Methods and materials for treating cancer

Abstract

This document provides method and materials for treating cancer. In some cases, methods and materials for treating estrogen receptor negative (ER-) cancers (e.g., ER- breast cancers such as triple-negative breast cancers (TNBCs)) are provided. For example, one or more endoxifen (ENDX) compounds can be administered to a mammal (e.g., a human) having an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal. For example, one or more ENDX compounds and one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell can be administered to a mammal (e.g., a human) having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal.

Description

[0001] Attorney Docket No. 07039-2296WO1 / 2023-600

[0002] METHODS AND MATERIALS FOR TREATING CANCER

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U. S. Patent Application Serial No. 63 / 552,505, filed on February 12, 2024. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.

[0005] STATEMENT REGARDING FEDERAL FUNDING

[0006] This invention was made with government support under CAI 16201 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] SEQUENCE LISTING

[0008] This application contains a Sequence Listing that has been submitted electronically as an XML file named “07039-2296W01_SL.xml.” The XML file, created on December 10, 2024, is 389,866 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0009] TECHNICAL FIELD

[0010] This document relates to methods and materials for treating cancer. In some cases, this document provides methods and materials for treating cancers including estrogen positive (ERposor ER+or ER+) cancers and / or estrogen receptor negative (ERnegor ER’ or ER-) cancers (e.g., ER- breast cancers such as triple-negative breast cancers (TNBCs)). For example, one or more endoxifen (ENDX) compounds can be administered to a mammal (e.g., a human) having an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal. For example, one or more ENDX compounds and one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell can be administered to a mammal (e.g., a human) having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal. Attorney Docket No. 07039-2296WO1 / 2023-600

[0011] BACKGROUND

[0012] Breast cancer is the most common cancer worldwide with over 2.3 million new cases and 685,000 deaths in 2020 (Arnold, Morgan et al. 2022). Breast cancer displays substantial heterogeneity in prognosis and therapy responses and is categorized into four primary subtypes based on an analysis of its molecular characteristics and immunohistochemical markers: luminal A breast cancers and luminal B breast cancers (both characterized by ER expression), HER2 -positive breast cancers, and TNBCs (characterized by the absence of ER, the absence of progesterone receptor (PR), and without HER2 amplification). TNBC exhibit aggressive clinical behavior and high rates of death.

[0013] SUMMARY

[0014] This document provides methods and materials for treating cancer (e.g., ER+ cancers and / or ER- cancers such as ER- breast cancers (e.g., TNBCs)). For example, one or more ENDX (also referred to as 4-hydroxy-N-desmethyltamoxifen) compounds can be administered to a mammal (e.g., a human) having an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal. For example, one or more ENDX and one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell can be administered to a mammal (e.g., a human) having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal. As demonstrated herein, ENDX can be used as an allosteric inhibitor of a protein kinase C beta 1 isoform (PKCβ1) polypeptide, thereby altering AKT signaling. Also as demonstrated herein, ENDX exhibits a dose dependent targeting of a PKCβ1 polypeptide.

[0015] In general, one aspect of this document features methods for treating a mammal having an ER- cancer. The methods can include, or consist essentially of, administering an ENDX compound to a mammal having an ER- cancer. The mammal can be a human. The mammal can be a female mammal. The mammal can be a pre-menopausal female human. The ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a Attorney Docket No. 07039-2296WO1 / 2023-600

[0016] leukemia, or a lymphoma. The ER- cancer can be a breast cancer. The breast cancer can be a triple negative breast cancer. The ENDX compound can be a Z-ENDX compound. The Z-ENDX compound can be a Z-ENDX salt. The Z-ENDX salt can be Z-ENDX hydrochloride. The method can include administering from about 20 milligrams per day (mg / day) to about 360 mg / day of said ENDX compound to said mammal. The method also can include administering to said mammal an agent that can alter the antigens presented on the surface of a cancer cell of said ER- cancer. The agent can be abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, or TQB-3602.

[0017] In another aspect, this document features methods for treating a mammal having an ER+ cancer or an ER- cancer. The methods can include, or consist essentially of, administering, to mammal having an ER+ cancer or an ER- cancer, (i) an ENDX compound, and (ii) an agent comprising the ability to alter the antigens presented on the surface of a cancer cell of said ER+ cancer or said ER- cancer. The mammal can be a human. The mammal can be a female mammal. The mammal can be a pre-menopausal female human. The mammal can have ER- cancer, and said ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate Attorney Docket No. 07039-2296WO1 / 2023-600

[0018] cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma. The ER- cancer can be a breast cancer. The breast cancer can be a triple negative breast cancer. The mammal can have ER+ cancer, and said ER+ cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, or a lung cancer. The ENDX compound can be a Z-ENDX compound. The Z-ENDX compound can be a Z-ENDX salt. The Z-ENDX salt can be Z-ENDX hydrochloride. The method can include administering from about 20 mg / day to about 360 mg / day of said ENDX compound to said mammal. The agent can be abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, or TQB-3602.

[0019] In another aspect, this document features uses of a composition comprising an ENDX compound to treat a mammal having an ER- cancer. The mammal can be a human. The mammal can be a female mammal. The mammal can be a pre-menopausal female human. The ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma. The ER- cancer can be a breast cancer. The breast cancer can be a triple negative breast cancer. The ENDX compound can be a Z-ENDX compound. The Z-ENDX compound can be a Z-ENDX salt. The Z-ENDX salt can be Z-ENDX hydrochloride. Attorney Docket No. 07039-2296WO1 / 2023-600

[0020] The composition can include from about 20 mg to about 360 mg of said ENDX compound. The composition also can include an agent that can alter the antigens presented on the surface of a cancer cell of said ER- cancer. The agent can be abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5 -fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, or TQB-3602.

[0021] In another aspect, this document features ENDX compounds for use in the preparation of a medicament to treat an ER- cancer. The ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma. The ER-cancer can be a breast cancer. The breast cancer can be a triple negative breast cancer. The ENDX compound can be a Z-ENDX compound. The Z-ENDX compound can be a Z-ENDX salt. The Z-ENDX salt can be Z-ENDX hydrochloride.

[0022] In another aspect, this document features ENDX compounds for use in the treatment of an ER- cancer. The ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin Attorney Docket No. 07039-2296WO1 / 2023-600

[0023] cancer, a leukemia, or a lymphoma. The ER- cancer can be a breast cancer. The breast cancer can be a triple negative breast cancer. The ENDX compound can be a Z-ENDX compound. The Z-ENDX compound can be a Z-ENDX salt. The Z-ENDX salt can be Z-ENDX hydrochloride.

[0024] In another aspect, this document features uses of a composition comprising (i) an ENDX compound, and (ii) an agent comprising the ability to alter the antigens presented on the surface of a cancer cell of said ER+ cancer or said ER- cancer to treat a mammal having an ER+ cancer or an ER- cancer. The mammal can be a human. The mammal can be a female mammal. The mammal can be a pre-menopausal female human. The mammal can have ER-cancer, and said ER- cancer be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma. The ER- cancer can be a breast cancer. The breast cancer can be a triple negative breast cancer. The mammal can have ER+ cancer, and said ER+ cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, or a lung cancer. The ENDX compound can be a Z-ENDX compound. The Z-ENDX compound can be a Z-ENDX salt. The Z-ENDX salt can be Z-ENDX hydrochloride. The composition can include from about 20 mg to about 360 mg of said ENDX compound. The agent can be abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, Attorney Docket No. 07039-2296WO1 / 2023-600

[0025] sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, or TQB-3602.

[0026] In another aspect, this document features ENDX compounds and agents comprising the ability to alter the antigens presented on the surface of a cancer cell for use in the preparation of a medicament to treat an ER+ cancer or ER- cancer. The ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma. The ER- cancer can be a breast cancer. The ER- breast cancer can be a triple negative breast cancer. The ER+ cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, or a lung cancer. The ENDX compound can be a Z-ENDX compound. The Z-ENDX compound can be a Z-ENDX salt. The Z-ENDX salt can be Z-ENDX hydrochloride. The agent abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5 -fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, TQB-3602.

[0027] In another aspect, this document features ENDX compounds and agents comprising the ability to alter the antigens presented on the surface of a cancer cell for use in the treatment of an ER- cancer. The ER- cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary Attorney Docket No. 07039-2296WO1 / 2023-600

[0028] tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, or a lymphoma. The ER- cancer can be a breast cancer. The ER- breast cancer can be a triple negative breast cancer. The ER+ cancer can be a breast cancer, an ovarian cancer, an endometrial cancer, or a lung cancer. The ENDX compound can be a Z-ENDX compound. The Z-ENDX compound can be a Z-ENDX salt. The Z-ENDX salt can be Z-ENDX hydrochloride. The agent abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5 -fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, TQB-3602.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Attorney Docket No. 07039-2296WO1 / 2023-600

[0030] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figures 1A-F. Structure of PKCβII reveals architecture of the auto-inhibited state. Figure 1A) PKCβI / II domain diagram with phosphorylation sites (phospho-S / T) indicated by circles. Figure 1B) 8% Acrylamide gel with or without Phos-tag 5 of purified PKCβII and purified PKCβII treated with phosphatase. Figure 1C) PKCβII crystal grown using sitting drop vapor diffusion method. Figure 1D) Overall structure of the PKCβII inactive state with the domains coloured as in Figure 1A. Figure 1E) In the inhibited state, many hydrophobic lipid binding residues are occluded by interaction with the catalytic domain. Figure 1F) Molecular details of the interdomain contacts between the Cla domain, kinase domain and C -terminal tail, and the three phosphorylation sites: activation loop (pT500), turn (pT641), and hydrophobic (pS660) with surrounding structures, and the bound AMPPNP nucleotide.

[0033] Figures 2A - 2C. Structure of PKCβI reveals an ordered active conformation. Figure 2A) Two PKCβI crystal forms with 1 or 2 PKCβI molecules per asymmetric unit (ASU) were aligned by their kinase domain. Their crystal -packing environments are shown (N-term 1, N-term 2, and N-term 3). Two domain arrangements corresponding to the active and inhibited state are common amongst the PKCβI molecules. The regulatory domain lipid-binding residues are in a plane for the activated state (upper) while the pseudosubstrate occupies the active site in the inhibited state. Figure 2B) Recombinant PKCβI protein with mutations engineered to disrupt the Clb-kinase and C2-kinase domain interactions. Figure 2C) Mutant PKCβI were assayed for kinase activity in vitro using the FRET probe CKAR. Error bars represent standard deviation.

[0034] Figures 3A - 3C. Allosteric activation of PKC is driven by binding to a phospholipid membrane. Figure 3 A) Lipid-lever model of PKC activation. A steric clash between the phospholipid bilayer and kinase domain drives PKCβI / II into its active conformation. Figure 3B) In vitro CKAR assay showing the kinase activity of PKCβI in the indicated concentrations 5 of PDBu or 40 pg / mL PS / DAG lipid micelles. Error bars represent standard Attorney Docket No. 07039-2296WO1 / 2023-600

[0035] deviation. Figure 3C) Limited proteolysis with elastase used to probe change in PKCβI / II conformation in the presence of the indicated ligands, with quantification of cleavage product (*) as a percentage of protein indicated at the bottom of each gel.

[0036] Figures 4A - 4C. Molecular basis for differential lipid affinities between PKCβI and PKCβII. Figure 4A) Comparison between the structural architecture of PKCβI and PKCβII inactive conformations and the relative locations of the lipid binding residues and pseudosubstrate. Figure 4B) Position of the 5 Clb domain differs between PKCβI and PKCβII with respect to the Cl a and kinase domains. Figure 4C) Intramolecular details of the residue interactions that lead to differential placement of the Clb domain, and the effect of this placement on the susceptibility of PKCβI (01), PKCβII (011), and PKCβI F648A (FA) to proteolytic cleavage by elastase (*).

[0037] Figures 5A - 5F. Endoxifen is an allosteric inhibitor of PKCβI. Figure 5A) / « vitro Z’-LYTE kinase assay shows differences in kinase activity of unstimulated (- lipids) compared to active (+ lipids) PKCβI and PKCβII in response to ENDX (E) and TAM (T) inhibition. The associated IC50 values are reported. Data 5 are mean ± SD for each data point (n=4 independent experiments). Figure 5B) In vitro Z’-LYTE kinase assay with 7.5 nM unstimulated PKCβI (- lipids) is more sensitive to inhibition by ENDX than 5nM activated PKCβI (+ lipids) and 0.3 nM PKCβI catalytic domain. Data are mean ± SD for each data point (n=4 independent experiments). Figure 5C) In vitro kinase assay (CKAR) on the catalytic domain of PKCβI and PKCβII reveals that ENDX as a non-competitive inhibitor with respect to ATP in contrast to the known ATP-competitive inhibitor ENZA. Error bars represent standard deviation (n=4 independent experiments). Figure 5D) SAXS scattering plot of PKCβI in the presence of the indicated ligands. Figure 5E) Limited proteolysis demonstrates dose-dependent conformational alteration of PKCβI by ENDX and not TAM. Data are presented as mean ± SD. Figure 5F) Confocal microscopy data shows plasma membrane recruitment of YFP-PKCβI in the presence of the indicated drug treatments.

[0038] Figure 6. Overall model of PKC allosteric regulation.

[0039] Figures 7A - 7D. Figure 7A) Sequence alignment of conventional PKCs with domains coloured as indicated. Sequences shown include, from top to bottom, PKCy (SEQ ID NO: 1), PKCa (SEQ ID NO:2), PKCβI (SEQ ID NO:3), and PKCβII (SEQ ID NO:4). The Attorney Docket No. 07039-2296WO1 / 2023-600

[0040] altered C-terminal tails derived from an alternative splicing event are coloured pink (PKCβI) or salmon (PKCβII). Figure 7B) Domain arrangement and second messenger sensitivity of the three PKC families. Figure 7C) Schematic diagram of YFP-PKCβII protein expression and affinity isolation using the anti-YFP nanobody system. Chromatograms and corresponding Coomassie-stained gels of PKCβII from FPLC purification steps. Figure 7D) Western blots with the indicated phospho-specific PKCβII antibodies detects phosphorylation at all three expected sites (T500 - activation, T642 - turn, and S660 -hydrophobic) in the purified protein.

[0041] Figures 8A - 8D. Figure 8A) Experimental 2F0 - Fc map contoured at 1σ indicating continuous electron density for the PKCβII Cla - Clb linker region and Clb - C2 linker region. Figure 8B) Experimental 2F0 - Fc map contoured at 1σ for residues 298 - 312 in the PKCβII C2-kinase domain linker region. Figure 8C) Experimental 2F0 - Fc of the three phosphorylation sites on PKCβII. Figure 8D) Location of Zn2+ ions (gray spheres) coordinated within the Cla and Clb domains, as well as a fifth Zn2+ ion that is part of the crystal lattice.

[0042] Figures 9A - 9C. Figure 9A) Measured length of the disordered region between C2 and kinase domain used to define pairing of N-terminal and kinase domains that belong to the same chain. Figure 9B) Domain-swapped arrangement of PKCBII with an adjacent identical monomer. Figure 9C) Molecular details of the N-terminal pseudosubstrate side chains and their specific interactions with the Clb and kinase domains labeled by residues and color-coded (hydrogen bonding, salt bridge, hydrophobic / Van der Waals).

[0043] Figures 10A - 10E. Figure 10A) Ion exchange and size exclusion chromatograms of PKCβI purification and SDS-PAGE analysis of indicated fractions. Figure 10B) Mass spectrometry of purified PKCβI protein detects phosphorylation at T500 and T642. The polypeptide sequence in the top spectrum is SEQ ID NO: 5 and the polypeptide sequence in the bottom spectrum is SEQ ID NO:6. Figure 10C) Individual copies of PKCβI within each crystal form. Figure 10D) Experimental 2F0 - Fc map of phosphorylation sites on PKCβI in each crystal form. Figure 10E) Experimental 2F0 - Fc map contoured at la for the PKCβI Cla - Clb linker region and Clb - C2 linker region. Attorney Docket No. 07039-2296WO1 / 2023-600

[0044] Figures 11 A - 1 IE. Figure 11 A) Pseudosubstrate of the adjacent monomer is present in the active site of crystal form 1. Figure 1 IB) Linkers between Cla and Clb domain and C2 and kinase domains are not observed in PKCβI crystal form 1. Figure 11C) PKCβI active site adopts a more closed conformation when occupied by AMPPNP nucleotide and remains open in its absence. Figure 1 ID) Local symmetry environment of the three copies of PKCβI reveals similar molecular interactions. Figure 1 IE) AlphaF old-predicted structure of PKCβI in its active form.

[0045] Figures 12A - 12E. Figure 12 A) Ligands coordinate in crystal form 1 and crystal form 2 of PKCβI. DAG binding site of PKCβI is occupied by glycerol molecules in crystal form 2. Figure 12B) 70 A distance between N-terminal tail and active site prevents reengagement of the pseudosubstrate. Figure 12C) Modeled mutations that disrupt C2-kinase and Clb-kinase domain interactions in the active conformation of PKCβI. Figure 12D) Aligned crystal structures of the Cla domain with PDBu (PDB 7KNJ) and PKCβI or PKCβII in their auto-inhibited conformation shows PDBu binding does not disrupt the inactive conformation. Figure 12E) Insertion of Cla domain hydrophobic residues in the phospholipid bilayer encourages kinase domain to disengage from the pseudosubstrate to prevent unfavourable charge interactions at the lipid bilayer.

[0046] Figures 13 A - 13D. Figure 13 A) Differential scanning fluorimetry of 1 pg full-length (FL), catalytic (CAT) domain or the N-terminal (NT) domain of PKCβI or of PKCβII with ddH2O as negative control or 40 pM ENDX. Presented melt curve and derivative melt plots are shown for each independent measurement. Average melting temperatures (TM) and standard deviations of each condition are reported. Figure 13B) In vitro Z’-LYTE kinase assay of other PKC isoforms and their associated IC50 values. Data are mean ± SD. Figure 13C) Confocal live cell microscopy images showing time-dependent cellular localization of YFP-tagged PKCβI in the presence of indicated drug treatment. Figure 13D) Confocal live cell microscopy images showing dose-dependent cellular localization of YFP-tagged PKCβI in the presence of indicated drug treatment.

[0047] Figures 14A - 14B. Z-endoxifen (ENDX) effects on cell viability and apoptosis in estrogen deprived ERa+ / HER2-MCF7ACl cells. Figure 14A) Cells grown in CSS medium were treated with vehicle control or the indicated ENDX concentrations for 48 hours. Cell Attorney Docket No. 07039-2296WO1 / 2023-600

[0048] viability was assessed by the crystal violet assay. Figure 14B) Cells were co-treated with vehicle control or the indicated ENDX concentrations, IncuCyte Annexin V green and NucLight red reagents in CSS medium for 48 hours. The apoptosis (%) graphs are presented as the green object count (which correspond to cells that are stained with the IncuCyte green fluorescence Annexin V reagent) divided by the red object count (which correspond to the total number of cells in the culture that are stained with the IncuCyte red fluorescence Nuclight Rapid Red Cell Labeling reagent that labels the nucleus of all cells without perturbing cell function or biology) and displayed as percentage using the IncuCyte S3 analysis software. Data represents the mean of six wells per treatment performed as biological duplicates ± s.d. **, / ? < 0.01; ***, p < 0.001; ****, p < 0.0001 by one-way ANOVA.

[0049] Figure 15. A schematic depicting the strategy used for quantitative proteomic and phosphoproteomic profiling of ENDX-treated MCF7AC1 cells. All experiments were performed in triplicate. Cells were treated with vehicle control or Z-endoxifen (ENDX) as specified dosages for 24 hours. After the ENDX treatment, cells were harvested and lysed in 8 M urea buffer, followed by trypsin digestion, desalting and tandem mass tag (TMT) labeling. The labeled peptides were fractionated, and phosphopeptides were enriched with immobilized metal affinity chromatography (IMAC) approach. Both fractionated peptides and IMAC-enriched phosphopeptides were analyzed by Orbitrap Lumos mass spectrometer.

[0050] Figures 16A- 16F. Effects of ENDX on the phosphoproteome ofMCF7ACl cells. Figure 16A) A pie chart showing the distribution of identified phosphorylation sites. Figures 16B - 16C) Volcano plots showing the total number of phosphosites, and the percentage that are upregulated (right side) and downregulated (left side) (Fold change (FC) 11.5|; p value < 0.05) in cells treated for 24 hours in CSS medium with 0.01 (Figure 16B), 0.1 (Figure 16C), or 5 pM (Figure 16D) ENDX relative to vehicle-treated cells, as detected by mass spectrometry analysis. Figure 16E) Venn diagram indicating the overlap of upregulated and downregulated phosphosites in ENDX-treated cells relative to vehicle-treated cells. Figure 16F) Heatmap indicating relative abundance of the phosphosites analyzed in the ENDX-treated cells relative to vehicle-treated cells. The hierarchical clustering of phosphosites is shown on the left. Attorney Docket No. 07039-2296WO1 / 2023-600

[0051] Figures 17A - 17C. Kinase enrichment analysis predicts AKT signaling is regulated by high-dose ENDX. 210 phospho-regulated proteins from 0.01 pM (Figure 17A), 224 phospho-regulated proteins from 0.1 pM (Figure 17B), and 347 phospho-regulated proteins from 5 pM (Figure 17C) ENDX treated cells were respectively used for KEA3 upstream kinase analysis. Left panels: Integrated rankings of most enriched kinases across libraries based on the MeanRank. The stacking bar chart shows the summation of the MeanRank derived from the libraries used for the KEA analysis. The libraries are color-coded. Right panels: Kinase co-regulatory networks constructed from kinase-kinase interactions between top-ranked kinase results for the integrated rankings. Directed edges indicate interactions supported by kinase-substrate evidence.

[0052] Figures 18A - 18D. Downregulated phosphosites following ENDX treatment are enriched for PKCp, CDK1, and AKT target sequences. Figure 18A) fuzzy c-means clustering showing the classification of ENDX treatment effects on the phosphoproteome into three regulatory clusters. Cluster 1 represents phosphosites downregulated by ENDX in a dosedependent manner. Cluster 2 represents phosphosites that are upregulated by ENDX at all concentrations examined. Cluster 3 represents phosphosites downregulated at 0.01 pM concentration but mostly unaffected at the 0.1 and 5 pM concentrations. Figure 18B) Molecular and cellular pathways potentially impacted by ENDX. Kyoto Encyclopedia of Genes and Genomes (KEGG) database analysis of the phosphosites altered by ENDX in cluster 1 showing the top biological pathways associated with these phosphosites. Figure 18C) Enriched phosphorylation motifs identified in cluster 1 phosphosites. Figure 18D) Graph showing the frequency of kinases known to phosphorylate cluster 1 phosphosites that were depleted by ENDX as assessed using NetworKIN and RoKAI prediction tools. Motifs shown include, from top to bottom, an AKT substrates motif (SEQ ID NO:7), a MAPK / CDK substrates motif (SEQ ID NO:8), and a CK2 substrates motif (SEQ ID NO:9).

[0053] Figures 19A - 19E. ENDX specifically downregulates pAKTSer473at 5 pM and inhibits PKC01 kinase activity. Figure 19A) MCF7AC1 cells in CSS medium were treated for 24 hours with vehicle control or 0.01, 0.1 and 5 pM ENDX. Immunoblot assays of pAKTSer473, pAKTThr308, AKT and p-AKT substrates are shown with -actin as a loading Attorney Docket No. 07039-2296WO1 / 2023-600

[0054] control. Figure 19B) Serum starved MCF7AC1 cells were pretreated with vehicle control, 0.01, 0.1, 5 pM ENDX, 0.1 pM tamoxifen (TAM) or 0.1 pM fulvestrant (ICI-182780) followed by the addition of 100 nM insulin for one hour as indicated. IB assays of pAKTSer473, pAKTThr308, AKT and P-actin are shown. Figure 19C) Serum starved MCF7AC1 cells were pretreated with vehicle control and 0.01, 0.1 and 5 pM ENDX for two hours followed by the addition of 100 nM insulin for one hour as indicated. IB assay of pAKT substrates and P-actin are shown. Figures 19D and 19E) In vitro kinase assay showing % PKCβI kinase activity in the presence of different concentrations of ENDX (Figure 19D) and TAM (Figure 19E). The broad-spectrum kinase inhibitor staurosporine serves as a positive control. The IC50 concentration of ENDX, TAM and staurosporine are indicated.

[0055] Figures 20A - 20F. Role of PKCβI in mediating ENDX inhibition of AKTSer473phosphorylation. Figure 20 A) Serum starved MCF7AC1 cells were treated with vehicle control or 20 and 200 nM PMA for 20 minutes. IB assays of pPKCβISer661, PKCβI, pAKTSer473, AKT, p-AKT substrates and P-actin are shown. Figure 20B) Serum starved MCF7AC1 cells were pretreated with vehicle control or 0.01, 0.1 and 5 pM ENDX for two hours followed by the addition of 200 nM PMA for 20 minutes as indicated. IB assays of pPKCβISer661, PKCβI, pAKTSer473, AKT, p-AKT substrates and P-actin are shown. Figure 20C) Serum starved MCF7AC1 cells were pretreated with vehicle control or 1 pM ENZA for two hours followed by the addition of 200 nM PMA for 30 minutes as indicated. IB assays of pPKCβISer661, PKCβI, pAKTSer473, AKT and P-actin are shown. Figure 20D) Serum starved MCF7AC1 cells were pretreated with vehicle control, 0.01, 0.1 and 5 pM ENDX, 0.1 pM TAM or 0.1 pM ICI followed by the addition of 100 nM insulin for one hour as indicated. IB assays of pPKCβISer661, PKCβI and P-actin are shown. Figure 20E) MCF7AC1 cells in CSS medium were transfected with non-targeting (siNT) or PKCP-targeting (siPKCP) siRNAs for 48 hours. IB assays of PKCβI, pAKTSer473and P-actin are shown. The histogram indicates the percentage (%) of PKCβI and pAKTSer473protein levels remaining upon PKCβI knockdown in siPKCP-treated cells relative to siNT-treated cells from two biological replicates ± s.d. The vertical lines indicate that different lanes of the same blot were juxtaposed to remove intervening lanes. Figure 20F) MCF7AC1 cells were treated with siNT or siPKCβI in CSS medium for six days. Cell viability was assessed by crystal violet assays. Attorney Docket No. 07039-2296WO1 / 2023-600

[0056] Data represents the mean of six wells per treatment performed as biological triplicates ± s.d. *,p < 0.05; **,p < 0.01; ***, p < 0.001 by one sample t-test.

[0057] Figures 21A - 21D. ENDX replicates the effects of the pan-AKT inhibitor, MK-2206, on apoptosis. Figures 21A and 21C) MCF7AC1 (Figure 21A) and T47D-LTED (Figure 21C) cells were co-treated with vehicle control or 0.01, 0.1 and 5 pM ENDX or MK-2206 in the presence of IncuCyte Annexin V green and NucLight red reagents in CSS medium for 48 hours. The apoptosis (%) graph was generated as described in Figure 14B. Data represents the mean of six wells per treatment performed as biological duplicates ± s.d. ***, > < 0.001; ****, p < 0.0001 by one-way ANOVA. Figures 21B and 21D) MCF7AC1 (Figure 21B) and T47D-LTED (Figure 21D) cells in CSS medium were treated with vehicle control or 0.01, 0.1 and 5 pM ENDX for 48 hours (Figure 21B) and 24 hours (Figure 21D), respectively. IB assay of pAKTSer473, AKT, PARP, cleaved PARP and -actin are shown.

[0058] Figures 22A - 22C. Expression of catalytically active AKT diminishes ENDX ability to induce apoptosis. Figure 22A) MCF7ACleaAKTcells were grown in FBS medium in the absence (-) or presence (+) of cumate for 48 hours. IB assay of C-terminally hemagglutinin tagged AKT (AKT-HA), endogenous AKT and P-actin. Figure 22B) MCF7AClcaAKTcells grown in FBS medium in the (-) or (+) of cumate for 48 hours. IB assay of pAKT-substrates (SEQ ID NO:10) and P-actin. Figure 22C) MCF7AClcaAKTcells grown in CSS medium were treated for 48 hours in the (-) or (+) of cumate, following which cells were co-treated with vehicle control or 5 pM ENDX and IncuCyte Annexin V green and NucLight red reagents for 48 hours. The percentage (%) of cells undergoing apoptosis was calculated as indicated in Fig. IB. Data represents the mean of six wells per treatment performed as biological duplicates ± s.d. ns: nonsignificant. **, / > < 0.01 by one-way ANOVA.

[0059] Figures 23 A - 23B. Summary of ENDX anticancer effects in ERa+ breast cancer cells. Figure 23 A) Activation of PKCβISer661by the PKC agonist PMA and / or insulin phosphorylates AKTSer473resulting in the activation of p-AKT downstream substrates, which mediates cell survival. Figure 23B) ENDX binds to PKCβI and facilitates PKCβI protein degradation, resulting in the attenuation of phosphorylation of AKTSer473as well as downstream p-AKT substrates, leading to induction of apoptosis. Attorney Docket No. 07039-2296WO1 / 2023-600

[0060] Figures 24A - 24C. Effects of ENDX on the global protein expression in MCF7AC1 cells. Figure 24A) Stacked barplot showing the number of total proteins and the percentage that are upregulated (red) and downregulated (blue) (Fold change (FC) |1.5|; p value < 0.05) in 0.01, 0.1 and 5 pM ENDX treated cells relative to vehicle treated cells for 24 hours in CSS medium, as identified by mass spectrometry analysis. Figure 24B) Venn diagram indicating the overlap of the total proteins in the 0.01, 0.1 and 5 pM ENDX treated cells relative to vehicle treated cells. Figure 24C) Venn diagram indicating the overlap of the list of the phosphosites and the list of the total proteins that are altered by ENDX treatment regardless of the concentration.

[0061] Figures 25A - 25B. ENDX effects on AKT phosphorylations in ERa+ breast cancer models in vivo and in vitro. Figure 25 A) MCF7AC1 xenograft tumors were treated with control, letrozole, tamoxifen (TAM) and 25 mg / kg and 75 mg / kg ENDX for four weeks in vivo. IB assay of pAKTSer473, pAKTThr308, AKT, p-AKT substrates are shown with P-actin as a loading control. Figure 25B) Serum starved T47D cells were pretreated with vehicle control or 0.01, 0.1 and 5 pM ENDX and 0.1 pM tamoxifen (TAM) and ICI-182780 (ICI) for two hours followed by the addition of 100 nM insulin treatment for one hour. IB assay of pAKTSer473, pAKTThr308, AKT and -actin.

[0062] Figures 26A - 26D. ENDX binds to PKCβI. Figures 26A and 26B) SPR sensograms (relative units, RU) of ENDX binding at the indicated concentrations to immobilized PKCβI. Figures 26C and 26D) Dot plots showing the affinity binding of ENDX to PKCβI corresponding to sensograms in Figures 26A and 26B, respectively.

[0063] Figures 27A - 27D. PKCβI knockdown by different strategies and the effects of PKCβI knockdown on PKC family members. Figure 27A) MCF7AC1 cells in CSS medium were transfected with non-targeting (siNT) or PKCβI -targeting (siPKCβI) siRNAs for 72 hours. IB assay of PKCβI and P-actin are shown. The histogram indicates the percentage (%) of PKCβI protein levels remaining in siPKCβI -transfected cells compared to siNT-transfected cells. Figure 27B) MCF7AC1 cells in CSS medium in the absence (-) or presence (+) of doxycycline (Dox) for 72 hours. IB assay of PKCβI and P-actin are shown. The histogram indicates the percentage (%) of PKCβI protein levels remaining in dox induced cells compared to noninduced cells. Figure 27C) IB assay of basal PKCP2 protein expression Attorney Docket No. 07039-2296WO1 / 2023-600

[0064] and P-actin in MCF7AC1 and K562 (a positive control for PKCP2) cells. Figure 27D) IB assay of the relative protein expression of PKC family members and P-actin in siNT and siPKCβI transfected MCF7AC1 cells. For Figures 27A and 27B, data represent mean of six wells per treatment performed as biological triplicates ± s.d. *,p < 0.05; ****,p < 0.0001 by one sample t test.

[0065] Figures 28A- 28D. Effects of ENDX on PKCβI, phospho-PKCBlSer661 and phospho- AKTSer473 expression levels in ERa- breast cancer cells. Figure 28A) Basal expression of PKCβI and ERa in the indicated cell lines. K562 cells serves as the positive control for PKCβI. Figure 28B) The effects of ENDX pretreatment for two hours followed by treatment in the presence or absence of 100 nM insulin for one hour on the protein expression levels of PKCβI, pAKTSer473, AKT and p-actin in MDAMB231 and HEK293F cells. Figure 28C) The effects of ENDX pretreatment for two hours followed by treatment in the presence or absence of 100 nM insulin for one hour on the protein expression levels of PKCβI, pAKTSer473, AKT, ERa and P-actin in MDAMB231-ERa cells. Dox was added 48 hours prior to the treatments to allow for induction of ERa protein expression. Figure 28D) The effects of ENDX pretreatment for two hours followed by treatment in the presence or absence of 20 nM PMA for 20 minutes on the protein expression levels of pPKCβISer661, PKCβI and P-actin in MDAMB231 and HEK293F cells.

[0066] Figures 29 A - 29 J. Effects of the pan-AKT inhibitor MK-2206 or ENDX on phenotypes in ERa+ breast cancer cells. Figure 29A) MCF7AC1 cells were treated with vehicle control or 0.01, 0.1 and 5 pM MK-2206 in CSS medium for six days. Cell viability is assessed by the crystal violet assay. Figure 29B) MCF7AC1 cells were co-treated with vehicle control or 0.01, 0.1 and 5 pM MK-2206 and IncuCyte Annexin V green and NucLight rapid red reagents in CSS medium for 48 hours. The apoptosis graphs are presented as the green object count divided by the red object count and displayed as percentage using the IncuCyte S3 analysis software. Cells were plated at a density of 2000 cells per well. Figure 29C) MCF7AC1 cells were treated with vehicle control or 0.01, 0.1 and 5 pM MK-2206 in CSS medium for 24 hours. IB assay of pAKTSer473, AKT, PARP, cleaved PARP and P-actin. Figure 29D) MCF7AC1 xenograft protein lysates were treated with the indicated drugs for four weeks. IB assay of cP ARP, PARP and P-actin. Figure 29E) Attorney Docket No. 07039-2296WO1 / 2023-600

[0067] T47D cells were cultured in FBS versus CSS medium for six days. Cell viability is assessed by the crystal violet assay. Figure 29F) Parental T47D and T47D-LTED cells were processed for protein lysates. IB assay of ERa, pAKTSer473 and P-actin. Figure 29G) T47D-LTED cells were treated with vehicle control or 0.01, 0.1 and 5 pM MK-2206 in CSS medium for six days. Cell viability is assessed by the crystal violet assay. Figure 29H) T47D-LTED cells were co-treated with vehicle control or 0.01, 0.1 and 5 pM MK-2206 and IncuCyte Annexin V green and NucLight rapid red reagents in CSS medium for 48 hours. Percentage of cells undergoing apoptosis was calculated as mentioned in Figure 29B. Figure 291) T47D-LTED cells were treated with vehicle control or 0.01, 0.1 and 5 pM MK-2206 in CSS medium for 24 hours. IB assay of pAKTSer473, AKT, PARP, cleaved PARP and P-actin. Figure 29J) T47D-LTED cells were treated with vehicle control or 0.01, 0.1 and 5 pM ENDX in CSS media for six days. Cell viability is assessed by the crystal violet assay. For Figures 29A, 29B, 29E, 29G, 29H, and 29 J, data represent mean of six wells per treatment performed as biological duplicates ± s.d. *,p < 0.05,

[0068]

[0069] < 0.0001 by one-way ANOVA for Figures 29 A, 29B, 29G, 29H, and 291 and unpaired t test for Figure 29E.

