WO2024178274A2

WO2024178274A2 - Methods of degrading raf(raf) protein in cells using mitogen-activated protein kinase kinase 1/2 (mek1/2) protein degraders

Info

Publication number: WO2024178274A2
Application number: PCT/US2024/016977
Authority: WO
Inventors: James Duncan
Original assignee: Institute for Cancer Research
Current assignee: Institute for Cancer Research
Priority date: 2023-02-24
Filing date: 2024-02-23
Publication date: 2024-08-29
Anticipated expiration: 2025-08-24
Also published as: WO2024178274A3

Abstract

Methods of treating cancer by administering a dual Raf/Mitogen-Activated Protein Kinase Kinase (RAF/MEK) degrader to a subject having a RAS-altered cancer are provided herein.

Description

Methods of Degrading Raf (RAF) Protein in Cells Using Mitogen-Activated Protein Kinase Kinase 1/2 (MEK1/2) Protein Degraders

Cross Reference to Related Applications

This application claims the benefit of U.S. Provisional Application No. 63/447,909, filed February 24, 2023. The content of this earlier filed application is hereby incorporated by reference herein in its entirety.

Statement Regarding Federally Funded Research

This invention was made with government support under CA211670 awarded by the National Institutes of Health. The government has certain rights in the invention.

Field

The present disclosure is directed, in part, to treating cancer in a subject in need thereof by administering a dual RAF/MEK degrader to the subj ect.

Background

Activating K-ras (KRAS) mutations frequently occur in several cancers including the majority of pancreatic ductal adenocarcinoma (96%), about 50% of colorectal cancers, about 30% of lung adenocarcinoma, and about 20% of endometrial carcinomas (Munoz- Maldonado et al.. Front. Oncol., 2019, 9. 1088). It has been shown that mutant KRAS preferentially signals through RAF-MEK-ERK signaling and that blockade of this pathway is essential for tumor regression (Lito et al., Cancer Cell, 2014, 25, 697-710). However, targeted MEK inhibitors (MEKi) have shown limited clinical benefit in the treatment of KRAS mutant cancers due to resistance caused by the feedback activation of the upstream kinase CRAF (RAFI). Consequently, there are no single agent MEKi trials ongoing for KRAS mutant cancers, with clinical trials focusing on combinations of CRAF and MEK inhibitors. Although genetic depletion of CRAF has been shown to cause tumor regression in KRAS mutant cancer models. CRAF inhibitors have shown limited efficacy for cancer treatment (Venkatanaray an et al.. Cell Reports, 2022, 38, 10351). It has been shown that depletion of CRAF leads to apoptosis in KRAS mutant cancers independent of MEK1/2 activity⁷, where CRAF promotes suppression of apoptosis and de-differentiation via kinase- independent functions (McCormick et al., Cancer Cell, 2018, 33, 158-159). Thus, the development of CRAF protein degraders that can block both kinase-dependent and independent functions of CRAF are needed for the treatment of RAS-altered cancers.

Summary

The present disclosure provides methods of treating a human disease that requires RAF-MEK in a subject in need thereof, the method comprising administering a dual RAF/MEK degrader to the subject.

The present disclosure also provides dual RAF/MEK degraders for use in treating a human disease that requires RAF-MEK.

Disclosed herein are methods of treating a human disease that requires RAF- Mitogen-Activated Protein Kinase Kinase (RAF-MEK) in a subject in need thereof, the methods comprising administering a dual RAF/MEK degrader to the subject.

Disclosed herein are dual RAF/Mitogen-Activated Protein Kinase Kinase (RAF/MEK) degraders for use in treating a human disease that requires RAF-Mitogen- Activated Protein Kinase Kinase (RAF-MEK).

Brief Description Of The Drawings

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIGS. 1 A-B show that MS934-treatment of KRAS mutant pancreatic cells reduces MEK1, MEK2, and CRAF protein levels. FIG. 1A shows PANC-1 cells treated with escalating doses of PD0325091 (0, 0.0001, 0.001 0.014, 0.041, 0.123, 0.370, 1 pM) for 24 hours, and MAPK signaling components determined by immunoblotting; blots that are representative of 3 independent blots. FIG. IB shows PANC-1 cells treated with escalating doses of MS934 (0, 0.014, 0.041, 0.123, 0.370, 1.1, 3.3, or 10 pM) for 24 hours, and MAPK signaling components determined by immunoblotting; blots are representative of 3 independent blots. FIG. 1C shows densitometric analysis of the MAPK protein immunoblots using ImageJ as means ± SD from 3 blots, each normalized to loading control, GAPDH; DCso values were determined in Prism Software by quantitating the dose of MS934 that reduces MAPK signaling protein levels 50% of the DMSO control treatment.

FIGS. 2A-J show CRAF protein is reduced by MS934-treatment across cancer cell lines, cancer cell lines treated with escalating doses of MS934 (each 0, 0.02, 0.05, 0.10, 0.20, or 0.50 pM) for 24 hours, and MAPK signaling components determined by immunoblotting (FIGS. 2A, 2B, 2C, and 2D); cancer cell lines treated with escalating doses of MS934 (each 0, 0.014, 0.041, 0.123, 0.370, 1.1. 3.3, or 10 pM) for 24 hours. and MAPK signaling components determined by immunoblotting (FIGS. 2E, 2F, 2G, 2H, and 21); and bar plot representing the DC50 values for CRAF across cell lines treated with MS934; DC50 values were determined in Prism Software by quantitating the dose of MS934 that reduces MAPK signaling protein levels 50% of the DMSO control treatment; significance determined by paired t-test using Prism software; ***P <0.001 (FIG. 2J).

FIGS. 3A-F show collateral degradation of CRAF by MS934 through a VHL- proteasome dependent mechanism in PANC-1 cells treated with control or VHL siRNAs for 48 hours and then treated with DMSO or 1 pM MS934 for 24 hours, and MAPK signaling components determined by immunoblotting; blots are representative of 3 independent blots (FIG. 3A); densitometric analysis of the MAPK protein immunoblots, using ImageJ as means ± SD from 3 blots, each normalized to loading control, GAPDH (FIG. 3B); PANC-1 cells treated with 1 pM MS934 and 1 pM PD0325901 or 100 nM bortezomib for 24 hours, and CRAF, MEK1 and MEK2 levels determined by immunoblotting; blots are representative of 3 independent blots (FIG. 3C); densitometric analysis of the MAPK protein immunoblots, using ImageJ as means ± SD from 3 blots, each normalized to loading control, GAPDH (FIG. 3D); TUBE1 pulldown for ubiquitinated CRAF in PANC-1 cells treated with DMSO, MS934 (1 pM), bortezomib (0. 1 pM) or the combination for 24 hours: blots are representative of 3 independent blots (FIG. 3E); and densitometric analysis of the CRAF levels using ImageJ as means ± SD from 3 blots, each normalized to loading control, GAPDH (FIG. 3F).

FIGS. 4A-H show feedback induced CRAF-MEK dimerization by MS934-treatment promotes collateral degradation of CRAF, MEK pulldown in PANC-1 cells treated with DMSO or PD0325901 (0. 1 pM) for 3 hours and CRAF binding to MEK1 determined by immunoblotting; blots are representative of 3 independent blots (FIG. 4A); densitometric analysis of CRAF protein levels using ImageJ as means ± SD from 3 blots, each normalized to loading control, GAPDH (FIG. 4B); PANC-1 cells treated with control or MEK1/2 siRNAs for 48 hours followed by MS934 (0. 1 pM) treatment for 24 hours and MAPK signaling components determined by immunoblotting; blots are representative of 3 independent blots (FIG. 4C); densitometric analysis of the CRAF protein levels using ImageJ as means ± SD from 3 blots, each normalized to loading control, GAPDH (FIG. 4D); PANC- 1 cells treated with DMSO, MS934 (0. 1 pM), RAF709 (1 pM), or the combination for 24 hours and MAPK signaling proteins determined by immunoblotting; blots are representative of 3 independent blots (FIG. 4E); densitometric analysis of CRAF, MEK1 or MEK2 protein levels using ImageJ as means ± SD from 3 blots, each normalized to loading control, GAPDH (FIGS. 4F and 4G).

FIGS. 5A-C show CRAF/MEK degradation reduces PLK1 and Rho GTPase signaling in MEK inhibitor-resistant KRAS mutant pancreatic cancer cells, circos plot of signaling pathways reduced in PANC-1 cells following 24 hours of treatment by PD0325901 (0.1 pM) or MS934 (10 pM) (FIG. 5 A); hierarchical clustering of pathways downregulated by PD0325901 (0.1 pM) or MS934 (10 pM) treatment PANC-1 cells; heat map color depicts P values for pathway enrichment (FIG. 5B); and hierarchical clustering of predicted transcription factors reduced by PD0325901 (0.1 pM) or MS934 (10 pM) treatment for 24 hours in PANC-1 cells; heat map color depicts P values for pathway enrichment; proteins repressed by PD0325901 or MS934 treatment (FDR<0.05) were imported into Metascape pathway analysis (FIG. 5C).

FIG. 6 shows CRAF/MEK degradation reduces growth of KRAS mutant cell lines; cell lines harboring KRAS mutations were treated with escalating doses of MS934 (each 0, 0.014, 0.041, 0.123, 0.370, 1.1, 3.3, or 10 pM) for 14 days and colony formation assessed by cry stal violet staining; images are representative of three independent assays.

FIGS. 7A-C show ARAF, BRAF and CRAF protein levels reduced at higher concentrations of MS 934; immunoblot of HT-29 (FIG. 7A), HCT116 (FIG. 7B), or NCI-H23 (FIG. 7C) cells treated with escalating doses of MS934 for 24 hours.

Description Of Embodiments

Unless defined otherwise, all technical and scientific terms have the same meaning as is commonly understood by one of ordinary skill in the art to which the embodiments disclosed belongs. As used herein, the terms “a” or “an” means that “at least one” of “one or more” unless the context clearly indicates otherwise.

As used herein, the term “about” means that the numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical value can vary +10% and remain with the scope of the disclosed embodiments.

As used herein, the terms “comprising” (and any form of comprising, such as “comprise”, “comprises” and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “contains” and “contain”), are inclusive or open-ended and do not exclude additional, unrecited elements of method steps.

As used herein, the phrase “in need thereof’ means that the animal or mammal has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, the animal or mammal can be in need thereof. For example, a subject who receives treatment with a dual RAF/MEK degrader, as described herein, in order to treat a cancer is “in need thereof’ (i.e., as opposed to receiving a RAF inhibitor or an MEK inhibitor to treat a cancer).

As used herein, the phrase “pharmaceutically acceptable” means those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with tissues of humans and animals. In some embodiments, “pharmaceutically acceptable” means approved by a regulatory agency of the Federal of a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

As used herein, the phrase “pharmaceutically acceptable salt(s),” includes, but is not limited to, salts of acidic or basic groups. Compounds that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. Acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions including, but not limited to, sulfuric, thiosulfuric, citric, maleic, acetic, oxalic, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, bisulfite, phosphate, acid phosphate, isonicotinate, borate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, bicarbonate, malonate, mesylate, esylate, napsydisylate, tosylate, besylate, orthophoshate, tri fluoroacetate, and pamoate (i.e., l,l'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds that include an amino moiety' may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include, but are not limited to, alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, ammonium, sodium, lithium, zinc, potassium, and iron salts. Salts also includes quaternary ammonium salts of the compounds described herein, where the compounds have one or more tertiary amine moiety.

