WO2025117928A1

WO2025117928A1 - Breast cancer treatment using pi3k inhibitor and cell cycle inhibitor

Info

Publication number: WO2025117928A1
Application number: PCT/US2024/058022
Authority: WO
Inventors: Tod Smeal; Sreeja SREEKUMAR; Maya RIDINGER
Original assignee: Cardiff Oncology Inc
Current assignee: Cardiff Oncology Inc
Priority date: 2023-12-01
Filing date: 2024-12-01
Publication date: 2025-06-05
Anticipated expiration: 2026-06-01

Abstract

Provided include methods, compositions and kits for treating hormone receptor positive (HR+) breast cancer, e.g., estrogen receptor positive (ER+) and progesterone receptor positive (PR+) breast cancer, in a subject. The method can comprise administrating a PI3K inhibitor and a cell cycle inhibitor, such as a PLK1 inhibitor, to the subject in a manner sufficient to inhibit or reduce progression of the cancer.

Description

BREAST CANCER TREATMENT USING PI3K INHIBITOR AND CELL CYCLE

INHIBITOR

RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No. 63/605,318, filed December 1, 2023. The entire content of this related application is incorporated herein by reference in its entirety.

BACKGROUND

Field

[0002] The present application generally relates to treatment for cancer, more specifically, combination therapies for treating breast cancer.

Description of the Related Art

[0003] Breast cancer is the most common form of cancer and the second leading cause of cancer death in women worldwide. Hormone receptor-positive breast cancer is a type of breast cancer that grows in response to hormones (e.g., estrogen or progesterone). Approximately 67%- 80% of breast cancers in women are estrogen receptor positive, while approximately 90% of breast cancers in men are estrogen receptor positive. Standard treatments of hormone receptor-positive breast cancer currently include chemotherapy, hormone therapy, surgery, radiation therapy, and targeted therapy.

[0004] There is an urgent need to develop effective treatment for breast cancer patients, particularly breast cancer patients that have or developed resistant forms including, for example, hormone resistant or kinase inhibitor resistant breast cancer.

SUMMARY

[0005] Disclosed herein include methods of treating hormone receptor positive (HR+) breast cancer. In some embodiments, the method comprises: administering a PI3K inhibitor and a G2/M cell cycle inhibitor to a subject with the HR+ breast cancer, thereby inhibiting or reducing progression of the HR+ breast cancer in the subject. The HR+ breast cancer can be estrogen receptor positive (ER+) and/or progesterone receptor positive (PR+).

[0006] In some embodiments, the HR+ breast cancer is a PIK3CA-mutated HR+ breast cancer. In some embodiments, the HR+ breast cancer is a PTEN-inactivated HR+ breast cancer. In some embodiments, the HR+ breast cancer is HER2 negative. In some embodiments, the ER+ breast cancer has a histological or cytological profile with ER > 1%, 10%, 20%, or higher.

[0007] In some embodiments, the subject with the HR+ breast cancer is resistant to or does not respond effectively to a hormone therapy. In some embodiments, the hormone therapy (SERM), an aromatase inhibitor, or a combination thereof. In some embodiments, the subject with the HR+ breast cancer is resistant to a kinase inhibitor. In some embodiments, the kinase inhibitor is a CDK inhibitor. In some embodiments, the CDK inhibitor is a CDK4/6 inhibitor. In some embodiments, the subject with the HR+ breast cancer developed stable disease, progressive disease or resistance to a CDK4/6 inhibitor and/or a SERD. In some embodiments, the subject with the HR+ breast cancer is resistant to palbociclib, fulvestrant, or both or developed stable or progressive disease following treatment with palbociclib, fulvestrant, or both. In some embodiments, the resistance is acquired resistance or intrinsic resistance.

[0008] In some embodiments, the subject with the HR+ breast cancer has received a prior PI3K inhibitor treatment, a prior Gl/S cell cycle inhibitor treatment or a prior G2/M cell cycle inhibitor treatment. In some embodiments, the subject with the HR+ breast cancer did not respond to the treatment with the prior PI3K inhibitor, the prior Gl/S cell cycle inhibitor or the prior G2/M cell cycle inhibitor. In some embodiments, the subject with the HR+ breast cancer developed stable or progressive disease following the treatment with the prior PI3K inhibitor, the prior Gl/S cell cycle inhibitor or the prior G2/M cell cycle inhibitor. In some embodiments, the subject with the HR+ breast cancer is known to be resistant to the PI3K inhibitor, the prior Gl/S cell cycle inhibitor or the G2/M cell cycle inhibitor alone.

[0009] In some embodiments, the PI3K inhibitor and the G2/M cell cycle inhibitor are co-administered simultaneously. In some embodiments, the PI3K inhibitor and the G2/M cell cycle inhibitor are administered sequentially. In some embodiments, the PI3K inhibitor, the G2/M cell cycle inhibitor, or both are administered to the subject in a cycle of 4 days, 7 days, 14 days, 28 days, 35 days, 42 days, or 49 days. In some embodiments, the PI3K inhibitor is administered to the subject about once a week and the G2/M cell cycle inhibitor is administered to the subject about 5 days a week. In some embodiments, each cycle of treatment is at least about 14 days. In some embodiments, each cycle of treatment is from about 14 days to about 28 days. In some embodiments, the G2/M cell cycle inhibitor is administered on at least five days, at least ten days, or at least fifteen days in a cycle. In some embodiments, the G2/M cell cycle inhibitor is not administered on at least one day, at least three days, or at least seven days in a cycle. In some embodiments, the PI3K inhibitor is administered once or twice weekly. In some embodiments, the PI3K inhibitor is administered once weekly for two, three, four, five, six or seven consecutive weeks in a cycle. In some embodiments, the subject undergoes at least two cycles of the administration of the PI3K inhibitor and the G2/M cell cycle inhibitor.

[0010] In some embodiments, the PI3K inhibitor is a PI3Ka inhibitor. In some embodiments, the PI3K inhibitor is idelalisib, copanlisib, duvelisib, alpelisib, umbralisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, tenalisib, voxtalisib, AMG 319, AZD8186, GSK2636771, SF1126, acalisib, omipalisib, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, or ZSTK474; or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof; or any combinations thereof. In some embodiments, the PI3Ka inhibitor is alpelisib. In some embodiments, the G2/M cell cycle inhibitor is a PLK1 inhibitor. In some embodiments, the PLK1 inhibitor is onvansertib (NMS-P937), BI2536, volasertib (BI 6727), GSK461364, adavosertib (AZDI 775), CYC 140, HMN-176, HMN-214, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960, Ro3280; or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof; and any combinations thereof. In some embodiments, the G2/M cell cycle inhibitor is onvansertib.

[0011] In some embodiments, the subject has received at least one prior cancer treatment. In some embodiments, the prior treatment does not comprise the use of a PI3K inhibitor, a PLK1 inhibitor, or both. In some embodiments, the PLK1 inhibitor is onvansertib. In some embodiments, the subject was in remission for cancer. In some embodiments, the subject in remission for cancer was in complete remission (CR) or in partial remission (PR).

[0012] The method can further comprise determining cancer status of the subject. The method can further comprise determining responsiveness of the subject to the treatment of the PI3K inhibitor and/or the G2/M cell cycle inhibitor. The method can further comprise administering one or more additional cancer therapeutics or therapies for the cancer. In some embodiments, the subject is human. In some embodiments, the subject achieves a complete response.

[0013] A kit, comprising: a PI3K inhibitor; a G2/M cell cycle inhibitor; and a manual providing instructions for co-administering the PI3K inhibitor and the G2/M cell cycle inhibitor to a subject in need thereof for treating HR+ breast cancer.

[0014] In some embodiments, the PI3K inhibitor is a PI3Ka inhibitor. In some embodiments, the PI3K inhibitor is idelalisib, copanlisib, duvelisib, alpelisib, umbralisib, buparlisib, copanlisib, dactolisib, leniolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, tenalisib, voxtalisib, AMG 319, AZD8186, GSK2636771, SF1126, acalisib, omipalisib, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, or ZSTK474; or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof; or any combinations thereof. In some embodiments, the G2/M cell cycle inhibitor is a PLK1 inhibitor. In some embodiments, the PLK1 inhibitor is onvansertib (NMS-P937), 214, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960, Ro3280; or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof; and any combinations thereof. In some embodiments, the PI3K inhibitor is alpelisib and/or the G2/M cell cycle inhibitor is onvansertib.

[0015] In some embodiments, the instructions comprise instructions for coadministrating the PI3K inhibitor and the G2/M cell cycle inhibitor simultaneously. In some embodiments, the instructions comprise instructions for co-administrating the PI3Ka inhibitor and the G2/M cell cycle inhibitor sequentially. In some embodiments, the instructions comprise instructions for administering to a subject that did not respond to treatment with the PI3Ka inhibitor or the G2/M cell cycle inhibitor alone. In some embodiments, the instructions comprise instructions for administering to a subject resistant to a CDK inhibitor, hormone therapy, or both. In some embodiments, the CDK inhibitor is a CDK4/6 inhibitor. In some embodiments, the CDK 4/6 inhibitor is palbociclib. In some embodiments, the hormone therapy comprises a selective estrogen receptor degrader (SERD), a selective estrogen receptor modulator (SERM), an aromatase inhibitor, or a combination thereof. In some embodiments, the SERD is fulvestrant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1A show Bliss synergy scores following a dose matrix (9 x 9) evaluation of alpelisib (Alp) and onvansertib (Onv) drug combination in ER+ breast cancer cell lines. The cells were treated with drugs, cell viability was assessed, and synergy scores were calculated. FIG. IB shows colony forming ability of the cells after being treated with the drugs (e.g., alpelisib and onvansertib). Results presented as mean ± SEM. One-way ANOVA was used to compare the means. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates pO.OOOl.

[0017] FIG. 2A-FIG. 2B show the effect of alpelisib (Alp) and onvansertib (Onv) on cell cycle distribution of ER+ breast cancer cell lines. The cells were treated with the drugs for 72-96 h, stained with DAPI and cell cycle distribution was assessed by flow cytometry. FIG. 2A is a plot showing the percentage of cells in Gl, S and G2/M phases. FIG. 2B is a plot showing the percentage of cells in G2/M phases. Results are the mean of three experiments and are presented as mean ± SEM. One-way ANOVA was used to compare the means. *** indicates p<0.001, and **** indicates pO.OOOl.

[0018] FIG. 3A-FIG. 3B show effect of alpelisib (Alp) and onvansertib (Onv) single agents and combination on apoptosis. The cells were treated with drugs for 72-96 h. FIG. 3A shows the percentage of cells undergoing apoptotic DNA fragmentation as analyzed by TUNEL assay. Results are presented as mean ± SEM. One-way ANOVA was used to compare the means. * indicates p<0.05, ** indicates p<0.01, *** indicates pO.001, and **** indicates pO.OOOl. FIG. of drugs for 24 h, as analyzed by Western blotting. P-actin was used as loading control.

[0019] FIG. 4A-FIG. 4C show the anti-tumor activity of the onvansertib and alpelisib combination in palbociclib-resistant PIK3CA-mutant estrogen receptor positive (ER+) breast cancer PDX models. ER+ breast cancer PDX models, HBCx-134palboR31, HBCx-86 and HBCx- 180, were treated with vehicle (Ctrl), onvansertib (Onv), alpelisib (Alp) or combination of onvansertib and alpelisib for the indicated duration (marked by the black lines along the horizontal axis). Tumors were measured, and the relative tumor volume (RTV) was calculated as RTV = (tumor volume on measured day)/(tumor volume on day 0). Individual tumor volume and RTV over time of HBCx-134palboR31 (FIG. 4A) and HBCx-86 (FIG. 4B) are shown. Tumor regression was reported if RTV < 0.5 in at least 1 tumor measurement. Unpaired t-test was used to compare relative tumor volume at last measurement. FIG. 4C shows the RTV and Kaplan- Meier survival curve for event-free survival (time to reach RTV = 4) of HBCx-180 PDX. Logrank Mantel Cox test was used for survival analyses. * indicates p<0.05, and ** indicates p<0.01.

[0020] FIG. 5A-FIG. 5C show the effects of onvansertib and alpelisib combination in inducing apoptosis in vivo. PDX models were treated as described in FIG. 4A-FIG. 4C for 32 days (HBCx-86) or 4 days (HBCx-134palboR31). Tumor samples were collected 3 h after the last treatment and fixed in formalin. FIG. 5A is hematoxylin and eosin stained photomicrographs (40x) showing apoptotic cells. For each sample, 5 fields of view were randomly selected in the tumor areas. Apoptotic cells were manually counted in these fields by a board-certified pathologist and mean values (apoptotic score) were calculated. FIG. 5B shows the apoptotic scores. Data is presented as mean ± SEM. * indicates p<0.05, and ** indicates p<0.01 Protein lysates were prepared from tumors obtained from HBCx-134palboR31. FIG. 5C shows the protein expression of cleaved-PARP as analyzed by Western blotting. P-actin was used as loading control.

DETAILED DESCRIPTION

[0021] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar 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 made part of the disclosure herein.

[0022] All patents, published patent applications, other publications, and sequences entirety with respect to the related technology.

Definitions

[0023] 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 the present disclosure belongs. See, e.g. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of the present disclosure, the following terms are defined below.

[0024] As used herein, a “subject” refers to an animal that is the object of treatment, observation or experiment. “Animals” include cold- and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles and, in particular, mammals. “Mammal” includes, without limitation, mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates, such as monkeys, chimpanzees, and apes, and, in particular, humans.

[0025] As used herein, a “patient” refers to a subject that is being treated by a medical professional, such as a Medical Doctor (z.e., Doctor of Allopathic medicine or Doctor of Osteopathic medicine) or a Doctor of Veterinary Medicine, to attempt to cure, or at least ameliorate the effects of, a particular disease or disorder or to prevent the disease or disorder from occurring in the first place. In some embodiments, the patient is a human or an animal. In some embodiments, the patient is a mammal.

[0026] As used herein, “administration” or “administering” refers to a method of giving a dosage of a pharmaceutically active ingredient to a vertebrate.

[0027] As used herein, a “dosage” refers to the combined amount of the active ingredients (e.g., alpelisib, and onvansertib).

[0028] As used herein, a “unit dosage” refers to an amount of therapeutic agent administered to a patient in a single dose.

[0029] As used herein, the term “daily dose” or “daily dosage” refers to a total amount of a pharmaceutical composition or a therapeutic agent that is to be taken within 24 hours.

[0030] As used herein, the term “delivery” refers to approaches, formulations, technologies, and systems for transporting a pharmaceutical composition or a therapeutic agent into the body of 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.

[0031] As used herein, the term “formulated” or “formulation” refers to the process in which different chemical substances, including one or more pharmaceutically active ingredients, active ingredients can be co-formulated into a single dosage form or combined dosage unit, or formulated separately and subsequently combined into a combined dosage unit. A sustained release formulation is a formulation which is designed to slowly release a therapeutic agent in the body over an extended period of time, whereas an immediate release formulation is a formulation which is designed to quickly release a therapeutic agent in the body over a shortened period of time.

[0032] As used herein, the term “pharmaceutically acceptable” indicates that the indicated material does not have properties that would cause a reasonably prudent medical practitioner to avoid administration of the material to a patient, taking into consideration the disease or conditions to be treated and the respective route of administration. For example, it is commonly required that such a material be essentially sterile.

[0033] As used herein, the term “pharmaceutically acceptable carrier” refers to pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition, or component thereof, from one organ, or portion of the body, to another organ, or portion of the body, or to deliver an agent to a diseased tissue or a tissue adjacent to the diseased tissue. Carriers or excipients can be used to produce compositions. The carriers or excipients can be chosen to facilitate administration of a drug or pro-drug. Examples of carriers include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents. Examples of physiologically compatible solvents include sterile solutions of water for injection (WFI), saline solution, and dextrose.

[0034] As used herein, the term “pharmaceutically acceptable salt” refers to any acid or base addition salt whose counter-ions are non-toxic to the patient in pharmaceutical doses of the salts. A host of pharmaceutically acceptable salts are well known in the pharmaceutical field. If pharmaceutically acceptable salts of the compounds of this disclosure are utilized in these compositions, those salts are preferably derived from inorganic or organic acids and bases. Included among such acid salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, hydrohalides salicylates, methylene-bis-b-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, ethanesulphonates, cyclohexylsulphamates, quinates, and the like. Pharmaceutically acceptable base addition salts include, without limitation, those derived from alkali 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 with organic bases, such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth.

[0035] As used herein, the term “hydrate” refers to a complex formed by combination of water molecules with molecules or ions of the solute. As used herein, the term “solvate” refers to a complex formed by combination of solvent molecules with molecules or ions of the solute. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Solvate is meant to include hydrate, hemi-hydrate, channel hydrate and the likes. Some examples of solvents include, but are not limited to, methanol, /f,/f-di methyl form am ide, tetrahydrofuran, dimethylsulfoxide, and water.