[0070] Figure 30. Unlike ENDX, TAM and ICI do not induce apoptosis. MCF7AC1 cells were co-treated with vehicle control or 5 pM ENDX, 0.1 pM TAM and 0.1 pM ICI and IncuCyte Annexin V green and NucLight rapid red reagents in CSS medium for 48 hours. The percentage (%) of cells undergoing apoptosis is calculated as described in Example 2. Cells were plated at a density of 2000 cells per well. Data represents mean of six wells per treatment performed as biological duplicates ± s.d. *,p< 0.05 by one-way ANOVA.

[0071] Figures 31 A - 31C. ENDX neither induces apoptosis nor inhibit growth of ER- breast cancer cells. Figure 31 A) MDAMB231 cells grown in CSS medium were were co-treated with vehicle control or the indicated ENDX concentrations, IncuCyte Annexin V green and NucLight red reagents in CSS medium for 48 hours. The apoptosis graphs are presented as the green object count divided by the red object count and displayed as percentage using the IncuCyte S3 analysis software. Cells were plated at a density of 2000 cells per well. Data represents the mean of six wells per treatment performed as biological duplicates ± s.d. ****, p < 0.0001 by one-way ANOVA. Figure 3 IB) Effects of ENDX on cell proliferation at the indicated concentrations on day seven of treatment. Cell viability was assessed by the crystal Attorney Docket No. 07039-2296WO1 / 2023-600

[0072] violet assay. Figure 31C) immunoblot of basal PKCβI protein expression in the indicated cell lines.

[0073] Figures 32A - 32B. Figure 32A) Endoxifen increased the expression of IFNy by CD8+cells. Peripheral blood mononuclear cells (PBMC) were isolated from normal donors’ apheresis cones and treated with endoxifen (2pM) or Enzastaurin (2pM) for 5 days. Control cells were left untreated. Cells were then treated with brefeldin for 4 hours, followed by staining with fluorochrome-conjugated antibodies for surface and intracellular markers and flow cytometry analysis. Bar graphs and representative dot plots depict the percentage of IFNy CD8+cells. n=8, *p<0.02 denotes significant difference. Figure 32B) IFNy expression can be used as a favorable prognostic marker in breast cancer. Kaplan-Meier (KM) survival analysis shows the correlation between the gene expression level of IFNy and overall survival (OS) in different subtypes of breast cancer tumors. Logrank P<0.05 denotes significance.

[0074] Figure 33. Endoxifen increased the expression of Granzyme B and IL-2, but not perforin, in CD8+and CD4+T cells. PBMCs were treated with endoxifen (2pM) or enzastaurin (2pM) for 5 days. Cells were then treated with brefeldin for 4 hours to stop secretion for 4 hours. Cells were stained with viable cell markers and Fluorochrome-conjugated surface and intracellular antibodies and analyzed by flow cytometry.

[0075] Figures 34A - 34C. CyTOF analysis showing that endoxifen altered the expression of immune cells markers. PBMCs were isolated from normal donors and treated with / without endoxifen (2pM) for 5 days. Cells were then treated with brefeldin for 4 hours, followed by staining with PBMC enhanced panel of immune markers and CyTOF analysis. Figure 34A) tSNE maps show the clusters of the cells that were altered in endoxifen treated cells. Figure 34B) Bar graphs represent the number of the cells in each cluster. Figure 34C) Heat map shows the expression of each of 36 immune markers within each cluster or population (Pop).

[0076] Figures 35A - 35B. Pre-treatment of PBMCs with endoxifen enhanced their ability to suppress the proliferation of MCF7 tumor cells. PBMCs were treated with endoxifen (2pM) or enzastaurin (2pM) for 5 days, followed by washing the cells and removing the drug and co-culturing with MCF7AC1 tumor cells in vitro. Cell proliferation was monitored by IncuCyte for a duration of 5 days (Figures 35A and 35B, left panels). In a similar assay set- Attorney Docket No. 07039-2296WO1 / 2023-600

[0077] up, MCF7AC1 or MCF7 endoxifen-resistant tumor cells were pretreated with abemaciclib (500 nM) for 5 days and then co-cultured with endoxifen-pretreated PBMCs, followed by IncuCyte analysis (Figures 35A and 35B, middle and left panels). Line graphs represent the proliferation of tumor cells over a duration of 5 days using live-cell analysis by IncuCyte.

[0078] Figures 36A-36D. CD3+ T cells were isolated via negative selection from healthy control donor PBMCs according to EasySep kit instructions (STEMCELL Cat. #19051) and cultured for 3-days under activating conditions using CD3 / CD28 Dynabeads (ThermoFisher Scientific Cat. 11131D). Figure 36A) Unstimulated cells at day-3 of in vitro culture. Figures 36B-36D) Stimulated cells at day-3 of in vitro culture. Figure 36B) No ENDX. Figure 36C) 2pM ENDX, Figure 36D) 5pM ENDX.

[0079] Figures 37A-37H. Gating strategy from Fig. 36 implemented to analyze CD4+ and CD8+ T cell subsets. Fulvestrant (FUL). Figure 37A) Frequency (%) of TIM-3+ CD4+ T cells. Figure 37B) Surface expression of TIM-3 (Geometric (g) MFI) on TIM-3+ CD4+ T cells. Figure 37C) Frequency (%) of TIM-3+ CD8+ T cells. Figure 37D) Surface expression of TIM-3 (gMFI) on TIM-3+ CD8+ T cells. Figures 37E-37F) CD4+ T cells. Figure 37E) Frequency (%) of PD-1+ cells. Figure 37F) Left: histogram of PD-1 expression. Right: PD-1 surface expression (gMFI) on PD-1+ CD4+ T cells. Figures 37G-37H) CD8+ T cells. Figure 37G) Frequency (%) of PD-1+ cells. Figure 37H)Left: histogram of PD-1 expression. Right: PD-1 surface expression (gMFI) on PD-1+ CD8+ T cells..

[0080] Figures 38A-38E. CD3+ T cells were isolated via negative selection from healthy control donor PBMCs according to EasySep kit instructions (STEMCELL Cat. #19051) and cultured for 5-hours under activating conditions using CD3 / CD28 Dynabeads (ThermoFisher Scientific Cat. 11131D) or PMA (50ng / mL) and lonomycin (Ipg / mL). Figure 38A) Representative gating strategy from unstimulated CD3+ negative selected cells. Figure 38B) Separate CD3 staining showing purity of EasySep T cell negative selection kit. Figure 38C) Plots showing CD4+ and CD8+ T cell gates under CD3 / CD28 Dynabeads (left) or PMA / Iono (right) activation methods. Figures 38D-38E) Representative gating for effector cytokines IL-2, IFN-y, and TNF-a. Figure 38D) CD4+ T cells. Figure 38E) CD8+ T cells.

[0081] Figures 39A-39B. Frequency of IFN-γ+ (left), TNF-a+ (middle), IL-2+ (right) cells. Figure 39A) CD4+ T cells. Figure 39B) CD8+ T cells. Attorney Docket No. 07039-2296WO1 / 2023-600

[0082] DETAILED DESCRIPTION

[0083] This document provides methods and materials for treating cancers (e.g., ER+ cancers and / or ER- cancers such as ER- breast cancers (e.g., TNBCs)). For example, one or more ENDX compounds can be administered to a mammal (e.g., a human) having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) to treat that mammal.

[0084] When treating a mammal (e.g., a human) having an ER+ cancer and / or an ER- cancer (e g., an ER- breast cancer such as a TNBC) as described herein, the mammal can have any type of cancer. In some cases, a cancer treated as described herein can include one or more solid tumors. In some cases, a cancer treated as described herein can be a blood cancer. In some cases, a cancer treated as described herein can be a primary cancer. In some cases, a cancer treated as described herein can be a metastatic cancer. In some cases, a cancer treated as described herein can be a refractory cancer. In some cases, a cancer treated as described herein can be a relapsed cancer. Examples of cancers that can be ER+ and that can be treated as described herein include, without limitation, breast cancers, ovarian cancers, endometrial cancers, and lung cancers. Examples of cancers that can be ER- and that can be treated as described herein include, without limitation, breast cancers (e.g., TNBCs), ovarian cancers, endometrial cancers, brain and / or central nervous system cancers (e.g., gliomas and glioblastomas), bone cancers (e.g., osteosarcomas and Ewing sarcomas, biliary tract cancers, thyroid cancers, lung cancers (e.g., mesotheliomas and non-small cell lung cancers), colorectal cancers, head and neck cancers, stomach cancers, pancreatic cancers, kidney cancers (e.g., clear cell renal carcinomas and rhabdoid cancers), liver cancers (e.g., hepatocellular carcinomas), prostate cancers, testicular cancers, skin cancers (e.g., melanomas), leukemias, and lymphomas (e.g., acute myeloid leukemias (AMLs)).

[0085] In some cases, the methods described herein can include identifying a mammal (e.g., a human) as having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC). Any appropriate method can be used to identify a mammal having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC). For example, imaging techniques, biopsy techniques, and molecular techniques (e.g., molecular techniques to detect RNA expression or protein expression such as immunohistochemistry) can be used to Attorney Docket No. 07039-2296WO1 / 2023-600

[0086] identify mammals (e.g., humans) having an ER+ cancer and / or an ER- cancer (e.g., an ER-breast cancer such as a TNBC).

[0087] Any type of mammal having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) can be treated as described herein. Examples of mammals that can have an ER- cancer (e.g., an ER- breast cancer such as a TNBC) and can be treated with one or more ENDX compounds as described herein include, without limitation, humans, nonhuman primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, rabbits, mice, and rats,. In some cases, a mammal having an ER+ cancer and / or an ER- cancer (e.g., an ER-breast cancer such as a TNBC) can be a female. For example, a premenopausal female having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) can be treated with one or more ENDX compounds as described herein. In some cases, a human having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) can be treated with one or more ENDX compounds as described herein.

[0088] A mammal having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) can be administered or instructed to self-administer one or more (e.g., one, two, three, four, or more) ENDX compounds described herein. An ENDX compound can be any type of ENDX compound. For example, an ENDX compound can be a trans isomer of ENDX (E-ENDX) or a cis isomer of ENDX (Z-ENDX). Attorney Docket No. 07039-2296WO1 / 2023-600

[0089] In some cases, an ENDX compound can have one of the following structures:

[0090]

[0091] In some cases, an ENDX compound can be in the form of a free base.

[0092] In some cases, an ENDX compound can be in the form of a salt. For example, an ENDX compound can be in the form of a hydrochloride salt (e.g., a Z-ENDX hydrochloride salt).

[0093] Any appropriate amount (e.g., any appropriate dose) of one or more ENDX compounds can be administered to a mammal (e.g., a human) having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC). An effective amount (e g., a therapeutically effective amount) of a composition containing one or ENDX compounds described herein can be any amount that can treat a mammal having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) as described herein without producing significant toxicity to the mammal. In some cases, an effective amount of one or more ENDX compounds can be about 40 milligrams (mg) or more. In some cases, an effective amount of one or more ENDX compounds can be a plasma concentration of from about 300 nM to about 3000 nM (e.g., from about 300 nM to about 2500 nM, from about 300 nM to about 2000 nM, from about 300 nM to about 1500 nM, from about 300 nM to about 1000 nM, from about 300 nM to about 750 nM, from about 300 nM to about 500 nM, from about 500 nM to about 3000 nM, from about 750 nM to about 3000 nM, from about 1000 nM Attorney Docket No. 07039-2296WO1 / 2023-600

[0094] to about 3000 nM, from about 1500 nM to about 3000 nM, from about 2000 nM to about 3000 nM, from about 2500 nM to about 3000 nM, from about 500 nM to about 2500 nM, from about 750 nM to about 2000 nM, from about 1000 nM to about 1500 nM, from about 500 nM to about 1500 nM, from about 1000 nM to about 2000 nM, or from about 1500 nM to about 2500 nM). In some cases, an effective amount of one or more ENDX compounds can be from about 20 mg / day to about 360 mg / day (e.g., from about 20 mg / day to about 340 mg / day, from about 20 mg / day to about 300 mg / day, from about 20 mg / day to about 250 mg / day, from about 20 mg / day to about 200 mg / day, from about 20 mg / day to about 150 mg / day, from about 20 mg / day to about 100 mg / day, from about 20 mg / day to about 50 mg / day, from about 50 mg / day to about 360 mg / day, from about 100 mg / day to about 360 mg / day, from about 150 mg / day to about 360 mg / day, from about 200 mg / day to about 360 mg / day, from about 250 mg / day to about 360 mg / day, from about 300 mg / day to about 360 mg / day, from about 50 mg / day to about 350 mg / day, from about 100 mg / day to about 300 mg / day, from about 150 mg / day to about 250 mg / day, from about 50 mg / day to about 150 mg / day, from about 100 mg / day to about 200 mg / day, from about 150 mg / day to about 250 mg / day, or from about 200 mg / day to about 300 mg / day). In some cases, an effective amount of one or more ENDX compounds can be as described elsewhere (see, e.g., Goetz et al., J. Clin. Oncol., 35:3391-3400 (2017)).

[0095] In some cases, one or more ENDX compounds described herein can be administered to a mammal (e.g., a human) having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) together with one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell. An agent that can alter the antigens presented on the surface of a cancer cell can be any appropriate type of molecule (e.g., small molecules and polypeptides such as antibodies). In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be a CDK4 / 6 inhibitor. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be a cell cycle checkpoint inhibitor. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be an ALK inhibitor. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be a RET inhibitor. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be an interferon. In some Attorney Docket No. 07039-2296WO1 / 2023-600

[0096] cases, an agent that can alter the antigens presented on the surface of a cancer cell can be an anthracyclin. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be a platinum-based agent. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be a HD AC inhibitor. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be a tyrosine kinase inhibitor. In some cases, an agent that can alter the antigens presented on the surface of a cancer cell can be a proteasome inhibitor. Examples of agents that can alter the antigens presented on the surface of a cancer cell include, without limitation, abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602.

[0097] Examples of therapies that can alter the antigens presented on the surface of a cancer cell include, without limitation, radiation therapies and oncolytic virus therapies.

[0098] One or more ENDX compounds described herein (and, optionally, one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be formulated into a composition (e g., a pharmaceutically acceptable composition) for administration to a mammal having an ER+ and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC). For example, a therapeutically effective amount of one or more ENDX compounds described herein can be formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. A pharmaceutical Attorney Docket No. 07039-2296WO1 / 2023-600

[0099] composition can be formulated for administration in solid or liquid form including, without limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.

[0100] A composition (e.g., a pharmaceutically acceptable composition) including one or more ENDX compounds described herein (and, optionally, one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be administered locally or systemically. A composition containing one or more ENDX compounds described herein (and, optionally, one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be designed for oral, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), or inhaled. For example, a composition containing one or more ENDX compounds described herein (and, optionally, one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be administered systemically by an oral administration to or inhalation by a mammal (e.g., a human). When being administered orally, a composition containing one or more ENDX compounds described herein (and, optionally, one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be in the form of a pill, tablet, or capsule.

[0101] In some cases, one or more ENDX compounds (and, optionally, one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC)) to reduce the size of the cancer present within a mammal. For example, the materials and methods described herein can be used to reduce the number of cancer cells present within a mammal having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, the materials and methods described herein can be used to reduce the size (e.g., volume) of one or more tumors present within a mammal having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. Attorney Docket No. 07039-2296WO1 / 2023-600

[0102] In some cases, one or more ENDX compounds (and, optionally, one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC)) to improve survival of the mammal. For example, disease-free survival (e.g., relapse-free survival) can be improved using the materials and methods described herein. For example, progression-free survival can be improved using the materials and methods described herein. In some cases, the materials and methods described herein can be used to improve the survival of a mammal having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

[0103] In some cases, one or more ENDX compounds described herein (and, optionally, one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be used as the sole active agent(s) to treat a mammal (e.g., a human) having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC). For example, a composition including one or more ENDX compounds (and, optionally, one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell) can include the one or more ENDX compounds (and, optionally, the one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell) as the sole active agent(s) to treat a mammal (e.g., a human) having an ER+ cancer and / or an ER- cancer (e g., an ER-breast cancer such as a TNBC).

[0104] In some cases, one or more ENDX compounds described herein (and, optionally, one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell described herein) can be administered to a mammal (e.g., a human) having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) together with one or more (e.g., one, two, three, four, or more) additional agents / therapies used to treat an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC). In some cases, an anticancer agent can be a targeted therapy. In some cases, an anti-cancer agent can be a hormone therapy. In some cases, an anti-cancer agent can be an immunotherapeutic agent. Examples of anti-cancer agents include, without limitation, trametinib, dabrafenib, binimetinib, selumntinib, vemurafenib, encorafenib, cobimetinib, goserelin, leuprolide, tamoxifen, Attorney Docket No. 07039-2296WO1 / 2023-600

[0105] letrozole, anastrozole, exemestane, bevacizumab, rucaparib, and any combinations thereof. In cases where one or more ENDX compounds (and, optionally, one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell) are used with one or more additional agents treat a cancer, the one or more additional agents can be administered at the same time (e.g., in a single composition) or independently. In some cases, one or more ENDX compounds (and, optionally, one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell) can be administered first, and the one or more additional agents administered second, or vice versa.

[0106] Examples of therapies that can be used to treat cancer include, without limitation, surgery, radiation therapy, carbon ion therapy, and proton therapy. In cases where one or more ENDX compounds (and, optionally, one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell) are used in combination with one or more additional therapies used to treat cancer, the one or more additional therapies can be performed at the same time or independently of the administration of one or more ENDX compounds (and, optionally, the one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell). For example, the one or more ENDX compounds (and, optionally, one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell) can be administered before, during, and / or after the one or more additional therapies are performed.

[0107] In certain instances, an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) within a mammal can be monitored to evaluate the effectiveness of the cancer treatment. Any appropriate method can be used to determine whether or not a mammal having an ER+ cancer and / or an ER- cancer (e.g., an ER- breast cancer such as a TNBC) is treated. For example, imaging techniques or laboratory assays can be used to assess the number of cancer cells and / or the size of a tumor present within a mammal. For example, imaging techniques or laboratory assays can be used to assess the location of cancer cells and / or a tumor present within a mammal.

[0108] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. Attorney Docket No. 07039-2296WO1 / 2023-600

[0109] EXAMPLES

[0110] Example 1: Allosteric Regulation and Pharmaceutical Targeting of Protein Kinase Cfi Protein Kinase C (PKC) enzymes are a family of ten kinases that mediate signal transduction in a wide range of cellular pathways, and altered PKC activity is implicated in many disease states, including cancer and neurodegenerative diseases (Leitges et al., Science, 273:788-791 (1996); Tagawa et al., Hum. Mol. Genet., 24:540-558 (2015); Zarate et al., CNS Drugs, 23:569-582 (2009); Geraldes et al., Circ. Res., 106:1319-1331 (2010); Palaniyandi et al., Cardiovasc. Res., 82:229-239 (2009); Garg et al., Oncogene, 33:5225-5237 (2014); and Sadeghi et al., Int. J. Mol. Sci., 22:5527 (2021)).

[0111] PKCs are ubiquitously expressed throughout the body, yet only a variable subset of the PKC enzymes is present in each tissue type. The four canonical PKC family members (a, pi, il, and y) are activated upon interacting with the plasma membrane in response to diacylglycerol (DAG) and Ca2+signals. Although PKCa, pi, pil, and y are >65% identical in sequence (Figure 7A), they perform distinct cellular functions and display varied sensitivity to lipid signals (Kedei et al., Cancer Res., 64:3243-3255 (2004); and Steinberg, Physiol. Rev., 88:1341-1378 (2008)). PKC i and PKC iI isoforms are expressed from the same gene and differ by only a 50 aa C-terminal segment that is derived from a regulated alternative splicing event (Figure 7A) (Chalfant et al., J. Biol. Chem., 270:13326-13332 (1995)), suggesting that even subtle sequence differences can impart altered activity on each isozyme.

[0112] Despite decades of investigation, the molecular mechanisms of PKC regulation and activation are not known. In spite of the considerable interest in developing drug candidates that inhibit (or activate) PKCs to treat an array of diseases including a variety of cancers, a strategy of using ATP-competitive inhibitors has, thus far, been unsuccessful.

[0113] Staurosporine and derivatives, which target the highly conserved ATP binding site, have demonstrated limited or no antitumor activity in multiple different cancers despite nanomolar affinity and are prone to off-target toxicity (Mina et al., Invest. New Drugs, 27:565-570 (2009); Clemons et al., Breast Cancer Res. Treat., 124:177-186 (2010); Millward et al., Br. J. Cancer, 95:829-834 (2006); Robertson et al., J. Clin. Oncol., 25:1741-1746 (2007); Attorney Docket No. 07039-2296WO1 / 2023-600

[0114] Macedo et al., Cancer Res., 68:3516-3522 (2008); and Kreisl et al., Neuro. Oncol., 12:181-189 (2010)).

[0115] This Example demonstrates that endoxifen (ENDX) can act as an allosteric inhibitor of PKC polypeptides.

[0116] Results

[0117] It was sought to improve on the established insect cell PKC expression system (Mukai et al., Methods Mol. Biol., 233:21-34 (2003)) by using HEK293F cells and the YFP-tag system (Schellenberg et al., Protein Sci., 27:1083-1092 (2018)) to generate recombinant PKCβII (Figure 7C). The final, purified PKCβII contains the phosphorylation sites (T500-activation loop, T641-turn motif, and S660-hydrophobic motif) (Figure 7D) and migrates as a single band on phos-tag SDS-PAGE (Figure 1B), indicating that the YFP-tagged purified PKC was fully phosphorylated at all three sites. Crystals of PKCβII in the presence of a non-hydrolysable ATP analogue AMPPNP were obtained and a 3.3 A structure was solved (Figures 1C and ID; Table 1). Attorney Docket No. 07039-2296WO1 / 2023-600

[0118] Table 1. X-ray diffraction data collection and structure refinement statistics

[0119] _ _

[0120] “PDB AcceZsiroi S’

[0121] Space group € 1 2 1 C 1 2 1 C 1 2 1 0 1 2 1 Cell dimensions

[0122] C i A? 139.6, 89.1, 160 7 162.7 157.8, 87.1 82.2, 153.2, 77.3 162.7, 157.9.85.1 <4 A 7 ' 90, 107.1, 90 90, 113.0, 90 90, 100.4, 90 90, 112.2, 90 Resolution (A)’ 50.00-3.35 50-2.7 50.00-2.60 50-2.95

[0123] (3.47-3.35) (2.80-2.70? { 2. „00 j (3.06-2.95) 0.299 {0.962? 0.141 (2.084) 0.149 (1.485) 0.115 (1.12) I / cZ 4.7 (1,5) 15.7 (1.1) 13.2 (1.4) 11 3 (1.3) 0.91 (0.46) 0.99 (0.48) 0.99 (0.49) 0.99 (0.48) Completeness {%) 99,9999.3? 99.5 (99.8) 99.2 (99 1) 98,8 (99.0) Redundancy 3.5 (3.7) 1(3 6 (10.6) 6.9 (7.1) 5.6 {3.6) Refinement

[0124] Resolution (A) 48 66-2.60 49.70-2 95 No reflections □i?? •'<> 55648 30355 4059

[0125] 0.199 / 0.256 0.206 / 0.258 0 107 / 0 j 2 / 0 19600,252 No atoms (non-H) 9685 4970

[0126] Protein 10077 9657 4871 Ligand / ion?"7 •4N Water I 71 s Average 5 factor (A2) 63.31? H si - -2 3

[0127] Riiniachrtnchsn plot’

[0128] Favoied {%) 94 4 94.4 Allowed (%s 81 4 5_ i 5.4 Outliers 0 5 0,2 t ms. deviations

[0129] Bond lengths (A) 0.906 G 0G2 0.005 Bond angles (*) 0.665 0510 0.805

[0130]

[0131] vw ’vVvxxaxxlxuxxevvsxvv inxxw pv.vavrxxewnvt.vhvxex'esvexvsvx V •eXXXtXeXVrXX tVoXX’V tXhXWsVX l'uVXsSSXhVXeWsVt. VV rXeXWstV. X hXVtXtXiVcXXn’VXX sXXhXXXeWlVl. WVXWV. WVXWV. XWV XVXX’VXXXXXXX'VXXWVX'eVXWVXWV. XVVXWW ■VXXXXXXX'VXXXVXXWV. WVXWV. XVVXWVXVVX*

[0132] Continuous electron density was visible for all four structured domains and the PS, as well as linker residues between the Cl a, Clb, and C2 domains (Figure 8A), and portions of the linker between the C2 domain and the kinase domain (Figure 8B). Density was observed for the three phosphorylated residues required for proper PKC folding and enzyme function (Figure 8C), Cl domain Zn2+ions (Figure 8D), and an active-site engaged Mg-AMPPNP (Figure 1F). Altogether, 91% and 94% of the residues in each of two chains respectively were observed, which represented the most complete picture of any PKC isoform to date. Attorney Docket No. 07039-2296WO1 / 2023-600

[0133] Molecular basis of PKC fill autoinhibition and regulation by phosphorylation

[0134] All domains (PS, Cla, Clb, C2, and kinase) were assigned to each of two chains based on the maximum length that the disordered residues of unobserved linkers could span (Figure 9A). Both chains contained a kinase active site occupied by the PS sequence, but chain A formed an intramolecular complex, and chain B formed a domain-swapped complex with the kinase domain of an adjacent monomer through a nearly identical molecular interface (Figure 9B). Thus, the structure revealed the complete molecular architecture of the inhibited, inactive PKCβII.

[0135] A complex network of inter-domain interactions comprised the inactive state of PKCβII (Figures IE and IF). The N-terminal PS residues (aa 19-30) were positioned in the substrate binding site with the A25 sidechain in a position equivalent to a Ser / Thr in a true substrate (Figure 1F), with specific recognition of the hydrophobic and positively charged residues contained within PKC substrate motifs (Figure 9C). The Clb domain interacted with the kinase domain and PS, which sequestered the hydrophobic lipid-binding residues and buttressed the PS in the kinase active site (Figure 1E). The Cla domain interacted with the kinase domain and C-terminal extension through a small hydrophobic patch and salt bridges such that most of its lipid binding residues and the DAG binding pocket remained solvent-accessible (Figure 1F). This configuration was supported through site-directed mutagenesis that showed F43A, D382K, or E655K mutations disrupted the Cla-Kinase interface and resulted in increased phospholipid interactions or a more extended conformation. A conserved PKC domain architecture consisting of a PS sequence immediately adjacent to a Cl domain in all PKC isozymes (Figure 7B) suggested the same regulatory mechanism was common to all PKC isoforms. The C2 domain interacted with the Cla and Clb domains such that its Ca2+binding site and lipid binding residues were accessible for interaction with the plasma membrane. The kinase domain contained three phosphorylated residues (pT500, pT641, and pS660), which were necessary for catalytic activity (Figure 1F). pT500 served a structural role mediated by salt bridges and a hydrogen bond to surrounding residues as well as to PS residues R27 and N30. The later suggests pT500 formed part of the substrate binding site and explains both the requirement of T500 phosphorylation for kinase activity and the strong counter-selection against negatively charged residues at the +2 and +5 Attorney Docket No. 07039-2296WO1 / 2023-600

[0136] substrate positions. pT641 formed salt bridges to two lysines located in the ATP-binding P-hairpin and ot-helix active-site motifs, as well as an additional hydrogen bond to Sill of the Clb domain. pS660 was located distal from the active site and played a structural role in stabilizing the C-terminal V5 domain by interacting with Q411 and R336 residues.

[0137] Phosphorylation of these three residues also regulated the affinity of PKC for lipid membranes despite being located outside of the lipid-binding domains. The pT500-PS and pT641-Clb interactions stabilized the inter-domain interfaces that sequestered the lipid binding surfaces of the Cla and Clb domains in an inaccessible conformation. Thus, in addition to the structural and substrate-binding roles, pT500 and pT641 also mediated molecular interactions that maintain PKC in its inactive state and in turn reduced the lipid binding affinity of the fully phosphorylated protein.

[0138] Crystal structures ofPKC l reveal active and inactive states

[0139] PKCβI and PKCβII isoforms differed by only 50 C-terminal amino acids (Figure 7A) derived from an alternative splicing event that is responsive to signals such as extracellular glucose levels and signaling by AKT2 kinase. Although the 50 residues were derived from different exons, many of the residues, including the two phosphorylation sites (pT642 and pS661) present in this region, were conserved. To determine the molecular basis of isoform-specific differences recombinant PKCβI (Figure 10A) was generated, and it was co-crystalized with AMPPNP in two crystal forms (Figures 2A and 10C; Table 1). Electron density was observed for all four domains (Cla, Clb, C2, and kinase) together with phosphorylation at the corresponding pi residues T500, T642, and S661 (Figure 10B and 10D), but the PS of only one monomer was visibly bound to the adjacent kinase domain through a domain swap in crystal form 1 (Figure 11 A). Similar to PKCβII, the hinge region between the C2 and kinase domain as well as the N-terminal 18 amino acids were disordered. The linkers between the Cla, Clb, and C2 domains of PKCβI were visible (Figure 10E) except for a gap of 12-13 amino acids linking the Cla and Clb domains in crystal form 1 (Figure 1 IB). However, this gap was small enough that it did affect the ability to unambiguously assign the domains to individual chains. AMPPNP nucleotide was only present in the monomer with a bound PS, and the active site was slightly more open when the Attorney Docket No. 07039-2296WO1 / 2023-600

[0140] nucleotide was absent (Figure 11C). Nonetheless, all monomers contained the DFG motif and helix aC “in” conformations which indicated the kinase domains were in the activated conformation.

[0141] To identify the biological unit of PKCβI from the crystal lattice, the kinase domain from the three asymmetric units were aligned to explore the local symmetry environment and to identify common interaction modalities present amongst the domain arrangements. Each kinase domain formed an interface with three N-terminal domain modules (Figure 1 ID), and two of them were comprised of similar molecular interactions (Figure 2A; N-term 1 & 2), while the third interface was variable amongst the three asymmetric units (N-term 3). The interface with N-term 2 describes a conformation that was similar to PKCβII (Figure 1) but with an altered location for the Clb domain, which was attributed to the auto-inhibited conformation for the PKCβI enzyme. The interface with N-term 1 positioned the Clb and C2 domains, so they interacted with the kinase domain distal from the active-site in an unexpected conformation. AlphaFold also predicted a very similar structural arrangement (Figure 1 IE), which prompted the exploration of possible functional roles for this second PKC conformation.

[0142] PKCβI active conformation

[0143] An unresolved mystery about PKC enzymes is how the activated state prevents the PS from re-engaging the active site. The domain arrangement comprised of the interface with N-term 1 cannot be attributed to any previously described structure of PKC isozymes and has a few striking differences compared to the inactive conformation. No PS was bound within the kinase domain of crystal form 2, and the active site appeared to be unobstructed and available to engage substrate. The Clb and C2 domains interacted with the kinase domain on the side opposite the active site, and the Cla domain was buttressed against the Clb and C2 domains (Figure 2A). The Cla and Clb domains contained a bound glycerol molecule in the DAG binding site (Figure 12A). Calcium was not included in the crystallization condition. The lipid binding surfaces of the Cla, Clb, and C2 domains aligned in the same plane, as would be the case were they bound or embedded in a plasma membrane. Furthermore, the PS Attorney Docket No. 07039-2296WO1 / 2023-600

[0144] would be anchored ~70A away from the active site and prevented from re-engaging the active site in this conformation (Figure 12B).

[0145] To test the hypothesis that PKC can form an active conformation upon binding to the lipid membrane, a site-directed mutagenesis strategy was employed, and a panel of mutants was generated to disrupt the observed interfaces between the kinase and Clb / C2 domains (Figure 2B). Kinase activity stimulated by Ca2+and DAG-containing phospholipids were assayed in vitro using the FRET-based CKAR substrate. All of the mutants that disrupted the C2-kinase and Clb-kinase interfaces impaired the kinase activity of PKCβI (Figures 2C and 12C). Since these mutations did not affect residues involved in the active-site or lipid binding, impaired kinase activity was attributed to disruption of the interfaces between the lipid binding domains and the kinase domain observed in the crystal structure. These findings indicated that PKCβI took on a defined and ordered conformation upon association with DAG-containing membranes that unmasked the kinase active site to activate PKCβI.

[0146] The Lipid-Lever mechanism of PKC activation

[0147] Another fundamental question about the mechanism of PKC activation is the role of the phospholipid membrane. In the presence of Ca2+, PKCs can be activated by a phospholipid bilayer containing DAG or an agonist such as phorbol 12, 13 -dibutyrate (PDBu). However, in the absence of a phospholipid bilayer, PDBu and Ca2+cannot activate PKCP kinase activity (Figure 3B). This suggested that it was not the DAG or phorbol agonist that was the activating ligand per se, but somehow the membrane itself drove a conformational change in PKC. The DAG / phorbol-binding cleft of the Cla domain is solvent-accessible, and PDBu binding would not disrupt the inactive conformation (Figure 12D), ruling out a direct competition mechanism and suggesting the phospholipid membrane itself is critical for driving the conformational change that activates PKC. Limited proteolysis was used to probe the conformation of PKCβI and increased elastase cleavage was observed in the presence of Ca2+and DAG-containing micelles but not in the presence of Ca2+and PDBu, even at 100 pM (Figure 3C), indicating that phorbol binding alone was insufficient to activate PKCβI. Modelling of the Cla domain engaged in a lipid bilayer revealed that the Cla-PSkinase domain architecture observed in the crystal structure would necessitate that the Attorney Docket No. 07039-2296WO1 / 2023-600

[0148] kinase domain become embedded into the membrane as well (Figure 3A). When the hydrophobic residues of the Cla domain engaged the membrane lipids, a clash between the charged kinase domain and hydrophobic membrane would prevent the kinase domain from remaining bound to the Cla and PS (Figure 12E). In this way, the lipid membrane acted as a lever that can pry PS from the catalytic domain and disrupt the interdomain interactions that preserve the inhibited state via a mechanism referred to herein as a “lipid-lever”. The kinase domain occluded some of the lipid-binding surface centered around F43 (Figure 1F) in the inactive conformation, providing a mechanistic basis by which a fully folded kinase domain moderated membrane binding by the Cl domains. The lipid-lever mechanism is likely shared across PKC isoforms as the PS was always found immediately N-terminal to a Cl domain across the family of PKCs (Figure 7B).

[0149] Molecular basis of isoform-specific differences between the splice variants PKC fl and PKCfill

[0150] One well-characterized difference between PKCβI and pil is the difference in affinity for phospholipid membranes (Kedei et al., Cancer Res., 64:3243-3255 (2004); and Truebestein et al., J. Mol. Biol., 428:121-141 (2016)), yet these splice variants contained identical lipid-binding domains, suggesting that a mechanism such as the lipid-lever links the variant C-termini to altered phospholipid binding. The crystal structures of PKCβI and PKCβII were examined to identify the molecular basis of isoform-specific effects. The active conformation did not contain any interactions between the unique C-terminal extension and lipid-binding domains, so the inhibited conformation of PKCβI (crystal form 1) was compared with that of PKCβII (Figure 4A). The most significant difference was the position of the Clb domain. In PKCβII, the Clb domain was sequestered through extensive contact with the catalytic domain but in contrast was displaced with its lipid-binding residues exposed in PKCβI (Figure 4B). This suggested a model whereby enhanced accessibility of Clb lipid-binding residues contributed to a stronger lipid interaction of the PKCβI isoform. The Clb domain only contacted pT641 and F633 of the PKCβII V5 domain, yet PKCβI contained spatially equivalent residues (pT642 and F634) at the same locations (Figure 7A). In PKCβII, Fl 14 of the Clb domain packed against helix aB, whereas this helix was shifted Attorney Docket No. 07039-2296WO1 / 2023-600

[0151] in PKCβI which occluded the Fl 14 binding pocket. The aB shift can be attributed to PKCβI F648, which corresponds to V647 in PKCβII. The larger F648 residue shifted helix aB closer to the PS and occluded the pocket occupied by Fl 14 in PKCβII, thus preventing Clb from making close contact with this region (Figure 4C). To evaluate this model, a PKCβI F648A mutant was engineered and the conformational state of PKCβI (WT and F648A) and PKCβII was probed using limited proteolysis. In the presence of Ca2+and activating lipids elastase cleavage was observe at a site corresponding to the hinge region (Figure 4C). PCKpi was more sensitive to elastase than PKCβII, indicating that PKCβI more easily adopted the active conformation in the presence of lipids. PKCβI F648A was less sensitive to elastase, consistent with a model whereby a smaller residue yields an enzyme with enhanced stability of the inactive state. Thus, the more potent lipid binding of the PKCβI isoform was attributed to a shift in the position of helix aB that displaced the Clb from its docked position.