As used herein, the terms ‘‘treat,’’ ‘‘treated,” or “treating” mean therapeutic treatment wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder or disease, or obtain beneficial or desired clinical results. For purposes herein, beneficial or desired clinical results include, but are not limited to. alleviation of symptoms: diminishment of extent of condition, disorder or disease; stabilized (i.e., not worsening) state of condition, disorder or disease; delay in onset or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder or disease. Treatment includes eliciting a clinically significant response, optionally without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

It has been discovered (as described herein) that Proteolysis-Targeting Chimeras (PROTACs), such as MS934 (that utilize the MEK inhibitor PD0325901 and the VHL E3 ligase ligand) and MS432, degrade MEK1, MEK2, as well as the protein CRAF. These observations led to the examination, as described herein, at the mechanistic level where it was discovered that the reduction of CRAF protein by MS934 required MEK proteins, whereby knockdown of MEK1/2 rescued CRAF protein loss, suggesting that the feedback induced interaction of CRAF and MEK was required for CRAF degradation. CRAF has been shown to have kinase-independent growth and survi val functions outside the MEK-ERK pathway in KRAS mutant cells, making CRAF degradation an attractive therapeutic avenue. Together, these findings support a method for degrading RAF protein in cells using MEK- degraders. MS934 is described in, for example. U.S. Patent Application Publication 2021/0395244.

In some of the embodiments, the human disease utilizes the RAF-MEK-ERK pathway. In some embodiments, the human disease is a cancer having an alteration in a receptor-tyrosine kinase. In some embodiments, the human disease is a RAS-altered cancer. In some embodiments, the RAS-altered cancer is a KRAS-altered cancer. In some embodiments, the RAS-altered cancer is an NRAS-altered cancer. In some embodiments, the RAS-altered cancer is an HRAS-altered cancer. In some embodiments, the RAS-altered cancer includes cancers with alterations in BRAF, NF1, MEK, ERK, MYC, EGFR, HER2, and/or FGFR. In some of the embodiments, the human disease is a RAF-altered cancer. In some of the embodiments, the RAF-altered cancer is a BRAF mutant cancer. In some of the embodiments, the BRAF mutant cancer is BRAF(V600E) cancer. In some of the embodiments, the BRAF(V600E) cancer is melanoma or colorectal cancer. In some of the embodiments, the RAF-altered cancer is a CRAF-altered cancer. In some of the embodiments, the human disease is an MEK-altered cancer. In some of the embodiments, the human disease is an ERK-altered cancer.

In some of the embodiments, the human disease is an RASopathy. In some of the embodiments, the RASopathy is Noonan syndrome. In some of the embodiments, the human disease is NF1 syndrome. In some of the embodiments, the human disease is NF2 syndrome. In some of the embodiments, the human disease is syndrome neurofibromatosis Type 1. Type 2, or Type 3 (Schwannomatosis). In some of the embodiments, the human disease is CoQ- deficiency kidney disease.

In some embodiments, the cancer is lung cancer (e.g., non-small-cell lung cancer, small-cell lung cancer), breast cancer, prostate cancer, ovarian cancer, testicular cancer, colon cancer, colorectal cancer, renal cancer (e.g., renal cell carcinoma), bladder cancer, pancreatic cancer, glioblastoma, neuroblastoma, retinoblastoma, leukemia (e.g., acute myeloid leukemia), melanoma, kidney cancer, bone cancer (e.g., osteosarcoma), appendiceal cancer, small bowel cancer, ampullary cancer, cervical cancer, endometrial cancer, gastrointestinal neuroendocrine tumor, uterine endometrioid carcinoma, germ cell tumor, oesophagogastric cancer, sex cord stromal tumor, hepatobiliary cancer, histiocytosis, anal cancer, thyroid cancer, mature B cell neoplasm, soft-tissue sarcoma, gastrointestinal stromal tumor, skin cancer (non-mel anoma), head and neck cancer, glioma, salivary gland cancer, mature B cell lymphoma, or a thymic tumor. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is testicular cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is renal cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is neuroblastoma. In some embodiments, the cancer is retinoblastoma. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is kidney cancer. In some embodiments, the cancer is osteosarcoma. In some embodiments, the cancer is colon cancer, non-small lung cancer, or pancreatic cancer. In some embodiments, the cancer is non-small lung cancer. In some embodiments, the cancer is appendiceal cancer. In some embodiments, the cancer is small bowel cancer. In some embodiments, the cancer is ampullary cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is gastrointestinal neuroendocrine tumor. In some embodiments, the cancer is uterine endometrioid carcinoma. In some embodiments, the cancer is germ cell tumor. In some embodiments, the cancer is oesophagogastric cancer. In some embodiments, the cancer is sex cord stromal tumor. In some embodiments, the cancer is hepatobiliary cancer. In some embodiments, the cancer is histiocytosis. In some embodiments, the cancer is anal cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is mature B cell neoplasm. In some embodiments, the cancer is soft-tissue sarcoma. In some embodiments, the cancer is gastrointestinal stromal tumor. In some embodiments, the cancer is skin cancer (non-melanoma). In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is glioma. In some embodiments, the cancer is salivary gland cancer. In some embodiments, the cancer is mature B cell lymphoma. In some embodiments, the cancer is a thymic tumor. In any of the embodiments described herein, the cancer can be one that requires inhibition or degradation of both CRAF and MEK1 and/or MEK2.

In some embodiments, the RAS-altered cancer is non-small lung cancer, breast cancer, prostate cancer, ovarian cancer, testicular cancer, colon cancer, colorectal cancer, renal cancer, bladder cancer, pancreatic cancer, glioblastoma, neuroblastoma, retinoblastoma, leukemia, melanoma, kidney cancer, or osteosarcoma. In some embodiments, the RAS- altered cancer is colon cancer, colorectal cancer, non-small lung cancer, or pancreatic cancer.

In some embodiments, the dual RAF/MEK degrader is an ARAF/MEK degrader. In some embodiments, the dual RAF/MEK degrader is a BRAF/MEK degrader. In some embodiments, the dual RAF/MEK degrader is a CRAF/MEK degrader. In some embodiments, the dual CRAF/MEK degrader comprises MS934, MS432, MS928. MS910, or any combination thereof. In some embodiments, the dual CRAF/MEK degrader is MS934 (which utilizes the MEK inhibitor PD0325901 and the VHL E3 ligase ligand). In some embodiments, the dual CRAF/MEK degrader is MS432. In some embodiments, the dual CRAF/MEK degrader is MS928. In some embodiments, the dual CRAF/MEK degrader is MS910. In some embodiments, the dual RAF/MEK degrader is any PROTAC that incorporates an MEK1/2 inhibitor that causes collateral degradation of RAF.

In any of the embodiments herein, the subject can also be administered an MEK inhibitor, a BRAF inhibitor, a PI3K inhibitor, an MTOR inhibitor, an SHP2 inhibitor, a KRAS inhibitor, an AKT inhibitor, an ERK inhibitor, an ERBB inhibitor, an ABCBl(MDRl) inhibitor, a CXCR1/2 inhibitor, a PD-L1/PD-1 inhibitor, an RTK inhibitor, an SOS1 inhibitor, a CDK4/6 inhibitor, an AURKA inhibitor, a WEE1 inhibitor, or an mTORCl inhibitor, a BET bromodomain protein inhibitor, an HD AC inhibitor, or any combination thereof. In any of the embodiments herein, the subject can also be administered an siRNA or a cancer vaccine. In any of the embodiments, described herein, the subject can also be administered an anti-cancer agent, such as a chemotherapeutic agent. In any of the embodiments, described herein, the subject can also be administered an immunotherapeutic agent. In any of the embodiments herein, the subject can also undergo radiation therapy.

In some embodiments, the subject can also be administered an MEK inhibitor. In some embodiments, the MEK inhibitor comprises trametinib (e.g., GSK1120212), cobimetinib (e.g., GDC-0973), binimetinib (e.g., MEK162), mirdametinib, TAK733, selumetinib (e.g., AZD6244), refametinib (e.g., BAY869766), pimasertib (e.g., AS703026), PD98059, U0126, Ro 09-2210, CI-1040, PD0325901, R04987655, R05126766, GDC-0623, G-573, E6201, AZD8330, or WX-554, or any combination thereof. In some embodiments, the MEK inhibitor comprises trametinib, cobimetinib, binimetinib. mirdametinib, TAK733. selumetinib, refametinib, pimasertib, PD98059, U0126, Ro 09-2210, CI-1040, PD0325901 , R04987655, R05126766, GDC-0623, G-573, E6201, AZD8330, or WX-554, or any combination thereof. In some embodiments, the MEK inhibitor comprises trametinib. In some embodiments, the MEK inhibitor comprises cobimetinib. In some embodiments, the MEK inhibitor comprises binimetinib. In some embodiments, the MEK inhibitor comprises mirdametinib. In some embodiments, the MEK inhibitor comprises refametinib. In some embodiments, the MEK inhibitor comprises pimasertib. In some embodiments, the MEK inhibitor comprises PD98059. In some embodiments, the MEK inhibitor comprises U0126. In some embodiments, the MEK inhibitor comprises Ro 09-2210. In some embodiments, the MEK inhibitor comprises CI- 1040. In some embodiments, the MEK inhibitor comprises PD0325901. In some embodiments, the MEK inhibitor comprises R04987655. In some embodiments, the MEK inhibitor comprises R05126766. In some embodiments, the MEK inhibitor comprises GDC-0623. In some embodiments, the MEK inhibitor comprises G-573. In some embodiments, the MEK inhibitor comprises E6201. In some embodiments, the MEK inhibitor comprises AZD8330. In some embodiments, the MEK inhibitor comprises WX- 554. In some embodiments, the MEK inhibitor comprises TAK733. In some embodiments, the MEK inhibitor comprises selumetinib.

