Use of cyclin E1 status as predictive biomarker for treatment of cancer with WEE1 inhibitors

Abstract

The present disclosure provides, inter alia, methods for treating cancer comprising administering to a subject selected to have a cyclin E1 state above a predetermined threshold an effective dose of Asensertib.

Description

[0001] Incorporation by reference into any priority application

[0002] Pursuant to 37 CFR 1.57 and sections 4.18 and 20.6, any and all applications for which foreign or domestic priority is identified in the Application Data Sheet filed with this application are hereby expressly incorporated by reference, including U.S. Provisional Application Nos. 63 / 382,817 filed on November 8, 2022, 63 / 485,764 filed on February 17, 2023, 63 / 459,520 filed on April 14, 2023, 63 / 504,166 filed on May 24, 2023, 63 / 506,023 filed on June 2, 2023, and 63 / 588,235 filed on October 5, 2023, each of which is incorporated by reference in its entirety, including any drawings. Background Art

[0003] DNA damage is typically resolved by repair proteins that rejoin or resynthesize the damaged DNA. However, incorrect substitutions of nucleotides into DNA can cause mutations and other genetic alterations, genetic diseases, and loss of protein function. Improper DNA repair can lead to cell death, tumor progression, and cancer. Cell cycle checkpoints are crucial for proper DNA repair, ensuring that cells do not undergo cell replication before their genomic integrity is restored. Cyclin E1 (encoded by the CCNE1 gene) participates in cell cycle regulation by binding to cyclin-dependent kinases (CDKs, including CDK2), thereby promoting cell cycle progression. WEE1 is a nuclear kinase involved in G2-M cell cycle checkpoint arrest for DNA repair prior to mitotic entry and is overexpressed in a variety of cancers. Summary of the Invention

[0004] The present disclosure is based in part on the discovery that certain cyclin E1 states, such as increased cyclin E1 protein expression levels, sensitize subjects with diseases involving defects (or deficiencies or alterations) in DNA damage repair (e.g., cancer) to treatment with the WEE1 inhibitor assensiti (also identified as ZN-c3) as a monotherapy or in combination with at least one second chemotherapeutic agent or a pharmaceutically acceptable salt thereof, and that cyclin E1 biomarker levels (e.g., cyclin E1 protein overexpression that may or may not be accompanied by CCNE1 gene amplification) can be used to select subjects for treatment with assensiti. Subjects selected based on predetermined thresholds of cyclin E1 biomarkers had significantly improved responses (e.g., tumor growth inhibition and increased progression-free survival (PFS)) when treated with assensiti or a pharmaceutically acceptable salt thereof as a monotherapy or in combination with at least one second chemotherapeutic agent or a pharmaceutically acceptable salt thereof.

[0005] The combination therapy of asenseti or its pharmaceutically acceptable salt and the second chemotherapeutic agent or its pharmaceutically acceptable salt can also provide synergistic effect and improve subject results.Therefore, the present disclosure especially provides using predetermined cyclin E1 state or cyclin E1 biomarker level above predetermined threshold value, with WEE1 inhibitor asenseti (including pharmaceutically acceptable salt) alone or with the second chemotherapeutic agent or its pharmaceutically acceptable salt combination treatment method for cancer.In certain embodiments, cyclin E1 state or cyclin E1 biomarker level are used as predictive biomarkers.

[0006] In a first aspect, the present disclosure provides a method for treating cancer comprising administering an effective dose of assensertin or a pharmaceutically acceptable salt thereof to a subject selected as having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold. In some embodiments, the predetermined cutoff value or predetermined threshold is a percentage of viable tumor cells with a cyclin E1 immunohistochemistry (IHC) staining intensity of 2+ greater than 30%. In some embodiments, the predetermined cutoff value or predetermined threshold is a cyclin E1 IHC H score greater than 125.

[0007] In a second aspect, the present disclosure provides a method for treating cancer, comprising administering an effective dose of assenserti or a pharmaceutically acceptable salt thereof, and a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof, to a subject selected as having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold. In some embodiments, the predetermined threshold is a percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 10%. In some embodiments, the predetermined threshold is a percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 30%. In some embodiments, the predetermined cutoff value or predetermined threshold is a cyclin E1 IHC H score greater than 50. In some embodiments, the predetermined cutoff value or predetermined threshold is a cyclin E1 IHC H score greater than 125.

[0008] In a fourth aspect, the present disclosure provides a method for treating ovarian cancer, comprising administering an effective dose of assensertin or a pharmaceutically acceptable salt thereof to a subject selected as having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold. In some embodiments, the predetermined threshold is a percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 30%. In some embodiments, the predetermined threshold is a cyclin E1 IHC H score greater than 125.

[0009] In a fifth aspect, the present disclosure provides a method for treating ovarian cancer, comprising: administering an effective dose of assensertin or a pharmaceutically acceptable salt thereof to a subject selected as having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold for a treatment cycle, and administering a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof one or more times during the treatment cycle. In some embodiments, the predetermined threshold is a percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 10%. In some embodiments, the predetermined threshold is a percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 30%. In some embodiments, the predetermined threshold is a cyclin E1 IHC H score greater than 50. In some embodiments, the predetermined threshold is a cyclin E1 IHC H score greater than 125.

[0010] Various embodiments of the first, second, third, fourth and fifth aspects of the present disclosure as described above are described in the following paragraphs of the Summary and in the Detailed Description.

[0011] In some embodiments, the predetermined cyclin E1 state is cyclin E1-positive or cyclin E1-high. In some embodiments, the predetermined cyclin E1 state is cyclin E1-positive (low) or cyclin E1-positive (high). In one embodiment, the predetermined cyclin E1 state is a cyclin E1 protein expression level above a predetermined cutoff value.

[0012] In some embodiments, the predetermined cyclin E1 state or cyclin E1 biomarker level is measured by cyclin E1 protein expression level. In some embodiments, cyclin E1 protein expression level is determined by CCNE1 mRNA or transcript levels. In some embodiments, cyclin E1 protein expression level is determined by protein level.

[0013] In some embodiments, the predetermined cyclin E1 status or cyclin E1 biomarker level is an immunohistochemistry (IHC) status.

[0014] In one embodiment, the predetermined cutoff value or predetermined threshold is measured by the percentage of viable tumor cells with a cyclin E1 immunohistochemistry (IHC) staining intensity of 2+.

[0015] In one embodiment, the predetermined cut-off value or predetermined threshold is cyclin E1 The percentage of viable tumor cells with an IHC staining intensity of 2+ is greater than 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 28%, 29%, 30%, 31%, 32%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61% or 62%.

[0016] In one embodiment, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 10%. In one embodiment, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 30%.

[0017] In some embodiments, the cyclin E1 expression level is measured by the cyclin E1 IHC H score.

[0018] In some embodiments, the predetermined cutoff value or predetermined threshold for the Cyclin El IHC H score is above 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, or 160.

[0019] In some embodiments, the predetermined cutoff value or predetermined threshold value for the Cyclin E1 IHC H score is above 50.

[0020] In some embodiments, the predetermined cutoff value or predetermined threshold value for the Cyclin E1 IHC H score is above 125.

[0021] In some embodiments, the predetermined cyclin 1 status or cyclin E1 biomarker level is unrelated to the subject's CCNE1 gene amplification status. In alternative embodiments, the predetermined cyclin 1 status or cyclin E1 biomarker level is concomitant with the subject's CCNE1 gene amplification status.

[0022] In some embodiments, CCNE1 gene amplification status is measured by CCNE1 gene copy number.

[0023] In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 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, 29, 30, 31, 32, 33, or 34.

[0024] In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 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, or 34.

[0025] In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 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, or 34.

[0026] In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 3. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 4. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 5. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 6. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 7. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 8. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 9. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 10. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 11. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 12. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 14.

[0027] In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 7.

[0028] In some embodiments, the subject is selected without determining the levels and status of other oncogenes.

[0029] In some embodiments, the additional oncogene is selected from BRCA1, BRCA2, TP53, PKMYT1, and PPP2R1A.

[0030] In some embodiments, the subject is selected without determining the level of BRCA1 and / or BRCA2.

[0031] In some embodiments, the subject is selected without determining the level of TP53.

[0032] In some embodiments, the cancer is a cyclin E1 driven cancer.

[0033] In some embodiments, the cancer is selected from the group consisting of glioblastoma (GBM), astrocytoma, meningioma, craniopharyngioma, medulloblastoma, other brain cancers, head and neck cancer, leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), skin cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, endometrium cancer, esophageal cancer, eye cancer, gallbladder cancer, stomach cancer, gastrointestinal cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, blood cancer, head cancer, blood malignancies, Kaposi's sarcoma, sarcoma), kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, non-small cell lung cancer (NSCLC), small cell, lymphoma, mesothelioma, melanoma, multiple myeloma, neuroblastoma, nasopharyngeal cancer, cervical cancer, ovarian cancer, osteosarcoma, sarcoma, gastrointestinal stromal tumor (GIST), pancreatic cancer, pituitary cancer, prostate cancer, kidney cancer, retinoblastoma, salivary gland cancer, skin cancer, stomach cancer, small intestine cancer, spleen cancer, sarcoma, testicular cancer, thymus cancer, thyroid cancer, uterine cancer, uterine sarcoma, uterine serous carcinoma (USC), uterine CS, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor tumor), solid tumor, or liquid tumor, HGSOC, invasive breast cancer, triple-negative breast cancer (TNBC), esophagogastric cancer, gastric cancer, esophageal cancer, pRCC, ccRCC, chromophobe RCC, head and neck cancer, adenoid cystic carcinoma (ACC), diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma (NHL), low-grade glioma (LGG), pheochromocytoma and paraganglioma (PCPG), bile duct cancer, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), thymoma, BRAF-mutant metastatic colorectal cancer, uveal melanoma, high-grade serous ovarian cancer, fallopian tube cancer, or primary peritoneal cancer, BRAF V600E mutant colorectal cancer, platinum-sensitive ovarian cancer, poly(ADP-ribose) polymerase inhibitor (PARPi)-resistant ovarian cancer, platinum-resistant ovarian cancer, platinum-refractory ovarian cancer, advanced pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, neuroendocrine tumors, neuroendocrine prostate cancer, pancreatic neuroendocrine tumors, small cell lung cancer (SCLC), germ cell cancer, and stromal cancer.

[0034] In some embodiments, the cancer is of an organ selected from the group consisting of adrenal gland, ampulla of Vater, bile duct, bladder / urinary tract, bone, intestine, breast, cervix, CNS / brain, esophagus / stomach, eye, head and neck, kidney, liver, lung, lymph, bone marrow, ovary / fallopian tube, pancreas, penis, peripheral nervous system, peritoneum, pleura, prostate, skin, soft tissue, testis, thymus, thyroid, uterus, vulva / vagina, adenocarcinoma in situ, extragonadal germ cell tumor (EGCT), mixed carcinoma types, high-grade neuroendocrine ovarian cancer, high-grade serous fallopian tube carcinoma (HGSFT), ovarian choriocarcinoma, and ovarian cancer NOS (OCNOS).

[0035] In some embodiments, the cancer is a solid tumor or a hematological malignancy.

[0036] In some embodiments, the cancer is a solid tumor.

[0037] In some embodiments, the solid tumor is selected from endometrial cancer, gallbladder cancer, ovarian cancer (e.g., HGSOC), endometrium cancer, melanoma, colorectal cancer, bladder cancer, breast cancer (e.g., invasive breast cancer, triple-negative breast cancer (TNBC)), prostate cancer, lung cancer (e.g., NSCLC, SCLC), esophagogastric cancer, gastric cancer, esophageal cancer, renal cancer (e.g., pRCC, ccRCC, chromophobe cell carcinoma RCC), head and neck cancer, osteosarcoma cancer, pancreatic cancer, brain cancer, uterine CS, uterine cancer, adenoid cystic carcinoma (ACC), mesothelioma, cervical cancer, diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma, liver cancer, glioblastoma (GBM), testicular cancer, low-grade glioma (LGG), pheochromocytoma and paraganglioma (PCPG), bile duct cancer, thyroid cancer, thymoma, and uveal melanoma.

[0038] In some embodiments, the cancer is acute myeloid leukemia (AML).

[0039] In some embodiments, the tumor is selected from the group consisting of SCLC, a neuroendocrine tumor, a neuroendocrine prostate cancer, and a pancreatic neuroendocrine tumor.

[0040] In some embodiments, the solid tumor is ovarian cancer.

[0041] In some embodiments, the ovarian cancer is epithelial ovarian cancer, germ cell cancer, or stromal cancer.

[0042] In some embodiments, the ovarian cancer is epithelial ovarian cancer.

[0043] In some embodiments, the ovarian cancer is high-grade serous ovarian cancer (HGSOC). In some embodiments, the ovarian cancer is platinum-resistant ovarian cancer (PROC). In some embodiments, the ovarian cancer is resistant to PARP inhibitors. In some embodiments, the ovarian cancer is CCNE1 gene-amplified ovarian cancer. In some embodiments, the ovarian cancer is a cyclin E1-overexpressing cancer. In some embodiments, the ovarian cancer is a cyclin E1-overexpressing / non-CCNE1 gene-amplified cancer.

[0044] In some embodiments, the cancer is confirmed histologically and / or cytologically, or the cancer is confirmed pathologically.

[0045] In some embodiments, the cancer is recurrent or persistent. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is unresectable.

[0046] In some embodiments, the subject has received no more than 1, at least 1, 1, 2, 3, 4, 1 or 2, 1 to 2, 1 to 3, or 1 to 4 prior lines of therapy, prior lines of therapy in the advanced or metastatic setting, prior line of chemotherapy, prior line of platinum-based chemotherapy, prior regimen, or prior treatment regimen.

[0047] In some embodiments, the cancer is platinum-resistant, platinum-sensitive, or platinum-refractory.

[0048] In some embodiments, the cancer is PARP inhibitor-resistant.

[0049] In one embodiment, the method of treatment comprises administering an effective dose of asensertib or a pharmaceutically acceptable salt thereof without combining with a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof. In an alternative embodiment, the method of treatment comprises administering an effective dose of asensertib or a pharmaceutically acceptable salt thereof in combination with a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof.

[0050] In some embodiments, the second chemotherapeutic agent is selected from carboplatin, cisplatin, paclitaxel, docetaxel, pegylated liposomal doxorubicin, doxorubicin, PLD), doxorubicin, gemcitabine, cytarabine, fludarabine, 5-fluorouracil (5-FU), irinotecan, topotecan, temozolomide, triapine, 5-azacytidine, capecitabine, AraC-FdUMP

[10] (CF-10), cladribine, decitabine, hydroxyurea, oxaliplatin, niraparib, encorafenib and cetuximab, or a pharmaceutically acceptable salt of any one of the foregoing.

[0051] In some embodiments, the second chemotherapeutic agent is selected from azacitidine, bendamustine, bortezomib, carfilzomib, ixazomib, busulfan, carboplatin, cytarabine, cyclophosphamide, cladribine, cisplatin, capecitabine, decitabine, dexamethasone, etoposide, fludarabine, gemcitabine, daunorubicin, doxorubicin, ifosfamide, methotrexate, and vincristine, or a pharmaceutically acceptable salt of any one of the foregoing.

[0052] In some embodiments, the second chemotherapeutic agent is carboplatin, paclitaxel, gemcitabine, or pegylated liposomal doxorubicin (PLD), or a pharmaceutically acceptable salt of any of the foregoing. In some embodiments, the second chemotherapeutic agent is carboplatin or a pharmaceutically acceptable salt thereof. In some embodiments, the second chemotherapeutic agent is paclitaxel or a pharmaceutically acceptable salt thereof. In some embodiments, the second chemotherapeutic agent is gemcitabine or a pharmaceutically acceptable salt thereof. In some embodiments, the second chemotherapeutic agent is pegylated liposomal doxorubicin (PLD) or a pharmaceutically acceptable salt thereof.

[0053] In some embodiments, the method comprises selecting a subject having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold.

[0054] In some embodiments, assenserti or a pharmaceutically acceptable salt thereof and a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof are administered concurrently.

[0055] In some embodiments, assenserti or a pharmaceutically acceptable salt thereof and the second chemotherapeutic agent or a pharmaceutically acceptable salt thereof are administered sequentially.

[0056] In some embodiments, assenserti or a pharmaceutically acceptable salt thereof and / or the second chemotherapeutic agent or a pharmaceutically acceptable salt thereof is administered intermittently.

[0057] In some embodiments, the methods of treatment comprise the step of selecting a subject having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold.

[0058] In some embodiments, the method of treatment comprises first determining the cyclin E1 status or cyclin E1 biomarker level prior to the selecting step.

[0059] In some embodiments, the treatment method results in an overall response rate (ORR) in the subject of the invention of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, the overall response rate is measured by a complete response (CR), a partial response (PR), a CA-125 50% response, or a combination thereof.

[0060] In some embodiments, the treatment method results in a median progression-free survival (mPFS) of 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or longer in the subject.

[0061] In a sixth aspect, the present disclosure provides a method for treating ovarian cancer, comprising: administering an effective dose of asensertib or a pharmaceutically acceptable salt thereof to a subject selected as having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold for a treatment cycle, and administering a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof one or more times during the treatment cycle. All of the various embodiments described above with respect to the first, second, third, fourth, and fifth aspects of the present disclosure are expressly applicable to this sixth aspect.

[0062] In some embodiments, the treatment cycle is 21 days or 28 days.

[0063] In some embodiments, treatment cycles are repeated.

[0064] Other features, purposes, and advantages are apparent in the following detailed description. However, it should be understood that although the embodiments are indicated, they are given in an illustrative and non-restrictive manner. According to the embodiments, various changes and modifications within the scope of the present disclosure will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The drawings are for purposes of illustration only and are not intended to be limiting.

[0066] Figure 1A-1G showed that overexpression of cyclin E1 protein was associated with increased sensitivity of ovarian cancer cell lines to assencillin. Figure 1A Exemplary results are shown demonstrating that assensertin sensitivity correlates with cyclin E1 protein expression in cancer cell lines OV90, Kuramochi, TYK-nu, and OVCAR3 as assessed by CellTiter Glo after 96 hours of culture. Figure 1B showed that OV90 cells (which have low endogenous expression levels of cyclin E1) transduced with a lentiviral vector expressing the CCNE1 gene had increased sensitivity to assensertin. Figure 1C Shown are cyclin E1 protein expression of the cell lines Kuramoto cells, COV362 and OV90 control (empty vector) and lentivirus-induced cyclin E1 protein overexpression as determined by Western blotting. Figure 1D Showing growth rate inhibition and IC of empty vector and cyclin E1 50 . Figure 1E Shown are the cyclin E1 protein expressions by Western blot analysis of cyclin E1 in the cell lines Kuramotomycin, COV362, JOM1, ES2, TYK-nu, CAVO3, OAW28, OVCAR4, OVCAR3 and OV90 control (empty vector). Figure 1F Graph showing the expression level of cyclin E1 protein H score as the level of cyclin E1 changes. 1G shows the growth rate (GR max ) values ​​as a function of cyclin E1 levels.

[0067] Figure 2 Demonstrated growth rate (GR) inhibition of OV90, Kuramoto, OVCAR8, TYK-nu, Cov362, OVCAR3, and Caov3 cells in the presence of assencillin.

[0068] Figure 3A Shown is the downregulation of CDK2 assessed by western blotting after treatment with siRNA. Figure 3BGraph showing percent viability with increasing concentration of assencillin. Figure 3C Graph showing the inhibition of growth rate with increasing concentrations of assencillin.