[0152] Endoxifen as an allosteric regulator of PKCfi

[0153] PKCs can be inhibited by tamoxifen (TAM) (Gundimeda et al., J. Biol. Chem., 271: 13504-13514 (1996)) and more potently by its metabolite endoxifen (ENDX) (Ali et al., Bioorg. Med. Chem. Lett., 20:2665-2667 (2010)). The effects of ENDX and TAM on PKCβI kinase activity were assessed in vitro in the presence and absence of activating lipids and it was found that ENDX was uniquely able to inhibit PKCβI at clinically achievable concentrations (IC50 = 1.49 pM; Goetz et al., J. Clin. Oncol., 35:3391-3400 (2017)), while TAM required much higher concentrations (IC50 = 5.9 pM, p<0.001), beyond that which is achievable with the FDA approved 20 mg / day dose (Figure 5A). Both ENDX and TAM were less effective inhibitors in the presence of Ca2+and activating phospholipid micelles, similar to when using a truncated, constitutively active PKC I consisting of only the kinase domain (Figure 5B). Additionally, differential scanning fluorimetry (DSF) analysis showed that both the N-terminal regulatory domain and the kinase domain bound to ENDX (Figure 13 A), and ENDX exhibited distinctly non-competitive inhibition with ATP (Figure 5C) in contrast to the established ATP-competitive PKC inhibitor enzastaurin (Faul et al., Bioorg. Med. Chem. Lett., 13:1857-1859 (2003)). Collectively, these data suggested ENDX inhibits PKCβI via a unique mechanism through binding to both the N-terminal and kinase domains. Furthermore, Attorney Docket No. 07039-2296WO1 / 2023-600

[0154] ENDX inhibited several PKC enzymes, albeit with a range of IC50 values, whereas the closely related AGC kinase PKA was completely unaffected by ENDX up to 100 pM, suggesting specificity for PKC enzymes (Figure 13B). The effect of ENDX on the solution conformation of PKCβI was evaluated using Small-Angle X-ray Scattering (SAXS) and it was found that ENDX but not TAM decreased the Rg and Dmax of PKCβI protein (Figure 5D). A limited proteolysis assay that monitors cleavage at a sensitive site located within the linker between the Clb and C2 domains by elastase with increasing concentrations of ENDX revealed that ENDX but not TAM caused a noticeable structural change with an apparent midpoint of 3.5 pM, as evidenced by a decrease in band intensity on SDS-PAGE gel (Figure 5E). Collectively, these data demonstrated that ENDX is a non-ATP-competitive inhibitor and an allosteric regulator of PKCβI, with the potential to target additional PKC enzymes at clinically achievable concentrations.

[0155] Live-cell imaging of MCF7AC1 cells expressing YFP-tagged PKC I was performed to examine whether ENDX also affects PKCβI translocation to the plasma membrane. It was found that in the presence of sub-saturating amounts of the PKC agonist phorbol myristate acetate (PMA), ENDX increased PKCβI localization to the membrane in response to PMA in both a time-dependent and dose-dependent manner (Figures 5F, 13C, and 13D). Furthermore, localization was mitigated by the addition of the pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP1 / 2) inhibitor NSC 117079. These data suggested that ENDX can induce dephosphorylation of PKCβI by PHLPP1 / 2, which in turn enhances recruitment to the plasma membrane (Figure 5F). Collectively, these results indicated that ENDX altered the conformation of PKC in a way that inhibited kinase activity but also promoted recruitment to the plasma membrane via a PHLPP1 / 2 -mediated mechanism.

[0156] Materials and Methods

[0157] Reagents, cell lines, antibodies, plasmids

[0158] HEK293F cells (Thermofisher) and MCF7AC1 cells were grown in DMEM with 10% (v / v) Fetal Bovine Serum (Gibco), 50 U / pL penicillin, 50 pg / mL streptomycin, and 0.5 mmol / L sodium pyruvate at 37°C in 5% CO2 atmosphere for adherent growth. Suspension cultures of HEK293F cells were grown in CDM4HEK (Cytiva) media supplemented with 20 Attorney Docket No. 07039-2296WO1 / 2023-600

[0159] mM glutamine, 12 U / pL penicillin, and 12 pg / mL streptomycin at 37°C in 8% CO2 atmosphere at 135 rpm in a Multitron shaker incubator (HT Infers) for suspension growth. Antibodies used in this study: Anti PKCP phospho-T500 (Abeam ab5817) Anti-phospho T642 (Abeam ab75657), and anti-phospho S661 (Abeam 192184). DNA encoding PKC constructs was cloned into pMCentr2 (DNASU) and recombined into mammalian protein expression vector pcDNA6.2 / NYFP-Dest using LR Clonase II (Invitrogen). Mutant PKCβI plasmids were generated using the Quickchange kit (Stratagene).

[0160] Expression and purification of PKC fl and PKC fill

[0161] Recombinant plasmid was mixed with 1 mg / mL polyethyleneimine pH 7.5

[0162] (Poly sciences, Inc.) in a 1:3 ratio to form a DNA-PEI mixture used to transfect HEK293F cells in suspension culture and grown in HyClone HyCell Transfx-H (Cytiva) medium supplemented with 200 mM Glutamine (Thermofisher) and 0.075% Plutonic (Gibco 24040-032). Transfected cells were grown for 72 hours at 37°C, while cell count and protein expression (GFP) level were monitored every 24 hours. PKC0 expressing culture was spun down at 5000xg for 10 minutes, followed by resuspension of pelleted cells in IX PBS containing 0.1X Roche EDTA-free protease inhibitor cocktail (Sigma). Centrifugation step was repeated to obtain cell pellet that was used directly for protein prep or frozen down at -80°C for storage.

[0163] Thawed HEK293F cells expressing PKCP were lysed at 4°C with a lysis buffer solution consisting of 50 mM Tris pH 7.4, 300 mM NaCl, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM P-glycerol phosphate, 1 mM TCEP, 2 mM benzamidine, 2 pg / mL leupeptin, 0.5 mM sodium orthovanadate, 0.5% CHAPS, and 1:100 protease inhibitor cocktail (Sigma P8849). Lysate was sonicated with a Branson sonicator at 50% power for 10 seconds (x2) and spun down in a Lynx 4000 centrifuge (Thermo Fisher) at 15,000 g, 4°C for 10 minutes. The clarified lysate was bound and recycled 3x over a GFP-enhancer nanobody linked NHS Sepharose (Cytiva 45002965) resin bed equilibrated in lysis buffer. Protein bound resin was washed in 3x resin volume with the same lysis buffer. TEV cleavage of the YFP tag was completed overnight by incubating the resin with 1.5x resin volume TEV cleavage buffer containing 50 mM Tris pH7.4, 50 mM NaF, 5 mM sodium pyrophosphate, Attorney Docket No. 07039-2296WO1 / 2023-600

[0164] 10 mM P-glycerol phosphate, 1 mM TCEP, 0.5 mM sodium orthovanadate, and 0.25% CHAPS, supplemented with 0.09 mg / mL TEV protease. Cleaved protein was eluted from the resin using buffer without TEV protease, and the presence of protein in eluted fractions was monitored using a Coomassie-stained SDS-PAGE gel.

[0165] Following purification, PKCβ protein was polished on an AKTA go FPLC system (Cytiva) to remove TEV protease and further purify protein for biochemical assays and crystallization. TEV eluted protein was diluted (1:3) with a low salt buffer containing 50 mM Tris pH 8.0 and 1 mM TCEP prior to being loaded onto a HiTrap Q HP anion exchange column (Cytiva). A salt gradient was introduced by the gradual addition of buffer supplemented with IM NaCl at a flow rate of 5 mL / min. Gradient fraction elution continued to a final of 60% or 600mM NaCl. Eluted fractions containing PKCP were combined and concentrated to 0.5 mL using a 10K cut off centrifugal filter (Sartorius, Vivaspin), pretreated overnight with 3% PEG 3350 (Jena Biosciences) at 4°C to avoid protein loss through binding to filter. Concentrated protein was subjected to final purification on a Superdex 200 Increase 10 / 300 GL column (Cytiva) using 20mM Tris pH 8.0, 100mM NaCl, 2mM MgCl2, and 1mM TCEP at a flow rate of 0.5 mL / min. All FPLC elution fractions were monitored for protein presence using Coomassie-stained SDS-PAGE gel. Fractions containing PKC were pooled and concentrated to 10 mg / mL using a 10K centrifugal filter (Sartorius, Vivaspin) for crystallization, or buffer exchanged into storage buffer (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM MgCl2, 0.5 mM TCEP, 25% (v / v) glycerol) and stored at -80 °C prior to use in kinase assays. Typical yield of final, purified PKCβI / II proteins (77kDa) was 3 to 6 mg per litre of culture.

[0166] Lambda Phosphatase Reactions

[0167] To generate dephosphorylated PKC, 7 pg PKC protein were incubated at 37°C for 2 hours with 1400U Lambda Phosphatase (New England BioLabs P0753S) and its lx reaction buffer according to the manufacturer’s protocol. As a control, 7 pg PKC protein was incubated under the same conditions in the absence of phosphatase. After the incubation period, loading dye was added and the reactions were heated to 75°C for 5 minutes prior to visualization via 8% SDS-PAGE with 0 pM PhosTag or 40pM Phostag (APExBIO). Attorney Docket No. 07039-2296WO1 / 2023-600

[0168] Crystallization and structure determination of PKCfil and PKCfill

[0169] Crystals of PKCβI and PKCβII were grown using the sitting-drop vapor diffusion method by mixing 200 nL of precipitant with 200 nL of protein mixture (PKCβI / II with 1 mM AMPPNP). Crystals of PKCβI crystal form 1 and 2 grew in 200-300 mM sodium citrate pH 7-8 and 8-10% (w / v) PEG3350. Crystals of PKCβII grew in 100-200 mM magnesium chloride, 0.1 M MES pH 6.5, and 6-10% (w / v) PEG8000. Crystals grew over a period of 2-6 weeks at 4°C and were transferred to a cryoprotectant containing crystallization condition supplemented with 25% (v / v) glycerol, then flash frozen in liquid nitrogen. For manganese soaks, the cryoprotectant also contained 1 mM MnCl2. X-ray diffraction datasets were collected at the NE-CAT beamlines (24-C and 24-E) at the Advanced Photon Source. X-ray diffraction data were processed and scaled using the HKL2000 suite. Structures were solved via molecular replacement using the Clb, C2, and kinase domains from PDB entry 3PFQ as search models using the PHENIX-PHASER. The C la domain and other missing regions were built manually using COOT, followed by refinement against the high-resolution datasets with PHENIX to produce the final models.

[0170] Limited Proteolysis

[0171] 0.4 mg / mL PKC protein was digested with 1.4 pg / mL elastase (Promega) in the presence and absence of activating factors (40 pg / mL lipids and 82 pM Ca2+). Additional reaction components, like drugs or phorbol dibutyrate, were added in the concentrations indicated. Reactions were incubated at room temperature for 45 minutes. Protease activity was quenched by adding SDS reducing dye and heating at 70°C for 10 minutes. The full mixture was loaded onto a SurePAGE Bis-Tris 4-12% gel (Genscript) and stained with Coomassie blue to visualize proteins.

[0172] Expression and purification of CKAR

[0173] pRSET-B-CKAR (Addgene) was transformed into JM109 DE3 E. coli (Promega). Cultures of pRSET-B-CKAR in JM109 DE3 were grown in 2 L of Terrific Broth (Research Products International), supplemented with 300 pL of antifoam 204 (Sigma-Aldrich) in a LEX-48 Bioreactor (Epiphyte3) at 37°C, until they reached an optical density (OD600) between 3.0 and 4.0. CKAR Protein expression was induced by the addition of 50 pM of Attorney Docket No. 07039-2296WO1 / 2023-600

[0174] isopropylthio-P-galactoside (IPTG, Goldbio) for 18 hours at 16°C. The bacterial cell pellet was harvested by centrifugation at 6000 g for 20 minutes at 4°C, and frozen at -80°C.

[0175] Cell pellets were thawed and lysed in Ni running buffer (20 mM Tris pH 7.5, 300 mM NaCl, 0.5 mM TCEP, 10 mM imidazole) supplemented with 0.1 mg / mL of lysozyme and a 1 / 2000 dilution of ethanol saturated phenylmethyl sulfonyl fluoride (PMSF, Goldbio) on ice for 30 minutes. Lysate was sonicated at 80% power for three 30-second intervals using a Branson 250 sonifier. Sonicated lysate was centrifuged at 25,000 g for 30 minutes and soluble fraction was passed over Ni-NTA resin (Qiagen) pre-equilibrated with lysis buffer. The resin was washed six times, each with 1 column volume of lysis buffer and CKAR was eluted with Ni running buffer supplemented with 250 mM imidazole. Protein was precipitated out of solution by the addition of 2x volume of 4 M ammonium sulfate and pellets were collected after centrifugation at 25,000 g for 30 minutes. The protein pellets were redissolved in 2 mL of milli-Q water and polished on a Superdex 200 16 / 60 column (Cytiva) in 20 mM Tris pH 7.5, 300 mM NaCl, and 0.5 mM TCEP buffer. Protein elution was monitored by absorbance at 280 nm, 460 nm, and 520 nm to identify fractions with full-length CKAR and both YFP and CFP modules. CKAR was further polished by ion exchange chromatography on a Hi-Trap Q HP column (Cytiva) using a 0-50% gradient between low salt (20 mM Tris pH 7.5, 3 mM DTT) and high salt buffers (20 mM Tris pH 8.0, IM NaCl). CKAR was concentrated and buffer exchanged into PKC Storage Buffer (20 mM Tris pH 8.0, 100 mM NaCl, 2mM MgCl2, ImM TCEP, and 25%(v / v) glycerol) using an Amicon 10K concentrator (Millipore). The final CKAR product was quantified using A520nm and an extinction coefficient of 70,000 M-lcm-1 for YFP.

[0176] CKAR Kinase Assay

[0177] Lipid vesicles were prepared fresh by dissolving 10 mg / mL bovine brain phosphatidylserine (PS) (Avanti) or / and 1 mg / mL 1,2-dioleoyl-glycerol (DAG) (Avanti) in chloroform. Lipids were mixed at the indicated ratio and chloroform was evaporated with a stream of dry air, then lipids were redissolved in water to a final concentration of 40 pg / mL, vortexed for 60 seconds, and sonicated 3 times for 30 seconds at 10% power on a Branson 250 sonifier with a microtip (12840498). Concentrated PKC protein was diluted into storage Attorney Docket No. 07039-2296WO1 / 2023-600

[0178] buffer containing 20 mM Tris pH 8.0, 100 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 25%(v / v) glycerol and 1 mg / mL BSA to create a 10X stock based on final assay concentration. Kinase reaction mixtures (100 pL) contained 20 mM HEPES pH 7.5, 2 mM MgCl2, 1 pM CKAR, 10 nM PKCβ protein and indicated concentrations of ENDX and ATP. Reaction mixtures were pre-heated at 30°C and ATP was mixed in last to initiate the reaction, just prior to placing the reaction plate in a CLARIOstarPlus plate reader (BMG Labtech). Forster Resonance Energy Transfer (FRET) was measured every 5 minutes using a 434-16 excitation filter with 476-16 and 528-16 emission filters at 30°C. Control reactions without PKC were used as a background measurement to correct for fluorophore decay throughout the experiment. The FRET ratio was calculated by dividing the emission measurement at 476 nm by 528 nm emission. PKC kinase activity was plotted as the change in FRET ratio over time (dFRET / dT).

[0179] Z ’-LYTE Kinase Assay

[0180] Kinase activity was measured using the Z’-LYTE Kinase Assay Kit - Ser / Thr 7 Peptide (ThermoFisher) following the manufacturer’s protocol. The reaction mix contained 250 mM HEPES pH 7.5, 50 mM MgCl2, 5 mM EGTA, 0.05% Brij-35, 40 pg / mL PS: DAG lipids, PKC protein at the indicated concentration, drug serial dilution at the respective concentration, 40 pM ATP and 2 pM Z’-LYTE Ser / Thr 7 peptide substrate. The peptide substrate and ATP mixture were added last to initiate the reaction. For reactions without lipids, the 40 pg / mL lipids were substituted with milli-Q H2O. Reactions were incubated at room temperature for 1 hour. The development solution, created using the manufacturer's instructions, was added and mixed with the kinase reaction prior to placing the plate in the CLARIOstarPlus plate reader (BMG Labtech). The extent of phosphorylation of the peptide substrate was calculated through the ratio of the coumarin emission at 445 nm to the fluorescein emission at 520 nm after a 1-hour incubation with development solution. Kinase activity was ploted as percent activity to the corresponding drug concentration. To determine inhibitor IC50 values, data were fitted to a 4-parameter dose-response model using Graphpad Prism 9. In cases where the IC50 could not be accurately determined due to precipitation of ENDX at very high concentrations, the model was constrained to a bottom value of 0 % and Attorney Docket No. 07039-2296WO1 / 2023-600

[0181] the resulting estimated ICso value was reported. Extra sum of F squares analysis was used to calculate P values between different drug treatments.

[0182] Expression and purification of PKA

[0183] DNA encoding PKA catalytic domain (Addgene 14921) was transformed into Rosetta2 cells (EMD) and inoculated in a 2 L culture of terrific broth (Research Products International) supplemented with 300 pL Antifoam 204, 100 pg / mL carbenicillin, and 34 pg / mL chloramphenicol. Culture was grown using a LEX-48 Bioreactor (Epiphyte) at 37°C and protein expression was induced with 100 pM IPTG overnight at 16°C. E. coli culture was pelleted, resuspended in lysis buffer (20 mM Tris pH 7.5, 300 mM NaCl, 10 mM imidazole, and 0.5 mM TCEP) supplemented with 10 mg of lysozyme (GoldBio) and 20 pL of saturated PMSF in ethanol (GoldBio). Cell lysate was incubated on ice for 30 minutes with occasional mixing and then sonicated by a Branson 250 sonicator at 80% power in five 15-second intervals. Clarified lysate was centrifuged at 25,000g for 30 minutes and passed over a Ni-NTA column equilibrated in lysis buffer. Ni-NTA resin was washed with column volumes lysis buffer and eluted with lysis buffer supplemented with 250 mM imidazole. Protein containing fractions were detected by a color change in a Bradford Assay (99 pL bradford reagent, 1 pL elution fraction), pooled and precipitated with two volumes of 4 M ammonium sulfate followed by centrifugation at 25,000g for 30 minutes at 4°C. Precipitated protein was dissolved in 3 mL Milli-Q water and loaded onto a Superdex S200 size exclusion column equilibrated in 20 mM Tris pH 7.5, 300 mM NaCl, and 0.5 mM TCEP. Fractions were analyzed by SDS-PAGE gel and PKA containing fractions were pooled and diluted 1:5 in Milli-Q water. The pH of the dilution was adjusted to pH 6 by addition of MES powder and loaded onto a Resource 15S cation exchange column (Cytiva) equilibrated in running buffer (20 mM NaH2PO4pH 6.0, 0.1 mM TCEP) and eluted with a linear gradient of 0-100% 1 M NaCl in 20 mM NaH2PO4pH 6.0. To increase the pH in PKA-containing fractions 20 mM Tris pH 8.0 was added to pooled protein and protein was then concentrated by Amicon ultrafiltration (Millipore). Concentrated protein was run over a Superdex 200 increase (Cytiva) equilibrated in running buffer (20 mM Tris pH 7.5, 300 mM NaCl, and 0.5 mM TCEP). Purified protein was analyzed by SDSPAGE gel and buffer exchanged during Attorney Docket No. 07039-2296WO1 / 2023-600

[0184] Amicon centrifugation to PKC storage buffer (20 mM Tris pH 8.0, 100 mM NaCl, 2 mM MgC12, ImM TCEP, and 25% (v / v) glycerol).

[0185] Live Cell Imaging

[0186] MCF7AC1 cells that stably expressed YFP-PKCβI were generated by transfecting plasmid DNA using Lipofectamine 2000 (Thermofisher) according to the manufacturer’s instructions, and then following the protocol described in the Expression and purification of PKCβI and PKCβII section for generating a stable expression cell line. For microscopy YFP+ cells were seeded in 35-mm glass bottom microwell dishes (MatTek Corporation) for at least 24 hours. Subsequently, cells were incubated with ENDX followed by 15 minutes of PMA incubation as indicated in figure legends. For the PHLPP inhibitor study, cells were treated with NSC117079 (MedChem Express) for the last 45 minutes of the total incubation prior to imaging. Successively, NucRed Live 647 Reagent (Invitrogen) was added for live cell nuclear staining and visualized using a Zeiss-LSM 780 confocal microscope. Confocal images were processed using Carl -Zeiss Blue / Black ZEN 3.0 SR software.

[0187] Quantification And Statistical Analysis

[0188] Standard statistical analyses were used to distinguish significant from non-significant results as indicated.

[0189] Example 2: Endoxifen Downregulates AKT Phosphorylation in ERa+ Breast Cancer Endoxifen (ENDX), a secondary tamoxifen (TAM) metabolite, is a potent antiestrogen exhibiting estrogen receptor alpha (ERa) binding at nanomolar concentrations. Phase 1 / 2 clinical trials identified clinical activity of Z-ENDX, in endocrine-refractory metastatic breast cancer as well as ERa+ solid tumors, raising the possibility that ENDX may have a second, ERa-independent, mechanism of action.

[0190] This Example describes the identification of PKCβI as a ENDX target whose engagement results in inhibition of AKT signaling and induction of apoptosis. Attorney Docket No. 07039-2296WO1 / 2023-600

[0191] Results

[0192] ENDX at 5 M inhibits growth and induces apoptosis in estrogen deprived ERa+ breast cancer cells

[0193] ENDX concentrations ranging from 0 - 10 pM were used to evaluate dose dependent effects of ENDX on cell viability under estrogen deprived conditions, i.e., in medium containing charcoal-stripped serum (CSS), to evaluate ENDX effects that may extend beyond ERa inhibition. ENDX concentrations > 2.5 pM significantly reduced cell viability (Figure 14A) and induced apoptosis in these cells (Figure 14B). These findings suggested that higher plasma concentrations of ENDX may elicit cytotoxic, and not just cytostatic, effects in ERa+ breast cancer cells.

[0194] ENDX concentration-dependent effects on the phosphoproteome of ERa+ breast cancer cells It was then sought to identify additional protein targets of ENDX that may contribute to its anticancer effects in estrogen deprived conditions. To this end, MCF7AC1 cells were treated with 0.01, 0.1, and 5 pM ENDX concentrations achieved in various clinical settings for 24 hours in CSS medium and subjected to TMT labeling-based LC-MS / MS mass spectrometry analysis to evaluate changes in the global protein expression and the phosphoproteome relative to vehicle treated cells (Figure 15). Assessment of the total proteome identified and quantified 8,894 unique proteins (accession number: PXD035007). The impact on global total protein expression induced by ENDX treatment for 24 hours in estrogen deprived cells was limited, with only 25, 34, and 65 total proteins differentially altered by treatment with 0.01, 0.1, and 5 pM ENDX, respectively, compared to vehicle treated cells (based on criteria of |1.5|-fold change and p value of < 0.05) (Table 2). Although ENDX impact on the total proteome was limited, ENDX at 5 pM downregulated two-fold more total proteins compared to 0.1 pM concentration (44 versus 22) and four-fold more total proteins compared to the 0.01 pM concentration (44 versus 11) (Figure 24A). Also, the number of total proteins uniquely downregulated by 5 pM ENDX (35) was greater than the number of total proteins uniquely downregulated by 0.01 pM (3) and 0.1 pM (9) concentrations (Figure 24B). Attorney Docket No. 07039-2296WO1 / 2023-600

[0195] Table 2.

[0196] Accession Gene Description Log FC FC Relative fold P value change

[0197] 0.01 pM ENDX:

[0198] Q9NRJ7 PCDHB16 Protocadherin beta-16 -0.85 0.554785 -1.802500925 0.0116 P52179 MYOM1 Myomesin-1 -0.692 0.618995 -1.615521555 0.0171 P48740 MASP1 Mannan-binding lectin serine -0.678 0.625031 -1.599920257 0.00014 protease 1 3 Q9Y680 FKBP7 Peptidyl-prolyl cis-trans isomerase -0.653 0.635957 -1.572434584 0.00356

[0199] FKBP7

[0200] Q96SR6 ZNF382 Zinc finger protein 382 -0624 0.648869 -1.541142217 0.00071 Q9C0D6 FHDC1 FH2 domain-containing protein 1 -0.601 0.659297 -1.516767545 0.0175 Q9C073 FAM117A Protein FAM117A -0.586 0.666187 -1.501079098 0.00879 043147 SGSM2 Small G protein signaling modulator -0.529 0.693035 -1.442928687 0.0047

[0201] 2

[0202] A4GXA9 EME2 Probable crossover junction -0.445 0.734584 -1.361314116 0.00737 endonuclease EME2

[0203] Q5HY98 ZNF766 Zinc finger protein 766 -0.323 0.799406 -1.25092908 0.0133 Q00987 MDM2 E3 ubiquitin-protein ligase Mdm2 =1 -0.314 0.804408 -1.243149669 0.0161 Q53HI1 UNC50 Protein unc-50 homolog 0.145 1.105731 1.105730653 0.0406 Q8TBK2 SETD6 N-lysine methyltransferase SETD6 0.245 1.185093 1.185092771 0.0373 Q9NPI6 DCP1A mRNA-decapping enzyme 1A 0.286 1.219255 1.219255094 0.00098

[0204] 9 Q96GV9 C5orf30 UNC119-binding protein C5orf30 0.447 1.363203 1.363202607 0.00943 Q9NZ53 P0DXL2 Podocalyxin-like protein 2 0.517 1.430977 1.43097652 0.0308 Q5SR56 MFSD14B Hippocampus abundant transcript0.624 1.541142 1.541142217 0.00080 like protein 1 4 Q9Y2C4 EXOG Nuclease EXOG, mitochondrial 0.636 1.554015 1.554014538 0.00332 Q7Z353 HDX Highly divergent homeobox 0.652 1.571345 1.571345033 0.0228 Q9Y3C7 MED31 Mediator of RNA polymerase II 0.76 1.693491 1.693490625 0.0139 transcription subunit 31

[0205] P29317 EPHA2 Ephrin type-A receptor 2 0.786 1.724287 1.72428709 1.54E- 05 Q14914 PTGR1 Prostaglandin reductase 1 0.881 1.841651 1.841651394 0.00193 Q9UKU6 TRHDE Thyrotropin-releasing hormone1.18 2.265768 2.265767771 0.0367 degrading ectoenzyme

[0206] Q9BX10 GTPBP2 GTP-binding protein 2 1.41 2.657372 2.657371628 0.00124 P20062 TCN2 Transcobalamin-2 1.71 3.271608 3.271608234 0.00704 0.1 pM ENDX:

[0207] Q53HI1 UNC50 Protein unc-50 homolog -1.3 0.406126 -2.462288827 1.31E-

[0208]

[0209] 05 Attorney Docket No. 07039-2296WO1 / 2023-600

[0210] Q9C0D6 FHDC1 FH2 domain-containing protein 1 -1.1 0.466516 -2.143546925 0.00428 Q8NE31 FAM 130 Protein FAM 130 -1 04 0.486327 -2.056227653 0.0349 Q8TBK2 SETD6 N-lysine methyltransferase SETD6 -0.951 0.517274 -1.933212194 0.00057

[0211] 7 P78310 CXADR Coxsackievirus and adenovirus -0.9 0.535887 -1.866065983 0.00703 receptor

[0212] 014526 FCH01 F-BAR domain only protein 1 -0.89 0.539614 -1.853176124 0.0233 Q96IP4 TENT5A Terminal nucleotidyltransferase 5A -0.853 0.553632 -1.80625302 0.0171 Q52M93 ZNF585B Zinc finger protein 585B -0807 0.571569 -1.74956953 0.0439 043147 SGSM2 Small G protein signaling modulator -0.781 0.581963 -1.71832151 0.0011

[0213] 2

[0214] Q9Y680 FKBP7 Peptidyl-prolyl cis-trans isomerase -0756 0.592136 -1.688801775 0.00304

[0215] FKBP7

[0216] Q08397 L0XL1 Lysyl oxidase homolog 1 -0.75 0.594604 -1.681792831 0.014 P48740 MASP1 Mannan-binding lectin serine -0.67 0.628507 -1.591072968 0.0344 protease 1

[0217] Q5HYA8 TMEM67 Meckelin -0.658 0.633756 -1.577893682 0.00998 Q14DG7 TMEM132B Transmembrane protein 132B -0.656 0.634635 -1.575707772 0.00546 Q9BVX2 TMEM106C Transmembrane protein 1060 -0637 0.643049 -1.555092072 0.0345 Q00987 MDM2 E3 ubiquitin-protein ligase Mdm2 -0.618 0.651574 -1.534746096 0.00052

[0218] 2 A4GXA9 EME2 Probable crossover junction -0586 0.666187 -1.501079098 0.00948 endonuclease EME2

[0219] Q3ZCT1 ZNF260 Zinc finger protein 260 -0.577 0.670356 -1.491744027 0.0197 014543 S0CS3 Suppressor of cytokine signaling 3 -0.575 0.671286 -1.489677463 0.00015 Q9BX10 GTPBP2 GTP-binding protein 2 -0.548 0.683968 -1.462057448 0.0131 Q9NRJ7 PCDHB16 Protocadherin beta-16 -0.475 0.719467 -1.38991822 0.042 A4QPH2 PI4KAP2 Putative phosphatidylinositol 4- -0.423 0.745872 -1.340712592 0.0154 kinase alpha-like protein P2

[0220] Q96QD8 SLC38A2 Sodium-coupled neutral amino acid 0.352 1.276329 1.276328769 0.00252 transporter 2

[0221] P27449 ATP6V0C V-type proton ATPase 16 kDa 0.449 1.365094 1.365093718 0.02 proteolipid subunit

[0222] Q969W9 PMEPA1 Protein TMEPAI 0.623 1.540074 1.540074348 0.0334 Q9Y3C7 MED31 Mediator of RNA polymerase II 0.683 1.605475 1.605474777 0.00321 transcription subunit 31

[0223] Q2M1P5 KIF7 Kinesin-like protein KIF7 0.73 1.658639 1.658639092 0.0416 Q9UPV0 CEP164 Centrosomal protein of 164 kDa 0.777 1.713564 1.71356391 0.0194 Q6PJG9 LRFN4 Leucine-rich repeat and fibronectin 0.795 1.735077 1.735077374 2.76E- type-ill domain-containing protein 4 05

[0224]

[0225] Q96J66 ABCC11 ATP-binding cassette sub-family C 0.847 1.798757 1.798756624 0.0215 Attorney Docket No. 07039-2296WO1 / 2023-600

[0226] member 11

[0227] Q96PQ0 SORCS2 VPS10 domain-containing receptor 1.52 2.86791 2.867910496 0.0123

[0228] SorCS2

[0229] Q7Z353 HDX Highly divergent homeobox 2.44 5.426417 5.42641731 0.00035

[0230] 9 Q9UKU6 TRHDE Thyrotropin-releasing hormone3.46 11.00433 11.00433455 0.00082 degrading ectoenzyme 2 P20062 TCN2 Transcobalamin-2 4.19 18.25222 18.25221945 7.10E- 07 5 pM ENDX:

[0231] A6NKB5 PCNX2 Pecanex-like protein 2 -1.66 0.316439 -3.160165247 0.00184 Q99456 KRT12 Keratin, type I cytoskeletal 12 -1 65 0.31864 -3.138336392 3.05E- 05 Q9H1X1 RSPH9 Radial spoke head protein 9 -1.52 0.348686 -2.867910496 0.00077 homolog 2 Q8NE31 FAM13C Protein FAM13C -1.51 0.351111 -2.848100391 0.00081

[0232] 1 Q8N118 CYP4X1 Cytochrome P4504X1 -1.47 0.360982 -2.770218936 0.00097 Q4G0N8 SLC9C1 Sodium / hydrogen exchanger 10 -1.41 0.376312 -2.657371628 0.00197 Q53HI1 UNC50 Protein unc-50 homolog -1.34 0.395021 -2.531513188 0.00017

[0233] 9 Q96BF6 NACC2 Nucleus accumbens-associated -1 25 0.420448 -2.37841423 0.00278 protein 2

[0234] Q8TBK2 SETD6 N-lysine methyltransferase SETD6 -1.21 0.432269 -2.313376368 1.50E- 05 Q52M93 ZNF585B Zinc finger protein 585B -1.07 0.476319 -2.099433367 0.00124 Q9HBK9 AS3MT Arsenite methyltransferase -1.03 0.48971 -2.042024251 0.0086 P78310 CXADR Coxsackievirus and adenovirus -1.02 0.493116 -2.02791896 0.00193 receptor

[0235] Q9UH03 SEPTIN3 Neuronal-specific septin-3 -0.969 0.51086 -1.957483301 0.00023

[0236] 2 Q08397 L0XL1 Lysyl oxidase homolog 1 -0.96 0.514057 -1.945309895 0.00479 Q8N126 CADM3 Cell adhesion molecule 3 -0.939 0.521594 -1.917198877 0.017 P48740 MASP1 Mannan-binding lectin serine -0.874 0.545632 -1.832737289 0.00159 protease 1

[0237] Q9C0D6 FHDC1 FH2 domain-containing protein 1 -0.865 0.549046 -1.821339667 0.00030

[0238] 1 Q8NAP3 ZBTB38 Zinc finger and BTB domain-0856 0.552482 -1.810012926 0.0117 containing protein 38

[0239] Q5TG30 ARHGAP40 Rho GTPase-activating protein 40 -0.846 0.556325 -1.797510253 0.0285

[0240]

[0241] Q14DG7 TMEM132B Transmembrane protein 132B -0.834 0.560972 -1.782620992 0.00483 Attorney Docket No. 07039-2296WO1 / 2023-600

[0242] 043147 SGSM2 Small G protein signaling modulator -0.822 0.565657 -1.767855062 0.00083

[0243] 2

[0244] P 19235 EPOR Erythropoietin receptor -0.82 0.566442 -1.765405993 0.0182 A4QPH2 PI4KAP2 Putative phosphatidylinositol 4- -0.76 0.590496 -1.693490625 0.00732 kinase alpha-like protein P2

[0245] Q5SZD1 C6orf141 Uncharacterized protein C6orf 141 -0.758 0.591316 -1.691144575 0.00801 P01042 KNG1 Kininogen-1 -0.753 0.593368 -1.685293659 0.0429 Q3ZCT1 ZNF260 Zinc finger protein 260 -0.75 0.594604 -1.681792831 0.00962 Q5T1B0 AXDND1 Axonemal dynein light chain domain-0744 0.597082 -1.674812975 0.0197 containing protein 1

[0246] Q00987 MDM2 E3 ubiquitin-protein ligase Mdm2 -0.728 0.60374 -1.656341323 0.011 Q8TBP5 FAM174A Membrane protein FAM174A -0716 0.608783 -1.642621402 0.00979 Q9Y680 FKBP7 Peptidyl-prolyl cis-trans isomerase -0.699 0.615999 -1.623379162 0.0015

[0247] FKBP7

[0248] Q5HY98 ZNF766 Zinc finger protein 766 -0.677 0.625465 -1.598811661 0.00103 O14771 ZNF213 Zinc finger protein 213 -0.667 0.629815 -1.587767862 0.0451 Q68CR1 SEL1L3 Protein sel-1 homolog 3 -0.664 0.631126 -1.584469622 0.00562 O95678 KRT75 Keratin, type II cytoskeletal 75 -0.66 0.632878 -1.580082624 0.00392 Q9H3M7 TXNIP Thioredoxin-interacting protein -0633 0.644834 -1.550786413 0.0018 P 10745 RBP3 Retinol-binding protein 3 -0.627 0.647521 -1.544350266 0.00027