In some embodiments, the subject can also be administered a BRAF inhibitor. In some embodiments, the BRAF inhibitor comprises vemurafenib (e.g., PLX4032), sorafenib (e.g., BAY 43-9006), dabrafenib (e.g., GSK2118436), encorafenib (e.g., LGX818), GDC- 0879, or PLX-4720. Raf inhibitor 2, GNE-9815, TBAP-001, Raf inhibitor 1, regorafenib (e.g., BAY 73-4506), doramapimod (e.g., BIRB 796), RAF265 (e.g., CHIR-265), NVP- BHG712, SB590885, ZM 336372, AZ 628, GW5074, regorafenib (e.g., BAY-734506), B- Raf IN 1, B -Raf inhibitor 1 (Compound 13) dihydrochloride, L-779450, MCP110, tovorafenib (e.g., MLN2480). RO5126766 (e.g., CH5126766), TAK-632, CCT196969, LY3009120, lifirafenib (e.g., BGB-283), PLX7904, PLX8394. agerafenib (e.g., RXDX-105), BAW2881 (e.g., NVP-BAW2881), RAF709, naporafemb (e.g., LXH254), AZ304, or belvarafenib (e.g., HM95573), or any combination thereof. In some embodiments, the BRAF inhibitor comprises vemurafenib. In some embodiments, the BRAF inhibitor comprises sorafenib. In some embodiments, the BRAF inhibitor comprises dabrafenib. In some embodiments, the BRAF inhibitor comprises encorafenib. In some embodiments, the BRAF inhibitor comprises GDC-0879. In some embodiments, the BRAF inhibitor comprises PLX- 4720. In some embodiments, the BRAF inhibitor comprises Raf inhibitor 2. In some embodiments, the BRAF inhibitor comprises GNE-9815. In some embodiments, the BRAF inhibitor comprises TBAP-001. In some embodiments, the BRAF inhibitor comprises Raf inhibitor 1. In some embodiments, the BRAF inhibitor comprises regorafenib. In some embodiments, the BRAF inhibitor comprises doramapimod. In some embodiments, the BRAF inhibitor comprises RAF265. In some embodiments, the BRAF inhibitor comprises NVP-BHG712. In some embodiments, the BRAF inhibitor comprises SB590885. In some embodiments, the BRAF inhibitor comprises ZM 336372. In some embodiments, the BRAF inhibitor comprises AZ 628. In some embodiments, the BRAF inhibitor comprises GW5074. In some embodiments, the BRAF inhibitor comprises regorafenib. In some embodiments, the BRAF inhibitor comprises B-Raf IN 1. In some embodiments, the BRAF inhibitor comprises B-Raf inhibitor 1 (Compound 13) dihydrochloride. In some embodiments, the BRAF inhibitor comprises L-779450. In some embodiments, the BRAF inhibitor comprises MCP110. In some embodiments, the BRAF inhibitor comprises tovorafenib. In some embodiments, the BRAF inhibitor comprises RO5126766. In some embodiments, the BRAF inhibitor comprises TAK-632. In some embodiments, the BRAF inhibitor comprises CCT196969. In some embodiments, the BRAF inhibitor comprises LY3009120. In some embodiments, the BRAF inhibitor comprises lifirafenib. In some embodiments, the BRAF inhibitor comprises PLX7904. In some embodiments, the BRAF inhibitor comprises PLX8394. In some embodiments, the BRAF inhibitor comprises agerafenib. In some embodiments, the BRAF inhibitor comprises BAW2881. In some embodiments, the BRAF inhibitor comprises RAF709. In some embodiments, the BRAF inhibitor comprises naporafenib. In some embodiments, the BRAF inhibitor comprises AZ304. In some embodiments, the BRAF inhibitor comprises belvarafenib.

In some embodiments, the subj ect can also be administered a PI3K inhibitor. In some embodiments, the PI3K inhibitor comprises ZSTK474. idelalisib, copanlisib (e.g.. BAY 80-6946), duvelisib (e.g., IPI-145), alpelisib (e.g., BYL719), umbralisib (e.g., TGR-1202), leniolisib (e.g., CDZ 173), parsaclisib (e.g., INCB050465), zandelisib, eganelisib (e.g., IPI- 549), linperlisib, nemiralisib, pilaralisib (e.g., XL147), seletalisib (e.g., UCB-5857), tenalisib (e.g., RP6530), AMG 319, AZD8186, AZD8835, CAL263, TG100-115, IC87114, dactolisib (e.g., BEZ235), pictilisib (e.g., GDC-0941), LY294002, buparlisib (e.g.. BKM120), PI-103. NU7441 (e.g.. KU-57788), TGX-221, IC-87114. Wortmannin (e.g., KY 12420), XL147 analogue, AS-605240, PIK-75 HC1, Rigosertib (e.g., ON-01910), 3-Methyladenine (3-MA), A66, voxtalisib (XL765) analogue, omipalisib (e.g., GSK2126458), PIK-90, AZD6482, PF- 04691502, apitolisib (e.g., GDC-0980), GSK1059615, gedatolisib (e.g., PKI-587), AS- 252424, NU7026. BGT226 (e.g., NVP-BGT226) maleate, fimepinostat (e.g., CUDC-907), PIK-294, AS-604850, GSK2636771, YM201636, CH5132799, CAY10505, PIK-293, PKI- 402, PI3K/Akt inhibitor library, TG100713, VS-5584 (e.g., SB2343), taselisib (e.g., GDC 0032), KU-0060648, CZC24832, demethyl-Coclaurine Oroxin B, Homosalate, cinobufagin, resibufogenin, hispidulin, GSK2292767, lanatoside C, PI3K/mTOR Inhibitor-2, zeaxanthin, disitertide (e.g.. P144), cafestol, paxalisib (e.g.. GDC-0084). SK1-V. Dichroa febrifuga extract, MTX-211 , Trichosanthis Pericarpium extract, Dioscoreae Nipponicae Rhizoma extract, 740 Y-P (e.g., PDGFR 740Y-P), GDC-0326, PIK-108, HS-173, SF2523, lupenone, serabelisib (e.g., TAK-117), quercetin (e.g., NSC 9221), bimiralisib (e.g., PQR309), VPS34 inhibitor 1 (Compound 19). IHMT-PI3K6-372. voxtalisib (e.g., XL765). autophinib, GNE- 317, (E)-Akt inhibitor-IV, notoginsenoside Rl, solasodine, galleon, pectolinarin, inavolisib (e.g., GDC-0077), SRX3207, a-linolenic acid, acalisib (e.g., GS-9820), ME-401, samotolisib (e.g., LY3023414), VPS34-IN1, ailanthone, Tripterygium wilfordii extract, IPI-3063, SAR405, PIK-III, PI-3065, quercetin dihydrate, SPP-86, erucic acid, trigonelline, deguelin, selective P13K5 Inhibitor 1 (compound 7n), amarogentin, Poria Cocos extract, loureirin A, YS-49, PF-4989216, GNE-477, or GNE-493, or any combination thereof. In some embodiments, the PI3K inhibitor comprises ZSTK474. In some embodiments, the PI3K inhibitor comprises idelalisib. In some embodiments, the PI3K inhibitor comprises copanlisib. In some embodiments, the PI3K inhibitor comprises duvelisib. In some embodiments, the PI3K inhibitor comprises alpelisib. In some embodiments, the PI3K inhibitor comprises umbralisib. In some embodiments, the PI3K inhibitor comprises leniolisib. In some embodiments, the PI3K inhibitor comprises parsaclisib. In some embodiments, the PI3K inhibitor comprises zandelisib. In some embodiments, the PI3K inhibitor comprises eganelisib. In some embodiments, the PI3K inhibitor comprises linperlisib. In some embodiments, the PI3K inhibitor comprises nemiralisib. In some embodiments, the PI3K inhibitor comprises pilaralisib. In some embodiments, the PI3K inhibitor comprises seletalisib. In some embodiments, the PI3K inhibitor comprises tenalisib. In some embodiments, the PI3K inhibitor comprises AMG 319. In some embodiments, the PI3K inhibitor comprises AZD8186. In some embodiments, the PI3K inhibitor comprises AZD8835. In some embodiments, the PI3K inhibitor comprises CAL263. In some embodiments, the PI3K inhibitor comprises TGI 00-115. In some embodiments, the PI3K inhibitor comprises IC87114. In some embodiments, the PI3K inhibitor comprises dactolisib. In some embodiments, the PI3K inhibitor comprises pictilisib. In some embodiments, the PI3K inhibitor comprises LY294002. In some embodiments, the PI3K inhibitor comprises buparlisib. In some embodiments, the PI3K inhibitor comprises PI-103. In some embodiments, the PI3K inhibitor comprises NU7441. In some embodiments, the PI3K inhibitor comprises TGX-221. In some embodiments, the PI3K inhibitor comprises IC- 87114. In some embodiments, the P13K inhibitor comprises Wortmannin. In some embodiments, the PI3K inhibitor comprises XL147 analogue. In some embodiments, the PI3K inhibitor comprises AS-605240. In some embodiments, the PI3K inhibitor comprises PIK-75 HC1. In some embodiments, the PI3K inhibitor comprises rigosertib. In some embodiments, the PI3K inhibitor comprises 3-Methyladenine (3-MA). In some embodiments, the PI3K inhibitor comprises A66. In some embodiments, the PI3K inhibitor comprises voxtalisib (XL765) analogue. In some embodiments, the PI3K inhibitor comprises omipalisib. In some embodiments, the PI3K inhibitor comprises PIK-90. In some embodiments, the PI3K inhibitor comprises AZD6482. In some embodiments, the PI3K inhibitor comprises PF-04691502. In some embodiments, the P13K inhibitor comprises apitolisib. In some embodiments, the PI3K inhibitor comprises GSK1059615. In some embodiments, the PI3K inhibitor comprises gedatolisib. In some embodiments, the PI3K inhibitor comprises AS-252424. In some embodiments, the PI3K inhibitor comprises NU7026. In some embodiments, the PI3K inhibitor comprises BGT226 maleate. In some embodiments, the PI3K inhibitor comprises fimepinostat. In some embodiments, the PI3K inhibitor comprises PIK-294. In some embodiments, the PI3K inhibitor comprises AS- 604850. In some embodiments, the PI3K inhibitor comprises GSK2636771. In some embodiments, the PI3K inhibitor comprises YM201636. In some embodiments, the PI3K inhibitor comprises CH5132799. In some embodiments, the PI3K inhibitor comprises CAY10505. In some embodiments, the PI3K inhibitor comprises PIK-293. In some embodiments, the PI3K inhibitor comprises PKI-402. In some embodiments, the PI3K inhibitor comprises PI3K/Akt inhibitor library. In some embodiments, the PI3K inhibitor comprises TG100713. In some embodiments, the PI3K inhibitor comprises VS-5584. In some embodiments, the PI3K inhibitor comprises taselisib. In some embodiments, the PI3K inhibitor comprises KU-0060648. In some embodiments, the PI3K inhibitor comprises CZC24832. In some embodiments, the PI3K inhibitor comprises demethyl-Coclaurine Oroxin B. In some embodiments, the PI3K inhibitor comprises homosalate. In some embodiments, the PI3K inhibitor comprises cinobufagin. In some embodiments, the PI3K inhibitor comprises resibufogenin. In some embodiments, the PI3K inhibitor comprises hispidulin. In some embodiments, the PI3K inhibitor comprises GSK2292767. In some embodiments, the PI3K inhibitor comprises lanatoside C. In some embodiments, the PI3K inhibitor comprises PI3K/mT0R Inhibitor-2. In some embodiments, the PI3K inhibitor comprises zeaxanthin. In some embodiments, the PI3K inhibitor comprises disitertide. In some embodiments, the PI3K inhibitor comprises cafestol. In some embodiments, the PI3K inhibitor comprises paxalisib. In some embodiments, the PI3K inhibitor comprises SKI-V. In some embodiments, the PI3K inhibitor comprises Dichroa febrifuga extract. In some embodiments, the PI3K inhibitor comprises MTX-211. In some embodiments, the PI3K inhibitor comprises Trichosanthis Pericarpium extract. In some embodiments, the PI3K inhibitor comprises Dioscoreae Nipponicae Rhizoma extract. In some embodiments, the PI3K inhibitor comprises 740 Y-P. In some embodiments, the PI3K inhibitor comprises GDC-0326. In some embodiments, the PI3K inhibitor comprises PIK-108. In some embodiments, the PI3K inhibitor comprises HS-173. In some embodiments, the P13K inhibitor comprises SF2523. In some embodiments, the PI3K inhibitor comprises lupenone. In some embodiments, the PI3K inhibitor comprises serabelisib. In some embodiments, the PI3K inhibitor comprises quercetin. In some embodiments, the PI3K inhibitor comprises bimiralisib. In some embodiments, the PI3K inhibitor comprises VPS34 inhibitor 1 (Compound 19). In some embodiments, the PI3K inhibitor comprises IHMT-PI3K5-372. In some embodiments, the PI3K inhibitor comprises voxtalisib. In some embodiments, the PI3K inhibitor comprises autophinib. In some embodiments, the PI3K inhibitor comprises GNE- 317. In some embodiments, the PI3K inhibitor comprises (E)-Akt inhibitor-IV. In some embodiments, the PI3K inhibitor comprises notoginsenoside Rl. In some embodiments, the PI3K inhibitor comprises solasodine. In some embodiments, the PI3K inhibitor comprises galleon. In some embodiments, the PI3K inhibitor comprises pectolinarin. In some embodiments, the PI3K inhibitor comprises inavolisib. In some embodiments, the PI3K inhibitor comprises SRX3207. In some embodiments, the PI3K inhibitor comprises a- linolenic acid. In some embodiments, the PI3K inhibitor comprises acalisib. In some embodiments, the PI3K inhibitor comprises ME-401 . In some embodiments, the PI3K inhibitor comprises samotolisib. In some embodiments, the PI3K inhibitor comprises VPS34- IN1. In some embodiments, the PI3K inhibitor comprises ailanthone. In some embodiments, the PI3K inhibitor comprises Tripterygium wilfordii extract. In some embodiments, the PI3K inhibitor comprises IPI-3063. In some embodiments, the PI3K inhibitor comprises SAR405. In some embodiments, the PI3K inhibitor comprises PIK-III. In some embodiments, the PI3K inhibitor comprises PI-3065. In some embodiments, the PI3K inhibitor comprises quercetin dihydrate. In some embodiments, the PI3K inhibitor comprises SPP-86. In some embodiments, the PI3K inhibitor comprises erucic acid. In some embodiments, the P13K inhibitor comprises trigonelline. In some embodiments, the PI3K inhibitor comprises deguelin. In some embodiments, the PI3K inhibitor comprises selective PI3K5 Inhibitor 1 (compound 7n). In some embodiments, the PI3K inhibitor comprises amarogentin. In some embodiments, the PI3K inhibitor comprises Poria Cocos extract. In some embodiments, the PI3K inhibitor comprises loureirin A. In some embodiments, the PI3K inhibitor comprises YS-49. In some embodiments, the PI3K inhibitor comprises PF-4989216. In some embodiments, the PI3K inhibitor comprises GNE-477. In some embodiments, the PI3K inhibitor comprises GNE-493.