[0036] As used herein, “therapeutically effective amount” or “pharmaceutically effective amount” refers to an amount of therapeutic agent, which has a therapeutic effect. The dosages of a pharmaceutically active ingredient which are useful in treatment when administered alone or in combination with one or more additional therapeutic agents are therapeutically effective amounts. Thus, as used herein, a therapeutically effective amount refers to an amount of therapeutic agent which produces the desired therapeutic effect as judged by clinical trial results and/or model animal studies. The therapeutically effective amount will vary depending on the compound, the disease, disorder or condition and its severity and the age, weight, etc., of the mammal to be treated. The dosage can be conveniently administered, e.g., in divided doses up to four times a day or in sustained-release form.

[0037] As used herein, the term “treat,” “treatment,” or “treating,” refers to administering a therapeutic agent or pharmaceutical composition to a subject for prophylactic and/or therapeutic purposes. The term “prophylactic treatment” refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition. The term “therapeutic treatment” refers to administering treatment to a subject already suffering from a disease or condition. As used herein, a “therapeutic effect” relieves, to some extent, one or more of the symptoms of a disease or disorder. For example, a therapeutic effect may be observed by a reduction of the subjective patient questionnaire).

[0038] As used herein, the term “prophylaxis,” “prevent,” “preventing,” “prevention,” and grammatical variations thereof as used herein refers the preventive treatment of a subclinical disease-state in a subject, e.g., a mammal (including a human), for reducing the probability of the occurrence of a clinical disease-state. The method can partially or completely delay or preclude the onset or recurrence of a disorder or condition and/or one or more of its attendant symptoms or barring a subject from acquiring or reacquiring a disorder or condition or reducing a subject’s risk of acquiring or requiring a disorder or condition or one or more of its attendant symptoms. The subject is selected for preventative therapy based on factors that are known to increase risk of suffering a clinical disease state compared to the general population. “Prophylaxis” therapies can be divided into (a) primary prevention and (b) secondary prevention. Primary prevention is defined as treatment in a subject that has not yet presented with a clinical disease state, whereas secondary prevention is defined as preventing a second occurrence of the same or similar clinical disease state.

[0039] As used herein, each of the terms “partial response,” “partial remission” and “PR” refers to the amelioration of a cancerous state, as measured by, for example, tumor size and/or cancer marker levels, in response to a treatment. In some embodiments, a “partial response” means that a tumor or tumor-indicating blood marker has decreased in size or level by about 50% in response to a treatment. The treatment can be any treatment directed against cancer, including but not limited to, chemotherapy, radiation therapy, hormone therapy, surgery, cell or bone marrow transplantation, and immunotherapy. The size of a tumor can be detected by clinical or by radiological means. Tumor-indicating markers can be detected by means well known to those of skill, e.g., ELISA or other antibody-based tests. A partial response of the target lesion can refer to at least a 30% decrease in the sum of the diameters of target lesions, taking as reference the baseline sum diameters.

[0040] As used herein, each of the terms “complete response” or “complete remission” or “CR” means that a cancerous state, as measured by, for example, tumor size and/or cancer marker levels, has disappeared following a treatment, including but are not limited to, chemotherapy, radiation therapy, hormone therapy, surgery, cell or bone marrow transplantation, and immunotherapy. The presence of a tumor can be detected by clinical or by radiological means. Tumor-indicating markers can be detected by means well known to those of skill, e.g., ELISA or other antibody-based tests. A “complete response” does not necessarily indicate that the cancer has been cured, however, a complete response may be followed by a relapse. A complete response of a target lesion includes disappearance of all target lesions and any pathological lymph nodes a non-target lesion includes disappearance of all non-target lesions and normalization of tumor marker level (all lymph nodes must be non-pathological in size (<10 mm short axis)). If tumor markers are initially above the upper normal limit, they need to normalize for a patient to be considered in complete clinical response of a nontarget lesion. The duration of overall CR is measured from the time measurement criteria are first met for CR until the first date that progressive disease is objectively documented, or death due to any cause. Participants without events reported are censored at the last disease evaluation.

[0041] As used herein, the term “stable disease” or “SD” means neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study. Duration of stable disease is measured from the start of the treatment until the criteria for progression are met, taking as reference the smallest measurements recorded since the treatment started, including the baseline measurements.

[0042] As used herein, the term “progressive disease” or “PD” when refers to a target lesion means at least a 20% increase in the sum of the diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progressions). When progressive disease or PD refers to a non-target lesion, it means the appearance of one or more new lesions and/or unequivocal progression of existing non-target lesions. Unequivocal progression should not normally trump target lesion status. It must be representative of overall disease status change, not a single lesion increase.

[0043] As used herein, the term “best overall response” means the best response recorded from the start of the treatment until disease progression/recurrence (taking as reference for progressive disease the smallest measurements recorded since the treatment started). The patient's best response assignment depends on the achievement of both measurement and confirmation criteria. The duration of an overall response is measured from the time measurement criteria are met for CR or PR (whichever is first recorded) until the first date that recurrent or progressive disease is objectively documented (taking as reference for progressive disease the smallest measurements recorded since the treatment started, or death due to any cause. Participants without events reported are censored at the last disease evaluation).

[0044] As used herein, the term “DLT rate” means dose-limiting toxicity rate.

[0045] As used herein, the term “ICso” means inhibitory drug concentration that produces 50% of the maximal effect.

[0046] As used herein, the term “SEM” means standard error of mean. is the starting time in hours and “y” is the ending time in hours.

[0048] As used herein, the term “Cavg” means average concentration. As used herein, the term “Cmax” means maximum concentration.

[0049] As used herein, the term “ANC” means absolute neutrophil count.

[0050] As used herein, the term “CT” means computed tomography.

[0051] As used herein, the term “ctDNA” means circulating tumor DNA.

[0052] As used herein, the term “MRI” means magnetic resonance imaging.

[0053] As used herein, the term “PK” means pharmacokinetic.

[0054] As used herein, the term “PBMC” means peripheral blood mononuclear cells.

[0055] As used herein, the term “tolerable” means a dose level where < 1/6 participants have experienced a DLT, or the dose level that is declared the RP2D.

[0056] As used herein, the term “adverse event” or “AE” means an untoward medical occurrence in a subject administered a medicinal product that does not necessarily have a causal relationship with this treatment. An AE can be an unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of an investigational product, whether or not related to the investigational medicinal product. An adverse event may include worsening or exacerbation of the disease under study; worsening or exacerbation of pre-existing conditions or events; intercurrent illnesses; or drug interactions. Anticipated fluctuations of pre-existing conditions that do not represent a clinically significant exacerbation or worsening are not considered AEs. Surgical procedures are not adverse events; they are therapeutic measures for conditions that require surgery. The condition, provided it develops or is a worsening of a pre-existing condition for which the surgery is required, is the AE. Disease progression is an efficacy endpoint and is not an AE. A clinical event in the setting of disease progression would be considered an AE if it could not be unequivocally attributed to or consistent with expected disease progression.

[0057] As used herein, the term “expected adverse event” means an adverse event that are listed or characterized in the current adverse event list, the Package Insert, the Investigator Brochure or is included in the informed consent document as a potential risk.

[0058] As used herein, the term “unexpected adverse event” means an adverse event that is not listed in the Package Insert (P.I.) or current Investigator Brochure (LB.) or not identified. This includes adverse events for which the specificity or severity is not consistent with the description in the P.I. or LB. For example, under this definition, hepatic necrosis would be unexpected.

[0059] As used herein, the term “severe adverse event” or “SAE” means an AE that AE, in the view of the investigator, places the subject at immediate risk of death, but does not include an AE that, had it occurred in a more severe form, might have caused death); (3) requires or prolongs inpatient hospitalization; (4) results in persistent or significant disability/incapacity (z.e., the AE results in substantial disruption of the subject’s ability to conduct normal life functions); or (5) results in a congenital anomaly /birth defect in a neonate/infant bom to a mother exposed to the IMP.

[0060] As used herein, the term “definite AE” means the AE is clearly related to the study treatment.

[0061] As used herein, the term “probable AE” means the AE is likely related to the study treatment.

[0062] As used herein, the term “possible AE” means the AE may be related to the study treatment.

[0063] As used herein, the term “unlikely AE” means the AE is doubtfully related to the study treatment.

[0064] As used herein, the term “unrelated AE” means the AE is clearly not related to the study treatment.

[0065] As used herein, the term “expected disease progression” means an event that is unequivocally related to disease progression, and that the clinical course is consistent with what would be expected for the patient’s disease.

[0066] As used herein, the term “measurable lesion” means a lesion that can be accurately measured in at least one dimension (longest diameter to be recorded) as > 20 mm by chest x-ray or >10 mm with CT scan, MRI, or calipers by clinical exam. Tumor lesions that are situated in a previously irradiated area might or might not be considered measurable. Cystic lesion thought to represent cystic metastases are measurable lesions if they meet the definition of measurability described above. However, they are target lesions if non-cystic lesions are also present in the same participant. Clinical lesions are measurable when they are superficial (e.g., skin nodules and palpable lymph nodes) and >10 mm in diameter as assessed using calipers (e.g., skin nodules).

[0067] As used herein, the term “malignant lymph node” means a pathologically enlarged and measurable lymph node with >15 mm in short axis when assessed by CT scan.

[0068] As used herein, the term “non-measurable disease” means a small lesion (or a site of disease) where the longest diameter <10 mm or pathological lymph nodes with >10 to <15 mm short axis. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, abdominal masses (not followed by the criteria for radiographically defined simple cysts are not malignant lesions (neither measurable nor non-measurable) and are simple cysts.

[0069] As used herein, the term “target lesion” means all measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, that is representative of all involved organs. Target lesions are selected on the basis of their size (lesions with the longest diameter), be representative of all involved organs, but in addition should be those that lend themselves to reproducible repeated measurements. When the largest lesion does not lend itself to reproducible measurement, the next largest lesion that can be measured reproducibly is the target lesion.

[0070] As used herein, the term “non-target lesions” means all lesions (or sites of disease) that are not target lesions. Non-target lesions include any measurable lesions over and above the 5 target lesions.

[0071] As used herein, the term “overall survival” or “OS” means the time from randomization (or registration) to death due to any cause, or censored at date last known alive.

[0072] As used herein, the term “progression-free survival” or “PFS” means the time from randomization (or registration) to the earlier of progression or death due to any cause. Participants alive without disease progression are censored at date of last disease evaluation.

[0073] As used herein, the term “time to progression” or “TTP” means the time from randomization (or registration) to progression, or censored at date of last disease evaluation for those without progression reported.

[0074] As used herein, the term “breast cancer” refers to a condition characterized by rapid proliferation of abnormal cells in one or both breasts of a subject. The abnormal cells often are referred to as “neoplastic cells,” which refers to, in some embodiments, transformed cells that can form a solid tumor. The term “tumor,” in some embodiments, refers to an abnormal mass or population of cells (i.e. two or more cells) that result from excessive or abnormal cell division, whether malignant or benign, and pre-cancerous and cancerous cells. Malignant tumors are distinguished from benign growths or tumors in that, in addition to uncontrolled cellular proliferation, they can invade surrounding tissues and can metastasize.

[0075] As used herein, the term “metastasis” refers to a process in which cancer cells travel from one organ or tissue to another non-adjacent organ or tissue. Cancer cells in the breast(s) can spread to tissues and organs of a subject, and conversely, cancer cells from other organs or tissue can invade or metastasize to a breast. Cancerous cells from the breast(s) may invade or metastasize to any other organ or tissue of the body. Breast cancer cells often invade lymph node cells and/or metastasize to the liver, brain and/or bone and spread cancer in these tissues and organs. The term “invasion,” in some embodiments, refers to the spread of cancerous cells to [0076] As used herein, the term “advanced breast cancer” refers to cancer that has spread to other places in the body and usually cannot be cured or controlled with current treatment.

[0077] Disclosed herein includes methods for treating breast cancer, e.g., estrogen positive breast cancer, using a PI3K inhibitor and a cell cycle inhibitor. In some embodiments, a method of treating hormone receptor positive (HR+) breast cancer (e.g., the HR+ cancer can be estrogen receptor positive (ER+) and/or progesterone receptor positive (PR+)) comprises administering a PI3K inhibitor and a G2/M cell cycle inhibitor to a subject with the HR+ breast cancer, thereby inhibiting or reducing progression of the HR+ breast cancer in the subject.

[0078] Disclosed herein also includes compositions and kits for treating estrogen positive breast cancer. In some embodiments, a kit comprises a PI3K inhibitor, a G2/M cell cycle inhibitor, and a manual providing instructions for co-administering the PI3K inhibitor and the G2/M cell cycle inhibitor to a subject in need thereof for treating HR+ breast cancer.

Hormone receptor positive (HR+) breast cancer

[0079] Methods, compositions and kits disclosed herein can be used for treating cancer and/or tumor such as hormone receptor positive (HR+) breast cancer. The HR+ cancer can be estrogen receptor positive (ER+) and/or progesterone receptor positive (PR+)).

[0080] The cancer and/or tumor described herein can be a breast cancer and/or breast tumor. In some embodiments, the breast cancer is hormone receptor positive breast cancer. Hormone receptor positive breast cancers include breast cancers in which a portion of the cancer cells express hormone receptors including estrogen receptor, progesterone receptor, or both. Hormone receptor positive breast cancers are typically susceptible to hormone therapies. Hormone therapy can slow or stop the growth of hormone-sensitive tumors by blocking the body’s ability to produce hormones or by interfering with effects of hormones on breast cancer cells. Exemplary hormone therapies include, but are not limited to, selective estrogen receptor modulators or SERMs (e.g., tamoxifen, toremifene), aromatase inhibitors (e.g., anastrozole, letrozole), or selective estrogen receptor degraders or SERDs (e.g., fulvestrant). A person skilled in the art would understand that hormone therapies such as aromatase inhibitors block production of estrogens in the body, whereas SERMs and SERDs block the proliferative action of estrogens on the breast cancer cells.

[0081] The breast cancer can be hormone receptor positive (HR+) breast cancer. In some embodiments, the breast cancer is estrogen receptor positive (ER+) breast cancer. The term “ER+ breast cancer” refers to breast cancer wherein at least a portion of the cancer cells express estrogen receptor (ER). In some embodiments, at least 1% of the cancer cells express ER. In some embodiments, the HR+ (e.g., ER+) breast cancer can comprise breast cancer cells also expressing progesterone receptor (PR). Accordingly, the HR+ or ER+ breast cancer can be PR positive (PR+). In some embodiments, the HR+ or ER+ breast cancer is PR negative (PR-). Immunohistochemistry (IHC) test can be performed to test if cancer cells have estrogen and/or progesterone receptors. In some embodiments, the breast cancer cells contain higher than normal levels of human epidermal growth factor receptor 2 (HER2) protein, thus referred to as HER2 positive (HER2+) cancer. In some embodiments, the breast cancer described herein is triple positive breast cancer. The term “triple positive breast cancer” refers to breast cancer cells that express estrogen receptors (ERs), progesterone receptors (PRs), and large amounts of human epidermal growth factor receptor 2 (HER2) protein. HER2 positive breast cancers are typically susceptible to HER2 kinase inhibitors (e.g., trastuzumab and lapatinib). Triple positive breast cancer can also be referred to as “ER+, PR+, HER2+ breast cancer.” In some embodiments, the breast cancer described herein are distinct from triple negative breast cancer (TNBC), which refers to breast cancer cells that do not have estrogen receptors, progesterone receptors, or large amounts of HER2/neu protein. In some embodiments, the HR+ or ER+ breast cancer is HER2 negative.

[0082] The ER+ breast cancer described herein can include breast cancer, advanced breast cancer, metastatic breast cancer, ER+/PR+ breast cancer, ER+/PR- breast cancer, ER+/PR+/HER2- breast cancer, triple positive breast cancer, ER+/PR-/HER2- breast cancer, ER+/PR-/HER2+ breast cancer, ER positive breast cancer with or without expression of androgen receptor (AR), refractory breast cancer, breast cancers that have failed or are resistant to hormone therapies (e.g., estrogen receptor modulators, aromatase inhibitors, and/or selective estrogen receptor degraders). The ER+ breast cancer described herein is not TNBC. In some embodiments, the ER+ breast cancer comprises cancer cells with unstable genome.