[0069] Figures 4A-4D Shown are changes in replication stress markers in SKOV3 cyclin E1-low cells after treatment with asenzel (80 mg / kg) and in OVCAR3 cyclin E1-high cells after treatment with asenzel (80 mg / kg). Figure 4A Shown are baseline cyclin E1 protein expression in SKOV3 cyclin E1-low cells and OVCAR3 cyclin E1-high cells as examined by immunohistochemistry (IHC). Figure 4B Shown are images depicting CDK1 before and after treatment with asenzeltin Y15 Horizontal reduction graph. Figure 4C Shown are the levels of γH2AX (a replication stress marker) in SKOV3 cyclin E1-low cells and OVCAR3 cyclin E1-high cells. Figure 4D Shown are γH2AX western blots of SKOV3 cyclin E1-low cells and OVCAR3 cyclin E1-high cells after treatment with asenzeltin.

[0070] Figures 5A-5D Demonstration of reduction in tumor volume in SKOV3 (non-CCNE1 amplified, CN=2) CDX mice treated with assensertin compared to vehicle control ( Figure 5A and 5C ) and weight changes ( Figure 5B and 5D ).

[0071] Figure 6A and Figure 6B Demonstration of a reduction in tumor volume in OVCAR8 (non-CCNE1 amplified) tumor-induced mice treated with asenzeltin compared to vehicle control ( Figure 6A ) and weight changes ( Figure 6B ).

[0072] Figure 7A and Figure 7B Demonstration of a reduction in tumor volume in HCC1806 (CCNE1 amplified, CN=7) CDX mice treated with asenzeltin compared to vehicle control ( Figure 7A ) and weight changes ( Figure 7B ).

[0073] Figures 8A-8DDemonstration of a reduction in tumor volume in OVCAR3 (CCNE1 amplified, CN=14) CDX mice treated with asenzel compared to vehicle control ( Figure 8A and Figure 8C ) and weight changes ( Figure 8B and Figure 8D ).

[0074] Figure 9 Demonstration of synergistic analysis of asensertib in combination with gemcitabine in OV90, OVCAR8, and OVCAR3 cells.

[0075] Figure 10A and Figure 10B Demonstration of reduction in tumor volume in A2780 (non-CCNE1 amplified, CN=2) tumor-induced mice treated with asensebest alone or in combination with paclitaxel compared to vehicle control ( Figure 10A ) and weight changes ( Figure 10B ).

[0076] Figure 11A and Figure 11B Demonstration of reduction in tumor volume in OVCAR3 (CCNE1 amplified, CN=14) tumor-induced mice treated with asensertin alone or in combination with paclitaxel compared to vehicle control ( Figure 11A ) and weight changes ( Figure 11B ). Figure 11C Shown is a heat map showing the synergistic effect of chemotherapy (paclitaxel) and asenzeltiel treatment in cyclin E1 high cells (OVCAR3) compared to cyclin E1 low cells (OV90 and TYL-nu).

[0077] Figures 12A-12C Demonstration of an exemplary correlation of cyclin E1 IHC H score with response in human subjects treated with assenserti in combination with carboplatin, paclitaxel, PLD, or gemcitabine.

[0078] Figures 13A-13E showed that cyclin E1 IHC H score correlates with tumor response in human subjects ( Figure 13A and Figure 13C ), progression-free survival ( Figure 13B ), CA125 reaction ( Figure 13D ). Subjects were grouped according to low cyclin E1 IHC H score (<70), medium cyclin E1 IHC H score (70-130), and high cyclin E1 IHC H score (>130). Figure 13E ).

[0079] Figures 14A-14C Shown are CCNE1 gene amplification status and cyclin E1 IHC H scores for subjects administered asenzeltiel. Figure 14D is an exemplary scatter plot demonstrating that cyclin E1 IHC H score is highly correlated with CCNE1 transcript levels. Figure 14E is an exemplary image of a tumor cell designated as cyclin E1-positive, and Figure 14F are exemplary images of tumor cells designated as cyclin E1-negative. Figure 14G and 14H Demonstration of correlation of cyclin E1 IHC H-score with response in human subjects treated with asenserati in combination with carboplatin, gemcitabine, paclitaxel, or PLD was shown, and subjects with partial or complete clinical response (PR or CR, respectively) were associated with cyclin E1 IHC protein expression levels and H-score >50. Figure 14I and 14J Demonstration correlation of the percentage of viable tumor cells with cyclin E1 IHC staining intensity of 2+ with response in human subjects treated with assenserti in combination with carboplatin, gemcitabine, paclitaxel, or PLD is shown, and subjects with partial or complete clinical response (PR or CR, respectively) correlated with cyclin E1 IHC protein expression levels and percentage of viable tumor cells >10%. Figure 14K Graph mapping the percentage of viable tumor cells with a staining intensity of 2+ to the H-score cutoff for cyclin E1 immunohistochemistry (IHC).

[0080] Figure 15A Systemic therapy and response prior to enrollment in the Asenzel study are shown. At the time of sample collection, subjects were classified as "platinum-sensitive" or "platinum-resistant." For each subject (y-axis), the timeline (x-axis) is centered on the time of sample collection. Each treatment line (data segment) and event (data point) is indicated. Treatment category + response and event type are coded by color (platinum = platinum) and shape, respectively. CR: complete response, PR: partial response, SD: stable disease; PD: progressive disease; NE: not evaluable; and NA: not available.

[0081] Figure 15BThe relative distribution of cyclin E1 protein expression status (i.e., cyclin E1-negative, cyclin E1-positive (low), cyclin E1-positive (high)) (y-axis) in key subsets (x-axis) is shown for the clinical variables: platinum response, previous platinum exposure, HRR status, and subject age at acquisition (panel). These data demonstrate that cyclin E1 protein expression is not significantly affected by platinum exposure, response, or HRR mutation status, meaning that cyclin E1 positivity is common in high-grade serous ovarian cancer and is not associated with prior platinum treatment.

[0082] Figure 16A and 16B Subjects with CCNE1-amplified platinum-resistant ovarian cancer at screening ( Figure 16A ) and after treatment with asensertib ( Figure 16B )'s exemplary pathology scan. DETAILED DESCRIPTION

[0083] definition

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. Unless otherwise indicated, all patents, applications, disclosed applications and other non-patent publications cited herein are incorporated by reference in their entirety. Where multiple definitions exist for a term in this article, those definitions in this section shall prevail unless otherwise stated.

[0085] As used herein, the term "about" has its ordinary meaning as understood by those skilled in the art, and thus indicates that a value includes the inherent variation of error for the method employed to determine the value or the variation that exists between multiple determinations.

[0086] As used herein, the term "modify" or "alter" or any form thereof means a modification, change, substitution, deletion, replacement, removal, variation, or conversion.

[0087] As used herein, the terms "function" and "functional" have their ordinary meaning as understood by those skilled in the art, and thus refer to a biological, enzymatic, or therapeutic function.

[0088] As used herein, the term "endogenous" has its ordinary meaning as understood by those skilled in the art, and therefore refers to the natural or wild-type nature of a gene, protein, or cell. In some embodiments, an endogenous gene is the wild-type sequence of the gene. In some embodiments, an endogenous protein is the wild-type sequence of the protein. In some embodiments, an endogenous protein function is the wild-type function and activity level of the protein. In some embodiments, an endogenous cell is a wild-type cell.

[0089] The term "mutation" has its ordinary meaning as understood by those skilled in the art and refers to an alteration in a gene sequence. In some embodiments, a cell has multiple mutations. In some embodiments, the mutation is located in a coding region of the genome. The size of the mutation can range from a single nucleotide to a large segment of a chromosome comprising multiple genes. In some embodiments, at least one mutation is silent and has no significant effect on gene expression or function. In some embodiments, at least one mutation has an effect on gene expression or function, such as gene amplification, overexpression, or increased copy number. In some embodiments, at least one mutation is silent and has no significant effect on protein expression or function. In some embodiments, at least one mutation has a small effect on protein expression or function. In some embodiments, at least one mutation has a moderate effect on protein expression or function. In some embodiments, at least one mutation has a large effect on protein expression or function. In some embodiments, at least one mutation prevents protein expression or function. Non-limiting examples of mutations include insertions, deletions, truncations, substitutions, duplications, transfers, and inversions. In some embodiments, the mutation is "somatic" or occurs in somatic cells and is not heritable. In some embodiments, a subset of somatic cells in an organism has at least one mutation that other somatic cells do not have. In some embodiments, the mutation is "germline" or occurs in germ cells and is heritable.

[0090] As disclosed herein, mutations can be monitored by a variety of sequencing, expression, or functional assays. Non-limiting examples include: DNA sequencing, RNA sequencing, DNA hybridization, protein sequencing, targeted genome sequencing, whole exome sequencing, whole genome sequencing, ATAC-sequencing, Sanger sequencing, PCR, qPCR, RT-PCR, RT-qPCR, next generation sequencing, protein truncation test, DNA microarray, heterodimer analysis, denaturing gradient gel electrophoresis, nucleotide sequencing, single-stranded conformational polymorphism, restriction enzyme digestion assay, fluorescence in situ hybridization (FISH), comparative genomic hybridization, restriction fragment length polymorphism, amplification refractory mutation system PCR, nested PCR, multiple ligation-dependent probe amplification, single-stranded conformational polymorphism, and oligonucleotide ligation assay. Mutations can also be monitored by a variety of antibody-based methods using biological samples, including but not limited to Western blotting, fluorescence-activated cell sorting, immunofluorescence, immunohistochemistry, immunocytochemistry, immunoprecipitation, enzyme-linked immunosorbent assays, radioimmunoassays, and electrochemiluminescence assays.

[0091] The term "cancer" is used herein in its ordinary biological sense and is understood by those skilled in the art. Thus, it can include cancers of any cell type, such as, but not limited to, glioblastomas, astrocytomas, meningiomas, craniopharyngiomas, medulloblastomas, and other brain cancers, leukemias, skin cancers, adrenal cancers, anal cancers, bile duct cancers, bladder cancers, bone cancers, breast cancers, cervical cancers, colorectal cancers, endometrial cancers, esophageal cancers, eye cancers, gallbladder cancers, gastrointestinal cancers, Hodgkin's lymphomas, hematological neoplasms, hematological malignancies, Kaposi's sarcoma, kidney cancers, laryngeal and hypopharyngeal cancers, liver cancers, lung cancers. , lymphoma, mesothelioma, melanoma, multiple myeloma, neuroblastoma, nasopharyngeal cancer, ovarian cancer, osteosarcoma, pancreatic cancer, pituitary cancer, retinoblastoma, salivary gland cancer, gastric cancer, small intestine cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer, uterine sarcoma, uterine serous carcinoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms' tumor, solid tumors, and / or liquid tumors.

[0092] As used herein, the term "tumor" has its usual meaning as understood by those skilled in the art, and refers to the abnormal growth of cells or tissues. In certain embodiments, the tumor is benign. In certain embodiments, the tumor is malignant. When the tumor metastasizes or spreads to other areas of the body, the tumor becomes cancer. As used herein, the term "solid tumor" has its usual meaning as understood by those skilled in the art, and refers to an abnormal tissue mass that does not contain a liquid area or cyst. Non-limiting examples of solid tumors include sarcomas, epithelial cell cancers (carcinomas), or lymphomas. Many cancer tissues can form solid tumors, such as but not limited to breast cancer, brain cancer, lung cancer, liver cancer, stomach cancer, spleen cancer, colon cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, neck cancer, sarcomas, neuroblastomas, and / or ovarian cancer. Unless the context clearly indicates that what is desired is a more specific meaning, the terms "cancer" and "tumor" can be used interchangeably.

[0093] As used herein, the term "cell" has its ordinary meaning as understood by those skilled in the art and may refer to any cell type. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0094] As used herein, the terms "individual," "subject," or "patient" have their ordinary meaning as understood by those skilled in the art, and thus include human and non-human mammals. The term "mammal" is used in its ordinary biological sense. Thus, it specifically includes, but is not limited to, primates (including simians (chimpanzees, apes, monkeys) and humans), cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or pigs. In some embodiments, the subject can be a human. In some embodiments, the subject can be a child and / or an infant. In other embodiments, the subject can be an adult.

[0095] As used herein, the term "cancer treatment" has its ordinary meaning as understood by those skilled in the art and refers to a therapeutic modality (e.g., surgery and / or radiation) or an anticancer agent, such as a small molecule, compound, protein, or other agent, used to treat, inhibit, or prevent cancer. Non-limiting examples of common classes of anticancer agents that can be used with any one or more of the alternatives described herein include alkylating agents, anti-EGFR antibodies, anti-Her-2 antibodies, antimetabolites, vinca alkaloids, platinum-based agents, anthracyclines, topoisomerase inhibitors, taxanes, antibiotics, immunomodulators, immune cell antibodies, interferons, interleukins, HSP90 inhibitors, antiandrogens, antiestrogens, antihypercalcemic agents, apoptosis inducers, Aurora kinase inhibitors, Bruton's tyrosine kinase inhibitors, kinase inhibitor), calcineurin inhibitor, CaM kinase II inhibitor, CD45 tyrosine phosphatase inhibitor, CDC25 phosphatase inhibitor, CHK kinase inhibitor, cyclooxygenase inhibitor, bRAF kinase inhibitor, cRAF kinase inhibitor, Ras inhibitor, cyclin-dependent kinase inhibitor, cysteine ​​protease inhibitor, DNA intercalator, DNA strand break agent, E3 ligase inhibitor, EGF pathway inhibitor, farnesyltransferase inhibitor, Flk-1 kinase inhibitor, glycogen synthase kinase-3 (GSK3) inhibitor, histone deacetylase (HDAC) inhibitor, I-κB-α kinase inhibitor, imidazotetrazinone, insulin tyrosine kinase inhibitor, c-Jun-N-terminal kinase inhibitor kinase (JNK) inhibitors, mitogen-activated protein kinase (MAPK) inhibitors, MDM2 inhibitors, MEK inhibitors, ERK inhibitors, MMP inhibitors, mTor inhibitors, NGFR tyrosine kinase inhibitors, p38 MAP kinase inhibitors, p56 tyrosine kinase inhibitors, PDGF pathway inhibitors, phosphatidylinositol 3-kinase inhibitors, phosphatase inhibitors, protein phosphatase inhibitors, PKC inhibitors, PKC delta kinase inhibitors, polyamine synthesis inhibitorsinhibitors), PTP1B inhibitors, protein tyrosine kinase inhibitors, SRC family tyrosine kinase inhibitors, Syk tyrosine kinase inhibitors, Janus (JAK-2 and / or JAK-3) tyrosine kinase inhibitors, retinoids, RNA polymerase II elongation inhibitors, serine / threonine kinase inhibitors, sterol biosynthesis inhibitors inhibitors), VEGF pathway inhibitors, chemotherapeutic agents, alitretinoin, hexamethylmelamine, aminopterin, aminolevulinic acid, amsacrine, asparaginase, atrasentan, bexarotene, carboquone, demecolcine, efaproxiral, elsamitrucin, etoglucid, hydroxycarbamide, leucovorin, lonidamine, lucanthone, masoprocol, methylaminolevulinate aminolevulinate), mitoguazone, mitotane, oblimersen, omacetaxine, pegaspargase, porfimer sodium, prednimustine, adenovirus vector site-coding gene (sitimageneExamples of chemotherapeutic agents that can be used for cancer treatment include carboplatin, cisplatin, paclitaxel, docetaxel, pegylated liposomal doxorubicin, doxorubicin, gemcitabine, cytarabine, fludarabine, fluorouracil (5-FU), irinotecan, topotecan, temozolomide, triamcinolone, 5-azacitidine, capecitabine, AraC-FdUMP

[10] (CF-10), cladribine, decitabine, hydroxyurea, and / or oxaliplatin, or a pharmaceutically acceptable salt of any one of the foregoing. Other examples of chemotherapeutic agents useful in cancer treatment include azacitidine, bendamustine, bortezomib, carfilzomib, ixazomib, busulfan, carboplatin, cytarabine, cyclophosphamide, cladribine, cisplatin, capecitabine, decitabine, dexamethasone, etoposide, fludarabine, gemcitabine, daunomycin, doxorubicin, ifosfamide, methotrexate, and / or vincristine, or a pharmaceutically acceptable salt of any of the foregoing.

[0096] The term "pharmaceutically acceptable salt" refers to a salt of a compound that does not cause significant irritation to the organism to which it is administered and does not invalidate the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of the compound. Pharmaceutical salts can be obtained by reacting the compound with an inorganic acid, such as a hydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), sulfuric acid, nitric acid, and phosphoric acid (e.g., 2,3-dihydroxypropyl dihydrogen phosphate). Pharmaceutical salts can also be obtained by reacting the compound with an organic acid, such as an aliphatic or aromatic carboxylic acid or sulfonic acid, such as formic acid, acetic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, benzoic acid, salicylic acid, 2-oxoglutaric acid, or naphthalenesulfonic acid. Pharmaceutical salts can also be obtained by reacting a compound with a base to form a salt, such as an ammonium salt, an alkali metal salt (e.g., a sodium salt, a potassium salt, or a lithium salt), an alkaline earth metal salt (e.g., a calcium salt or a magnesium salt), a carbonate salt, a bicarbonate salt, a salt with an organic base (e.g., dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, C1-C7 alkylamine, cyclohexylamine, triethanolamine, ethylenediamine), and a salt with an amino acid (e.g., arginine and lysine).

[0097] It is understood that where compounds disclosed herein have unfilled valencies, the valencies are filled with hydrogen or an isotope thereof, for example, hydrogen-1 (protium) and hydrogen-2 (deuterium).

[0098] It should be understood that the compounds described herein may be isotopically labeled. Substitution with isotopes such as deuterium can provide certain therapeutic advantages resulting from higher metabolic stability, such as increased half-life in vivo or reduced dosage requirements. Each chemical element represented in a compound structure may include any isotope of that element. For example, in a compound structure, a hydrogen atom may be explicitly disclosed or understood to be present in the compound. At any position of a compound where a hydrogen atom may be present, the hydrogen atom may be any isotope of hydrogen, including but not limited to hydrogen-1 (protium) and hydrogen-2 (deuterium). Therefore, reference to a compound herein encompasses all potential isotopic forms unless the context clearly indicates otherwise.

[0099] It should be understood that the compounds described herein include crystalline forms (also known as polymorphs, which include different crystal packing arrangements of the same elemental composition of the compound), amorphous phases, salts, solvates, and hydrates. In some embodiments, the compounds described herein exist in a solvated form with a pharmaceutically acceptable solvent (e.g., water, ethanol, etc.). In other embodiments, the compounds described herein exist in a non-solvated form. Solvates contain stoichiometric or non-stoichiometric amounts of solvents and can be formed with pharmaceutically acceptable solvents (e.g., water, ethanol, etc.) during a crystallization procedure. When the solvent is water, a hydrate is formed, and when the solvent is alcohol, an alcoholate is formed. In addition, the compounds provided herein can exist in a non-solvated form as well as a solvated form. In general, for the purposes of the compounds and methods provided herein, the solvated form is considered to be equivalent to the non-solvated form.

[0100] When a range of values ​​is provided, it is understood that the upper and lower limits of the range and every intervening value between the upper and lower limits are encompassed within the embodiments.

[0101] Unless expressly stated otherwise, the terms, phrases, and variations thereof used in this application (especially in the appended claims) should be understood as open-ended and not restrictive. As an example, the term "including" should be interpreted to mean "including, without limitation," "including but not limited to," etc.; as used herein, the term "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unlisted elements or method steps; the term "having" should be interpreted as "having at least"; the term "include" should be interpreted as "includes but is not limited to"; to); the term “example” is used to provide illustrative examples of the items being discussed rather than an exhaustive or limiting list thereof; and the use of terms such as “preferably,” “preferred,” or “desired,” or “desirable,” and words of similar import should not be construed to imply that certain features are critical, necessary, or even essential to structure or function, but are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment. Furthermore, the term “comprising” should be interpreted synonymously with the phrases “having at least” or “including at least.” When used in the context of a compound, composition, or device, the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.