In some embodiments, the subj ect can also be administered an MTOR inhibitor. In some embodiments, the MTOR inhibitor comprises PI3K/mT0R Inhibitor-2, mTOR inhibitor-1, dactolisib (e.g., BEZ235), rapamycin (e.g., AY-22989), everolimus (e.g., RAD001). AZD8055, temsirolimus (e.g., CCI-779), PI-103, NU7441 (e.g., KU-57788), KU- 0063794, torkinib (e.g.. PP242), ridaforolimus (e.g., deforolimus, MK-8669), sapanisertib (e.g., MLN0128), voxtalisib (XL765) analogue, torin 1, omipalisib (e.g., GSK2126458), OSI- 027, PF-04691502, apitohsib (e.g., GDC-0980), GSK1059615, gedatolisib (e.g., PKI-587), WYE-354, vistusertib (e.g., AZD2014), torin 2, WYE-125132 (e.g., WYE-132), BGT226 (e.g., NVP-BGT226) maleate, palomid 529 (P529), PP121, WYE-687, nitazoxanide (e.g.. NSC 697855), WAY-600, GDC-0349, XL388, 4EGI-1, JR-AB2-011, rotundic acid, lanatoside C, Compound 401, astragaloside IV, ginkgolide K, CC-115, paxalisib (e.g., GDC- 0084), CZ415, SF2523, bimiralisib (e.g., PQR309), voxtalisib (e.g., XL765), chry sophanic acid, onatasertib (e.g., CC 223), MTI-31, ABTL-0812, MHY-1685, GNE-477, GNE-493, ETP-46464, zotarolimus (e.g., ABT-578), or PQR620, or any combination thereof. In some embodiments, the MTOR inhibitor comprises PI3K/mT0R Inhibitor-2. In some embodiments, the MTOR inhibitor comprises mTOR inhibitor-1. In some embodiments, the MTOR inhibitor comprises dactolisib. In some embodiments, the MTOR inhibitor comprises rapamycin. In some embodiments, the MTOR inhibitor comprises everolimus. In some embodiments, the MTOR inhibitor comprises AZD8055. In some embodiments, the MTOR inhibitor comprises temsirolimus. In some embodiments, the MTOR inhibitor comprises PI- 103. In some embodiments, the MTOR inhibitor comprises NU7441. In some embodiments, the MTOR inhibitor comprises KU-0063794. In some embodiments, the MTOR inhibitor comprises torkinib. In some embodiments, the MTOR inhibitor comprises ridaforolimus. In some embodiments, the MTOR inhibitor comprises sapanisertib. In some embodiments, the MTOR inhibitor comprises voxtalisib (XL765) analogue. In some embodiments, the MTOR inhibitor comprises torin 1. In some embodiments, the MTOR inhibitor comprises omipalisib. In some embodiments, the MTOR inhibitor comprises OSI-027. In some embodiments, the MTOR inhibitor comprises PF-04691502. In some embodiments, the MTOR inhibitor comprises apitolisib. In some embodiments, the MTOR inhibitor comprises GSK1059615. In some embodiments, the MTOR inhibitor comprises gedatolisib. In some embodiments, the MTOR inhibitor comprises WYE-354. In some embodiments, the MTOR inhibitor comprises vistusertib. In some embodiments, the MTOR inhibitor comprises torin 2. In some embodiments, the MTOR inhibitor comprises WYE-125132. In some embodiments, the MTOR inhibitor comprises BGT226 maleate. In some embodiments, the MTOR inhibitor comprises palomid 529 (P529). In some embodiments, the MTOR inhibitor comprises PP121. In some embodiments, the MTOR inhibitor comprises WYE-687. In some embodiments, the MTOR inhibitor comprises nitazoxanide. In some embodiments, the MTOR inhibitor comprises WAY-600. In some embodiments, the MTOR inhibitor comprises GDC-0349. In some embodiments, the MTOR inhibitor comprises XL388. In some embodiments, the MTOR inhibitor comprises 4EGI-1. In some embodiments, the MTOR inhibitor comprises JR-AB2-011. In some embodiments, the MTOR inhibitor comprises rotundic acid. In some embodiments, the MTOR inhibitor comprises lanatoside C. In some embodiments, the MTOR inhibitor comprises Compound 401. In some embodiments, the MTOR inhibitor comprises stragaloside IV. In some embodiments, the MTOR inhibitor comprises ginkgolide K. In some embodiments, the MTOR inhibitor comprises CC-115. In some embodiments, the MTOR inhibitor comprises paxalisib. In some embodiments, the MTOR inhibitor comprises CZ415. In some embodiments, the MTOR inhibitor comprises SF2523. In some embodiments, the MTOR inhibitor comprises bimiralisib. In some embodiments, the MTOR inhibitor comprises voxtalisib. In some embodiments, the MTOR inhibitor comprises chrysophanic acid. In some embodiments, the MTOR inhibitor comprises onatasertib. In some embodiments, the MTOR inhibitor comprises MTI-31. In some embodiments, the MTOR inhibitor comprises ABTL-0812. In some embodiments, the MTOR inhibitor comprises MHY-1685. In some embodiments, the MTOR inhibitor comprises GNE-477. In some embodiments, the MTOR inhibitor comprises GNE-493. In some embodiments, the MTOR inhibitor comprises ETP -46464. In some embodiments, the MTOR inhibitor comprises zotarolimus. In some embodiments, the MTOR inhibitor comprises PQR620.

In some embodiments, the subj ect can also be administered an SHP2 inhibitor. In some embodiments, the SHP2 inhibitor comprises GDC-1971, TNO155, SHP099, sodium stibogluconate, IACS-13909, RMC-4550, SHP099 HC1, PHPS1. NSC87877, BVT-948. SPI- 112, RMC-4630, JAB-3068, JAB-3312, or ERAS-601, or any combination thereof. In some embodiments, the SHP2 inhibitor comprises GDC-1971. In some embodiments, the SHP2 inhibitor comprises TNO155. In some embodiments, the SHP2 inhibitor comprises SHP099. In some embodiments, the SHP2 inhibitor comprises sodium stibogluconate. In some embodiments, the SHP2 inhibitor comprises IACS-13909. In some embodiments, the SHP2 inhibitor comprises RMC-4550. In some embodiments, the SHP2 inhibitor comprises SHP099 HC1. In some embodiments, the SHP2 inhibitor comprises PHPS1. In some embodiments, the SHP2 inhibitor comprises NSC87877. In some embodiments, the SHP2 inhibitor comprises BVT-948. In some embodiments, the SHP2 inhibitor comprises SPI-112. In some embodiments, the SHP2 inhibitor comprises RMC-4630. In some embodiments, the SHP2 inhibitor comprises JAB-3068. In some embodiments, the SHP2 inhibitor comprises JAB-3312. In some embodiments, the SHP2 inhibitor comprises ERAS-601.