[0083] In some embodiments, the HR+ breast cancer is refractory breast cancer that does not respond to treatment such as hormone drugs, kinase inhibitors or drugs that target HER2. Refractory breast cancer can also be referred to as “resistant cancer.” The HR+ breast cancer may be resistant at the beginning of the treatment (intrinsic resistance), or it may become resistant during treatment (acquired or induced resistance). In some embodiments, the HR+ breast cancer is resistant or does not respond to hormone therapies such as SERMs (e.g., tamoxifen, toremifene, raloxifene), aromatase inhibitors (e.g., anastrozole, letrozole, exemestane), or SERDs (e.g., fulvestrant). For example, the subject HR+ breast cancer can develop stable or progressive disease following hormone therapies. In some embodiments, the HR+ breast cancer has or developed resistance to fulvestrant, anastrozole, letrozole, exemestane, tamoxifen, toremifene, raloxifene, or a combination thereof. In some embodiments, the HR+ breast cancer has or developed resistance [0084] In some embodiments, the HR+ breast cancer is resistant or does not respond to kinase inhibitors. For example, the HR+ breast cancer is resistant or does not respond to cyclin- dependent kinase (CDK) inhibitors that inhibit a family of proline-binding serine/threonine protein kinases known as CDKs. In some embodiments, the CDK inhibitor comprises CDK4 and/or CDK6 inhibitor. The CDK4/6 complex acts as a checkpoint during the cell cycle transition from cell growth (Gl) to DNA synthesis (S) phase and its deregulation or overexpression induces abnormal cell proliferation and cancer development. Exemplary FDA-approved CDK inhibitors include, but are not limited to, palbociclib, ribociclib, and abemaciclib. In some embodiments, the HR+ breast cancer is resistant or does not respond to (e.g., develops stable or progressive disease following treatment) CDK4/6 inhibitors (e.g., palbociclib) alone or in combination with hormone drugs such as SERDs (e.g., fulvestrant) or aromatase inhibitors. The resistance can be acquired resistance or intrinsic resistance. In some embodiments, the HR+ breast cancer is resistant to palbociclib. In some embodiments, the HR+ breast cancer develops stable disease, progressive disease, or acquired resistance to palbociclib and fulvestrant.

[0085] The HR+ breast cancer and/or tumor can be a cancer and/or tumor having abnormal alterations to PLK1 gene or protein. It has been identified that polo-like kinase 1 (PLK1) is an important gene for growth and survival of breast cancer cells with unstable genome. For example, the abnormal alterations can include one or more PLK1 alterations and/or PLK1 aberrant activation such as copy number alteration (CNA), single-nucleotide variation (SNV), and gene rearrangement or fusions. Non-limiting exemplary cancer and/or tumor with PLK1 alterations include cancer with PLK1 gene or protein amplification, PLK1 gene or protein modification, PLK1 gene deletion, PLK1 gene or protein overexpression, elevated PLK1 gene or protein expression, and/or a combination thereof. In some embodiments, the cancer and/or tumor can be a PLK1 -amplified cancer in which PLK1 gene and/or protein is amplified, for example, as a result of gene duplication and/or aberrant gene transcriptional control. For example, the cancer with PLK1 amplification can be a cancer with higher PLK1 mRNA and/or protein levels as compared to healthy tissues. In heterogenous cancer types, the HR+ breast cancer/or tumor can include a subtype that has an abnormal high expression of PLK1 gene and/or protein. In some embodiments, the HR+ breast cancer and/or tumor with amplified PLK1 can be node-positive tumors, aggressive tumors and/or invasive tumors. In some embodiments, the HR+ breast cancer and/or tumor with amplified PLK1 can have a shorter disease-free survival as compared to cancer and/or tumor with normal levels of PLK1. In some embodiments, the HR+ breast cancer and/or tumor exhibits a high relapse and/or resistance to traditional and/or mono-therapies, such as hormone therapy, chemotherapy and/or radiotherapy. abnormal alterations to phospatidylinositol-3 kinase (PI3K) and/or PTEN genes or proteins. The PI3K pathway is a key regulatory hub for cell growth, survival, and metabolism. PI3K has four catalytic subunits encoded: pl 10a and pl lop encoded by PIK3CA and PIK3CB, respectively, and pl 105 and pl lOy encoded by PIK3CD and PIK3CG, respectively. Catalytic subunits pl 10a and pl iop are ubiquitously expressed, while pl 105 and pl lOy are restricted to immune lineages. Activation of PI3K is a frequent hallmark of cancer. The PI3K pathway is activated via alterations of the PI3K genes (e.g., PIK3CA) or inactivation of modulator(s) of PI3K (e.g., the phosphatase and tensin homolog (PTEN)). For example, the abnormal alterations can include one or more PI3K alterations and/or PI3K aberrant activation such as copy number alteration (CNA), singlenucleotide variation (SNV), and gene rearrangement or fusions. The pl 10a subunit of PIK3CA is frequently mutated and amplified (-30%) in a variety of cancers. Exemplary hotspot mutations include: E545K, E542K, and Hl 047. In some embodiments, the abnormal alteration can be a point mutation in PIK3CA. PTEN is a well-characterized tumor suppressor which is a negative modulator of the PI3K pathway. The loss of or gain in function of PTEN is also seen in several cancers including breast cancer. Loss of function or mutation in PTEN results in Increased PI3K signaling and tumorigenesis. It is also known that loss of PTEN can lead to clinical resistance to PI3K inhibitor (e.g., alpelisib) in breast cancer, in some embodiments, the cancer and/or tumor with PTEN alterations is a PTEN-inactivated cancer, resulting in resistant to PI3Ka inhibitors. Non-limiting exemplary cancer and/or tumor with PI3K alterations include cancer with PI3K gene or protein amplification, PI3K gene or protein modification, PI3K gene deletion, PI3K gene or protein overexpression, elevated PI3K gene or protein expression, and/or a combination thereof. In some embodiments, cancer and/or tumor with PTEN alterations include cancer with PTEN gene or protein amplification, PTEN gene or protein modification, PTEN gene deletion, PTEN gene or protein under-expression, decreased PTEN gene or protein expression, and/or a combination thereof. In some embodiments, the cancer and/or tumor can be a PIK3CA-mutated cancer in which PIK3CA gene and/or protein has a point mutation, resulting in aberrant regulation of PI3K activities. In heterogenous cancer types, the HR+ breast cancer/or tumor can include a subtype that has an abnormal high expression of PI3K genes and/or activities of PI3K protein. In some embodiments, the HR+ breast cancer and/or tumor with amplified PI3K can be node-positive tumors, aggressive tumors and/or invasive tumors. In some embodiments, the HR+ breast cancer and/or tumor with amplified PI3K can have a shorter disease-free survival as compared to cancer and/or tumor with normal activities/level of PI3K.

[0087] In some embodiments, the breast cancer is resistant to or does not respond effectively to (e.g., develops stable or progressive disease) mono-treatment with a single cell cycle inhibitors capable of inhibiting cell cycle progression from cell growth phase (Gl) to DNA replication phase (S). Gl/S cell cycle inhibitors can include CDK inhibitor (e.g., CDK4/6 inhibitor), kinase inhibitors (e.g., ATPase inhibitors), Ca²⁺ channel inhibitors, GSK3P inhibitors, compounds capable of inhibiting ribonucleotide reductase activities, and others as will be understood by a person skilled in the art and described herein in the present disclosure. In some embodiments, a Gl/S cell cycle inhibitor is a CDK inhibitor (e.g., palbociclib). G2/M cell cycle inhibitors refer to cell cycle inhibitors capable of inhibiting cell cycle progression through the G2 in which the cell grows and prepares to divide and mitosis (M) phases. Exemplary G2/M cell cycle inhibitors include, but are not limited to, CDK inhibitors (e.g., CDK1 inhibitor), PLK1 inhibitors, mitotic inhibitors that inhibit mitosis or cell division, and DNA intercalating agents such as DNA topoisomerase II inhibitors. In some embodiments, a G2/M cell cycle inhibitor is a PLK1 inhibitor (e.g., onvansertib).

[0088] In some embodiments, administering to the subject having an HR+ breast cancer that is resistant to or does not respond effectively to a single cell cycle inhibitor with another cell cycle inhibitor, such as a cell cycle inhibitor capable of arresting cells in a same or different cell cycle (e.g., a Gl/S or G2/M cell cycle), can unexpectedly enhance the therapeutic effect in treating the HR+ breast cancer. When combined, surprisingly, the two cell cycle inhibitors (e.g., a Gl/S cell cycle inhibitor and a G2/M cell cycle inhibitor or two G2/M cell cycle inhibitors) can obtain complete cell inhibition and resulting tumor regression and cancer survival rate/duration by the combination can be surprisingly synergistic (z.e., more than additive, superior to the cumulated anti-tumor efficacy caused by the two cell cycle inhibitors separately).

Cell cycle inhibitors

[0089] Methods, compositions and kits disclosed herein can be used for treating breast cancer and/or breast tumor (e.g., HR+ breast cancer). In some embodiments, a method for treating HR+ breast cancer comprises administrating a PI3K inhibitor and a cell cycle inhibitor to a subject with HR+ breast cancer. The cell cycle inhibitor can inhibit or damage cell cycle events and arrest cell in a phase of the cell cycle. In some embodiments, a method for treating HR+ breast cancer comprises administering a PI3K inhibitor (e.g., a PI3Ka inhibitor), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, and a G2/M cell cycle inhibitor (e.g., a PLK1 inhibitor), or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, and to a subject with HR+ breast cancer. In some embodiments, a method for treating HR+ breast cancer comprises administering a PI3K inhibitor, or a pharmaceutically acceptable salt, solvate, stereoisomer thereof, and a G2/M cell cycle inhibitor, or a pharmaceutically acceptable salt, [0090] Cell cycle inhibitors such as G2/M cell cycle inhibitor can inhibit or damage cell cycle events. Cell cycle comprises a set of coordinated events that culminate in the formation of two cells from one mother cell. A cell cycle is composed of four major phases: G1 (growth phase 1), S (DNA synthesis phase), G2 (growth phase 2), and M (mitosis), which function to integrate environment sensing signaling pathways with cell growth and proliferation. Cancer cells often deregulate the cell cycle and undergo unscheduled cell divisions, therefore inhibition of the cell cycle represents an opportunity for therapeutic intervention in treating proliferative diseases such as cancer. The cell cycle inhibitors described herein can perturb the proliferation cycle of tumor cells by inhibiting/damaging cell cycle events, activate checkpoints, arrest cells and induce apoptosis of cancer cells. The cell cycle inhibitors described herein can target different cell cycle events and arrest cells in different cell cycles. For example, some inhibitors can target DNA replication (e.g., 5 -fluorouracil). In some embodiments, the cell cycle inhibitors described here are Gl/S cell cycle inhibitors. Gl/S cell cycle inhibitors refer to a range of cell cycle inhibitors that can inhibit or damage cell cycle events and arrest cells in G1 -phase or S-phase of a cell cycle. In some embodiments, the cell cycle inhibitors described herein are G2/M cell cycle inhibitors. G2/M cell cycle inhibitors refer to a range of cell cycle inhibitors that can inhibit or damage cell cycle events and arrest cells in G2-phase or M-phase of a cell cycle. Methods of identifying cell cycle specific inhibitors are known to a person skilled in the art. For example, a cancer cell-based high-throughput chemical screening method for cell cycle modulators is described in Senese et al., “Chemical dissection of the cell cycle: probes for cell biology and anti-cancer drug development” Cell Death & Disease volume 5, page el462 (2014), the content of which is incorporated herein by reference in its entirety. Senese identified Gl, S, G2, and M-phase specific inhibitors with drug-like properties and diverse chemotypes likely targeting a broad array of processes in a cell cycle.

[0091] Exemplary Gl cell cycle inhibitors include, but are not limited to, CDK inhibitors (e.g., CDK4/6 inhibitors), kinase inhibitors (e.g., ATPase inhibitor) such as Staurosporine, Tyrphostin, and their analogs that mimic the ATP substrate of PKC and EGFR, which are known to block the MAPK signaling pathway for tumor proliferation, PI3K inhibitor, which are known to block the PI3K/AKT/mT0R Pathway and compounds capable of modulating the intracellular calcium concentration including the ion channel inhibitors Thapsigargin (scarco- endoplasmic reticulum Ca²⁺ ATPase inhibitor), Ouabain (Na⁺/K⁺ ATPase inhibitor). Exemplary S cell cycle inhibitors include, but are not limited to, compounds capable of inhibiting ribonucleotide reductase activities and GSK3P inhibitors capable of regulating cyclin DI expression required for S-phase entry and progression. Exemplary G2 cell cycle inhibitors include, including Etoposide and Amsacrine-like analogs.

[0092] In some embodiments, the Gl/S cell cycle inhibitors described herein comprise CDK inhibitors, such as CDK4/6 inhibitors. The CDK4/6 inhibitors act at the Gl-to-S cell cycle checkpoint and prevent progression through this checkpoint, leading to cell cycle arrest. Exemplary CDK4/6 inhibitors include, for example, carboxamide-based analogues, pyrimidine- based analogues, quinazoline-based analogs, acridone-based analogs, indole conjugates analogues, and others identifiable to a person skilled in the art. Exemplar FDA-approved CDK inhibitors include, but are not limited to, Palbociclib, Riboci clib, and Abemaciclib.

[0093] In some embodiments, a CDK inhibitor can be a G2/M cell cycle inhibitor. For example, CDK1 inhibitor plays a role in cell cycle progression through the G2/M phase transition and activation of homologous recombination DNA repair pathway. Various CDK1 inhibitors have been developed for cancer therapy that induce prolonged G2 arrest and/or sensitize cells to DNA damaging agents in tumor cells, resulting in cell death.

[0094] In some embodiments, the Gl/S cell cycle inhibitors used in the methods, compositions and kits described herein are PI3K inhibitors (e.g., PI3Ka inhibitors). PI3K inhibitors arrest cycle through the PI3K/protein kinase B (AKT) pathway, by inhibiting the activities of PI3K. PI3K is an enzyme involved in lipid synthesis and is composed of pl 10 catalytic, p55 regulatory subunit and p85 regulatory subunits. PI3K can be divided into 3 classes: classes I, II, and III. Class I PI3Ks comprised of class IA and class IB PI3Ks. Class IA PI3K is a heterodimer of p58 regulatory subunit and pl 10 catalytic subunit. Class IA PI3K contains pl 10a, pl iop and pl 105 catalytic subunits produced from different genes PIK3CA, PIK3CB and PIK3CD, respectively. Subunit pl lOy produced by PIK3CG represents the catalytic subunit in class IB PI3K. PI3K inhibitors can inhibit one or more pl 10 isoforms of the class I PI3Ks. In some embodiments, the PI3K inhibitor described herein is a PI3Ka inhibitor, a PI3KP inhibitor, a PI3K5 inhibitor, or a PI3Ky inhibitor. In some embodiments, the PI3K inhibitor described herein is a Pan-PI3K inhibitor, an isoform-specific inhibitor, or a dual PI3K inhibitor. Examples of PI3K inhibitors include, but not limited to, fimepinostat, rigosertib, buparlisib, CH5132799, pilaralisib, ZSTK474, sonolisib, pictilisib, copanlisib, B591, TG-100-115, RIDR-PI- 103, dactolisib, apitolisib, gedatolisib, SF1126, omipalisib, samotolisib, bimiralisib, paxalisib, voxtalisib, GSK1059615, MEN1611, ZSTK474. The PI3K inhibitors can be isoform-specific inhibitors such as a PI3Ka inhibitor (e.g., inavolisib, alpelisib, AZD8835, PWT33597, taselisib, and/or serabelisib), a PI3KP inhibitor (e.g., AZD8186 and/or GSK2636771), a PI3K5 inhibitor (e.g., AZD8835, AZD8186, nemiralisib, seletalisib, acalisib, CAL263, TG100-115, duvelisib, idelalisib, tenalisib, taselisib, zandelisib, AMG319, linperlisib, parsaclisib, umbralisib, and/or [0095] The PI3K/AKT pathway may impact cell proliferationby through a downstream effector, mammalian target of rapamycin complex 1 (mTORCl), one type of complex formed by mammalian target of rapamycin (mTOR). Overexpression of mTOR is commonly observed in various types of cancers. Inhibition of mTOR is known to induce cell cycle arrest in the G1 phase, as downstream effectors of mTOR are required for G1 phase progression. The arresting of cell cycle in G1 phase may be achieved through the transcriptional regulation of G1 cyclins (D- and E-type cyclins) or the cytoplasmic sequestration of cyclin-dependent kinase inhibitor 1 (p21CIPl/WAFl) and cyclin-dependent kinase inhibitor IB (p27Kipl). Without being bound to any particular theory, several PI3K inhibitors function to inhibit the activation of the PI3K/AKT/mTOR signaling pathway. For example, alpelisib induces G0/G1 phase arrest through mTORCl inhibition by repressing the activity of PI3K catalytic subunit pl 10a, therefore, demonstrates effects against breast cancer.

[0096] In some embodiments, the PI3K inhibitor is alpelisib, an alpelisib derivative or analog (e.g., docetaxel), or a pharmaceutically acceptable salt thereof. Alpelisib, also known as BYL719, (S)~ pyrrolidine- 1,2-dicarboxylic acid 2-amide l-({4-methyl-5-[2-(2,2,2-trifhroro-l,l- dimethyl-ethyl)- pyridin-4-yl]-thiazol-2-yl}-amide) or (5)-Nl-(4-methyl-5-(2-(l,l,l-trifluoro-2- methylpropan-2-yl)pyridin-4-yl)thiazol-2-yl)pyrrolidine-l,2-dicarboxamide, has the empirical formula C19H22F3N5O2S and a molecular weight of 441.5. The structure and methods of preparing alpelisib are described in PCT Patent Application No. WO2010/029082, which is hereby incorporated by reference in its entirety.