[0102] With respect to the use of substantially any plural and / or singular terms herein, those skilled in the art can translate from the plural to the singular and / or from the singular to the plural as appropriate to the context and / or application. Various singular / plural permutations and combinations may be explicitly set forth herein for the sake of clarity. The indefinite article "a" or "an" does not exclude plurality. The mere fact that certain measures are recited in mutually different clauses does not indicate that a combination of these measures cannot be used to advantage. Any reference to an element in the claims should not be construed as limiting the scope.

[0103] As used herein, the term "equivalent dose" refers to an effective amount of a compound such as assensertin in other salt forms as described above.

[0104] The term "break" or "break day" refers to a period of time or a non-dosing day, a day of discontinuation of therapy, or a rest day when Asenzel is not administered. For example, a break refers to the period after a dosing cycle or the period between dosing weeks when Asenzel is suspended.

[0105] When referring to cancer, the term "platinum-resistant" or "platinum-refractory" refers to a cancer that initially responds to treatment with a drug containing the metal platinum, but then recurs within a certain period of time. For example, ovarian cancer that recurs within 6 months of treatment is considered platinum-resistant. In one embodiment, a cancer is platinum-refractory when progression occurs within 90 days of the last administered dose of any line of a platinum-based regimen.

[0106] The term "cyclin E1" refers to a protein that participates in the regulation of the cell cycle by binding to cyclin-dependent kinases (including CDK2). The protein cyclin E1 is encoded by the "CCNE1" gene, which is an oncogene in many cancers. The term "cyclin E1 status" refers to the expression level of cyclin E1 protein, which can be classified as, for example, cyclin E1-negative, cyclin E1-low, cyclin E1-positive, cyclin E1-positive (low), cyclin E1-positive (high) or cyclin E1-high. Overexpression of cyclin E1 refers to the overexpression of the protein, rather than the overexpression or amplification of the CCNE1 gene. According to the experimenter's stratification, in one embodiment, the overexpression of cyclin E1 is indicated by the cyclin E1 status of cyclin E1-positive, cyclin E1-positive (low), cyclin E1-positive (high) and cyclin E1-high, or by the cyclin E1 status of cyclin E1-high, or by the cyclin E1 status of cyclin E1-positive. In a similar manner, in one embodiment, the lack of cyclin E1 expression or overexpression is indicated by the cyclin E1 status of cyclin E1-negative or cyclin E1-low. In a clinical setting, the level of cyclin E1 protein expression can be determined by, for example, immunohistochemistry (IHC) or Western blot, whereby a sample (e.g., a subject tumor tissue, a subject tumor cell) is contacted with an anti-cyclin E1 antibody, stained, and histologically evaluated by a board-certified pathologist who scores the nuclear staining intensity of viable tumor cells in the sample as 0 (negative), 1+ (negative, weak or low), 2+ (positive, weakly positive, unclear, weak to moderate or moderate), or 3+ (positive, strongly positive or high). In some embodiments, the percentage of viable tumor cells with a staining intensity of 0, 1+, 2+, or 3+ is obtained. In some embodiments, the percentage is used to obtain a histological score or H score. As used herein, the terms "H score," "IHC H score," and "cyclin E1 IHC H score" and their unabbreviated and / or plural forms are used interchangeably.

[0107] Each aspect is described in detail in the following sections. The use of sections is not intended to limit the present disclosure. Each section can be applied to any aspect of the present disclosure. In this application, unless otherwise stated, the use of "or" means "and / or".

[0108] Detailed description

[0109] The present disclosure especially provides a method for treating a cancer of a subject, the subject being selected as having a predetermined cyclin E1 state or a cyclin E1 biomarker level higher than a predetermined threshold value. In particular, the present disclosure provides a method for treating a disease (such as cancer) characterized by excessive cell proliferation by administering WEE1 inhibitor Asenseti to the subject. In some aspects, the present disclosure provides a method for treating a disease (such as cancer) characterized by excessive cell proliferation, which is by administering Asenseti (WEE1 inhibitor) or a pharmaceutically acceptable salt thereof to the subject, whether as a monotherapy or administered in combination with one or more chemotherapeutic agents of an effective dose. The present disclosure also relates to a cancer for the treatment of a subject, the patient being identified as a subject with a cyclin E1 biomarker level higher than a predetermined threshold value, but the level of other cancer biomarkers in the subject is not determined.

[0110] The present disclosure also provides a method for treating ovarian cancer, comprising: administering an effective dose of asensertib or a pharmaceutically acceptable salt thereof to a subject selected as having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold. The present disclosure also provides a method for treating ovarian cancer, comprising: administering an effective dose of asensertib or a pharmaceutically acceptable salt thereof to a subject selected as having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold for a treatment cycle, and administering a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof one or more times during the treatment cycle.

[0111]

[0112] The compound Asenseti and its pharmaceutically acceptable salts are WEE1 inhibitors. The chemical structure of the compound Asenseti is depicted above. The compound Asenseti and its pharmaceutically acceptable salts can be prepared in various ways. See, for example, WO2019 / 173082. WO 2019 / 173082 and WO 2021 / 231653 describe the compound Asenseti and methods of using it to treat cancer.

[0113] Cyclin E1 (encoded by the CCNE1 gene) participates in cell cycle regulation by binding to cyclin-dependent kinases (CDKs, including CDK2), thereby promoting cell cycle progression. Cyclin E1 and cyclin E2 are encoded by the CCNE1 gene at 19q12 and the CCNE2 gene at 8q22.1, respectively. Cyclin E1 plays a crucial role in cell proliferation and tumorigenesis, while cyclin E2 is largely considered functionally redundant with cyclin E1. Cyclin E accumulates at the G1-S phase boundary and is degraded as cells progress through S phase. Cyclin E has multiple functions in cell cycle progression, both CDK2-dependent and CDK2-independent. The cyclin E / CDK2 complex controls the G1 / S transition and S phase progression by phosphorylating multiple proteins. It also regulates the apoptotic response to DNA damage through FOXO1 phosphorylation and plays a role in epigenetic regulation through EZH2 phosphorylation.

[0114] WEE1 is a tyrosine kinase that is a key component of ATR-mediated G2 cell cycle checkpoint control, which prevents entry into mitosis in response to cellular DNA damage. WEE1 activation leads to selective phosphorylation of CDK2, thereby regulating the CDK2-cyclin A / E complex that controls G1 / S phase progression. Inhibition of WEE1 can lead to excessive replicative activity, resulting in catastrophic replication events. WEE1 inhibition has the potential to sensitize tumors to induce tumor cell death.

[0115] Subject history and selection

[0116] The CCNE1 gene is overexpressed and / or amplified in various cancers. Altered CCNE1 levels (e.g., gene amplification and / or gene overexpression) dysregulate cell cycle progression, making cells more susceptible to WEE1 inhibition. Increased CCNE1 levels increase sensitivity to WEE1 inhibitors (e.g., assensertin), thereby improving the efficacy of WEE1 inhibitors in treating cancer.

[0117] Thus, the methods described herein use CCNE1 gene amplification status, predetermined cyclin E1 status, and / or cyclin E1 biomarker levels to select subjects for treatment of cancer with asenzeltiel or a pharmaceutically acceptable salt thereof. In some embodiments, the methods described herein comprise the step of selecting a subject having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold.

[0118] In some embodiments, the method further comprises first determining the level of a cyclin E1 biomarker prior to the selecting step.

[0119] In some embodiments, the subject has received one or more prior lines of therapy. In some embodiments, the subject has received 2 prior lines of therapy. In some embodiments, the subject has received 3 prior lines of therapy. In some embodiments, the subject has received 3 or more prior lines of therapy.

[0120] In some embodiments, the subject has a relapsed or refractory cancer. In some embodiments, the cancer is platinum-resistant. In some embodiments, the cancer is platinum-refractory. In some embodiments, the cancer is resistant to a PARP inhibitor.

[0121] Predetermined cyclin E1 status and predetermined cutoff value Cyclin E1 biomarker level and predetermined threshold value

[0122] Cyclin E1 status and its predetermined cutoff value and cyclin E1 biomarker predetermined threshold value can be determined by a variety of methods. In certain embodiments, the predetermined threshold value is an absolute value or a standard. In certain embodiments, the predetermined threshold value is obtained from a literature source. In certain embodiments, the predetermined threshold value is obtained from the historical cyclin E1 status cyclin E1 biomarker level of the subject himself. In certain embodiments, the predetermined threshold value is obtained from the cyclin E1 status and cyclin E1 biomarker level of a subject without cancer.

[0123] In some embodiments, the predetermined threshold is represented by comparison with a reference or control. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the cyclin E1 biomarker level in the test subject. In embodiments, the reference or control is a historical reference or control. In some embodiments, the reference or control can be based on the cyclin E1 level of the subject before treatment with asencillin or a pharmaceutically acceptable salt thereof.

[0124] Cyclin E1 overexpression

[0125] In some embodiments, the predetermined cyclin E1 state or predetermined cyclin E1 biomarker threshold is measured by cyclin E1 protein expression level.

[0126] In some embodiments, the cyclin E1 protein expression level is determined by detecting the amount of CCNE1 mRNA (transcript) or cyclin E1 protein. In some embodiments, the cyclin E1 protein overexpression level is determined by mRNA or transcript levels. In some embodiments, the cyclin E1 protein expression level is determined by protein level. In some embodiments, the cyclin E1 protein expression level is a cyclin E1 protein expression level above a predetermined cutoff value. In one embodiment, the predetermined cutoff value or predetermined threshold value I is measured by the percentage of viable tumor cells with a cyclin E1 immunohistochemistry (IHC) staining intensity of 2+. In some embodiments, the cyclin E1 protein expression level is measured by a cyclin E1 IHC H score.

[0127] CCNE1 mRNA levels or Cyclin E1 protein expression levels can be measured by any method known in the art, including but not limited to reporter gene, Northern blot, Western blot, fluorescence in situ hybridization (FISH), reverse transcription PCR, or RNA-Seq based assays.

[0128] In some embodiments, RNA detection of CCNE1 mRNA or transcripts is used to determine cyclin E1 protein expression. In some embodiments, RNA sequencing methods are used to determine cyclin E1 protein expression.

[0129] In some embodiments, cyclin E1 protein expression is measured by a quantitative readout. In some embodiments, cyclin E1 protein expression is measured by a qualitative readout.

[0130] In some embodiments, cyclin E1 protein expression is measured by signal intensity. In some embodiments, signal intensity is determined using Western blot. In some embodiments, relative signal intensity is quantified.

[0131] IHC percentage of viable tumor cells with staining intensity of 0, 1+, 2+, or 3+ and H score

[0132] In some embodiments, cyclin E1 protein expression levels are measured by the percentage of live tumor cells with a certain cyclin E1 immunohistochemistry (IHC) staining intensity (i.e., IHC staining intensity is 0, 1+, 2+, or 3+). In some embodiments, cyclin E1 protein expression levels are measured by cyclin E1 IHC H scoring. In some embodiments, cyclin E1 protein expression levels are measured by a combination of the percentage of live tumor cells with a certain cyclin E1 IHC staining intensity and cyclin E1 IHC H scoring. In some embodiments, the H score is calculated using the method described in Diar Aziz et al., Gynecologic Oncology 151, 327-336 (2018). In short, the H score is a semi-quantitative measurement obtained by immunohistochemical staining of subject tumor cells. Using Vetana Bench Mark ULTRA TM Automated staining platform and Optiview TM The test kit stains the subject's tumor cells with a cyclin E1 antibody. Trained and qualified observers assess the expression of each protein using a staining intensity of 0, 1+, 2+, or 3+, and categorize ambiguous cases confirmed by a pathologist. In the case of cyclin E1, the percentage of tumor cells with a cyclin E1 IHC staining intensity (SI) of 0, 1+, 2+, or 3+ is obtained and used to determine cyclin E1 protein expression levels. Cyclin E1 expression is assessed based on nuclear staining, while URI1 expression is assessed based on cytoplasmic staining.

[0133] The H score of the subject is determined by adding 3 times the percentage of strongly stained cells (SI = 3+) + 2 times the percentage of moderately stained cells (SI = 2+) + 1 times the percentage of weekly stained cells (SI = 1+). Therefore, the H score of the subject can range from 0 to 300. The formula for calculating the histological score (H score) is <score = a×f1+b×f2+c×f3>, where fi is the fraction of cells with a staining intensity of i, and i=1 (for 1+), 2 (for 2+) or 3 (for 3+). In some embodiments, a=1, b=2, c=3. In some embodiments, a=0, b=1, c=1, and the score is the fraction of cells with a staining intensity of 2+. In some embodiments, a=0, b=0, c=1, and the score is the fraction of cells with a staining intensity of 3+.

[0134] In some embodiments, the predetermined cutoff value for cyclin E1 status and the predetermined threshold value for cyclin E1 biomarker level are measured by the percentage of viable tumor cells with a cyclin E1 immunohistochemistry (IHC) staining intensity of 2+. In some embodiments, the predetermined cutoff value or predetermined threshold value is a cyclin E1 biomarker level. The percentage of viable tumor cells with an IHC staining intensity of 2+ is greater than 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 28%, 29%, 30%, 31%, 32%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61% or 62%. In some embodiments, the predetermined cutoff value or predetermined threshold is that the percentage of viable tumor cells with a Cyclin E1 IHC staining intensity of 2+ is greater than 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 28%, 29%, 30%, 31%, 32%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40%.

[0135] In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 10%. In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 8-12%, or greater than 8-11%, or greater than 8-10%, or greater than 8-9%, or greater than 9-12%, or greater than 9-11%, or greater than 9-10%, or greater than 10-12%, or greater than 10-11%, or greater than 11-12%.

[0136] In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 15%. In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 13-17%, or greater than 13-16%, or greater than 13-15%, or greater than 13-14%, or greater than 14-16%, or greater than 14-16%, or greater than 14-15%, or greater than 15-17%, or greater than 15-16%, or greater than 16-17%.

[0137] In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 20%. In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 18-22%, or greater than 18-21%, or greater than 18-20%, or greater than 18-19%, or greater than 19-22%, or greater than 19-21%, or greater than 19-20%, or greater than 20-22%, or greater than 20-21%, or greater than 21-22%.

[0138] In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 30%. In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 28-32%, or greater than 28-31%, or greater than 28-30%, or greater than 28-29%, or greater than 29-32%, or greater than 29-31%, or greater than 29-30%, or greater than 30-32%, or greater than 30-31%, or greater than 31-32%.

[0139] In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 35%. In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 33-37%, or greater than 33-36%, or greater than 33-35%, or greater than 33-34%, or greater than 34-36%, or greater than 34-36%, or greater than 34-35%, or greater than 35-37%, or greater than 35-36%, or greater than 36-37%.

[0140] In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 40%. In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 38-42%, or greater than 38-41%, or greater than 38-40%, or greater than 38-39%, or greater than 39-42%, or greater than 39-41%, or greater than 39-40%, or greater than 40-42%, or greater than 40-41%, or greater than 41-42%.

[0141] In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 45%. In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 43-47%, or greater than 43-46%, or greater than 43-45%, or greater than 43-44%, or greater than 44-46%, or greater than 44-46%, or greater than 44-35%, or greater than 45-47%, or greater than 45-46%, or greater than 46-47%.

[0142] In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 50%. In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 48-52%, or greater than 48-51%, or greater than 48-50%, or greater than 48-49%, or greater than 49-52%, or greater than 49-51%, or greater than 49-50%, or greater than 50-52%, or greater than 50-51%, or greater than 51-52%.

[0143] In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 55%. In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 53-57%, or greater than 53-56%, or greater than 53-55%, or greater than 53-54%, or greater than 54-56%, or greater than 54-56%, or greater than 54-55%, or greater than 55-57%, or greater than 55-56%, or greater than 56-57%.

[0144] In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 60%. In some embodiments, the predetermined cutoff value or predetermined threshold value is that the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ is greater than 58-62%, or greater than 58-61%, or greater than 58-60%, or greater than 58-69%, or greater than 59-62%, or greater than 59-61%, or greater than 59-60%, or greater than 60-62%, or greater than 60-61%, or greater than 61-62%.

[0145] In some embodiments, the predetermined cutoff value for cyclin E1 status and the predetermined threshold for cyclin E1 biomarker level are measured by the percentage of viable tumor cells with a cyclin E1 immunohistochemistry (IHC) staining intensity of 3+.

[0146] In some embodiments, the predetermined cutoff value or predetermined threshold is a Cyclin El IHC H score above 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300. In some embodiments, the threshold value of the Cyclin El IHC H score is above 40, 50, 60, 65, 70, 75, 80, 90, 95, 130, or 180.

[0147] In some embodiments, the predetermined cutoff value or predetermined threshold is a cyclin E1 IHC H score above 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, or 160.

[0148] In some embodiments, the predetermined cutoff value or predetermined threshold value is a cyclin E1 IHC H score greater than 40. In some embodiments, the predetermined cutoff value or predetermined threshold value is a cyclin E1 IHC H score greater than 50. In some embodiments, the predetermined cutoff value or predetermined threshold value is a cyclin E1 IHC H score greater than 60. In some embodiments, the predetermined cutoff value or predetermined threshold value is a cyclin E1 IHC H score greater than 65. In some embodiments, the predetermined cutoff value or predetermined threshold value is a cyclin E1 IHC H score greater than 70. In some embodiments, the predetermined cutoff value or predetermined threshold value is a cyclin E1 IHC H score greater than 75. In some embodiments, the threshold value for the cyclin E1 IHC H score is greater than 80. In some embodiments, the predetermined cutoff value or predetermined threshold value is a cyclin E1 IHC H score greater than 90. In some embodiments, the predetermined cutoff value or predetermined threshold value is a cyclin E1 IHC H score greater than 95. In some embodiments, the threshold value for the cyclin E1 IHC H score is greater than 125. In some embodiments, the predetermined cutoff value or predetermined threshold value is a cyclin E1 IHC H score greater than 130. In some embodiments, the predetermined cutoff value or predetermined threshold is a cyclin E1 IHC H score above 135. In some embodiments, the threshold for the cyclin E1 IHC H score is above 150.

[0149] In some embodiments, the predetermined cutoff value or predetermined threshold value is a cyclin E1 IHC H score greater than 25. In some embodiments, the predetermined cutoff value or predetermined threshold value is a cyclin E1 IHC H score greater than 70. In some embodiments, the predetermined cutoff value or predetermined threshold value is a cyclin E1 IHC H score greater than 130.

[0150] In some embodiments, a predetermined cutoff or threshold value for the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ and / or a predetermined cutoff or threshold value for the cyclin E1 IHC H score are predictive biomarkers for treating a subject with a WEE1 inhibitor, such as assenserti (including a pharmaceutically acceptable salt thereof), whether as a monotherapy or in combination with one or more second chemotherapeutic agents or a pharmaceutically acceptable salt thereof, for example, to predict the subject's sensitivity to treatment, responsiveness (including predicting the subject's tumor response), overall response rate (ORR) and / or median progression-free survival (mPFS).

[0151] It should be understood that in the cyclin E1 IHC H score and the percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ scoring system, a strict inequality is typically used to represent the comparison with a score threshold or cutoff value X to determine a positive state (e.g., cyclin E1-positive) versus a negative state (e.g., cyclin E1-negative). Since scores are typically rounded to the nearest integer, using a score strictly greater than X to determine a positive is equivalent to using a score greater than or equal to X+1 to determine a positive.