[0249] 1 Q9HBH9 MKNK2 MAP kinase-interacting -0.621 0.65022 -1.537940831 0.00347 serine / threonine-protein kinase 2

[0250] Q9BY08 EBPL Emopamil-binding protein-like -0.614 0.653383 -1.53049677 0.0115 Q2M1Z3 ARHGAP31 Rho GTPase-activating protein 31 -0.606 0.657016 -1.522033381 0.0479 P53634 CTSC Dipeptidyl peptidase 1 -0.602 0.65884 -1.517819253 0.0129 Q8NBQ5 HSD17B11 Estradiol 17-beta-dehydrogenase 11 -0.598 0.660669 -1.513616793 0.0356 O14543 SOCS3 Suppressor of cytokine signaling 3 -0.597 0.661127 -1.512567997 4.26E-05 A4GXA9 EME2 Probable crossover junction -0.536 0.68968 -1.449946833 0.00239 endonuclease EME2

[0251] Q96SR6 ZNF382 Zinc finger protein 382 -0.532 0.691595 -1.445932295 0.0429 Q14914 PTGR1 Prostaglandin reductase 1 0.164 1.120389 1.120389214 0.027 Q5SR56 MFSD14B Hippocampus abundant transcript0.348 1.272795 1.272794935 0.0449 like protein 1

[0252] Q96GV9 C5orf30 UNC119-binding protein C5orf30 0.587 1.50212 1.502119927 0.019 Q86YB7 ECHDC2 Enoyl-CoA hydratase domain0.602 1.517819 1.517819253 0.0146 containing protein 2, mitochondrial

[0253] =2

[0254] Q96QD8 SLC38A2 Sodium-coupled neutral amino acid 0.607 1.523089 1.52308874 5.70E-

[0255]

[0256] transporter 2 05 Attorney Docket No. 07039-2296WO1 / 2023-600

[0257] Q9NZ53 PODXL2 Podocalyxin-like protein 2 0.616 1.53262 1.53261996 0.00501 P27449 ATP6V0C V-type proton ATPase 16 kDa 0.618 1.534746 1.534746096 0.00457 proteolipid subunit

[0258] P29317 EPHA2 Ephrin type-A receptor 2 0.644 1.562656 1.56265576 0.0256 P36941 LTBR Tumor necrosis factor receptor 0.677 1.598812 1.598811661 0.00554 superfamily member 3

[0259] Q96CS7 PLEKHB2 Pleckstrin homology domain0.702 1.626758 1.626758396 0.00361 containing family B member 2

[0260] Q15582 TGFBI Transforming growth factor-beta- 0.71 1.635804 1.635804117 0.0184 induced protein ig-h3

[0261] Q9BT67 NDFIP1 NEDD4 family-interacting protein 1 0.721 1.648324 1.64832417 3.21E- 06 O95807 TMEM50A Transmembrane protein 50A 0.734 1.663244 1.663244197 0.0326 Q 15858 SCN9A Sodium channel protein type 9 0.752 1.684126 1.684125907 0.0178 subunit alpha

[0262] Q96J66 ABCC11 ATP-binding cassette sub-family C 0.818 1.76296 1.762960316 0.016 member 11

[0263] Q96DR4 STARD4 StAR-related lipid transfer protein 4 0.89 1.853176 1.853176124 0.00014

[0264] 6 Q9NPI6 DCP1A mRNA-decapping enzyme 1A 0.964 1.950711 1.950710923 3.08E- 05 Q8TAP9 MPLKIP M-phase-specific PLK1 -interacting 1.01 2.013911 2.0139111 0.012 protein

[0265] P20062 TCN2 Transcobalamin-2 2.08 4.228072 4.228072162 0.00199 Q7Z353 HDX Highly divergent homeobox 2.54 5.81589 5.815890069 3.46E- 05 Q9UKU6 TRHDE Thyrotropin-releasing hormone-3.91 15.03236 15.03236399 0.00010

[0266]

[0267] degrading ectoenzyme 2

[0268] ENDX displayed a much greater impact on the phosphoproteome compared to the total proteome during the course of the 24-hour treatment. Phosphoproteomic analyses identified 14,715 unique phosphosites derived from 4,480 proteins (accession number:

[0269] PXD035007). Out of all sites, 10,046 (82%) were phospho-Serine (pS) sites, 2,042 (17%) were phospho-Threonine (pT) sites, and 134 (1%) were phospho-Tyrosine (pY) sites, as shown in Figure 16A. In MCF7-AC1 cells, treatment with 0.01 pM ENDX resulted in the downregulation of 109 phosphosites and upregulation of 132 phosphosites (|1.5|-fold change and a p value of < 0.05) (Figure 16B). Similarly, treatment with 0.1 pM ENDX led to the Attorney Docket No. 07039-2296WO1 / 2023-600

[0270] downregulation of 94 phosphosites and the upregulation of 150 phosphosites (Figure 16C). Finally, 5 pM ENDX treatment downregulated 341 phosphosites and upregulated 164 phosphosites (Figure 16D). Remarkably, ENDX at 5 pM downregulated three-fold more phosphosites compared to 0.1 pM (341 versus 94) and 0.01 pM (341 versus 109, Figures 16B-16D). In addition, the number of phosphosites uniquely downregulated by 5 pM ENDX (289) was also three-fold greater than the number uniquely downregulated by 0.01 pM (87) and 0.1 pM (59, Figure 16E). Heat-map analysis suggested an ENDX concentrationdependent downregulation for a subset of these phosphosites (Figure 16F). A comparison of the phosphosite list with the total protein list altered by ENDX revealed minimal overlap (Figure 24C).

[0271] In order to identify protein phosphorylation signaling pathways regulated by ENDX, a kinase enrichment analysis (KEA3) was performed, which integrated multiple databases covering kinase-substrate interactions (KSI), kinase-protein interactions (KPI), and interactions supported by co-expression and co-occurrence data to infer the overrepresentation of upstream kinases whose putative substrates were among the phosphorylated proteins altered by ENDX treatments. Three separate analyses were performed for 210 proteins, 224 proteins, and 347 proteins differentially phosphorylated in cells treated with 0.01 pM, 0.1 pM, and 5 pM ENDX, respectively. Figure 17 shows the top enriched upstream kinases predicted to regulate the protein phosphorylation changes induced by ENDX at different concentrations. Casein kinase (CSNK1A1), serine / arginine-rich protein-specific kinase (SRPK1, SRPK2), and mitogen-activated protein kinases (MAPK1 and MAPK8) were identified as putative kinases regulated by low-dose (0.01 and 0.1 pM) ENDX (Figures 17A-17B). This analysis suggested that proteins (MTOR, RPS6K and AKT1) involved in the AKT signaling pathway can be regulated by high-dose (5 pM) ENDX (Figure 17C).

[0272] In addition to recognizing potential upstream kinases, based on the effects of ENDX on phosphorylation, fuzzy-C mean clustering was also performed to identify the regulation patterns induced by different ENDX concentrations. This analysis identified three clusters depicting three different regulatory patterns (Figure 18A). Cluster 1 represented 325 phosphosites downregulated by ENDX in a dose-dependent manner, cluster 2 represented Attorney Docket No. 07039-2296WO1 / 2023-600

[0273] 201 phosphosites upregulated by ENDX at all concentrations and cluster 3 represented 73 phosphosites downregulated at 0.01 pM concentration but mostly unaffected at the 0.1 and 5 pM concentrations (Table 3). Attorney Docket No. 07039-2296WO1 / 2023-600

[0274] Table 3.

[0275] prt_site_ind Ph_Site_Window Gene symbol SEQ ID NO ETOH ETOH ETOH 0.01 pM 0.01 pM 0.01 pM 0.1 pM 0.1 pM 0.1 pM 5 pM 5 pM 5 pM (1) (2) (3) ENDX ENDX ENDX ENDX ENDX ENDX ENDX ENDX ENDX (1) (2) (3) (1) (2) (3) (1) (2) (3) 0.01 pM EN DX:

[0276] S54 VGMGQKDsYVGDEAQ ACTA1 11 33.21 22.69 24.60 21.31 23.00 20.93 13.47 26.07 16.49 15.49 21.34 12.35 S54 VGMGQKDsYVGDEAQ ACTA2 11 33.21 22.69 24.60 21.31 23.00 20.93 13.47 26.07 16.49 15.49 21.34 12.35 S199 ILTERGYsFTTTAER ACTB 12 43.12 32.29 37.63 19.77 35.52 34.10 14.97 45.56 20.91 14.46 24.55 13.97 T202 ERGYSFTtTAEREIV ACTB 13 52.64 37.71 41.39 31.97 47.84 43.51 23.39 54.00 32.33 27.27 30.23 21.12 S52 VGMGQKDsYVGDEAQ ACTB 11 33.21 22.69 24.60 21.31 23.00 20.93 13.47 26.07 16.49 15.49 21.34 12.35 S54 VGMGQKDsYVGDEAQ ACTC1 11 33.21 22.69 24.60 21.31 23.00 20.93 13.47 26.07 16.49 15.49 21.34 12.35 S199 ILTERGYsFTTTAER ACTG1 12 43.12 32.29 37.63 19.77 35.52 34.10 14.97 45.56 20.91 14.46 24.55 13.97 T202 ERGYSFTtTAEREIV ACTG1 13 52.64 37.71 41.39 31.97 47.84 43.51 23.39 54.00 32.33 27.27 30.23 21.12 S52 VGMGQKDsYVGDEAQ ACTG1 11 33.21 22.69 24.60 21.31 23.00 20.93 13.47 26.07 16.49 15.49 21.34 12.35 S53 VGMGQKDsYVGDEAQ ACTG2 11 33.21 22.69 24.60 21.31 23.00 20.93 13.47 26.07 16.49 15.49 21.34 12.35 S329 IGIHHKNsPPKVTVP AGGF1 14 14.03 11.75 13.89 11.78 9.75 12.16 9.26 12.40 9.76 8.47 8.68 9.10 S1549 LLSDLTLsPVPRDSL AHDC1 15 1.48 4.70 2.22 2.36 3.39 3.20 2.81 2.87 2.58 1.19 1.23 1.25 S134 WYAKCDsSPDSAED AHSG 16 25.23 17.07 39.56 18.85 40.34 47.26 15.34 39.32 16.40 12.39 18.87 10.62 T260 AEVSKLVtDLTKVHT ALB 17 18.79 18.09 25.57 15.47 26.38 27.61 10.20 30.71 11.88 10.95 14.80 9.21 S443 TKKVPQVsTPTLVEV ALB 18 44.53 47.52 92.04 37.60 81.61 103.03 20.02 84.12 31.96 17.04 41.81 10.40 T446 VPQVSTPtLVEVSRN ALB 19 22.52 21.66 43.70 21.62 36.44 50.19 11.04 43.37 16.12 13.53 18.65 4.66 S451 TPTLVEVsRNLGKVG ALB 20 34.37 40.88 64.06 33.71 68.57 75.79 23.30 65.55 23.86 18.38 35.27 10.07 S132 TQGLDGLsERCAQYK ALDOA 21 8.37 6.85 6.85 6.97 6.88 6.67 4.77 7.42 5.25 2.89 6.33 4.12 S309 YGRALQAsALKAWGG ALDOA 22 10.43 3.68 9.07 3.38 6.57 8.41 1.50 10.71 4.42 2.38 4.50 2.06 S322 VNSTSEEsHDEDEIR ATAD1 23 13.64 10.42 13.60 10.66 11.70 7.59 9.73 9.36 9.40 7.95 6.43 9.64 S939 VLPLALPsPPRQLSE ATAD2B 24 3.35 2.35 2.12 2.97 1.13 2.29 2.43 0.93 2.21 1.14 1.18 2.38

[0277]

[0278] S419 LSVSRVGsAAQTRAM ATP5F1A 25 16.22 10.63 12.64 7.17 9.96 13.26 6.17 14.43 8.66 6.61 9.54 5.85

[0279]

[0280] Attorney Docket No. 07039-2296WO1 / 2023-600

[0281] S76 EETGRVLsIGDGIAR ATP5F1A 26 18.79 14.51 18.14 10.86 14.37 15.08 11.60 17.30 13.17 7.23 11.90 7.69 T330 AAPAHRGtPDTDLEV ATXN1L 27 36.17 51.40 33.09 33.91 40.55 35.20 29.28 30.88 27.63 20.76 28.52 25.57 T123 NRHFRHDtPDSSPRR BUD13 28 11.71 16.96 20.07 12.81 13.55 14.08 11.13 10.29 12.99 10.84 10.61 8.02 S126 FRHDTPDsSPRRVRH BUD13 29 8.88 8.89 13.12 9.02 10.27 7.31 8.70 6.33 10.04 5.37 7.83 5.42 S197 PRRARHDsPDPSPPR BUD13 30 10.04 10.42 8.59 7.79 7.19 5.49 4.68 8.61 4.05 5.16 6.00 6.82 S201 RHDSPDPsPPRRPQH BUD13 31 10.04 10.42 8.59 7.79 7.19 5.49 4.68 8.61 4.05 5.16 6.00 6.82 S80 RFYALSAsFEPFSNK CALR 32 5.02 3.78 4.63 4.10 3.08 5.94 1.68 5.40 4.70 2.89 3.00 0.97 S585 EAEAGAGsPTSTPAP CAMSAP3 33 1.67 3.78 2.80 1.33 3.59 4.02 1.22 3.04 2.39 1.34 1.39 1.41 S769 RVPATRRsPGPGPSQ CAMSAP3 34 10.81 10.22 8.20 8.40 6.06 7.13 4.87 8.18 6.45 5.58 7.50 5.85 S699 LVAAAPTsPDHSP- CBARP 35 4.76 3.68 3.28 2.46 3.29 2.47 4.12 1.86 2.49 1.14 1.18 1.19 S703 APTSPDHsP- CBARP 36 4.76 3.68 3.28 2.46 3.29 2.47 4.12 1.86 2.49 1.14 1.18 1.19 S138 LRSQPDAsKEELRLL CCDC183 37 19.69 10.73 16.88 12.91 22.38 20.57 4.96 21.43 10.32 5.58 11.26 4.87 S190 SSTGSVGsPDQLPLA CCDC85B 38 3.22 3.78 3.67 0.92 2.46 1.65 2.34 2.02 3.87 0.93 0.96 0.97 S23 NDMKVRKsSTPEEVK CFL1 39 51.23 28.10 46.31 35.14 42.60 46.17 21.05 57.54 24.13 20.24 33.45 11.81 S203 KKAEAAAsALADADA CHMP2A 40 7.47 5.52 6.95 5.53 4.82 5.58 3.46 4.89 3.68 5.16 2.57 5.74 S86 RDNLAQQsFNMEQAN CHMP5 41 5.92 6.13 5.31 6.76 7.19 5.03 4.21 5.91 4.88 4.34 2.68 4.33 S100 EDPCHPDsPPAPRAT CREB3L4 42 3.35 3.88 4.92 4.41 3.90 4.02 4.68 4.72 2.03 3.20 1.18 2.60 T21 LLGGPAGtPPGGGAL DBP 43 4.63 6.13 4.05 2.97 1.33 4.48 4.87 4.30 2.39 3.82 1.39 1.41 S315 PTYTIPLsPVLSPTL DCP1A 44 4.12 4.29 2.41 1.28 5.65 3.20 2.99 1.05 4.24 2.58 1.34 1.35 S754 QGHNSPDsPVTSAAK DDX42 45 4.38 5.82 5.98 2.36 5.44 7.95 5.33 5.48 4.88 2.27 3.97 4.22 T261 YKIGGIGtVPVGRVE EEF1A1 46 19.44 15.43 16.59 13.42 15.71 15.91 9.82 23.29 14.28 9.81 13.08 6.07 T261 YKIGGIGtVPVGRVE EEF1A1P5 46 19.44 15.43 16.59 13.42 15.71 15.91 9.82 23.29 14.28 9.81 13.08 6.07 T261 YKIGGIGtVPVGRVE EEF1A2 46 19.44 15.43 16.59 13.42 15.71 15.91 9.82 23.29 14.28 9.81 13.08 6.07 T86 TIVQEEDtQPLTEPI EFTUD2 47 5.15 8.58 9.36 3.59 7.70 3.84 6.27 6.67 4.33 5.37 2.14 3.47 Y189 MRIGAEVyHNLKNVI ENO1 48 18.28 16.04 15.63 13.52 16.12 12.52 10.39 16.71 12.99 8.99 14.04 8.99 S419 RIEEELGsKAKFAGR ENO1 49 55.22 27.08 46.79 28.48 43.83 51.19 18.15 60.41 30.12 22.10 26.91 17.87 Y774 ENSSDPTyTSSLGGK EPHB4 50 7.34 8.89 8.10 6.56 8.42 5.58 7.86 8.69 7.00 6.82 4.50 4.44

[0282]

[0283] S489 WEKTGSHsEPQARGD FAM120A 51 1.29 2.04 2.03 1.02 3.59 2.10 0.94 3.29 0.92 1.03 1.07 1.08

[0284]

[0285] Attorney Docket No. 07039-2296WO1 / 2023-600

[0286] T34 APPAPEAtPPPASAA FAM207A 52 1.35 2.25 3.38 1.08 2.16 0.96 0.98 0.89 2.12 1.08 1.13 1.14 S324 QEPLLIGsTKSNMGH FASN 53 7.08 5.01 6.37 5.33 5.44 4.66 3.37 8.10 3.32 4.03 4.40 3.68 S962 PIPKSPFsVAVSPSL FLNA 54 1.29 2.55 2.80 2.25 1.03 2.56 0.94 1.69 0.92 1.03 1.07 1.08 S488 GRGDRRHsSDINHLV FNBP1L 55 20.98 13.49 19.49 12.50 14.58 13.90 16.75 12.74 18.98 10.74 10.29 13.00 S148 DNSLKIIsNASCTTN GAPDH 56 24.71 16.45 25.86 13.22 21.97 25.32 8.05 27.67 13.72 9.29 12.33 6.17 S266 KKWKQAsEGPLKGI GAPDH 57 43.12 16.25 39.46 24.08 31.00 35.01 11.88 41.76 20.54 15.28 19.94 9.53 S746 ESCSGLGsTSDDTDV GAPVD1 58 21.62 19.72 23.35 17.62 13.45 17.19 17.59 14.85 15.01 14.05 13.40 15.49 T846 YMAKRLEtHYRILFR GLDC 59 13.00 3.07 10.71 10.66 10.16 7.59 6.36 4.98 3.22 1.55 1.61 1.62 Y848 AKRLETHyRILFRGA GLDC 60 13.00 3.07 10.71 10.66 10.16 7.59 6.36 4.98 3.22 1.55 1.61 1.62 S623 FGGCFGRsESPQPKA GNAS 61 2.96 3.07 3.09 4.10 2.16 2.10 2.15 0.89 3.68 2.27 1.13 1.14 S187 KGNEAVAsRDLSENN GOLM1 62 12.74 21.87 17.37 16.19 18.89 13.90 15.91 16.20 13.82 9.60 9.65 7.91 S908 SRRHSKRsHDSDDSD GPATCH8 63 10.17 10.73 8.78 7.79 8.11 6.40 7.95 8.27 7.46 6.40 4.61 7.04 T466 GDSPDSStPKLSRAQ GTSE1 64 13.51 12.06 15.53 10.04 13.55 16.73 12.91 13.25 12.53 11.88 6.86 8.99 S113 ELAKHAVsEGTKAVT H2BFS 65 30.89 12.16 27.40 15.68 26.69 17.55 18.52 21.43 20.45 10.95 15.12 7.80 S56 VHPDTGIsSKAMGlM H2BFS 66 20.59 13.90 17.66 11.78 17.25 16.64 10.67 19.91 10.78 11.05 13.62 7.80 T97 ITSREIQtAVRLLLP H2BFS 67 44.66 22.99 36.56 24.08 30.69 27.70 14.03 39.15 16.67 19.11 25.73 13.00 T46 PHRYRPGtVALREIR H3F3A 68 25.36 12.16 13.99 10.04 18.68 13.16 6.83 22.27 10.78 7.33 10.51 4.01 T45 PHRYRPGtVALREIR H3F3C 68 25.36 12.16 13.99 10.04 18.68 13.16 6.83 22.27 10.78 7.33 10.51 4.01 S105 QTKGTGAsGSFKLNK HIST1H1A 69 59.34 41.08 47.37 36.06 64.37 45.71 35.46 48.51 37.21 28.81 33.24 28.49 S105 QTKGTGAsGSFKLNK HIST1H1B 69 59.34 41.08 47.37 36.06 64.37 45.71 35.46 48.51 37.21 28.81 33.24 28.49 S116 KLNKKAAsGEAKPKA HIST1H1B 70 57.15 22.38 31.55 24.90 48.56 32.09 20.40 33.75 28.92 17.76 23.16 16.03 S102 QTKGTGAsGSFKLNK HIST1H1C 69 59.34 41.08 47.37 36.06 64.37 45.71 35.46 48.51 37.21 28.81 33.24 28.49 S113 KLNKKAAsGEAKPKV HIST1H1C 71 57.15 22.38 31.55 24.90 48.56 32.09 20.40 33.75 28.92 17.76 23.16 16.03 S41 KASGPPVsELITKAV HIST1H1C 72 44.53 33.21 44.67 37.09 57.38 47.72 29.66 45.31 38.60 27.99 29.38 20.64 S103 QTKGTGAsGSFKLNK HIST1H1D 69 59.34 41.08 47.37 36.06 64.37 45.71 35.46 48.51 37.21 28.81 33.24 28.49 S42 KASGPPVsELITKAV HIST1H1D 72 44.53 33.21 44.67 37.09 57.38 47.72 29.66 45.31 38.60 27.99 29.38 20.64 S102 QTKGTGAsGSFKLNK HIST1H1E 69 59.34 41.08 47.37 36.06 64.37 45.71 35.46 48.51 37.21 28.81 33.24 28.49

[0287]

[0288] S113 KLNKKAAsGEAKPKA HIST1H1E 70 57.15 22.38 31.55 24.90 48.56 32.09 20.40 33.75 28.92 17.76 23.16 16.03

[0289]

[0290] Attorney Docket No. 07039-2296WO1 / 2023-600

[0291] S41 KASGPPVsELITKAV HIST1H1E 72 44.53 33.21 44.67 37.09 57.38 47.72 29.66 45.31 38.60 27.99 29.38 20.64 S106 QTRGTGAsGSFKLSK HIST1H1T 73 59.34 41.08 47.37 36.06 64.37 45.71 35.46 48.51 37.21 28.81 33.24 28.49 S114 ELAKHAVsEGTKAVT HIST1H2BA 65 30.89 12.16 27.40 15.68 26.69 17.55 18.52 21.43 20.45 10.95 15.12 7.80 S57 VHPDTGIsSKAMSIM HIST1H2BA 74 20.59 13.90 17.66 11.78 17.25 16.64 10.67 19.91 10.78 11.05 13.62 7.80 T98 ISSREIQtAVRLLLP HIST1H2BA 75 44.66 22.99 36.56 24.08 30.69 27.70 14.03 39.15 16.67 19.11 25.73 13.00 S113 ELAKHAVsEGTKAVT HIST1H2BB 65 30.89 12.16 27.40 15.68 26.69 17.55 18.52 21.43 20.45 10.95 15.12 7.80 S37 RKRSRKEsYSlYVYK HIST1H2BB 76 10.94 5.93 7.53 4.61 6.98 10.42 2.99 6.75 3.78 1.65 3.65 1.73 S39 RSRKESYslYVYKVL HIST1H2BB 77 16.35 11.75 13.89 11.07 13.45 12.98 9.64 19.74 8.11 5.06 8.79 8.34 S56 VHPDTGIsSKAMGlM HIST1H2BB 66 20.59 13.90 17.66 11.78 17.25 16.64 10.67 19.91 10.78 11.05 13.62 7.80 T97 ITSREIQtAVRLLLP HIST1H2BB 67 44.66 22.99 36.56 24.08 30.69 27.70 14.03 39.15 16.67 19.11 25.73 13.00 S113 ELAKHAVsEGTKAVT HIST1H2BC 65 30.89 12.16 27.40 15.68 26.69 17.55 18.52 21.43 20.45 10.95 15.12 7.80 S56 VHPDTGIsSKAMGlM HIST1H2BC 66 20.59 13.90 17.66 11.78 17.25 16.64 10.67 19.91 10.78 11.05 13.62 7.80 T97 ITSREIQtAVRLLLP HIST1H2BC 67 44.66 22.99 36.56 24.08 30.69 27.70 14.03 39.15 16.67 19.11 25.73 13.00 S113 ELAKHAVsEGTKAVT HIST1H2BD 65 30.89 12.16 27.40 15.68 26.69 17.55 18.52 21.43 20.45 10.95 15.12 7.80 S56 VHPDTGIsSKAMGlM HIST1H2BD 66 20.59 13.90 17.66 11.78 17.25 16.64 10.67 19.91 10.78 11.05 13.62 7.80 T97 ITSREIQtAVRLLLP HIST1H2BD 67 44.66 22.99 36.56 24.08 30.69 27.70 14.03 39.15 16.67 19.11 25.73 13.00 S113 ELAKHAVsEGTKAVT HIST1H2BH 65 30.89 12.16 27.40 15.68 26.69 17.55 18.52 21.43 20.45 10.95 15.12 7.80 S56 VHPDTGIsSKAMGlM HIST1H2BH 66 20.59 13.90 17.66 11.78 17.25 16.64 10.67 19.91 10.78 11.05 13.62 7.80 T97 ITSREIQtAVRLLLP HIST1H2BH 67 44.66 22.99 36.56 24.08 30.69 27.70 14.03 39.15 16.67 19.11 25.73 13.00 S113 ELAKHAVsEGTKAVT HIST1H2BJ 65 30.89 12.16 27.40 15.68 26.69 17.55 18.52 21.43 20.45 10.95 15.12 7.80 S37 RKRSRKEsYSlYVYK HIST1H2BJ 76 10.94 5.93 7.53 4.61 6.98 10.42 2.99 6.75 3.78 1.65 3.65 1.73 S39 RSRKESYslYVYKVL HIST1H2BJ 77 16.35 11.75 13.89 11.07 13.45 12.98 9.64 19.74 8.11 5.06 8.79 8.34 S56 VHPDTGIsSKAMGlM HIST1H2BJ 66 20.59 13.90 17.66 11.78 17.25 16.64 10.67 19.91 10.78 11.05 13.62 7.80 T97 ITSREIQtAVRLLLP HIST1H2BJ 67 44.66 22.99 36.56 24.08 30.69 27.70 14.03 39.15 16.67 19.11 25.73 13.00 S113 ELAKHAVsEGTKAVT HIST1H2BK 65 30.89 12.16 27.40 15.68 26.69 17.55 18.52 21.43 20.45 10.95 15.12 7.80 S56 VHPDTGIsSKAMGlM HIST1H2BK 66 20.59 13.90 17.66 11.78 17.25 16.64 10.67 19.91 10.78 11.05 13.62 7.80 T97 ITSREIQtAVRLLLP HIST1H2BK 67 44.66 22.99 36.56 24.08 30.69 27.70 14.03 39.15 16.67 19.11 25.73 13.00

[0292]

[0293] S113 ELAKHAVsEGTKAVT HIST1H2BL 65 30.89 12.16 27.40 15.68 26.69 17.55 18.52 21.43 20.45 10.95 15.12 7.80 Attorney Docket No. 07039-2296WO1 / 2023-600

[0294] S56 VHPDTGIsSKAMGlM HIST1H2BL 66 20.59 13.90 17.66 11.78 17.25 16.64 10.67 19.91 10.78 11.05 13.62 7.80 T97 ITSREIQtAVRLLLP HIST1H2BL 67 44.66 22.99 36.56 24.08 30.69 27.70 14.03 39.15 16.67 19.11 25.73 13.00 S113 ELAKHAVsEGTKAVT HIST1H2BM 65 30.89 12.16 27.40 15.68 26.69 17.55 18.52 21.43 20.45 10.95 15.12 7.80 S56 VHPDTGIsSKAMGlM HIST1H2BM 66 20.59 13.90 17.66 11.78 17.25 16.64 10.67 19.91 10.78 11.05 13.62 7.80 T97 ITSREIQtAVRLLLP HIST1H2BM 67 44.66 22.99 36.56 24.08 30.69 27.70 14.03 39.15 16.67 19.11 25.73 13.00 S113 ELAKHAVsEGTKAVT HIST1H2BN 65 30.89 12.16 27.40 15.68 26.69 17.55 18.52 21.43 20.45 10.95 15.12 7.80 S56 VHPDTGIsSKAMGlM HIST1H2BN 66 20.59 13.90 17.66 11.78 17.25 16.64 10.67 19.91 10.78 11.05 13.62 7.80 T97 ITSREIQtAVRLLLP HIST1H2BN 67 44.66 22.99 36.56 24.08 30.69 27.70 14.03 39.15 16.67 19.11 25.73 13.00 S113 ELAKHAVsEGTKAVT HIST1H2BO 65 30.89 12.16 27.40 15.68 26.69 17.55 18.52 21.43 20.45 10.95 15.12 7.80 S37 RKRSRKEsYSlYVYK HIST1H2BO 76 10.94 5.93 7.53 4.61 6.98 10.42 2.99 6.75 3.78 1.65 3.65 1.73 S39 RSRKESYslYVYKVL HIST1H2BO 77 16.35 11.75 13.89 11.07 13.45 12.98 9.64 19.74 8.11 5.06 8.79 8.34 S56 VHPDTGIsSKAMGlM HIST1H2BO 66 20.59 13.90 17.66 11.78 17.25 16.64 10.67 19.91 10.78 11.05 13.62 7.80 T97 ITSREIQtAVRLLLP HIST1H2BO 67 44.66 22.99 36.56 24.08 30.69 27.70 14.03 39.15 16.67 19.11 25.73 13.00 T46 PHRYRPGtVALREIR HIST1H3A 68 25.36 12.16 13.99 10.04 18.68 13.16 6.83 22.27 10.78 7.33 10.51 4.01 T31 RDNIQGItKPAIRRL HIST1H4A 78 63.58 28.31 49.78 30.84 49.17 46.62 15.53 61.76 27.73 17.35 32.27 7.47 S48 RGGVKRIsGLIYEET HIST1H4A 79 55.73 44.25 44.96 45.80 43.83 53.84 27.13 55.94 34.08 29.64 36.88 27.08 Y52 KRISGLIyEETRGVL HIST1H4A 80 33.98 17.99 20.36 12.60 22.07 20.39 14.13 24.21 15.20 12.81 15.12 10.51 S37 RKRSRKEsYSlYVYK HIST2H2BC 76 10.94 5.93 7.53 4.61 6.98 10.42 2.99 6.75 3.78 1.65 3.65 1.73 S39 RSRKESYslYVYKVL HIST2H2BC 77 16.35 11.75 13.89 11.07 13.45 12.98 9.64 19.74 8.11 5.06 8.79 8.34 S37 RKRSRKEsYSlYVYK HIST2H2BD 76 10.94 5.93 7.53 4.61 6.98 10.42 2.99 6.75 3.78 1.65 3.65 1.73 S39 RSRKESYslYVYKVL HIST2H2BD 77 16.35 11.75 13.89 11.07 13.45 12.98 9.64 19.74 8.11 5.06 8.79 8.34 S113 ELAKHAVsEGTKAVT HIST2H2BE 65 30.89 12.16 27.40 15.68 26.69 17.55 18.52 21.43 20.45 10.95 15.12 7.80 S37 RKRSRKEsYSlYVYK HIST2H2BE 76 10.94 5.93 7.53 4.61 6.98 10.42 2.99 6.75 3.78 1.65 3.65 1.73 S39 RSRKESYslYVYKVL HIST2H2BE 77 16.35 11.75 13.89 11.07 13.45 12.98 9.64 19.74 8.11 5.06 8.79 8.34 S56 VHPDTGIsSKAMGlM HIST2H2BE 66 20.59 13.90 17.66 11.78 17.25 16.64 10.67 19.91 10.78 11.05 13.62 7.80 T97 ITSREIQtAVRLLLP HIST2H2BE 67 44.66 22.99 36.56 24.08 30.69 27.70 14.03 39.15 16.67 19.11 25.73 13.00 S113 ELAKHAVsEGTKAVT HIST2H2BF 65 30.89 12.16 27.40 15.68 26.69 17.55 18.52 21.43 20.45 10.95 15.12 7.80

[0295]

[0296] S56 VHPDTGIsSKAMGlM HIST2H2BF 66 20.59 13.90 17.66 11.78 17.25 16.64 10.67 19.91 10.78 11.05 13.62 7.80

[0297]

[0298] Attorney Docket No. 07039-2296WO1 / 2023-600

[0299] T97 ITSREIQtAVRLLLP HIST2H2BF 67 44.66 22.99 36.56 24.08 30.69 27.70 14.03 39.15 16.67 19.11 25.73 13.00 T46 PHRYRPGtVALREIR HIST2H3A 68 25.36 12.16 13.99 10.04 18.68 13.16 6.83 22.27 10.78 7.33 10.51 4.01 S113 ELAKHAVsEGTKAVT HIST3H2BB 65 30.89 12.16 27.40 15.68 26.69 17.55 18.52 21.43 20.45 10.95 15.12 7.80 S37 RKRGRKEsYSlYVYK HIST3H2BB 81 10.94 5.93 7.53 4.61 6.98 10.42 2.99 6.75 3.78 1.65 3.65 1.73 S39 RGRKESYslYVYKVL HIST3H2BB 82 16.35 11.75 13.89 11.07 13.45 12.98 9.64 19.74 8.11 5.06 8.79 8.34 S56 VHPDTGIsSKAMGlM HIST3H2BB 66 20.59 13.90 17.66 11.78 17.25 16.64 10.67 19.91 10.78 11.05 13.62 7.80 T46 PHRYRPGtVALREIR HIST3H3 68 25.36 12.16 13.99 10.04 18.68 13.16 6.83 22.27 10.78 7.33 10.51 4.01 S185 TPLPSLAsPAVPAPG HJURP 83 6.56 5.52 5.11 3.69 5.95 5.21 6.08 7.85 3.87 2.38 3.65 3.79 S397 TAVQYIEsSDSEEIE HLTF 84 6.31 5.72 6.56 7.38 2.77 5.03 5.05 5.91 3.41 3.10 3.22 2.60 S398 AVQYIESsDSEEIET HLTF 85 6.31 5.72 6.56 7.38 2.77 5.03 5.05 5.91 3.41 3.10 3.22 2.60 S400 QYIESSDsEEIETSE HLTF 86 6.31 5.72 6.56 7.38 2.77 5.03 5.05 5.91 3.41 3.10 3.22 2.60 S121 KGEHPGLsIGDVAKK HMGB1 87 36.55 15.02 28.27 15.68 25.66 28.07 6.92 32.57 12.07 12.60 15.76 5.20 S121 KGEHPGLsIGDVAKK HMGB1P1 87 36.55 15.02 28.27 15.68 25.66 28.07 6.92 32.57 12.07 12.60 15.76 5.20 S365 GGSSSSSsYGSGRR- HNRNPA1 88 9.65 6.44 7.72 8.91 6.26 5.21 4.58 5.48 4.88 5.89 4.61 4.77 T110 GKPGAHVtVKKLFVG HNRNPA2B1 89 11.20 10.42 15.53 10.55 11.81 14.72 7.77 12.15 6.08 6.30 8.36 3.36 S391 FIRGWDsEDLPLNI HSP90AA1 90 19.56 15.43 17.17 12.70 18.17 14.44 4.30 20.59 11.97 7.54 11.26 5.74 S68 YESLTDPsKLDSGKE HSP90AA1 91 33.08 17.99 34.06 20.49 23.00 26.97 9.82 36.19 16.95 15.90 21.44 11.05 S68 YESLTDPsKLDSGKE HSP90AA2P 91 33.08 17.99 34.06 20.49 23.00 26.97 9.82 36.19 16.95 15.90 21.44 11.05 S383 FIRGWDsEDLPLNI HSP90AB1 90 19.56 15.43 17.17 12.70 18.17 14.44 4.30 20.59 11.97 7.54 11.26 5.74 S434 KKFYEAFsKNLKLGI HSP90AB1 92 6.44 2.55 4.82 0.97 3.08 3.84 2.43 5.48 2.86 1.96 2.57 1.03 S48 RELISNAsDALDKIR HSP90AB1 93 32.56 19.82 31.16 21.31 29.57 24.59 14.78 34.76 16.58 11.98 19.62 7.80 S63 YESLTDPsKLDSGKE HSP90AB1 91 33.08 17.99 34.06 20.49 23.00 26.97 9.82 36.19 16.95 15.90 21.44 11.05 S48 WELISNAsDALDKIR HSP90AB2P 94 32.56 19.82 31.16 21.31 29.57 24.59 14.78 34.76 16.58 11.98 19.62 7.80 S63 YESLTDPsKLDSGKE HSP90AB2P 91 33.08 17.99 34.06 20.49 23.00 26.97 9.82 36.19 16.95 15.90 21.44 11.05 S325 FIHGWDsEDLPLNI HSP90AB3P 95 19.56 15.43 17.17 12.70 18.17 14.44 4.30 20.59 11.97 7.54 11.26 5.74 S48 QELISNAsDALDKIR HSP90AB3P 96 32.56 19.82 31.16 21.31 29.57 24.59 14.78 34.76 16.58 11.98 19.62 7.80 S63 YESLTDPsKLDSGKE HSP90AB3P 91 33.08 17.99 34.06 20.49 23.00 26.97 9.82 36.19 16.95 15.90 21.44 11.05