(Alpelisib)

[0097] Alpelisib can be obtained via a synthetic process from imidazole- 1 -carboxylic acid [5-(2-te/f-butyl-pyridin-4-yl)-4-methyl-thiazol-2-yl]-amide. In some embodiments, alpelisib is administered by intravenous injection. In some embodiments, alpelisib is administered orally. In some embodiments, alpelisib is combined with additives.

[0098] Alpelisib can be formulated as a tablet or granular formulation. In some embodiments, in the granular formulation, alpelisib is formulated as free flowing granules in an As free flowing granules, alpelisib can free flow in dray state during dosing, though for administration to a patient it may be administered to a fluid.

[0099] In some embodiments, alpelisib is administered orally. For example, in a dose escalation phase, a subject (e.g., a subject with cancer with PIK3CA alteration) can be orally administered with alpelisib either (a) at a dosage ranging from 30 mg to 450 mg once per day (q.d.) on a continuous daily schedule for 28-days, or (b) at a dosage ranging from 120 mg to 200 mg twice per day (b.i.d.) on a continuous daily schedule for 28- days, as guided by Bayesian logistic regression model with overdose control. In some embodiments, the subject has PIK3CA wildtype HR+/HER2- breast cancer. In some embodiments, alpelisib is administered to the subject at > 270 mg/day, including e.g., subject suffering from breast cancer, colorectal cancer, endometrial cancer and/or cervical cancer.

[0100] The G2/M cell cycle inhibitor used in the methods, compositions and kits described herein can be a polo-like kinases (PLK) inhibitor. Polo-like kinases (PLK) are a family of five highly conserved serine/threonine protein kinases. PLK1 is a master regulator of mitosis and is involved in several steps of the cell cycle, including mitosis entry, centrosome maturation, bipolar spindle formation, chromosome separation, and cytokinesis. It is also critical for the entry and progression through mitosis, regulates progression of cells through the G2 phase of the cell cycle by phosphorylating forkhead box protein Ml (F0XM1), which then regulates the expression of cyclins and other genes necessary for cells to progress through the cell cycle. PLK1 has been shown to be overexpressed in solid tumors and hematologic malignancies, including breast cancers. Overall survival of breast cancer patients with high PLK1 expression is lower than breast cancer patients with low PLK1 expression.

[0101] In a randomized phase II study of patients with AML who were treatment naive yet unsuitable for induction therapy, the pan-PLK inhibitor, volasertib (BI6727), administered intravenously in combination with LDAC showed a significant increase in OS when compared with LDAC alone. A subsequent randomized phase III study identified no benefit of the combination and described an increased risk of severe infections. PLK1 facilitates HR during Double Strand DNA Break (DSB) Repair. PLK1 phosphorylates Rad51 and BRCA1, facilitating their recruitment to DSB sites and thereby HR-mediated DNA repair.

[0102] Onvansertib (also known as PCM-075, NMS-1286937, NMS-937, “compound of formula (I)” in U.S. Patent No. 8,927,530; IUPAC name l-(2-hydroxyethyl)-8-{[5-(4- methylpiperazin-l-yl)-2-(trifluoromethoxy) phenyl] amino}-4,5-dihydro-lH-pyrazolo[4,3-h] quinazoline-3 -carboxamide), or a pharmaceutically acceptable salt, is a selective ATP- competitive PLK1 inhibitor. Onvansertib can be formulated, for example, with an additive such embodiments, the onvansertib is formulated for oral administration, such as in a hard gelatin capsule

[0103] Biochemical assays demonstrated high specificity of onvansertib for PLK1 among a panel of 296 kinases, including other PLK members. Onvansertib has potent in vitro and in vivo antitumor activity in models of both solid and hematologic malignancies. Onvansertib is the first PLK1 specific ATP competitive inhibitor administered by oral route to enter clinical trials with proven antitumor activity in different preclinical models. Onvansertib has shown a promising safety profile in a phase 1 clinical trial as single agent. In addition, clinical investigations of onvansertib includes onvansertib in combination with abiraterone and prednisone in adult patients with metastatic castration-resistant prostate cancer, onvansertib in combination with FOLFIRI and bevacizumab in adult patients with KRAS-mutated metastatic colorectal cancer, and onvansertib in combination with nanoliposomal irinotecan and 5-FU in patients with metastatic pancreatic cancer. As described herein, in presence of genomic instability, onvansertib can synergize with alpelisib, and thereafter achieve good anti-tumor activity at a lower dose compared to single agent, and without drug specific toxicity.

[0104] Onvansertib also inhibited cell proliferation at nanomolar concentrations in AML cell lines and tumor growth in xenograft models of AML. In addition, onvansertib significantly increased cytarabine antitumor activity in disseminated models of AML.

Onvansertib

[0105] Onvansertib shows high potency in proliferation assays having low nanomolar activity on a large number of cell lines, both from solid as well as hematologic tumors. Onvansertib has a relative short half-life of 24 h and is highly potent against the PLK1 enzyme ([IC50] = 2 nM). In comparison, low or no activity was observed on a panel of 63 kinases (ICso > 500 nM), including the PLK members PLK2 and PLK3 (ICso > 10 pM). Onvansertib potently causes a mitotic cell-cycle arrest followed by apoptosis in cancer cell lines and inhibits xenograft oral administration. In addition, onvansertib shows activity in combination therapy with approved cytotoxic drugs, such as irinotecan, in which there is enhanced tumor regression in HT29 human colon adenocarcinoma xenografts compared to each agent alone, and shows prolonged survival of animals in a disseminated model of AML in combination therapy with cytarabine. Onvansertib has favorable pharmacologic parameters and good oral bioavailability in rodent and nonrodent species, as well as proven antitumor activity in different nonclinical models using a variety of dosing regimens, which may potentially provide a high degree of flexibility in dosing schedules, warranting investigation in clinical settings. Onvansertib has several advantages over volasertib (BI6727, another PLK1 inhibitor), including a higher degree of potency and specificity for the PLK1 isozyme, and oral bioavailability. In addition, onvansertib has proven antitumor activity in different nonclinical models using a variety of dosing regimens, which can provide flexibility in dosing schedules, and therefore, warrants investigation in clinical settings.

[0106] A phase I, first-in-human, dose-escalation study of onvansertib in patients with advanced/metastatic solid tumors identified neutropenia and thrombocytopenia as the primary dose-limiting toxicities. These hematologic toxicities were anticipated on the basis of the mechanism of action of the drug and were reversible, with recovery occurring within 3 weeks. The half-life of onvansertib was established between 20 and 30 hours. The oral bioavailability of onvansertib plus its short half-life provide the opportunity for convenient, controlled, and flexible dosing schedules with the potential to minimize toxicities and improve the therapeutic window. Pharmacodynamics and biomarker studies, including baseline genomic profiling, serial monitoring of mutant allele fractions in plasma, and the extent of PLK1 inhibition in circulating blasts, have been performed to identify biomarkers associated with clinical response and are described in PCT Publication No. WO2021/146322, the content of which is incorporated herein by reference in its entirety.

[0107] The major metabolic pathways found in the different animal species were N- oxidation of the N methyl-piperazine ring to give N-oxide M2 and hydroxylation on an aliphatic carbon atom of the methylene bridge of the pyrazoloquinazoline moiety to give metabolite Ml. Qualitatively, no marked differences in the metabolism of onvansertib were observed between species and, quantitatively, some differences were observed cross-species.

[0108] The potential inhibitory capacity of onvansertib towards the major human cytochrome P450 (CYP) isoforms that are responsible for hepatic drug metabolism in man (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) was investigated using human liver microsomes. Onvansertib was able to inhibit the metabolic activities of CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 isoforms to different extents, with 50% inhibitory against CYP1 A2 were detected. Considering that the concentrations relevant to achieve significant anti-tumoral activity of the compound in mice were in the order of 1 pM, the likelihood that onvansertib would show clinically relevant metabolic drug-drug interactions is considered low.

[0109] To date, a single Phase 1 safety study with onvansertib has been completed in adult patients with advanced/metastatic solid tumors at a single study site in the U.S. First cycle dose-limiting toxi cities (DLTs) and the maximum tolerated dose (MTD) of onvansertib administered orally for 5 consecutive days every 3 weeks (z.e., a 21-day treatment cycle) was conducted. Safety profile of onvansertib, to determine the pharmacokinetics (PK) of onvansertib in plasma (at the MTD), and to document any antitumor activity has been determined. In one study, a total of 21 patients were enrolled, and 19 patients were treated. No DLTs occurred at the first 3 dose levels (doses of 6, 12, and 24 mg/m²/day). At the subsequent dose level (dose of 48 mg/m²/day), 2 of 3 patients developed DLTs. An intermediate dose level of 36 mg/m²/day was investigated. At the intermediate dose level, 4 patients were treated and 2 DLTs were observed. After further cohort expansion, the MTD was determined to be 24 mg/m²/day. The best observed treatment response was stable disease (SD); SD occurred in 5 of the 16 evaluable patients. The study identified thrombocytopenia and neutropenia as the primary toxicities; this is consistent with the expected mechanism of action of onvansertib and with results from the preclinical studies. These hematologic toxicities were reversible, with recovery usually occurring within 3 weeks. No other clinically relevant safety findings emerged with treatment with onvansertib as a single agent. Other mechanism-related, possibly expected events such as gastrointestinal disorders, mucositis, and alopecia were not observed, confirming that with this schedule, the bone marrow is the most sensitive target of onvansertib in humans.

Treating HR+ breast cancer with cell cycle inhibitors

[0110] Provided herein include methods, compositions and kits for treating cancer in a subject, for example, a human patient suffering from HR+ breast cancer. The method comprises administrating a PI3K inhibitor and a G2/M cell cycle inhibitor to a subject with the HR+ breast cancer, thereby inhibiting or reducing progression of the HR+ breast cancer in the subject. The PI3K inhibitor and the G2/M cell cycle inhibitor can be administered to the subject with the HR+ breast cancer in a manner sufficient to inhibit or reduce progression of the cancer. For example, the PI3K inhibitor and the G2/M cell cycle inhibitor can be administrated to a subject with cancer simultaneously, separately, or sequentially. The PI3K inhibitor and the G2/M cell cycle inhibitors can be administered in any suitable order. For example, the PI3K inhibitor can be administered followed by the G2/M cell cycle. Alternatively or in combination, the G2/M cell cycle inhibitor using the PI3K inhibitor and the G2/M cell cycle inhibitor can result in significantly enhanced efficacy against HR+ breast cancer, causing tumor regression and cancer survival. The resulted tumor regression and cancer survival rate/duration by the combination can be surprisingly synergistic (z.e., more than additive, superior to the cumulated anti -turn or efficacy caused by the PI3K inhibitor and the G2/M cell cycle inhibitor separately). In an exemplary embodiment, as described herein, onvansertib in combination of alpelisib showed synergy in HR+ breast cancer models that are resistant to hormone therapy treatments and a Gl/S or G2/M cell cycle inhibitor treatment alone. Without being bound to any particular theory, it is believed that the G2/M cell cycle inhibitor can increase the responsiveness of the cancer cells that may have escaped from the PI3K inhibitor treatment. It is also believed that in some instances the G2/M cell cycle inhibitor can sensitize cancer cells to the PI3K inhibitor to achieve enhanced effective cancer treatment. In some embodiments, the PI3K inhibitor and the G2/M cell cycle inhibitor can arrest cancer cells by inhibiting different cell cycle events in a same cell cycle or different cell cycles of a cell cycle, thus resulting in enhanced tumor suppressing activity and more effective cancer treatment.

[OHl] In some embodiments, the inhibition or reduction of cancer progression is not merely additive, but is enhanced or synergistic (that is, the inhibition is greater than the combined inhibition of progression caused by the PI3K inhibitor and the G2/M cell cycle inhibitor alone). The enhanced or synergistic efficacy or inhibition of any combination of the PI3K inhibitor and the G2/M cell cycle inhibitor of the present disclosure can be different in different embodiments. In some embodiments, the enhanced or synergistic efficacy or inhibition of any combination of a PI3K inhibitor and a G2/M cell cycle inhibitor of the present disclosure is, is about, is at least, is at least about, is at most, or is at most about, 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 a range between any two of these values, higher than the combined inhibition of progression caused by the PI3K inhibitor and the G2/M cell cycle inhibitor alone.

[0112] The molar ratio of the G2/M cell cycle inhibitor (e.g., onvansertib) to the PI3K inhibitor (e.g., alpelisib) can be, for example, 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, or 2000: 1, or a number or a range between any two of these values. In some embodiments, the enhanced or synergistic efficacy or inhibition of cancer progression caused by a combination of the PI3K inhibitor (e.g., alpelisib) to the G2/M cell cycle inhibitor (e.g., onvansertib) is, is about, is at least, is at least about, is at most, or is at most about 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 a range the PI3K inhibitor (e.g., alpelisib) alone plus the G2/M cell cycle inhibitor (e.g., onvansertib) alone. For example, a combination of the PI3K inhibitor (e.g., alpelisib) and the G2/M cell cycle inhibitor (e.g., onvansertib) can cause a 50%, 60%, 70%, 80%, 90%, or more, inhibition of cancer progression (cancer cell viability of 50%, 40%, 30%, 20%, 10%, or less), whereas under the same conditions the combined inhibition of the PI3K inhibitor (e.g., alpelisib) alone plus the G2/M cell cycle inhibitor (e.g., onvansertib) alone can cause a 10%, 20%, 25%, 30%, or less inhibition of cancer progression (cancer cell viability of 90%, 80%, 75%, 70%, or more). Thus, the enhanced or synergistic efficacy or inhibition of cancer progression caused by the combination of the PI3K inhibitor (e.g., alpelisib) and the G2/M cell cycle inhibitor (e.g., onvansertib) is, for example, 50%, 60%, 70%, 80%, 90%, 100%, or more higher than the combined inhibition of progression caused by the PI3K inhibitor (e.g., alpelisib) alone plus the G2/M cell cycle inhibitor (e.g., onvansertib) alone. In some embodiments, the PI3K inhibitor is alpelisib and the G2/M cell cycle inhibitor is onvansertib.

[0113] As described herein, the patient can achieve complete response or partial response after treatment with the PI3K inhibitor and the G2/M cell cycle inhibitor. In some embodiments, the patient achieves a complete response. In some embodiments, the patient achieves a partial response. In some embodiments, the patient did not respond to or developed stable or progressive disease following treatment with the PI3K inhibitor (without the G2/M cell cycle inhibitor).

[0114] The PI3K inhibitor and the G2/M cell cycle inhibitor can be administered to the patient in any manner deemed effective to treat the cancer. The PI3K inhibitor can be administered together with, or separately from, the G2/M cell cycle inhibitor. When administered separately, the PI3K inhibitor can be administered before or after the G2/M cell cycle inhibitor, or in different administration cycles.

[0115] The PI3K inhibitor and the G2/M cell cycle inhibitor can each be administered in any schedule, e.g., once or multiple times per day or week; once, twice, three times, four times, five times, six times or seven times (daily) per week; for one or multiple weeks etc. In some embodiments, the G2/M cell cycle inhibitor (e.g., onvansertib) is, or is only, administered to a patient daily for 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 consecutive days during a cycle, for example, on the first 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 consecutive days of the cycle; and the PI3K inhibitor (e.g., alpelisib) is, or is only, administered to the patient once in each of the weeks that onvansertib is administered. The cycle can be, for example, 21-28 days in length. In some embodiments, the G2/M cell cycle inhibitor is administered to a patient daily for the first 21 consecutive days during a 28-day cycle, and the cycle. In some embodiments, no G2/M cell cycle inhibitor nor PI3K inhibitor is administered to the patient in the last 7 days of the 28-day cycle. The patient can undergo one or more cycles of treatment/administration, for example at least two cycles of treatment/administration. The administration schedule of the PI3K inhibitor and the G2/M cell cycle inhibitor can be the same or different in each of the cycles of treatment/administration.

[0116] In some embodiments, the PI3K inhibitor and/or the G2/M cell cycle inhibitor is administered in a cycle of 4-50 days of administration. In some embodiments, the PI3K inhibitor and/or the G2/M cell cycle inhibitor is administered in a cycle of 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, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, or 50 days. In some embodiments, the PI3K inhibitor and/or the G2/M cell cycle inhibitor is administered in 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, 31 days, 32 days, 33 days, 34 days, 35 days,

36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days,

47 days, 48 days, 49 days, or 50 days of a cycle. In some embodiments, the PI3K inhibitor and/or the G2/M cell cycle inhibitor is administered in 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, and/or day 50. In some embodiments, the PI3K inhibitor and/or the G2/M cell cycle inhibitor is not administered in 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, and/or day 50. For example, alpelisib and/or onvansertib can be administered in a cycle of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 days.