In some embodiments, the subject can also be administered an KRAS inhibitor. In some embodiments, the KRAS inhibitor comprises zoledronic acid (e.g., ZOL 446), lonafamib (e.g., SCH66336), K-Ras(G12C) inhibitor 9, salirasib, MRTX1133, alamandine, sotorasib (e.g., AMG510), LC-2, (Rac)-Antineoplaston, A10, K-Ras-IN-1, GDC6036, deltarasin, APS6-45, BAY-293, MCP110, 6H05, K-Ras(G12C) inhibitor 12, Kobe0065, K- Ras(G12C) inhibitor 6, BQU57, ARS-1620, JDQ443, Kobe2602, BI-2852, NAV-2729, antineoplaston A10. fendiline hydrochloride, adagrasib (e.g.. MRTX849). KRpep-2d. ARS- 853 (e.g., ARS853), BI-3406, perillyl alcohol, ASP2453, RBC8, sotorasib (e.g., AMG510) racemate, Pan-RAS-IN-1, MRTX-1257, KY1220, CID-1067700, zoledronic acid monohydrate, D-1553, RG6330/GDC-6036, BI-1823911, JAB-21822, LY3537982, JNJ- 74699157, MK-1084, JAB-22000, JAB-23000. RM-018. RMC-6291, RMC-9805/RM-036, RMC-8839, RMC-6236, JAB-23400, or BBP-454, or any combination thereof. In some embodiments, the KRAS inhibitor comprises zoledronic acid. In some embodiments, the KRAS inhibitor comprises lonafamib. In some embodiments, the KRAS inhibitor comprises K-Ras(G12C) inhibitor 9. In some embodiments, the KRAS inhibitor comprises salirasib. In some embodiments, the KRAS inhibitor comprises MRTX1133. In some embodiments, the KRAS inhibitor comprises alamandine. In some embodiments, the KRAS inhibitor comprises sotorasib. In some embodiments, the KRAS inhibitor comprises LC-2. In some embodiments, the KRAS inhibitor comprises (Rac)-Antineoplaston. In some embodiments, the KRAS inhibitor comprises Alt). In some embodiments, the KRAS inhibitor comprises K-Ras-IN-1. In some embodiments, the KRAS inhibitor comprises GDC6036. In some embodiments, the KRAS inhibitor comprises deltarasin. In some embodiments, the KRAS inhibitor comprises APS6-45. In some embodiments, the KRAS inhibitor comprises BAY-293. In some embodiments, the KRAS inhibitor comprises MCP110. In some embodiments, the KRAS inhibitor comprises 6H05. In some embodiments, the KRAS inhibitor comprises K- Ras(G12C) inhibitor 12. In some embodiments, the KRAS inhibitor comprises Kobe0065. In some embodiments, the KRAS inhibitor comprises K-Ras(G12C) inhibitor 6. In some embodiments, the KRAS inhibitor comprises BQU57. In some embodiments, the KRAS inhibitor comprises ARS-1620. In some embodiments, the KRAS inhibitor comprises JDQ443. In some embodiments, the KRAS inhibitor comprises Kobe2602. In some embodiments, the KRAS inhibitor comprises BI-2852. In some embodiments, the KRAS inhibitor comprises NAV-2729. In some embodiments, the KRAS inhibitor comprises antineoplaston A10. In some embodiments, the KRAS inhibitor comprises fendiline hydrochloride. In some embodiments, the KRAS inhibitor comprises adagrasib. In some embodiments, the KRAS inhibitor comprises KRpep-2d. In some embodiments, the KRAS inhibitor comprises ARS-853. In some embodiments, the KRAS inhibitor comprises BI- 3406. In some embodiments, the KRAS inhibitor comprises perillyl alcohol. In some embodiments, the KRAS inhibitor comprises ASP2453. In some embodiments, the KRAS inhibitor comprises RBC8. In some embodiments, the KRAS inhibitor comprises sotorasib racemate. In some embodiments, the KRAS inhibitor comprises Pan-RAS-IN-1 . In some embodiments, the KRAS inhibitor comprises MRTX-1257. In some embodiments, the KRAS inhibitor comprises KYI 220. In some embodiments, the KRAS inhibitor comprises CID- 1067700. In some embodiments, the KRAS inhibitor comprises zoledronic acid monohydrate. In some embodiments, the KRAS inhibitor comprises D-1553. In some embodiments, the KRAS inhibitor comprises RG6330/GDC-6036. In some embodiments, the KRAS inhibitor comprises BI- 1823911. In some embodiments, the KRAS inhibitor comprises JAB-21822. In some embodiments, the KRAS inhibitor comprises LY3537982. In some embodiments, the KRAS inhibitor comprises JNJ-74699157. In some embodiments, the KRAS inhibitor comprises MK-1084. In some embodiments, the KRAS inhibitor comprises JAB-22000. In some embodiments, the KRAS inhibitor comprises JAB-23000. In some embodiments, the KRAS inhibitor comprises RM-018. In some embodiments, the KRAS inhibitor comprises RMC-6291. In some embodiments, the KRAS inhibitor comprises RMC- 9805/RM-036. In some embodiments, the KRAS inhibitor comprises RMC-8839. In some embodiments, the KRAS inhibitor comprises RMC-6236. In some embodiments, the KRAS inhibitor comprises JAB-23400. In some embodiments, the KRAS inhibitor comprises BBP- 454.

In some embodiments, the subject can also be administered an AKT inhibitor. In some embodiments, the AKT inhibitor comprises MK-2206 2HC1, perifosine (e.g., KRX- 0401), GSK690693, rigosertib (e.g., ON-01910), ipatasertib (e.g., GDC-0068), capivasertib (e.g., AZD5363), PF-04691502, AT7867, triciribine (e.g., NSC 154020), CCT128930, A- 674563. PHT-427, miltefosine, honokiol (e.g., NSC 293100), PI3K/Akt Inhibitor library, TIC 10 analogue, demethyl-coclaurine, oroxin B, homosalate, daphnoretin, rotundic acid, cinobufagin, neferine, alobresib (e.g., GS-5829), borussertib, resibufogenin, hispidulin, lanatoside C, astragaloside IV, zeaxanthin, A-443654, uprosertib (e.g., GSK2141795), TIC10 (e.g., ONC201). akti-1/2, SC66, Dichroa febrifuga extract, MAZ51, usnic acid, Dioscoreae Nipponicae Rhizoma extract, Cinnamomi Ramulus extract, miransertib (e.g., ARQ 092) HCL lupenone, miransertib (e.g., ARQ-092), (E)-Akt inhibitor-IV, notoginsenoside Rl, Alpiniae Katsumadai extract, afuresertib (e.g., SK2110183), praeruptorin A, Weigela Grandiflora Fortune extract, LM22B-10, solasodine, pectolinarin, a-Linolenic acid, BIA, ailanthone, methyl-hesperidin, RPI-1, BAY1125976. oridonin (e.g., NSC-250682), hematein, scutellarin, ABTL-0812, ML-9 HC1, SPP-86. ATI 3148, urolithin B. trigonelline, deguelin, SC79. amarogentin, loureirin A, or YS-49, or any combination thereof. In some embodiments, the AKT inhibitor comprisesMK-2206 2HC1. In some embodiments, the AKT inhibitor comprises perifosine. In some embodiments, the AKT inhibitor comprises GSK690693. In some embodiments, the AKT inhibitor comprises rigosertib. In some embodiments, the AKT inhibitor comprises ipatasertib. In some embodiments, the AKT inhibitor comprises capivasertib. In some embodiments, the AKT inhibitor comprises PF-04691502. In some embodiments, the AKT inhibitor comprises AT7867. In some embodiments, the AKT inhibitor comprises triciribine. In some embodiments, the AKT inhibitor comprises CCT128930. In some embodiments, the AKT inhibitor comprises A-674563. In some embodiments, the AKT inhibitor comprises PHT-427. In some embodiments, the AKT inhibitor comprises miltefosine. In some embodiments, the AKT inhibitor comprises honokiol. In some embodiments, the AKT inhibitor comprises PI3K/Akt Inhibitor library. In some embodiments, the AKT inhibitor comprises TIC 10 analogue. In some embodiments, the AKT inhibitor comprises demethyl -coclaurine. In some embodiments, the AKT inhibitor comprises oroxin B. In some embodiments, the AKT inhibitor comprises homosalate. In some embodiments, the AKT inhibitor comprises daphnoretin. In some embodiments, the AKT inhibitor comprises rotundic acid. In some embodiments, the AKT inhibitor comprises cinobufagin. In some embodiments, the AKT inhibitor comprises neferine. In some embodiments, the AKT inhibitor comprises alobresib. In some embodiments, the AKT inhibitor comprises borussertib. In some embodiments, the AKT inhibitor comprises resibufogenin. In some embodiments, the AKT inhibitor comprises hispidulin. In some embodiments, the AKT inhibitor comprises lanatoside C. In some embodiments, the AKT inhibitor comprises astragaloside IV. In some embodiments, the AKT inhibitor comprises zeaxanthin. In some embodiments, the AKT inhibitor comprises A-443654. In some embodiments, the AKT inhibitor comprises uprosertib. In some embodiments, the AKT inhibitor comprises TIC 10. In some embodiments, the AKT inhibitor comprises akti-1/2. In some embodiments, the AKT inhibitor comprises SC66. In some embodiments, the AKT inhibitor comprises Dichroa febrifuga extract. In some embodiments, the AKT inhibitor comprises MAZ51. In some embodiments, the AKT inhibitor comprises usnic acid. In some embodiments, the AKT inhibitor comprises Dioscoreae Nipponicae Rhizoma extract. In some embodiments, the AKT inhibitor comprises Cinnamomi Ramulus extract. In some embodiments, the AKT inhibitor comprises miransertib HC1. In some embodiments, the AKT inhibitor comprises lupenone. In some embodiments, the AKT inhibitor comprises miransertib. In some embodiments, the AKT inhibitor comprises (E)-Akt inhibitor-IV. In some embodiments, the AKT inhibitor comprises notoginsenoside Rl. In some embodiments, the AKT inhibitor comprises Alpiniae Katsumadai extract. In some embodiments, the AKT inhibitor comprises afuresertib. In some embodiments, the AKT inhibitor comprises praeruptorin A. In some embodiments, the AKT inhibitor comprises Weigela Grandiflora Fortune extract. In some embodiments, the AKT inhibitor comprises LM22B-10. In some embodiments, the AKT inhibitor comprises solasodine. In some embodiments, the AKT inhibitor comprises pectolinarin. In some embodiments, the AKT inhibitor comprises a- Linolenic acid. In some embodiments, the AKT inhibitor comprises BIA. In some embodiments, the AKT inhibitor comprises ailanthone. In some embodiments, the AKT inhibitor comprises methyl-hesperidin. In some embodiments, the AKT inhibitor comprises RPI-1. In some embodiments, the AKT inhibitor comprises BAY1125976. In some embodiments, the AKT inhibitor comprises oridonin. In some embodiments, the AKT inhibitor comprises hematein. In some embodiments, the AKT inhibitor comprises scuteliarin. In some embodiments, the AKT inhibitor comprises ABTL-0812. In some embodiments, the AKT inhibitor comprises ML-9 HC1. In some embodiments, the AKT inhibitor comprises SPP-86. In some embodiments, the AKT inhibitor comprises ATI 3148. In some embodiments, the AKT inhibitor comprises urolithin B. In some embodiments, the AKT inhibitor comprises trigonelline. In some embodiments, the AKT inhibitor comprises deguelin. In some embodiments, the AKT inhibitor comprises SC79. In some embodiments, the AKT inhibitor comprises amarogentin. In some embodiments, the AKT inhibitor comprises loureirin A. In some embodiments, the AKT inhibitor comprises YS-49.

In some embodiments, the subj ect can also be administered an ERK inhibitor. In some embodiments, the ERK inhibitor comprises 3'-Hydroxypterostilbene, selumetinib (e.g., AZD6244), SU1498, SCH772984, alobresib (e.g., S-5829), astragaloside IV, DC260126. senkyunolide I, ASN007, cucurbitacin lib, mirdametinib (e.g., PD0325901), MK-8353 (e.g., SCH900353), AZD0364 (e.g., ATG-017), usnic acid, ezatiostat, SL-327, BM213, 3- Deazaadenosine hydrochloride, DMU-212, CGP 57380, notoginsenoside Rl, NLX-204, CC- 90003, honokiol (e.g., NSC 293100), 1-191, KO-947, doramapimod (e.g., BIRB 796), WP1066, tanzisertib (e.g., CC-930), 2',5'-Dihydroxyacetophenone, Iramelinib (e.g., GSK1120212), PD98059. magnolin, PLX7904, STAT5-IN-2, temuterkib (e.g., LY3214996), pluripotin (e.g., SCI), ulixertinib (e.g., BVD-523), methylthiouracil, pamoic acid disodium, FR 180204, AG-126, NSC87877, corynoxeine, TAK-715, agerafenib (e.g., RXDX-105), NSC95397, tracheloside, or ravoxertinib (e.g., GDC-0994), or any combination thereof. In some embodiments, the ERK inhibitor comprises 3'-Hydroxypterostilbene. In some embodiments, the ERK inhibitor comprises selumetinib. In some embodiments, the ERK inhibitor comprises SU1498. In some embodiments, the ERK inhibitor comprises SCH772984. In some embodiments, the ERK inhibitor comprises alobresib. In some embodiments, the ERK inhibitor comprises astragaloside IV. In some embodiments, the ERK inhibitor comprises DC260126. In some embodiments, the ERK inhibitor comprises senkyunolide I. In some embodiments, the ERK inhibitor comprises ASN007. In some embodiments, the ERK inhibitor comprises cucurbitacin lib. In some embodiments, the ERK inhibitor comprises mirdametinib. In some embodiments, the ERK inhibitor comprises MK- 8353. In some embodiments, the ERK inhibitor comprises AZD0364. In some embodiments, the ERK inhibitor comprises usnic acid. In some embodiments, the ERK inhibitor comprises ezatiostat. In some embodiments, the ERK inhibitor comprises SL-327. In some embodiments, the ERK inhibitor comprises BM213. In some embodiments, the ERK inhibitor comprises 3 -Deazaadenosine hydrochloride. In some embodiments, the ERK inhibitor comprises DMU-212. In some embodiments, the ERK inhibitor comprises CGP 57380. In some embodiments, the ERK inhibitor comprises notoginsenoside Rl. In some embodiments, the ERK inhibitor comprises NLX-204. In some embodiments, the ERK inhibitor comprises CC-90003. In some embodiments, the ERK inhibitor comprises honokiol. In some embodiments, the ERK inhibitor comprises 1-191. In some embodiments, the ERK inhibitor comprises KO-947. In some embodiments, the ERK inhibitor comprises doramapimod. In some embodiments, the ERK inhibitor comprises WP1066. In some embodiments, the ERK inhibitor comprises tanzisertib. In some embodiments, the ERK inhibitor comprises 2',5'-Dihydroxyacetophenone. In some embodiments, the ERK inhibitor comprises trametinib. In some embodiments, the ERK inhibitor comprises PD98059. In some embodiments, the ERK inhibitor comprises magnolin. In some embodiments, the ERK inhibitor comprises PLX7904. In some embodiments, the ERK inhibitor comprises STAT5- IN-2. In some embodiments, the ERK inhibitor comprises temuterkib. In some embodiments, the ERK inhibitor comprises pluripotin. In some embodiments, the ERK inhibitor comprises ulixertinib. In some embodiments, the ERK inhibitor comprises methylthiouracil. In some embodiments, the ERK inhibitor comprises pamoic acid disodium. In some embodiments, the ERK inhibitor comprises FR 180204. In some embodiments, the ERK inhibitor comprises AG-126. In some embodiments, the ERK inhibitor comprises NSC87877. In some embodiments, the ERK inhibitor comprises corynoxeine. In some embodiments, the ERK inhibitor comprises TAK-715. In some embodiments, the ERK inhibitor comprises agerafenib. In some embodiments, the ERK inhibitor comprises NSC95397. In some embodiments, the ERK inhibitor comprises tracheloside. In some embodiments, the ERK inhibitor comprises ravoxertinib.