[0152] CCNE1 gene amplification

[0153] CCNE1 gene amplification is a differential increase in the CCNE1 portion of the genome relative to the genome as a whole. In some embodiments, "gene amplification" or "increased CCNE1 gene amplification level" refers to any increase in gene copies relative to endogenous copies.

[0154] The CCNE1 gene amplification level can be determined by methods known in the art. In some embodiments, the CCNE1 gene amplification level is determined using in situ hybridization (ISH) assay. In some embodiments, the CCNE1 gene amplification level is determined using amplification by fluorescence in situ hybridization (FISH). In some embodiments, the CCNE1 gene amplification level is determined using quantitative polymerase chain reaction methods. In some embodiments, the CCNE1 gene amplification level is determined using next-generation sequencing methods.

[0155] Copy number

[0156] In some embodiments, the CCNE1 gene amplification level or CCNE1 gene amplification status of a subject is measured by CCNE1 gene copy number. In some embodiments, CCNE1 gene copy number is determined using the ISH assay described by Aziz et al. (supra). Briefly, in Vetana ULTRA TM In an ISH assay of tumor cells from a subject optimized on the platform, CCNE1 gene amplification is measured using a pre-diluted, ready-to-use 19q12 DNP ISH probe (covering the coding sequences of CCNE1 and URI1) in combination with an ISNR DIG ISH probe (serving as a surrogate reference for diploid copy number at 19p13.2). Copy number can be determined when interpretable black (19q12) and red (INSR) signals are present in normal and malignant cells, with at least 50 malignant cells and minimal background staining, and is the average number of interpretable black signals per cell. In some embodiments, CCNE1 gene copy number is determined using whole-genome or whole-exome sequencing methods.

[0157] In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 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, 29, 30, 31, 32, 33, or 34. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 7. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 8. In some embodiments, the CCNE1 gene amplification status is a CCNE1 gene copy number of at least 14.

[0158] In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 3. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 4. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 5. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 6. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 7. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 8. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 9. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 10. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 11. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 12. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 13. In some embodiments, the CCNE1 gene amplification state is a CCNE1 gene copy number of at least 14.

[0159] In some embodiments, a CCNE1 copy number of 2 (CN=2) is non-amplified. In some embodiments, a CCNE1 copy number between 2 and 5 (CN=2-5) is a copy number gain. In some embodiments, a CCNE1 copy number greater than 5 (CN>5) is amplified.

[0160] In some embodiments, the CCNE1 gene amplification status based on a CCNE1 gene copy number cutoff is a predictive biomarker. In some embodiments, the CCNE1 gene copy number cutoff is a predictive biomarker for treating cancer with assencillin or a pharmaceutically acceptable salt thereof. In some embodiments, the CCNE1 gene copy number cutoff is selected based on progression-free survival. In some embodiments, the CCNE1 gene copy number cutoff is selected based on tumor response. In some embodiments, the CCNE1 gene copy number cutoff is selected based on clinical benefit rate (CBR). In some embodiments, the CCNE1 gene copy number cutoff is selected based on disease control rate (DCR). In some embodiments, the CCNE1 gene copy number cutoff is selected based on overall survival (OS).

[0161] Additional biomarkers

[0162] In certain embodiments, subject is selected based on the level of cyclin E1 predetermined state or cyclin E1 biomarker level and one or more other biomarkers.In certain embodiments, subject has been identified as having one or more other biomarkers.In certain embodiments, other biomarkers are included in the selection criteria.In certain embodiments, other biomarkers are not included in the selection criteria.

[0163] In some embodiments, the subject is selected without determining the levels of other cancer biomarkers. In some embodiments, the subject is selected based solely on the level of the cyclin E1 biomarker. In some embodiments, the subject is selected without determining the levels of BRCA1 and / or BRCA2. In some embodiments, the subject is selected without determining the level of TP53. In some embodiments, the subject is selected without determining the level of CA125.

[0164] In other embodiments, subjects are selected without determining the levels of other cancer biomarkers. In some embodiments, subjects are selected for a predetermined level of a cancer biomarker other than cyclin E1. In some embodiments, subjects are selected for a level of BCRA1 and / or BRCA2 biomarkers below a predetermined threshold. In some embodiments, subjects are selected for a level of TP53 biomarker below a predetermined cutoff or threshold. In some embodiments, subjects are selected for a level of CA125 biomarker below a predetermined threshold. In some embodiments, subjects are selected for a level of BCRA1 and / or BRCA2 biomarkers above a predetermined cutoff or threshold. In some embodiments, subjects are selected for a level of TP53 biomarker above a predetermined cutoff or threshold. In some embodiments, subjects are selected for a level of CA125 biomarker above a predetermined threshold.

[0165] Treatment

[0166] The present disclosure provides a method of treating cancer using a compound called assenseti or a pharmaceutically acceptable salt thereof, wherein a subject having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold is selected. Assenseti is a WEE1 inhibitor having the following formula:

[0167]

[0168] WO 2019 / 173082 and WO 2021 / 231653 describe the compound asenseti, both of which are incorporated herein by reference in their entirety. Asenseti is also known as ZN-c3, and these terms are used interchangeably.

[0169] In an embodiment, the methods described herein produce a therapeutic effect (e.g., a desired pharmacological and / or physiological effect). The therapeutic effect can include partial or complete cure of the disease, alleviation of one or more adverse symptoms attributable to the disease, and / or delayed progression of the disease. To this end, the methods of the present invention comprise administering a therapeutically effective amount of a therapeutic agent (e.g., asenseti or a pharmaceutically acceptable salt thereof, and / or a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof). A therapeutically effective amount can be an amount that effectively achieves a desired therapeutic outcome (e.g., tumor growth inhibition, progression-free survival, complete response, partial response, etc.) within the necessary dosage and time period. A therapeutically effective amount can vary according to factors such as the individual's disease state, age, sex, and weight, as well as the ability of the binding agent to induce a desired response in the individual.

[0170] Route of administration

[0171] In some embodiments, the effective dose of Asenseti or its pharmaceutically acceptable salt is oral, intravenous, or subcutaneous administration. In some embodiments, the effective dose of Asenseti or its pharmaceutically acceptable salt is oral administration. Alternative suitable techniques for administering an effective dose of Asenseti or its pharmaceutically acceptable salt known to those skilled in the art can also be used, including but not limited to oral, rectal, pulmonary, external, aerosol, injection, infusion, and parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal, and intraocular injection. In other embodiments, Asenseti or its pharmaceutically acceptable salt and / or chemotherapeutic agent can be administered orally.

[0172] In some embodiments, the effective dose of asenseti or a pharmaceutically acceptable salt thereof is administered orally, intravenously, subcutaneously, intrathecally, intramuscularly, intracavitaryly, intrapleurally, intralesionally, or intraarterially. In some embodiments, the effective dose of asenseti or a pharmaceutically acceptable salt thereof is administered orally, intravenously, or subcutaneously. In some embodiments, the effective dose of asenseti or a pharmaceutically acceptable salt thereof is administered intrathecally, intramuscularly, intracavitaryly, intrapleurally, intralesionally, or intraarterially.

[0173] In some embodiments, the effective dose of asensertib or a pharmaceutically acceptable salt thereof is administered orally.

[0174] Asensertib dosage and schedule

[0175] In some embodiments, the methods described herein comprise intermittent dosing in one or more dosing cycles comprising a rest week, i.e., comprising consecutive dosing days followed by a rest day. In some embodiments, the methods described herein comprise continuous dosing. In some embodiments, the methods described herein comprise combination therapy comprising continuous dosing of one of the agents described. In some embodiments, the methods described herein comprise combination therapy comprising continuous dosing of one of the agents described and intermittent dosing of asensertib or a pharmaceutically acceptable salt thereof.

[0176] In some embodiments, asensettin or a pharmaceutically acceptable salt thereof is administered based on the subject's weight. In some embodiments, the effective dose of asensettin or a pharmaceutically acceptable salt thereof is between 2 mg / kg and 20 mg / kg. In some embodiments, the effective dose of asensettin or a pharmaceutically acceptable salt thereof is between 2-18 mg / kg, 2-16 mg / kg, 2-14 mg / kg, 2-12 mg / kg, 2-10 mg / kg, 2-8 mg / kg, 2-6 mg / kg, 3-4 mg / kg, 3-5 mg / kg, or 4-6 mg / kg. In some embodiments, the effective dose is at least 2 mg / kg, at least 3 mg / kg, at least 4 mg / kg, at least 5 mg / kg, at least 6 mg / kg, at least 7 mg / kg, at least 8 mg / kg, at least 9 mg / kg, at least 10 mg / kg, at least 11 mg / kg, at least 12 mg / kg, at least 13 mg / kg, at least 14 mg / kg, at least 15 mg / kg, at least 16 mg / kg, at least 17 mg / kg, at least 18 mg / kg, or at least 19 mg / kg.

[0177] In some embodiments, Asencolor can also be in the form of an equivalent dose (for example, a compound in the form of other salts). In some embodiments, the effective dose is a uniform dose. In some embodiments, the effective dose range of Asencolor or its pharmaceutically acceptable salt is 200-800 mg or its equivalent once a day. In some embodiments, the effective dose range of Asencolor or its pharmaceutically acceptable salt is 200-600 mg or its equivalent once a day. In some embodiments, the effective dose range of Asencolor or its pharmaceutically acceptable salt is 300-600 mg or its equivalent once a day. In some embodiments, the effective dose range of Asencolor or its pharmaceutically acceptable salt is 400-600 mg or its equivalent once a day. In some embodiments, the effective dose range of Asencolor or its pharmaceutically acceptable salt is 400-800 mg or its equivalent once a day. In some embodiments, the effective dosage range of asenseti or a pharmaceutically acceptable salt thereof is 50-350 mg, 50-290 mg, 100-290 mg, 100-250 mg, 150-250 mg or 180-220 mg once daily, or their equivalents. In some embodiments, the effective dosage range of asenseti or a pharmaceutically acceptable salt thereof is 50-400 mg, 100-400 mg, 150-400 mg, 200-400 mg, 200-375 mg, 200-350 mg, 200-300 mg, 200-400 mg or 400-600 mg, or their equivalents, once daily.

[0178] In some embodiments, the effective dose of asensertib or a pharmaceutically acceptable salt thereof is 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 450 mg, 500 mg, 550 mg or 600 mg, or the equivalent thereof, once daily.

[0179] In some embodiments, the effective dose of asensetti or a pharmaceutically acceptable salt thereof is 200 mg or its equivalent once a day. In some embodiments, the effective dose of asensetti or a pharmaceutically acceptable salt thereof is 300 mg or its equivalent once a day. In some embodiments, the effective dose of asensetti or a pharmaceutically acceptable salt thereof is 350 mg or its equivalent once a day. In some embodiments, the effective dose of asensetti or a pharmaceutically acceptable salt thereof is 400 mg or its equivalent once a day. In some embodiments, the effective dose of asensetti or a pharmaceutically acceptable salt thereof is 450 mg or its equivalent once a day. In some embodiments, the effective dose of asensetti or a pharmaceutically acceptable salt thereof is 500 mg or its equivalent once a day. In some embodiments, the effective dose of asensetti or a pharmaceutically acceptable salt thereof is 600 mg or its equivalent once a day. In some embodiments, the effective dose of asensetti or a pharmaceutically acceptable salt thereof is 700 mg or its equivalent once a day. In some embodiments, the effective dose of asensetti or a pharmaceutically acceptable salt thereof is 800 mg or its equivalent once a day.

[0180] In some embodiments, the daily dose of asenseti or a pharmaceutically acceptable salt thereof is equal to or greater than 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 550 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, or its equivalent. In some embodiments, the present disclosure provides for the administration of a high dose of asenseti or a pharmaceutically acceptable salt thereof, for example, wherein the dose is equal to or greater than 375 mg.

[0181] Treatment cycle

[0182] The methods of the present disclosure include administering asenseti or a pharmaceutically acceptable salt thereof and / or a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof in a suitable dosing schedule. For example, asenseti or a pharmaceutically acceptable salt thereof and / or a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof as described herein can be administered once or more daily (e.g., once, twice, or three times a day) for a period of days, followed by a period of no administration. This treatment cycle (including dosing days and no administration days) can then be repeated.

[0183] In some embodiments, the treatment cycle is a period of 3-28 days. In some embodiments, the treatment cycle is 5, 7, 10, or 14 days. In some embodiments, the treatment cycle is 21 or 28 days. In some embodiments, the treatment cycle is repeated.

[0184] In some aspects, provided herein is a method of treating cancer comprising

[0185] A daily dose of 350 mg or more of asensetib or a pharmaceutically acceptable salt thereof, or its equivalent, is administered to a subject in need thereof according to an intermittent dosing cycle, wherein the intermittent dosing cycle comprises one or more dosing weeks, and each dosing week comprises at least three consecutive dosing days and at least one day without dosing. In some embodiments, the daily dose of asensetib or a pharmaceutically acceptable salt thereof is 400 mg. In some embodiments, the daily dose of asensetib or a pharmaceutically acceptable salt thereof is 450 mg.

[0186] In some aspects, the present disclosure provides an intermittent dosing regimen for administering a high dose of asenseti or a pharmaceutically acceptable salt thereof (e.g., between about 350 mg and about 800 mg once daily, or between about 175 mg and about 400 mg twice daily), such as 5 days of administration ("dosing" day) followed by 2 days of rest ("off" day) (i.e., 5 / 2), 4 days of administration followed by 3 days of rest (i.e., 4 / 3), or 3 days of administration followed by 4 days of rest (i.e., 3 / 4), or 6 days of administration followed by 1 day of rest (i.e., 6 / 1). Alternatively, the intermittent dosing regimen of asenseti or a pharmaceutically acceptable salt thereof is also represented by an intermittent frequency of, for example, 5 days of administration / 2 days of rest, 4 days of administration / 2 days of rest, 3 days of administration / 4 days of rest, etc., administered once daily at a dose of between about 350 mg and about 800 mg, or administered twice daily at a dose of between about 175 mg and about 400 mg.

[0187] In certain embodiments, one or more dosing weeks are separated by the rest of at least one week.In certain embodiments, intermittent dosing regimen as described herein (such as 7 / 0, 6 / 1, 5 / 2, 4 / 3 or 3 / 4) carries out 2 weeks of rest followed by a week, or carries out a week of rest followed by a week, so as to achieve high efficacy, while increasing the safety and tolerance for treating cancer.In certain embodiments, intermittent dosing regimen as described herein (such as 7 / 0, 5 / 2, 6 / 1, 4 / 3 or 3 / 4) carries out 3 weeks of rest followed by a week or carries out a week of rest followed by a week, so as to achieve high efficacy, while increasing the safety and tolerance for treating cancer.In certain embodiments, intermittent dosing regimen as described herein (such as 7 / 0, 6 / 1, 5 / 2, 4 / 3 or 3 / 4) carries out more than 3 weeks of rest followed by a week, or carries out a week of rest followed by a week, so as to achieve high efficacy, while increasing the safety and tolerance for treating cancer.

[0188] In some aspects, provided herein is a method of treating cancer comprising

[0189] According to an intermittent dosing cycle, a daily dose of 100 mg or more of Asenseti or a pharmaceutically acceptable salt thereof, or its equivalent, is administered to a subject in need thereof, wherein the intermittent dosing cycle comprises one or more dosing weeks, and each dosing week comprises at least three consecutive dosing days and at least one day without dosing, followed by at least one week of rest. In some embodiments, the daily dose of Asenseti or a pharmaceutically acceptable salt thereof is equal to or greater than 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, or its equivalent. In some embodiments, Asenseti or a pharmaceutically acceptable salt thereof is administered at a dose of about 200 mg once daily in an intermittent dosing regimen. In some embodiments, Asenseti or a pharmaceutically acceptable salt thereof is administered at a dose of about 225 mg once daily in an intermittent dosing regimen. In some embodiments, Asenseti or a pharmaceutically acceptable salt thereof is administered at a dose of about 250 mg once daily in an intermittent dosing regimen. In some embodiments, asenseti or a pharmaceutically acceptable salt thereof is administered at a dose of about 275 mg once daily in an intermittent dosing regimen. In some embodiments, asenseti or a pharmaceutically acceptable salt thereof is administered at a dose of greater than about 300 mg once daily in an intermittent dosing regimen. In some embodiments, asenseti or a pharmaceutically acceptable salt thereof is administered at a dose of about 300 mg once daily in an intermittent dosing regimen. In some embodiments, asenseti or a pharmaceutically acceptable salt thereof is administered at a dose of about 350 mg once daily in an intermittent dosing regimen.

[0190] In some embodiments, the daily dose of asensetti or a pharmaceutically acceptable salt thereof is equal to or greater than 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 550 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, or their equivalents. In some embodiments, the daily dose of asensetti or a pharmaceutically acceptable salt thereof is equal to or greater than 375 mg. In some embodiments, the daily dose of asensetti or a pharmaceutically acceptable salt thereof is equal to about 400 mg. In some embodiments, the daily dose of asensetti or a pharmaceutically acceptable salt thereof is equal to about 425 mg. In some embodiments, the daily dose of asensetti or a pharmaceutically acceptable salt thereof is equal to about 450 mg. In some embodiments, the daily dose of asensetti or a pharmaceutically acceptable salt thereof is equal to about 475 mg. In some embodiments, the daily dose of asensetti or a pharmaceutically acceptable salt thereof is equal to about 500 mg. In some embodiments, the daily dose of asenseti or a pharmaceutically acceptable salt thereof is equal to about 550 mg. In some embodiments, the daily dose of asenseti or a pharmaceutically acceptable salt thereof is equal to about 600 mg. In some embodiments, the daily dose of asenseti or a pharmaceutically acceptable salt thereof is equal to about 625 mg. In some embodiments, the daily dose of asenseti or a pharmaceutically acceptable salt thereof is equal to about 650 mg. In some embodiments, the daily dose of asenseti or a pharmaceutically acceptable salt thereof is equal to about 675 mg. In some embodiments, the daily dose of asenseti or a pharmaceutically acceptable salt thereof is equal to about 700 mg. In some embodiments, the daily dose of asenseti or a pharmaceutically acceptable salt thereof is equal to about 725 mg. In some embodiments, the daily dose of asenseti or a pharmaceutically acceptable salt thereof is equal to about 750 mg. In some embodiments, the daily dose of asenseti or a pharmaceutically acceptable salt thereof is equal to about 775 mg. In some embodiments, the daily dose of asenseti or a pharmaceutically acceptable salt thereof is equal to about 800 mg or its equivalent.

[0191] In some embodiments, the daily dose of asensertib or a pharmaceutically acceptable salt thereof is administered once daily.

[0192] In some embodiments, the daily dose of asensertib or a pharmaceutically acceptable salt thereof is divided into two doses per day.

[0193] In some embodiments, each dosing week comprises at least four, five, or six consecutive dosing days.

[0194] In some embodiments, each dosing week comprises five consecutive dosing days and two days without dosing.

[0195] In some embodiments, each dosing week comprises four consecutive dosing days and three days without dosing.

[0196] In some embodiments, each dosing week comprises three consecutive dosing days and four days without dosing.

[0197] In some embodiments, each dosing week comprises seven consecutive dosing days and seven days without dosing.

[0198] In some embodiments, each intermittent dosing cycle is comprised between about 7 to about 10 consecutive dosing days. In some embodiments, each intermittent dosing cycle is comprised between about 8 consecutive dosing days. In some embodiments, each intermittent dosing cycle is comprised between about 9 consecutive dosing days. In some embodiments, each intermittent dosing cycle is comprised between about 10 consecutive dosing days.

[0199] In some embodiments, an intermittent dosing cycle comprises twenty-one consecutive dosing days and seven days without dosing.