[0117] The PI3K inhibitor (e.g., the PI3Ka inhibitor) can be administrated to the patient once weekly or twice weekly. In some embodiments, the PI3K inhibitor (e.g., alpelisib) is administrated weekly on each week or on selected weeks of the administration cycle. In some three weeks (e.g., on Day 1, 8 and 15) and no administration for the remaining days of the cycle, including Days 16-28. In some embodiments, alpelisib is administered in a cycle of 18 days with a weekly administration for three weeks (e.g., on Day 1, 8 and 15) and no administration for the remaining days of the cycle. In some embodiments, alpelisib is administered in a cycle of 32 days with a weekly administration for five weeks (e.g., on Day 1, 8, 15, 22, and 29) and no administration for the remaining days of the cycle. In some embodiments, alpelisib is administered in a cycle of 39 days with a weekly administration for six weeks (e.g., on Day 1, 8, 15, 22, 29, and 36) and no administration for the remaining days of the cycle. In some embodiments, there is no administration of alpelisib in one or more weeks of a cycle.

[0118] The PI3K inhibitor such as the PI3Ka inhibitor can be administered to the patient at any appropriate dosage in different embodiments. In some embodiments, the PI3K inhibitor (e.g., the PI3Ka inhibitor) can be administered to the patient at a dosage of about, at least or at most 5 mg/m² 10 mg/m², 15 mg/m², 20 mg/m², 25 mg/m², 30 mg/m², 35 mg/m², 40 mg/m², 45 mg/m², 50 mg/m², 55 mg/m², 60 mg/m², 65 mg/m², 70 mg/m², 75 mg/m², 80 mg/m², 85 mg/m², 90 mg/m², 95 mg/m², 100 mg/m², 105 mg/m², 110 mg/m², 115 mg/m², 120 mg/m², 125 mg/m², 130 mg/m², 135 mg/m², 140 mg/m², 145 mg/m², 150 mg/m², 155 mg/m², 160 mg/m², 165 mg/m²,

170 mg/m², 175 mg/m², 180 mg/m², 185 mg/m², 190 mg/m², 195 mg/m², 200 mg/m², 205 mg/m²,

210 mg/m², 215 mg/m², 220 mg/m², 225 mg/m², 230 mg/m², 235 mg/m², 240 mg/m², 245 mg/m²,

250 mg/m², 255 mg/m², 260 mg/m², 265 mg/m², 270 mg/m², 275 mg/m², 280 mg/m², 285 mg/m², or a number between any two of these values. The dosage unit based on the body surface area (e.g., mg/m²) can be converted to the dosage unit based on body weight (mg/kg) using a conversion chart such as the body surface area (BSA) conversion chart as will be understood by a person of skill in the art. In some embodiments, the PI3Ka inhibitor is alpelisib. Alpelisib can be administered at a dosage of about, at least or at most 38 mg/m², 39 mg/m², 40 mg/m², 41 mg/m², 42 mg/m², 43 mg/m² 44 mg/m², 45 mg/m², 46 mg/m², 47 mg/m², 48 mg/m², 49 mg/m², 50 mg/m², 51 mg/m² 52 mg/m², 53 mg/m² 54 mg/m², 55 mg/m² 56 mg/m², 57 mg/m² 58 mg/m², 59 mg/m² 60 mg/m², 61 mg/m², 62 mg/m², 63 mg/m², 64 mg/m², 65 mg/m², 66 mg/m², 67 mg/m², 68 mg/m², 69 mg/m², 70 mg/m², 71 mg/m² 72 mg/m², 73 mg/m², 74 mg/m², 75 mg/m², 76 mg/m², 77 mg/m², 78 mg/m², 79 mg/m², 80 mg/m², 81 mg/m², 82 mg/m², 83 mg/m², 84 mg/m², 88 mg/m², 86 mg/m², 87 mg/m², 88 mg/m², 89 mg/m² 90 mg/m², or a number between any two of these values. In some embodiments, alpelisib is administered at a dosage of about, at least or at most 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, or a number between any two of these values. In some embodiments, alpelisib is administered at a dosage from about 10 mg/kg of body weight to about 30 mg/kg of body weight, optionally at a dose from about [0119] Similarly, the G2/M cell cycle inhibitor such as the PLK1 inhibitor can be administered to the patient at any appropriate dosage in different embodiments. In some embodiments, the G2/M cell cycle inhibitor (e.g., PLK1 inhibitor) can be administered to the patient at a dosage of about, at least or at most 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 105 mg/kg, 110 mg/kg, 115 mg/kg, 125 mg/kg, 130 mg/kg, 135 mg/kg, 140 mg/kg, 145 mg/kg, 150 mg/kg, 155 mg/kg, 160 mg/kg, 165 mg/kg, 170 mg/kg, 175 mg/kg, 180 mg/kg, 185 mg/kg, 190 mg/kg, 195 mg/kg, 200 mg/kg or a number between any two of these values.

[0120] In some embodiments, the PLK1 inhibitor is onvansertib. Onvansertib can be administered to the patient at any appropriate dosage, e.g., a dosage of less than 12 mg/m², less than or equal to 24 mg/m², or greater than 24 mg/m². In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered at a dosage from about 10 mg/kg of body weight to about 80 mg/kg of body weight, optionally at a dose from about 20 mg/kg of body weight to about 60 mg/kg of body weight, optionally at a dose from about 30 mg/kg of body weight to about 50 mg/kg of body weight. In some embodiments, onvansertib is administered to the patient daily. In some embodiments, onvansertib is administered in a cycle of 5-14 days of daily onvansertib administration with 2-16 days with no onvansertib administration. For example, in some embodiments, onvansertib is administered daily for 21 consecutive days followed by no onvansertib administration for 7 days in a cycle. In some embodiments, onvansertib is administered for 5 consecutive days a week followed by no onvansertib administration for 2 days each week or on selected weeks of an administration cycle.

[0121] In some embodiments, the combination treatment with the PI3K inhibitor and the G2/M cell cycle inhibitor is administered in the same dose as single treatment with the PI3K inhibitor and the G2/M cell cycle inhibitor. In some embodiments, the combination treatment with the PI3K inhibitor and the G2/M cell cycle inhibitor is administered with the PI3K inhibitor and the G2/M cell cycle inhibitor separately.

[0122] As can be appreciated by one of skill in the art, the amount of co-admini strati on of the PI3K inhibitor and the G2/M cell cycle inhibitor, and the timing of co-administration, can depend on the type (species, gender, age, weight, etc.) and condition of the subject being treated and the severity of the disease or condition being treated. The PI3K inhibitor and the G2/M cell cycle inhibitor can be formulated into a single pharmaceutical composition, or two separate pharmaceutical compositions. The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interracial polymerization, for example, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.

[0123] The PI3K inhibitor and the G2/M cell cycle inhibitor can be administered by any suitable routes, including but not limited to oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal, epidural, and intranasal administration. Parenteral administration (e.g., injection) can include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the PI3K inhibitor (e.g., alpelisib) can be, for example, administered by intravenous infusion (e.g., over about 30 minutes) and the G2/MS cell cycle inhibitor (e.g., onvansertib) can be, for example, administered orally.

[0124] Methods, compositions, kits and systems disclosed herein can be applied to different types of subjects. For example, the subject can be a subject receiving a cancer treatment, a subject at cancer remission, a subject has received one or more cancer treatment, or a subject suspected of having cancer. The subject can have a stage I cancer, a stage II cancer, a stage III cancer, and/or a stage IV cancer. The methods can further comprise administering an additional therapeutic intervention to the subject. The additional therapeutic intervention can comprise a different therapeutic intervention than administering the PI3K inhibitor and the G2/M cell cycle inhibitor, such as an antibody, an adoptive T cell therapy, a chimeric antigen receptor (CAR) T cell therapy, an antibody-drug conjugate, a cytokine therapy, a cancer vaccine, a checkpoint inhibitor, a radiation therapy, surgery, a chemotherapeutic agent, or any combination thereof. The therapeutic intervention can be administered at any time of the treatment, for example at a time when the subject has an early-stage cancer, and wherein the therapeutic intervention is more effective that if the therapeutic intervention were to be administered to the subject at a later time.

Dosing and Pharmacokinetics

[0125] The treatment described in the present disclosure can comprise administration of a PI3K inhibitor (e.g., alpelisib) and a G2/M cell cycle inhibitor (e.g., onvansertib) for a desired duration in one or more cycles of treatment.

[0126] Daily or weekly administration of a PI3K inhibitor such as a PI3Ka inhibitor (e.g., intravenous administration) can be at, or be about, 0.01 mg, 0.05 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, 1 mg, 5mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, or a number or a range between any two of these values. The daily or weekly dose of the PI3K inhibitor can daily or weekly administration of the PI3K inhibitor can be at different amounts on different days or during different weeks. For example, the treatment can comprise daily or weekly administration of the PI3K inhibitor at 0.1 mg to 20 mg during week 1, 0.25 mg to 50 mg during week 2, 0.5 mg to 100 mg during week 3, 1 mg to 200 mg during week 4, and 2 mg to 400 mg during week 5 and beyond. For example, the treatment can comprise daily or weekly administration of the PI3K inhibitor at 0.1 mg to 100 mg on day 1, 0.2 mg to 200 mg on day 2, 0.4 mg to 400 mg on day 3, and 0.4 mg to 400 mg or 0.6 mg to 600 mg on day 4 and beyond. For example, the PI3K inhibitor is alpelisib and is administered at a daily or weekly dose of about 0.01 mg, 0.05 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, or a number or a range between any two of these values.

[0127] In some embodiments, the PI3K inhibitor can be administered daily or weekly at a drug/body surface area unit dose of about 15 mg/m² to about 275 mg/m². For example, the PI3K inhibitor is a PI3Ka inhibitor, and the PI3Ka inhibitor (e.g., alpelisib) can be administered at, or at about 5 mg/m², 10 mg/m², 15 mg/m², 20 mg/m², 25 mg/m², 30 mg/m², 35 mg/m², 40 mg/m², 45 mg/m², 50 mg/m², 55 mg/m², 60 mg/m², 65 mg/m², 70 mg/m², 75 mg/m², 80 mg/m², 85 mg/m², 90 mg/m², 95 mg/m², 100 mg/m², 105 mg/m², 110 mg/m², 115 mg/m², 120 mg/m², 125 mg/m², 130 mg/m², 135 mg/m², 140 mg/m², 145 mg/m², 150 mg/m², 155 mg/m², 160 mg/m², 165 mg/m², 170 mg/m², 175 mg/m², 180 mg/m², 185 mg/m², 190 mg/m², 195 mg/m², 200 mg/m², 205 mg/m², 210 mg/m², 215 mg/m², 220 mg/m², 225 mg/m², 230 mg/m², 235 mg/m², 240 mg/m², 245 mg/m², 250 mg/m², 255 mg/m², 260 mg/m², 265 mg/m², 270 mg/m², 275 mg/m², 280 mg/m², 285 mg/m², or a number or a range between any two of these values. In some embodiments, the PI3Ka inhibitor can be administered daily or weekly at a drug/body surface area unit dose of at, or at about, 38 mg/m², 39 mg/m², 40 mg/m², 41 mg/m², 42 mg/m², 43 mg/m², 44 mg/m², 45 mg/m², 46 mg/m², 47 mg/m², 48 mg/m², 49 mg/m², 50 mg/m², 51 mg/m², 52 mg/m², 53 mg/m², 54 mg/m², 55 mg/m², 56 mg/m², 57 mg/m², 58 mg/m², 59 mg/m², 60 mg/m², 61 mg/m², 62 mg/m², 63 mg/m², 64 mg/m², 65 mg/m², 66 mg/m², 67 mg/m², 68 mg/m², 69 mg/m², 70 mg/m², 71 mg/m², 72 mg/m², 73 mg/m², 74 mg/m², 75 mg/m², 76 mg/m², 77 mg/m², 78 mg/m², 79 mg/m², 80 mg/m², 81 mg/m², 82 mg/m², 83 mg/m², 84 mg/m², 88 mg/m², 86 mg/m², 87 mg/m², 88 mg/m², 89 mg/m², 90 mg/m² or a number or a range between any two of these values.

[0128] Each cycle of treatment/administration can have various lengths, for example, 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, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or more. In some embodiments, the PI3K inhibitor (e.g., PI3Ka exemplary embodiments, the PI3Ka inhibitor is administered for 1 to 10 cycles, for example, 1 to 9 cycles, 1 to 8 cycles, 1 to 7 cycles, 1 to 6 cycles, 1 to 5 cycles, 1 to 4 cycles, 1 to 3 cycles, 1 to 2 cycles, or 1 cycle. The administration of the PI3Ka inhibitor (and/or the one or more chemotherapeutic agents) can be daily or weekly and/or with break(s) between the administrations. The break can 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. In some embodiments, the breaks are 6 days and/or 13 days. In some embodiments, the daily or weekly dose of the PI3K inhibitor (e.g., PI3Ka inhibitor) can be adjusted (e.g., increased or decreased with the range) during the treatment of the subject. The daily or weekly administration of the PI3K inhibitor (e.g., PI3Ka inhibitor) can be at different amounts on different days or during different weeks. For example, the treatment can comprise weekly administration of the PI3K inhibitor (e.g., PI3Ka inhibitor) at 80 mg/m² on day 1, 64 mg/m² on day 8, and 48 mg/m² on day 15. For example, the treatment can comprise daily or weekly administration of the PI3Ka inhibitor at 0.1 mg to 20 mg during week 1, 0.25 mg to 50 mg during week 2, 0.5 mg to 100 mg during week 3, 1 mg to 200 mg during week 4, and 2 mg to 400 mg during week 5 and beyond. For example, the treatment can comprise daily or weekly administration of the PI3Ka inhibitor at 0.1 mg to 100 mg on day 1, 0.2 mg to 200 mg on day 2, 0.4 mg to 400 mg on day 3, and 0.4 mg to 400 mg or 0.6 mg to 600 mg on day 4 and beyond. For example, the PI3Ka inhibitor can be administered at a daily or weekly dose of about 0.01 mg, 0.05 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, or a number or a range between any two of these values. In some embodiments, the daily or weekly dose of the PI3K inhibitor (e.g., PI3Ka inhibitor) can be, or be about, 0.005 mg/m², 0.01 mg/m², 0.05 mg/m², 0.1 mg/m², 0.15 mg/m², 0.2 mg/m², 0.25 mg/m², 0.3 mg/m², 0.35 mg/m², 0.4 mg/m², 0.45 mg/m², 0.5 mg/m², 0.55 mg/m², 0.6 mg/m², 0.65 mg/m², 0.7 mg/m², 0.75 mg/m², 0.8 mg/m², 0.85 mg/m², 0.9 mg/m², 0.95 mg/m², 1 mg/m², 2 mg/m², 3 mg/m², 4 mg/m², 5 mg/m², 6 mg/m², 7 mg/m², 8 mg/m², 9 mg/m², 10 mg/m², or a number or a range between any two of these values. In some embodiments, a patient is administered an effective dose of a corticosteroids (e.g., dexamethasone), a diphenhydramine, and/or H2 antagonists (e.g., cimetidine or famotidine) prior to administering the PI3Ka inhibitor.

[0129] In some embodiments, the PI3K inhibitor is a PI3Ka inhibitor. A maximum concentration (C_max) of the PI3Ka inhibitor in a blood of the subject (during the treatment or after the treatment) when the PI3Ka inhibitor is administered alone or in combination with the G2/M cell cycle inhibitor (e.g., the PLK1 inhibitor) can be from about 1 pg/mL (picogram per mL) to about 10 pg/mL (microgram per mL). For example, the Cmax of the PI3Ka inhibitor in a blood of inhibitor can be, or be about, 1 pg/mL, 5 pg/mL, 10 pg/mL, 20 pg/mL, 30 pg/mL, 40 pg/mL, 50 pg/mL, 60 pg/mL, 70 pg/mL, 80 pg/mL, 90 pg/mL, 100 pg/mL, 150 pg/mL, 200 pg/mL, 250 pg/mL, 300 pg/mL, 350 pg/mL, 400 pg/mL, 450 pg/mL, 500 pg/mL, 1000 pg/mL, 5000 pg/mL,

10000 pg/mL, 50000 pg/mL, 100000 pg/mL (0.1 pg/mL), 0.2 pg/mL, 0.3 pg/mL, 0.4 pg/mL, 0.5 pg/mL, 0.6 pg/mL, 0.7 pg/mL, 0.8 pg/mL, 0.9 pg/mL, 1 pg/mL, 1.1 pg/mL, 1.2 pg/mL, 1.3 pg/mL, 1.4 pg/mL, 1.5 pg/mL, 1.6 pg/mL, 1.7 pg/mL, 1.8 pg/mL, 1.9 pg/mL, 2 pg/mL, 2.1 pg/mL, 2.2 pg/mL, 2.3 pg/mL, 2.4 pg/mL, 2.5 pg/mL, 2.6 pg/mL, 2.7 pg/mL, 2.8 pg/mL, 2.9 pg/mL, 3 pg/mL, 3.1 pg/mL, 3.2 pg/mL, 3.3 pg/mL, 3.4 pg/mL, 3.5 pg/mL, 3.6 pg/mL, 3.7 pg/mL, 3.8 pg/mL, 3.9 pg/mL, 4 pg/mL, 4.1 pg/mL, 4.2 pg/mL, 4.3 pg/mL, 4.4 pg/mL, 4.5 pg/mL, 4.6 pg/mL, 4.7 pg/mL, 4.8 pg/mL, 4.9 pg/mL, 5 pg/mL, 5.1 pg/mL, 5.2 pg/mL, 5.3 pg/mL, 5.4 pg/mL, 5.5 pg/mL, 5.6 pg/mL, 5.7 pg/mL, 5.8 pg/mL, 5.9 pg/mL, 6 pg/mL, 6.1 pg/mL, 6.2 pg/mL, 6.3 pg/mL, 6.4 pg/mL, 6.5 pg/mL, 6.6 pg/mL, 6.7 pg/mL, 6.8 pg/mL, 6.9 pg/mL, 7 pg/mL, 7.1 pg/mL, 7.2 pg/mL, 7.3 pg/mL, 7.4 pg/mL, 7.5 pg/mL, 7.6 pg/mL, 7.7 pg/mL, 7.8 pg/mL, 7.9 pg/mL, 8 pg/mL, 8.1 pg/mL, 8.2 pg/mL, 8.3 pg/mL, 8.4 pg/mL, 8.5 pg/mL, 8.6 pg/mL, 8.7 pg/mL, 8.8 pg/mL, 8.9 pg/mL, 9 pg/mL, 9.1 pg/mL, 9.2 pg/mL, 9.3 pg/mL, 9.4 pg/mL, 9.5 pg/mL, 9.6 pg/mL, 9.7 pg/mL, 9.8 pg/mL, 9.9 pg/mL, 10 pg/mL, a range between any two of these values, or any value between 1 pg/mL to 10 pg /mL.