In some embodiments, the subject can also be administered an ERBB inhibitor. In some embodiments, the ERBB inhibitor comprises erlotinib (e.g., OSI-774) HC1, gefitinib (e.g., ZD1839), lapatinib (e.g., GW-572016), ditosylate, afatinib (e.g., BIBW2992), saracatinib (e.g., AZD0530), vandetanib (e g., ZD6474), neratinib (e.g., HKI-272), canertinib (e.g., CI-1033), lapatinib (e.g., GW-572016). AG-490 (e.g., Tyrphostin B42), CP-724714, dacomitinib (e.g., PF-00299804), WZ4002, sapitimb (e.g., AZD8931), CUDC-101, AG-1478 (e.g., Tyrphostin AG-1478), PD153035 HC1, pehtinib (e.g., EKB-569), AC480 (e.g., BMS- 599626), AEE788 (e.g., NVP-AEE788), AP26113-analog (e.g., ALK-IN-1). OSI-420, WZ3146, HER2-Inhibitor-1, WZ8040, allitinib tosylate, rociletinib (e.g., CO-1686), genistein (e.g., NP1 031L), varhtinib. icotimb (e.g.. BPI-2009H). TAK-285. WH1-P154. daphnetm. PD168393, tyrphostin 9, CNX-2006, AG-18, licochalcone D, canertinib dihydrochloride, AG 555, BLU-945, CH7233163, AZ5104, lazertinib, osimertinib (e.g., AZD9291), CL-387785 (e.g., EKI-785), tyrphostin AG-528, AG 494. AG 556, tucatinib, olmutinib (e.g., BI 1482694). necitumumab (anti-EGFR), RG14620, panitumumab (anti-EGFR), PD153035, EBE-A22, MTX-21 1, mobocertinib (e.g., TAK788), (-)-Epigallocatechin Gallate, erlotinib (e.g., OSI-774), gefitinib-based PROTAC 3, gefitinib hydrochloride, tyrphostin AG30 (e.g., AG30), alflutinib (e.g., AST2818) mesylate, afatinib (e.g., BIBW2992) Dimaleate, zorifertinib (e.g., AZD3759), ErbB2 inhibitor, poziotinib (e.g.. HM781-36B), brigatinib (e.g., AP26113), osimertinib mesylate, TQB3804 (e.g., EGFR-IN-7), EGFR Inhibitor, naquotinib (e.g., ASP8273), chrysophanic acid, kaempferide, AV -412 free base, nazartinib (e.g., EGF816), AST-1306, (Rac)-JBJ-04-125-02, JND3229, BI-4020, norcanthandin, AG99, theliatinib (e.g., HMPL-309), AG-1557, BDTX-189, RG 13022, lifirafenib (e.g., BGB-283), lidocaine hydrochloride, sunvozertinib, cetuximab (anti-EGFR), falnidamol. pyrotinib (e.g.. SHR-1258) dimaleate, O-Demethyl-Gefitinib, butein, EAI045. NSC228155. zipalertinib, cyasterone, epertinib hydrochloride, SU5214, almonertinib (e g., HS-10296), avitinib (e.g., AC0010), EGF Rabbit Recombinant mAb, herceptin, or trastuzumab, or any combination thereof. In some embodiments, the ERBB inhibitor comprises erlotinib HC1. In some embodiments, the ERBB inhibitor comprises gefitinib. In some embodiments, the ERBB inhibitor comprises lapatinib. In some embodiments, the ERBB inhibitor comprises ditosylate. In some embodiments, the ERBB inhibitor comprises afatinib. In some embodiments, the ERBB inhibitor comprises saracatinib. In some embodiments, the ERBB inhibitor comprises vandetanib. In some embodiments, the ERBB inhibitor comprises neratinib. In some embodiments, the ERBB inhibitor comprises canertinib. In some embodiments, the ERBB inhibitor comprises lapatinib. In some embodiments, the ERBB inhibitor comprises AG-490. In some embodiments, the ERBB inhibitor comprises CP- 724714. In some embodiments, the ERBB inhibitor comprises dacomitinib. In some embodiments, the ERBB inhibitor comprises WZ4002. In some embodiments, the ERBB inhibitor comprises sapitinib. In some embodiments, the ERBB inhibitor comprises CUDC- 101. In some embodiments, the ERBB inhibitor comprises AG-1478. In some embodiments, the ERBB inhibitor comprises PD153035 HC1. In some embodiments, the ERBB inhibitor comprises pelitinib. In some embodiments, the ERBB inhibitor comprises AC480. In some embodiments, the ERBB inhibitor comprises AEE788. In some embodiments, the ERBB inhibitor comprises AP26113-analog. In some embodiments, the ERBB inhibitor comprises OSI-420. In some embodiments, the ERBB inhibitor comprises WZ3146. In some embodiments, the ERBB inhibitor comprises HER2-Inhibitor- 1. In some embodiments, the ERBB inhibitor comprises WZ8040. In some embodiments, the ERBB inhibitor comprises allitinib tosylate. In some embodiments, the ERBB inhibitor comprises rociletinib. In some embodiments, the ERBB inhibitor comprises genistein. In some embodiments, the ERBB inhibitor comprises varlitinib. In some embodiments, the ERBB inhibitor comprises icotinib. In some embodiments, the ERBB inhibitor comprises TAK-285. In some embodiments, the ERBB inhibitor comprises WHI-P154. In some embodiments, the ERBB inhibitor comprises daphnetin. In some embodiments, the ERBB inhibitor comprises PD168393. In some embodiments, the ERBB inhibitor comprises tyrphostin 9. In some embodiments, the ERBB inhibitor comprises CNX-2006. In some embodiments, the ERBB inhibitor comprises AG- 18. In some embodiments, the ERBB inhibitor comprises licochalcone D. In some embodiments, the ERBB inhibitor comprises canertinib dihydrochloride. In some embodiments, the ERBB inhibitor comprises AG 555. In some embodiments, the ERBB inhibitor comprises BLU-945. In some embodiments, the ERBB inhibitor comprises CH7233163. In some embodiments, the ERBB inhibitor comprises AZ5104. In some embodiments, the ERBB inhibitor comprises Lazertinib. In some embodiments, the ERBB inhibitor comprises Osimertinib. In some embodiments, the ERBB inhibitor comprises CL- 387785. In some embodiments, the ERBB inhibitor comprises tyrphostin AG-528. In some embodiments, the ERBB inhibitor comprises AG 494. In some embodiments, the ERBB inhibitor comprises AG 556. In some embodiments, the ERBB inhibitor comprises tucatinib. In some embodiments, the ERBB inhibitor comprises olmutinib. In some embodiments, the ERBB inhibitor comprises necitumumab (anti-EGFR). In some embodiments, the ERBB inhibitor comprises RG14620. In some embodiments, the ERBB inhibitor comprises panitumumab (anti-EGFR). In some embodiments, the ERBB inhibitor comprises PD153035. In some embodiments, the ERBB inhibitor comprises EBE-A22. In some embodiments, the ERBB inhibitor comprises MTX-211. In some embodiments, the ERBB inhibitor comprises mobocertinib. In some embodiments, the ERBB inhibitor comprises (-)-Epigallocatechin Gallate. In some embodiments, the ERBB inhibitor comprises erlotinib. In some embodiments, the ERBB inhibitor comprises gefitinib-based PROTAC 3. In some embodiments, the ERBB inhibitor comprises gefitinib hydrochloride. In some embodiments, the ERBB inhibitor comprises tyrphostin AG30. In some embodiments, the ERBB inhibitor comprises alflutinib mesylate. In some embodiments, the ERBB inhibitor comprises afatinib dimaleate. In some embodiments, the ERBB inhibitor comprises zorifertinib. In some embodiments, the ERBB inhibitor comprises ErbB2 inhibitor. In some embodiments, the ERBB inhibitor comprises poziotinib. In some embodiments, the ERBB inhibitor comprises brigatinib. In some embodiments, the ERBB inhibitor comprises osimertinib mesylate. In some embodiments, the ERBB inhibitor comprises TQB3804. In some embodiments, the ERBB inhibitor comprises EGFR inhibitor. In some embodiments, the ERBB inhibitor comprises naquotinib. In some embodiments, the ERBB inhibitor comprises chrysophanic acid. In some embodiments, the ERBB inhibitor comprises kaempferide. In some embodiments, the ERBB inhibitor comprises AV-412 free base. In some embodiments, the ERBB inhibitor comprises nazartinib. In some embodiments, the ERBB inhibitor comprises AST-1306. In some embodiments, the ERBB inhibitor comprises (Rac)-JBJ-04-125-02. In some embodiments, the ERBB inhibitor comprises JND3229. In some embodiments, the ERBB inhibitor comprises BI-4020. In some embodiments, the ERBB inhibitor comprises norcantharidin. In some embodiments, the ERBB inhibitor comprises AG99. In some embodiments, the ERBB inhibitor comprises theliatinib. In some embodiments, the ERBB inhibitor comprises AG-1557. In some embodiments, the ERBB inhibitor comprises BDTX- 189. In some embodiments, the ERBB inhibitor comprises RG 13022. In some embodiments, the ERBB inhibitor comprises lifirafenib. In some embodiments, the ERBB inhibitor comprises lidocaine hydrochloride. In some embodiments, the ERBB inhibitor comprises sunvozertinib. In some embodiments, the ERBB inhibitor comprises cetuximab (anti-EGFR). In some embodiments, the ERBB inhibitor comprises falnidamol. In some embodiments, the ERBB inhibitor comprises pyrotinib dimaleate. In some embodiments, the ERBB inhibitor comprises O-Demethyl-Gefitinib. In some embodiments, the ERBB inhibitor comprises butein. In some embodiments, the ERBB inhibitor comprises EAI045. In some embodiments, the ERBB inhibitor comprises NSC228155. In some embodiments, the ERBB inhibitor comprises zipalertinib. In some embodiments, the ERBB inhibitor comprises cyasterone. In some embodiments, the ERBB inhibitor comprises epertinib hydrochloride. In some embodiments, the ERBB inhibitor comprises SU5214. In some embodiments, the ERBB inhibitor comprises almonertinib. In some embodiments, the ERBB inhibitor comprises avitinib. In some embodiments, the ERBB inhibitor comprises EGF Rabbit Recombinant mAb. In some embodiments, the ERBB inhibitor comprises Herceptin. In some embodiments, the ERBB inhibitor comprises trastuzumab.

