mu.g/mL, 8.3. Mu.g/mL, 8.4. Mu.g/mL, 8.5. Mu.g/mL, 8.6. Mu.7. Mu.g/mL, 8.8. Mu.g/mL, 8.9. Mu.9. Mu.4. Mu.g/mL, 9.5. Mu.g/mL, 9.6. Mu.g/mL, 9.2. Mu.g/mL, 9.9.1. Mu.g/mL, any value between these two or any value between these values between them, 10.1. Mu.g/mL, 10. Mu.g/mL.

When PARP inhibitors are administered alone or in combination with PLK1 inhibitors, the area under the curve (AUC) of the plot of concentration of PARP inhibitor (e.g., olapari or NMS-293) in the blood of a subject over time (e.g., AUC 24 hours prior to administration _0-24 ) May be about 1pg.h/mL to about 100. Mu.g.h/mL. For example, when the PARP inhibitor is administered alone or in combination with the PLK1 inhibitor, the concentration of the PARP inhibitor (e.g., olapari) in the blood of the subject varies over time over the AUC of the plot (e.g., AUC 24 hours after administration) _0-24 ) The following may be or about: 1pg.h/mL, 5pg.h/mL, 10pg.h/mL, 20pg.h/mL, 30pg.h/mL, 40pg.h/mL, 50pg.h/mL, 60pg.h/mL, 70pg.h/mL, 80pg.h/mL, 90pg.h/mL, 100pg.h/mL, 200pg.h/mL, 300pg.h/mL, 400pg.h/mL, 500pg.h/mL, 600pg.h/mL, 700pg.h/mL, 800pg.h/mL, 900pg.h/mL, 1000pg.h/mL, 2000pg.h/mL, 3000pg.h/mL, 4000pg.h/mL, 5000pg.h/mL, 700pg.h/mL 8000pg.h/mL, 9000pg.h/mL, 10000pg.h/mL, 50000pg.h/mL, 100000pg.h/mL, 500000pg.h/mL, 1000000pg.h/mL (1. Mu.g.h/mL), 2. Mu.g.h/mL, 3. Mu.g.h/mL, 4. Mu.g.h/mL, 5. Mu.g.h/mL, 6. Mu.g.h/mL, 7. Mu.g.h/mL, 8. Mu.g.h/mL, 9. Mu.g.h/mL, 10. Mu.g.h/mL, 11. Mu.g.h/mL, 12. Mu.g.h/mL, 13. Mu.g.h/mL, 14. Mu.g.h/mL, 15. Mu.g.h/mL, 16. Mu.g.h/mL, 17. Mu.g.h/mL, 18. Mu.g.h/mL, 19. Mu.g.h/mL, 20. Mu.g.h/mL, 21. Mu.g.h/mL, 22. Mu.g.h/mL, 23. Mu.g.h/mL, 24. Mu.g.h/mL, 25. Mu.g.h/mL, 26. Mu.g.h/mL, 27. Mu.g.h/mL, 28. Mu.g.h/mL, 29. Mu.g.h/mL, 30. Mu.h/mL, 31. Mu.g.h/mL, 32. Mu.g.h/mL, 33. Mu.g.h/mL, 34. Mu.g.h/mL, 35. Mu.g.h/mL, 36. Mu.g.h/mL, 37. Mu.g.h/mL, 38. Mu.g.g.g.h/mL, 39. Mu.g.g.g.g.h/mL, 40. Mu.g.g.g.g.h/mL, 41. Mu.g.g.g.g.g.h/mL, 43, and 43. Mu.g.g.g.g.g.g.g.g.g.g.g.L, 4.g.g.g.g.L, and L, 45 μg.h/mL, 46 μg.h/mL, 47 μg.h/mL, 48 μg.h/mL, 49 μg.h/mL, 50 μg.h/mL, 51 μg.h/mL, 52 μg.h/mL, 53 μg.h/mL, 54 μg.h/mL, 55 μg.h/mL, 56 μg.h/mL, 57 μg.h/mL, 58 μg.h/mL, 59 μg.h/mL, 60 μg.h/mL, 61 μg.h/mL, 62 μg.h/mL, 63 μg.h/mL, 64 μg.h/mL, 65 μg.h/mL, 66 μg.h/mL, 67 μg.h/mL, 68 μg.h/mL, 69 μg.h/mL, 70 μg.h/mL, 71 μg.h/mL, 72 μg.h/mL, 73 μg.h/mL, 74 μg.h/mL, 75 μg.h/mL 76 μg.h/mL, 77 μg.h/mL, 78 μg.h/mL, 79 μg.h/mL, 80 μg.h/mL, 81 μg.h/mL, 82 μg.h/mL, 83 μg.h/mL, 84 μg.h/mL, 85 μg.h/mL, 86 μg.h/mL, 87 μg.h/mL, 88 μg.h/mL, 89 μg.h/mL, 90 μg.h/mL, 91 μg.h/mL, 92 μg.h/mL, 93 μg.h/mL, 94 μg.h/mL, 95 μg.h/mL, 96 μg.h/mL, 97 μg.h/mL, 98 μg.h/mL, 99 μg.h/mL, 100 μg.h/mL, a range between any two of these values, or any value between 10 μg.h/mL and 100 μg.h/mL. For example, the PARP inhibitor is olapari, and when olapari is administered alone or in combination with the PLK1 inhibitor, the AUC of the plot of the concentration of olapari in the blood of the subject over time (e.g., AUC 10 hours prior to administration _0-10 h) The following may be or about: 0.5. Mu.g.h/mL, 1. Mu.g.h/mL,1.5 μg.h/mL, 2 μg.h/mL, 2.5 μg.h/mL, 3 μg.h/mL, 3.5 μg.h/mL, 4 μg.h/mL, 4.5 μg.h/mL, 5 μg.h/mL, 5.5 μg.h/mL, 6 μg.h/mL, 6.5 μg.h/mL, 7 μg.h/mL, 8 μg.h/mL, 9 μg.h/mL, 10 μg.h/mL, 20 μg.h/mL, 30 μg.h/mL, 40 μg.h/mL, 50 μg.h/mL, 100 μg.h/mL, or numbers or ranges between any two of these values.

When the PARP inhibitor is administered alone or in combination with the PLK1 inhibitor, the time (T) to maximum concentration of PARP inhibitor (e.g., olapari or NMS-293) in the blood of the subject _max ) And may be about 3 hours to 10 hours. For example, when the PARP inhibitor is administered alone or in combination with the PLK1 inhibitor, the time (T) to the maximum concentration of the PARP inhibitor (e.g., olapari) in the blood of the subject _max ) The following may be or about: 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, a range between any two of these values, or any value between 2 hours and 24 hours. For example, the PARP inhibitor is olapari, and the time to maximum concentration of olapari in the blood of the subject (T _max ) The following may be or about: 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 18 hours, or numbers or ranges between any two of these values.

The elimination half-life (T) of a PARP inhibitor (e.g., olaparib or NMS-293) in the blood of a subject when the PARP inhibitor is administered alone or in combination with a PLK1 inhibitor _1/2 ) And may be from about 10 hours to about 100 hours. For example, when the PARP inhibitor is administered alone or in combination with the PLK1 inhibitor, the elimination half-life (T) of the PARP inhibitor (e.g., olapari) in the blood of the subject _1/2 ) The following may be or about: 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, 65 hours, 70 hours, 75 hours, 80 hours, 85 hours, 90 hours, 95 hours, 100 hours, ranges between any two of these valuesOr any value between 10 hours and 100 hours. For example, the PARP inhibitor is olapari, and when olapari is administered alone or in combination with a PLK1 inhibitor, the elimination half-life (T _1/2 ) The following may be or about: 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours or numbers or ranges between any two of these values.

Treatment of the present disclosure may include administration of a PLK1 inhibitor (onvansertib) for a desired duration of time over a period. Administration of the PLK inhibitor (and/or one or more chemotherapeutic agents) may be daily, or with one or more interruptions between administration days. The interruption may be, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or more. When the PLK1 inhibitor (and/or one or more chemotherapeutic agents) is administered to a patient, the administration may be once, twice, three times, four times or more a day. The administration may be, for example, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days. The length of the desired duration may vary, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or more. Each treatment cycle may have various lengths, for example, at least 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or more. For example, a single cycle of treatment may include administration of PLK1 inhibitor (e.g., onvansertib) and/or one or more chemotherapeutic agents for 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or more over a cycle (e.g., over a cycle of at least 21 days (e.g., 21 to 28 days)). In some embodiments, treatment may include administration of a PLK1 inhibitor (e.g., onvansertib) and/or one or more chemotherapeutic agents for at least 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or a range between any two of these values for a period (e.g., a period of at least 21 days (e.g., 21 to 28 days)). Administration of a PLK1 inhibitor (e.g., onvansertib) and/or one or more chemotherapeutic agents may be continuous or have one or more intervals (e.g., one or two days of interruption) in a single cycle of treatment. In some embodiments, the treatment comprises administration of a PLK1 inhibitor (e.g., onvansertib) for 5 days over a period of 21 to 28 days.