[0130] The treatment of the present disclosure can comprise administration of a G2/M cell cycle inhibitor such as a PLK1 inhibitor (e.g., onvansertib) for a desired duration in one or more cycles. In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered for 1 to 10 cycles, for example, 1 to 9 cycles, 1 to 8 cycles, 1 to 7 cycles, 1 to 6 cycles, 1 to 5 cycles, 1 to 4 cycles, 1 to 3 cycles, 1 to 2 cycles, or 1 cycle. Each cycle of treatment can have various lengths, for example, 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, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or more.

[0131] In some embodiments, the administration of the G2/M cell cycle inhibitor (e.g., the PLK1 inhibitor) can be daily or with break(s) between days of administrations. The break can 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. The administration can be once, twice, three times, four times, or more on a day when the G2/M cell cycle inhibitor (e.g., the PLK1 inhibitor) is administered to the patient. The administration can be, for example, once every two days, every three days, every four days, every five days, every six days, or every seven days. The length of the desired duration can vary, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or more days. Each cycle of treatment can have various days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or more. For example, a single cycle of the treatment can comprise administration of the G2/M cell cycle inhibitor (e.g., the PLK1 inhibitor) for four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, thirteen days, fourteen days, fifteen days, sixteen days, seventeen days, eighteen days, nineteen days, twenty days, twenty-one days, twenty-two days, twenty-three days, twenty-four days, twenty-five days, twenty-six days, twenty-seven days, twenty-eight days, or more in a cycle (e.g., in a cycle of at least 21 days (e.g., 21 to 28 days)). In some embodiments, the treatment can comprise administration of the PLK1 inhibitor (e.g., onvansertib) and/or one or more chemotherapeutic agents for, or for at least, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, thirteen days, fourteen days, fifteen days, sixteen days, seventeen days, eighteen days, nineteen days, twenty days, or a range between any two of these values, in a cycle (e.g., a cycle of at least 21 days (e.g., 21 to 28 days)). The administration of the G2/M cell cycle inhibitor (e.g., the PLK1 inhibitor) in a single cycle of the treatment can be continuous or with one or more intervals (e.g., one day or two days of break). In some embodiments, the treatment comprises administration of the PLK1 inhibitor (e.g., onvansertib) for five days in a cycle of 14 to 28 days. In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered daily for about 14 days, followed by a 7-day off. In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered orally. In some embodiments the PLK1 inhibitor (e.g., onvansertib) is administered without any catch-up doses.

[0132] In some embodiments, the G2/M cell cycle inhibitor is a PLK1 inhibitor (e.g., onvansertib). The PLK1 inhibitor (e.g., onvansertib) can be administered to the subject in need thereof on twenty days (e.g., Days 1-10 and 15-24) during a 28-day cycle. The twenty days can be, for example, a continuous daily administration for ten days (e.g., Days 1-10) and another continuous daily administration (e.g., Days 15-24) for ten days, or a continuous daily administration for four sets of five days (e.g., Days 1-5, 8-12, 15-19 and 22-26). In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered to the subject in need thereof on twenty-one days (e.g., Days 1-21) during a 28-day cycle. In some embodiments, for example when the patient is identified to have low tolerance to the PLK1 inhibitor (e.g., onvansertib), the PLK1 inhibitor is administered to the subject in need thereof on ten days (e.g., Days 1-5 and 15- 19) during a 28-day cycle. The ten days can be, for example, a continuous daily administration for ten days (e.g., Days 1-10) or two continuous daily admiration for five days each (e.g., Days 1-5 and Days 15-19). In some embodiments, the PLK1 inhibitor (e.g., onvansertib) is administered to the subject in need thereof daily throughout the whole cycle (e.g., daily for 28 days in a cycle of 28 days). Depending on the needs of inhibition/reversion of cancer progression in the subject, the treatment, the administration cycles, dosing schedules, and/or dosage amounts of the PI3K inhibitor (e.g., the PI3Ka inhibitor) and the PLK1 inhibitor can be the same or different. For combination treatment, the administration cycle, dosing schedule, and/or dosage amount of the PI3K inhibitor (e.g., the PI3Ka inhibitor) can be adjusted according to the administration cycle, dosing schedule, and/or dosage amount of the PLK1 inhibitor. For example, the PI3Ka inhibitor (e.g., alpelisib) can be administered three times in a 28-day cycles (e.g., daily dose on Days 1, 8 and 15), which corresponds to a 28-day cycle for administration of the PLK1 inhibitor (e.g., onvansertib).

[0133] The treatment can comprise administration of the PLK1 inhibitor (e.g., onvansertib) at, or at about, 6 mg/m² - 90 mg/m² drug/body surface area, for example, as a daily dose. For example, the treatment can comprise daily administration of the PLK1 inhibitor (e.g., onvansertib) at, or at about, 6 mg/m², 8 mg/m², 10 mg/m², 12 mg/m², 14 mg/m², 16 mg/m², 18 mg/m², 20 mg/m², 23 mg/m², 27 mg/m², 30 mg/m², 35 mg/m², 40 mg/m², 45 mg/m², 50 mg/m², 55 mg/m², 60 mg/m², 65 mg/m², 70 mg/m², 80 mg/m², 85 mg/m², 90 mg/m², a number or a range between any two of these values, or any value between 8 mg/m² - 90 mg/m². In some embodiments, the daily dose of the PLK1 inhibitor (e.g., onvansertib) can be adjusted (e.g., increased or decreased with the range) during the treatment, or during a single cycle (e.g., the first cycle, the second cycle, the third cycle, and a subsequent cycle) of the treatment, for the subject. In some embodiments, the PLK inhibitor (e.g., onvansertib) is administered at 12 mg/m² on twenty days (e.g., Days 1-10 and 15-24) during a 28-day cycle. In some embodiments, the PLK inhibitor (e.g., onvansertib) is administered at 15 mg/m² on ten days (e.g., Days 1-5 and 15-19) during a 28-day cycle. In some embodiments, the PLK inhibitor (e.g., onvansertib) is administered at 8 mg/m² or 10 mg/m² everyday (e.g., Days 11-28) during a 28-day cycle. In some embodiments, the PLK inhibitor (e.g., onvansertib) is administered at 45 mg/kg 5 days a week during an 18-day cycle. In some embodiments, the PLK inhibitor (e.g., onvansertib) is administered at 45 mg/kg 5 days a week during a 32-day cycle. In some embodiments, the PLK inhibitor (e.g., onvansertib) is administered at 45 mg/kg 5 days a week during a 39-day cycle. In some embodiments, the PLK inhibitor (e.g., onvansertib) is administered at 45 mg/kg 5 days a week during a 45-day cycle. In some embodiments, the PLK inhibitor (e.g., onvansertib) is administered at 45 mg/kg 5 days a week during a cycle (e.g., 30-day, 31-day, 32-day, 33-day, 34-day, 35-day, 36-day, 37-day, 38- day, 39-day or 40-day cycle) with no administration of the PLK inhibitor (e.g., onvansertib) for one week.

[0134] In some embodiments, the daily dose of the G2/M cell cycle inhibitor (e.g., PLK1 inhibitor) can be adjusted (e.g., increased or decreased with the range) during the treatment, cycle) of the treatment, for the subject.

[0135] In some embodiments, the G2/M cell cycle inhibitor is a PLK1 inhibitor. A maximum concentration (C_max) of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject (during the treatment or after the treatment) when the PLK1 inhibitor is administered alone or in combination with the PI3K inhibitor (e.g., the PI3Ka inhibitor) can be from about 100 nmol/L to about 1500 nmol/L. For example, the Cmax of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject when the PLK1 inhibitor is administered alone or in combination with the PI3Ka inhibitor can be, or be about, 100 nmol/L, 200 nmol/L, 300 nmol/L, 400 nmol/L, 500 nmol/L, 600 nmol/L, 700 nmol/L, 800 nmol/L, 900 nmol/L, 1000 nmol/L, 1100 nmol/L, 1200 nmol/L, 1300 nmol/L, 1400 nmol/L, 1500 nmol/L, a range between any two of these values, or any value between 200 nmol/L to 1500 nmol/L.

[0136] An area under curve (AUC) of a plot of a concentration of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject over time (e.g., AUC0-24 for the first 24 hours after administration) when the PLK1 inhibitor is administered alone or in combination with the PI3Ka inhibitor can be from about 1000 nmol/L. hour to about 400000 nmol/L. hour. For example, the AUC of a plot of a concentration of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject over time (e.g., AUC0-24 for the first 24 hours after administration) when the PLK1 inhibitor is administered alone or in combination with the PI3Ka inhibitor can be, or be about, 1000 nmol/L. hour, 5000 nmol/L. hour, 10000 nmol/L. hour, 15000 nmol/L. hour, 20000 nmol/L. hour, 25000 nmol/L. hour, 30000 nmol/L. hour, 35000 nmol/L. hour, 40000 nmol/L. hour, a range between any two of these values, or any value between 1000 nmol/L. hour and 400000 nmol/L. hour.

[0137] A time (T max ) to reach a maximum concentration of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject when the PLK1 inhibitor is administered alone or in combination with the PI3Ka inhibitor can be from about 1 hour to about 5 hours. For example, the time (Tmax) to reach a maximum concentration of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject when the PLK1 inhibitor is administered alone or in combination with the PI3Ka inhibitor can be, or be about, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, a range between any two of these values, or any value between 1 hour and 5 hours.

[0138] An elimination half-life (T1/2) of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject when the PLK1 inhibitor is administered alone or in combination with the PI3Ka inhibitor can be from about 10 hours to about 60 hours. For example, the elimination halflife (T1/2) of the PLK1 inhibitor (e.g., onvansertib) in a blood of the subject when the PLK1 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, a range between any two of these values, or any value between 10 hours and 60 hours.

[0139] Patients administered one or more dose cycles of the PI3K inhibitor (e.g., the PI3Ka inhibitor) in combination with one or more dose cycles of the G2/M cell cycle inhibitor (e.g., the PLK1 inhibitor) can exhibit very tolerable AE, including in some cases undetectable definite AE or definite SAE. A remarkable, but unlikely result is the finding that the patient has no probable or even possible AE or SAE. In some embodiments, treated with the combined therapy of PI3Ka inhibitor and PLK1 inhibitor can lead to remarkable therapeutic effect. A therapeutic effect greater than the therapeutic effect predicted from in vitro or in silico is indicative of a surprising result. A therapeutic dose lower than the therapeutic dose predicted from in vitro or in silico is indicative of a surprising result. It is expected that the combination treatment can mitigate disease progression in patients. A highly positive result is the finding that the combination therapy can lead to stable disease. A remarkable, but unlikely result is the finding of a complete response or complete remission of the cancer, a progression-free survival, an overall survival rate exceeding values predicted from in vitro or in silico analysis, is free of any measurable lesion, free of any target lesion, or free of any malignant lymph nodes

Additional Cancer Therapeutics or Therapy

[0140] Methods, compositions and kits disclosed herein can be used for treating cancer, for example HR+ breast cancer. In some embodiments, a method for treating cancer comprises administrating a PI3K inhibitor and a G2/M cell cycle inhibitor (e.g., a PLK1 inhibitor such as onvansertib) to a subject (e.g., a patient) in need thereof. The method can comprise administering a therapeutically effective amount of the PI3K inhibitor (e.g., a PI3Ka inhibitor) and a therapeutically effective amount of the G2/M cell cycle inhibitor (e.g., a PLK1 inhibitor). The treatment can comprise administration of at least one additional cancer therapeutics or cancer therapy. The treatment can comprise administration a therapeutically effective amount of at least one additional cancer therapeutics or cancer therapy. The PI3K inhibitor (e.g., the PI3Ka inhibitor) and the cancer therapeutics or cancer therapy can, for example, co-administered simultaneously or sequentially. The G2/M cell cycle inhibitor (e.g., the PLK1 inhibitor) and the cancer therapeutics or cancer therapy can, for example, co-administered simultaneously or sequentially. Additional cancer therapeutics or therapies in treating breast cancer (e.g., HR+ breast cancer) are identifiable to a person skilled in the art. Exemplary additional cancer therapeutics or therapies in treating breast cancer include, but are not limited to, surgery, chemotherapy, targeted therapy, immunotherapy, radiation therapy, hormone therapy, and neoadjuvant systemic therapy. [0141] Also disclosed herein include methods, compositions, kits, and systems for predicting/determining clinical outcome for a combination treatment of cancer of the present disclosure, monitoring of the combination treatment, predicting/determining responsiveness of a subject to the combination treatment, determining the status of the cancer in a subject, and improving combination treatment outcome. The methods, compositions, kits and systems can be used to guide the combination treatment, provide combination treatment recommendations, reduce or avoid unnecessary ineffective combination treatment for patients. ctDNA can be analyzed to predict/determine clinical outcome for cancer treatment using a combination of a PI3K inhibitor (e.g., an PI3Ka inhibitor) and a G2/M cell cycle inhibitor (e.g., a PLK1 inhibitor) of the present disclosure, monitor the combination treatment, predict/determine responsiveness of a subject to the combination treatment, determine cancer status in a subject, improve combination treatment outcome, guide combination treatment, provide combination treatment recommendations, and/or to reduce or avoid ineffective combination treatment. ctDNA can be analyzed to predict/determine clinical outcome for cancer treatment, monitor cancer treatment, predict/determine responsiveness of a subject to a cancer treatment, determine cancer status in a subject, improve cancer treatment outcome, guide cancer treatment, provide treatment recommendations, and/or to reduce or avoid ineffective cancer treatment. Such analysis of ctDNA has been described in PCT Publication No. WO/2021/146322, the content of which is incorporated herein by reference in its entirety.

[0142] A method of determining responsiveness of a subject to a combination treatment comprising a PI3K inhibitor (e.g., an PI3Ka inhibitor) and a G2/M cell cycle inhibitor (e.g., a PLK1 inhibitor) of the disclosure can comprise, for example, analyzing circulating tumor DNA (ctDNA) of a subject with cancer, the subject is undergoing a treatment and/or has received the combination treatment, thereby determining the responsiveness of the subject to the combination treatment. In some embodiments, determining the responsiveness of the subject comprises determining if the subject is a responder of the treatment, if the subject is or is going to be in CR, or if the subject is or is going to be in partial remission (PR). For example, analyzing ctDNA can comprise detecting variant allele frequency in the ctDNA in a first sample obtained from the subject at a first time point, detecting variant allele frequency in the ctDNA obtained from the subject at one or more additional time points in one or more additional samples, and determining the difference of the variant allele frequency in ctDNA between the first 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 indicates the subject as responsive to the cancer treatment.

[0143] In some embodiments, the first time point is prior to or immediately prior to end of or after at least a cycle of the combination treatment. In some embodiments, the cycle of the combination treatment is the first cycle of the combination treatment. In some embodiments, the first time point is prior or immediately prior to a first cycle of the combination treatment, and the one or more additional time points are at the end of or after a second cycle of the combination treatment.

[0144] In some embodiments, the first cycle of the combination treatment is immediately prior to the second cycle of the combination treatment. In some embodiments, the method comprises continuing the combination treatment to the subject if the subject is indicated as responsive to the combination treatment. In some embodiments, the method comprises discontinuing the combination treatment to the subject and/or starting a different combination treatment to the subject if the subject is not indicated as responsive to the combination treatment.