[0200] In some embodiments, the intermittent dosing cycle comprises two consecutive dosing weeks.

[0201] In some aspects, provided herein is a method of treating cancer comprising

[0202] A daily dose of 350 mg or more of asensertib or a pharmaceutically acceptable salt thereof, or its equivalent, is administered to a subject in need thereof according to an intermittent dosing cycle comprising at least two consecutive dosing days and at least one day without dosing.

[0203] In some embodiments, the intermittent dosing cycle comprises at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen consecutive dosing days. In some embodiments, the intermittent dosing cycle comprises greater than fourteen consecutive dosing days. In some embodiments, the intermittent dosing cycle comprises twenty-one consecutive dosing days. In some embodiments, the intermittent dosing cycle comprises twenty-eight consecutive dosing days. In some embodiments, the intermittent dosing cycle comprises thirty-two consecutive dosing days. In some embodiments, the intermittent dosing cycle comprises forty-two consecutive dosing days.

[0204] In some embodiments, the intermittent dosing cycle comprises at least one, two, three, four, five, six, or seven days without dosing. In some embodiments, the intermittent dosing cycle comprises one day without dosing. In some embodiments, the intermittent dosing cycle comprises between about two and seven days without dosing. In some embodiments, the intermittent dosing cycle comprises two days without dosing. In some embodiments, the intermittent dosing cycle comprises three days without dosing. In some embodiments, the intermittent dosing cycle comprises four days without dosing. In some embodiments, the intermittent dosing cycle comprises five days without dosing. In some embodiments, the intermittent dosing cycle comprises six days without dosing. In some embodiments, the intermittent dosing cycle comprises seven days without dosing.

[0205] In some embodiments, an intermittent dosing cycle comprises between about two and seven consecutive dosing days ("dosing" days) followed by between about one and seven rest periods ("off" days).

[0206] In some embodiments, an intermittent dosing cycle comprises five consecutive dosing days and two days without dosing.

[0207] In some embodiments, an intermittent dosing cycle comprises four consecutive dosing days and three days without dosing.

[0208] In some embodiments, an intermittent dosing cycle comprises three consecutive dosing days and four days without dosing.

[0209] In some embodiments, an intermittent dosing cycle comprises six consecutive dosing days and one day without dosing.

[0210] In some embodiments, the intermittent dosing cycle comprises seven consecutive dosing days and seven days without dosing.

[0211] In some embodiments, the intermittent dosing cycle comprises fourteen consecutive dosing days and seven days without dosing.

[0212] In some embodiments, an intermittent dosing cycle comprises twenty-one consecutive dosing days and seven days without dosing.

[0213] In some embodiments, the daily dose of asenseti or a pharmaceutically acceptable salt thereof is equal to or greater than 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 550 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, or its equivalent. In some embodiments, the present disclosure provides the administration of a high dose of asenseti or a pharmaceutically acceptable salt thereof, for example, wherein the dose is equal to or greater than 375 mg. In some embodiments, asenseti or a pharmaceutically acceptable salt thereof is administered at a dose of about 400 mg once daily in an intermittent dosing regimen. In some embodiments, asenseti or a pharmaceutically acceptable salt thereof is administered at a dose of about 450 mg once daily in an intermittent dosing regimen. In some embodiments, asenseti or a pharmaceutically acceptable salt thereof is administered at a dose of about 500 mg once daily in an intermittent dosing regimen. In some embodiments, Asenseti or its pharmaceutically acceptable salt is administered at a dose of about 550 mg once daily in an intermittent dosing regimen. In some embodiments, Asenseti or its pharmaceutically acceptable salt is administered at a dose of about 600 mg once daily in an intermittent dosing regimen. In some embodiments, Asenseti or its pharmaceutically acceptable salt is administered at a dose greater than about 600 mg once daily in an intermittent dosing regimen. In some embodiments, Asenseti or its pharmaceutically acceptable salt is administered at a dose of about 650 mg once daily in an intermittent dosing regimen. In some embodiments, Asenseti or its pharmaceutically acceptable salt is administered at a dose of about 700 mg once daily in an intermittent dosing regimen. In some embodiments, Asenseti or its pharmaceutically acceptable salt is administered at a dose of about 750 mg once daily in an intermittent dosing regimen. In some embodiments, Asenseti or its pharmaceutically acceptable salt is administered at a dose of about 775 mg once daily in an intermittent dosing regimen. In some embodiments, Asenseti or its pharmaceutically acceptable salt is administered at a dose of about 800 mg once daily in an intermittent dosing regimen.

[0214] In some embodiments, the daily dose of asensertib or a pharmaceutically acceptable salt thereof is administered once daily.

[0215] In some embodiments, the daily dose of asensertib or a pharmaceutically acceptable salt thereof is divided equally into two doses daily.

[0216] In some embodiments, the daily dose of asensertib or a pharmaceutically acceptable salt thereof is divided into three doses per day. In some embodiments, the daily dose of asensertib or a pharmaceutically acceptable salt thereof is divided into four doses per day.

[0217] In some embodiments, the twice daily dosage of asensertib or a pharmaceutically acceptable salt thereof is equal to or greater than 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, or the equivalent thereof.

[0218] In some aspects, provided herein is a method of treating cancer comprising

[0219] A daily dose of 400 mg or more of asensertib or a pharmaceutically acceptable salt thereof, or its equivalent, is administered to a subject in need thereof according to an intermittent dosing cycle comprising five consecutive dosing days and two consecutive non-dosing days.

[0220] In some aspects, provided herein is a method of treating cancer comprising

[0221] A daily dose of 450 mg or more of asensertib or a pharmaceutically acceptable salt thereof, or its equivalent, is administered to a subject in need thereof according to an intermittent dosing cycle comprising five consecutive dosing days and two consecutive non-dosing days.

[0222] In some aspects, provided herein is a method of treating cancer comprising

[0223] A daily dose of 400 mg or more of asensertib or a pharmaceutically acceptable salt thereof, or its equivalent, is administered to a subject in need thereof according to an intermittent dosing cycle comprising four consecutive dosing days and three consecutive non-dosing days.

[0224] In some aspects, provided herein is a method of treating cancer comprising

[0225] A daily dose of 450 mg or more of asensertib or a pharmaceutically acceptable salt thereof, or its equivalent, is administered to a subject in need thereof according to an intermittent dosing cycle comprising four consecutive dosing days and three consecutive non-dosing days.

[0226] In some embodiments, the intermittent dosing cycles are repeated.

[0227] In some embodiments, the method further comprises administering a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof during an intermittent dosing cycle. Without wishing to be bound by any particular theory, administering asenseti or a pharmaceutically acceptable salt thereof in combination with a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof enables a subject resistant to treatment with the second chemotherapeutic agent or a pharmaceutically acceptable salt thereof alone to respond, or prevents or reduces the toxicity of the agent compared to monotherapy, and / or improves the efficacy of the treatment. Combination therapy using an intermittent dosing cycle further benefits administration by requiring, for example, a lower effective dose of the second chemotherapeutic agent or a pharmaceutically acceptable salt thereof and / or asenseti or a pharmaceutically acceptable salt thereof.

[0228] In some embodiments, assenserti or a pharmaceutically acceptable salt thereof is administered in combination with one or more second chemotherapeutic agents (including pharmaceutically acceptable salts thereof) in an intermittent dosing cycle.

[0229] Cancer type

[0230] The methods of the present disclosure can be used to treat cancer.

[0231] In some embodiments, the cancer is glioblastoma (GBM), astrocytoma, meningioma, craniopharyngioma, medulloblastoma, other brain cancers, head and neck cancer, leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), skin cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, endometrium cancer, cancer), esophageal cancer, eye cancer, gallbladder cancer, stomach cancer, gastrointestinal cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, blood cancer, head cancer, blood malignancies, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, non-small cell lung cancer (NSCLC), small cell, lymphoma, mesothelioma, melanoma, multiple myeloma, neuroblastoma, nasopharyngeal cancer, neck cancer, ovarian cancer, osteosarcoma, sarcoma, gastrointestinal stromal tumor (GIST), pancreatic cancer, pituitary Body cancer, prostate cancer, kidney cancer, retinoblastoma, salivary gland cancer, skin cancer, stomach cancer, small intestine cancer, spleen cancer, sarcoma, testicular cancer, thymic cancer, thyroid cancer, uterine cancer, uterine sarcoma, uterine serous carcinoma (USC), uterine CS, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, solid tumors or liquid tumors, HGSOC, invasive breast cancer, triple-negative breast cancer (TNBC), esophagogastric cancer, gastric cancer, esophageal cancer, pRCC, ccRCC, suspected Chromocyte RCC, head and neck cancer, adenoid cystic carcinoma (ACC), diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma (NHL), low-grade glioma (LGG), pheochromocytoma and paraganglioma (PCPG), cholangiocarcinoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), thymoma, BRAF mutant metastatic colorectal cancer Colorectal cancer, uveal melanoma, high-grade serous ovarian cancer, fallopian tube cancer, or primary peritoneal cancer, BRAFV600E mutant colorectal cancer, platinum-sensitive ovarian cancer, poly(ADP-ribose) polymerase inhibitor (PARPi)-resistant ovarian cancer, platinum-resistant ovarian cancer, platinum-refractory ovarian cancer, advanced pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, neuroendocrine tumors, neuroendocrine prostate cancer, pancreatic neuroendocrine tumors, small cell lung cancer (SCLC), germ cell cancer, and stromal cancer.

[0232] In some embodiments, the subject has cancer. In some embodiments, the cancer is breast cancer, brain cancer, lung cancer, liver cancer, stomach cancer, spleen cancer, colon cancer, kidney cancer, pancreatic cancer, prostate cancer, uterine cancer, skin cancer, head cancer, neck cancer, sarcoma, neuroblastoma, or ovarian cancer.

[0233] In some embodiments, the cancer is glioblastoma, astrocytoma, meningioma, craniopharyngioma, medulloblastoma, and other brain cancers, leukemia, skin cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal cancer, Hodgkin's lymphoma, hematological cancer, hematological malignancies, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, lymphoma, mesothelioma, melanoma, multiple myeloma, neuroblastoma, nasopharyngeal cancer, ovarian cancer, osteosarcoma, pancreatic cancer, pituitary cancer, retinoblastoma, salivary gland cancer, stomach cancer, small intestine cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer, uterine sarcoma, uterine serous carcinoma (USC), vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, solid tumors, or liquid tumors.

[0234] In certain embodiments, cancer is a solid tumor or a hematological malignancy.In certain embodiments, cancer is a solid tumor.In certain embodiments, solid tumors are selected from endometrial cancer (endometrial cancer), gallbladder cancer, ovarian cancer, HGSOC, endometrium cancer (endometrium cancer), melanoma, colorectal cancer, bladder cancer, breast cancer, invasive breast cancer, triple negative breast cancer (TNBC), prostate cancer, lung cancer, NSCLC, SCLC, esophagogastric cancer, gastric cancer, esophageal cancer, renal cancer, pRCC, ccRCC, chromophobe cell carcinoma RCC, head and neck cancer, osteosarcoma cancer, pancreatic cancer, brain cancer, uterine CS, uterine cancer, adenoid cystic carcinoma (ACC), mesothelioma, cervical cancer, diffuse large B cell lymphoma (DLBCL), non-Hodgkin's lymphoma, liver cancer, glioblastoma (GBM), testicular cancer, low grade glioma (LGG), pheochromocytoma and paraganglioma (PCPG), bile duct cancer, thyroid cancer, thymoma and uveal melanoma.

[0235] In some embodiments, the solid tumor is ovarian cancer. In some embodiments, the ovarian cancer is epithelial ovarian cancer, germ cell cancer, or stromal cancer. In some embodiments, the ovarian cancer is epithelial ovarian cancer. In some embodiments, the epithelial ovarian cancer is high-grade serous ovarian cancer (HGSOC).

[0236] In some embodiments, the cancer is associated with "homologous recombination deficiency," "homologous recombination repair deficiency," "homologous repair deficiency," or "HRD," which refers to a reduction or impairment in the homologous recombination process. In some embodiments, the cancer is associated with increased levels of cyclin E1 or a cyclin E1 biomarker (e.g., CCNE1 gene amplified cancer, cyclin E1 overexpressing / non-CCNE1 gene amplified cancer, cyclin E1 driven cancer).

[0237] In some embodiments, the cancer has a homologous recombination deficiency (HRD) positive status. In some embodiments, the cancer is an HRD-positive cancer selected from the group consisting of ovarian cancer (including recurrent ovarian cancer), breast cancer (e.g., triple-negative breast cancer and / or metastatic breast cancer), prostate cancer (e.g., metastatic castration-resistant prostate cancer), fallopian tube cancer, and primary peritoneal cancer.

[0238] In some embodiments, the cancer is of an organ selected from the group consisting of adrenal gland, ampulla of Vater, bile duct, bladder / urinary tract, bone, intestine, breast, cervix, cns / brain, esophagus / stomach, eye, head and neck, kidney, liver, lung, lymph, bone marrow, ovary / fallopian tube, pancreas, penis, peripheral nervous system, peritoneum, pleura, prostate, skin, soft tissue, testis, thymus, thyroid, uterus, vulva / vagina, adenocarcinoma in situ, extragonadal germ cell tumor (EGCT), mixed carcinoma types, high-grade neuroendocrine ovarian cancer, high-grade serous fallopian tube carcinoma (HGSFT), ovarian choriocarcinoma, and ovarian cancer NOS (OCNOS).

[0239] In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is uterine cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is prostate cancer.

[0240] In some embodiments, the cancer is a primary cancer originating from a related organ. In some embodiments, the cancer is primary peritoneal cancer.

[0241] In some embodiments, the cancer has metastasized to related organs.

[0242] In some embodiments, the cancer is a solid tumor or a hematological malignancy.

[0243] In some embodiments, the cancer is a solid tumor.

[0244] In some embodiments, the solid tumor is selected from endometrial cancer, gallbladder cancer, ovarian cancer (e.g., HGSOC), endometrium cancer, melanoma, colorectal cancer, bladder cancer, breast cancer (e.g., invasive breast cancer, triple-negative breast cancer (TNBC)), prostate cancer, lung cancer (e.g., NSCLC, SCLC), esophagogastric cancer, gastric cancer, esophageal cancer, renal cancer (e.g., pRCC, ccRCC, chromophobe cell carcinoma RCC), head and neck cancer, osteosarcoma cancer, pancreatic cancer, brain cancer, uterine CS, uterine cancer, adenoid cystic carcinoma (ACC), mesothelioma, cervical cancer, diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma, liver cancer, glioblastoma (GBM), testicular cancer, low-grade glioma (LGG), pheochromocytoma and paraganglioma (PCPG), bile duct cancer, thyroid cancer, thymoma and uveal melanoma.

[0245] In some embodiments, the cancer is acute myeloid leukemia (AML).

[0246] In some embodiments, the tumor is a neuroendocrine tumor, a neuroendocrine prostate cancer, and a pancreatic neuroendocrine tumor.

[0247] In some embodiments, the solid tumor is ovarian cancer.

[0248] In some embodiments, the ovarian cancer is epithelial ovarian cancer, germ cell cancer, or stromal cancer.

[0249] In some embodiments, the ovarian cancer is epithelial ovarian cancer.

[0250] In some embodiments, the ovarian cancer is high-grade serous ovarian cancer (HGSOC). In some embodiments, the ovarian cancer is platinum-resistant ovarian cancer (PROC). In some embodiments, the ovarian cancer is cyclin E amplified ovarian cancer. In some embodiments, the ovarian cancer is a cyclin E1 overexpressed cancer. In some embodiments, the ovarian cancer is a cyclin E1 overexpressed / non-amplified cancer.

[0251] In some embodiments, the cancer is platinum-resistant. In some embodiments, the cancer is resistant to one or more chemotherapy regimens. In some embodiments, the cancer is refractory to one or more chemotherapy regimens.

[0252] In some embodiments, the cancer is uterine serous carcinoma (USC).

[0253] In some embodiments, the cancer is osteosarcoma.

[0254] In some embodiments, the solid tumor is uterine serous carcinoma, ovarian cancer, peritoneal cancer, fallopian tube cancer, osteosarcoma, pancreatic cancer, or BRAF mutant metastatic colorectal cancer.

[0255] In some embodiments, the cancer is acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), chronic myelomonocytic leukemia (CMML), cutaneous B-cell lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, or multiple myeloma (MM).

[0256] In some embodiments, the cancer is platinum-refractory or platinum-resistant. In some embodiments, the cancer is platinum-resistant. In some embodiments, the cancer is platinum-resistant. In some embodiments, the cancer is resistant to one or more chemotherapy regimens. In some embodiments, the cancer is refractory to one or more chemotherapy regimens.

[0257] Combination therapy

[0258] The present disclosure provides methods of using asenzelti or a pharmaceutically acceptable salt thereof in combination with one or more additional agents (e.g., combination therapy with a chemotherapeutic agent). In one aspect, the present disclosure provides a method of treating cancer comprising administering an effective dose of asenzelti or a pharmaceutically acceptable salt thereof, and a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof, to a subject selected as having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold.

[0259] Combination therapy refers to a clinical intervention in which a subject is exposed to two or more treatment regimens (e.g., assencillin or a pharmaceutically acceptable salt thereof, and a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof) simultaneously. In some embodiments, the two or more chemotherapeutic regimens can be administered simultaneously. In some embodiments, the two or more chemotherapeutic regimens can be administered sequentially (e.g., a first regimen is administered before administering any dose of a second regimen). In some embodiments, the two or more chemotherapeutic regimens are administered with overlapping dosing regimens.

[0260] In some embodiments, the combination therapy does not necessarily require that the individual agents be administered together (or even simultaneously) in the form of a single composition. In some embodiments, the two or more treatment regimens of the combination therapy (e.g., assencillin or a pharmaceutically acceptable salt thereof and a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof) are administered to the subject via separate routes of administration (e.g., one agent is administered orally and the other agent is administered intravenously) and / or separately at different time points (e.g., in the form of separate compositions). In some embodiments, the two or more chemotherapeutic agents may be administered together via the same route of administration, and / or simultaneously in the form of a combined composition, or even in the form of a combined compound (e.g., as part of a single chemical complex or covalent entity).

[0261] In some embodiments, assenseti or a pharmaceutically acceptable salt thereof and a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof are administered concurrently. In some embodiments, assenseti or a pharmaceutically acceptable salt thereof and a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof are administered sequentially. In some embodiments, assenseti or a pharmaceutically acceptable salt thereof is administered before the second chemotherapeutic agent or a pharmaceutically acceptable salt thereof. In other embodiments, assenseti or a pharmaceutically acceptable salt thereof is administered after the second chemotherapeutic agent or a pharmaceutically acceptable salt thereof. In some embodiments, assenseti or a pharmaceutically acceptable salt thereof and a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof are administered intermittently.

[0262] In some embodiments, the second chemotherapeutic agent is selected from bendamustine, bortezomib, carfilzomib, ixazomib, busulfan, carboplatin, cisplatin, cyclophosphamide, cladribine, paclitaxel, docetaxel, pegylated liposomal doxorubicin (PLD), dexamethasone, doxorubicin, gemcitabine, cytarabine, fludarabine, fluorouracil (5-FU), irinotecan, topotecan, temozolomide, triamcinolone, azacitidine, 5-azacytidine, capecitabine, AraC-FdUMP

[10] (CF-10), cladribine, etoposide, decitabine, daunorubicin, doxorubicin, ifosfamide, methotrexate, vincristine, hydroxyurea and oxaliplatin, or a pharmaceutically acceptable salt of any one of the foregoing.