In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered to a subject in need thereof on day 20 (e.g., days 1-10 and days 15-24) during a 28-day period. The 20 days may be, for example, continuous daily administration for 10 days (e.g., days 1-10) and another continuous daily administration (e.g., days 15-24) for 10 days, or continuous daily administration for four groups of five days (e.g., days 1-5, 8-12, 15-19, and 22-26). In some embodiments, for example, when a patient is identified as having low tolerance to a PLK1 inhibitor (e.g., onvansertib), the PLK1 inhibitor is administered to a subject in need thereof on days 10 (e.g., days 1-5 and 15-19) during a 28-day period. These 10 days may be, for example, continuous daily administration for 10 days (e.g., days 1-10) or two continuous daily administrations each for five days (e.g., days 1-5 and 15-19). In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered daily throughout the cycle (e.g., daily administration for 28 days in a 28 day cycle) to a subject in need thereof. Depending on the need for inhibition/reversal of cancer progression in the subject, the subject may receive one, two, three, four, five, six or more cycles of treatment. For combination therapy, the administration periods, dosing schedules, and/or amounts of dosages of PARP inhibitors and PLK1 inhibitors may be the same or different. For combination therapy, the period of administration, dosing regimen and/or amount of dose of PARP inhibitor may be adjusted according to the period of administration, dosing schedule and/or amount of dose of PLK1 inhibitor. For example, a PARP inhibitor (e.g., olapari or NMS-293) may be administered in four 7-day cycles (e.g., daily on days 1-5 and not on days 6-7, repeated for 4 weeks), which corresponds to a 28-day cycle of PLK1 inhibitor (e.g., onvansertib) administration.

Treatment may include administration of the PLK1 inhibitor (e.g., onvansertib) at or about the following, for example, as a daily dose: 6mg/m ² -90mg/m ² . For example, treatment may include daily administration of the following or about the following PLK1 inhibitors (e.g., onvansertib): 6mg/m ² 、8mg/m ² 、10mg/m ² 、12mg/m ² 、14mg/m ² 、16mg/m ² 、18mg/m ² 、20mg/m ² 、23mg/m ² 、27mg/m ² 、30mg/m ² 、35mg/m ² 、40mg/m ² 、45mg/m ² 、50mg/m ² 、55mg/m ² 、60mg/m ² 、65mg/m ² 、70mg/m ² 、80mg/m ² 、85mg/m ² 、90mg/m ² Numbers or ranges between any two of these values or 8mg/m ² -90mg/m ² Any value in between. In some embodiments, the daily dose of PLK1 inhibitor (e.g., onvansertib) may be adjusted (e.g., increased or decreased in range) during treatment or during a single cycle of treatment (e.g., first cycle, second cycle, third cycle, and subsequent cycles) for a subject. In some embodiments, the PLK inhibitor (e.g., onvansertib) is at 12mg/m ² Administered on days 20 (e.g., days 1-10 and 15-24) during a 28 day period. In some embodiments, the PLK inhibitor (e.g., onvansertib) is at 15mg/m ² Administered on days 10 (e.g., days 1-5 and 15-19) during the 28 day period. In some embodiments, the PLK inhibitor (e.g., onvansertib) is at 8mg/m ² Or 10mg/m ² Daily (e.g., days 11-28) administration during a 28 day period. In some embodiments, for a subject, a PLK1 inhibitor (e.g., onvansertib) may be during the treatment or a single cycle of treatment (e.g., a first period, a second period, The third and subsequent cycles) during which adjustments are made (e.g., an increase or decrease in range). In some embodiments, the PLK1 inhibitor is at 12mg/m ² Or at about 12mg/m ² And (3) application. In some embodiments, the PLK1 inhibitor is at 15mg/m ² Or at about 15mg/m ² And (3) application. In some embodiments, the PLK1 inhibitor is at 18mg/m ² Or at about 18mg/m ² And (3) application.

When the PLK1 inhibitor is administered alone or in combination with the PARP inhibitor, the maximum concentration (C) of PLK1 inhibitor (e.g., onvansertib) in the blood of the subject _max ) And may be about 100nmol/L to about 1500nmol/L (during or after treatment). For example, when the PLK1 inhibitor is administered alone or in combination with a PARP inhibitor, C of the PLK1 inhibitor (e.g., onvansertib) in the blood of the subject _max The following may be or about: 100nmol/L, 200nmol/L, 300nmol/L, 400nmol/L, 500nmol/L, 600nmol/L, 700nmol/L, 800nmol/L, 900nmol/L, 1000nmol/L, 1100nmol/L, 1200nmol/L, 1300nmol/L, 1400nmol/L, 1500nmol/L, any value between any two of these values, or between 200nmol/L and 1500nmol/L.

When PLK1 inhibitors are administered alone or in combination with PARP inhibitors, the area under the curve (AUC) of the plot of concentration of PLK1 inhibitor (e.g., onvansertib) in the blood of a subject versus time (e.g., AUC 24 hours after administration _0-24 ) May be about 1000nmol/L.h to about 400000nmol/L.h. For example, when the PLK1 inhibitor is administered alone or in combination with the PARP inhibitor, the AUC of the graph of the concentration of PLK1 inhibitor (e.g., onvansertib) in the blood of the subject over time (e.g., AUC 24 hours after administration) _0-24 ) The following may be or about: 1000nmol/L.h, 5000nmol/L.h, 10000nmol/L.h, 15000nmol/L.h, 20000nmol/L.h, 25000nmol/L.h, 30000nmol/L.h, 35000nmol/L.h, 40000nmol/L.h, any value between any two of these values, or any value between 1000nmol/L.h and 400000nmol/L.h.

When the PLK1 inhibitor is administered alone or in combination with the PARP inhibitor, it reaches the blood of the subjectTime (T) of maximum concentration of PLK1 inhibitor (e.g., onvansertib) _max ) May be from about 1 hour to about 5 hours. For example, when the PLK1 inhibitor is administered alone or in combination with the PARP inhibitor, the time (T) to the maximum concentration of PLK1 inhibitor (e.g., onvansertib) in the blood of the subject _max ) The following may be or about: 1 hour, 1.5 hour, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, any range between any two of these values, or any value between 1 hour and 5 hours.

When PLK1 inhibitors are administered alone or in combination with PARP inhibitors, the elimination half-life (T) of PLK1 inhibitors (e.g., onvansertib) in the blood of a subject _1/2 ) And may be from about 10 hours to about 60 hours. For example, when the PLK1 inhibitor is administered alone or in combination with the PARP inhibitor, the elimination half-life (T) of the PLK1 inhibitor (e.g., onvansertib) in the blood of the subject _1/2 ) The following may be or about: 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, any range between any two of these values, or any value between 10 hours and 60 hours.

Additional cancer therapeutic agents or therapies

The methods, compositions and kits disclosed herein can be used to treat cancer. In some embodiments, a method for treating cancer comprises administering a PARP inhibitor and a PLK1 inhibitor (e.g., onvansertib) to a subject (e.g., patient) in need thereof. The method may comprise administering a therapeutically effective amount of a PARP inhibitor and a therapeutically effective amount of a PLK1 inhibitor. The treatment may include administration of at least one additional cancer therapeutic or cancer therapy. The treatment may comprise administering a therapeutically effective amount of at least one additional cancer therapeutic or cancer therapy. PARP inhibitors and cancer therapeutic agents or cancer therapies may be co-administered, for example, simultaneously or sequentially. The PLK1 inhibitor (e.g., onvansertib) and the cancer therapeutic agent or cancer therapy may be co-administered, e.g., simultaneously or sequentially. In some embodiments, the additional cancer therapeutic agent is cytarabine Low dose cytarabine (LDAC) and/or decitabine. The safety, pharmacokinetics and preliminary clinical activity of the combination of onvansertib with LDAC or decitabine have been determined in patients with R/R AML and are described in PCT application published as WO2021146322, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the treatment comprises LDAC at 20mg/m ² Or at about 20mg/m ² Subcutaneous (SC) administration once daily (qd) for 7, 8, 9, 10, 11, 12 or 13 days in one cycle. In some embodiments, the treatment comprises decitabine at 20mg/m ² Or at about 20mg/m ² Intravenous (IV) qd administration lasts for 3 days, 4 days, 5 days, 6 days, or 7 days in one cycle. In some embodiments, the treatment comprises LDAC at 20mg/m ² Or at about 20mg/m ² Subcutaneous (SC) once daily (qd) administration for 10 days in one cycle, and decitabine at 20mg/m ² Intravenous (IV) qd administration for 5 days in one cycle.

Methods for predicting/determining the efficacy and status of cancer treatments

Also disclosed herein are methods, compositions, kits, and systems for predicting/determining clinical outcome of a cancer combination therapy of the present disclosure, monitoring the combination therapy, predicting/determining responsiveness of a subject to the combination therapy, determining the status of cancer in the subject, and improving the outcome of the combination therapy. Methods, compositions, kits, and systems can be used to guide combination therapy, provide combination therapy advice, reduce or avoid unnecessary ineffective combination therapy for a patient. ctDNA can be analyzed to predict/determine clinical outcome of cancer treatment using a combination of PARP inhibitor and PLK1 inhibitor of the present disclosure, monitor combination therapy, predict/determine responsiveness of a subject to combination therapy, determine cancer status in a subject, improve combination therapy outcome, guide combination therapy, provide combination therapy recommendations, and/or reduce or avoid ineffective combination therapy. ctDNA can be analyzed to predict/determine clinical outcome of a cancer treatment, monitor a cancer treatment, predict/determine responsiveness of a subject to a cancer treatment, determine a cancer status in a subject, improve cancer treatment outcome, guide a cancer treatment, provide treatment recommendations, and/or reduce or avoid ineffective cancer treatment. Analysis of such ctDNA has been described in PCT application published as WO2021146322, the contents of which are incorporated herein by reference in their entirety.