[0145] Disclosed herein include methods of determining cancer status of a subject, comprising analyzing circulating tumor DNA (ctDNA) of a subject, thereby determining cancer status of the subject. The subject can be a subject undergoing a current combination treatment comprising a PI3K inhibitor (e.g., a PI3Ka inhibitor) and a G2/M cell cycle inhibitor (e.g., a PLK1 inhibitor) of the present disclosure, a subject that has received a prior combination treatment of the present disclosure, and/or a subject that is in remission for the cancer. The subject in remission for cancer can be in complete remission (CR), or in partial remission (PR).

[0146] In some embodiments, analyzing the ctDNA comprises detecting variant allele frequency in the ctDNA. In some embodiments, analyzing the ctDNA comprises detecting variant allele frequency in the ctDNA obtained from the subject at a first time point in a first sample, detecting variant allele frequency in the ctDNA obtained from the subject at one or more additional time points in one or more additional samples, and determining the difference of the variant allele frequency in ctDNA between the first and at least one of the one or more additional samples, an increase in the variant allele frequency at the additional sample(s) relative to the first sample indicates that the subject is at risk of cancer relapse or is in cancer relapse.

[0147] In some embodiments, the first time point is prior or immediately prior to the combination treatment, and the one or more additional time points are at the end of or after at least a cycle of the combination treatment, optionally the cycle of the combination treatment is the first cycle of the combination treatment. In some embodiments, the first time point is prior or immediately prior to a first cycle of the combination treatment, and the one or more additional time points are at the end of or after a second cycle of the combination treatment, optionally the first cycle of the combination treatment is immediately prior to the second cycle of the combination treatment. to the subject if the subject is indicated as in cancer relapse. The additional treatment can be the same or different from the current or prior combination treatment.

[0149] The variant allele frequency in ctDNA can be determined, for example, by total mutation count in the ctDNA in each of the first sample and one or more additional samples, or by the mean variant allele frequency in each of the first sample and one or more additional samples. In some embodiments, the variant allele frequency is mutant allelic frequency (MAF) for a driver mutation of the 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 MAF for one or more driver mutations of the cancer (e.g., ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof). In some embodiments, Log2(Ci/Co) < a MAF threshold indicates a decrease in ctDNA MAF Co is ctDNA MAF in the first sample and Ci is ctDNA MAF in one of the additional samples. In some embodiments, the MAF threshold is, or is about, 0.01 to -0.10. In some embodiments, the MAF threshold is, or is about, 0.06. In some embodiments, the MAF threshold is, or is about, 0.05.

[0150] In some embodiments, the first sample comprises ctDNA from the subject before treatment, and the one of additional samples comprises ctDNA from the subject after treatment. In some embodiments, the driver mutation is a mutation in one of the below 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, LUC7L2, 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, one or more of the driver mutations are mutations in the 75 genes.

[0151] The driver mutation or at least one of the one or more driver mutations can be in a gene selected from the group consisting of TP53, ASXL1, DNMT3A, NRAS, SRSF2, TET2, SF3B1, FLT3, FLT3 ITD, IDH2, NPM1, RUNX1, CDKN2A, KRAS, STAG2, CALR, CBL, CSF3R, DDX41, GATA2, JAK2, PHF6, and SETBP1. In some embodiments, the driver mutation or at least one of the one or more driver mutations is in a gene selected from the group consisting of DNMT3A, TET2, NPM1, SRSF2, NRAS, CDKN2A, SF3B1, FLT3, ASXL1, SRSF2, IDH2, NRAS, and SF3B1. In some embodiments, the method further comprises determining variant allele frequency in one or more of the ctDNA, PBMCs and BMMCs of the subject. (PCR), next generation sequencing (NGS), and/or droplet digital PCR (ddPCR). The sample disclosed herein can be derived from, for example, whole blood of the subject, plasma of the subject, serum of the subject, or a combination thereof. In some embodiments, the ctDNA is from whole blood of the subject, plasma of the subject, serum of the subject, or a combination thereof.

[0153] In some embodiments, the method comprises analyzing ctDNA of the subject before the treatment. In some embodiments, the treatment comprises one or more cycles, and the ctDNA is analyzed before, during and after each cycle of the treatment. Each cycle of treatment can be at least 21 days. In some embodiments, each cycle of treatment is from about 21 days to about 28 days. In some embodiments, the subject is human.

[0154] Disclosed herein include methods of improving treatment outcome for the cancer. The method can comprise: detecting variant allele frequency in circulating tumor DNA (ctDNA) obtained from a subject at a first time point in a first sample before the subject undergoes a combination treatment comprising a PI3K inhibitor (e.g., a PI3Ka inhibitor) and a G2/M cell cycle inhibitor (e.g., a PLK1 inhibitor) of the present disclosure; detecting variant allele frequency in ctDNA obtained from the subject at one or more additional time points in one or more additional samples after the subject undergoes the combination treatment; determining the difference of the variant allele frequency in ctDNA between the first 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 indicates the subject as responsive to the combination treatment; and continuing the combination treatment to the subj ect if the subj ect is indicated as responsive to the combination treatment, or discontinuing the combination treatment to the subject and/or starting a different cancer treatment to the subject if the subject is not indicated as responsive to the combination treatment.

[0155] Also disclosed herein include methods of treating cancer (e.g., HR+ breast cancer). The method can comprise: administering a combination treatment comprising a PI3K inhibitor (e.g., a PI3Ka inhibitor) and a G2/M cell cycle inhibitor (e.g., a PLK1 inhibitor) of the present disclosure to a subject in need thereof; determining a decrease, relative to a variant allele frequency in a first sample of the subject obtained at a first time point before the subject receives the combination treatment, in a variant allele frequency in a second sample of the subject obtained at a second time point after the subject receives the combination treatment; and continuing with the combination treatment. In some embodiments, the subject is a subject newly diagnosed with cancer, for example a subject that has not received any prior cancer treatment before the combination treatment. In some embodiments, the subject has received prior cancer treatment and was in remission for the cancer, for example a subject in complete remission (CR), or in partial [0156] The first time point can be, for example, prior or immediately prior to the combination treatment. The at least one of the one or more additional time points can be, for example, at the end of or after at least a cycle of the combination treatment. In some embodiments, the cycle of the combination treatment is the first cycle of the combination treatment. In some embodiments, the first time point is prior or immediately prior to a first cycle of the combination treatment, and the one or more additional time points are at the end of or after a second cycle of the combination treatment. In some embodiments, the first cycle of the combination treatment is immediately prior to the second cycle of the combination treatment.

[0157] The variant allele frequency in ctDNA can be determined, for example, by total mutation count in the ctDNA in each of the first sample and one or more additional samples, and/or by the mean variant allele frequency in each of the first sample and one or more additional samples. In some embodiments, the variant allele frequency is mutant allelic frequency (MAF) for a driver mutation of the 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 mutant allelic frequency (MAF) for one or more driver mutations of the cancer (e.g., ovarian cancer, breast cancer, prostate cancer, colorectal cancer, pancreatic cancer, or a combination thereof). In some embodiments, Log2(Ci/Co) < a MAF threshold indicates a decrease in ctDNA MAF Co is ctDNA MAF in the first sample and Ci is ctDNA MAF in one of the additional samples. In some embodiments, the MAF threshold is -0.05.

[0158] The driver mutation can be, for example, a mutation in at least one of the one or more driver mutations is a mutation in one of the below 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, LUC7L2, 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, and/or one or more of the driver mutations are mutations in the 75 genes. In some embodiments, the driver mutation or at least one of the one or more driver mutations is in a gene selected from the group consisting of TP53, ASXL1, DNMT3 A, NRAS, SRSF2, TET2, SF3B1, FLT3, FLT3 ITD, IDH2, NPM1, RUNX1, CDKN2A, KRAS, STAG2, CALR, CBL, CSF3R, DDX41, GATA2, JAK2, PHF6, and SETBP1. In some embodiments, the driver mutation or at least one of the one or more driver mutations is in a gene selected from the group consisting of DNMT3A, TET2, NPM1, SRSF2, NRAS, CDKN2A, [0159] In some embodiments, the method further comprises determining variant allele frequency in one or more of the ctDNA, PBMCs and BMMCs of the subject. The variant allele frequency in ctDNA can be detected, for example, using polymerase chain reaction (PCR) or next generation sequencing (NGS). In some embodiments, the variant allele frequency in ctDNA is detected using droplet digital PCR (ddPCR).

[0160] At least one of the first sample, the one or more additional samples, and the second sample can be derived from whole blood of the subject, plasma of the subject, serum of the subject, or a combination thereof. In some embodiments, the ctDNA is from whole blood of the subject, plasma of the subject, serum of the subject, or a combination thereof.

[0161] In some embodiments, the subject whose ctDNA is analyzed is undergoing or will be undergoing treatment for the cancer. The method can comprise analyzing ctDNA of the subject before the treatment. The treatment can comprise one or more cycles, and the ctDNA is analyzed before, during and after one or more cycles of the treatment. For example, the ctDNA can be analyzed before, during and after two or more cycle of the treatment, three or more cycle of the treatment, or each cycle of the treatment. Each cycle of treatment can be at least 21 days, for example, 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 from about 21 days to about 28 days. In some embodiments, each cycle of treatment is from 21 days to 28 days. In some embodiments, the subject is human.

Compositions and Kits

[0162] Disclosed herein include compositions and kits for treating cancer (e.g., HR+ breast cancer). In some embodiments, a kit comprises: a PI3K inhibitor, a G2/M cell cycle inhibitor, and a manual providing instructions for co-administering the PI3K inhibitor and the G2/M cell cycle inhibitor to a subject in need thereof for treating HR+ breast cancer. In some embodiments, the PI3K inhibitor is a PI3Ka inhibitor (e.g., alpelisib). In some embodiments, the G2/M cell phase inhibitor is a PLK1 inhibitor (e.g., onvansertib).

[0163] In some embodiments, the instructions comprise instructions for coadministrating the PI3K inhibitor and the G2/M cell phase inhibitor simultaneously. In some embodiments, the instructions comprise instructions for co-administrating the PI3K inhibitor and the G2/M cell cycle inhibitor sequentially. In some embodiments, the instructions comprise instructions for administering the PI3K inhibitor orally. In some embodiments, the instructions comprise instructions for administrating the G2/M cell cycle inhibitor orally. In some embodiments, the instructions comprise instructions for administering the PI3K inhibitor and/or instructions for administering the PI3K inhibitor (e.g., the PI3Ka inhibitor) intravenously and the G2/M cell cycle inhibitor (e.g., the PLK1 inhibitor) orally.

[0164] In some embodiments, the instructions comprise instructions for subjects who have received a prior PI3K inhibitor or G2/M cell cycle inhibitor treatment. In some embodiments, the instructions comprise instructions for subjects who did not respond to treatment with the PI3K inhibitor or G2/M cell cycle inhibitor alone. In some embodiments, the instructions comprise instructions for subjects who are known to be resistant to a PI3K inhibitor or G2/M cell cycle inhibitor therapy. In some embodiments, the prior G2/M cell cycle inhibitor is a CDK inhibitor such as a CDK 4/6 inhibitor (e.g., palbociclib).

[0165] In some embodiments, the instructions comprise instructions for subjects who have received a hormone therapy treatment. In some embodiments, the instructions comprise instructions for subjects who did not respond to treatment with the hormone therapy. In some embodiments, the instructions comprise instructions for subjects who are known to be resistant to a hormone therapy. In some embodiments, the hormone therapy comprises using one or more of selective estrogen receptor modulators or SERMs (e.g., tamoxifen, toremifene), aromatase inhibitors (e.g., anastrozole), or selective estrogen receptor degraders or SERDs (e.g., fulvestrant).

[0166] In some embodiments, the instructions comprise instructions for the subject who has received at least one prior treatment for the cancer. In some embodiments, the prior treatment does not comprise the use of a PI3K inhibitor, a PLK inhibitor, or both. In some embodiments, the instructions comprise instructions for the subject who was in remission for the cancer. In some embodiments, the subj ect in remission for cancer was in complete remission (CR), or in partial remission (PR).

[0167] The instructions can comprise instructions for administering each of the PI3K inhibitor and the G2/M cell cycle inhibitor to the subject in a cycle of at least once or twice within a week. In some embodiments, the instructions comprise instructions for administering each of the PI3K inhibitor and the G2/M cell cycle inhibitor to the subject in a cycle of at least five times within a week. In some embodiments, the instructions comprise instructions for administering the

PI3K inhibitor and the G2/M cell cycle inhibitor, or both are in a cycle of at least 7 days. In some embodiments, each cycle of treatment is at least about 14 days to about 21 days. In some embodiments, each cycle of treatment is from about 21 days to about 28 days. In some embodiments, each cycle of treatment is from about 28 days to about 35 days. In some embodiments, each cycle of treatment is from about 35 days to about 42 days. In some embodiments, each cycle of treatment is from about 42 days to about 49 days. In some embodiments, the instructions comprise instructions for administering the G2/M cell cycle instructions comprise instructions for not administering the G2/M cell cycle inhibitor (e.g., the PLK1 inhibitor) on at least one day in the cycle. In some embodiments, the instructions comprise instructions for administrating the PI3K inhibitor or G2/M cell cycle inhibitor daily. In some embodiments, the instructions comprise instructions for administrating the PI3K inhibitor (e.g., the PI3Ka inhibitor) once or twice a week. In some embodiments, the instructions comprise instructions for administrating the PI3K inhibitor (e.g., the PI3Ka inhibitor) and the G2/M cell cycle inhibitor (e.g., the PLK1 inhibitor) for at least two cycles.

[0168] In some embodiments, the PI3K inhibitor is a PI3Ka inhibitor. In some embodiments, the PI3K inhibitor is a compound targeting at least one catalytic subunit of PI3K, such as idelalisib, copanlisib, duvelisib, alpelisib, umbralisib, buparlisib, copanlisib, dactolisib, leniolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, tenalisib, voxtalisib, AMG 319, AZD8186, GSK2636771, SF1126, acalisib, omipalisib, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, or ZSTK474; or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof; or any combinations thereof. The PI3Ka inhibitor can be alpelisib, a derivative of alpelisib, or a pharmaceutically acceptable salt thereof. In some embodiments, the G2/M cell cycle inhibitor is a PLK1 inhibitor. The PLK1 inhibitor can be selective and/or specific for PLK1. In some embodiments, the PLK1 inhibitor is a dihydropteridinone, a pyridopyrimidine, a aminopyrimidine, a substituted thiazolidinone, a pteridine derivative, a dihydroimidazo[l,5-f]pteridine, a metasubstituted thiazolidinone, a benzyl styryl sulfone analogue, a 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, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960 or Ro3280. In some embodiments, the PLK1 inhibitor is onvansertib.

[0169] In some embodiments, the instructions comprise dosing guidelines for administering the PI3K inhibitor and the G2/M cell cycle inhibitor. In some embodiments, the instructions comprise instructions for administering the PLK1 inhibitor at 8 mg/m² - 90 mg/m². In some embodiments, the instructions comprise instructions for administering the PLK1 inhibitor (e.g., onvansertib) at a dose from about 10 mg/kg of body weight to about 80 mg/kg of body weight, optionally at a dose from about 20 mg/kg of body weight to about 60 mg/kg of body weight, optionally at a dose from about 30 mg/kg of body weight to about 50 mg/kg of body weight. In some embodiments, the instructions comprise instructions for administering the PI3Ka inhibitor at 0.01 mg - 1200 mg (e.g., daily dose of at 0.01 mg - 10 mg administered orally). In some embodiments, the instructions comprise instructions for administering the PI3Ka inhibitor (e.g., optionally at a dose from about 10 mg/kg of body weight to about 30 mg/kg of body weight, optionally at a dose from about 15 mg/kg of body weight to about 25 mg/kg of body weight.

EXAMPLES

[0170] Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure.

Example 1

Onvansertib synergizes with alpelisib in ER+ breast cancer PDXs

[0171] CDK4/6 inhibitors (e.g., palbociclib) in combination with endocrine therapy is the first line standard-of-care for ER+/HER2- breast cancer. Though the standard treatment is effective, most patients progress due to intrinsic or acquired resistance to CDK4/6 inhibitors or endocrine therapy. PIK3CA gene mutation is one of the most prevalent (-40%) gene alteration in HR+ breast cancer and is implicated in resistance to therapy.

[0172] In this example, three palbociclib-resistant ER+ breast cancer PDX models were treated with a PI3K inhibitor (e.g., alpelisib), a G2/M cell cycle inhibitor (e.g., onvansertib), or a combination of both. ER+ palbociclib-resistant breast cancer PDX models were established from bone metastasis biopsies or primary breast tumors. Specifically, PDX models were developed from patients who progressed on palbociclib, patients with intrinsic resistance to palbociclib, or PDXs with induced resistance by prolonged treatment with palbociclib. Two out of three PDXs displayed resistance to fulvestrant and to the combined treatment of fulvestrant and palbociclib. Table 1 below summarizes the characteristics of the ER+ breast cancer PDX models and their responses to paclitaxel/onvansertib single treatment as well as combined treatment. In

Table 1, ful is short for fulvestrant and palbo is short for palbociclib.