[0300]

[0301] S24 QELISNAsDALDKIR HSP90AB4P 96 32.56 19.82 31.16 21.31 29.57 24.59 14.78 34.76 16.58 11.98 19.62 7.80 Attorney Docket No. 07039-2296WO1 / 2023-600

[0302] S109 RELISNAsDALDKIR HSP90B1 93 32.56 19.82 31.16 21.31 29.57 24.59 14.78 34.76 16.58 11.98 19.62 7.80 S98 TADRWRVsLDVNHFA HSPB1 97 31.41 21.26 33.09 16.80 28.33 24.59 13.00 34.42 17.50 11.46 20.37 10.07 S67 RTVIlEQsWGSPKVT HSPD1 98 11.97 13.28 9.55 7.48 14.89 13.71 10.48 11.39 11.51 10.64 6.33 6.17 S423 AGClHGLsNVKLNEH IDH2 99 26.26 18.90 18.91 11.07 12.11 17.00 13.19 20.16 17.69 10.02 12.33 10.29 S258 EDLCKIGsERSLVLD IKZF1 100 23.55 25.75 16.11 12.70 21.97 21.67 12.72 19.24 11.79 10.64 13.29 11.81 S274 PPRTGGPsPAGPAAA IRX5 101 39.77 42.82 35.02 31.04 32.03 29.80 32.93 25.06 23.58 23.96 23.80 25.24 S25 DLGHTPLsKKEGIKW KCNIP3 102 6.56 5.62 7.53 3.89 5.65 6.22 2.53 5.65 2.95 3.00 3.97 1.46 T713 RSCDEPLtPPPHSPT KDM2A 103 4.12 5.01 3.86 3.18 4.52 2.29 4.21 3.46 5.90 4.03 1.34 1.35 S731 QLIHDPVsPRGMVTR KDM2A 104 4.12 5.01 3.86 3.18 4.52 2.29 4.21 3.46 5.90 4.03 1.34 1.35 S570 PGASVSSsLTSLCSS KIAA1522 105 17.12 10.32 9.17 12.60 8.93 7.95 7.39 7.51 7.00 8.68 9.54 6.28 S193 SLNNQFAsFIDKVRF KRT1 106 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 S159 NLNDRLAsYLDKVRA KRT10 107 73.75 31.17 53.54 27.77 42.09 47.26 16.19 72.81 29.57 18.38 39.67 13.87 S138 NLNDRLAsYLDKVRA KRT12 107 73.75 31.17 53.54 27.77 42.09 47.26 16.19 72.81 29.57 18.38 39.67 13.87 S128 NLNDRLAsYLDKVRA KRT14 107 73.75 31.17 53.54 27.77 42.09 47.26 16.19 72.81 29.57 18.38 39.67 13.87 S118 NLNDRLAsYLDKVRA KRT15 107 73.75 31.17 53.54 27.77 42.09 47.26 16.19 72.81 29.57 18.38 39.67 13.87 S130 NLNDRLAsYLDKVRA KRT16 107 73.75 31.17 53.54 27.77 42.09 47.26 16.19 72.81 29.57 18.38 39.67 13.87 S97 NLNDRLAsYLDKVRA KRT17 107 73.75 31.17 53.54 27.77 42.09 47.26 16.19 72.81 29.57 18.38 39.67 13.87 T11 TTRSTFStNYRSLGS KRT18 108 35.14 26.57 37.72 22.95 30.08 32.54 15.34 41.09 19.62 16.83 18.01 13.11 S319 SMRNLKAsLENSLRE KRT18 109 40.67 44.55 28.56 19.36 31.00 34.10 20.68 39.32 23.30 19.73 25.19 12.89 S323 LKASLENsLREVEAR KRT18 110 28.70 25.44 30.78 14.86 23.71 19.75 15.44 28.18 15.38 10.95 20.05 10.94 T425 WSETNDtKVLR- KRT18 111 27.93 16.96 18.52 16.80 19.92 18.65 7.67 24.72 13.36 10.74 12.54 7.80 S93 SLNDRLAsYLDRVRS KRT18 112 89.71 35.36 62.90 28.89 54.10 51.47 26.38 68.68 35.19 24.48 31.95 12.89 S48 GGRGVSVsSARFVSS KRT19 113 14.93 14.31 16.02 10.04 13.76 15.36 8.79 18.98 7.74 7.54 9.22 9.53 S93 NLNDRLAsYLDKVRA KRT19 107 73.75 31.17 53.54 27.77 42.09 47.26 16.19 72.81 29.57 18.38 39.67 13.87 S191 TLNNKFAsFIDKVRF KRT2 114 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 S211 TLNNKFAsFIDKVRF KRT3 114 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 S181 TLNNKFAsFIDKVRF KRT5 114 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27

[0303]

[0304] S176 TLNNKFAsFIDKVRF KRT6A 114 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 Attorney Docket No. 07039-2296WO1 / 2023-600

[0305] S176 TLNNKFAsFIDKVRF KRT6B 114 117.13 5998 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 S176 TLNNKFAsFIDKVRF KRT6C 114 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 S104 TLNNKFAsFIDKVRF KRT7 114 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 S143 ALNNKFAsFIDKVRF KRT71 115 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 S138 ALNNKFAsFIDKVRF KRT72 115 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 S145 VLNNKFAsFIDKVRF KRT73 116 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 S153 VLNDKFAsFIDKVRF KRT74 117 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 S162 TLNNKFAsFIDKVRF KRT75 114 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 Y327 lAEVKAQyEDIANRS KRT75 118 29.22 20.34 25.95 16.29 25.66 25.60 9.08 34.09 21.37 10.33 12.33 7.69 S196 TLNNKFAsFIDKVRF KRT76 114 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 S177 VLNNKFAsFIDKVRF KRT77 116 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 S124 TLNNQFAsFIDKVRF KRT78 119 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 S155 TLNNKFAsFIDKVRF KRT79 114 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 S104 TLNNKFAsFIDKVRF KRT8 114 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 Y267 lAEVKAQyEDIANRS KRT8 118 29.22 20.34 25.95 16.29 25.66 25.60 9.08 34.09 21.37 10.33 12.33 7.69 S291 IKYEELQsLAGKHGD KRT8 120 15.32 9.50 14.09 12.50 12.22 12.43 8.51 20.08 12.16 7.54 9.76 5.52 S58 GGGYGGAsGMGGITA KRT8 121 14.42 14.20 10.71 6.56 11.91 10.24 4.21 11.31 7.37 5.78 4.82 4.55 S178 TLNNKFAsFIDKVRF KRT84 114 117.13 59.98 89.72 55.43 79.05 81.82 43.69 106.47 60.43 46.16 63.68 28.27 T865 TVGSYGCtPQSLPKF LARP1 122 8.88 8.89 9.17 12.50 7.08 8.23 7.11 7.34 8.47 7.85 3.97 5.74 S144 LGFSSDEsDVEASPR LEMD3 123 66.54 74.09 48.14 46.00 58.62 59.88 34.52 59.90 34.27 28.71 35.17 35.64 S140 EGSEKAHsDDEKWGR LE01 124 13.13 12.57 13.51 11.78 14.06 13.53 6.74 5.82 7.09 10.74 5.25 5.52 S301 QSRIRIDsLSAQLSQ LMNA 125 24.84 16.55 21.13 17.42 15.40 22.40 15.16 16.37 9.95 17.87 13.72 13.54 S1616 IQDRFLNsFEELQAE LTBP1 126 23.17 34.64 31.55 21.72 23.10 19.47 20.96 24.64 17.59 20.76 17.58 18.09 S214 SGSTYTPsEAGNELD MAF1 127 8.69 12.77 8.59 9.78 8.01 8.41 10.85 8.69 9.40 6.30 5.63 7.64 S507 LNGERDGsLCQQQNE MAP4K3 128 4.25 2.76 3.67 4.82 2.77 2.29 0.98 1.77 2.39 3.20 1.13 2.27 S410 HKVQRSVsSSQKQRR MARK3 129 57.02 39.44 30.58 37.19 37.67 26.15 32.75 28.85 26.53 30.36 24.12 23.72 S1384 SPLARTPsPTPQPTS MAST4 130 12.36 13.18 8.30 10.04 15.91 11.43 7.77 8.69 5.53 10.02 8.15 5.85

[0306]

[0307] T1425 TSTDQPVtPEPTSQA MDC1 131 13.39 11.85 10.71 10.76 15.71 12.98 6.92 11.39 5.71 9.91 7.18 7.91

[0308]

[0309] Attorney Docket No. 07039-2296WO1 / 2023-600

[0310] S1119 SPQVSGPsPAARMPG MED14 132 16.09 12.77 15.15 12.91 10.88 13.62 8.51 21.09 11.97 7.02 11.26 6.17 Y47 VSGKRPDyAPMESSD MFAP1 133 15.70 15.84 8.39 12.19 17.76 11.70 6.46 8.77 3.96 10.95 5.47 7.69 S1406 LSLPTPRsPSDRELR MICAL3 134 10.43 5.72 9.36 7.68 7.39 3.11 6.36 5.32 5.90 4.65 5.15 6.17 S732 PPGPPNAsSNPDLRR MINK1 135 41.06 81.65 33.86 43.44 36.34 58.78 21.61 60.58 25.33 28.61 25.19 29.68 T117 EVAATTAtPDGGPRA NAA30 136 7.47 7.87 6.85 6.35 8.32 5.21 4.21 10.12 6.82 6.92 3.65 2.49 S145 APSSEPQsPVAQTSG NACC1 137 1.61 3.37 3.76 1.28 1.28 3.57 2.34 2.36 1.15 1.29 1.34 1.35 S282 RNLVYDQsPNRTGGP NDE1 138 16.09 15.94 14.18 14.65 18.99 18.37 13.75 16.03 12.44 8.88 9.11 11.37 S106 TSLANLIsPVRNGAV NET1 139 1.48 2.86 2.89 1.18 3.80 1.05 1.08 1.94 1.06 1.19 1.23 1.25 S44 AMKFLRAsEEHLKQH NME2 140 18.15 9.71 18.43 9.22 13.14 12.89 5.80 14.43 13.17 5.68 9.43 5.20 S29 AMKFLPAsEEHLKQH NME2P1 141 18.15 9.71 18.43 9.22 13.14 12.89 5.80 14.43 13.17 5.68 9.43 5.20 S43 DENEHQLsLRTVSLG NPM1 142 19.44 11.04 15.82 13.52 17.25 20.39 3.74 16.62 9.03 4.96 11.15 5.74 S948 RGDCSTNsPVGVSKV NSD1 143 10.43 11.96 11.09 7.89 10.37 9.96 9.36 5.74 7.18 7.13 6.33 10.07 S259 SDRCALSsPSLAFTP NUP35 144 74.78 79.35 66.47 52.46 62.00 69.48 40.32 69.10 45.09 42.24 43.53 43.76 T65 IITLAGPtNAIFKAF PCBP2 145 1.48 2.86 2.22 1.18 2.36 2.10 1.08 2.28 1.06 1.19 1.23 1.25 S232 NRYGMGTsVERAAAS PDHA1 146 50.97 43.63 53.83 43.85 49.07 42.23 38.83 32.48 38.87 29.54 32.27 33.26 S415 LPGVDALsN- PGK1 147 11.58 5.82 6.85 5.74 8.32 7.13 3.74 8.69 2.58 3.10 5.57 4.44 S259 NMTSGHSsAPPKETS PM20D1 148 10.55 14.51 11.67 10.76 10.06 13.99 9.92 10.55 9.58 7.44 7.29 9.10 S512 GKIRRTQsGNFYTDT PPFIBP2 149 8.37 7.15 7.81 7.07 7.19 6.58 6.08 5.82 4.24 5.58 4.40 4.22 S150 AGKDTNGsQFFITTV PPIB 150 11.33 11.34 14.28 9.12 13.24 9.51 9.73 11.81 7.46 4.85 8.47 5.31 T256 VAAPGDAtPPAEKKY PPP1R10 151 11.46 13.49 16.88 12.29 10.68 10.24 7.30 14.17 15.57 9.29 10.72 7.58 S437 RGAPHRHsMPELGLR PRICKLE3 152 7.08 7.05 5.21 5.12 4.62 5.39 5.52 3.80 2.67 5.47 2.25 4.77 S34 GDGERRLsGSSLCSG PRPF40A 153 7.08 10.93 8.10 7.79 9.65 8.23 7.48 9.36 7.83 4.85 6.11 5.96 S778 HNVFKRHsMREEDFI PTK2B 154 23.81 11.96 10.42 16.80 29.46 14.44 12.54 10.12 7.46 7.95 5.68 7.47 S255 LCAATGPsIKIWDLE RACK1 155 3.73 2.35 5.50 4.92 3.39 3.47 1.08 5.06 3.04 1.19 1.23 1.25 S93 LDPAPAVsEAGPETH RASEF 156 1.48 2.35 2.41 1.18 2.57 4.02 3.56 0.97 1.06 1.19 1.23 1.25 S78 RSEDGYHsDGDYGEH RBM5 157 7.08 8.17 6.46 6.45 3.49 7.50 3.74 3.63 4.61 3.10 2.79 6.39 S120 SETVSEAsPGSTASQ RFX1 158 8.75 7.66 6.13 4.35 6.06 6.03 7.44 7.76 5.43 4.91 4.34 4.50

[0311]

[0312] S329 ATGEGGAsDLPEDPD RNF113A 159 3.73 1.23 2.89 1.23 2.46 1.10 1.12 1.01 1.11 1.24 1.29 1.30

[0313]

[0314] Attorney Docket No. 07039-2296WO1 / 2023-600

[0315] S77 VRAGRGFsLEELRVA RPL13 160 23.04 16.45 20.94 15.88 19.71 19.65 16.65 22.36 17.32 12.19 14.47 10.07 S233 GITLLNVsKLNILKL RPL4 161 3.09 2.86 2.22 1.08 2.16 2.47 0.98 3.71 0.97 1.08 1.13 1.14 S206 PRGTGIVsAPVPKKL RPS2 162 4.63 3.88 6.27 4.30 3.18 1.83 2.25 3.97 3.22 2.58 1.07 1.08 S6 RPS6 4.89 4.29 6.85 4.41 4.41 7.04 3.46 7.59 3.50 1.08 2.25 2.38 S82 LLLSKGHsCYRPRRT RPS6 163 22.27 15.33 22.38 14.65 21.66 21.21 10.57 20.08 17.04 8.78 14.15 10.62 S35 DGYNYTLsKTEFLSF S100A11 164 38.36 30.15 36.18 31.45 28.95 25.78 26.01 39.40 23.95 22.51 24.87 17.77 S73 EKIANLGsCNDSKLE S100A14 165 50.33 49.87 42.16 37.60 46.09 39.58 29.94 39.91 36.29 34.60 27.55 32.28 S138 SDDDGGDsPVQDIDT SAP30 166 9.85 1083 9.36 7.12 11.04 10.42 5.47 10.71 10.32 8.26 670 4.82 S1630 RGLGPVPs- SCRIB 167 5.02 2.15 3.18 6.04 3.49 3.20 4.30 3.71 3.13 2.58 1.13 1.14 T1185 APEPPPAtPPQAKFP SETD1A 168 3.60 5.31 2.70 1.95 3.39 3.47 3.56 2.36 1.66 0.93 0.96 0.97 S45 CEERNLLsVAYKNW SFN 169 31.28 26.77 28.75 21.11 28.03 26.42 14.22 36.87 22.01 18.28 17.05 9.42 S63 RAAWRVLsSIEQKSN SFN 170 45.31 30.15 34.35 26.95 33.16 35.84 18.62 42.35 20.73 21.07 27.45 18.20 S34 LPPPAPGsPAAPAAV SIK3 171 8.75 8.69 5.69 5.84 5.34 3.93 4.77 6.50 4.05 6.40 3.75 3.57 S2 SLC33A1 7.59 7.15 9.07 6.04 5.95 9.87 5.24 5.57 7.74 6.61 3.75 5.31 S216 KDLDLLAsVPSPSSS SMAP2 172 10.68 8.79 11.38 10.04 11.09 10.33 9.73 8.10 7.09 7.33 450 7.15 S451 PRAGPGSsPLFSLLP SMG9 173 10.04 12.26 14.86 10.35 10.68 15.27 7.86 11.56 9.76 6.09 10.61 7.15 S258 PVPAVAHsPPATVEA SOWAHB 174 14.54 17.27 9.74 13.11 10.47 9.87 15.16 13.75 7.65 10.02 6.97 7.26 S180 KQGEVPEsPEARKES SP140L 175 9.78 11.55 9.07 12.81 7.08 9.60 6.08 5.82 7.37 4.65 7.08 10.40 S1822 RGGSGYHsRSPARQE SRRM2 176 7.08 5.62 4.73 5.63 3.70 5.58 4.30 4.39 3.32 4.44 3.54 2.82 S1824 GSGYHSRsPARQESS SRRM2 177 7.08 5.62 4.73 5.63 3.70 5.58 4.30 4.39 3.32 4.44 3.54 2.82 S67 RNRRERFsPPRHELS SRRT 178 4.12 3.58 4.15 3.79 2.87 4.39 3.37 2.19 2.21 2.48 1.29 2.82 S342 RRCTVDGsPHELESR STRN4 179 19.95 17.58 14.57 13.63 14.68 16.27 17.03 16.87 12.53 11.98 13.19 8.45 S41 IKRHTPLsKLMKAYC SUMO2 180 6.56 5.62 7.53 3.89 5.65 6.22 2.53 5.65 2.95 3.00 3.97 1.46 S40 IKRHTPLsKLMKAYC SUMO3 180 6.56 5.62 7.53 3.89 5.65 6.22 2.53 5.65 2.95 3.00 3.97 1.46 S1039 NRGSRHSsAPPKKKR SUPT16H 181 10.55 14.51 11.67 10.76 10.06 13.99 9.92 10.55 9.58 7.44 7.29 9.10 S425 SSDDEVYsRPSSLVS TBC1D9 182 27.03 26.67 35.60 27.05 25.25 25.96 22.55 27.17 28.65 15.70 19.62 19.61 S91 DPAGPAAsPVLADGL TEX2 183 2.57 2.15 2.32 1.02 2.77 3.11 4.87 1.69 0.92 2.27 1.07 1.08

[0316]

[0317] S224 PKDGSNKsGAEEQGP TGOLN2 184 21.62 24.22 28.94 27.05 19.81 21.67 25.45 26.32 30.21 14.98 15.55 17.01

[0318]

[0319] Attorney Docket No. 07039-2296WO1 / 2023-600

[0320] S266 PDYERAYsPEYRRGA TJP2 185 174.27 171.98 162.66 166.59 119.29 122.13 85.89 98.88 71.48 90.68 89.74 104.43 S271 ARRDLVDsPASLASS TLE2 186 10.43 6.54 7.04 7.38 4.82 6.58 5.89 5.32 4.05 6.92 4.07 4.98 S393 FIRGWDsEDIPLNL TRAP1 187 18.79 14.41 13.60 12.19 17.25 12.80 8.14 20.59 7.18 5.68 10.72 8.77 T109 NYARGHYtIGKEIID TUBA1A 188 14.29 8.89 11.87 9.53 9.24 8.87 6.74 17.38 9.67 6.51 7.61 4.12 T223 NLDIERPtYTNLNRL TUBA1A 189 23.30 19.52 19.78 14.55 15.50 20.29 11.98 23.54 15.66 10.22 14.69 9.42 S277 ATYAPVIsAEKAYHE TUBA1A 190 10.17 10.42 14.47 8.40 11.91 10.88 8.33 16.11 7.83 6.82 6.75 3.79 T109 NYARGHYtIGKEIID TUBA1B 188 14.29 8.89 11.87 9.53 9.24 8.87 6.74 17.38 9.67 6.51 7.61 4.12 T223 NLDIERPtYTNLNRL TUBA1B 189 23.30 19.52 19.78 14.55 15.50 20.29 11.98 23.54 15.66 10.22 14.69 9.42 S277 ATYAPVIsAEKAYHE TUBA1B 190 10.17 10.42 14.47 8.40 11.91 10.88 8.33 16.11 7.83 6.82 6.75 3.79 T109 NYARGHYtIGKEIID TUBA1C 188 14.29 8.89 11.87 9.53 9.24 8.87 6.74 17.38 9.67 6.51 7.61 4.12 T223 NLDIERPtYTNLNRL TUBA1C 189 23.30 19.52 19.78 14.55 15.50 20.29 11.98 23.54 15.66 10.22 14.69 9.42 S277 ATYAPVIsAEKAYHE TUBA1C 190 10.17 10.42 14.47 8.40 11.91 10.88 8.33 16.11 7.83 6.82 6.75 3.79 T109 NYARGHYtIGKElVD TUBA3C 191 14.29 8.89 11.87 9.53 9.24 8.87 6.74 17.38 9.67 6.51 7.61 4.12 T223 NLDIERPtYTNLNRL TUBA3C 189 23.30 19.52 19.78 14.55 15.50 20.29 11.98 23.54 15.66 10.22 14.69 9.42 S277 ATYAPVIsAEKAYHE TUBA3C 190 10.17 10.42 14.47 8.40 11.91 10.88 8.33 16.11 7.83 6.82 6.75 3.79 T109 NYARGHYtIGKElVD TUBA3D 191 14.29 8.89 11.87 9.53 9.24 8.87 6.74 17.38 9.67 6.51 7.61 4.12 T223 NLDIERPtYTNLNRL TUBA3D 189 23.30 19.52 19.78 14.55 15.50 20.29 11.98 23.54 15.66 10.22 14.69 9.42 S277 ATYAPVIsAEKAYHE TUBA3D 190 10.17 10.42 14.47 8.40 11.91 10.88 8.33 16.11 7.83 6.82 6.75 3.79 T109 NYARGHYtIGKElVD TUBA3E 191 14.29 8.89 11.87 9.53 9.24 8.87 6.74 17.38 9.67 6.51 7.61 4.12 T223 NLDIERPtYTNLNRL TUBA3E 189 23.30 19.52 19.78 14.55 15.50 20.29 11.98 23.54 15.66 10.22 14.69 9.42 S277 ATYAPVIsAEKAYHE TUBA3E 190 10.17 10.42 14.47 8.40 11.91 10.88 8.33 16.11 7.83 6.82 6.75 3.79 T109 NYARGHYtIGKEIID TUBA4A 188 14.29 8.89 11.87 9.53 9.24 8.87 6.74 17.38 9.67 6.51 7.61 4.12 T223 NLDIERPtYTNLNRL TUBA4A 189 23.30 19.52 19.78 14.55 15.50 20.29 11.98 23.54 15.66 10.22 14.69 9.42 S277 ATYAPVIsAEKAYHE TUBA4A 190 10.17 10.42 14.47 8.40 11.91 10.88 8.33 16.11 7.83 6.82 6.75 3.79 T162 NLDIERPtYTNLNRL TUBA4B 189 23.30 19.52 19.78 14.55 15.50 20.29 11.98 23.54 15.66 10.22 14.69 9.42 T48 NYAWGHYtIGKEFID TUBA4B 192 14.29 8.89 11.87 9.53 9.24 8.87 6.74 17.38 9.67 6.51 7.61 4.12 T223 NLDIERPtYTNLNRL TUBA8 189 23.30 19.52 19.78 14.55 15.50 20.29 11.98 23.54 15.66 10.22 14.69 9.42

[0321]

[0322] S48 DLQLDRIsVYYNEAT TUBB 193 6.95 4.80 9.45 6.76 11.19 9.78 4.77 7.76 5.62 2.17 4.61 4.44

[0323]

[0324] Attorney Docket No. 07039-2296WO1 / 2023-600

[0325] S288 LIDASEDsQLEAAIR UBXN7 194 9.40 11.14 6.85 7.89 7.08 6.76 8.98 6.50 6.08 4.96 7.61 5.31 Y541 HKVRGYRyLEEDNSD UNC93B1 195 4.38 4.91 2.60 5.12 0.92 3.11 3.37 3.21 3.68 3.00 0.96 1.95 S680 VNEKTIGsPPNEFYC USP38 196 10.17 9.40 11.19 9.02 7.39 6.49 4.21 7.68 6.54 6.40 8.47 4.98 S407 EAHGVSEsEGEERPV UTP14A 197 8.37 8.69 7.62 6.04 8.32 7.59 8.79 7.85 4.15 4.65 5.36 5.85 S569 QNLLTTQsPSVKSLA UTP14A 198 5.41 6.03 6.37 4.71 4.52 4.75 3.46 4.22 3.78 3.30 4.50 4.22 S356 EDFSPFGsGGGLFSG WASHC2A 199 5.02 4.70 5.50 4.92 4.72 4.85 3.18 2.53 2.39 3.10 2.57 3.90 S356 EDFSPFGsGGGLFSG WASHC2C 199 5.02 4.70 5.50 4.92 4.72 4.85 3.18 2.53 2.39 3.10 2.57 3.90 S544 LFSDEEDsEDLFSSQ WASHC2C 200 1.48 2.66 2.80 3.28 1.18 2.10 2.43 0.97 1.06 1.19 1.23 1.25 S47 NEERNLLsVAYKNW YWHAB 201 31.28 26.77 28.75 21.11 28.03 26.42 14.22 36.87 22.01 18.28 17.05 9.42 S46 VEERNLLsVAYKNVI YWHAE 202 31.28 26.77 28.75 21.11 28.03 26.42 14.22 36.87 22.01 18.28 17.05 9.42 S46 NEERNLLsVAYKNW YWHAG 201 31.28 26.77 28.75 21.11 28.03 26.42 14.22 36.87 22.01 18.28 17.05 9.42 S46 NEDRNLLsVAYKNW YWHAH 203 31.28 26.77 28.75 21.11 28.03 26.42 14.22 36.87 22.01 18.28 17.05 9.42 S45 NEERNLLsVAYKNW YWHAQ 201 31.28 26.77 28.75 21.11 28.03 26.42 14.22 36.87 22.01 18.28 17.05 9.42 S114 PNASQAEsKVFYLKM YWHAZ 204 15.45 10.01 19.30 7.48 11.39 11.88 7.77 16.87 7.65 8.06 11.26 5.85 S156 YQEAFEIsKKEMQPT YWHAZ 205 14.29 14.82 13.02 14.96 16.43 13.80 8.51 20.08 11.61 8.68 12.44 5.52 S45 NEERNLLsVAYKNW YWHAZ 201 31.28 26.77 28.75 21.11 28.03 26.42 14.22 36.87 22.01 18.28 17.05 9.42 S63 RSSWRWsSIEQKTE YWHAZ 206 35.78 22.07 28.75 19.47 29.15 28.07 15.72 33.07 21.00 18.28 22.62 15.06 S345 SLLRRSLsMDSQVPV ZBTB21 207 29.86 32.70 21.42 18.34 25.36 34.83 12.26 23.88 13.26 12.70 12.01 13.54 S1182 GEEEAPPsRSDPDGG ZNF687 208 6.44 4.91 4.53 4.61 3.29 4.48 3.84 4.98 4.88 2.89 4.82 2.38 S1184 EEAPPSRsDPDGGDS ZNF687 209 6.44 4.91 4.53 4.61 3.29 4.48 3.84 4.98 4.88 2.89 4.82 2.38 Y483 IDIRPRPyTCSECGK ZNF749 210 9.27 9.30 8.97 10.55 10.37 8.78 6.08 9.28 8.47 4.65 5.90 7.15 T484 DIRPRPYtCSECGKA ZNF749 211 9.27 9.30 8.97 10.55 10.37 8.78 6.08 9.28 8.47 4.65 5.90 7.15 S82 PAAPRSRsLGGAVGS ZNRF2 212 66.93 73.27 59.04 57.99 53.07 59.42 41.73 47.42 38.23 41.83 42.99 42.14 0.1 pM ENDX:

[0326] S1410 PQDPDNVsLQEVEAE ABCA2 213 1.29 1.02 0.96 2.05 1.03 0.91 2.34 3.21 2.39 1.03 2.25 2.17 S404 EKFRQKAsIHEAWTD ACTN1 214 13.13 13.18 18.52 18.34 17.55 21.30 22.64 25.31 24.87 23.86 24.98 22.32 S423 EKFRQKAsIHEAWTD ACTN4 214 13.13 13.18 18.52 18.34 17.55 21.30 22.64 25.31 24.87 23.86 24.98 22.32

[0327]

[0328] T217 RSQSAAVtPSSTTSS ADRM1 215 8.37 9.91 6.95 10.76 8.93 15.45 11.13 10.63 8.38 11.98 13.40 13.97 Attorney Docket No. 07039-2296WO1 / 2023-600

[0329] S62 TLPSIWDsPTKQLSV ALG5 216 4.38 5.42 5.69 4.92 7.90 5.58 8.05 7.17 11.15 7.44 515 6.61 S100 PPLQSARsLPGPAPC AMPD2 217 4.76 3.47 2.70 6.86 5.95 3.11 4.87 4.81 4.33 6.71 5.68 5.42 S669 EAEGAPGsPEREPPA ANO8 218 1.29 2.04 0.96 2.56 2.05 1.92 3.27 4.13 2.95 3.41 3.00 2.60 S96 QQQGEDGsPQAHRRG AR 219 1.29 1.02 0.96 1.02 1.03 2.29 2.90 3.12 0.92 2.89 2.14 2.17 S592 IEEEIPDSPGIEKHD ARHGAP12 220 1.48 5.31 2.22 3.48 5.34 3.38 5.80 5.32 5.71 4.54 6.22 4.87 S227 QERGLNTsQESDDDI ARHGEF16 221 7.98 6.95 9.26 9.94 8.32 8.87 14.31 8.61 14.37 11.88 12.65 9.32 S413 DDRPSCPs- ARRDC3 222 1.29 1.02 0.96 1.02 2.05 3.20 0.94 2.02 2.86 3.30 2.47 2.60 S728 AMPIPAAsPTPASPA ATXN2 223 13.71 9.81 12.98 17.83 13.96 15.72 19.41 15.40 14.74 18.54 19.89 16.90 S733 AASPTPAsPASNRAV ATXN2 224 1.35 1.07 1.01 3.18 3.39 3.47 0.98 2.95 2.21 2.17 4.61 2.38 S134 LYQVPGPsPQFQSPP BCAR1 225 4.25 3.27 6.46 6.45 6.06 9.69 6.36 9.20 9.40 6.51 6.97 5.20 S165 GAELEYDsEHSDWHG C20orf27 226 5.41 7.15 8.88 10.04 12.22 12.16 10.67 10.21 8.38 11.77 7.40 12.46 S168 LEYDSEHsDWHGF- C20orf27 227 5.41 7.15 8.88 10.04 12.22 12.16 10.67 10.21 8.38 11.77 7.40 12.46 S643 ISEEI IGsPIPEPRQ C2CD5 228 1.54 2.45 3.38 4.00 4.52 3.11 4.12 4.89 4.70 4.13 2.68 4.55 S280 SGSSGCPsPTPQSSD CBX6 229 2.45 5.42 4.44 3.89 5.34 6.49 5.33 4.98 7.74 8.88 6.00 7.26 S240 AQRQRAHsVDVEKNR CCSAP 230 5.66 5.11 7.53 8.81 6.98 5.85 12.35 7.09 9.21 11.67 6.22 6.61 T177 KPSRDPEtPRSSGSM CDCA3 231 4.12 4.09 7.81 9.22 4.62 9.60 10.95 6.58 9.86 8.06 6.86 10.94 S793 DVAPTLMsVPRYLPR CDH1 232 1.42 4.50 2.60 5.12 2.26 4.66 6.64 4.13 3.68 4.34 5.47 6.61 S12 KKIKRQLsMTLRGGR CDK16 233 6.69 7.05 4.82 7.68 6.06 7.50 8.70 7.26 8.01 10.33 8.36 9.86 T417 PEPVSSPtTPPEYKA CDR2 234 1.29 1.02 1.93 3.38 2.57 2.56 2.25 2.28 5.71 3.72 3.32 1.08 T418 EPVSSPTtPPEYKAL CDR2 235 1.29 1.02 1.93 3.38 2.57 2.56 2.25 2.28 5.71 3.72 3.32 1.08 S164 NENQLLNsQD- CGGBP1 236 1.48 2.35 3.09 6.15 4.31 4.48 3.93 5.32 5.16 4.03 3.43 4.66 S756 GDSGSLFsPSKEEAR CPSF1 237 4.25 2.35 6.08 3.79 6.57 7.77 6.36 6.83 11.51 7.33 8.15 5.31 S533 PPLSTERsPEAVGSE CRYBG1 238 2.70 2.15 2.60 3.28 4.31 4.30 2.81 4.81 4.15 4.65 3.00 2.71 S320 DPRRRLRsYEDMIGE CTNND1 239 2.70 3.17 3.76 3.18 4.11 5.12 5.24 5.06 4.88 3.30 5.47 3.68 S560 GKVSSPLsPLSPGIK CTTNBP2NL 240 5.92 3.99 5.60 7.58 8.42 7.04 8.79 7.85 6.91 10.43 9.11 4.87 S381 KVELQHIsQKDCQED DBF4 241 5.15 6.74 4.24 8.61 7.60 5.12 6.36 7.00 5.25 7.44 9.76 7.47 S413 EKKLLFIsEPIPHPS DBF4 242 3.35 2.25 4.82 5.63 4.93 5.85 4.68 4.89 5.34 4.13 4.61 5.96

[0330]

[0331] S420 SEPIPHPsNELRGLN DBF4 243 3.35 2.25 4.82 5.63 4.93 5.85 4.68 4.89 5.34 4.13 4.61 5.96

[0332]