In some embodiments, the subject can also be administered an ABCBl(MDRl) inhibitor. In some embodiments, the ABCBl(MDRl) inhibitor comprises any one or more of the following: afatinib, dacomitinib, dovitinib, erdafitinib, erlotinib, everolimus, gefitinib, imatinib, lapatinib, neratinib, nintedanib, ponatinib, regorafenib, sirolimus, sorafenib, sunitinib, temsirolimus, vemurafenib, osimertinib, pelitinib, WZ3146, WZ4002, deforolimus, rapamycin, WYE-687, WAY-600, BEZ235, cabozantinib, canertinib, pazopanib, icotinib. abemaciclib, alectinib, avapritinib, axitinib, bosutinib, crizotinib, entrectinib, fedratinib, idelalisib, lenvatinib, mobocertinib, nilotinib, palbociclib, ripretinib, selpercatinib, sotorasib, tepotinib, tucatinib, umbralisib, upadacitinib, vandetanib, fostamatinib, voruciclib, purvalanol A, olomoucine II, roscovitine, NVP-TAE684, SNS-314, LY2603618, MP -470, masitinib, K18751, BIBW2992, TG101209, CI-1033, NVP-ADW742, NVP-BSK805, crenolanib, BI2536. GSK461364. BI6727, AZD0530, cediranib. AV-951, ceritinib, motesanib, saracatinib, vatalanib, apatinib, dasatinib, ibrutinib, linsitinib, quizartinib, tandutinib, vatalinib, telatinib, acetaminophen, alfentanil, alpelisib, amiodarone, amlodipine, amodiaquine, amoxapine, amsacrine, annamycin, arsenic trioxide, astemizole, asunaprevir, atazanavir, atorvastatin. atovaquone, azelastine, azilsartan medoxomil. azithromycin, belumosudil, benzocaine, benzquinamide, bepridil, berotralstat, bicalutamide, biricodar, bisoprolol, boceprevir, brefeldin A, bromocriptine, buprenorphine, buspirone, cabazitaxel, canagliflozin, candesartan, candesartan cilexetil, capmatinib, captopril, carfilzomib, carvedilol, caspofungin, ceftriaxone, cethromycin, cetirizine, chloroquine, chlorpromazine, chlorprothixene, cholesterol, cilazapril. citalopram, clarithromycin, clofazimine, clomipramine, clotrimazole, cobicistat, colforsin, concanamycin A, conivaptan, curcumin, cyclosporine, daclatasvir, dactinomycin, darunavir, daunorubicin, desipramine, desloratadine, desmethylsertraline, dexamethasone, dexamethasone acetate, dexniguldipine, dexverapamil, digoxin, dihydroergotamine, diltiazem, diosmin, dipyridamole, dofequidar, doxazosin, doxorubicin, dronabinol, dronedarone, duloxetine, econazole, elacridar, elagolix, elbasvir, elexacaftor, eliglustat, emopamil, enalapril, enasidenib, enzalutamide, ergometrine, ergotamine, erythromycin, esomeprazole, estramustine, etoposide, etravirine, favipiravir, felodipine, fenofibrate, fentanyl, fmgolimod, flibanserin, fluconazole, fluoxetine, flupentixol, fluphenazine, flurazepam, fluvoxamme, galantamine, galloparml, genistein, glasdegib, glecaprevir, glyburide, gramicidin D, grepafloxacin, haloperidol, HM-30181, hy canthone, hydroxychloroquine, ibuprofen, indinavir, indomethacin, infigratinib, isavuconazole, isavuconazonium. isradipine, istradefylline, itraconazole, ivacaftor, ivermectin, ivosidenib, ixabepilone. ketoconazole, lamotrigine, laniquidar. lansoprazole, lasmi ditan, ledipasvir. letermovir, levofloxacin, levoketoconazole, lidocaine, linagliptin, lomerizine, lomitapide, lonafamib, lopinavir, loratadine, losartan, lovastatin, loxapine, lumacaftor, lurbinectedin, medroxyprogesterone acetate, mefloquine, megestrol acetate, methadone, methylene blue, metronidazole, mibefradil, miconazole, mifepristone, mirabegron, mitotane, mitoxantrone, monensin, naproxen, nefazodone, nelfmavir, netupitant, nicardipine, nifedipine, niguldipine, nimodipine, nisoldipine, nitrendipine, norethisterone, norgestimate, olaparib, omeprazole, ONT-093, paclitaxel, paliperidone, pantoprazole, paritaprevir, paroxetine, pemigatinib, pibrentasvir, pimozide, piperine, polyethylene glycol, polyethylene glycol 400, posaconazole, pralsetinib, prazosin, prednisone, primaquine, progesterone, promethazine, propafenone, propranolol, protriptyline, quercetin, quinacrine, quinidine, quinine, ranitidine, ranolazine, reboxetine, relugolix, reserpine, reversin 121, rifamycin, rilpivirine, ritonavir, rolapitant, rucaparib, salinomycin, sapropterin, saquinavir, sarecycline, selegiline, sertraline, sildenafil, simeprevir, simvastatin, staurosporine, suvorexant, tacrolimus, tamoxifen, tariquidar, taurocholic acid, telaprevir, telmisartan, tenofovir disoproxil, terazosin, terfenadine, tesmilifene, testosterone, testosterone cypionate, testosterone enanthate, testosterone undecanoate, tetrandrine, tezacaftor, ticagrelor, tipifamib, tipranavir, tolvaptan, toremifene, trifluoperazine, triflupromazine, trimethoprim, troleandomycin, valinomycin, valspodar, vardenafil, velpatasvir, venetoclax, venlafaxine, verapamil, vinblastine, vincristine, voacamine, voclosporin, vorapaxar. voxilaprevir. yohimbine, zimelidine, zonisamide. zosuquidar, elagolix-estradiol norethindrone, elexacaftor-tezacaftor-ivacaftor, ombitasvir- paritaprevir-ritonavir (technivie), ritonavir and ritonavir-containing coformulations, tamoxifen, tezacaftor-ivacaftor, glecaprevir-pibrentasvir, valspdar, encequidar, XR9051, YS- 370. or any combination thereof.

In some embodiments, the subject can also be administered an RTK inhibitor. In some embodiments, the RTK inhibitor is erlotinib, afatinib, panitumumab, or cetuximab, or any combination thereof. In some embodiments, the RTK inhibitor is erlotinib. In some embodiments, the RTK inhibitor is afatinib. In some embodiments, the RTK inhibitor is panitumumab. In some embodiments, the RTK inhibitor is cetuximab.

In some embodiments, the subject can also be administered an S0S1 inhibitor. In some embodiments, the S0S1 inhibitor is BI-1701963, BI-3406, RMC-5845, or BAY-293, or any combination thereof. In some embodiments, the S0S1 inhibitor is BI-1701963. In some embodiments, the S0S1 inhibitor is BI-3406. In some embodiments, the S0S1 inhibitor is RMC-5845. In some embodiments, the S0S1 inhibitor is BAY-293.

In some embodiments, the subject can also be administered a CDK4/6 inhibitor. In some embodiments, the CDK4/6 inhibitor is palbociclib, abemaciclib, or ribociclib, or any combination thereof. In some embodiments, the CDK4/6 inhibitor is Palbociclib. In some embodiments, the CDK4/6 inhibitor is abemaciclib. In some embodiments, the CDK4/6 inhibitor is ribociclib.

In some embodiments, the subj ect can also be administered an AURKA inhibitor. In some embodiments, the AURKA inhibitor is VIC-1911.

In some embodiments, the subject can also be administered a WEE1 inhibitor. In some embodiments, the WEE1 inhibitor is adavosertib or LY3295668, or a combination thereof. In some embodiments, the WEE1 inhibitor is adavosertib. In some embodiments, the WEE1 inhibitor is LY3295668.

In some embodiments, the subj ect can also be administered an siRNA. In some embodiments, the siRNA comprises iExosomes.

In some embodiments, the subject can also be administered a cancer vaccine. In some embodiments, the cancer vaccine is mRNA-5671/V941, ELI-002, or mDC3/8-KRAS vaccine, or any combination thereof. In some embodiments, the cancer vaccine is mRNA- 5671/V941. In some embodiments, the cancer vaccine is ELI-002. In some embodiments, the cancer vaccine is mDC3/8-KRAS vaccine.

Examples of anti-cancer therapeutic agents that may be used in any of the methods described herein include, but are not limited to, alkylating agents, alkyl sulfonates, anastrozole, amanitins, aziridines, ethylenimines and methylamelamines, acetogenins. a camptothecin. BEZ-235, bortezomib. bryostatin, callystatin, CC-1065. ceritinib. crizotinib, cryptophycins, dolastatin, duocarmycin, eleutherobin, erlotinib, pancratistatin, a sarcodictyin, spongistatin, nitrogen mustards, antibiotics, enediyne dynemicin, bisphosphonates, esperamicin, chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, canfosfamide, carabicin. carminomycin, carzinophilin. chromomycinis, cyclosphosphamide, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, exemestane, fluorouracil, fulvestrant, gefitinib, idarubicin, lapatinib, letrozole, lonafamib, marcellomycin, megestrol acetate, mitomycins, mycophenolic acid, nogalamycin, olivomycins, pazopanib, peplomycin. potfiromycin, puromycin, quelamycin, rapamycin, rodorubicin, sorafenib, streptonigrin, streptozocin, tamoxifen, tamoxifen citrate, temozolomide, tepodina, tipifamib, tubercidin, ubenimex, vandetanib, vorozole, XL- 147, zinostatin, zorubicin; anti-metabolites, folic acid analogues, purine analogs, androgens, antiadrenals, folic acid replenisher such as frolinic acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfomithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, polysaccharide complex, razoxane; rhizoxin; SF-1126, sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside; cyclophosphamide; thiotepa; taxoids, chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, vinblastine; platinum; etoposide; ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan, topoisomerase inhibitor RFS 2000; difluorometlhylomithine; retinoids; capecitabine; combretastatin; leucovorin; oxaliplatin; XL518, inhibitors of PKC-alpha, Raf, H-Ras, EGFR and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts or solvates, acids or derivatives thereof Also included are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor antibodies, aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, and anti-androgens; as well as troxacitabine (a 1.3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, ribozymes such as a VEGF expression inhibitor and a HER2 expression inhibitor; vaccines, PROLEUKIN® rIL-2; LURTOTEC AN® topoisomerase 1 inhibitor; ABARELIX® rmRH; Vinorelbine and Esperamicins and pharmaceutically acceptable salts or solvates, acids or derivatives thereof.