TABLE 1 : OVERVIEW OF THE PDX MODELS

[0173] P13Ka inhibitor (e.g., alpelisib) is approved tor PlK3CA-mutated, advanced ER+/HER2- breast cancer. However, drug-associated toxicides and resistance mechanisms may limit its clinical benefit. PLK1 (e.g., onvansertib) is a serine/threonine protein kinase and key regulator of mitosis and cell cycle progression. PLK1 is overexpressed in breast cancer and has been shown to mediate resistance to palbociclib in HR+ breast cancer. Onvansertib is an oral and selective inhibitor of PLK1, currently in clinical development for solid tumors and hematological malignancies. In this example, the anti-tumor activities of the onvansertib and alpelisib combination were evaluated. The results show that onvansertib and alpelisib combination exhibits robust anti-tumor activity and prolongs the benefit of alpelisib in palbociclib-resistant PIK3CA- mutant ER+ breast cancer PDX models. Moreover, the onvansertib and alpelisib combination also induces apoptosis in vivo.

Onvansertib and alpelisib in HBCx-134palboR31 PDX model

[0174] HBCx-134palboR31 PDX model was treated with onvansertib at a concentration of about 45 mg/kg, alpelisib at a concentration of about 25 mg/kg, or a combination of onvansertib at 45 mg/kg and alpelisib at 25 mg/kg. Tumor volume and relative tumor volume were monitored for a period of up to 80 days. FIG. 4A are plots showing tumor volume and relative tumor volume, respectively, in HBCx-134palboR31 PDX model treated with onvansertib, alpelisib, or both. FIG. 5A-FIG. 5C show the level of in vivo apoptosis induced by alpelisib and onvansertib single agents and combination.

[0175] The data suggests that alpelisib or onvansertib alone had limited anti-tumor activity in the HBCx-134palboR31 model. Conversely, the combination of alpelisib and onvansertib induced tumor regression in most mice (6/8) (Table 2). Moreover, the combination also induced higher level of apoptosis in vivo in HBCx-134palboR31 PDX model (FIG. 5A-FIG. 5C).

Onvansertib and alpelisib in HBCx-86 primary breast tumor PDX model

[0176] HBCx-86 primary breast tumor PDX model was treated with onvansertib at a concentration of about 45 mg/kg, alpelisib at a concentration of about 25 mg/kg, or a combination of onvansertib at 45 mg/kg and alpelisib at 25 mg/kg. Tumor volume and relative tumor volume were monitored for a period of up to 80 days. FIG. 4B are plots showing tumor volume and relative tumor volume, respectively, in HBCx-86 primary breast tumor PDX model treated with onvansertib, alpelisib, or both. FIG. 5A and FIG. 5B show the level of in vivo apoptosis induced by alpelisib and onvansertib single agents and combination.

[0177] The data suggests that combination of onvansertib and alpelisib induced stable disease (4/10 PDXs) in HBCx-86 PDX model, compared to 0/6 PDXs induced by alpelisib the combination, compared to alpelisib monotherapy. The combination of onvansertib and alpelisib induced tumor regression and delayed tumor recurrence compared to alpelisib monotherapy. Moreover, the combination also induced higher level of apoptosis in vivo in HBCx- 86 PDX model (FIG. 5A and FIG. 5B).

Onvansertib and alpelisib in HBCx-180 PDX model

[0178] HBCx-180 model was treated with onvansertib at a concentration of about 45 mg/kg, alpelisib at a concentration of about 25 mg/kg, or a combination of onvansertib at 45 mg/kg and alpelisib at 25 mg/kg. Tumor volume and relative tumor volume were monitored for a period of up to 80 days. FIG. 4C are plots showing tumor volume and relative tumor volume, respectively, in HBCx-180 PDX model treated with onvansertib, alpelisib, or both.

[0179] The data suggests that alpelisib or onvansertib alone had limited anti-tumor activity in the HBCx-180 model. Conversely, the combination of alpelisib and onvansertib induced tumor regression in most mice (4/7) (Table 2).

TABLE 2: ANTI-TUMOR ACTIVITY OF THE COMBINATION

[0180] Collectively, these results demonstrated an enhanced or synergistic efficacy in inhibiting cancer progression using a combination of alpelisib and onvansertib in ER+ breast cancer. In all the PIK3CA-mutant palbociclib-resistant ER+ breast cancer PDX models tested (n=3), the combination of onvansertib and alpelisib exhibited robust anti-tumor activity. Specifically, in the 4 models that showed no to minimal sensitivity to monotherapies, the combination induced tumor regression in all 3 models and tumor stasis in 1 model. An increase in apoptosis was also observed in tumors from mice (2/3 PDX models) treated with the combination compared to the monotherapies.

Example 2

Onvansertib synergizes with alpelisib in ER+ breast cancer cell lines

[0181] In this Example, the effects of onvansertib in increasing the efficacy of alpelisib in PIK3CA-mutant HR+ breast cancer preclinical models were evaluated. Six ER+ breast cancer cell lines were treated with varying doses of onvansertib or alpelisib for 6-7 days and cell viability was assessed using CellTiter-Glow® assay. ICso values were also calculated. Table 3 below provides the mutational status and ICso values of the six exemplary ER+ breast cancer cell lines.

TABLE 3: MUTATIONAL STATUS AND ICso VALUES OF ONVANSERTIB AND

[0182] After the cells were treated with onvansertib and alpelisib, cell viability was assessed using CellTiter-Glow® assay and synergy scores were calculated using the Bliss model and colony forming ability was also assessed. FIG. 1A shows the Bliss synergy scores of the dose matrix (9 x 9) evaluation of alpelisib and onvansertib drug combination in ER+ breast cancer cell lines. FIG IB shows the colony forming ability. The combination of onvansertib and alpelisib reduced the colony forming abilities of the 3 out of 3 breast cancer cell lines tested. The experiment was run in triplicates.

[0183] FIG. 2A-FIG. 2B are plots showing the effect of alpelisib and onvansertib single agents and combination on cell cycle. FIG. 2A is a plot showing the percentage of cells in Gl, S and G2/M phases. FIG. 2B is a plot showing the percentage of cells of four cell lines (e.g., MCF7, T-47D, EFM-19 and ZR-75-1) in G2/M phases. Results are the mean of three different experiments and are presented as Mean ± SEM.

[0184] FIG. 3A are plots showing effect of alpelisib and onvansertib single agents and combination on apoptosis. The percentage of cells undergoing apoptotic DNA fragmentation as analyzed by TUNEL assay is shown. Results are the mean of three different experiments and are presented as Mean ± SEM. FIG. 3B shows the cleaved-PARP protein expression as analyzed by Western blotting. The poly (ADP-ribose) polymerase (PARP) is a DNA repair enzyme that is cleaved during apoptosis by activated caspases. Therefore, increased level of cleaved PARP is a hallmark of apoptosis.

[0185] The data indicated that onvansertib in combination with alpelisib synergistically inhibited the viability of PIK3CA-mutant and PTEN-loss ER+ breast cancer cell lines. Compared to monotherapies, the combination induced more pronounced G2/M arrest (3 out of 4 cell lines) and apoptosis (4/4 cell lines) in ER+ breast cancer cell lines. Thus, co-targeting PLK1 and PI3Ka with onvansertib and alpelisib respectively, can constitute a promising therapeutic combination strategy for patients with PIK3CA-mutant HR+ breast cancer failing to respond to first-line standard of care therapies.

[0186] In at least some of the previously described embodiments, one or more a replacement is not technically feasible. It will be appreciated by those skilled in the art 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, as defined by the appended claims.

[0187] 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. The various singular/plural permutations may be expressly set forth herein for sake of clarity. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated.

[0188] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art 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 holds true for 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 two or more recitations). Furthermore, in those instances where a convention analogous 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 where a convention analogous 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 will be further 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.

[0189] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0190] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0191] While various aspects and embodiments have been 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 hormone receptor positive (HR+) breast cancer, the method comprising: administering a PI3K inhibitor and a G2/M cell cycle inhibitor to a subject with the ER+ breast cancer, thereby inhibiting or reducing progression of the ER+ breast cancer in the subj ect.

2. The method of claim 1, wherein the ER+ breast cancer is a PIK3CA-mutated HR+ breast cancer.

3. The method of any one of claims 1-2, wherein the HR+ breast cancer is a PTEN- inactivated ER+ breast cancer.

4. The method of any one of claims 1-3, wherein the HR+ breast cancer is HER2 negative.

5. The method of any one of claims 1-4, wherein the HR+ breast cancer has a histological or cytological profile with ER > 1%, 10%, 20%, or higher.

6. The method of any one of claims 1-5, wherein the HR+ breast cancer is estrogen receptor positive (ER+).

7. The method of any one of claims 1-5, wherein the HR+ breast cancer is progesterone receptor positive (PR+).

8. The method of any one of claims 1-7, wherein the subject with the HR+ breast cancer is resistant to or does not respond effectively to a hormone therapy, optionally the hormone therapy comprises a selective estrogen receptor degrader (SERD), a selective estrogen receptor modulator (SERM), an aromatase inhibitor, or a combination thereof.

9. The method of any one of claims 1-8, wherein the subject with the HR+ breast cancer is resistant to a kinase inhibitor, optionally the kinase inhibitor is a CDK inhibitor, further optionally the CDK inhibitor is a CDK4/6 inhibitor.

10. The method of any one of claims 1-9, wherein the subject with the HR+ breast cancer developed stable disease, progressive disease or resistance to a CDK4/6 inhibitor and/or a SERD.

11. The method of any one of claims 1-10, wherein the subject with the HR+ breast cancer is resistant to palbociclib, fulvestrant, or both or developed stable or progressive disease following treatment with palbociclib, fulvestrant, or both.

12. The method of claim 11, wherein the resistance is acquired resistance or intrinsic resistance.

13. The method of any one of claims 1-12, wherein the subject with the HR+ breast cancer has received a prior PI3K inhibitor treatment, a prior Gl/S cell cycle inhibitor treatment,

14. The method of claim 13, wherein the subject with the HR+ breast cancer did not respond to the treatment with the prior PI3K inhibitor, the prior Gl/S cell cycle inhibitor or the prior G2/M cell cycle inhibitor.

15. The method of claim 14, wherein the subject with the HR+ breast cancer developed stable or progressive disease following the treatment with the prior PI3K inhibitor, the prior Gl/S cell cycle inhibitor and/or the prior G2/M cell cycle inhibitor.

16. The method of any one of claims 1-15, wherein the subject with the HR+ breast cancer is known to be resistant to the PI3K inhibitor, the prior Gl/S cell cycle inhibitor or the G2/M cell cycle inhibitor alone.

17. The method of any one of claims 1-16, wherein the PI3K inhibitor and the G2/M cell cycle inhibitor are co-administered simultaneously.

18. The method of any one of claims 1-16, wherein the PI3K inhibitor and the G2/M cell cycle inhibitor are administered sequentially.

19. The method of any one of claims 1-18, wherein the PI3K inhibitor, the G2/M cell cycle inhibitor, or both are administered to the subject in a cycle of 4 days, 7 days, 14 days, 28 days, 35 days, 42 days, or 49 days.

20. The method of any one of claims 1-19, wherein the PI3K inhibitor is administered to the subject about once a week and the G2/M cell cycle inhibitor is administered to the subject about 5 days a week.

21. The method of any one of claims 1-20, wherein each cycle of treatment is at least about 14 days.

22. The method of any one of claims 1-21, wherein each cycle of treatment is from about 14 days to about 28 days.

23. The method of any one of claims 1-22, wherein the G2/M cell cycle inhibitor is administered on at least fourteen days, at least twenty-one days, or at least twenty-eight days in a cycle.

24. The method of any one of claims 1-23, wherein the G2/M cell cycle inhibitor is not administered on at least one day, at least three days, or at least seven days in a cycle.

25. The method of any one of claims 1-24, wherein the PI3K inhibitor is administered once or twice weekly.

26. The method of any one of claims 1-25, wherein the PI3K inhibitor is administered once weekly for two, three, four, five, six or seven consecutive weeks in a cycle.

27. The method of any one of claims 1-26, wherein the subject undergoes at least two cycles of the administration of the PI3K inhibitor and the G2/M cell cycle inhibitor. inhibitor.

29. The method of any one of claims 1-27, wherein the PI3K inhibitor is idelalisib, copanlisib, duvelisib, alpelisib, umbralisib, buparlisib, copanlisib, dactolisib, leniolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, tenalisib, voxtalisib, AMG 319, AZD8186, GSK2636771, SF1126, acalisib, omipalisib, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, or ZSTK474; or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof; or any combinations thereof.

30. The method of any one of claims 1-28, wherein the PI3Ka inhibitor is alpelisib.

31. The method of any one of claims 1-30, wherein the G2/M cell cycle inhibitor is a PLK1 inhibitor, optionally the PLK1 inhibitor is onvansertib (NMS-P937), BI2536, volasertib (BI 6727), GSK461364, adavosertib (AZDI 775), CYC 140, HMN-176, HMN-214, rigosertib (ON- 01910), MLN0905, TKM-080301, TAK-960, Ro3280; or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof; and any combinations thereof.

32. The method of any one of claims 1-31, wherein the G2/M cell cycle inhibitor is onvansertib.

33. The method of any one of claims 1-32, wherein the subject has received at least one prior cancer treatment.

34. The method of claim 33, where the prior treatment does not comprise the use of a PI3K inhibitor, a PLK1 inhibitor, or both; and optionally the PLK1 inhibitor is onvansertib.

35. The method of any one of claims 1-34, wherein the subject was in remission for cancer.

36. The method of any one of claims 1-35, wherein the subject in remission for cancer was in complete remission (CR) or in partial remission (PR).

37. The method of any one of claims 1-36, further comprising determining cancer status of the subject.

38. The method of any one of claims 1-37, further comprising determining responsiveness of the subject to the treatment of the PI3K inhibitor and/or the G2/M cell cycle inhibitor.

39. The method of any one of claims 1-38, further comprising administering one or more additional cancer therapeutics or therapies for the cancer.

40. The method of any one of claims 1-39, wherein the subject is human.

41. The method of any one of claims 1-40, wherein the subject achieves a complete response. a PI3K inhibitor; a G2/M cell cycle inhibitor; and a manual providing instructions for co-administering the PI3K inhibitor and the G2/M cell cycle inhibitor to a subject in need thereof for treating ER+ breast cancer.

43. The kit of claim 42, wherein the PI3K inhibitor is a PI3Ka inhibitor.

44. The method of any one of claims 42-43, wherein the PI3K inhibitor is idelalisib, copanlisib, duvelisib, alpelisib, umbralisib, buparlisib, copanlisib, dactolisib, leniolisib, parsaclisib, paxalisib, taselisib, zandelisib, inavolisib, apitolisib, bimiralisib, eganelisib, fimepinostat, gedatolisib, linperlisib, nemiralisib, pilaralisib, samotolisib, seletalisib, serabelisib, sonolisib, tenalisib, voxtalisib, AMG 319, AZD8186, GSK2636771, SF1126, acalisib, omipalisib, AZD8835, CAL263, GSK1059615, MEN1611, PWT33597, TG100-115, or ZSTK474; or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof; or any combinations thereof.

45. The kit of claim 42 or 43, wherein the G2/M cell cycle inhibitor is a PLK1 inhibitor, optionally the PLK1 inhibitor is onvansertib (NMS-P937), BI2536, volasertib (BI 6727), GSK461364, adavosertib (AZDI 775), CYC 140, HMN-176, HMN-214, rigosertib (ON-01910), MLN0905, TKM-080301, TAK-960, Ro3280; or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof; and any combinations thereof.

46. The kit of any one of claims 42-45, wherein the PI3K inhibitor is alpelisib and/or the G2/M cell cycle inhibitor is onvansertib.

47. The kit of any one of claims 42-46, wherein the instructions comprise instructions for co-administrating the PI3K inhibitor and the G2/M cell cycle inhibitor simultaneously.

48. The kit of any one of claims 42-47, wherein the instructions comprise instructions for co-administrating the PI3Ka inhibitor and the G2/M cell cycle inhibitor sequentially.

49. The kit of any one of claims 42-48, wherein the instructions comprise instructions for administering to a subject that did not respond to treatment with the PI3Ka inhibitor or the G2/M cell cycle inhibitor alone.

50. The kit of any one of claims 42-49, wherein the instructions comprise instructions for administering to a subject resistant to a CDK inhibitor, hormone therapy, or both.

51. The kit of claim 50, wherein the CDK inhibitor is a CDK4/6 inhibitor, optionally the CDK4/6 inhibitor is palbociclib and wherein the hormone therapy comprises a selective estrogen receptor degrader (SERD), a selective estrogen receptor modulator (SERM), an aromatase inhibitor, or a combination thereof, optionally the SERD is fulvestrant.