Methods of determining responsiveness of a subject to a combination therapy comprising a PARP inhibitor and a PLK1 inhibitor of the present disclosure may include, for example, analyzing circulating tumor DNA (ctDNA) of a subject having cancer that is undergoing treatment and/or has received the combination therapy, thereby determining responsiveness of the subject to the combination therapy. In some embodiments, determining responsiveness of the subject includes determining whether the subject is a responder to the treatment, whether the subject is or will be in CR, or whether the subject is or will be in Partial Remission (PR). For example, analyzing ctDNA may include: detecting a variant allele frequency in ctDNA in a first sample obtained from the subject at a first time point, detecting a variant allele frequency in ctDNA obtained from the subject in one or more additional samples at one or more additional time points, and determining a difference in the variant allele frequency in ctDNA between the first sample and at least one of the one or more additional samples, a decrease in the variant allele frequency in at least one of the additional samples relative to the first sample being indicative of the subject being responsive to the cancer treatment.

In some embodiments, the first time point is before or immediately before the combination therapy, and at least one of the one or more additional time points is at or after the end of at least one cycle of the combination therapy. In some embodiments, the period of combination therapy is the first period of combination therapy. In some embodiments, the first time point is before or immediately before the first cycle of the combination therapy, and the one or more additional time points are at or after the end of the second cycle of the combination therapy.

In some embodiments, the first cycle of the combination therapy immediately precedes the second cycle of the combination therapy. In some embodiments, the method comprises continuing the combination therapy for the subject if the subject is indicated as responsive to the combination therapy. In some embodiments, the method comprises stopping the combination therapy to the subject and/or starting a different combination therapy to the subject if the subject is not indicated as responsive to the combination therapy.

Disclosed herein are methods comprising determining a cancer status in a subject, comprising analyzing circulating tumor DNA (ctDNA) of the subject, thereby determining the cancer status of the subject. The subject may be a subject undergoing current combination therapy comprising a PARP inhibitor and a PLK1 inhibitor of the present disclosure, a subject who has received prior combination therapy of the present disclosure, and/or a subject in cancer remission. A subject in cancer remission may be in Complete Remission (CR) or Partial Remission (PR).

In some embodiments, analyzing ctDNA comprises detecting variant allele frequencies in ctDNA. In some embodiments, analyzing ctDNA comprises: detecting a variant allele frequency in ctDNA obtained from the subject in a first sample at a first time point, detecting a variant allele frequency in ctDNA obtained from the subject in one or more additional samples at one or more additional time points, and determining a difference in the variant allele frequency in ctDNA between the first sample and at least one of the one or more additional samples, an increase in the variant allele frequency in the one or more additional samples relative to the first sample being indicative of the subject being at risk of cancer recurrence or being at cancer recurrence.

In some embodiments, the first time point is before or immediately before the combination therapy, and the one or more additional time points are at or after the end of at least one cycle of the combination therapy, optionally, the cycle of the combination therapy is the first cycle of the combination therapy. In some embodiments, the first time point is before or immediately before the first period of the combination therapy and the one or more additional time points are at or after the end of the second period of the combination therapy, optionally, the first period of the combination therapy immediately before the second period of the combination therapy.

In some embodiments, the method comprises initiating additional treatment of the subject if the subject is indicated to be in a recurrence of cancer. The additional treatment may be the same as or different from the current combination treatment or the prior combination treatment.

The variant allele frequency in ctDNA may be determined, for example, by the total mutation count in ctDNA of each of the first sample and one or more additional samples, or by the average variant allele frequency of each of the first sample and one or more additional samples. In some embodiments, the variant allele frequency is the Mutant Allele Frequency (MAF) of a driven mutation of a cancer (e.g., ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof). In some embodiments, the variant allele frequency is one or more MAFs driving mutations of cancer (e.g., ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof). In some embodiments, log ₂ (C ₁ /C ₀ )<The MAF threshold indicates a decrease in ctDNA MAF. C (C) ₀ Is ctDNA MAF in the first sample, and C ₁ Is ctDNA MAF in one of the other samples. In some embodiments, the MAF threshold is 0.01 to-0.10 or about 0.01 to-0.10. In some embodiments, the MAF threshold is 0.06 or about 0.06. In some embodiments, the MAF threshold is 0.05 or about 0.05.

In some embodiments, the first sample comprises ctDNA from the subject prior to treatment, and one of the additional samples comprises ctDNA from the subject after treatment. In some embodiments, the driving mutation is a mutation in one of the following 75 genes: ABL1, ANKRD26, ASXL1, ATRX, BCOR, BCORL1, BRAF, BTK, CALR, CBL, CBLB, CBLC, CCND, CDC25C, CDKN2A, CEBPA, CSF3R, CUX1, CXCR4, DCK, DDX41, DHX15, DNMT3A, ETNK1, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, IKZF1, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, LUC L2, MAP2K1, MPL, MYC, MYD88, NF1, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PPM1D, PTEN, PTPN11, RAD21, RBBP6, RPS14, RUNX1, SETBP1, SF3B1, SH2B3, SLC29A1, SMC1A, SMC3, SRSF2, STAG2, STAT3, TET2, TP53, U2AF1, U2AF2, WT1, XPO1, and ZRSR2. In some embodiments, at least one of the one or more driver mutations is a mutation in the 75 genes. In some embodiments, the one or more driving mutations are mutations in the 75 genes.

At least one of the driving mutation or the one or more driving mutations may be located in a gene selected from the group consisting of: TP53, ASXL1, DNMT3A, NRAS, SRSF, TET2, SF3B1, FLT3 ITD, IDH2, NPM1, RUNX1, CDKN2A, KRAS, STAG2, CALR, CBL, CSF3R, DDX41, GATA2, JAK2, PHF6, and SETBP1. In some embodiments, at least one of the driving mutation or the one or more driving mutations is located in a gene selected from the group consisting of: DNMT3A, TET, NPM1, SRSF2, NRAS, CDKN2A, SF B1, FLT3, ASXL1, SRSF2, IDH2, NRAS, and SF3B1. In some embodiments, the method further comprises determining variant allele frequencies in one or more of ctDNA, PBMC, and BMMC of the subject.

ctDNA can be analyzed using, for example, polymerase Chain Reaction (PCR), next Generation Sequencing (NGS), and/or drop digital PCR (ddPCR). The samples disclosed herein may be derived from, for example, whole blood of a subject, plasma of a subject, serum of a subject, or a combination thereof. In some embodiments, ctDNA is from whole blood of a subject, plasma of a subject, serum of a subject, or a combination thereof.

In some embodiments, the method comprises analyzing ctDNA of the subject prior to treatment. In some embodiments, the treatment comprises one or more cycles, and ctDNA is analyzed before, during, and after each cycle of the treatment. Each cycle of treatment may be at least 21 days. In some embodiments, each cycle of treatment is about 21 days to about 28 days. In some embodiments, the subject is a human.

Disclosed herein include methods of improving the outcome of cancer treatment. The method may include: detecting in a first sample a variant allele frequency in circulating tumor DNA (ctDNA) obtained from a subject at a first time point prior to the subject undergoing a combination therapy comprising a PARP inhibitor and a PLK1 inhibitor of the present disclosure; detecting in one or more additional samples the variant allele frequencies in ctDNA obtained from the subject at one or more additional time points after the subject has undergone combination therapy; determining a difference in variant allele frequency in ctDNA between the first sample and at least one of the one or more additional samples, a decrease in variant allele frequency in at least one of the additional samples relative to the first sample being indicative of the subject responding to the combination therapy; and continuing the combination therapy with the subject if the subject is indicated as responsive to the combination therapy, or stopping the combination therapy with the subject and/or starting a different cancer therapy with the subject if the subject is not indicated as responsive to the combination therapy.

Also disclosed herein are methods comprising treating cancer. The method may include: administering to a subject in need thereof a combination therapy comprising a PARP inhibitor and a PLK1 inhibitor of the present disclosure; determining a decrease in the frequency of the variant allele in a second sample of the subject obtained at a second time point after the subject received the combination therapy relative to the frequency of the variant allele in a first sample of the subject obtained at a first time point before the subject received the combination therapy; and the combination therapy is continued. In some embodiments, the subject is a subject newly diagnosed with cancer, e.g., a subject who did not receive any prior cancer treatment prior to the combination treatment. In some embodiments, the subject has received a prior cancer treatment and is in cancer remission, e.g., the subject is in Complete Remission (CR) or Partial Remission (PR) after receiving the prior combination treatment.

The first point in time may be, for example, before or immediately before the combination therapy. At least one of the one or more additional points in time may be, for example, at or after the end of at least one cycle of the combination therapy. In some embodiments, the period of combination therapy is the first period of combination therapy. In some embodiments, the first time point is before or immediately before the first cycle of the combination therapy, and the one or more additional time points are at or after the end of the second cycle of the combination therapy. In some embodiments, the first cycle of the combination therapy immediately precedes the second cycle of the combination therapy.