[0333] Attorney Docket No. 07039-2296WO1 / 2023-600

[0334] S287 EAGAETRsPGKAEAE DDX24 244 1.48 1.18 1.11 1.18 2.77 1.05 2.15 4.30 2.39 3.51 1.23 1.25 S109 FHSKPVDsDSDDDPL DDX42 245 29.73 21.66 37.14 35.81 33.06 34.28 35.27 31.81 39.06 48.33 41.71 45.93 S111 SKPVDSDsDDDPLEA DDX42 246 29.73 21.66 37.14 35.81 33.06 34.28 35.27 31.81 39.06 48.33 41.71 45.93 S160 KADSEPEsPLNASYV DDX59 247 13.77 9.91 15.05 19.77 21.04 21.03 14.88 15.02 16.86 20.04 14.37 17.87 S265 STSFMSVsPSKEIKI DEPTOR 248 23.17 22.28 25.57 35.14 28.44 36.11 31.53 27.34 30.58 40.17 34.63 41.38 S460 TKQSMDMsPIKIVKN DHX8 249 54.96 30.96 65.41 77.25 54.61 64.72 72.70 63.28 77.10 84.07 71.72 77.78 S454 NSRKRREsASSSSSV DMAP1 250 3.22 4.19 3.67 5.33 6.98 4.85 9.73 5.23 6.17 7.75 7.29 3.68 S370 ENPLDDNsEEEMEDA DNAJC21 251 59.46 30.25 63.19 68.85 50.30 65.00 76.06 67.33 72.22 80.35 71.08 89.37 S196 RKEDRSAsSGAEGDV DTD1 252 3.86 5.82 4.15 8.09 4.72 5.67 7.58 7.34 7.37 5.37 7.40 6.28 T55 TNTERPDtPTNTPNA EED 253 3.93 3.63 7.24 7.07 5.18 7.86 7.02 9.28 9.58 5.89 5.25 6.39 S1194 SKEVEERsRERPSQP EIF4G1 254 9.01 5.82 7.91 10.96 10.88 10.97 11.79 8.86 13.91 10.64 10.08 9.86 S66 FREWSFsPDPLPVR ENGASE 255 1.42 2.55 1.06 4.10 2.57 3.38 2.53 3.80 3.68 2.27 3.43 1.19 T148 HFRFPPStPSEVLSP ERF 256 2.51 1.99 3.71 3.89 3.29 2.93 3.23 2.45 3.68 3.61 4.72 5.74 S150 RFPPSTPsEVLSPTE ERF 257 2.51 1.99 3.71 3.89 3.29 2.93 3.23 2.45 3.68 3.61 4.72 5.74 S161 SPTEDPRsPPACSSS ERF 258 2.51 1.99 3.71 3.89 3.29 2.93 3.23 2.45 3.68 3.61 4.72 5.74 S369 SRMKPAGsVNDMALD EVL 259 4.63 6.34 6.08 6.04 8.32 8.32 9.36 9.03 8.11 4.44 9.11 9.32 S270 TTTDSPVsPAQAASP FAM 122 A 260 4.18 6.85 8.20 8.61 7.49 6.49 8.61 7.00 9.03 9.71 10.40 10.72 S276 VSPAQAAsPFIPLDE FAM 122 A 261 2.90 4.80 4.44 6.15 5.34 4.39 5.15 3.80 4.88 6.09 7.08 5.09 S436 VSRQTFLsHGDDFRF FAM83H 262 8.37 5.42 4.53 9.53 8.52 12.43 11.51 7.09 7.92 7.02 10.51 10.29 S461 VGVQSCPsPFSGQAC FBXL18 263 1.29 2.96 0.96 2.25 5.13 4.48 3.65 5.57 5.07 3.10 2.14 6.50 S135 DSLLFDTsDDEELRE FEZ2 264 1.29 1.02 2.41 1.02 2.87 3.20 0.94 2.19 2.03 2.07 3.22 3.03 S2107 TRTSRAPsVATVGSI FLNB 265 8.37 10.73 13.60 16.39 17.76 19.56 21.61 16.79 19.62 18.07 25.62 21.99 S13 PPPQDYEsDDDSYEV FUNDC1 266 2.83 3.58 2.22 5.12 4.52 2.65 3.18 4.81 5.16 3.92 3.54 4.66 S23 SPPPKARsNENLDKI FYTTD1 267 9.78 15.74 12.16 20.39 13.35 15.08 18.06 18.81 15.84 20.04 18.65 18.20 S10 WMFKEDHsLEHRCVE GABARAPL2 268 2.96 3.78 4.15 5.23 7.19 6.31 5.89 4.64 5.71 7.44 7.50 6.82 S620 GPLDAPGsPLACTEP GAS2L1 269 3.35 4.50 4.05 6.97 5.95 5.21 4.30 5.57 8.29 4.96 5.57 6.82 S508 RRDRQAFsMYEPGSA GIT1 270 11.71 7.66 9.07 13.01 13.55 10.15 17.31 10.46 12.71 15.70 15.22 13.76

[0335]

[0336] S9 SGRPRTTsFAESCKP GSK3B 271 27.16 34.23 28.07 39.44 30.39 36.84 41.26 47.84 37.77 46.58 40.53 48.42 Attorney Docket No. 07039-2296WO1 / 2023-600

[0337] T427 AKRLRLDtGPQSLSG GTF2F1 272 11.97 13.28 11.29 11.78 17.25 15.08 18.99 21.01 18.05 13.84 18.01 19.17 S784 GVPFRRPsTFGIPRL GTF2I 273 2.70 3.37 4.44 2.25 5.95 4.94 5.05 4.56 7.18 5.78 5.04 7.91 S454 VERTRKRsEGFSMDR HDGFL2 274 19.44 17.47 21.03 21.82 23.20 26.51 30.31 29.19 28.37 24.79 24.77 30.66 S1003 LLPPEPPsPARAPVP INPPL1 275 1.93 3.37 4.44 7.17 4.00 5.76 4.40 5.91 4.24 4.03 5.47 3.25 T33 PGPSGSEtPPPPRRA ITPKB 276 5.92 5.11 5.02 6.35 9.85 7.13 7.30 8.44 9.03 7.54 6.65 5.52 T104 METGIAEtPEGRRTS KDM1A 277 1.16 0.92 2.51 3.79 1.85 3.66 3.09 2.36 3.50 3.82 0.96 3.03 S17 KKASFDHsPDSLPLR KDM1B 278 17.38 17.17 27.11 25.10 17.66 23.86 26.85 24.55 28.55 30.05 28.30 32.71 S730 GRSSSPTsSLTQPIE KDM3B 279 3.99 6.03 6.56 7.07 5.85 8.96 9.26 10.71 7.18 7.33 5.79 6.39 T259 GTRAPPLtRPMSLAV KIAA1522 280 2.00 1.58 2.99 3.89 5.54 3.11 4.30 2.78 4.15 3.82 4.07 3.79 S177 TELAMRQsVENDIHG KRT18 281 1.35 1.07 2.60 2.15 4.21 0.96 2.34 4.72 3.87 2.58 2.68 3.03 S64 SASERLPsVEEAEVP LAD1 282 5.28 6.23 7.53 10.45 6.06 7.59 10.85 11.64 10.32 5.78 6.97 11.16 S324 DSGDKRWsGNEPTDE LRCH3 286 2.96 2.04 4.05 3.89 6.88 4.66 4.49 6.16 3.41 2.48 6.22 5.42 S592 SLRCGEAsPPSAACG MAP1S 287 1.87 1.48 1.40 1.49 1.49 3.75 4.58 3.71 2.86 1.50 3.54 3.14 S696 PNKELPPsPEKKTKP MAP4 288 8.37 6.95 13.89 11.27 9.65 10.79 14.50 12.82 16.21 13.32 14.90 10.29 S42 HDSERAVsADPLPPP MARVELD2 289 1.35 2.15 2.80 3.18 3.29 3.38 4.49 4.05 3.78 2.79 407 2.60 T474 KPQKRPLtPFHHRVS MED13 290 4.89 2.86 4.34 5.94 4.62 6.40 7.20 5.23 6.82 6.71 4.50 5.63 T821 PVILEPEtEESENEF MIS18BP1 291 3.35 3.07 2.41 6.04 2.57 2.29 3.84 3.04 3.87 5.58 4.93 4.55 S824 LEPETEEsENEFYIK MIS18BP1 292 3.35 3.07 2.41 6.04 2.57 2.29 3.84 3.04 3.87 5.58 4.93 4.55 S357 QYSQQQNsPQKHKNK MKI67 293 3.09 4.60 6.37 7.68 6.88 7.50 6.36 7.51 7.92 5.68 5.90 4.66 S649 EVIEVDEsDVEEDIF MRE11 294 2.70 2.15 2.03 3.28 4.41 5.03 6.27 3.21 1.93 2.17 4.50 2.27 S1307 PSEDRRWsTELVPEG MYO9A 295 3.86 4.50 7.14 7.89 6.36 8.41 7.30 6.58 10.04 7.95 9.43 10.51 S1317 LVPEGLQsPRGTPDS MYO9A 296 7.34 9.40 9.84 11.58 11.09 11.06 9.82 10.71 13.45 13.32 13.83 13.87 S69 TPTGYIEsLPRWKR NAP1L1 297 1.54 1.23 2.32 3.07 2.77 3.38 3.84 3.37 2.76 2.58 5.47 3.25 T1867 NLGEVPLtPTEEASL NBEAL2 298 7.59 5.72 9.17 7.68 9.03 7.04 12.07 9.53 13.08 9.81 9.43 11.70 S674 CEGTEINsDDEQESK NCAPG 299 6.69 5.52 6.66 9.02 8.52 7.40 11.88 9.96 8.66 8.78 9.97 6.93 S1299 SLSDRANsTESVRNT NE01 300 10.55 12.57 13.12 13.22 13.35 12.52 16.93 18.56 18.88 15.80 15.33 13.43 S53 PNAHKVAsPPSGPAY NFATC2 301 7.34 11.60 8.54 9.48 12.01 13.35 11.04 12.66 13.45 16.01 11.63 14.84

[0338]

[0339] S295 DFYPSPSsPAAGSRT NFIB 302 2.45 0.97 0.92 5.94 4.93 4.39 2.62 4.56 3.59 5.27 4.07 2.92 Attorney Docket No. 07039-2296WO1 / 2023-600

[0340] T709 FFQPPPLtPTSKVYT OGA 303 6.05 6.03 5.02 7.38 6.47 7.04 7.30 8.35 8.75 9.50 997 7.04 S203 VSRQRVEsLRKKRPL PANK2 304 7.85 3.07 4.24 7.79 4.82 4.57 11.51 8.86 6.63 8.78 8.79 7.26 T883 ARYGPQFtLQHVPDY PCDHGA1 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S899 QNVYIPGsNATLTNA PCDHGA1 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T888 ARYGPQFtLQHVPDY PCDHGA10 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S904 QNVYIPGsNATLTNA PCDHGA10 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T887 ARYGPQFtLQHVPDY PCDHGA11 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S903 QNVYIPGsNATLTNA PCDHGA11 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T884 ARYGPQFtLQHVPDY PCDHGA12 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S900 QNVYIPGsNATLTNA PCDHGA12 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T884 ARYGPQFtLQHVPDY PCDHGA2 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S900 QNVYIPGsNATLTNA PCDHGA2 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T884 ARYGPQFtLQHVPDY PCDHGA3 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S900 QNVYIPGsNATLTNA PCDHGA3 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T914 ARYGPQFtLQHVPDY PCDHGA4 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S930 QNVYIPGsNATLTNA PCDHGA4 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T883 ARYGPQFtLQHVPDY PCDHGA5 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S899 QNVYIPGsNATLTNA PCDHGA5 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T884 ARYGPQFtLQHVPDY PCDHGA6 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S900 QNVYIPGsNATLTNA PCDHGA6 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T884 ARYGPQFtLQHVPDY PCDHGA7 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S900 QNVYIPGsNATLTNA PCDHGA7 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T884 ARYGPQFtLQHVPDY PCDHGA8 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S900 QNVYIPGsNATLTNA PCDHGA8 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T884 ARYGPQFtLQHVPDY PCDHGA9 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S900 QNVYIPGsNATLTNA PCDHGA9 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T879 ARYGPQFtLQHVPDY PCDHGB1 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10

[0341]

[0342] S895 QNVYIPGsNATLTNA PCDHGB1 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10

[0343]

[0344] Attorney Docket No. 07039-2296WO1 / 2023-600

[0345] T883 ARYGPQFtLQHVPDY PCDHGB2 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S899 QNVYIPGsNATLTNA PCDHGB2 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T881 ARYGPQFtLQHVPDY PCDHGB3 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S897 QNVYIPGsNATLTNA PCDHGB3 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T875 ARYGPQFtLQHVPDY PCDHGB4 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S891 QNVYIPGsNATLTNA PCDHGB4 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T875 ARYGPQFtLQHVPDY PCDHGB5 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S891 QNVYIPGsNATLTNA PCDHGB5 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T882 ARYGPQFtLQHVPDY PCDHGB6 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S898 QNVYIPGsNATLTNA PCDHGB6 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T881 ARYGPQFtLQHVPDY PCDHGB7 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S897 QNVYIPGsNATLTNA PCDHGB7 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T886 ARYGPQFtLQHVPDY PCDHGC3 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S902 QNVYIPGsNATLTNA PCDHGC3 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T890 ARYGPQFtLQHVPDY PCDHGC4 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S906 QNVYIPGsNATLTNA PCDHGC4 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 T896 ARYGPQFtLQHVPDY PCDHGC5 305 6.44 6.03 7.43 8.50 12.42 9.51 11.88 10.46 10.59 11.77 10.19 9.10 S912 QNVYIPGsNATLTNA PCDHGC5 306 17.50 16.35 18.81 21.21 21.46 25.60 23.95 23.45 20.36 25.92 33.66 22.10 S1909 LPGPPPAsPIPTEGP PCNX3 307 1.22 2.76 1.83 4.00 2.05 3.20 5.15 2.36 5.62 3.10 4.72 5.96 S1097 SALCNADsPKDPVLP PDS5A 308 6.82 7.56 7.81 9.84 7.29 8.78 7.95 7.76 9.40 12.08 11.15 11.48 T657 VDRKRRDtSGLERSH PNN 309 4.38 4.39 4.15 2.97 6.26 6.03 7.58 7.59 9.76 7.75 4.40 8.88 T707 VPVSSNDtPPSALQE POGZ 310 1.22 0.97 0.92 2.15 2.87 0.87 0.89 0.80 2.58 1.96 2.25 2.49 T8 MGRKRLItDSYPWK P0LD4 311 1.16 2.55 0.87 0.92 1.85 3.29 5.24 2.95 2.49 4.34 2.14 4.22 S527 IPLDEECsMDETPYV PPP1R10 312 1.29 1.02 1.93 3.07 1.03 2.10 1.87 0.84 2.95 2.48 3.54 2.82 S545 EPGGSGGsPDGAGGS PPP1R10 313 5.28 5.62 4.24 7.07 10.88 5.30 4.68 7.09 5.34 8.16 6.75 11.59 S78 RARSLPSsPERRQKA PPP1R3D 314 47.49 44.86 50.36 47.95 47.22 49.00 80.18 65.72 75.99 56.60 65.61 69.65 S2079 EWRVRAIsAANLHLR PRPF8 315 3.86 2.86 2.80 3.28 5.34 5.39 2.90 3.54 5.07 5.78 7.18 5.20

[0346]

[0347] S277 TVGTPIAsVPGSTNT PSMD1 316 8.62 8.48 9.74 9.94 11.09 9.05 16.09 12.32 13.08 11.77 10.72 10.51

[0348]

[0349] Attorney Docket No. 07039-2296WO1 / 2023-600

[0350] S98 EQLGGGGsGGGGYNN PUM1 317 2.96 1.18 6.17 5.23 6.88 5.76 7.20 7.68 7.37 4.75 6.86 7.26 S773 STRVRHFsQSEETGN RALGAPA1 318 14.03 7.82 8.73 11.37 14.27 19.38 14.13 16.37 15.57 11.46 12.28 14.62 S2628 KKPEDSPsDDDVLIV RANBP2 319 5.02 2.55 3.96 6.76 3.80 4.21 4.96 4.47 4.88 6.30 4.61 7.69 S583 QIKQEPEsEEEEEEK RBM25 320 72.85 66.83 89.24 128.79 62.72 128.99 78.31 134.74 87.51 152.13 79.98 132.27 T208 FNGKFIKtPPGVSAP RBMS1 321 4.25 5.01 2.89 5.02 6.67 6.67 4.58 5.91 7.18 5.47 5.25 5.20 S350 EKAEVYGsENESERN RIOK2 322 5.79 8.07 6.27 7.99 6.67 8.87 10.01 8.52 15.29 10.33 10.08 10.51 S501 SQPSQPSsPLPGSHG RNF214 323 3.22 3.58 4.63 6.25 8.42 5.03 4.58 4.30 5.80 6.51 2.89 6.28 S9 GHQQLYWsHPRKFGQ RPS29 324 2.70 3.47 4.24 3.07 3.80 4.02 5.71 4.56 5.16 5.68 6.75 4.12 S452 PVSPVKFsPGDFWGR RPS6KB1 325 37.84 37.91 32.51 42.01 42.81 45.16 55.20 55.01 56.37 47.51 44.71 50.26 S503 LPIRQPNsGPYKKQA RPS6KB1 326 1.87 2.96 7.53 6.97 7.49 8.32 9.92 6.92 11.15 6.92 8.26 7.80 S528 QEKIEPGsLNEEPFM RPS6KC1 327 5.53 7.46 3.86 6.25 5.34 5.76 9.92 8.02 8.66 5.47 9.11 8.45 T77 GKSEAPEtPMEEEAE RRP12 328 1.29 1.02 1.93 2.77 2.05 2.19 2.62 0.84 3.22 3.10 1.07 2.60 S212 AEDGVRGsPPVPSGP SENP3 329 7.92 10.32 10.42 15.06 8.88 13.99 12.35 12.91 12.34 19.11 10.51 15.71 T14 TQFINPEtPGYVGFA SEPTIN2 330 1.42 1.12 2.12 3.48 1.13 1.01 2.43 2.36 2.58 3.10 1.18 1.19 T473 SSTGRRHtLAEVSTR SIK1 331 5.66 2.96 2.80 9.12 5.34 7.59 5.43 6.67 7.55 5.99 6.43 7.15 T473 SSTGRRHtLAEVSTR SIK1B 331 5.66 2.96 2.80 9.12 5.34 7.59 5.43 6.67 7.55 5.99 6.43 7.15 S534 NFLEDNPsLKDIMLA SIK2 332 2.77 2.20 3.04 4.66 3.85 3.98 3.88 4.43 4.10 4.39 4.93 3.52 T171 QPGIHPKtPNKFKKY SLBP 333 20.34 10.42 22.86 28.48 20.63 20.84 34.15 28.35 30.21 20.76 25.84 21.56 S417 KPLRRNNsYTSYTMA SLC20A1 334 1.42 1.12 1.06 1.13 1.13 2.47 4.02 2.19 1.01 2.27 3.11 3.14 T478 GASQSDKtPEELFHP SLC2A1 335 12.48 10.53 11.67 19.77 14.47 19.56 15.44 16.87 14.28 15.08 13.83 16.14 S297 EGHKLCLsTVDLEVK SLC43A2 336 4.38 4.19 4.24 4.10 5.65 6.76 8.05 5.65 6.45 3.30 4.72 7.69 S709 TQTHENMsQLSEEEQ SLC4A1AP 337 5.41 9.81 10.32 10.35 10.57 10.06 10.85 11.47 12.25 11.26 12.65 14.41 T479 PQLHFLDtDDEVSPT SMG6 338 2.70 1.07 1.01 4.30 2.57 2.10 2.53 3.21 2.95 2.48 2.57 1.14 S484 LDTDDEVsPTSWGDS SMG6 339 2.70 1.07 1.01 4.30 2.57 2.10 2.53 3.21 2.95 2.48 2.57 1.14 S1323 ELNQAWSsLGKRADQ SPTAN1 340 7.59 4.09 6.66 10.86 4.31 5.21 8.98 6.41 6.91 9.40 9.86 8.88 S17 DASQRRRsLEPAENV SRC 341 1.61 3.17 2.60 2.56 3.59 3.57 4.02 4.64 3.32 1.29 5.36 3.36 S837 PSRQSHSsSSPHPKV SRRM2 342 4.25 1.69 6.85 9.02 8.42 6.58 4.02 6.24 9.76 8.99 7.40 10.18

[0351]

[0352] T714 KTKFICVtPTTCSNT STAT3 343 1.29 2.04 2.99 1.02 5.95 3.38 3.37 3.88 3.32 3.82 3.43 3.79

[0353]

[0354] Attorney Docket No. 07039-2296WO1 / 2023-600

[0355] S281 NRRQLAFsTVGTPDY STK38 344 8.11 4.39 4.53 11.47 7.80 7.50 9.92 6.50 7.18 11.57 9.11 6.93 S823 AAQAKQRsPSKVKPP TCOF1 345 9.27 4.60 7.91 13.01 11.09 13.99 13.19 13.08 14.09 7.64 13.51 9.86 S153 FPRGELEsPKPLVTP TGIF2 346 8.11 8.58 11.29 10.45 10.47 14.90 13.10 15.19 13.91 13.84 10.83 12.78 S307 LFKPPEDsQDDESDS THOC5 347 1.48 3.17 2.41 4.41 4.00 5.30 1.08 3.97 3.96 5.16 3.65 2.49 S403 KYLQDQLsPLTRQRE TNIP1 348 1.48 1.18 2.51 1.18 2.57 2.38 2.25 0.97 4.70 2.38 4.18 3.03 S1585 NSSTSPAsPPGSIGD TNRC6A 349 1.67 4.19 2.51 4.61 5.44 3.75 3.46 4.64 3.87 2.89 4.29 4.22 S154 QKRKRTEsSCVKSGS TRIP12 350 23.94 16.35 22.09 25.00 25.66 21.67 31.53 26.49 38.60 29.54 24.12 28.60 S8 MSSDRQRsDDESPST UBE2E3 351 1.80 1.43 2.80 4.51 3.18 3.11 4.40 3.71 4.33 3.20 3.00 4.66 S130 LEESVSMsPEERARY UCHL3 352 3.22 2.96 3.76 4.92 3.70 5.03 3.74 6.92 6.08 5.68 6.33 5.85 S42 RADGGGHsPTEVAGT VAX2 353 1.42 2.66 1.06 1.13 2.57 2.38 2.34 1.86 2.21 3.10 3.22 2.82 S779 DMEEEKEsEDSDEEN WDR75 354 13.06 13.69 21.13 16.50 20.12 16.91 19.18 14.85 22.20 24.89 21.55 29.14 S782 EEKESEDsDEENDFT WDR75 355 13.06 13.69 21.13 16.50 20.12 16.91 19.18 14.85 22.20 24.89 21.55 29.14 S1978 KKEGPVAsPPFMDLE WNK1 356 1.93 1.53 3.09 3.07 3.18 3.29 5.05 2.95 5.62 4.65 3.97 5.85 S883 TDAGGGLsSDEEEGT XPC 357 7.59 11.75 9.55 13.01 19.81 14.54 15.06 10.55 11.42 14.87 10.29 12.02 S884 DAGGGLSsDEEEGTS XPC 358 7.59 11.75 9.55 13.01 19.81 14.54 15.06 10.55 11.42 14.87 10.29 12.02 S164 AQHLRQSsFElPDDV YAP1 359 70.53 33.31 81.91 73.15 60.77 78.98 85.33 75.76 86.31 92.23 107.43 121.33 S61 IVHVRGDsETDLEAL YAP1 360 10.43 7.87 13.80 12.70 13.04 16.18 17.78 12.06 19.62 13.32 16.40 15.71 S189 RFIPRPPsVAPPPMV YBX2 361 3.86 4.70 4.82 5.94 4.93 5.58 7.02 7.51 8.20 7.02 7.08 6.39 S293 TYRPRYRsRGPPRPR YBX3 362 1.67 2.66 4.44 5.23 2.87 6.03 6.17 5.32 9.76 5.89 8.58 3.47 S1053 SHRCDAGsPSKDSAA ZBED4 363 2.70 1.07 1.01 4.82 2.16 6.03 4.68 2.87 2.67 4.03 4.18 4.98 T168 QYLEAFAtATASGVP ZBTB7B 364 1.35 1.07 2.51 2.15 2.26 4.21 2.43 0.89 2.03 2.48 2.47 3.90 S2801 LSPRTLLsPSSlKVE ZFHX3 365 1.54 2.96 2.41 3.69 2.87 3.75 2.99 2.02 2.30 3.61 4.29 4.33 S426 AGAAAPPsPPFSFQL ZFP36L2 366 3.41 2.71 2.56 6.30 2.72 2.42 5.47 4.68 3.91 4.18 5.84 4.22 S1348 NAENNVPsCHHSQPA ZGRF1 367 1.54 2.86 2.41 2.87 2.46 4.94 4.12 4.13 7.00 3.82 2.57 5.20 S1352 NVPSCHHsQPAKLVM ZGRF1 368 1.54 2.86 2.41 2.87 2.46 4.94 4.12 4.13 7.00 3.82 2.57 5.20 S551 ASEYSPAsLDAFGAF ZKSCAN1 369 2.83 2.76 3.38 3.48 5.34 4.02 4.49 5.48 4.97 6.51 5.47 5.96

[0356]

[0357] S301 LLTSEEDsGFSTSPK ZNF148 370 5.41 4.09 5.50 7.07 5.44 7.95 7.30 9.11 6.91 8.26 4.72 5.20

[0358]

[0359] Attorney Docket No. 07039-2296WO1 / 2023-600

[0360] S412 LEQNQTIsPLSTYEE ZNF148 371 17.12 13.90 25.95 22.44 22.48 20.75 29.66 23.88 32.79 27.47 29.80 23.18 S452 PSTPERQsSPSGSEQ ZNF185 372 3.35 1.33 3.38 4.30 5.34 5.12 7.20 3.97 6.26 6.61 5.79 3.79 5 pM ENDX:

[0361] S166 PKEPAPPsPESHSPR ACBD4 373 8.75 5.82 5.69 3.38 5.65 4.02 5.05 5.23 5.62 3.10 5.25 9.21 S332 PKAGLRVsAPEVSVG AHNAK 374 15.32 13.18 12.06 8.91 8.93 4.94 17.78 12.74 12.34 15.18 11.79 11.92 S5237 KIKFPKFsMPKIGIP AHNAK 375 57.66 41.08 36.18 28.79 28.54 29.62 57.63 46.07 46.89 61.14 44.28 61.31 T444 RYFDDEFtAQSITIT AKT2 376 3.99 2.66 3.28 1.95 0.98 0.87 2.81 2.87 2.12 4.85 2.14 3.90 T1023 RRMLRRHtVEDAWS ANKRD27 377 49.81 26.77 35.70 22.64 29.57 15.63 23.95 12.91 16.86 23.24 44.60 23.18 S675 AQLGAPTsPlPDSAI ANKZF1 378 3.86 3.17 4.53 1.95 2.67 2.93 1.96 5.48 5.71 3.30 4.93 2.60 S782 EKLCRANsRDGEAGR ARHGEF2 379 147.12 109.44 82.39 77.35 59.64 54.67 114.42 102.93 89.90 108.44 82.66 103.78 S557 VGESNLPsPSPTVSV ASH1L 380 4.12 5.11 5.40 3.18 1.75 3.20 3.37 3.29 3.87 5.58 2.36 3.79 S381 GKLFWKKsVKEDSVP BCAS1 381 23.17 11.55 19.39 11.58 11.09 9.14 17.50 17.55 17.78 14.46 12.22 16.14 S332 STPSRTSsGLFPRIP C17orf53 382 13.13 12.16 12.73 8.30 7.08 8.14 11.70 9.87 11.70 14.05 8.26 12.57 S878 PPPKRLSsVSGPSPE CASKIN2 383 15.32 15.33 16.98 12.09 9.14 10.33 13.19 12.32 13.54 12.60 13.51 14.84 S250 KSKLDWEsFKEEEGI CFDP1 384 16.99 13.80 13.22 10.96 8.01 6.95 13.57 14.26 19.62 16.01 17.80 14.62 S512 RSPRRGPsPACSDSS CROCC 385 5.41 5.62 7.33 4.20 3.29 3.02 3.74 7.17 4.61 6.40 3.54 4.77 T85 STSSSSStPPLQPRD CUL4B 386 8.88 10.01 6.66 6.15 5.75 4.21 7.20 7.85 7.65 7.85 8.15 7.04 T172 CPHIWGItPGRILAL DDX39B 387 8.88 10.73 7.72 6.56 6.57 4.94 6.55 4.13 6.54 7.95 5.25 9.53 S14 PNIPKAKsLDGVTND DHCR7 388 180.45 176.07 122.91 114.65 101.01 92.88 162.33 143.34 132.92 170.92 146.02 171.59 S184 ESHTEAIsDAETSSS DNTTIP2 389 3.99 7.15 4.82 2.66 3.49 3.20 2.81 6.50 3.87 1.34 5.79 4.33 S896 LNLNRSRsLSNSNPD DOCK7 390 12.48 16.66 11.77 9.32 5.24 8.04 8.98 11.81 8.84 10.43 8.47 15.38 S470 YSNRKYEsDEDSLGS EEF2K 391 5.28 3.78 5.50 2.66 2.57 2.10 3.74 2.19 4.97 3.51 4.61 3.90 S1257 GVRLRRPsVNGEPGS EHBP1L1 392 28.32 33.11 30.97 19.77 19.50 22.21 19.93 24.72 22.20 26.23 17.90 26.11 S597 ASKYAALsVDGEDEN EIF4B 393 25.48 15.33 17.08 12.09 12.63 9.23 13.94 20.75 14.74 18.80 15.76 16.03 S348 LGSRFRYsGKTEYQT EPB41L5 394 92.29 78.38 56.05 53.89 45.48 43.24 68.21 56.36 57.20 62.17 57.57 58.82 S435 AGEVPARsPGAFDMS EPN1 395 4.50 4.70 3.86 3.79 2.52 2.24 3.37 4.05 5.43 5.06 2.63 4.55 S358 SGTVLSRsQPWDLTP EPN3 396 4.89 4.39 2.51 2.46 2.26 2.38 2.25 5.40 2.30 5.68 3.86 4.01

[0362]

[0363] S177 DKILPPPsPWPKSSI GGA2 397 2.45 3.58 2.51 0.97 0.98 0.87 2.81 3.71 4.33 2.38 3.43 4.33

[0364]

[0365] Attorney Docket No. 07039-2296WO1 / 2023-600

[0366] S3016 ASPETSAsPDGSQNL GOLGB1 398 3.73 4.70 5.40 3.18 2.57 1.74 3.09 3.21 3.13 3.82 3.00 3.47 S1160 PQGEHARsPQPRGPA GREB1 399 7.59 11.85 11.09 6.04 7.29 4.85 4.58 6.24 9.58 13.63 11.79 8.77 S600 KLVSSAVsPSIIPQE INTS4 400 5.79 7.05 4.92 3.28 4.72 3.38 4.68 4.47 4.33 2.79 4.07 6.17 S180 SNMRMQFsFEGPEKV IQSEC1 401 19.18 19.72 23.25 14.14 12.22 14.72 23.67 16.11 20.45 18.28 19.51 24.59 S253 QVKSLAEsIDDALNC IQSEC1 402 4.50 4.50 3.76 2.97 2.57 2.83 3.74 4.22 4.88 4.54 3.75 4.77 S832 SSANLRHsPRVLVQH KDM2A 403 1.35 5.93 2.80 1.08 1.08 0.96 0.98 1.77 0.97 1.08 3.54 3.14 S897 ALASLPSsPGLLQSL KDM5C 404 7.98 4.80 6.66 3.48 2.77 4.57 5.43 4.64 4.70 5.37 6.22 4.98 S12 VEKLTHLsYKEVPTA LRATD2 405 28.96 28.51 22.58 18.65 17.14 12.34 25.54 24.64 21.00 30.47 22.09 25.13 S148 AVPCTTRsPREGEVP MAGI1 406 5.02 4.50 5.11 2.87 2.26 2.83 4.30 3.37 3.50 4.34 2.89 3.57 S775 SKSRRSAsPPTSLPS MAP3K21 407 4.76 5.21 3.09 3.38 1.13 2.01 5.05 5.15 6.26 4.75 4.29 6.39 S639 PVLSRRDsPLQGSGQ MAP4K4 408 10.17 6.95 9.55 7.27 5.24 4.57 8.79 8.10 7.74 7.13 9.97 8.23 S156 ACDGDKEsEVEDVET MIER3 409 14.93 12.88 10.71 9.84 6.98 8.59 8.61 13.25 11.05 13.53 11.15 9.64 S1030 FLLGSPGs- MMS19 410 3.48 2.76 2.51 1.28 2.57 1.14 4.49 2.95 2.49 1.29 4.29 2.92 S107 TDMQRKRsSECLDGT MON1A 411 12.48 13.18 9.94 8.50 6.36 7.50 8.23 8.18 11.61 7.75 8.68 9.86 S145 KNAKKEDsDEEEDDD NCL 412 3.73 3.27 2.70 2.36 2.26 1.01 1.03 3.21 3.22 4.34 2.68 2.82 S153 DEEEDDDsEEDEEDD NCL 413 3.73 3.27 2.70 2.36 2.26 1.01 1.03 3.21 3.22 4.34 2.68 2.82 S1329 VITSQSDsPTRATDV NHS 414 2.83 4.19 4.63 1.13 1.13 1.01 3.18 5.23 2.58 4.44 3.86 1.19 S411 PGHKRTPsEAERWLE NUMBL 415 10.81 10.73 9.94 6.56 7.08 6.12 6.36 8.10 10.50 9.40 11.04 8.99 S648 VTPKRSHsPSIDGTP PAPOLG 416 11.20 10.93 12.83 5.12 9.55 8.32 8.51 8.35 12.07 10.84 10.51 14.41 S116 KDIIRQPsEEEIIKL PEA15 417 605.45 447.79 424.40 356.23 300.37 296.47 514.02 465.72 476.68 478.17 463.90 533.51 S537 PSLEPQKsLGDEGLN PLCB3 418 25.74 24.93 15.63 14.04 13.45 14.54 21.71 22.95 19.44 26.13 23.48 23.07 S77 FGESSTEsDEEEEEG PPP1R11 419 3.86 6.85 5.98 2.46 4.62 5.21 3.18 3.97 3.41 5.47 3.86 6.17 S710 GEKSFRRsWGTPAY PRKD2 420 24.45 17.88 13.41 13.93 8.42 10.15 19.37 16.28 17.23 20.04 15.22 22.64 S973 GKPGESRsASSDTIE PRRT3 421 6.56 8.28 8.39 4.51 4.93 4.30 5.43 4.56 6.26 7.13 6.75 7.04 T978 SRSASSDtlE- PRRT3 422 6.56 8.28 8.39 4.51 4.93 4.30 5.43 4.56 6.26 7.13 6.75 7.04 S449 PLQEGPKsFDGNTLL PTPN12 423 53.03 42.61 40.62 22.23 19.71 21.57 36.30 44.72 38.23 53.08 38.70 50.81 S564 RSPIKRRsGLFPRLH RAP1GAP2 424 13.13 12.16 12.73 8.30 7.08 8.14 11.70 9.87 11.70 14.05 8.26 12.57

[0367]

[0368] S971 STVEEPVsPMLPPSA REST 425 2.83 4.60 2.60 1.08 2.77 0.96 3.46 1.77 3.22 3.72 2.25 4.33

[0369]

[0370] Attorney Docket No. 07039-2296WO1 / 2023-600

[0371] S608 KTEELIEsPKLESSE SCAF11 426 13.51 9.30 14.37 7.79 10.68 6.22 12.63 8.86 9.76 10.43 10.61 10.40 S330 FVLHKSKsEEAHAED SERBP1 427 28.44 36.07 20.55 16.39 14.37 14.08 26.48 21.35 22.38 28.19 24.55 23.72 S21 NMKKQSDsVKGKRRD SERF2 428 80.96 61.21 55.38 40.98 37.98 36.75 51.36 61.76 59.69 84.48 62.40 74.20 S1021 EQKKRSYsEPEKMNE SHROOM3 429 8.24 7.36 8.78 5.63 4.31 5.49 6.46 5.74 5.80 6.30 7.29 7.26 S1441 AHAAREDsLPEESSA SHROOM3 430 35.27 38.01 27.50 25.10 22.38 18.01 30.78 31.39 27.17 28.09 28.52 36.29 T148 LPPPLIPtPPPDDPR SNX15 431 2.57 1.02 2.41 1.02 1.03 0.91 0.94 2.19 0.92 2.17 4.40 2.27 S136 SSNNGSAsPTKTKSG SP4 432 5.02 7.36 7.91 4.20 4.52 4.30 7.30 6.24 5.71 7.75 5.79 4.33 S13 FRRILRLsTFEKRKS STK10 433 34.75 29.94 22.38 21.00 17.45 14.17 36.77 29.87 33.07 34.70 33.13 34.88 S707 LFREMEKsFDEQNVP SVIL 434 63.33 58.55 48.14 42.31 35.42 34.74 67.55 51.89 50.75 65.99 56.93 68.03 S509 SAARKMPsKSLEDIS SYTL2 435 47.62 32.70 26.92 18.34 16.43 14.90 36.11 30.71 33.62 44.51 33.66 31.96 S222 KTLVKSLsTDTSRQE TEX2 436 78.90 71.43 51.61 53.69 35.62 37.12 64.84 54.67 57.29 67.03 62.08 84.93 S263 DPSSPRAsPAHSPRE TLE1 437 9.65 10.93 10.13 6.56 4.93 6.49 8.33 7.93 9.21 7.02 10.51 10.29 S267 PRASPAHsPRENGID TLE1 438 7.08 9.91 8.01 5.53 3.90 5.58 6.17 5.99 8.29 4.96 6.65 9.21 S24 VKYSRLSsTDDGYID TMEM230 439 23.81 22.89 17.17 14.86 14.99 11.52 26.01 22.70 19.80 24.68 21.98 23.07 S46 RHGVLRHsVDLIGRP TRMT61A 440 5.28 5.11 5.02 2.56 3.90 2.93 5.05 3.80 5.99 4.44 5.47 4.66 S29 LPKDYLLsESEDEGD UTP14A 441 4.12 4.91 6.27 4.00 2.57 2.56 5.33 4.39 4.42 4.75 5.90 3.79 S31 KDYLLSEsEDEGDND UTP14A 442 4.12 4.91 6.27 4.00 2.57 2.56 5.33 4.39 4.42 4.75 5.90 3.79 S158 PIVSKSLsSSLDDTE VAPB 443 9.91 8.99 10.23 3.48 4.62 7.13 6.36 8.27 9.12 4.23 7.18 7.91 S284 QALPGDLsGLFPRLR VASN 444 13.13 12.16 12.73 8.30 7.08 8.14 11.70 9.87 11.70 14.05 8.26 12.57

[0372]

[0373] S480 RGQAEEEsPSQEETV ZBTB37 445 5.15 6.34 5.02 2.87 3.80 3.66 4.40 3.54 3.13 4.44 3.22 6.50

[0374]

[0375] Attorney Docket No. 07039-2296WO1 / 2023-600

[0376] Given the interest in ENDX dose-dependent effects and the mechanistic basis for induction of apoptosis at 5 pM ENDX, cluster 1 was focused on. KEGG pathway enrichment analysis using DAVID, an online gene functional annotation tool, identified viral carcinogenesis, systemic lupus erythematosus, phagosome, PI3K-AKT signaling pathway and gap junction as the top five biological pathways impacted by ENDX (Figure 18B).