[0263] In some embodiments, the cancer treatment is an alkylating agent, an anti-EGFR antibody, an anti-Her-2 antibody, an antimetabolite, a vinca alkaloid, a platinum-based agent, an anthracycline, a topoisomerase inhibitor, a taxane, an antibiotic, an immunomodulator, an immune cell antibody, an interferon, an interleukin, an HSP90 inhibitor, an anti-androgen, an anti-estrogen, an anti-hypercalcemic agent, an apoptosis inducer, an Aurora kinase inhibitor, a Bruton's tyrosine kinase inhibitor, a calcineurin inhibitor, a CaM kinase II inhibitor, a CD45 tyrosine phosphatase inhibitor, a CDC25 phosphatase inhibitor, a CHK kinase inhibitor, a cyclooxygenase inhibitor, a bRAF kinase inhibitor, a cRAF kinase inhibitor inhibitors, Ras inhibitors, cyclin-dependent kinase inhibitors, cysteine ​​protease inhibitors, DNA intercalators, DNA strand breakers, E3 ligase inhibitors, EGF pathway inhibitors, farnesyl transferase inhibitors, Flk-1 kinase inhibitors, glycogen synthase kinase-3 (GSK3) inhibitors, histone deacetylase (HDAC) inhibitors, I-κB-α kinase inhibitors, tetrazabenzimidazoles, insulin tyrosine kinase inhibitors, c-Jun-N-terminal kinase inhibitors, mitogen-activated protein kinase (MAPK) inhibitors, MDM2 inhibitors, MEK inhibitors, ERK inhibitors, MMP inhibitors, mTor inhibitors, NGFR tyrosine kinase inhibitors, p38 MAP kinase inhibitors, p56 tyrosine kinase inhibitors, PDGF pathway inhibitors, phosphatidylinositol 3-kinase inhibitors, phosphatase inhibitors, protein phosphatase inhibitors, PKC inhibitors, PKCδ kinase inhibitors, polyamine synthesis inhibitors, PTP1B inhibitors, protein tyrosine kinase inhibitors, SRC family tyrosine kinase inhibitors, Syk tyrosine kinase inhibitors, Janus (JAK-2 and / or JAK-3) tyrosine kinase inhibitors, retinoids, RNA polymerase II elongation inhibitors, serine / threonine kinase inhibitors, sterols Biosynthesis inhibitors, VEGF pathway inhibitors, chemotherapeutic agents, alitretinoin, altretinoin, aminopterin, aminolevulinic acid, amsacrine, asparaginase, atrasentan, bexarotene, carboquinone, colcemid, ethoxycycline, elsamitrucin, ethoxydimidine, hydroxyurea, folinic acid, lonidamine, lucanthone, masopropol, methylaminolevulinate, mitoguanidine, mitotane, oblimersen, omacitaxin, pegaspargase, porfimer sodium, prednimustine, adenovirus vector site-directed coding gene, talaporfin, temoporfin, trabectedin, or verteporfin.

[0264] In some embodiments, the chemotherapeutic agent is carboplatin, cisplatin, paclitaxel, docetaxel, pegylated liposomal doxorubicin, doxorubicin, gemcitabine, cytarabine, fludarabine, fluorouracil (5-FU), irinotecan, topotecan, temozolomide, triamcinolone, 5-azacitidine, capecitabine, AraC-FdUMP

[10] (CF-10), cladribine, decitabine, hydroxyurea and oxaliplatin, or a pharmaceutically acceptable salt of any one of the foregoing. In other embodiments, the chemotherapeutic agent is azacitidine, bendamustine, bortezomib, carfilzomib, ixazomib, busulfan, carboplatin, cytarabine, cyclophosphamide, cladribine, cisplatin, capecitabine, decitabine, dexamethasone, etoposide, fludarabine, gemcitabine, daunomycin, doxorubicin, ifosfamide, methotrexate, and vincristine, or a pharmaceutically acceptable salt of any one of the foregoing.

[0265] In some embodiments, the second chemotherapeutic agent is carboplatin, paclitaxel, gemcitabine, or pegylated liposomal doxorubicin (PLD), or a pharmaceutically acceptable salt of any of the foregoing.

[0266] In some embodiments, the second chemotherapeutic agent is encofenib or a pharmaceutically acceptable salt thereof. In some embodiments, the second chemotherapeutic agent is cetuximab or a pharmaceutically acceptable salt thereof. In some embodiments, the second chemotherapeutic agent consists of a combination of encofenib and cetuximab, or a pharmaceutically acceptable salt of either of the foregoing.

[0267] Dosage of the second chemotherapeutic agent

[0268] In some embodiments, the second chemotherapeutic agent is carboplatin, paclitaxel, gemcitabine, or pegylated liposomal doxorubicin (PLD), or a pharmaceutically acceptable salt of any of the foregoing.

[0269] In some embodiments, the second chemotherapeutic agent is carboplatin or a pharmaceutically acceptable salt thereof, and wherein carboplatin is administered intravenously over 15 minutes or more once during a treatment cycle at a dose of 1-10 mg / mL×min. In some embodiments, the second chemotherapeutic agent is carboplatin or a pharmaceutically acceptable salt thereof, and wherein carboplatin or a pharmaceutically acceptable salt thereof is administered intravenously over 15 minutes or more once during a treatment cycle at a dose of 3-6 mg / mL×min. In some embodiments, the second chemotherapeutic agent is carboplatin or a pharmaceutically acceptable salt thereof, and wherein the carboplatin or a pharmaceutically acceptable salt thereof is administered intravenously over 15 minutes or more once during a treatment cycle at a dose of 1-10 mg / mL×min, 2-10 mg / mL×min, 3-10 mg / mL×min, 4-10 mg / mL×min, 5-10 mg / mL×min, 6-10 mg / mL×min, 7-10 mg / mL×min, 8-10 mg / mL×min, 9-10 mg / mL×min, 2-8 mg / mL×min, 2-7 mg / mL×min, 3-7 mg / mL×min, 4-7 mg / mL×min, 5-7 mg / mL×min, 4-6 mg / mL×min, 2-6 mg / mL×min, 3-8 mg / mL×min, 9-10 mg / mL×min.

[0270] In some embodiments, the second chemotherapeutic agent is PLD or a pharmaceutically acceptable salt thereof, and wherein PLD or a pharmaceutically acceptable salt thereof is administered at a dose of 10-100 mg / m during the treatment cycle. 2 In some embodiments, the second chemotherapeutic agent is PLD or a pharmaceutically acceptable salt thereof, and wherein PLD or a pharmaceutically acceptable salt thereof is administered intravenously once over 60 minutes at a dose in the range of 5-50 mg / m2 during the treatment cycle. In some embodiments, the second chemotherapeutic agent is PLD or a pharmaceutically acceptable salt thereof, and wherein PLD or a pharmaceutically acceptable salt thereof is administered intravenously once over 60 minutes during the treatment cycle at a dose in the range of 10-40 mg / m2.

[0271] In some embodiments, the second chemotherapeutic agent is PLD or a pharmaceutically acceptable salt thereof, and wherein PLD or a pharmaceutically acceptable salt thereof is administered at a dose of 10-100 mg / m during the treatment cycle. 2 10-90 mg / m 2 10-80 mg / m 2 10-70 mg / m 2 10-60 mg / m 2 10-50 mg / m 210-40 mg / m 2 10-30 mg / m 2 10-20 mg / m 2 20-90 mg / m 2 30-90 mg / m 2 40-90 mg / m 2 50-90 mg / m 2 , 60-90mg / m 2 70-90 mg / m 2 20-80 mg / m 2 20-70 mg / m 2 20-60 mg / m 2 20-50 mg / m 2 20-40 mg / m 2 or 30–40 mg / m 2 Doses within the range were administered once intravenously over 60 minutes.

[0272] In some embodiments, the second chemotherapeutic agent is paclitaxel or a pharmaceutically acceptable salt thereof, and wherein paclitaxel or a pharmaceutically acceptable salt thereof is administered intravenously three times over 60 minutes (+10 minutes) during a treatment cycle at a dose in the range of 10-120 mg / m2. In some embodiments, the second chemotherapeutic agent is paclitaxel or a pharmaceutically acceptable salt thereof, and wherein paclitaxel or a pharmaceutically acceptable salt thereof is administered intravenously three times over 60 minutes (+10 minutes) during a treatment cycle at a dose in the range of 10-120 mg / m2. 2 20-100mg / m 2 30-100mg / m 2 40-100 mg / m 2 50-100mg / m 2 , 60-100mg / m 2 70-100mg / m 2 80-100mg / m 2 90-100mg / m 2 10-90 mg / m 2 10-80 mg / m 2 10-70 mg / m 2 10-60 mg / m 2 10-50 mg / m 2 10-40 mg / m 2 10-30 mg / m 2 30-70 mg / m 2 40-70 mg / m 2 50-70 mg / m 2, 60-70mg / m 2 30-90 mg / m 2 30-80 mg / m 2 Doses within the range were administered intravenously three times over a period of up to 3 hours.

[0273] In some embodiments, the second chemotherapeutic agent is paclitaxel or a pharmaceutically acceptable salt thereof, and wherein paclitaxel or a pharmaceutically acceptable salt thereof is administered intravenously up to three times over up to 3 hours during a treatment cycle at a dose in the range of 40-100 mg / m2.

[0274] In some embodiments, the second chemotherapeutic agent is gemcitabine or a pharmaceutically acceptable salt thereof, and wherein gemcitabine or a pharmaceutically acceptable salt thereof is administered at a dose of 500-1500 mg / m during a treatment cycle. 2 Doses within the range are administered once intravenously over a period of 15 minutes or longer.

[0275] In some embodiments, the second chemotherapeutic agent is gemcitabine or a pharmaceutically acceptable salt thereof, and wherein gemcitabine or a pharmaceutically acceptable salt thereof is administered at a dose of 100 to 1000 mg / m during a treatment cycle. 2 100 to 1000 mg / m 2 100 to 900 mg / m 2 100 to 800 mg / m 2 100 to 700 mg / m 2 100 to 600 mg / m 2 100 to 500 mg / m 2 100 to 400 mg / m 2 100 to 300 mg / m 2 100 to 200 mg / m 2 200 to 1000 mg / m 2 300 to 1000 mg / m 2 400 to 1000 mg / m 2 500 to 1000 mg / m 2 , 600 to 1000 mg / m 2 700 to 1000 mg / m 2 800 to 1000 mg / m 2 200 to 800 mg / m 2 200 to 700 mg / m 2 200 to 600 mg / m 2 200 to 500 mg / m 2 300 to 900 mg / m 2 300 to 800 mg / m2 400 to 700 mg / m 2 500 to 700 mg / m 2 500 to 800 mg / m 2 , 600 to 900 mg / m 2 Doses within the range were administered intravenously up to 3 times over 15 minutes or longer.

[0276] In some embodiments, the second chemotherapeutic agent is gemcitabine or a pharmaceutically acceptable salt thereof, and wherein gemcitabine or a pharmaceutically acceptable salt thereof is administered at a dose of 100 to 1000 mg / m during a treatment cycle. 2 Doses within the range were administered intravenously up to 3 times over 15 minutes or longer.

[0277] Reactivity

[0278] In some embodiments, the treatment methods described herein result in a response rate equal to or greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, the response rate is measured by complete response (CR), partial response (PR), CA-125 50% response, or a combination thereof. In some embodiments, the response is determined based on progression-free survival. In some embodiments, the response is determined based on tumor response. In some embodiments, the response is determined based on clinical benefit rate (CBR). In some embodiments, the response is determined based on disease control rate (DCR). In some embodiments, the response is determined based on overall survival (OS).

[0279] Progression-free survival (PFS) refers to the period of time during which a subject with a disease (e.g., cancer) survives and the disease state does not significantly worsen. Progression-free survival can be assessed as the period of time during which there is no progression of tumor growth and / or during which the subject's disease state is not determined to be a progressive disease. In an embodiment, progression-free survival of a subject with cancer is assessed by assessing tumor size, number of tumors, and / or metastasis.

[0280] In some embodiments, treatment results in a progression-free survival (PFS) of 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more. In some embodiments, treatment results in a progression-free survival (PFS) of 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or more. In some embodiments, treatment results in a median progression-free survival (mPFS) of 1 year, 1.5 years, 2 years, 2.5 years or more.

[0281] As used herein, the term "progression" of tumor growth, or the term "progressive disease" (PD) as used herein with reference to a cancer state, refers to an increase in the sum of the diameters of the tumors of interest. For the purpose of determining progression-free survival, progression may also be determined if at least one of the following criteria is met: 1) unequivocal demonstration of progressive disease by CT / MRI tumor assessment according to RECIST 1.1 criteria; or 2) identification of a new tumor or confirmation of an existing tumor by additional diagnostic testing (e.g., histology / cytology, ultrasound, endoscopy, positron emission tomography) according to the Gynecologic Cancer Intergroup (GCIG) criteria (see Rustin et al., Int J Gynecol Cancer 2011;21:419-423, which is incorporated herein in its entirety); or 3) definitive clinical signs and symptoms of PD unrelated to non-malignant or iatrogenic causes according to the GCIG criteria ([i] refractory cancer-related pain; [ii] worsening malignant bowel obstruction / dysfunction; or [iii] unequivocal symptomatic worsening of ascites or pleural effusion) and / or CA-125-progression.

[0282] As used herein, the term "partial response" or "PR" refers to a decrease in tumor progression in a subject, as indicated by a decrease in the sum of the diameters of the target tumor, taking the baseline total diameter as a reference. In an embodiment, PR refers to a decrease in the sum of the diameters by at least 30%, taking the baseline total diameter as a reference. Exemplary methods for assessing partial responses are identified by RECIST guidelines. See EA Eisenhauer et al., "New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1.)," Eur. J. of Cancer, 45: 228-247 (2009).

[0283] As used herein, tumor growth "stabilization" or "stable disease" (SD) refers to neither shrinking enough to meet PR nor increasing enough to meet PD. In an embodiment, stability refers to a change (increase or decrease) of less than 30%, 25%, 20%, 15%, 10%, or 5% in the sum of the diameters of the target tumors, taking the baseline total diameter as a reference. Exemplary methods for assessing tumor growth stabilization or stable disease are identified by RECIST guidelines. See EA Eisenhauer et al., "New response evaluation criteria insolid tumors: Revised RECIST guideline (version 1.1.)," Eur. J. of Cancer, 45: 228-247 (2009).

[0284] As used herein, the term "complete response" or "CR" is used to refer to the disappearance of all or substantially all target lesions. In an embodiment, CR refers to the reduction in the diameter sum of the target tumor (i.e., tumor disappearance) by about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% when the baseline total diameter is considered as a reference. In an embodiment, CR indicates that after treatment, there is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less total lesion diameter remaining. The exemplary method for assessing complete response is identified by RECIST guidelines. See EA Eisenhauer et al., " New response evaluation criteria in solid tumors: Revised RECIST guideline (version 1.1.), " Eur. J. of Cancer, 45: 228-247 (2009).

[0285] Examples

[0286] Additional embodiments are disclosed in further detail in the following examples, which are not intended to limit the scope of the claims in any way.

[0287] Example 1. Cancer cell lines overexpressing cyclin E1 are sensitive to asenzeltin

[0288] Overexpression of cyclin E1 (encoded by the CCNE1 gene) is associated with increased sensitivity of ovarian cancer cell lines to the WEE1 inhibitor assencillin. Figure 1A As shown, assencillin sensitivity correlated with cyclin E1 protein expression in OV90, Kuramochi, TYK-nu, and OVCAR3 cells. Protein expression and assencillin sensitivity were assessed by CellTiter Glo after 96 hours of culture.

[0289] To confirm whether cyclin E1 expression levels cause increased sensitivity to assensertin, OV90, Kuramochi, and COV362 cells (which have low endogenous expression levels of cyclin E1) were transduced with a lentiviral vector carrying the CCNE1 gene or with an empty vector control. Stable OV90, Kuramochi, and COV362 cell lines were established by puromycin selection. Cyclin E1 overexpression in the stable OV90 cell line was confirmed by Western blotting compared to empty vector control cells ( Figure 1C ).like Figure 1B As shown in , overexpression increased sensitivity to assencillin. Figure 1DIn this study, we evaluated the sensitivity of HGSOC cell lines to assencillin due to cyclin E1 overexpression by plotting growth rate (GR) inhibition. We observed that cyclin E1 overexpression sensitized HGSOC cell lines to assencillin by further reducing growth rate and cell viability compared to the control empty vector cell line ( Figure 1D The GR of Asenseti was determined using the CellTiter-Glo (CTG) assay and the GR calculator. 50 and IC 50 value.

[0290] Similar assays were performed in other cell lines, e.g., JOM1, ES2, TYK-nu, CAOV3, OAW28, OVCAR4, OVCAR3. OVCAR3 (CCNE1 amplification, CN=12), OAW28, and OVCAR4 cells expressed high levels of cyclin E1, as determined by Western blot (WB) and immunohistochemistry (IHC). Cyclin E1 expression was normalized to focal adhesion protein. Figure 1E Cyclin E1 protein overexpression status was shown by Western blotting. These values ​​were plotted using the H score as a function of cyclin E1 levels ( Figure 1F ). Figure 1G Graph showing changes in GR values ​​as a function of cyclin E1 levels.

[0291] The growth rate (GR) inhibition of HGSOC cells was assessed in the presence of assensettin (relative to untreated controls). GR values ​​were independent of cell division rate and were determined as described in Hafner et al. (Curr. Protoc. Chem. Biol. 9, 96-116 (2017)). HGSOC cells with high cyclin E1 protein expression were most sensitive to assensettin. Cells with low cyclin E1 expression or cells without cyclin E1 expression showed reduced sensitivity or resistance to assensettin. ( Figure 2 In cells treated with assensetin, inhibitory effects were observed in OV90 and Kuramochi cells, cytotoxic effects were observed in ES2 and TYK-nu cells, and cytotoxic effects were observed in COV362, JHOMI, OAW28, OVCAR3, CAOV3, and OVCAR4 cells. Ovarian cancer cell lines overexpressing cyclin E1 protein (cyclin E1-high) were more sensitive to assensetin, inducing significant cytotoxic effects (GR) compared to ovarian cancer cell lines with lower levels of cyclin E1 protein (cyclin E1-low). max In cyclin E1-low ovarian cancer cell lines, lentiviral overexpression of cyclin E1 increased sensitivity to assencillin.

[0292] Taken together, these data indicate that high cyclin E1 protein expression is associated with sensitivity to assencillin and that artificial overexpression of cyclin E1 in cell lines with low endogenous cyclin E1 expression sensitizes cells to assencillin.

[0293] Example 2. CDK2-dependent sensitivity of cyclin E1-high HGSOC to asenzeltin

[0294] The role of CDK2 in sensitizing cyclin E1-high HGSOC to assencillin was assessed by knocking down CDK2 protein expression using two different siRNAs. Downregulation of CDK2 was assessed by Western blotting at 3 and 6 days after transfection ( Figure 3A Three days after CDK2 siRNA transfection, OVCAR4 cells were treated with ascension for three days. Viability and growth rate values ​​were determined using the CellTiter-Glo assay and a growth rate calculator. Figure 3B and Figure 3C The results showed that the percentage of viability and growth rate after siRNA treatment was reduced after treatment with asenzeltin. The sensitivity of cyclin E1-high HGSOC to asenzeltin was observed to be CDK2-dependent.

[0295] These data provide fundamental details on the mechanistic basis for the sensitivity of cyclin E1 to Wee1 inhibition, including that cyclin E1 protein overexpression leads to the accumulation of replication stress biomarkers and that assensitivity is mediated by CDK2 activity.