The variant allele frequency in ctDNA may be determined, for example, by the total mutation count in ctDNA of each of the first sample and the one or more additional samples, and/or by the average variant allele frequency of each of the first sample and the one or more additional samples. In some embodiments, the variant allele frequency is the Mutant Allele Frequency (MAF) of a driven mutation of a cancer (e.g., ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof). In some embodiments, the variant allele frequency is the Mutant Allele Frequency (MAF) of one or more driving mutations of a cancer (e.g., ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof). In some embodiments, log ₂ (C ₁ /C ₀ )<The MAF threshold indicates a decrease in ctDNA MAF. C (C) ₀ Is ctDNA MAF in the first sample, and C ₁ Is ctDNA MAF in one of the other samples. In some embodiments, the MAF threshold is-0.05.

The driving mutation may be, for example, a mutation in one of the 75 genes listed in table 3, at least one of the one or more driving mutations being a mutation in one of the following 75 genes: ABL1, ANKRD26, ASXL1, ATRX, BCOR, BCORL1, BRAF, BTK, CALR, CBL, CBLB, CBLC, CCND2, CDC25C, CDKN2A, CEBPA, CSF3R, CUX1, CXCR4, DCK, DDX41, DHX15, DNMT3A, ETNK1, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, IKZF1, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, LUC L2, MAP2K1, MPL, MYC, MYD, NF1, NOTCH1, NPM1, NRAS, PDGFRA, PHF6, PPM1D, PTEN, PTPN11, RAD21, RBBP6, RPS14, RUNX1, SETBP1, SF3B1, SH2B3, SLC29A1, SMC1A, SMC3, SRSF2, STAG2, STAT3, TET2, TP53, U2AF1, U2AF2, WT1, XPO1 and ZRSR2, and/or mutations in one or more of these genes. In some embodiments, at least one of the driving mutation or the one or more driving mutations is located in a gene selected from the group consisting of: TP53, ASXL1, DNMT3A, NRAS, SRSF, TET2, SF3B1, FLT3 ITD, IDH2, NPM1, RUNX1, CDKN2A, KRAS, STAG2, CALR, CBL, CSF3R, DDX41, GATA2, JAK2, PHF6, and SETBP1. In some embodiments, at least one of the driving mutation or the one or more driving mutations is located in a gene selected from the group consisting of: DNMT3A, TET, NPM1, SRSF2, NRAS, CDKN2A, SF B1, FLT3, ASXL1, SRSF2, IDH2, NRAS, and SF3B1.

In some embodiments, the method further comprises determining variant allele frequencies in one or more of ctDNA, PBMC, and BMMC of the subject. For example, variant allele frequencies in ctDNA can be detected using Polymerase Chain Reaction (PCR) or Next Generation Sequencing (NGS). In some embodiments, the variant allele frequencies in ctDNA are detected using droplet digital PCR (ddPCR).

At least one of the first sample, the one or more additional samples, and the second sample may be derived from whole blood of the subject, plasma of the subject, serum of the subject, or a combination thereof. In some embodiments, ctDNA is from whole blood of a subject, plasma of a subject, serum of a subject, or a combination thereof.

In some embodiments, the subject whose ctDNA is being analyzed is undergoing or will undergo cancer treatment. The method may comprise analyzing ctDNA of the subject prior to the treatment. Treatment may include one or more cycles, and ctDNA is analyzed before, during, and after one or more cycles of treatment. For example, ctDNA may be analyzed before, during, and after two or more cycles of treatment, three or more cycles of treatment, or each cycle of treatment. Each cycle of treatment may be at least 21 days, e.g., 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or more, or a range between any two of these values. In some embodiments, each cycle of treatment is about 21 days to about 28 days. In some embodiments, each cycle of treatment is 21 days to 28 days. In some embodiments, the subject is a human.

Compositions and kits

Disclosed herein include compositions and kits for treating cancer. In some embodiments, the kit comprises: a Polo-like kinase 1 (PLK 1) inhibitor; and providing a manual for instructions for co-administering the PLK1 inhibitor and the PARP inhibitor to the subject for treating the cancer. In some embodiments, the kit comprises a PARP inhibitor. The cancer may be, for example, ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof.

In some embodiments, the subject has cancer (e.g., ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof). In some embodiments, the instructions comprise instructions for co-administering the PLK inhibitor and the PARP inhibitor simultaneously. In some embodiments, the instructions comprise instructions for sequentially co-administering the PLK inhibitor and the PARP inhibitor. In some embodiments, the instructions comprise instructions for orally administering the PLK1 inhibitor. In some embodiments, the instructions comprise instructions for orally administering a PARP inhibitor.

In some embodiments, the instructions comprise instructions that the subject has received prior PARP inhibitor treatment. In some embodiments, the instructions comprise instructions that the subject does not respond to treatment with PARP inhibitor alone. In some embodiments, the instructions comprise instructions for a subject known to be resistant to PARP inhibitor therapy.

In some embodiments, the instructions comprise instructions that the subject has received at least one prior treatment for cancer. In some embodiments, the prior treatment does not include the use of PARP inhibitors, PLK inhibitors, or both. In some embodiments, the instructions comprise instructions that the subject is in cancer remission. In some embodiments, the subject in cancer remission is in Complete Remission (CR) or Partial Remission (PR).

In some embodiments, the instructions comprise instructions for administering each of the PARP inhibitor and the PLK1 inhibitor to the subject at least twice a week. In some embodiments, the instructions comprise instructions for administering each of the PARP inhibitor and the PLK1 inhibitor to the subject at least five cycles per week. In some embodiments, the instructions comprise instructions for administering the PARP inhibitor, the PLK1 inhibitor, or both, at a period of at least 7 days. In some embodiments, each cycle of treatment is at least about 21 days. In some embodiments, each cycle of treatment is about 21 days to about 28 days, e.g., 28 days. In some embodiments, the instructions comprise instructions for administering the PLK1 inhibitor on at least four days of the cycle. In some embodiments, the instructions comprise instructions for not administering the PLK1 inhibitor for at least one day of the cycle. In some embodiments, the instructions comprise instructions for daily administration of the PARP inhibitor. In some embodiments, the instructions comprise instructions for administering the PARP inhibitor and the PLK1 inhibitor for at least two cycles.

In some embodiments, the PARP inhibitor is selective and/or specific for PARP inhibition (e.g., PARP1 inhibitor, PARP2 inhibitor, or both). In some embodiments, the PARP inhibitor is iniparib (BSI 201), tazopanib (BMN-673), AZD5305, olaparib (AZD-2281), rupa line (AG 014699, PF-01367338), ABT-888, vitamin Li Pali (ABT-888), nilaparib, CEP 9722, MK 4827, BGB-290 (pamipril), BSI-201, CEP-8983, E7016, 3-aminobenzamide, or a combination thereof. In some embodiments, the PARP inhibitor is olapari. In some embodiments, the PARP inhibitor is NMS-293.

In some embodiments, the PLK1 inhibitor is selective and/or specific for PLK 1. In some embodiments, the PLK1 inhibitor is a dihydropteridinone, pyridopyrimidine, aminopyrimidine, substituted thiazolinone, pteridine derivative, dihydroimidazo [1,5-f ] pteridine, meta-substituted thiazolinone, benzylstyryl sulfone analog, stilbene derivative, or any combination thereof. In some embodiments, the PLK1 inhibitor is onvansertib, BI2536, volasertib (BI 6727), GSK461364, AZD1775, CYC140, HMN-176, HMN-214, rigossitib (ON-01910), MLN0905, TKM-080301, TAK-960, or Ro3280. In some embodiments, the PLK1 inhibitor is onvansertib. In some embodiments, the PARP inhibitor is olaparib and the PLK1 inhibitor is onvansertib.

In some embodiments, the instructions include instructions for use at 12mg/m ² -90mg/m ² Instructions for administration of PLK1 inhibitors. In some embodiments, the instructions comprise instructions for administering the PARP inhibitor at 20mg-1200 mg.

The methods, compositions and kits disclosed herein may also be used to sensitize cancer cells to one or more PARP inhibitors. The method may comprise contacting the cancer cell with a composition comprising a PLK1 inhibitor (e.g., onvansertib) or a pharmaceutically acceptable salt, solvate, stereoisomer, or prodrug thereof, thereby sensitizing the cancer cell to one or more PARP inhibitors (e.g., olapari or NMS-293). Contacting the cancer cells with the composition may occur in vitro, ex vivo, in vivo, or any combination. In some embodiments, contacting the cancer cells with the composition is in the body of the subject. In some embodiments, the cancer cells are contacted with the composition in a cell culture. The subject may be a mammal, such as a human. Sensitization of cancer cells may increase the responsiveness of cancer cells to one or more PARP inhibitors by or about: 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or a range between any two of these values. Sensitization of cancer cells may increase the responsiveness of the cancer cells to one or more PARP inhibitors by at least the following or at least about the following: 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or a range between any two of these values. In some embodiments, the increase in cancer cell responsiveness is relative to untreated cancer cells. Sensitization of cancer cells may increase the responsiveness of a subject having cancer cells to one or more PARP inhibitors by or about: 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or a range between any two of these values. Sensitization of cancer cells may increase responsiveness of a subject having cancer cells to one or more PARP inhibitors by at least the following or at least about the following: 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or a range between any two of these values. In some embodiments, the increase in responsiveness of the subject with cancer cells is relative to a subject not treated with the composition.