[0377] ENDX downregulated phosphosites are enriched for PKCfl, CDK1 andAKTl target sequences

[0378] It was postulated that the observed ENDX effects on cluster 1 phosphosites were due to effects on kinase mediators of these phosphorylation events. To identify these kinases, NetworKIN and RoKAI kinase prediction tools were used. Using the 325 phosphosites from cluster 1 as an input, these two tools collectively identified protein kinase C beta (PKC ) and cyclin-dependent kinase 1 (CDK1) followed by AKT1 and PKC0 / PKCa as the top five most frequently predicted kinases involved (Figure 18C; Table 4). Further, motif enrichment analysis identified that RXXpS, pS / pTP and pSXXE as prevalent motifs in the regulated cluster 1 phosphosites. These motifs mapped to the AKT, MAPK / CDK and CK2 kinase substrate motifs, respectively (Figure 18D). Attorney Docket No. 07039-2296WO1 / 2023-600

[0379] Table 4.

[0380] NETWORKIN:

[0381] 1 metphorest_gmetphore»t_s:

[0382] ^substrates position id (kinase) networkin_score;roup >core:string_score sequence iCALR_HUMAN 80 PKCepsilon 4.065470903 iPKCjroup i 0.137074 io.8 YALSAsFEPFS iSRRMZJIUMAN j1824 CLK1 2.875628747 iCLKjroup 50.362799 10.518 GYHSRsPARQE OCP1A_HUMAN 315 MAPK1 2 19639563! MAPK3_MAP!0310604 '0400591 YTIPLsPVLSP tJURPJdUMAN 185 CDK1 6.421757393! CDK2_CDK3jo.109025 >0784 LPSLAsPAVPA 1CHMP5J4UMAN 86 PKCbeta 2.140616152 iPKC_group >0.21809 '0.17949 NLAQQsFNMEQ > LTBP1_HUMAN 1616 TGFbR2 2.299561278 iACTR2jACTfi0.063905 iO 599 DRFLNsFEELQ > IKZF1_HUMAN 258 PKCbeta 249482! PKC_group I0337303 io 355444 LCKIGsERSLV ACTBJ1UMAN 202 CKtalpha 6.51385129 'CKIjroup:0.091339 iO.577 GYSFTtTAERE ACT B_H OMAN 199 CaMKJIalpha 2.633651739 iCaMKII_group(0.136666 >0.429647 TERGYsFTTTA ACTBJ4UMAN 199 PKCeta 4.6791866 iPKCjroup >0.105486!08 TERGYsFTTTA ACTBJtJMAN 199 PKCtheta 4.6791866 lPKC_group J0 105486 ' 08 TERGYsFTTTA ACTBJtJMAN 199 PKCdeita 2.463258217 iPKC^group >0.105486 >0918 TERGYsFTTTA ACT B_H UM AN 199 PKCalpha 2.416426046 'pKCjroup A 105486 jO.88 TERGYsFTTTA ACTBJ4UMAN 199 PKCgamma 4.6791866 jPKCjroup i0.105486 10.8 TERGYsFTTTA ACTB_HUMAN 52 PKCeta 3.720939899! PKC_group >007651!08 MGQKDsYVGDE ACTBJ4UMAN 52 PKCtheta 3.720939899 iPKCjroup >0.07651 >08 MGQKDsYVGDE ACT B_H OMAN 52 PKCdeita 2.288892878 jPKCjroup >0.07651 '0.918 MGQKDsYVGDE ACTBJ4UMAN 52 PKCgamma 3.720939899 iPKCTgroup >0.07651 '0.8 MGQKDsYVGOE 'NDE1_HUMAN 282 CDK1 2373356046! CDK2_CDK3J0265051 I0979 LVYDQsPNRTG LEO 1J1UMAM 140 CK2alptia 3.070586115! CK2_group?0.472848 iO 382 SEKAHsDDEKW H2BFSJHUMAN 56 PKCbeta 2.49482 iPKCTgroup >0.27139 >0.0948168 PDTGIsSKAMG iHMGBVHUMAN 121 CK1 alpha 3.716011139 iCKtjgroup >0.080802 I0584 EHPGLsIGDVA > GTSE INHUMAN 466 CDK1 7^897446404! CDK2jCOK3j0.303688 '0.485 SPDSStPKLSR ELNAJtJMAN 962 MAP2K4 3.895767008? MAP2K_grou{i0.08979 '0918 PKSPFsVAVSP ELNA_HUMAN 962 PAK1 7.594150906 > PAK_group 0.138624 >0.815 PKSPFsVAVSP IFLNAJIUMAN 962 PKCtheta 3.712592031 iPKCTgroup 50.071264 10.914 PKSPFsVAVSP 'HSP81_HUMAN 98 OMPK1 5.820349691 IBM PKjroup >0 176467 ' 0864 DRWRVsLDVNH SCRIB_HUMAN 1630 GRK2 3.629764457 iGRKjgroup >0 116805 >0447223 LGPVPs - ALDOAJUJMAN 132 PKCbeta 2.401351502 IPKCTgroup >0.233593 '0.277559 GLDGLsERCAQ TRAP CTHU MAN 393 TGF6R2 2.770734565 ACTR2jACTH0.064374 10.741 RGVVDsEDIPL IKRT84J-IUMAN 178 PKCalpha 2.150968509! PKC_group J0.358835 10173082 NNKFAsFIOKV

[0383]

[0384] lKRT84_HUMAN 178 PKCbeta 249482 iPKCTgroup I0358835 '0152893 NNKFAsFIDKV ROKAI;

[0385] KWD KinName 'KinGene SubsPrctein iSubsGene ^Position Rankinfl iQuanSicaton ZScore PValue FDR i P31749 AKt1: AKT1 P02545 ’LMNA iS301 QSFUR! DSLSAQLSQ U0.47 -1.732 0.0833 0.0835 1. P31749 AKJ1 iAKTI P06899 IH2SC11; S37 RKRSR^SlyV>^ M TO -6.597K42le-nKt37e-9 s; Q6P2M8 CAMK18 iPHCK P07900 iHSP9GAA1 |S391 HfGWDsEDLPLNI i-1.09 <017 0.000059 0.000347!.015530 PDK1 1POPK1 P08559 5PQHA1 IS232 NRyGMGtsVERAAAs i-064 -2.359 00183 0.0204 i. Q151 IS PDHK1 iPOKI P08559 iPDHAI |S232: NRyGMGtsVEPAAAS i-O.&l -2.359 00183 0.0204 }.015119 PDHK2 1PDK2 PG8559 5PQHA1 iS232 NRyGMGtsVERAAAS 1-064 -2.359 00183 0.0204 i; QI 6654 PDHK4 iPDK4 P08559; PDHA1! S232 NRyGMGtsVERAAAS i-G.64 -2.359 00183 0.0204 }. P49841 GSK3B 1GSK3B P22670 SRFX1 >3120 SETVsEAsPGStAsQ i-071 -2.616 000888 0.0118 i P17252 PKCA iPRKCA P23528; CFL1! S23 NDMKVRKsstPEEVK 5-0.94 -3.464 0000532 000113 i ^043318 TAK1 1MAP3K7 P68104 ’EEF1A1; T261 yMGGGWPVGTVE i-0.83 -3.059 0.00222 0.00355 j. P17612 PKACA iPRKACA Q14289 IPTK2S; S778 HNVFKRHSMREEQH i-113 <164 00000312 0.000209 s ^31749 AKt1 lAKH Q71D13 ’H3C14 JT46 PHiyjPGtVAL-ElR U1.24 <57 0.00000489 o.oooow i P45983 JJiKI ISO21MAPKS Q9NPI6 iDCPIA; S315 PlytPLsPVLsPtL i-lM -3.833 0000127 0.000472 s

[0386]

[0387] P06493 CDK1 iCDKI Q^XRI; NDE1; S282 RMLVyDQsPNRiGGP j-0.65 -2.395 0.0166 00188 1 Attorney Docket No. 07039-2296WO1 / 2023-600

[0388] ENDX at 5 uM downregulates AKT^er473phosphorylation in ERa I breast cancer cells Pathway analysis studies identified PI3K-AKT signaling as one of the top biological pathways targeted by ENDX (Figure 18B), with AKT1 frequently predicted as atop kinase for phosphosites downregulated by ENDX (Figure 18C). Therefore, the effect of ENDX on AKTSer473phosphorylation was examined in estrogen deprived MCF7AC1 cells. Immunoblot assays revealed that ENDX at 5 pM, but not at 0.01 and 0.1 pM, atenuated AKTSer473phosphorylation compared to vehicle treated cells (Figure 19A). Given that phosphorylation of AKT is initiated at Thr308 followed by phosphorylation at Ser473 for full AKT activation (Cicenas, Int. J. Biol. Markers, 23:1-9 (2008)), the effects of ENDX on AKT™08phosphorylation were also evaluated. While 0.01 and 0.1 pM concentrations of ENDX stimulated KT™08phosphorylation, ENDX at 5 pM did not alter AKT1111'308phosphorylation under estrogen deprived conditions (Figure 19A). Because commercially available phosphosite specific antibodies for predicted AKT-mediated phosphorylations in cluster 1 were limited, to an alternate approach where we evaluated ENDX effects on the levels of total phospho-AKT substrates was used. A commercially available antibody that specifically recognizes the RXXpS / pT AKT motif in AKT substrates was used to determine whether downregulation of AKTSer473phosphorylation by 5 pM ENDX had a global impact on the phosphorylation of AKT substrates. Consistent with reduced AKTSer473phosphorylation, ENDX at 5 pM, but not 0.01 and 0.1 pM, also reduced the phosphorylation of AKT substrates compared to vehicle treated estrogen deprived MCF7AC1 cells (Figure 19A; arrows). The effects of ENDX on AKTSer473and AKT™08phosphorylations were next evaluated in the endocrine-sensitive MCF7AC1 xenograft model in vivo. Treatment of mice harboring MCF7AC1 tumors with high-dose ENDX (75 mg / kg) but not low-dose ENDX (25 mg / kg), TAM or letrozole, atenuated AKTSer473phosphorylation and reduced the levels of AKT phosphorylated substrates but had no appreciable impact on AKT11"’308phosphorylation compared to control treatment (Figure 25A). These data support a dose and concentration dependent effect of ENDX on AKT signaling both in vitro and in vivo.

[0389] Next, it was examined whether ENDX can block ligand stimulated AKTSer473phosphorylation. Insulin, a known activator of AKTSer473phosphorylation, robustly stimulated AKTSer473phosphorylation in serum starved MCF7AC1 cells. Pretreatment with Attorney Docket No. 07039-2296WO1 / 2023-600

[0390] ENDX at 5 pM, but not at 0.01 and 0.1 pM, for two hours prior to insulin stimulation blocked AKTSer473phosphorylation (Figure 19B). Insulin treatment also stimulated AKT'11"'308phosphorylation, albeit modestly compared to AKTSer473phosphorylation in serum starved MCF7AC1 cells (Figure 19B). Interestingly, pretreatment with ENDX at 5 pM was also able to block AKT™08phosphorylation (Figure 19B). Additionally, ENDX at 5 pM also diminished insulin-stimulated phosphorylation of AKT substrates (Figure 19C). In contrast, treatment with clinically attainable concentrations of 0.1 pM TAM or 0.1 pM ICI, failed to inhibit insulin stimulated AKTSer473and AKT™08phosphorylations (Figure 19B), an effect also observed with ERa-targeting 0.1 pM concentration of ENDX. The ability of ENDX at 5 pM, but not at 0.01 or 0.10 pM, to block insulin-stimulated AKTSer473phosphorylation was also observed in the ERa+ / HER2- T47D breast cancer cells under serum starved conditions, with TAM and ICI again failing to block insulin-stimulated AKTScr473phosphorylation (Figure 25B). However, contrary to the observation in serum starved MCF7AC1 cells (Figure 19B), insulin induced stimulation of AKT™08phosphorylation was not blocked by ENDX at 5 pM in serum starved T47D cells (Figure 25B). Collectively, these findings suggest that ENDX attenuates AKT signaling primarily through attenuation of AKTSer473phosphorylation in ERa+ breast cancer cells at clinically relevant 5 pM concentration, a unique effect not observed with other SERM’s at clinically relevant concentrations.

[0391] ENDX inhibits PKC / 31 kinase activity and binds to PKC l

[0392] To examiner wheter ENDX might mediate its effects on AKT through PKC01, it was first sought to evaluate ENDX effects on PKC i kinase activity. To this end, the concentration-dependent effects of ENDX on a kinase panel composed of 12 PKC isoforms were evaluated, since multiple PKC family members including PKCp were identified in the kinase prediction analysis (Figure 18C). While ENDX inhibited the kinase activity of PKC01 with an IC50 concentration of 360 nM (Figure 19D), ENDX did not inhibit other PKC family members as potently (Table 5). TAM, a known PKC inhibitor, also inhibited PKCβI kinase activity, but at higher concentrations (IC50 = 4.9 pM) (Figure 19E), a concentration not achievable with the 20 mg / day dose. These findings suggest that ENDX may inhibit PKC i kinase activity in vitro. Attorney Docket No. 07039-2296WO1 / 2023-600

[0393] Table 5.

[0394] PKC family kinase Symbol ENDX IC50* (M) Staurosporine IC50 (M) PKC alpha PKC a >5.00E-05 <1.00E-09 PKC beta 2 PKCb2 4.33E-05 <1.00E-09 PKC delta PKCd n / a* <1.00E-09 PKC epsilon PKCe n / a* <1.00E-09 PKC eta PKCh >5.00E-05 <1.00E-09 PKC gamma PKCg 4.97E-05 1.65E-09 PKC iota PKCi >5.00E-05 1.84E-08 PKC mu PKCm / PRKDl 3.16E-05 1.24E-09 PKC nu PKCn / PRKD3 2.64E-05 C1.00E-09 PKC thetha PKCq 4.18E-05 1.54E-09

[0395]

[0396] PKC zeta PKCz 4.81E-05 7.72E-08

[0397] It was next determined whether ENDX directly bound PKC01. By employing surface plasmon resonance (SPR), a widely used method for assessing protein-ligand interactions and using a wide range of ENDX concentrations (100 - 8000 nM), we demonstrate that ENDX binds PKCβI (Figures 26A and 26B). Taken together, these findings establish PKCβI as a potential ENDX substrate.

[0398] ENDX downregulates AKl^er473phosphorylation through PKC [31 inhibition in ERa breast cancer cells

[0399] To determine the role of PKCβI in mediating ENDX effects on AKTSer473phosphorylation, it was first assessed whether PKC i activation can impact AKTSer473phosphorylation. In serum starved MCF7AC1 cells, the PKC agonist phorbol myristyl acetate (PMA) stimulated PKCβISer661auto-phosphorylation and AKTSer473phosphorylation, which was associated with increased levels of AKT substrate phosphorylation (Figure 20A). It was next evaluated the effects of ENDX on PKCβI under PMA-stimulated conditions. Pretreatment with ENDX at 0.01, 0.1 and 5 |_iM had either no (0.01, 0.1 pM) or minimal (5 M) effect to block PMA-stimulated PKCβISer661phosphorylation, respectively. In contrast, only ENDX 5 pM robustly reduced PKCβI total protein levels, which correlated with reduced AKTSer473phosphorylation and AKT substrate phosphorylation (Figure 20B). In contrast, while treatment with the potent and selective ATP competitive PKCP kinase Attorney Docket No. 07039-2296WO1 / 2023-600

[0400] inhibitor enzastaurin reduced PKCβISer661phosphorylation, it neither impacted the expression of PKCβI nor downregulated AKTSer473phosphorylation (Figure 20C). Further, in insulin treated MCF7AC1 cells, ENDX pretreatment also reduced PKCβI total protein levels, with no effects on PKCβIScr661phosphorylation. In contrast, both TAM and ICI pretreatments failed to diminish PKCβI total protein expression in insulin treated MCF7AC1 cells (Figure 20D), which correlated with the lack of attenuation of AKTSer473phosphorylation (Figure 19B).

[0401] Given that ENDX robustly blocked PMA- and insulin-stimulated AKTSer473phosphorylation and additionally targeted PKCβI for degradation (Figure 19B, Figure 20B), it was sought to determine the effects of downregulating PKCβI protein expression on AKTSer473phosphorylation using three different approaches to silence PKCβI expression in MCF7AC1 cells. In the first approach, a commercially available siRNA that targets an mRNA sequence common to both PKCβI and PKCP2 isoforms (siPKCP) was used (Figure 20E). In the second, a custom designed siRNA from Dharmacon that specifically targets nucleotides 2049-2067 of the PKCβI mRNA, a target sequence that is unique and distinct from PKCP2, was used (Figure 27A). In the third approach, a doxycycline (dox)-inducible SMART vector inducible human PRKCB mCMV-TurboGFP shRNA for PRKCB gene silencing (shPKCβIdox) was utilized (Figure 27B). Of these approaches, siPKCP resulted in the greatest reduction in PKCβI protein levels (Figure 20E). Accordingly, it was sought to assess the biological effects of PKCβI knockdown using siPKCp. Even though PKCb2 is theoretically targeted by this reagent, PKCP2 protein expression was undetectable in MCF7AC1 cells (Figure 27C), indicating that effects on expression of PKCb2 are unlikely to contribute to effects of siPKCp. Reduction in PKCβI protein expression by siPKCp resulted in a 51% reduction in AKTSer473phosphorylation levels compared to non-targeting (siNT) control at 48 hours (Figure 20E). PKCP siRNA did not affect the expression of other PKC family members (Figure 27D), suggesting that the decrease in AKTSer473phosphorylation was due to PKCβI alone. Downregulation of PKCβI protein levels also significantly inhibited growth of MCF7AC1 cells (Figure 20F). Thus, PKCb siRNA recapitulated both the signaling and growth inhibitory effects of ENDX. Attorney Docket No. 07039-2296WO1 / 2023-600

[0402] To determine whether the observed effects of ENDX on PKCβI degradation and AKTSer473phosphorylation inhibition are dependent on the presence of ERa, ENDX effects on PKCβI degradation and AKTSer473phosphorylation in the ER negative (ER-) MDAMB231 breast cancer cells and nonbreast HEK293F cells, a human embryonic kidney cell line, both of which express higher amounts of PKCβI compared to ER+ MCF7AC1 cells, were additionally evaluated (Figure 28A). Pretreatment with 5 pM ENDX for two hours followed by treatment with 100 nM insulin for one hour did not impact PKCβI protein levels in either cell line (Figure 28B). While insulin treatment induced AKTSer473phosphorylation in ER- cells, pretreatment with ENDX did not inhibit this phosphorylation (Figure 28B). To determine whether the addition of ERa to the MDAMB231 cell line could facilitate ENDX effects on PKCβI, doxycycline (dox)-induced ERa protein expression was performed in MDAMB231 cells and ENDX effects on PKCβI were evaluated using the above-mentioned experimental conditions. While forced expression of ERa in dox-induced cells modestly decreased PKCβI protein levels compared to nondox-induced cells, ENDX pretreatment displayed no impact on PKCβI protein expression in the presence of ERa (Figure 28C). ERa overexpression also resulted in increased AKTSer473phosphorylation that remained unaffected by ENDX pretreatment in these cells. Additionally, MDAMB231 and HEK293F cells were pretreated with or without 5 pM ENDX for two hours followed by 0 or 200 nM PMA treatment for 20 minutes and effects on PKCβI were evaluated. Treatment with PMA had minimal effects on PKCβI phosphorylation and ENDX pretreatment in the presence of PMA displayed no impact on PKCβI protein expression (Figure 28D). Taken together, these data demonstrate that in ER- cells, while AKT signaling may be further activated by insulin, PMA does not result in meaningful activation of PKCβI, suggesting that activation of AKT signaling in ER- cells may not be mediated through PKCβI nor blocked by ENDX.

[0403] ENDX at 5 pM replicates apoptotic effects of the pan-AKT inhibitor MK-2206 in estrogen deprived ERa+ breast cancer cells

[0404] To assess the impact of AKT inhibition on estrogen deprived MCF7AC1 cells, MK-2206 was administered at a variety of concentrations. These studies showed that growth was Attorney Docket No. 07039-2296WO1 / 2023-600

[0405] inhibited at MK-2206 concentrations > 0.1 pM, AKTSer473phosphorylation was reduced at MK-2206 concentrations > 1 pM, and apoptosis as manifested by both annexin V binding and caspase-mediated PARP1 cleavage was induced at 5 pM MK-2206 (Figure 21A, Figures 29A-29C). In these same cells, treatment with ENDX at 5 pM induced apoptosis (Figure 21 A). Lower (0.01 and 0.1 pM) and higher (5 pM) ENDX concentrations were also evaluated and it was demonstrated that only the 5 pM concentrations reduced AKTSer473phosphorylation and increased PARP cleavage (Figure 2 IB). Consistent with these in vitro finding, in the MCF7AC1 xenograft model, in vivo treatment with ENDX at 75 mg / kg but not at 25 mg / kg also increased PARP cleavage (Figure 29D).

[0406] It was next asked whether ENDX had similar effects on T47D cells. Unlike MCF7AC1 cells, parental T47D cells failed to proliferate in CSS medium (Figure 29E). Therefore, ENDX effects on the growth of the ERa+ / HER2- LTED T47D cell line model (T47D-LTED) that proliferates well in CSS medium were examined. Evaluation of basal protein expression revealed reduced ERoc levels and a modest decrease in AKTSer473phosphorylation in the T47D-LTED cell line compared to the parental T47D cell line (Figure 29F). As noted with MCF7AC1 cells, treatment of T47D-LTED cells with MK-2206 significantly inhibited growth starting at 0.1 pM (Figure 29G). At 1 and 5 pM MK-2206, but not 0.1 pM, reduced AKTSer473phosphorylation and increased apoptosis, as indicated by both annexin V staining and increased PARP cleavage were observed (Figures 29H and 291), phenocopying the reported biological effects of MK-2206. Treatment of T47D-LTED cells with 5 pM ENDX likewise attenuated AKTSer473phosphorylation, inhibited growth, and induced apoptosis as manifested by annexin V binding and PARP1 cleavage (Figures 21C and 2 ID, and Figure 29J), replicating the MCF7AC1 response. ENDX (5 pM) was then compared with clinically attainable concentrations of TAM and ICI (0.1 pM) in terms of their effects on apoptosis. Unlike ENDX, both TAM and ICI failed to induce apoptosis (Figure 30). Attorney Docket No. 07039-2296WO1 / 2023-600

[0407] Expression of constitutively active AKT attenuates ENDX-induced apoptosis in ERa \ breast cancer cells

[0408] To confirm the role of AKT inhibition in ENDX-induced apoptosis, a cumate inducible expression system was utilized to overexpress a C-terminally HA-tagged constitutively active AKT in MCF7AC1 cells (MCF7AClcaAKTcells). Immunoblot assays with an anti-HA antibody confirmed cumate induced expression of caAKT (Figure 22A) that was associated with increased phosphorylation of AKT substrates (Figure 22B, arrows). While ENDX at 5 pM induced apoptosis in the absence of cumate in this cell model, expression of caAKT significantly diminished the ability of ENDX to induce apoptosis (Figure 22C). Taken together, these findings established that ENDX not only inhibited proliferation, but at higher concentrations, induced apoptosis of ERa+ breast cancer cells, and this may occur in part through inhibition of PKCβI and the resulting decrease in AKT kinase activity.

[0409] Evaluation of ENDX dose response effects (0-10 pM) on apoptosis in MDAMB231 cells cultured in CSS medium showed that ENDX did not induce apoptosis in these cells until it reached the highest concentration of 10 pM (Figure 31 A). Concurrent studies performed to evaluate ENDX effects on growth of MDAMB231 cells at the above-mentioned concentrations, revealed that ENDX did not inhibit growth until it reached concentrations of > 7.5 pM (Figure 3 IB). Given the lack of MDAMB231 response to ENDX treatment, ENDX response in the ER- BT549 and MDAMB436 breast cancer cells was evaluated. Similar to MDAMB231 cells, ENDX did not inhibit growth of these cells until concentrations > 7.5 pM (Figure 3 IB). Immunoblot confirmed basal expression of PKCβI in all three cell lines (Figure 31C). Collectively, the lack of an effect of ENDX on apoptosis in ER- cells was consistent with the null findings regarding ENDX’s effects on AKT signaling in ER- cells, and suggested that unlike ER+ cells, ENDX did not elicit pharmacodynamic effects on apoptosis through PKCβI targeting. Attorney Docket No. 07039-2296WO1 / 2023-600

[0410] Methods

[0411] Cell culture

[0412] MCF7 human breast cancer cells stably transfected with the aromatase gene (MCF7AC1) were cultured in phenol-red free IMEM medium (Gibco #A10488-01) supplemented with 10% fetal bovine serum (FBS) (Gemini #900-108), 600 pg / mL geneticin (G418) (Gibco #10131-027) and 1% Antibiotic-Antimycotic (AA) (Gibco #15240-062). To maintain an estrogen deprived state, MCF7AC1 cells were cultured in IMEM medium containing 10% charcoal-stripped serum (CSS) (Hyclone #SH30068), 600 pg / mL G418 and 1% AA. T47D cells were cultured in DMEM / F12 medium (Coming #16-405-V) containing 10% FBS and 1% AA. T47D-1 ong-term estrogen deprived (LTED) cells were cultured in DMEM / F12 medium containing 10% CSS and 1% AA. C-terminally hemagglutinin (HA)-tagged, catalytically active AKT expressing MCF7AC1 (MCF7AClcaAKT) cells were cultured in IMEM containing 10% FBS, 600 pg / mL G418, 1% AA and 0.5 pg / mL puromycin (Gibco #A11138-03). The ER- MDAMB231, BT549 and MDAMB436 breast cancer cells were cultured in DMEM / F12 medium containing 10% FBS and 1% AA. The HEK293F cells were cultured in DMEM medium (Corning #34722014) containing 10% FBS and IX penicillinstreptomycin (Sigma #P0781). The doxycycline-inducible ERa-expressing MDAMB231 cell line was established using the T-REx™ system (InVitrogen) and were maintained in DMEM / F12 medium containing 10% FBS 1% AA, 5 mg / L Blasticidin S (Sigma #15205) and 500 mg / L Zeocin (InVivoGen #ant-zn-5b).

[0413] The Z-endoxifen hydrochloride utilized in this study was synthesized. Estrogen deprived MCF7AC1 were treated with vehicle control or ENDX (National Cancer Institute) for 24 hours. For the phorbol 12-myristate 13-acetate (PMA) (LC Laboratories, #P-1680) experiments, MCF7AC1 cells were maintained in serum-free medium for 24 hours prior to pretreatment with vehicle or ENDX for two hours followed by treatment with 20 or 200 nM PMA for 20 minutes. For the insulin (Sigma-Aldrich, #10516) experiments, MCF7AC1 and T47D cells were maintained in serum-free medium for 24 hours prior to pretreatment with vehicle control or drugs for two hours followed by treatment with 100 nM insulin for one hour. Attorney Docket No. 07039-2296WO1 / 2023-600

[0414] Proliferation assay

[0415] Cells were plated at a density of 2000 cells per well. Cell viability of (i) vehicle or drug treated MCF7AC1 and T47D-LTED cells in CSS medium, (ii) siNT or siPKCP-transfected MCF7AC1 cells in CSS medium, and (iii) T47D cells in FBS versus CSS medium were analyzed by crystal violet staining assay after six days of treatment or siRNA transfection. Cell viability was calculated as the average absorbance of the drug treated cells divided by the average absorbance of the vehicle treated cells x 100.

[0416] Apoptosis assay

[0417] MCF7AC1 and T47D-LTED cells were plated at a density of 2000 cells per well in CSS medium for 24 hours. Cells were then co-treated for 48 hours with vehicle or drug, IncuCyte Annexin V green reagent (#4642, 1:300), an early-stage apoptosis marker, and IncuCyte NucLight rapid red reagent (#4717, 1:500), a dye that stains all cell nuclei red, in CSS medium. MCF7AClcaAKTcells were plated at a cell density of 2000 cells per well in CSS medium in the absence or presence of cumate for 48 hours and then co-treated with vehicle or drug, Annexin V green and NucLight rapid red reagents in the absence or presence of cumate for an additional 48 hours in CSS medium. The apoptosis graphs are presented as the green object count (which correspond to cells that are stained with the IncuCyte green fluorescence Annexin V reagent) divided by the red object count (which correspond to the total number of cells in the culture that are stained with the IncuCyte red fluorescence Nuclight Rapid Red Cell Labeling reagent that labels the nucleus of all cells without perturbing cell function or biology) and displayed as percentage using the IncuCyte S3 analysis software.

[0418] Protein sample preparation for mass spectrometry-based quantitative proteomics analysis (i). Cell lysis and in-solution trypsin digestion. Following treatment of MCF7AC1 cells with vehicle control or 0.01, 0.1, or 5 pM ENDX for 24 hours in CSS media, cells were harvested and lysed in 8 M urea buffer (8 M urea, 20 mM HEPES pH 8.0, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM P-glycerophosphate, and 5 mM sodium fluoride), followed by sonication, and centrifugation at 15,000 x g at 4°C for 20 minutes to clear cell debris. BCA Protein Assay was used to measure the protein Attorney Docket No. 07039-2296WO1 / 2023-600

[0419] concentration. 2 mg of protein lysates from each treatment condition was used for digestion with trypsin. Briefly, the protein lysates were reduced with 5 mM dithiothreitol at 37°C for 1 hour and alkylated with 10 mM iodoacetamide at room temperature in dark for 30 minutes. The protein lysates were then diluted in 20 mM HEPES pH 8.0 to a final concentration < 2 M urea and digested with TPCK-treated trypsin (Worthington Biochemical Corp. Lakewood, NJ) overnight at room temperature. Digested peptides were acidified with 20% trifluoroacetic acid (TFA) to a final concentration of 1% TFA. The tryptic peptides were desalted using SepPak Cis cartridge (Waters Corporation, Milford, MA). Eluted peptides were lyophilized and stored at -80°C prior to TMT labeling.

[0420] (ii). Tandem Mass Tag (TMT) labeling of peptides and basic reversed-phase liquid chromatography (bRPLC) fractionation. The lyophilized tryptic peptides were reconstituted in 150 pL 100 mM tri ethylammonium bicarbonate (TEABC) and measured with peptide BCA assay (Thermo Scientific). 1 mg peptides from each sample in a final volume of 100 uL 1 mM TEABC were mixed with 1 mg pg TMTpro reagent that was dissolved in 20 pL anhydrous acetonitrile. After 1 hour incubation at RT, 10 pL of 5% hydroxylamine was added and incubated for 15 minutes at room temperature to quench the labeling reaction. Peptides labeled by different TMT reagents were then mixed and dried with Speed-Vac.

[0421] The dried TMT -labeled peptides were reconstituted in 20 mM ammonium formate and fractionated by high-pH reversed-phase liquid chromatography on Dionex Ultimate 3000 (Thermo Scientific). Peptides (12 mg) were separated on a 4.6 mm x 50 cm x 3.5 pm Xbridge column (Waters) with a 2-hour gradient from 2 to 40% mobile phase B (MPB). Mobile phase A was composed of 20 mM ammonium formate in water, and MPB was composed of 20 mM ammonium formate in 80% acetonitrile. A total of 96 fractions were collected and concatenated into 24 fractions. A 20-pg equivalent of each fraction was set aside for global proteome analysis, and the rest of each sample was concentrated into 12 fractions and dried before phosphopeptide enrichment.

[0422] (iii). Phosphopeptide enrichment. Each fraction was reconstituted in 1 mL of 80% acetonitrile in 0.1% TFA. Phosphopeptides were enriched using an immobilized metal affinity chromatography (IMAC) approach. In brief, nickel-nitrilotriacetic (Ni-NTA) Attorney Docket No. 07039-2296WO1 / 2023-600

[0423] superflow agarose beads were stripped of nickel with 100 mM EDTA, incubated with 10 mM FeCE solution and equilibrated in 80% ACN / 0.1%TFA. 10 pL IMAC beads were mixed with each fractionated peptide in 80% acetonitrile / 0.1% TFA and rotated for 30 minutes at room temperature. Subsequently, incubated IMAC beads were washed with 500 pL 80% ACN / 0.1%TFA four times and 500 pL 0.1% FA one time. Phosphopeptides were eluted from IMAC beads with 200 pL of 500 mM dibasic sodium phosphate (pH 7.0) for three times. The eluted phosphopeptides were desalted with Cl 8 Stage Tips and Speed-Vac dried.