[0296] Example 3. Effect of Asensertib in a Cyclin E1-High Tumor Mouse Model with Increased Replication Stress

[0297] This example demonstrates that the stronger antitumor effect of assensertin in a cyclin E1-high tumor model is associated with increased replication stress.

[0298] NOD / SCID mice (SKOV3-cyclin E1-low and OVCAR3-cyclin E1-high) or BALB / c nude mice bearing the corresponding tumors (HCC1806) were orally administered daily for Figure 5A and 8A Treatment was well tolerated and changes in tumor volume were plotted relative to the time of administration. Baseline cyclin E1 protein expression was examined in each model by IHC and is shown in Figure 4A Figure 2. Target engagement of assencillin (pCDK1 reduction) and γH2AX in tumors was examined by IHC 12 hours after assencillin treatment in SKOV3-cyclin E1-low and OVCAR3-cyclin E1-high cells. The Y axis represents the sum of the H scores assessed by 3 independent pathologists ( Figure 4B and4C Replication stress markers pCHK1 and γH2AX were determined by Western blotting in OVCAR3 tumor samples with or without 12 h asenzel treatment ( Figure 4D ).

[0299] Example 4. Mouse model with CCNE1-amplified cell line-derived xenografts (CDX) demonstrates efficacy against Asenia Increased sensitivity to .

[0300] This example demonstrates that treatment with asensertib results in a reduction in tumor volume and that increased CCNE1 gene amplification is associated with increased sensitivity in the CDX model.

[0301] SKOV3 cells (non-CCNE1 amplification, wild-type CCNE1CN=2) were used to co-culture with 95% viable tumor cells (1×10 7 ) were inoculated into the right flank of mice with a serum-free monocytic suspension in 100 μL of McCoy's 5a and Matrigel mixture (1:1 ratio) for tumor development. Animals were randomly divided into groups and the average tumor volume reached 215 mm 3 At 4 hr, mice were treated with either vehicle (20% HP-β-CD) or 80 mg / kg of assencillin daily for 21 days. Tumor growth inhibition (TGI) was calculated using the following equation: TGI = (1-(Td-T0) / (Cd-C0)) x 100%. Td and Cd are the mean tumor volumes of treated and control animals, and T0 and C0 are the mean tumor volumes of treated and control animals at the start of the experiment. Mice treated with assencillin showed reduced tumor growth ( ) compared to vehicle-treated control animals. Figure 5A and 5B ). As summarized in Table 1 below and Figure 5C and 5D Additional doses and schedules are included as shown in the Table 1. Treatment with 80 mg / kg of asenzeltin daily resulted in a TGI of 28 days. (80mpk，第28 sky ) The average tumor growth inhibition was 51.5%. Similarly, the CDX model of OVCAR8 (non-CCNE1 amplification) was administered with 80 mg / kg of asenciltin daily for 39 days, and the results showed that TGI (80 mpk, day 28 ) The average tumor growth inhibition was 52.8%. ( Figure 6A and 6B These results indicate that assencillin moderately inhibits tumor growth in a non-CCNE1 amplified ovarian model.

[0302] Table 1. Asenzeltiel dosage and schedule

[0303]

[0304]

[0305] 1×10 6 HCC1806 human triple-negative breast cancer cells (CCNE1 amplification, CN=7) were inoculated into the right flank of ten 6-8 week old female BALB / c nude mice. The animals were randomly divided into groups and the average tumor volume reached 155 mm. 3 Treatment was started at 4 hr. Animals were treated daily with vehicle (20% HP-β-CD) or 80 mg / kg of asenzeltin, and tumor volume and body weight were measured twice a week. Mice treated with asenzeltin showed significantly reduced tumor growth (63.5%) after 28 days compared to mice treated with vehicle ( Figure 7A and 7B ).

[0306] OVCAR-3 cells (CCNE1 expanded, CN=14) were inoculated into the right flank of 6-8 week old female NOD / SCID mice. Animals were randomly divided into groups and stratified when the mean tumor volume reached 111 mm. 3 At 28 days, mice treated with 80 mg / kg of asenzeltin vehicle (20% HP-β-CD) or 80 mg / kg of asenzeltin were treated daily. Tumor volume and body weight were measured twice a week. Mice treated with asenzeltin showed significantly reduced tumor growth (88%) compared to vehicle controls after 28 days (TGI (80mpk，第28天) )( Figure 8A and 8B A reduction in tumor growth was also observed in animals treated with 40 mg / kg of asenzel per day, 60 mg / kg of asenzel per day, and 80 mg / kg of asenzel per day. Figure 8C and 8D ).

[0307] Taken together, these results suggest that CCNE1 gene amplification (eg, high copy number) results in increased efficacy of asenzeltine treatment (eg, improved tumor growth inhibition).

[0308] Example 5. CCNE1 gene amplification increases sensitivity to the combination of asenzeltib and a second chemotherapeutic agent

[0309] The sensitivity of ovarian cancer cell lines to the combination of asenzeltib and gemcitabine was evaluated. Figure 9 As shown, in OVCAR3 (CCNE1 amplified, CN=14), 37% of the tested combination conditions exhibited synergistic effects, compared with 16% and 21% in OV90 (non-CCNE1 amplified, CN=2) and OVCAR8 (non-CCNE1 amplified, CN=2), respectively.

[0310] To evaluate the effect of asenzeltiel in combination with paclitaxel, A2780 (non-CCNE1 amplified, CN=2) ( Figure 10A and 10B ) or OVCAR3 (CCNE1 amplification, CN=14) ( Figure 11A and Figure 11B ) cells were inoculated into mice. Animals were randomized into groups and, once the mean tumor volume reached the designated size, they were treated as outlined in Table 2. Mice treated with ascension and paclitaxel showed a reduction in tumor growth compared to vehicle-treated control animals. Tumor regression was observed in animals with CCNE1-amplified tumors, as shown in Table 2. Figure 11A and 11B As shown in Table 2, the cyclin E1-high OVCAR3 model (46% reduction in initial tumor volume / 104% TGI) and the cyclin E1-low A2780 model (85% TGI) were significantly more sensitive to combination therapy (e.g., asenzeltine and paclitaxel) than non-CCNE1-amplified tumors.

[0311] Table 2. Dosing schedule for the asencilti and paclitaxel combination study

[0312]

[0313] Cyclin E1-high OVCAR3 cells showed greater synergistic effects than cyclin E1-low OV90 and TYK-nu cells in all chemotherapy and assencillin combinations (Loewe synergy score >10 for synergy and <-10 for antagonism) ( Figure 11C ). The drug combination effect was evaluated by measuring cell viability and 4 calculation methods (ZIP, Bliss, Loewe and HSA) according to the SynergyFinder guide. The Loewe score is consistent with other methods for calculating synergy. For visualization purposes, the score is capped at 30. Each plot represents the summary of 3 to 6 replicates. The chemotherapy concentrations were rank transformed and their ranges are cell line and chemotherapy specific: oxaliplatin: 0-10μM (OVCAR3, OV90), 0-3.3μM (TYK-nu); paclitaxel: 0-0.005μM (OVCAR3), 0-0.02μM (OV90, TYK-nu); gemcitabine: 0-1μM (OVCAR3), 0-0.02μM (OV90), 0-0.01μM (TYK-nu).

[0314] Example 6. Clinical Trial for Treating Cancer in Subjects with CCNE1 Gene Amplification

[0315] A clinical trial was conducted to evaluate the effectiveness of asenzeltib in combination with a second chemotherapeutic agent in human subjects with CCNE1 gene amplification. The selected subjects had high-grade serous ovarian cancer (HGSOC) that was platinum-resistant or refractory, had received one or two prior chemotherapies, and had measurable disease. The subjects were administered asenzeltib in combination with PLD, carboplatin, paclitaxel, and gemcitabine.

[0316] Cyclin E1 expression levels were determined using immunohistochemistry (IHC) and summarized as an H score for each tumor sample from 63 subjects enrolled in the clinical study. Of the 63 subjects included in the IHC test, a total of 58 subjects were evaluated for best overall response (BOR), including partial response or PR (N=17), stable disease or SD (N=34), progressive disease or PD (N=7), and unevaluable NE (N=5). These included subjects from all four arms of the study: carboplatin (N=18), paclitaxel (N=11), PLD (N=28), and gemcitabine (N=6). Each IHC microscopy image was reviewed by a board-certified pathologist and used to assess cyclin E1 expression levels. Epithelial cells from the tumor area were classified according to cyclin E1 staining intensity (SI): absent (SI=0), low (SI=1+), moderate (SI=2+), and high (SI=3+). ( Figure 12A ) Calculate a summary H score from the proportion of cells in each category weighted by their SI:

[0317] H score = f1 + 2 × f2 + 3 × f3

[0318] where fi is the fraction of cells with SI = i (where i = 1+, 2+, or 3+)

[0319] Best overall response was assessed using the (PR) versus (PD+SD+NE+uPR+) criteria. Subjects with a cyclin E1 H score greater than 130 were more likely to respond (ORR = 47% vs. 16%, p = 0.01 Fisher's exact test), and PR subjects had higher cyclin E1 H scores ( Figure 12B and 12C ). Subjects were evaluated based on the change in tumor size from baseline. Figure 13AAs shown in , subjects with intermediate (70-130) and high (>130) cyclin E1 H scores exhibited superior tumor responses compared to subjects with low cyclin E1 H scores (<70). Subjects with the highest expression (H score>130) had a greater tumor response (-34% vs. -12%, p=0.001 Wilcoxon test). Low expression subjects (H<70, N=10) had a significantly shorter progression-free survival (PFS) (3.25 vs. 10.35 months, p=0.0027 log-rank test ( Figure 13B Overall responders ( Figure 13C ) or CA125 responders ( Figure 13D ) scores decreased with increasing H scores. Based on the best correlation with tumor response (high group) and PFS (low group), the groups ( Figure 13E ).

[0320] Example 7. Clinical trial for treating cancer in subjects with CCNE1 gene amplification.

[0321] This example describes a clinical trial evaluating the safety and efficacy of asensertib in combination with a second exemplary chemotherapeutic agent in subjects with metastatic high-grade epithelial ovarian, peritoneal, or fallopian tube cancer (EOC) after two or fewer lines of chemotherapy (including platinum chemotherapy, in some embodiments).

[0322] Azenosertib is administered continuously or intermittently once daily in a 21 or 28 day cycle together with a second chemotherapeutic agent. In some embodiments, the second chemotherapeutic agent is selected from pegylated liposomal doxorubicin, carboplatin, paclitaxel and gemcitabine. In addition to clinical activity, this study is designed to evaluate safety and determine the maximum tolerated dose of each combination. In one embodiment, Azenosertib is tested in combination with paclitaxel. Azenosertib is administered orally once daily in a 28 day treatment cycle in two phases with an intermittent dosing regimen (starting with 200 mg QD 5 days of administration / 2 days of withdrawal (5 / 2), followed by 300 mg QD 5 / 2). Pacific paclitaxel is administered at 80 mg / m2 on D1, D8, and D15 of each 28 day cycle. 2 The dose was administered intravenously over 60 minutes (± 10 minutes).

[0323] In one embodiment, azenosertib was tested in combination with carboplatin. Azenosertib was administered orally once daily in four doses over a 28-day treatment cycle using an intermittent dosing regimen (starting with a second dose of 300 mg QD 5 / 2, followed by a second dose of 200 mg QD 5 / 2). Carboplatin was administered intravenously at 5 mg / mL x min on day 1 of each 21-day cycle (± 3 days) for 15 minutes or longer.

[0324] In one embodiment, Azenosertib was tested in combination with gemcitabine. Azenosertib was administered orally once daily in a 28-day treatment cycle in four doses with an intermittent dosing regimen (starting with three doses of 200 mg QD, followed by 200 mg QD 5 / 2). Gemcitabine was administered in two intravenous doses of 1000 mg / m on the 1st and 8th days of each 21-day cycle. 2 and 600 mg / m 2 Administer intravenously over 30 minutes or more.

[0325] In one embodiment, Azenosertib was tested in combination with pegylated liposomal doxorubicin (PLD). Azenosertib was administered orally once daily in a 28-day treatment cycle in three doses with an intermittent dosing regimen (starting with 200 mg QD, followed by 400 mg QD 5 / 2). PLD was administered every 4 weeks at 40 mg / m2 on day 1 of each 28-day cycle. 2 The dose was administered intravenously over 60 minutes.

[0326] The endpoints were to determine the recommended phase 2 dose (RP2D), safety, and preliminary clinical activity. Based on these clinical studies, the RP2D was determined to be: (a) asenzeltiel 300 mg QD 5 / 2 with paclitaxel 80 mg / m 2 (on D1, D8, and D15 of a 28-day cycle); (b) a combination of 200 mg QD 5 / 2 and carboplatin AUC 5 mg / mL×min (on D1 of a 21-day cycle); (c) a combination of 400 mg QD 5 / 2 and PLD 40 mg / m 2 The combination of asenzeltib and gemcitabine has sustained activity and is being studied in dose-cohorts to determine the maximum tolerated dose (MTD).

[0327] The overall response rate and median progression-free survival of the study are shown in Table 3 below. Overall response rate (ORR) refers to the proportion of subjects in the trial whose tumors were significantly reduced or destroyed after treatment. Median progression-free survival (mPFS) refers to the length of time from the date of diagnosis or the start of treatment to when half of the subjects diagnosed with the disease or tumor are still alive. It provides an indication of treatment success.

[0328] Table 3. Combination therapy of asenciltin and chemotherapeutic agents

[0329]

[0330] Of the 103 subjects enrolled, 26.6% had a partial response, and the median progression-free survival was 9.03 months (95% CI: 5.52-11.01). The results showed that assencillin and paclitaxel demonstrated the highest overall response rate (ORR) (9 / 18 (50%)), followed by carboplatin (9 / 27 (33.3%)).

[0331] The overall response rate for assenserti was 14.3% when administered in combination with pegylated liposomal doxorubicin or when administered in combination with gemcitabine (5 / 35, 2 / 14, respectively).

[0332] Cyclin E1 expression data of 80 subjects were evaluated by immunohistochemistry (IHC), and higher cyclin E1 (using a threshold of H score> 50) was associated with a higher overall response rate (ORR=31.3% vs. 7.7%) and a longer progression-free survival (PFS=10.35 vs. 3.25 months, HR=0.3). For example, a hazard ratio (HR) of 0.5 means that half of the subjects in the active group had adverse events at any time point compared to placebo. The frequently observed ≥ grade 3 treatment-emergent adverse events (TEAEs) (%) were neutropenia (44.4), thrombocytopenia (30.3), anemia (12.1), leukopenia (11.1), fatigue (10.1), diarrhea (6.1), nausea (5.1), and vomiting (5.1).

[0333] At another time point, 115 subjects were enrolled in the study, and 94 subjects were evaluable for efficacy, with a median progression-free survival (mPFS) of 9.0 months (95% CI: 5.8-13.7). The combination of assensertib and paclitaxel showed the highest confirmed ORR of 50% (mPFS of 7.4m), followed by gemcitabine 38.5% (mPFS of 10.4m), carboplatin 35.7% (mPFS of 8.3m) and PLD 19.4% (mPFS of 6.3). A total of 82 response-evaluable subjects had available cyclin E1 expression data by IHC testing. Cyclin E1-positive status (H score>50) was associated with higher ORR and longer PFS (ORR=40.0% vs. 8.3%; PFS=9.86 vs. 3.25 months, HR=0.37; P value was 0.0078). Frequently occurring grade ≥3 related TEAEs (%) in the intermittent asenzel treatment group were thrombocytopenia (12.2), neutropenia (11.3), anemia (7.0), fatigue (4.3), nausea (1.7), vomiting (1.7), and diarrhea (0.9).

[0334] Subject samples were also evaluated for CCNE1 gene amplification. Figures 14A-14C As shown, an H score > 50 includes all CCNE1 amplified tumors. The H score was calculated by multiplying the percentage of cells (0 to 100%) by the intensity of cyclin E1 expression (0, 1, 2+, 3+).

[0335] Subjects were classified as either cyclin E1-positive (H>50) or cyclin E1-high (H>135, the lowest score observed in samples with CCNE1 gene amplification). 90% (151 / 167) were cyclin E1-positive, 59% (99 / 167) were cyclin E1-high, 9% (13 / 141) of the evaluable samples were CCNE1 amplified, and 85% (71 / 84) of the cyclin E1-high evaluable samples were not CCNE1 amplified. Cyclin E1-positive expression was highly prevalent, including in subjects without CCNE1 gene amplification. CCNE1 transcript levels were assessed in subject samples and were highly correlated with cyclin E1 H scores (rho=0.5, p<0.001) ( Figure 14D ).

[0336] Cyclin E1 status, including CCNE1 gene amplification, predicted benefit of asenzeltib in addition to chemotherapy, suggesting that asenzeltib restored chemotherapy sensitivity in heavily pretreated platinum-resistant ovarian cancer.

[0337] These results show that the combination of asensertib and chemotherapeutic agents is well tolerated and clinically active, with durable responses in subjects with platinum-resistant or refractory metastatic high-grade epithelial ovarian, peritoneal, or fallopian tube cancer. Subjects with cyclin E1-overexpressing tumors (cyclin E1-positive), a subgroup known to benefit less from chemotherapy, showed significant improvements in ORR and PFS compared to subjects with tumors that had low cyclin E1 expression (cyclin E1-negative). Figure 14E is an exemplary image of a tumor cell designated as cyclin E1-positive, and Figure 14F are exemplary images of tumor cells designated as cyclin E1-negative.

[0338] The combination of asencil and chemotherapy showed strong anti-tumor activity in the heavily pre-treated population, with an ORR of 50% in combination with paclitaxel, 35.7% in combination with carboplatin, and 38.5% in combination with gemcitabine. Asencil had a higher objective response rate than previous chemotherapy alone or in combination with other WEE1 inhibitors. Regardless of the type of chemotherapy used, the response to the asencil combination increased. Subjects with cyclin E1-positive tumors (e.g., H-score greater than 50) benefited from the chemotherapy combination arm, illustrating the synergistic effect between asencil and chemotherapy in this subject population. Figures 14G to 14J As shown, subjects with an IHC H score greater than 50 or a percentage of viable tumor cells with a cyclin E1 IHC staining intensity of 2+ greater than 10% achieved a partial response after receiving the combination therapy with asensertib. Figure 14K Figure 2 is a graph mapping the percentage of viable tumor cells with a staining intensity of 2+ to the H-score cutoff for cyclin E1 immunohistochemistry (IHC), showing that an H-score of >50 across the subject population is associated with >10% of viable tumor cells having a staining intensity of 2+, and an H-score of >125 across the subject population is associated with >30% of viable tumor cells having a staining intensity of 2+.

[0339] Asencil is well tolerated in combination with various types of chemotherapy and has shown encouraging clinical activity, including durable objective responses in subjects with platinum-resistant ovarian cancer. The addition of Asencil increases the objective response rate (ORR) and median progression-free survival (mPFS) compared to historical observations of chemotherapy alone or chemotherapy combined with Asencil. Particularly promising is that improvements in ORR and mPFS were observed in subjects with cyclin E1-positive tumors, who are considered to be a subgroup with poor prognosis and difficult to treat by chemotherapy. In addition, the tolerability and durable efficacy of Asencil in combination with paclitaxel or carboplatin are favorable compared to historical data of paclitaxel-carboplatin or PLD-carboplatin doublet chemotherapy.