The method may comprise determining sensitization of the cancer cells to the one or more PARP inhibitors after contact with the PLK1 inhibitor. The method may comprise contacting the cancer cell with the PLK1 inhibitor simultaneously with and/or after contacting the cancer cell with the PLK1 inhibitor with one or more PARP inhibitors. In some embodiments, contacting the cancer cell with one or more PARP inhibitors occurs in the subject. The subject may be a mammal, such as a human. The subject may be, for example, a subject that does not respond to or is known to have resistance to PARP inhibitor alone. The subject may be, for example, a subject having prior treatment with one of the one or more PARP inhibitors. In some embodiments, the method comprises determining the response of the subject to one or more PARP inhibitors.

Examples

Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not intended to limit the scope of the present disclosure in any way.

Example 1

Determination of antitumor Activity of Olaparib and onvansertib in BRCA1 Defect model

PLK1 inhibitors may be of therapeutic value as a single agent in BRCA deficient tumors (triple negative, BRCA1-2 mutated breast and ovary). In this example, the efficacy of the combination of onvansertib and the PARP inhibitor olapari was evaluated.

Step 1 toxicology study

Immunocompetent mice are orally treated with a combination of 75mg/kg of olapari and 40mg/kg of onvansertib for 5 days a week for two weeks, or with a combination of 100mg/kg of olapari and 40mg/kg of onvansertib for 5 days a week for two weeks.

Mice (5 animals per group) were weighed twice a week and clinically examined daily. Mice were then observed for an additional 1 week, and if no toxicity was noted, the dose of onvansertib was scaled up to 50mg/kg, and mice were treated for an additional two weeks.

The treated mice were resistant to a combination of onvansertib (40 mg/kg or 50 mg/kg) and olapari (75 mg/kg and 100 mg/kg) (body weight loss no > 20%).

Step 2. Evaluation of the antitumor Activity of the combination

The combination of onvansertib and olapari was tested in the following two BRCA1 mutant High Grade Serous Ovarian Cancer (HGSOC) PDX (patient-derived xenograft) models with resistance to olapari: pdx#hoc22 and pdx#hoc266. Their main features are summarized in table 1. Included are both BRCA1 and TP53 status, as well as characteristics of platinum and Olaparib tumor responsiveness.

TABLE 1 molecular and pharmacological characterization of BRCA1 mutant PDX

++++: very sensitive; -: resistance; i.p.: intraperitoneal implantation; mut = mutant

Transplanting mice ip 5X 10 ⁶ Individual tumor cells and randomized two weeks after tumor implantation to receive a given treatment (as described belowAs described above).

Mice (8 mice/group) were treated for 4 weeks with: (1) a vehicle; (2) Olapari (OLA), 5 days a week, for 4 weeks. In one embodiment, 100mg/kg is administered to PDX #HOC22 and 75mg/kg is administered to PDX #HOC 266; (3) Onvansertib (ONVA) (50 mg/kg), day 5 of week for 4 weeks; or (4) a combination of ola+onva.

On the day of administration of both onvansertib and olapari to mice, onvansertib was administered 2 hours before olapari (see fig. 2).

An additional 8 mice per group were transplanted for pharmacodynamic studies. Four weeks after tumor implantation, mice were treated as described above for one week and sacrificed 2 hours and 24 hours after the last dose of olapari.

Ascites fluid was collected, cells were recovered, counted, and portions of the cells were flash frozen (for protein, DNA and RNA extraction) and portions of the cells were paraffin embedded. IHC Ki67, rad51 focal spot expression was measured. Gamma H2AX (a marker for both DNA damage and apoptosis induction) and caspase activation (readout of apoptosis induction) were measured by western blotting. When the sample is available, additional markers are evaluated.

Fig. 3A is a Kaplan Meier curve showing the probability of survival in the ovarian BRCA1 mutant pdx#hoc22 model, and fig. 3B is a Kaplan Meier curve showing the probability of survival in the ovarian BRCA 1-mutant pdx#hoc266 model. The data indicate that the combination of onvansertib with olaparib significantly increased mouse survival compared to vehicle treatment and single agent treatment (olaparib or onvansertib) in the two BRCA1 mutated olaparib resistant PDX models. In particular, in the pdx#hoc266 model, 2 out of 8 mice in the combination group survived at day 300 after tumor implantation. Mice were sacrificed on day 300 and 1 mouse was tumor-free (fig. 6D). Without being bound by any particular theory, it is believed that onvansertib as a PLK1 inhibitor sensitizes tumor cells to PARP inhibitors such as olapari to achieve an effective cancer treatment.

Example 2

Determination of OlaparibAntitumor Activity of onvansertib in BRCA1 Defect model

The combination of onvansertib and olapari was tested in the following two PDX models with resistance to olapari using a similar protocol as described in example 1: pdx#218ola and pdx#154. Their main features are summarized in table 2. PDX #218ola is PDX obtained from PDX #218, which is highly sensitive to olapari, through different in vivo cycles of olapari treatment until the olapari treatment is no longer effective.

Table 2 molecular and pharmacological characterization of pdx

Mice were s.c. transplanted with PDX #218ola and PDX #154 tumor fragments (3 mm. Times.3 mm) and when tumor mass reached 100-150mm ³ When randomized to receive treatment as described below.

Mice (10 mice/group) were treated with the following for 4 to 5 weeks: (1) a vehicle; (2) Olaparib (OLA), 80mg/kg, 5 days a week for 4 to 5 weeks; (3) Onvansertib (ONVA) 45mg/kg, 5 days a week, for 4 weeks to 5 weeks; (4) ONVA 30mg/kg, day 5 a week for 4 to 5 weeks; (5) OLA 80mg/kg+ONVA 45mg/kg for 4-5 weeks on 5 days of week; or (6) a combination of OLA 80mg/kg+ONVA 30mg/kg, 5 days a week for 4-5 weeks

The onvansertib was administered 2 hours before the olaparib.

For pharmacodynamic experiments, mice were transplanted and treated as described above for 5 days, and sacrificed at the following time points:

1-vehicle: 4 hours (n=4) and 24 hours (n=4) after the last treatment (total=8)

2-OLA: 2 hours (n=4) and 22 hours (n=4) after the last dose of olapari (total=8)

3-ONVA45mg/kg: 4 hours (n=4), 8 hours (n=3) and 24 hours (n=4) after the last dose of onvansertib (total=11)

4-ONVA 30mg/kg: 4 hours (n=4), 8 hours (n=3) and 24 hours (n=4) after the last dose of onvansertib (total=11)

5-OLA 80mg/kg+ONVA 45mg/kg combination: 4 hours (n=4), 8 hours (n=3) and 24 hours (n=4) after the last dose of onvansertib (total=11)

Combination of 6-OLA 80mg/kg+ONVA 30 mg/kg: 4 hours (n=4), 8 hours (n=3) and 24 hours (n=4) after the last dose of onvansertib (total=11)

At the same time point where tumors were collected, blood was collected from all mice for pharmacokinetic analysis. At 4 hours and 24 hours, tumors can be collected and split into two parts: one part may be flash frozen for protein, DNA and RNA extraction and the other part may be paraffin embedded. IHC Ki67 may be measured. By western blotting, γh2ax, caspase activation and phosphoh 3 (M2 blocked readout) can be measured. In addition, activation of the DNA damage response pathway can be assessed by the ATR/Chk1 axis.

Example 3

Antitumor Activity of Olaparib and onvansertib in BRCA1 wild-type model

In this example, the efficacy of onvansertib alone and in combination with olapari in BRCA1 wild type model was evaluated. The same scheme as in example 1 was used in this example, unless otherwise specified.

The combination of onvansertib and olapari was tested in the following three BRCA1-WT HGSOC PDX models that are resistant to olapari: pdx#124, pdx#239, and pdx#76. Their main features are summarized in table 3.

TABLE 3 molecular and pharmacological characterization of BRCA1-WT PDX

++++: very sensitive; -: resistance; i.p.: intraperitoneal, s.c. subcutaneous implantation

Mice were transplanted with tumor fragments and randomized to receive treatment. For example, smallMice can be transplanted 3mm s.c ³ Tumor fragments or i.p. transplants 5X 10 ⁶ Individual cells, and then 2 weeks after tumor implantation or when the tumor reaches 100-150mm ³ Size, randomized to receive treatment. Mice were s.c. transplanted 3mm in PDX #124 model ³ Tumor fragments, and when the tumor reaches 100-150mm ³ Size was randomized to receive treatment.

Mice (8 mice/group) were treated for 4 weeks with: (1) a vehicle; (2) Onvansertib (ONVA) (50 mg/kg), day 5 of the week; (3) Olaparib (OLA) (100 mg/kg), 5 days of the week; or (4) OLA (100 mg/kg) +ONVA (50 mg/kg), 5 days a week.