[0424] (iv). LC-MS / MS analysis. The peptide fractions were loaded on a 2 cm trap column (Acclaim PepMap 100, Cis, 5 pm particle size, 100 pm i.d. 100 A pore size, Thermo Scientific, San Jose, CA) using 0.1% formic acid with a flow rate 20 pL / minute for 4 minutes. The peptides were separated on a 50 cm analytical column (Acclaim PepMap 100, Cis, 2 pm particle size, 75 pm i.d. 100 A pore size, Thermo Scientific, San Jose, CA) with a 135 minute gradient from 3% to 40% acetonitrile in 0.1% formic acid at a flow rate of 0.3 pL / minute. The spray voltage was set to 2.3 kV while capillary temperature was set to 275°C. The samples were analyzed on an Orbitrap Fusio Lumos mass spectrometer (Thermo Scientific, Bremen, Germany). The MS instrument was operated in data-dependent acquisition mode. A survey full scan MS (from 350-1,500 m / z) was acquired in the Orbitrap with resolution 120,000 at m / z 200 with a maximum AGC target value of 800,000 ions. The data-dependent MS / MS was carried out using Top Speed method with a duty cycle of 2 seconds. Singly charged precursor ions were excluded while precursor ions with charge states 2-7 were sequentially isolated and fragmented in the higher-energy collisional dissociation (HCD) cell using 34% normalized collision energy (NCE). The maximum ion injection time for MS and MS / MS were set to 50 ms. Fragment ion spectra were detected in Orbitrap mass analyzer with a resolution 30,000 atrn z 200. Dynamic exclusion was enabled one event of fragmentation followed by exclusion of the precursor for next 45 seconds within 7 ppm of the selected m / z. For all measurements with the Orbitrap detector, a lock-mass ion from ambient air (m z 445.120025) was used for internal calibration. Attorney Docket No. 07039-2296WO1 / 2023-600

[0425] Mass spectrometry data analysis

[0426] Proteome Discoverer software suite (v 2.5; Thermo Fisher Scientific, San Jose, CA) was used for quantitation and database searches. The MS / MS data were searched using the SEQUEST search algorithm against a Human Uniport protein database supplemented with frequently observed contaminants. Search parameters included trypsin as a protease with full specificity and a maximum of two allowed missed cleavages; carbamidomethylation of cysteine and TMTpro tag (+304.207 Da) on lysine residues or peptide N-terminus as a fixed modification; oxidation at methionine and phosphorylation at serine / threonine / tyrosine as variable modifications. The precursor tolerance was set at 10 ppm, while the fragment match tolerance was set to 0.02 Da. The PSMs, peptides and proteins were filtered at 1% false discovery rate cut-off calculated using target-decoy database searches. The probability of an identified phosphorylation of specific Ser / Thr / Tyr residue on each identified phosphopeptide was determined from the PhosphoRS algorithm.

[0427] Phosphoproteome data analysis

[0428] The intensities of TMT reporter ions were normalized based on the average total phosphopeptide intensity detected in each TMT-labeling channel. Differentially phosphorylated sites were identified with an empirical Bayesian moderated t-statistics test as implemented in the R limma package. Multiple comparison correction was performed with Benjamini -Hochberg procedure. Phosphorylation sites with log2 fold change > 1.5 and unadjusted p-value < 0.05 were selected for downstream analysis.

[0429] DAVID, an integrated online functional annotation tool, was used to annotate the functions of the differentially modulated phosphoproteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) database was selected to identify enriched signaling pathways. The ggplot package in R was used to generate the bubble plot depicting the enriched pathways. The Fuzzy C-means clustering showing the dynamic regulation patterns of phosphosites were generated using ggplot and mfuzz packages in R. Kinase substrate enrichment analysis (KSEA), PhosphoSitePlus, NetworKIN, and RoKAI datasets were used to predict upstream kinases of regulated phosphosites, as described below. To identify the motifs enriched in the ENDX-regulated phosphosites, MoMo program with motif-x algorithm were used. Attorney Docket No. 07039-2296WO1 / 2023-600

[0430] Upstream kinase prediction analysis

[0431] The 325 phosphosites in Cluster 1 was provided as an input in the NetworKIN and RoKAI kinase prediction tools. While NetworKIN predicted upstream kinases for 32 (10%) of the 325 phosphosites, RoKAI predicted upstream kinases for 14 (4%) of the 325 phosphosites. For the remaining 279 phosphosites no upstream kinase predictions were provided by these prediction tools. Taken together, both NetworKIN and RoKAI predicted a total of 46 upstream kinases for only 14% of the phosphosites in Cluster 1 and these kinases were graphed in the X-axis of Figure 16A. The number of counts in the Y-axis refers to the total number of phosphosites substrates for which the given kinase is predicted as a potential upstream kinase. For example, PKCB is predicted as the upstream kinases for 5 phosphosites, CDK1 is predicted as the upstream kinase for 4 phosphosites and AKT1 is predicted as the upstream kinase for 3 phosphosites, respectively in Cluster 1.

[0432] Immunoblot analysis

[0433] Protein lysates were prepared using the RIPA lysis buffer system (ChemCruz #sc-24948) and quantified using the DC™ Protein Assay reagents (Bio-Rad #5000112). Equal amounts of protein lysates were separated on 10% Criterion gels (Bio-Rad #3450112), transferred to PVDF membranes (Bio-Rad #1620177), blocked in TBST-5% milk and probed with primary antibodies listed in Table 6, at the indicated dilutions. Membranes were incubated with HRP-conjugated anti-rabbit (CST #7074) or anti-mouse (CST #7076) secondary antibodies and visualized using chemiluminescent West Pico (Thermo Scientific #34580) or West Femto (Thermo Scientific #34096) reagents and a Li-Cor Odyssey® XF imager. Protein lysates from the MCF7AC1 xenograft model were obtained as described elsewhere (Jayaraman et al., Breast Cancer Res. 22:51 (2020)). Quantitation of the protein bands signal intensity was performed using the National Institute of Health (NIH) ImageJ image analysis software (imagej.nih.gov / ij / / / index.html). For the quantitation of the phospho protein levels, total protein levels were first normalized to β-actin loading control and these values were used to normalize the phospho protein levels and compared change in protein expression levels relative to vehicle control normalized to 1.0. For the quantitation of total protein levels, the total protein levels were normalized to β-actin and change in expression Attorney Docket No. 07039-2296WO1 / 2023-600

[0434] levels was compared relative to vehicle control normalized to 1.0. All blots were derived from the same experiment and processed in parallel.

[0435] Table 6.

[0436] Protein name {Primary Antibody Species [Vendor and Catalog # [Dilution phospho-AKTSer473[Rabbit [CST #9271 [ 1;1000 phospho-AKTThr308[Rabbit [CST#9275 [ 1:1000 AKT [Rabbit jCST #9272 j 1:1000 phospho-AKT substrate {Rabbit [CST *9614 [ 1:1000 pliospho-PKCβISc!fiM{Rabbit [Abeam #192184 [ 1:1000 ERα Mouse [SantaCruz #8002 [ 1:500 PKCPI {Rabbit [Abeam #136917 [ 1:1000 PKCβ2 Mouse SantaCruz #sc-13149 [ 1:200 cleaved PARP {Rabbit [CST #5625 [ 1:500 PARP [Rabbit [CST #9542 [ 1:1000 β-actin Mouse Sigma #A2228 1:20,000 HA-tag Rat Roche #11867423001 1:4,000

[0437]

[0438] ‘ ’

[0439] In vitro kinase assay

[0440] For the evaluation of ENDX effects on the kinase activity of the protein kinase C (PKC) family members and tamoxifen (TAM) effects on the kinase activity of PKC beta 1 (PKCβI), the drug compounds were tested in a 10-dose IC50 mode with three-fold serial dilution starting at 50 pM, in the presence of 10 pM ATP. Staurosporine, a broad-spectrum kinase inhibitor and a positive control, was also tested as described above. The IC50 concentrations of these drugs in inhibiting PKC kinase activity are provided in Table 5.

[0441] Affinity measurements by Surface Plasma Resonance (SPR)

[0442] Binding assays were performed at 25°C on a Biacore T200 biosensor (GE healthcare). Purified PKCβI protein were immobilized on a CM5 S sensor chip using amino coupling and immobilization buffer (10 mM HEPES, 150 mM NaCl, pH 7.4, P20 0.01% (w / w)) and acetate pH 5.0 at a flow rate of 10 pL / minute and reaching 10,000-12,000 resonance units (RUs). ENDX at concentrations ranging from 0- 8000 nM in phosphate buffer (Gibco) with 2% DMSO (v / v) and 0.01% (w / w) P20 were run over the chips at a flow rate of 50 pL / minute for 30 seconds. Binding kinetics were derived from sensograms using Biacore BIA evaluation software (GE). Sensograms were subtracted for background Attorney Docket No. 07039-2296WO1 / 2023-600

[0443] contributions, and affinity constants were derived using a steady state affinity fitting of a 1: 1 interaction model.

[0444] siRNA transfection

[0445] MCF7AC1 cells were maintained in CSS medium and transfected with non-targeting siRNA (siNT) (SI03650325) or a pool of two different siRNA’ s targeting total PKCp (Hs_PRKCpi_6 SI00605948 and Hs_PRKCpi_4 SI00042273) (siPKCP) (Qiagen) at 5 nM concentration in the presence of lipofectamine RNAiMAX transfection reagent (13778-075, Invitrogen) for up to 48 hours for IB analysis and for up to six days for proliferation assays in biological triplicates.

[0446] The custom made siPKCβI siRNA (sense: 5’-AAGCCAAAAGCUAGAGACAUU-3’ (SEQ IDNO:446); antisense: 5’-UGUCUCUAGCUUUUGGCUUUU-3’ (SEQ ID NO:447)) or the siGENOME non-Targeting siRNA Pool #1 (#D-001206-13-20) from Dharmacon were transfected into MCF7AC1 cells maintained in CSS medium at 40 nM concentration in the presence of DharmaFECT 1 (Dharmacon #T-2001-03) for up to 72 hours for IB assay.

[0447] A SMART vector inducible human PRKCB mCMV-TurboGFP lentiviral shRNA (Horizon Discovery #V3SH7675-01EG5579) was used for the generation of doxycycline (dox)-inducible PRKCB gene silencing. MCF7AC1 cells grown in 6-well plate in IMEM medium containing 10% FBS, 600 pg / mL G418 and 1% amino acids and at a confluency of 50% were infected with the lentivirus at a multiplicity of infection (MOI) of 5.0 in the presence of 1 pg / mL polybrene. After 48 hours, 10 mg / mL puromycin (Gibco #A11138-03) was added to cells at a dilution of 1 pL per 10 mL to allow for the selection of lentivirus-transfected cells. The expression of the shRNA sequence was induced in the presence of 1 pg / mL of doxycycline (Sigma-Aldrich #D3072) for 72 hours for IB assay. The target PRKCB gene sequence used is CAGTGTTGATGGCTGGTTT (SEQ ID NO:448; Horizon Discovery #V3IHSMCG_9696026).

[0448] Molecular cloning

[0449] Molecular cloning was performed for the generation of catalytically active AKT-expressing MCF7AC1 cells. For this purpose, vector pCDNA3.1+ containing an N-terminal Attorney Docket No. 07039-2296WO1 / 2023-600

[0450] SRCMyr signal AKT ORF and C-terminal hemagglutinin (HA)-tag was used. The constitutively active AKT (caAKT)-HA insert was excised using Nhel / EcoRV restriction digest, gel purified and cloned into the Nhel / / Pmel site of an SBI (System biosciences, Palo Alto, CA) vector with modified restriction sites. Sanger sequencing was performed by Azenta / Genewiz, (South Plainfield, NJ) to confirm in frame sequence. After viral transduction of the caAKT-HA construct into MCF7AC1 cells, cells were selected beginning 48 hours later for a mixed population (MCF7AClcaAKTcells) using puromycin (Invitrogen) 0.5 pg / mL for several weeks. Expression of caAKT-HA was induced by adding 60 pg / mL cumate (Sigma Aldrich 268402, 4-Isopropylbenzoic acid) for 48 hours prior to drug treatment.

[0451] Statistical analysis

[0452] Differences in cell proliferation in the drug treated MCF7AC1 and T47D-LTED cells compared to vehicle treated cells, the % of apoptosis in the drug treated MCF7AC1 and T47D-LTED cells compared to vehicle treated cells and the % of apoptosis in the vehicle or 5 pM ENDX treated MCF7AClcaAKTcells in the presence and absence of cumate were analyzed by one-way ANOVA. Differences in the % of PKCβI and AKTSer473protein levels remaining upon PKCβI knockdown in the siNT versus siPKCp or siPKCβI transfected MCF7AC1 cells and in the doxycycline-induced versus noninduced MCF7AC1 cells as well as differences in cell proliferation in the siNT versus siPKCp transfected MCF7AC1 cells were analyzed by one sample t-test. Comparison of proliferation rates of T47D cells cultured in FBS medium versus CSS medium were analyzed by unpaired t-test. All statistical analysis was performed in Graphpad Prism imaging software (Version 9). A p value of < 0.05 was considered statistically significant.

[0453] Example 3: ENDX and Immune Function in Patients with ER+ Breast Cancer

[0454] ENDX upregulates the expression of IFNy, IL-2 and Granzyme B expression by CD4+ and CD8+ T cells in vitro

[0455] In multiple ER+ cell lines, the ability of PKCβI to bind ENDX was linked to decreases in PKCβI protein levels and reduction in Akt activity, resulting in the induction of Attorney Docket No. 07039-2296WO1 / 2023-600

[0456] apoptosis (see, Example 2 and Jayaraman et al., NPJ Breast Cancer, 9(1): 101 (2023)). Given that PKCβI is highly expressed in immune cells, the potential impact of ENDX on immune function was investigated. Using peripheral blood mononuclear cells (PBMC) from apheresis cones derived from healthy premenopausal female donors, concentrations of ENDX (0.1 uM), tamoxifen (1 uM), and fulvestrant (0.1 uM) known to solely target the ER were evaluated. When using these concentrations, no effect on immune cell proliferation or function was observed. Based on data demonstrating that ENDX inhibits PKC 1 kinase and targets PKCβI protein for degradation at concentrations of > 1.5 uM, 2 pM ENDX concentrations and its effects on PBMCs were evaluated. As a control, the PBMCs were also treated with enzastaurin, a potent and selective serine / threonine kinase inhibitor of PKCβI previously studied in phase II metastatic BC setting which did not demonstrate sufficient antitumor activity for further development (Mina et al., Invest. New Drugs, 27(6): 565-570 (2009)). 5-day treatment of PBMCs with 2 pM ENDX significantly (p=0.02) increased IFNy expression compared to untreated or 2 pM enzastaurin-treated cells (Figure 32A). The expression of several cytokines by PBMCs following treatment with ENDX or enzastaurin was additionally assessed. The findings revealed that ENDX significantly induced the expression of GranzymeB and IL-2 in both CD4+ and CD8+ T cells. However, it had no impact on perforin expression (Figure 33). Conversely, enzastaurin treatment led to a reduction in the expression of these cytokines, suggesting distinct regulatory mechanisms (Figure 33).

[0457] Within the context of the tumor microenvironment, tumor-infiltrating lymphocytes (TILs) constitute a predominant source of IFNy secretion, thereby playing a pivotal role in tumor immune surveillance and cytotoxicity. While both pro- and anti-tumorigenic roles are attributed to IFNy, its gene expression profiles have been correlated with enhanced overall survival outcomes across distinct subtypes of BC (Figure 32B), most likely via distinct mechanisms. These in vitro data showing increased IFNy secretion in response to ENDX suggested the drug may exhibit an immune modulatory role in patients receiving this drug. Attorney Docket No. 07039-2296WO1 / 2023-600

[0458] ENDX modulates the phenotypic profile of immune cells

[0459] The influence of ENDX on IFNy, GranzymeB, and IL-2 prompted expansion of the analysis and the drug’s regulatory impact on various immune markers, encompassing immune checkpoint molecules, across different T cell populations was examined. To accomplish this, peripheral blood mononuclear cells (PBMCs) from healthy donors were subjected to treatment with ENDX (2 pM). Simultaneously, these cells were stimulated with CD3 / CD28 Dynabeads for 5 days. Subsequently, a panel consisting of 37 immune markers was employed for cellular staining, followed by analysis utilizing mass cytometry (CyTOF) on the Fluidigm® Helios™ platform. The acquired data using FlowJo-vl0.8.1 software detected notable changes in specific cell subpopulations in response to drug treatment (Figure 34). The data provide further evidence that ENDX treatment increased the population of CD4+ and CD8+ T cells exhibiting a memory phenotype, as evidenced by the expression of specific markers such as CD28, CD45RO, CCR4, CXCR3, CD25, CDlla, CD38, and TIGIT. Conversely, there was a decrease in the CD8+ T cell population characterized by low CD45RO expression but high levels of CD25, CD27, CD28, CCR7, CXCR3, and TIM3. These findings provide further in vitro support demonstrating that ENDX alters the phenotypic profile of immune cells, thereby potentially contributing to their function.

[0460] ENDX primes immune cells to regulate ER+ breast cancer cell line growth

[0461] To investigate the potential impact of ENDX-induced alterations in immune cell functionality on their anti-tumor efficacy, PBMCs were pre-treated with either 2 pM of ENDX or enzastaurin for 5 days, and subsequently co-cultured with either MCF7AC1 cells sensitive to ENDX or MCF7 cells resistant to ENDX, followed by monitoring real-time tumor proliferation using IncuCyte over 5 days (Figure 35A). In a series of parallel experiments, tumor cells were subjected to prior treatment with abemaciclib, a potent CDK4 / 6 inhibitor, after which they were co-cultured with PBMCs, followed by a real-time proliferation assay using IncuCyte (Figure 35). It was investigated whether synergistic immune killing may be present when ER+ tumor cells are co-cultured with PBMCs that have been treated with both ENDX and abemaciclib. The data indicated that co-culture of PBMCs previously exposed to ENDX along with either aromatase expressing MCF7AC1 cells or Attorney Docket No. 07039-2296WO1 / 2023-600

[0462] MCF7 ENDX-resistant cells, resulted in a significant decrease in tumor cell proliferation as compared to untreated control cells (Figure 35B). In contrast, PBMCs exposed to enzastaurin demonstrated no discernible impact on proliferation. Furthermore, when either MCF7AC1 or MCF7 ENDX-resistant cells were pre-treated with abemaciclib (500 nM) before ENDX exposed PBMCs, nearly complete inhibition of tumor growth was observed, demonstrating the effectiveness of this combination therapy in reducing BC cell proliferation (Figure 35, middle and right panels).

[0463] Example 4: Z-Endoxifen (ENDX) mediated CD4+ and CD8+ T cell alterations at physiological achievable drug concentrations

[0464] This Example demonstrates that endoxifen negatively regulated inhibitory receptors PD-1 and TIM-3 on CD4+ and CD8+ T cells.

[0465] A flow cytometry gating strategy for of CD4+ and CD8+ T cells is shown in Figure 36. Endoxifen negatively regulated inhibitory receptors PD-1 and TIM-3, T cell exhaustion markers that are upregulated in poor prognosis ER+ / HER2- breast cancer, on CD4+ and CD8+ T cells (Figure 37). These data were not seen with fulvestrant, a pure antiestrogen (Figure 37). These data suggest that endoxifen may have the ability to activate the immune system by eradicating a subset of T cells associated with immune exhaustion.

[0466] A flow cytometry gating strategy for T cell effector cytokines is shown in Figure 38. T cell IFN-y+, TNF-a+, and IL-2+ expression was preserved in presence of endoxifen (Figure 39).

[0467] Example 5: Treating ER- Cancer

[0468] A human identified as having an ER- cancer (e.g., an ER- breast cancer such as a TNBC) is administered from about 20 mg / day to about 360 mg / day of one or more ENDX compounds (e.g., a Z-ENDX such as Z-ENDX hydrochloride). The administered one or more ENDX compounds can reduce the number of cancer cells present in the human.

[0469] Example 6: Treating ER- Cancer

[0470] A human identified as having an ER+ cancer and / or an ER- cancer (e.g., an ER-breast cancer such as a TNBC) is administered one or more ENDX compounds (e.g., a Z- Attorney Docket No. 07039-2296WO1 / 2023-600

[0471] ENDX such as Z-ENDX hydrochloride) together with one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell (e.g., abemaciclib) of the ER+ cancer and / or the ER- cancer. The administered one or more ENDX compounds and one or more agents / therapies that can alter the antigens presented on the surface of a cancer cell can reduce the number of cancer cells present in the human.

[0472] Example 7: Exemplary Embodiments

[0473] Embodiment 1. A method for treating a mammal having an estrogen receptor negative (ERneg) cancer, wherein said method comprises administering, to said mammal, an endoxifen (ENDX) compound.

[0474] Embodiment 2. The method of embodiment 1, wherein said mammal is a human.

[0475] Embodiment 3. The method of any one of embodiments 1-2, wherein said mammal is a female mammal.

[0476] Embodiment 4. The method of any one of embodiments 1-3, wherein said mammal is a pre-menopausal female human.

[0477] Embodiment 5. The method of any one of embodiments 1-4, wherein said ERnegcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma.

[0478] Embodiment 6. The method of embodiment 5, wherein said ERnegcancer is a breast cancer. Attorney Docket No. 07039-2296WO1 / 2023-600

[0479] Embodiment 7. The method of embodiment 6, wherein said breast cancer is a triple negative breast cancer.

[0480] Embodiment 8. The method of any one of embodiments 1-7, wherein said ENDX compound is a Z-ENDX compound.

[0481] Embodiment 9. The method of embodiment 8, wherein said Z-ENDX compound is a Z-ENDX salt.

[0482] Embodiment 10. The method of embodiment 9, wherein said Z-ENDX salt is Z-ENDX hydrochloride.

[0483] Embodiment 11. The method of any one of embodiments 1-10, wherein said method comprising administering from about 20 milligrams per day (mg / day) to about 360 mg / day of said ENDX compound to said mammal.

[0484] Embodiment 12. The method of any one of embodiments 1-11, said method further comprising administering to said mammal an agent that can alter the antigens presented on the surface of a cancer cell of said ERnegcancer.

[0485] Embodiment 13. The method of embodiment 12, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5 -fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, Attorney Docket No. 07039-2296WO1 / 2023-600

[0486] REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602.

[0487] Embodiment 14. A method for treating a mammal having an ERposcancer or an ERnegcancer, wherein said method comprises administering, to said mammal, (i) an ENDX compound, and (ii) an agent comprising the ability to alter the antigens presented on the surface of a cancer cell of said ERposcancer or said ERnegcancer.

[0488] Embodiment 15. The method of embodiment 14, wherein said mammal is a human.

[0489] Embodiment 16. The method of any one of embodiments 14-15, wherein said mammal is a female mammal.

[0490] Embodiment 17. The method of any one of embodiments 14-16, wherein said mammal is a pre-menopausal female human.

[0491] Embodiment 18. The method of any one of embodiments 14-17, wherein said mammal has said ERnegcancer, and wherein said ERnegcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma.

[0492] Embodiment 19. The method of embodiment 18, wherein said ERnegcancer is a breast cancer. Attorney Docket No. 07039-2296WO1 / 2023-600

[0493] Embodiment 20. The method of embodiment 19, wherein said breast cancer is a triple negative breast cancer.

[0494] Embodiment 21. The method of any one of embodiments 14-17, wherein said mammal has said ERposcancer, and wherein said ERposcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, and a lung cancer.

[0495] Embodiment 22. The method of any one of embodiments 14-21, wherein said ENDX compound is a Z-ENDX compound.

[0496] Embodiment 23. The method of embodiment 22, wherein said Z-ENDX compound is a Z-ENDX salt.

[0497] Embodiment 24. The method of embodiment 23, wherein said Z-ENDX salt is Z-ENDX hydrochloride.

[0498] Embodiment 25. The method of any one of embodiments 14-24, wherein said method comprising administering from about 20 mg / day to about 360 mg / day of said ENDX compound to said mammal.

[0499] Embodiment 26. The method of any one of embodiments 14-25, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, Attorney Docket No. 07039-2296WO1 / 2023-600

[0500] abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602.

[0501] Embodiment 27. The use of a composition comprising an ENDX compound to treat a mammal having an ERnegcancer.

[0502] Embodiment 28. The use of embodiment 27, wherein said mammal is a human.

[0503] Embodiment 29. The use of any one of embodiments 27-28, wherein said mammal is a female mammal.

[0504] Embodiment 30. The use of any one of embodiments 27-29, wherein said mammal is a pre-menopausal female human.

[0505] Embodiment 31. The use of any one of embodiments 27-30, wherein said ERnegcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma.

[0506] Embodiment 32. The use of embodiment 31, wherein said ERnegcancer is a breast cancer.

[0507] Embodiment 33. The use of embodiment 32, wherein said breast cancer is a triple negative breast cancer. Attorney Docket No. 07039-2296WO1 / 2023-600

[0508] Embodiment 34. The use of any one of embodiments 27-33, wherein said ENDX compound is a Z-ENDX compound.

[0509] Embodiment 35. The use of embodiment 34, wherein said Z-ENDX compound is a Z- ENDX salt.

[0510] Embodiment 36. The use of embodiment 35, wherein said Z-ENDX salt is Z-ENDX hydrochloride.

[0511] Embodiment 37. The use of any one of embodiments 27-36, wherein said composition comprises from about 20 mg to about 360 mg of said ENDX compound.

[0512] Embodiment 38. The use of any one of embodiments 27-37, said composition further comprises an agent that can alter the antigens presented on the surface of a cancer cell of said ERnegcancer.

[0513] Embodiment 39. The use of embodiment 38, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5 -fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, Attorney Docket No. 07039-2296WO1 / 2023-600

[0514] bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602.

[0515] Embodiment 40. An ENDX compound for use in the preparation of a medicament to treat an ERnegcancer.

[0516] Embodiment 41. An ENDX compound for use in the treatment of an ERnegcancer.

[0517] Embodiment 42. The ENDX compound of any one of embodiments 40-41, wherein said ERnegcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma.

[0518] Embodiment 43. The ENDX compound of embodiment 42, wherein said ERnegcancer is a breast cancer.

[0519] Embodiment 44. The ENDX compound of embodiment 43, wherein said breast cancer is a triple negative breast cancer.

[0520] Embodiment 45. The ENDX compound of any one of embodiments 40-44, wherein said ENDX compound is a Z-ENDX compound.

[0521] Embodiment 46. The ENDX compound of embodiment 45, wherein said Z-ENDX compound is a Z-ENDX salt.

[0522] Embodiment 47. The ENDX compound of embodiment 46, wherein said Z-ENDX salt is Z-ENDX hydrochloride. Attorney Docket No. 07039-2296WO1 / 2023-600

[0523] Embodiment 48. The use of a composition comprising (i) an ENDX compound, and (ii) an agent comprising the ability to alter the antigens presented on the surface of a cancer cell of said ERposcancer or said ERnegcancer to treat a mammal having an ER+ cancer or an ERnegcancer.

[0524] Embodiment 49. The use of embodiment 48, wherein said mammal is a human.

[0525] Embodiment 50. The use of any one of embodiments 48-49, wherein said mammal is a female mammal.

[0526] Embodiment 51. The use of any one of embodiments 48-50, wherein said mammal is a pre-menopausal female human.

[0527] Embodiment 52. The use of any one of embodiments 48-51, wherein said mammal has said ERnegcancer, and wherein said ERnegcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma.

[0528] Embodiment 53. The use of embodiment 52, wherein said ERnegcancer is a breast cancer.

[0529] Embodiment 54. The use of embodiment 53, wherein said breast cancer is a triple negative breast cancer.

[0530] Embodiment 55. The use of any one of embodiments 48-51, wherein said mammal has said ERposcancer, and wherein said ERposcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, and a lung cancer. Attorney Docket No. 07039-2296WO1 / 2023-600

[0531] Embodiment 56. The use of any one of embodiments 48-55, wherein said ENDX compound is a Z-ENDX compound.

[0532] Embodiment 57. The use of embodiment 56, wherein said Z-ENDX compound is a Z- ENDX salt.

[0533] Embodiment 58. The use of embodiment 57, wherein said Z-ENDX salt is Z-ENDX hydrochloride.

[0534] Embodiment 59. The use of any one of embodiments 48-58, wherein said composition comprises from about 20 mg to about 360 mg of said ENDX compound.

[0535] Embodiment 60. The use of any one of embodiments 48-59, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602. Attorney Docket No. 07039-2296WO1 / 2023-600

[0536] Embodiment 61. An ENDX compound and an agent comprising the ability to alter the antigens presented on the surface of a cancer cell for use in the preparation of a medicament to treat an ERposcancer or ERnegcancer.

[0537] Embodiment 62. An ENDX compound and an agent comprising the ability to alter the antigens presented on the surface of a cancer cell for use in the treatment of an ERnegcancer.

[0538] Embodiment 63. The ENDX compound of any one of embodiments 61-62, wherein said ERnegcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma.

[0539] Embodiment 64. The ENDX compound of embodiment 63, wherein said ERnegcancer is a breast cancer.

[0540] Embodiment 65. The ENDX compound of embodiment 64, wherein said breast cancer is a triple negative breast cancer.

[0541] Embodiment 66. The ENDX compound of any one of embodiments 61-62, wherein said ERposcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, and a lung cancer.

[0542] Embodiment 67. The ENDX compound of any one of embodiments 61-66, wherein said ENDX compound is a Z-ENDX compound.

[0543] Embodiment 68. The ENDX compound of embodiment 67, wherein said Z-ENDX compound is a Z-ENDX salt. Attorney Docket No. 07039-2296WO1 / 2023-600

[0544] Embodiment 69. The ENDX compound of embodiment 68, wherein said Z-ENDX salt is Z-ENDX hydrochloride.

[0545] Embodiment 70. The ENDX compound of any one of embodiments 61-69, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5 -fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602.

[0546] OTHER EMBODIMENTS

[0547] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 07039-2296WO1 / 2023-600WHAT IS CLAIMED IS:

1. A method for treating a mammal having an estrogen receptor negative (ERneg) cancer, wherein said method comprises administering, to said mammal, an endoxifen (ENDX) compound.

2. The method of claim 1, wherein said mammal is a human.

3. The method of claim 1, wherein said mammal is a pre-menopausal female human.

4. The method of claim 1, wherein said ERnegcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma.

5. The method of claim 4, wherein said ERnegcancer is a breast cancer.

6. The method of claim 5, wherein said breast cancer is a triple negative breast cancer.

7. The method of claim 1, wherein said ENDX compound is a Z-ENDX compound.

8. The method of claim 1, wherein said method comprising administering from about 20 milligrams per day (mg / day) to about 360 mg / day of said ENDX compound to said mammal.

9. The method of claim 1, said method further comprising administering to said mammal an agent that can alter the antigens presented on the surface of a cancer cell of said ERnegcancer.Attorney Docket No. 07039-2296WO1 / 2023-60010. The method of claim 9, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5 -fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602.

11. A method for treating a mammal having an ERposcancer or an ERnegcancer, wherein said method comprises administering, to said mammal, (i) an ENDX compound, and (ii) an agent comprising the ability to alter the antigens presented on the surface of a cancer cell of said ERposcancer or said ERnegcancer.

12. The method of claim 11, wherein said mammal is a human.

13. The method of claim 11, wherein said mammal is a pre-menopausal female human.

14. The method of claim 11, wherein said mammal has said ERnegcancer, and wherein said ERnegcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma.Attorney Docket No. 07039-2296WO1 / 2023-60015. The method of claim 14, wherein said ERnegcancer is a breast cancer.

16. The method of claim 15, wherein said breast cancer is a triple negative breast cancer.

17. The method of claim 11, wherein said mammal has said ERposcancer, and wherein said ERposcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, and a lung cancer.

18. The method of claim 11, wherein said ENDX compound is a Z-ENDX compound.

19. The method of claim 11, wherein said method comprising administering from about 20 mg / day to about 360 mg / day of said ENDX compound to said mammal.

20. The method of claim 11, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5 -fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602.Attorney Docket No. 07039-2296WO1 / 2023-60021. The use of a composition comprising an ENDX compound to treat a mammal having an ERnegcancer.

22. The use of claim 21, wherein said mammal is a human.

23. The use of claim 21, wherein said mammal is a pre-menopausal female human.

24. The use of claim 21, wherein said ERnegcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma.

25. The use of claim 24, wherein said ERnegcancer is a breast cancer.

26. The use of claim 22, wherein said breast cancer is a triple negative breast cancer.

27. The use of claim 21, wherein said ENDX compound is a Z-ENDX compound.

28. The use of claim 21, wherein said composition comprises from about 20 mg to about 360 mg of said ENDX compound.

29. The use of claim 21, said composition further comprises an agent that can alter the antigens presented on the surface of a cancer cell of said ERnegcancer.

30. The use of claim 29, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib,Attorney Docket No. 07039-2296WO1 / 2023-600brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602.

31. An ENDX compound for use in the preparation of a medicament to treat an ERnegcancer.

32. An ENDX compound for use in the treatment of an ERnegcancer.

33. The ENDX compound of any one of claims 31-32, wherein said ERnegcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma.

34. The ENDX compound of claim 33, wherein said ERnegcancer is a breast cancer.

35. The ENDX compound of claim 34, wherein said breast cancer is a triple negative breast cancer.

36. The ENDX compound of any one of claims 31-32, wherein said ENDX compound is a Z-ENDX compound.Attorney Docket No. 07039-2296WO1 / 2023-60037. The use of a composition comprising (i) an ENDX compound, and (ii) an agent comprising the ability to alter the antigens presented on the surface of a cancer cell of said ERposcancer or said ERnegcancer to treat a mammal having an ER+ cancer or an ERnegcancer.

38. The use of claim 37, wherein said mammal is a human.

39. The use of claim 37, wherein said mammal is a pre-menopausal female human.

40. The use of claim 37, wherein said mammal has said ERnegcancer, and wherein said ERnegcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma.

41. The use of claim 40, wherein said ERnegcancer is a breast cancer.

42. The use of claim 41, wherein said breast cancer is a triple negative breast cancer.

43. The use of claim 37, wherein said mammal has said ERposcancer, and wherein said ERposcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, and a lung cancer.

44. The use of claim 37, wherein said ENDX compound is a Z-ENDX compound.

45. The use of claim 37, wherein said composition comprises from about 20 mg to aboutAttorney Docket No. 07039-2296WO1 / 2023-60046. The use of claim 37, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5 -fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602.

47. An ENDX compound and an agent comprising the ability to alter the antigens presented on the surface of a cancer cell for use in the preparation of a medicament to treat an ERposcancer or ERnegcancer.

48. An ENDX compound and an agent comprising the ability to alter the antigens presented on the surface of a cancer cell for use in the treatment of an ERnegcancer.

49. The ENDX compound of any one of claims 47-48, wherein said ERnegcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, a brain and / or central nervous system cancer, a bone cancer, a biliary tract cancer, a thyroid cancer, a lung cancer, a colorectal cancer, a head and neck cancer, a stomach cancer, a pancreatic cancer, a kidney cancer, a liver cancer, a prostate cancer, a testicular cancer, a skin cancer, a leukemia, and a lymphoma.

50. The ENDX compound of claim 49, wherein said ERnegcancer is a breast cancer.Attorney Docket No. 07039-2296WO1 / 2023-60051. The ENDX compound of claim 50, wherein said breast cancer is a triple negative breast cancer.

52. The ENDX compound of any one of claims 47-48, wherein said ERposcancer is selected from the group consisting of a breast cancer, an ovarian cancer, an endometrial cancer, and a lung cancer.

53. The ENDX compound of any one of claims 47-48, wherein said ENDX compound is a Z-ENDX compound.

54. The ENDX compound of any one of claims 47-48, wherein said agent is selected from the group consisting of abemaciclib, palbociclib, ribociclib, dalpiciclib, trilaciclib, birociclib, lerociclib, BEBT-209, BPI-16350, FCN-437c, TQB-3616, 1-022, milciclib, auceliciclib, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, iruplinalkib, ensartinib, entrectinib, lorlatinib, brigatinib, alectinib, ceritinib, crizotinib, envonalkib, repotrectinib, unecritinib, conteltinib, foritinib, alkotinib, ficonalkib, pralsetinib, selpercatinib, lenvatinib, alectinib, cabozantinib, regorafenib, vandetanib, sorafenib, sitravatinib, enbezotinib, vepafestinib, interferon alfa-2b, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, oxaliplatin, cisplatin, carboplatin, 5-fluorouracil, panobinostat, chidamide, belinostat, romidepsin, vorinostat, entinostat, abexinostat, givinostat, sulforadex, REC-2282, citarinostat, domatinostat, axitinib, bosutinib, cabozantinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, vandetanib, vemurafenib, ixazomib, carfilzomib, bortezomib, marizomib, ACU-D1, CX-13-608, oprozomib, zetomipzomib, GSK-3494245, M-3258, and TQB-3602.

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