[0340] Example 8. Cyclin E1-Positive Status in High-Grade Serous Ovarian Cancer Is Not Associated with Prior Platinum Therapy

[0341] HGSOC tumor samples from 167 subjects were obtained from an ongoing clinical trial of Asenseti (N=111) (NCT04516447) that evaluates the combination of Asenseti and chemotherapy in subjects with platinum-resistant ovarian cancer, peritoneal cancer, or fallopian tube cancer. Cyclin E1 protein expression levels were measured by immunohistochemistry using an anti-cyclin E1 mouse monoclonal antibody (Abcam Cyclin E1 / 2460). The H score is defined as the percentage (pc) of cells stained with increased intensity or the weighted sum of scores (1×pc1+2×pc2+3×pc3). Subjects were classified as cyclin E1-positive (H>50, which is the predictive threshold previously reported in this study) or cyclin E1-high (H>135, which is the lowest score observed in samples with CCNE1 gene amplification).

[0342] CCNE1 gene copy number (N=141) and homologous recombination and repair (HRR) gene mutation status (N=86) were obtained from tissue-based genomic profiling. CCNE1 gene amplification status (amplified, unamplified, or non-amplified) was obtained from clinical testing or using a minimum of 6 copies to determine gene amplification (research testing). Transcript abundance (N=49) was obtained using the central Caris MI profile test and measured as transcripts per million reads (TPM). Clinical and pathological variables (including treatment modalities and outcomes) were primarily obtained from clinical data in this study and supplemented when available by data associated with the obtained samples (age, collection method, etc.). There was no statistically significant association between the histological variables examined (anatomical location, collection site, collection method, tumor cellularity, accepted form, or tissue age at IHC testing) and cyclin E1 IHC H scores (data not shown). The distribution of cyclin E1 expression measured by IHC was independent of pre-analysis variables.

[0343] Platinum response categories were established based on analysis of the duration and response of previous systemic therapy ( Figure 15A Subjects whose specimens met any of the following criteria were classified as platinum-sensitive at the time of specimen collection (PS-aCT, N=63 / 107):

[0344] Samples collected before any platinum exposure

[0345] Subjects whose samples were collected after the first platinum exposure but before subsequent platinum therapy and who had a platinum-free interval (PFI) of at least 6 months after the last dose

[0346] ● Subjects whose samples were collected during their first or second platinum treatment period and who have a PFI of at least 6 months after the last dose

[0347] ● Subjects undergoing intervening surgery between neoadjuvant and adjuvant platinum therapy and with a PFI of at least 6 months after the last dose

[0348] All other evaluable cases were classified as platinum-resistant / refractory at the time of collection (PR-aCT, N=44 / 107). Figure 15B As shown, platinum exposure, response, or HRR mutation status had no significant impact on the classification of subjects based on cyclin E1 expression.

[0349] Example 9. Treatment of a subject selected for having a cyclin E1 biomarker level above a predetermined threshold The subject's cancer

[0350] As described above, CCNE1 gene amplification and / or cyclin E1 expression serve as markers to enrich the subject population for treatment with assensert. These data demonstrate that assensert drives cancer cell death in cyclin E1-high tumor cells in vitro and substantially inhibits the growth of an in vivo tumor model derived from cyclin E1-high subjects. Additionally, these data support the use of CCNE1 gene copy number and / or cyclin E1 protein expression as predictive markers to significantly improve subject outcomes by enabling the selection of optimal subjects for treatment with assensert.

[0351] This example demonstrates the use of an effective dose of assensertin (e.g., 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 450 mg, or 600 mg) alone or in combination with a second chemotherapeutic agent to treat subjects selected for having a predetermined cyclin E1 status or a cyclin E1 biomarker level above a predetermined threshold. Subjects are selected based on whether the subject's tumor tissue has: (1) CCNE1 gene amplification (e.g., copy number equal to or greater than 5); (2) cyclin E1 overexpression (e.g., mRNA or IHC H score greater than 50 or the percentage of viable tumor cells with cyclin E1 IHC staining intensity of 2+ greater than 10%). Additional selection criteria may include subjects having a specific cancer type (eg, high-grade serous ovarian cancer (HGSOC)), being platinum-resistant or refractory, or having received 1-3 prior lines of therapy (eg, bevacizumab).

[0352] Subjects were also selected based on the following: having high-grade serous ovarian cancer; ECOGPS 0-1; being platinum-resistant (excluding platinum-refractory); having received 1-3 prior lines of chemotherapy; having measurable disease according to RECIST v1.1; being cyclin E1-positive (e.g., IHC+) and / or being CCNE1 amplified.

[0353] This phase 3 study compared asenzeltib plus carboplatin or paclitaxel with traditional doublet chemotherapy in patients with platinum-sensitive ovarian cancer. The phase 3 trial focused on cyclin E1-positive ovarian cancer and was supported by positive phase 1b clinical data. Patients with cyclin E1-positive tumors have been shown to be refractory to chemotherapy alone and generally have a poor prognosis.

[0354] The subjects were administered 400 mg QD of asenzel for 5 days in a week, followed by 2 days without asenzel (5:2). The ORR of the subjects was evaluated after treatment and divided into the following treatment groups:

[0355] Cohort 1 (N=30) subjects were confirmed to be cyclin E1-positive (e.g., IHC+) and / or CCNE1 amplified

[0356] Cohort 2A (N=60) subjects had confirmed CCNE1 amplification.

[0357] Cohort 2B (N=80) subjects had non-CCNE1 amplified tumors and were cyclin E1-positive (IHC+).

[0358] Cohort 2C (N=40) subjects had non-CCNE1 amplified tumors and were cyclin E1-low (eg, IHC-low or IHC-negative (IHC-)).

[0359] In an additional group of subjects with recurrent, platinum-sensitive ovarian cancer that is cyclin E1-positive, assensertin and chemotherapy were administered. Eligibility criteria included subjects who had confirmed high-grade serous ovarian cancer; an ECOG performance status of 0-1; had received ≥1L prior line of platinum-based chemotherapy; were platinum-sensitive (platinum-free interval ≥6 months); were eligible and had previously received bevacizumab and PARPi according to regional standard of care; and were cyclin E1-positive (CCNE1 amplification and / or cyclin E1 IHC+).

[0360] Subjects receiving combination therapy were stratified based on stratification factors, including prior line therapy (1 vs. 2-3); prior PARPi (yes vs. no); and CCNE1 amplification (yes vs. no). Subjects were randomly divided into two groups. Group 1 received asenseti + chemotherapy (paclitaxel or carboplatin) for 6 cycles, followed by asenseti maintenance therapy, which was 400 mg QD 5:2. Group 2 received asenseti + carboplatin doublet therapy (paclitaxel or pegylated liposomal doxorubicin) for 6 cycles, without a subsequent asenseti maintenance therapy period. Exemplary dosing for the combination therapy group is shown in Table 4 below.

[0361] Table 4. Exemplary Combination Therapy Dosages

[0362] Asensetti chemotherapy Pacific taxol 300mg once daily 5:2 <![CDATA[80mg / m 2 (D1, D8, and D15 of a 28-day cycle) Carboplatin 200mg once daily 5:2 AUC = 5 (D1 of a 21-day cycle) PLD 400mg once daily 5:2 <![CDATA[40mg / m 2 (D1 of the 28-day cycle)]]>

[0363] Subjects were assessed for primary and secondary endpoints, including progression-free survival and overall survival, as determined by blinded independent central review.

[0364] Example 10. Asensertib Demonstrates Cancer Response in Heavily Pretreated Subjects

[0365] Asenzel has demonstrated efficacy in exemplary human subjects with CCNE1-amplified, platinum-resistant ovarian cancer. Subjects with CCNE1 amplification status (confirmed by Foundation testing) were selected and administered an intermittent dosing schedule of at least 400 mg of asenzel once daily for five consecutive days, followed by two days of rest, for 11 months. The subject was a 73-year-old female who had received 10 prior lines of therapy: (1) avelumab (SD); (2) liposomal doxorubicin (PD); (3) topotecan / bevacizumab (PD); (4) cyclophosphamide / bevacizumab (unk); (5) XMT1536 (NaPi2b ADC) (PR); (6) APG115 (MDM2inh) / pembrolizumab (SD); (7) ABBV-155 (CD275 ADC) (PD); (8) NC318 (Siglec-15mAB) (SD); (9) SM08502 (CLK inhibitor) (PD); (10) NBMBMX (HDAC8 inh) (SD). After treatment with asenseti, the subject demonstrated a durable cPR of -71% and a visible reduction in target lesions ( Figure 16A and 16B The exemplary subjects further demonstrated the feasibility of using asenoseltib and CCNE1 gene amplification status as biomarkers to treat platinum-resistant cancers.

[0366] Equivalents and scope

[0367] Furthermore, although the foregoing has been described in considerable detail by way of illustration and example for purposes of clarity and understanding, it will be appreciated by those skilled in the art that numerous and various modifications may be made without departing from the spirit of the present disclosure. It should therefore be clearly understood that the forms disclosed herein are merely illustrative and are not intended to limit the scope of the present disclosure, but rather are intended to cover all modifications and alternatives that come within the true scope and spirit of the present disclosure. The scope of the present disclosure is not intended to be limited to the foregoing description, but is as set forth in the following claims.

Claims

1. A method for treating cancer, comprising: An effective dose of Azenosertib or a pharmaceutically acceptable salt thereof is administered to a subject selected as having a predetermined cyclin E1 status.

2. A method for treating cancer, comprising: An effective dose of asensertib or a pharmaceutically acceptable salt thereof is administered to a subject selected as having a cyclin E1 biomarker level above a predetermined threshold.

3. A method for treating cancer, comprising: An effective dose of asensertib, or a pharmaceutically acceptable salt thereof, and a second chemotherapeutic agent, or a pharmaceutically acceptable salt thereof, is administered to a subject selected as having a cyclin E1 biomarker level above a predetermined threshold.

4. The method of claim 1, wherein the predetermined cyclin E1 status is cyclin E1-positive, cyclin E1-positive (low), cyclin E1-positive (high), or cyclin E1-high. The method of claim 4 , wherein the predetermined cyclin E1 status is cyclin E1-positive. The method of claim 4 , wherein the predetermined cyclin E1 state is cyclin E1-high.

7. The method according to any one of claims 1 to 6, wherein the cyclin E1 status or the cyclin E1 biomarker level is measured by cyclin E1 protein expression level. 8 . The method according to claim 7 , wherein the Cyclin E1 protein expression level is determined by CCNE1 mRNA or transcript level.

9. The method according to claim 7, wherein the cyclin E1 protein expression level is determined by protein level.

10. The method according to any one of claims 1 and 5 to 9, wherein the predetermined cyclin E1 status is a cyclin E1 protein expression level above a predetermined cut-off value.

11. The method according to any one of claims 1 and 5 to 10, wherein the predetermined cyclin E1 status is an immunohistochemistry (IHC) status.

12. The method according to any one of claims 2, 4 and 8 to 12, wherein the predetermined cut-off value or the predetermined threshold value is measured by the percentage of viable tumor cells with a cyclin E1 immunohistochemistry (IHC) staining intensity of 2+.

13. The method according to claim 12, wherein the predetermined cut-off value or the predetermined threshold value is cyclin E1 The percentage of viable tumor cells with an IHC staining intensity of 2+ IHC is greater than 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 28%, 29%, 30%, 31%, 32%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61% or 62%. The method according to claim 13 , wherein the predetermined cutoff value or the predetermined threshold value is that the percentage of live tumor cells with a cyclin E1 IHC staining intensity of 2+IHC is higher than 10%. The method according to claim 13 , wherein the predetermined cutoff value or the predetermined threshold value is that the percentage of live tumor cells with a cyclin E1 IHC staining intensity of 2+IHC is higher than 30%.

16. The method according to any one of claims 2, 4 and 10 to 15, wherein the predetermined cut-off value or the predetermined threshold value is measured by cyclin E1 immunohistochemistry (IHC) H scoring.

17. The method of claim 16, wherein the predetermined cutoff value or the predetermined threshold value is a cyclin E1 IHC H score above 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155 or 160.

18. The method according to claim 17, wherein the predetermined cut-off value or the predetermined threshold value is a cyclin E1 IHC H score above 50. The method according to claim 17 , wherein the predetermined cut-off value or the predetermined threshold value is a cyclin E1 IHC H score above 125.

20. The method of any one of claims 1 to 19, wherein the predetermined cyclin 1 status or the cyclin E1 biomarker level is not associated with CCNE1 gene amplification in the subject.

21. The method of any one of claims 1 to 19, wherein the predetermined cyclin 1 status or the cyclin E1 biomarker level is accompanied by the subject's CCNE1 gene amplification status.

22. The method according to claim 20 or claim 21, wherein the CCNE1 gene amplification or the CCNE1 gene amplification status is measured by CCNE1 gene copy number.

23. The method of claim 22, wherein the CCNE1 gene amplification status is that the CCNE1 gene copy number is at least 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, 29, 30, 31, 32, 33 or 34. The method according to claim 23 , wherein the CCNE1 gene amplification state is that the CCNE1 gene copy number is at least 7.

25. The method of any one of claims 1 to 24, wherein the subject is selected without determining the levels and status of other oncogenes.

26. The method of claim 25, wherein the additional oncogene is selected from the group consisting of BRCA1, BRCA2, TP53, PKMYT1, and PPP2R1A.

27. The method of any one of claims 1 to 26, wherein the cancer is a solid tumor or a hematological malignancy.

28. The method of any one of claims 1 to 27, wherein the cancer is a cyclin E1 driven cancer.

29. The method of any one of claims 1 to 28, wherein the cancer is selected from the group consisting of glioblastoma (GBM), astrocytoma, meningioma, craniopharyngioma, medulloblastoma, other brain cancers, head and neck cancer, leukemia, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), skin cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, endometrium cancer, esophageal cancer, eye cancer, gallbladder cancer, stomach cancer, gastrointestinal cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, blood cancer, head cancer, blood malignancies, Kaposi's sarcoma, sarcoma), kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, non-small cell lung cancer (NSCLC), small cell, lymphoma, mesothelioma, melanoma, multiple myeloma, neuroblastoma, nasopharyngeal cancer, cervical cancer, ovarian cancer, osteosarcoma, sarcoma, gastrointestinal stromal tumor (GIST), pancreatic cancer, pituitary cancer, prostate cancer, kidney cancer, retinoblastoma, salivary gland cancer, skin cancer, stomach cancer, small intestine cancer, spleen cancer, sarcoma, testicular cancer, thymus cancer, thyroid cancer, uterine cancer, uterine sarcoma, uterine serous carcinoma (USC), uterine CS, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor tumor), solid or liquid tumors, HGSOC, invasive breast cancer, triple-negative breast cancer (TNBC), esophagogastric cancer, gastric cancer, esophageal cancer, pRCC, ccRCC, chromophobe RCC, head and neck cancer, adenoid cystic carcinoma (ACC), diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma (NHL), low-grade glioma (LGG), pheochromocytoma and paraganglioma (PCPG), bile duct cancer, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), thymoma, BRAF-mutant metastatic colorectal cancer, uveal melanoma, high-grade serous ovarian cancer, fallopian tube cancer, or primary peritoneal cancer, BRAF V600E mutant colorectal cancer, platinum-sensitive ovarian cancer, poly(ADP-ribose) polymerase inhibitor (PARPi)-resistant ovarian cancer, platinum-resistant ovarian cancer, platinum-refractory ovarian cancer, advanced pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma, neuroendocrine tumors, neuroendocrine prostate cancer, pancreatic neuroendocrine tumors, small cell lung cancer (SCLC), germ cell cancer, and stromal cancer.

30. The method of any one of claims 1 to 29, wherein the cancer is confirmed histologically or cytologically, or the cancer is confirmed pathologically.

31. The method of any one of claims 1 to 30, wherein the cancer is recurrent or persistent.

32. The method of any one of claims 1 to 31, wherein the cancer is metastatic.

33. The method of any one of claims 1 to 32, wherein the cancer is unresectable.

34. The method of any one of claims 1 to 33, wherein the subject has received no more than 1, at least 1, 1, 2, 3, 4, 1 or 2, 1 to 2, 1 to 3, or 1 to 4 prior lines of therapy, prior lines of therapy in the advanced or metastatic setting, prior line of chemotherapy, prior line of platinum-based chemotherapy, prior regimen, or prior treatment regimen.

35. The method of any one of claims 1 to 34, wherein the cancer is platinum-resistant, platinum-sensitive, or platinum-refractory.

36. The method of any one of claims 1 to 35, wherein the cancer is PARP inhibitor-resistant.

37. The method of any one of claims 1, 2, and 4 to 36, comprising administering an effective dose of asensertib or a pharmaceutically acceptable salt thereof without combination with a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof.

38. The method of any one of claims 1 and 4 to 36, comprising administering an effective dose of assenserti or a pharmaceutically acceptable salt thereof in combination with a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof.

39. The method of any one of claims 3 to 38, wherein the second chemotherapeutic agent is selected from bendamustine, bortezomib, carfilzomib, ixazomib, busulfan, carboplatin, cisplatin, cyclophosphamide, cladribine, paclitaxel, docetaxel, pegylated liposomal doxorubicin, doxorubicin (PLD), dexamethasone, doxorubicin, gemcitabine, cytarabine, fludarabine, fluorouracil (5-FU), irinotecan, topotecan, temozolomide, triapine, azacitidine, 5-azacytidine, capecitabine tabine), AraC-FdUMP[10](CF-10), cladribine, etoposide, decitabine, daunorubicin, doxorubicin, ifosfamide, methotrexate, vincristine, hydroxyurea, oxaliplatin, niraparib, encorafenib and cetuximab, or a pharmaceutically acceptable salt of any one of the foregoing.

40. The method of claim 39, wherein the second chemotherapeutic agent is carboplatin, paclitaxel, gemcitabine, or pegylated liposomal doxorubicin (PLD), or a pharmaceutically acceptable salt of any of the foregoing.

41. The method of any one of claims 3 to 36 and 38 to 40, wherein assenserti or a pharmaceutically acceptable salt thereof and the second chemotherapeutic agent or a pharmaceutically acceptable salt thereof are administered concurrently.

42. The method of any one of claims 3 to 36 and 38 to 40, wherein assenserti or a pharmaceutically acceptable salt thereof and the second chemotherapeutic agent or a pharmaceutically acceptable salt thereof are administered sequentially.

43. The method of any one of claims 3 to 36 and 38 to 42, wherein assenserti or a pharmaceutically acceptable salt thereof and / or the second chemotherapeutic agent or a pharmaceutically acceptable salt thereof is administered intermittently.

44. The method according to any one of claims 1 to 43, wherein the method comprises the step of selecting a subject having the predetermined cyclin E1 status or the cyclin E1 biomarker level above the predetermined threshold.

45. The method of claim 44, wherein the method further comprises first determining the cyclin E1 status or the cyclin E1 biomarker level prior to the selecting step.

46. ​​The method of any one of claims 1 to 45, wherein the method results in an overall response rate (ORR) in subjects equal to or greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

47. The method of claim 46, wherein the overall response rate is measured by complete response (CR), partial response (PR), CA-125 50% response, or a combination thereof.

48. The method of claim 47 or claim 48, wherein the method results in a median progression-free survival (mPFS) of 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or longer in the subjects.

49. A method for treating ovarian cancer, comprising: administering an effective dose of asensertib or a pharmaceutically acceptable salt thereof to a subject selected as having a predetermined cyclin E1 status for a treatment period, and Optionally, a second chemotherapeutic agent or a pharmaceutically acceptable salt thereof is administered one or more times during the treatment cycle.

50. A method for treating ovarian cancer, comprising: administering an effective dose of asensertib or a pharmaceutically acceptable salt thereof to a subject selected as having a cyclin E1 biomarker level above a predetermined threshold for a treatment cycle, and The second chemotherapeutic agent or a pharmaceutically acceptable salt thereof is administered one or more times during the treatment cycle.

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