FIG. 4A is a graph showing tumor volumes in the BRCA1-WT HGSOC PDX model (PDX # 124). In comparison to the control, the combination of onvansertib and olapari showed significant tumor growth inhibition (Kruskal-Wallis test, p=0.04), whereas the single dose (onvansertib or olapari) did not show significant anti-tumor activity.

For the combination group, treatment was resumed for one week on day 80. Fig. 4B shows tumor volumes in the same PDX #124 model that restored treatment in the combination group. The data indicate that recovery from treatment with the combination of onvansertib and olapari slowed tumor growth after the 32 day drug holiday, supporting that combination with the drug holiday schedule may be effective.

Example 4

Antitumor Activity of Olaparib and onvansertib in BRCA1 deficiency and wild type models

In this example, the efficacy of onvansertib alone and in combination with olapari in three HGSOC models was evaluated. The same scheme as in example 1 was used in this example, unless otherwise specified.

The patient-derived xenograft (PDX) used in this example was part of a human ovarian xenograft library recently established in Milan Mario Negri Institute (IT) and described in Ricci f et al, cancer res.2014, the contents of which are incorporated herein by reference. Three models were selected for which the molecular and pharmacological characteristics are reported in fig. 5. MNHOC316DDP derived from cisplatin (DDP) sensitive PDX develops resistance through multiple cycles of in vivo DDP treatment.

Selected PDX was transplanted in situ in NCr-nu/nu mice and randomized to: 1) Control/vehicle treated group; 2) Olaparib (100 mg/kg-MNHOC 22 and MNHOC316 DDP-or 80 mg/kg-MNHOC 266-per os)); 3) onvansertib (50 mg/kg, oral); 4) Combination (Combo), 5 days/week for 4 weeks. For MNHOC316DDP, DDP treated mice (5 mg/kg q 7X 3) were considered as controls. Antitumor activity was assessed by calculating the increase in longevity (ILS%) = [ (median survival control group-median survival treated group)/(-median survival treated group) ]x100.

For Pharmacodynamic (PD) studies, mice carrying MNHOC22 and MNHOC266 were treated with previously reported doses for four consecutive days and then euthanized 2 hours and 24 hours after the last treatment. Both ascites cells (ascitic cells) were Formalin Fixed Paraffin Embedded (FFPE) and flash frozen for PD studies. PD studies include: proliferation was measured by Ki67 IHC staining, byApoptosis was measured by 3/7 kit (Promega), mitosis was quantified by counting mitotic events for FFPE, and anti-pH 3-Ser10 expression was quantified by WB, DNA damage/apoptosis was quantified by WB using anti- γh2ax antibodies. RAD51 foci were quantified by using an IF-based method as described in guffnti f. Et al, BJC 2022, and scoring the percentage of RAD 51/biproteins (GMN) -positive tumor cells (rad51+/gmn+) with 5 or more foci per nucleus by blindness. At least 100 GMN positive cells in three different regions of the tissue section were analyzed. Using a predefined threshold to determine a qualitative score: RAD 51-positive tumors are>10% RAD51+/GMN+ cells.

For survival analysis, kaplan-Mayer curves were reported and the Mantel-Cox test was used; unpaired t-test was performed on all other comparisons. p-values <0.05 were considered significant.

Fig. 6A, 6C and 6E are graphs showing changes in mouse body weight in the three PDX models (MNHOC 22, MNHOC26 and MNHOC316DDP, respectively) shown in fig. 5 treated with control, olapari, onvansertib, or a combination of onvansertib and olapari. The data show that the olapari/onvansertib combination is well tolerated in vivo in all three models; even if weight loss is observed, it never exceeds 20% and resumes after drug withdrawal.

Fig. 6B, 6D and 6F are Kaplan Meier curves showing the probability of survival in three PDX models (MNHOC 22, MNHOC26 and MNHOC316DDP, respectively) treated with control, olapari, onvansertib, or a combination of onvansertib and olapari. Figure 7 shows the median survival time and lifetime Increase (ILS) for three PDXs (MNHOC 22, MNHOC266 and MNHOC316 DDPs). All three PDXs are resistant to olapari; onvansertib is slightly active in the MNHOC266 model, but not in both MNHOC22 and MNHOC316 DDP. In contrast, the combination was very effective compared to control and single dose treatments, as shown by the significant increase in survival.

Fig. 8 shows evaluation of Ki67 positivity (panel a), mitosis (panel B), apoptosis (panel C) and RAD51 foci formation (panel D) in PDX MNHOC22 and MNHOC 266. No difference in the percentage of Ki67 positive cells between the different groups was observed (fig. 8, panel a); the onvansertib treatment induced an increase in mitotic events at 2 hours as both a single agent and in combination with olapari (fig. 8, panel B); at 24 hours, higher apoptosis was observed in both models (fig. 8, panel C). High basal levels of RAD51 focus positive cells were observed in both models. A trend towards a RAD51 focus positive cell decrease following onvansertib treatment can be observed (fig. 8, panel D).

FIG. 9 shows the levels of pSer10-H3 and pSer139- γH2AX in tumors of PDX MNOCC 22 and MNOCC 266 treated with control, olaparib, onvansertib or a combination of onvansertib and Olaparib. In both models, higher levels of pSer10-H3 and pSer139- γH2AX were observed in the combined group at both 2 and 24 hours (FIG. 9), indicating G2/M blockade and increased apoptotic death/DNA damage, confirming data on mitotic counts and caspase 3/7 activity (FIG. 8, panels B and C).

The results show strong therapeutic efficacy of the olapari/onvansertib combination in olapari-resistant ovarian cancer PDX. The combination induced higher G2/M blockade and apoptosis/DNA damage.

Example 5

PLK1 inhibition sensitizes cancer cells to PARP inhibitors

In vitro preclinical studies, PLK1 inhibition sensitizes cells to genotoxic stress (i.e., radiation) and PARP inhibitors by damaging HR. PLK1 inhibition also sensitizes tumor cells to PARP inhibition in vivo. For example, the combination of the PLK1 inhibitor BI2536 and the PARP inhibitor olapari synergistically inhibited the growth of prostate BRCA2 mutant xenograft tumors (fig. 10). As shown herein (e.g., fig. 3A-3B), the combination of onvansertib and olapari significantly increased survival of mice (2.7-fold and 6.5-fold compared to control or olapari single agent, respectively) in the ovarian BRCA1 mutant PDX model that is resistant to olapari.

As shown in this example, onvansertib may sensitize tumors that are resistant to PARP inhibitors.

Example 6

onvansertib and olapari cell line selection

In this example, the synergy between onvansertib and olapari was tested in vitro on breast, ovarian, pancreatic and prostate cancer cell lines. The series of cells tested in this example are shown in table 4. Table 5 provides a list of cell lines with BRCA mutations.

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Briefly, cells were thawed from a liquid nitrogen storage state. After the cells have been expanded and divided by their expected number of doublings, the screen begins. Cells were seeded in growth medium in black 384-well tissue culture treated plates and equilibrated via centrifugation. After inoculation, the plates were placed in standard 5% co before and after compound treatment ₂ During incubation. At the time of treatment, a set of "time zero" assay plates (untreated) were collected and ATP levels were measured using CellTiter-Glo 2.0 (Promega). Therapeutic combinations were collected using a 9 x 9 full dose matrix. The 9 x 9 full dose matrix includes: treatment-free controls of 8 dose points plus enhanced agent (enhancement); non-therapeutic controls of 8 dose points plus enhancer (enhancement); 64 combining ratio points. The compound is added at time zero and will not be administered repeatedly. Two-and three-fold serial dilutions of onvansertib and olapari were performed, respectively, to bring the concentration of onvansertib to 0.7nM (from 1500nM to 0.7 nM) and the concentration of olapari to 13.7nM (from 30 μm to 13.7 nM).

The treated assay plates were incubated with the compounds for 6 days. After the treatment time, the plates were subjected to endpoint analysis using CellTiter-Glo 2.0. Up to three replicates were collected to account for assay variability. The data (inhibition and growth inhibition) were analyzed using software from Horizon Discovery.

Fig. 11 depicts two pie charts showing the cancer types of the cell lines tested in this example. 63 cell lines (22 breast cancer cell lines, 24 ovarian cancer cell lines, 14 pancreatic cancer cell lines, and 3 prostate cancer cell lines) were tested in a two-dimensional cell culture (2D), and 46 cell lines (18 breast cancer cell lines, 18 ovarian cancer cell lines, 9 pancreatic cancer cell lines, and 3 prostate cancer cell lines) were tested in a three-dimensional cell culture (3D). The results of the onvansertib and olapari cell line screens in 2D and 3D are shown in fig. 12A and 12B, respectively.

Fig. 12A-12B depict the synergy scores for each of the test cell lines. As shown in the 2D cell line screen (fig. 12A), 32 of the 63 cell lines tested (51%) had a synergy score of greater than 2 and were considered likely synergistic based on manufacturer guidelines. Cell lines KP-4 (human pancreatic ductal carcinoma cell line), PSN1 (human pancreatic adenocarcinoma cell line), HMC-1-8 (target cells of T cell killing clone T (cHMC-1) derived from breast cancer), DU-4475 (breast ductal carcinoma cell line), caov-3 (primary ovarian carcinoma cell line with epithelial morphology), BT-474 (cell line isolated from solid invasive ductal carcinoma from breast cancer patients), OVCAR-3 (high grade serous ovarian adenocarcinoma cell line), and SNU-119 (high grade serous ovarian carcinoma cell line) have a synergy score of greater than 5. Synergy was observed in breast, ovarian, prostate and pancreatic cancer cell lines, as well as in BRCA1/2 wild-type and mutant cell lines. 8 of the 12 cell lines with BRCA1/2 mutations (67%) had a synergy score of greater than 2, including BT-474, MDA-MD-436 (metastatic breast cancer cell line), COV362 (high grade serous ovarian adenocarcinoma cell line), DU-145 (prostate cancer cell line with epithelial morphology), OC-316 (serous ovarian adenocarcinoma cell line), MDA-MD-468 (cell line with epithelial morphology isolated from patients with breast metastatic adenocarcinoma), HCC-1954 (epithelial breast cancer cell line isolated from primary IIA stage 3 invasive ductal carcinoma without lymph node metastasis) and HCC38 (epithelial cells isolated from the breast of patients with prior history of leiomyosarcoma). Information about each cell line used in the examples, including origin, morphology, sequence variation and disease type, can be found, for example, in www.atcc.org and www.expasy.org.

As shown in 3D cell line screening (fig. 12B), 17 of the 48 cell lines tested (35%) had a synergy score of greater than 1 (e.g., OC-316, MDA-MD-231, CAMA-1, bxPC-3, KP-4, HCC38, BT-20, OV7, MDA-MD-436, SNU-324, kurami, ZR-75-1, TOV-21G, PA-1, MDA-MD-175-VII, BT-474, and HPAF-II) and were considered likely synergistic based on manufacturer guidelines. Synergy was observed in both breast, ovarian and pancreatic cancer cell lines, BRCA1/2 wild-type and mutant cell lines.

These results show that PLK1 inhibition can sensitize the activity of olaparib, leading to a synergistic effect between onvansertib and olaparib.

Example 7

In vitro evaluation of antitumor Activity of combinations of Olaparib and onvansertib

In this example, the activity of the combination of olapari and onvansertib was evaluated in two different murine isogenic ovarian cell lines: ID8 System (Walton et al 2016; walton et al 2017) and the recently available murine isogenic model (Lyer et al).

The ID8 system consists of: ID8 murine cell lines (p 53 and BRCA1/2 wt), ID8 p53-/- (p 53 deleted), ID8 p53-/-, BRCA1-/- (p 53 and BRCA1 deleted) and ID8 p53-/-, BRCA2-/- (p 53 and BRCA2 deleted). The murine isogenic model system consists of: BPPNM cell (TP 53) ^-/-R172H 、BRCA1 ^-/ -；PTEN ^-/- 、NF1 ^-/ -、Myc ^OE )；PPNM(TP53 ^-/-R172H 、PTEN ^-/- 、NF1 ^-/ -、Myc ^OE ) And BPCA (CCNE) ^OE 、AKT2 ^OE 、KRAS ^GD2V )。

Cytotoxicity experiments were performed using the MTS assay. Each cell line was treated with a concomitant administration of a series of drug concentrations (5 to 7 different drug concentrations).

Treatment can last for 72 hours and cell viability is analyzed by MTS assay system (Promega). MTS reagent was added to the cells and after a constant incubation time for all plates, absorbance was obtained using a plate reader (Infinite M200, TECAN). The data can be checked by isobologram analysis using Calcusyn software (Biosoft, cambridge, UK) and IC can be calculated ₅₀ Combination Index (CI) values at (i) to evaluate the efficacy of the combination. All experiments were performed at least twice and each experimental group had three replicates.

Based on these results, further studies can be performed in cell lines that combine to show synergy. In particular, cell cycle analysis can be performed in untreated cells, cells treated with onvansertib, cells treated with olapari, and cells treated with a combination at different time points (e.g., 8 hours, 24 hours, 48 hours, and 72 hours) after the start of the treatment. Briefly, about 2×10 at different time points can be used ⁶ Individual cells were fixed in 70% ethanol, stained, and double parametric analysis could be performed as described previously (Lupi et al). For each sample, 10,000 events were obtained using a FACS Calibur (Becton Dickinson, san Jose, calif.) flow cytometer.

Furthermore, apoptosis at these time points can be assessed (e.g., by caspase activation in cell extracts), and the status of DNA damage pathway (ATR/CHK 1 axis), pRPA and pRAD51 can be studied by western blot analysis along with activation of γh2ax and p-S10 histone H3.

In at least some of the previously described embodiments, one or more elements used in an embodiment may be used interchangeably in another embodiment unless such substitution is technically not feasible. Those skilled in the art will appreciate that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. Various singular/plural permutations may be explicitly set forth herein for clarity. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Any reference herein to "or" is intended to include "and/or" unless otherwise specified.

Those skilled in the art will understand that in general, terms used herein, and especially terms used in the appended claims (e.g., bodies of the appended claims), are generally intended to mean "open" terms (e.g., the term "including" should be understood to mean "including but not limited to," the term "having" should be understood to mean "having at least," the term "including" should be understood to mean "including but not limited to," etc.). Those skilled in the art will also understand that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should be interpreted to mean "at least one" or "one or more"); the same applies to the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or means two or more recitations). Further, in those instances where a convention similar to "at least one of A, B and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B and C" would include but not be limited to systems that have a alone, B alone, C alone, a and B together, a and C together, B and C together, and/or A, B and C together, etc.). In those instances where a convention similar to "at least one of A, B or C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B or C" would include but not be limited to systems that have a alone, B alone, C alone, a and B together, a and C together, B and C together, and/or A, B and C together, etc.). It should also be understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms.

Further, when features or aspects of the present disclosure are described in terms of markush groups, those skilled in the art will recognize that the present disclosure is thereby also described in terms of any individual member or subgroup of members of the markush group.

As will be understood by those skilled in the art, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof for any and all purposes, such as in providing a written description. Any listed range can be readily identified as sufficiently descriptive and that the same range can be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each of the ranges discussed herein can be readily broken down into a lower third, a middle third, an upper third, and the like. As will also be understood by those skilled in the art, all language such as "up to", "at least", "greater than", "less than" and the like include the stated numbers and refer to ranges that may be subsequently broken down into subranges as discussed above. Finally, as will be appreciated by those skilled in the art, a range includes each individual member. Thus, for example, a group of 1-3 items refers to a group of 1, 2, or 3 items. Similarly, a group having 1-5 items refers to a group having 1 item, 2 items, 3 items, 4 items, or 5 items, and so forth.

While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. A method of treating cancer, the method comprising: administering a poly (ADP-ribose) polymerase (PARP) inhibitor and a Polo-like kinase 1 (PLK 1) inhibitor to a subject having cancer, thereby inhibiting progression of the cancer.

2. The method of claim 1, wherein the cancer is ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof.

3. The method of any one of claims 1-2, wherein the cancer is a homologous recombination deficient cancer.

4. The method of any one of claims 1-3, wherein the cancer is BRCA1 mutant cancer, BRCA2 mutant cancer, or both.

5. The method of any one of claims 1-4, wherein the PLK1 inhibitor and the PARP inhibitor are co-administered simultaneously.

6. The method of any one of claims 1-4, wherein the PLK1 inhibitor and the PARP inhibitor are administered sequentially.

7. The method of claim 6, wherein the PLK1 inhibitor is administered prior to the PARP inhibitor administration, and optionally, wherein the PLK1 inhibitor is administered prior to the PARP inhibitor administration, daily of the PLK1 inhibitor and the PARP inhibitor to the subject.

8. The method of claim 7, wherein the PLK1 inhibitor is administered about 30 minutes to about 5 hours prior to administration of the PARP inhibitor on a given day.

9. The method of any one of claims 1-8, wherein the administration of the PLK1 inhibitor is oral administration, the administration of the PARP inhibitor is oral administration, or both are oral administration.

10. The method of any one of claims 1-9, wherein the inhibition of cancer progression is greater than the combined inhibition of progression caused by the PARP inhibitor alone plus the PLK1 inhibitor alone.

11. The method of any one of claims 1-10, wherein the subject achieves a complete response.

12. The method of any one of claims 1-11, wherein the subject has received prior PARP inhibitor treatment.

13. The method of any one of claims 1-12, wherein the subject is not responsive to treatment with the PARP inhibitor alone.

14. The method of any one of claims 1-13, wherein the subject is known to be resistant to PARP inhibitor therapy.

15. The method of any one of claims 1-14, wherein the PARP inhibitor and the PLK1 inhibitor are each administered to the subject at least twice or at least five cycles per week.

16. The method of any one of claims 1-15, wherein the PARP inhibitor, the PLK1 inhibitor, or both are administered at a period of at least 7 days; optionally, each treatment cycle is at least about 21 days; and also optionally, each treatment cycle is about 21 days to about 28 days.

17. The method of any one of claims 15-16, wherein the PLK1 inhibitor is administered on at least four days of the cycle.

18. The method of any one of claims 16-17, wherein the PLK1 inhibitor is not administered for at least one day of the cycle.

19. The method of any one of claims 1-18, wherein the PARP inhibitor is administered daily.

20. The method of claims 1-19, wherein the subject experiences administration of the PARP inhibitor and the PLK1 inhibitor for at least two cycles.

21. The method of any one of claims 1-20, wherein the PARP inhibitor is selective and/or specific for PARP inhibition.

22. The method of any one of claims 1-20, wherein the PARP inhibitor is iniparib (BSI 201), taprazopanib (BMN-673), olaparib (AZD-2281), AZD5305, NMS-293, rukapali (AG 014699, PF-01367338), ABT-888, vitamin Li Pali (ABT-888), nilaparib, CEP 9722, MK 4827, BGB-290 (pamipril), BSI-201, CEP-8983, E7016, 3-aminobenzamide, or a combination thereof; optionally the PARP inhibitor is olapari.

23. The method of any one of claims 1-22, wherein the PLK1 inhibitor is selective and/or specific for PLK 1.

24. The method of any one of claims 1-22, wherein the PLK1 inhibitor is a dihydropteridinone, a pyridopyrimidine, an aminopyrimidine, a substituted thiazolinone, a pteridine derivative, a dihydroimidazo [1,5-f ] pteridine, a meta-substituted thiazolinone, a benzylstyryl sulfone analog, a stilbene derivative, or any combination thereof.

25. The method of any one of claims 1-22, wherein the PLK1 inhibitor is onvansertib, BI2536, volaserib (BI 6727), GSK461364, AZD1775, CYC140, HMN-176, HMN-214, rigoserib (ON-01910), MLN0905, TKM-080301, TAK-960, or Ro3280; and optionally the PLK1 inhibitor is onvansertib.

26. The method of claim 25, wherein onvansertib is at 12mg/m ² -90 mg/m ² Is applied.

27. The method of any one of claims 25-26, wherein the maximum concentration of onvansertib (C _max ) Is about 100nmol/L to about 1500nmol/L.

28. The method of any one of claims 25-27, wherein the area under the curve (AUC) of the plot of concentration of onvansertib in the subject's blood over time is from about 1000nmol/l.h to about 400000nmol/l.h.

29. The method of any one of claims 25-28, wherein the time to maximum concentration of onvansertib in the subject's blood (T _max ) Is from about 1 hour to about 5 hours.

30. The method of any one of claims 25-29, wherein the elimination half-life (T _1/2 ) Is from about 10 hours to about 60 hours.

31. The method of any one of claims 1-30, wherein the PARP inhibitor is olapari and the PLK1 inhibitor is onvansertib.

32. The method of any one of claims 1-31, wherein the subject has received at least one prior cancer treatment, and optionally wherein the prior treatment does not include use of a PARP inhibitor, a PLK inhibitor, or both.

33. The method of any one of claims 1-32, wherein the subject is in cancer remission, optionally wherein the subject in cancer remission is in Complete Remission (CR) or Partial Remission (PR).

34. The method of any one of claims 1-33, further comprising one or more of: (1) determining the cancer status of the subject, (2) determining the responsiveness of the subject to treatment with a PLK1 inhibitor, and (3) administering one or more cancer therapeutic agents or therapies for cancer.

35. The method of any one of claims 1-34, wherein the subject is a human.

36. A method of sensitizing a cancer cell to a PARP inhibitor, the method comprising: contacting a cancer cell with a composition comprising a Polo-like kinase 1 (PLK 1) inhibitor, thereby sensitizing the cancer cell to the PARP inhibitor.

37. The method of claim 36, wherein the PLK1 inhibitor is onvansertib, the PARP inhibitor is olapari, or the PLK1 inhibitor is onvansertib and the PARP inhibitor is olapari.

38. The method of any one of claims 36-37, wherein contacting cancer cells with the composition occurs in vitro, ex vivo, and/or in vivo.

39. The method of any one of claims 36-38, wherein contacting a cancer cell with the composition is in a subject, and optionally wherein the subject is not responsive to, or is known to be resistant to, the PARP inhibitor.

40. The method of claim 39, wherein the subject has prior treatment with the PARP inhibitor.

41. The method of any one of claims 39-40, wherein the subject is a mammal, and optionally the mammal is a human.

42. The method of any one of claims 36-41, comprising determining sensitization of the cancer cells to a PARP inhibitor after contact with the composition.

43. The method of any one of claims 36-42, comprising contacting the cancer cell with the PARP inhibitor, and optionally, wherein contacting the cancer cell with the PARP inhibitor occurs in the subject.

44. The method of claim 43, comprising determining the response of the subject to the PARP inhibitor.

45. The method of any one of claims 43-44, wherein contacting the cancer cell with the PARP inhibitor is simultaneous with contacting the cancer cell with the composition or after contacting the cancer cell with the composition.

46. A kit comprising:

a Polo-like kinase 1 (PLK 1) inhibitor; and

a manual for instructions for co-administering the PLK1 inhibitor with a poly (ADP-ribose) polymerase (PARP) inhibitor to a subject for treating cancer is provided.

47. The kit of claim 46, wherein the subject has ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof.

48. The kit of claim 46 or 47, wherein the cancer is a Homologous Recombination (HR) deficient cancer.

49. The kit of any one of claims 46-48, wherein the cancer is BRCA1 mutant cancer, BRCA2 mutant cancer, or both.

50. The kit of any one of claims 46-49, wherein the instructions comprise instructions for co-administering the PLK1 inhibitor and the PARP inhibitor simultaneously.

51. The kit of any one of claims 46-49, wherein the instructions comprise instructions for sequentially co-administering the PLK1 inhibitor and the PARP inhibitor.

52. The kit of any one of claims 46-51, wherein the instructions comprise (1) instructions for orally administering the PLK1 inhibitor, (2) instructions for orally administering the PARP inhibitor, or both.

53. The kit of any one of claims 46-52, wherein the instructions comprise instructions that the subject has received prior PARP inhibitor treatment.

54. The kit of any one of claims 46-53, wherein the instructions comprise instructions for the subject not responding to treatment with the PARP inhibitor alone.

55. The kit of any one of claims 46-54, wherein the instructions comprise instructions for the subject being known to be resistant to PARP inhibitor therapy.

56. The kit of any one of claims 46-55, wherein the instructions comprise instructions for administering each of the PARP inhibitor and the PLK1 inhibitor to the subject at least twice or at least five cycles a week.

57. The kit of any one of claims 46-56, wherein the instructions comprise instructions for administering the PARP inhibitor, the PLK1 inhibitor, or both, at a period of at least 7 days; and optionally wherein each treatment cycle is at least about 21 days, and further optionally, each treatment cycle is from about 21 days to about 28 days of instruction.

58. The kit of claim 57, wherein the instructions comprise instructions for administering the PLK1 inhibitor on at least four days of the cycle.

59. The kit of any one of claims 57-58, wherein the instructions comprise instructions for not administering the PLK1 inhibitor for at least one day of the cycle.

60. The kit of any one of claims 36-59, wherein the instructions comprise instructions for daily administration of the PARP inhibitor.

61. The kit of any one of claims 46-60, wherein the instructions comprise instructions for administering the PARP inhibitor and the PLK1 inhibitor for at least two cycles.

62. The kit of any one of claims 46-61, wherein the PARP inhibitor is selective and/or specific for PARP1 inhibition and/or PARP2 inhibition.

63. The kit of any one of claims 46-62, wherein the PARP inhibitor is iniparib (BSI 201), taprazopanib (BMN-673), olaparib (AZD-2281), AZD5305, rupa's (AG 014699, PF-01367338), ABT-888, vitamin Li Pali (ABT-888), nilaparib, CEP 9722, MK 4827, BGB-290 (pamipril), BSI-201, CEP-8983, E7016, 3-aminobenzamide, NMS-293, or a combination thereof; and optionally wherein the PARP inhibitor is olapari.

64. The kit of any one of claims 46-63, wherein the PLK1 inhibitor is selective and/or specific for PLK 1.

65. The kit of any one of claims 46-64, wherein the PLK1 inhibitor is a dihydropteridinone, a pyridopyrimidine, an aminopyrimidine, a substituted thiazolinone, a pteridine derivative, a dihydroimidazo [1,5-f ] pteridine, a meta-substituted thiazolinone, a benzylstyryl sulfone analog, a stilbene derivative, or any combination thereof.

66. The kit of any one of claims 46-64, wherein the PLK1 inhibitor is onvansertib, BI2536, volasertib (BI 6727), GSK461364, AZD1775, CYC140, HMN-176, HMN-214, rigossentib (ON-01910), MLN0905, TKM-080301, TAK-960, or Ro3280; and optionally wherein the PLK1 inhibitor is onvansertib.

67. The kit of claim 66, wherein the instructions comprise instructions for use at 12mg/m ² -90mg/m ² Instructions for administering onvansertib.

68. The kit of any one of claims 46-67, wherein the PARP inhibitor is olapari and the PLK1 inhibitor is onvansertib.

69. The kit of any one of claims 46-68, wherein the instructions comprise that the subject has received at least one prior treatment for the cancer, and optionally wherein the prior treatment does not comprise instructions for use of a PARP inhibitor, a PLK inhibitor, or both.

70. The kit of any one of claims 46-69, wherein the instructions comprise instructions that the subject is in cancer remission, and optionally wherein the subject in cancer remission is in Complete Remission (CR) or Partial Remission (PR).

71. The kit of any one of claims 46-70, further comprising the PARP inhibitor.