Suitable anti-cancer agents comprise commercially or clinically available compounds such as TARCEVA® (erlotinib), TAXOTERE® (docetaxel), 5-FU (fluorouracil, 5- fluorouracil, CAS No. 51-21-8), GEMZAR® (gemcitabine), PD-0325901 (CAS No. 391210- 10-9), cisplatin (cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), TAXOL® (paclitaxel), HERCEPTIN® (trastuzumab), TEMODAR® and TEMODAL® (temozolomide; 4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo [4.3.0] nona-2,7,9- triene-9-carboxamide, CAS No. 85622-93-1), NOLVADEX®, ISTUBAL®, and VALODEX® (tamoxifen; (Z)-2-[4-( 1.2-diphenylbut- 1 -enyl)phenoxy] -N,N-dimethylethanamine), and ADRIAMYCIN® (doxorubicin). Additional commercially or clinically available anti-cancer agents comprise ELOXATIN® (oxaliplatin), VELCADE® (bortezomib), SUNITINIB® (sutent; SU11248), FEMARA® (letrozole), GLEEVEC® (imatinib mesylate), XL-518 (Mek inhibitor, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244), SF-1126 (PI3K inhibitor), BEZ-235 (PI3K inhibitor), XL-147 (PI3K inhibitor), PTK787/ZK 222584, FASLODEX® (fulvestrant), leucovorin (folinic acid), RAPAMLTNE® (rapamycin; sirolimus), TYKERB® (lapatinib; GSK572016), SARASAR™ (lonafamib, SCH 66336), NEXAVAR® (sorafenib; BAY43-9006), IRESSA® (gefitinib), CAMPTOSAR® (irinotecan; CPT-11 ), ZARNESTRA™ (tipifamib), ABRAXANE™ (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel, ZACTIMA® (vandetanib; rINN, ZD6474,), chloranmbucil, AG1478, AG1571 (SU 5271), TORISEL® (temsirolimus), pazopanib, TELCYTA® (canfosfamide), CYTOXAN® (thiotepa) and NEOSAR® (cyclosphosphamide); NAVELBINE® (vinorelbine); XELODA® (capecitabine), NOLVADEX® (tamoxifen); FARESTON® (tamoxifen citrate), MEGASE® (megestrol acetate), AROMASIN® (exemestane), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole), and AR1MIDEX® (anastrozole).

Particularly suitable anti-cancer chemotherapeutic agents include, but are not limited to, Mechlorethamine hydrochloride, Cyclophosphamide, Ifosfamide, Chlorambucil, Melphalan, Busulfan, Thiotepa (Triethylenethiophosphoramide). Carmustine, Lomustine, Streptozocin, Vincristine, Vinblastine, Paclitaxel. Methotrexate, Mercaptopurine, Thioguanine, Fluorouracil, Cytarabine, Azacitidine, Dactinomycin, Doxorubicin, Daunorubicin, Idarubicin, Bleomycin, Picamycin, Mitomycin, Hydroxyurea, Procarbazine, Dacarbazine, Cisplatin, Carboplatin, Asparaginase, Etoposide, Amsarcrine, Mitotane, Shreptozoin, Altretamine. Teniposde, Plcamydin. Fluorodeoxyuridine, CB3717, Floxuridine, Pentostatin. Cyctrabine. Fludarabine. Irinotecan, Adriamycin, Camptothecin, a-. 0-. or y- Interferon, Interleukin-2, Docetaxel, Topotecan, and Mitoxantrone, etc., or adjuvant therapies that further stimulate the immune response.

In some embodiments, the subj ect is a non-responder or resistant to treatment with an MEK inhibitor. In some embodiments, the subject is a non-responder or resistant to treatment with a RAS inhibitor. In some embodiments, the subject is a non-responder or resistant to treatment with a KRAS inhibitor. In some embodiments, the subject is anon- responder or resistant to treatment with a RAF inhibitor. In some embodiments, the subject is a non-responder or resistant to treatment with an ERK inhibitor. In some embodiments, the subject is a non-responder or resistant to treatment with a PI3K inhibitor. In some embodiments, the subj ect is a non-responder or resistant to treatment with an AKT inhibitor. In some embodiments, the subject is a non-responder or resistant to treatment with an MTOR inhibitor. In some embodiments, the subject is a non-responder or resistant to treatment with a BET bromodomain inhibitor. In some embodiments, the subject is a non-responder or resistant to treatment with an SHP2 inhibitor. In some embodiments, the subject is a non- responder or resistant to treatment with an SOS inhibitor. In some embodiments, the subject is a non-responder or resistant to treatment with an RTK inhibitor.

In some embodiments, the pharmaceutical composition is an oral dosage formulation, an intravenous dosage formulation, a topical dosage formulation, an intraperitoneal dosage formulation, or an intrathecal dosage form. In some embodiments, the pharmaceutical composition is an oral dosage formulation. In some embodiments, the pharmaceutical composition is an intravenous dosage formulation. In some embodiments, the pharmaceutical composition is a topical dosage formulation. In some embodiments, the pharmaceutical composition is an intrapentoneal dosage formulation. In some embodiments, the pharmaceutical composition is an intrathecal dosage form.

In some embodiments, the pharmaceutical composition is an oral dosage formulation in the form of a pill, tablet, capsule, cachet, gel-cap, pellet, powder, granule, or liquid.

In some embodiments, the pharmaceutical composition the oral dosage formulation is protected from light and present within a blister pack, bottle, or intravenous bag.

The compounds and compositions described herein can be formulated for parenteral administration by injection, such as by bolus injection or continuous infusion. The compounds and compositions can be administered by continuous infusion subcutaneously over a period of about 15 minutes to about 24 hours. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulary agents such as suspending, stabilizing and/or dispersing agents. In some embodiments, the injectable is in the form of short-acting, depot, or implant and pellet forms injected subcutaneously or intramuscularly. In some embodiments, the parenteral dosage form is the form of a solution, suspension, emulsion, or dry powder.

For oral administration, the compounds and compositions described herein can be formulated by combining the compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, emulsions, liquids, gels, syrups, caches, pellets, powders, granules, slurries, lozenges, aqueous or oily suspensions, and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by, for example, adding a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fdlers such as sugars, including, but not limited to, lactose, sucrose, mannitol, and sorbitol; cellulose preparations including, but not limited to, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, including, but not limited to, the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Orally administered compositions can contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. Moreover, when in tablet or pill form, the compositions may be coated to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compounds. Oral compositions can include standard vehicles such as, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such vehicles are suitably of pharmaceutical grade.

Dragee cores can be provided with suitable coatings. For this purpose, concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include, but are not limited to, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added.

For buccal administration, the compositions can take the form of. such as, tablets or lozenges formulated in a conventional manner.

For administration by inhalation, the compounds and compositions described herein can be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, such as gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

In transdermal administration, the compounds and compositions can be applied to a plaster, or can be applied by transdermal, therapeutic systems that are consequently supplied to the organism. In some embodiments, the compounds and compositions are present in creams, solutions, powders, fluid emulsions, fluid suspensions, semi-solids, ointments, pastes, gels, jellies, and foams, or in patches containing any of the same.

The compounds and compositions described herein can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Depot injections can be administered at about 1 to about 6 months or longer intervals. Thus, for example, the compounds and compositions can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

In some embodiments, the compounds and compositions can be delivered in a controlled release system. In some embodiments, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng., 1987, 14, 201; Buchwald et al., Surgery, 1980, 88, 507 Saudek et al., N. Engl. J. Med., 1989, 321, 574). In some embodiments, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger et al., J. Macromol. Sci. Rev. Macromol. Chem., 1983, 23, 61; see, also Lexy et al., Science, 1985, 228, 190; During et al., Ann. Neurol., 1989, 25, 351; Howard et al., J. Neurosurg., 1989, 71, 105). In some embodiments, a controlled-release system can be placed in proximity of the target of the compounds and compositions described herein, such as the liver, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems discussed in the review by Langer. Science, 1990, 249, 1527-1533) may be used.

The compounds and compositions described herein can be contained in formulations with pharmaceutically acceptable diluents, fillers, disintegrants, binders, lubricants, surfactants, hydrophobic vehicles, water soluble vehicles, emulsifiers, buffers, humectants, moisturizers, solubilizers, preservatives and the like. The pharmaceutical compositions can also comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to. calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. In some embodiments, the compounds described herein can be used with agents including, but not limited to, topical analgesics (e.g., lidocaine), barrier devices (e.g., GelClair), or rinses (e.g., Caphosol). Pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. The pharmaceutical carriers can also be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used.

In some embodiments, the compounds and compositions described herein can be delivered in a vesicle, in particular a liposome (see, Langer, Science, 1990, 249, 1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

The amount of compound to be administered may be that amount which is therapeutically effective. The dosage to be administered may depend on the characteristics of the subject being treated, e.g., the particular animal treated, age, weight, health, types of concurrent treatment, if any, and frequency of treatments, and on the nature and extent of the disease, condition, or disorder, and can be easily determined by one skilled in the art (e.g., by the clinician). The selection of the specific dose regimen can be selected or adjusted or titrated by the clinician according to methods know n to the clinician to obtain the desired clinical response. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions may also depend on the route of administration, and should be decided according to the judgment of the practitioner and each patient’s circumstances. In some embodiments, the amount of any of the compounds described herein to be administered to a subject can be from about 1 mg to about 1000 mg, from about 10 mg to about 900 mg, from about 50 mg to about 750 mg, from about 100 mg to about 500 mg. from about 200 mg to about 400 mg, or from about 250 mg to about 350 mg.

The compounds and compositions described herein can be administered by any route of administration including, but not limited to, oral, sublingual, buccal, rectal, intranasal, inhalation, eye drops, ear drops, epidural, intracerebral, intracerebroventricular, intrathecal, epicutaneous or transdermal, subcutaneous, intradermal, intravenous, intraarterial, intraosseous infusion, intramuscular, intracardiac, intraperitoneal, intravesical infusion, and intravitreal. In some embodiments, the administration is oral, sublingual, buccal, rectal, intranasal, inhalation, eye drops, or ear drops. In some embodiments, the administration is oral, sublingual, buccal, rectal, intranasal, or inhalation. In some embodiments, the administration is epidural, intracerebral, intracerebroventricular, or intrathecal. In some embodiments, the administration is epicutaneous or transdermal, subcutaneous, or intradermal. In some embodiments, the administration is intravenous, intraarterial, intraosseous infusion, intramuscular, intracardiac, intraperitoneal, intravesical infusion, or intravitreal. In some embodiments, the administration is intravenous, intramuscular, or intraperitoneal. The route of administration can depend on the particular disease, disorder, or condition being treated and can be selected or adjusted by the clinician according to methods known to the clinician to obtain desired clinical responses. Methods for administration are known in the art and one skilled in the art can refer to various pharmacologic references for guidance (see, for example. Modem Pharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman & Gilman’s The Pharmaceutical Basis of Therapeutics, 6th Edition, MacMillan Publishing Co., New' York (1980)).

In some embodiments, it may be desirable to administer one or more compounds, or a pharmaceutically acceptable salt thereof, or compositions comprising the same, to a particular area in need of treatment. This may be achieved, for example, by local infusion (for example, during surgery), topical application (for example, with a wound dressing after surgery), by injection (for example, by depot injection), catheterization, by suppository⁷, or by an implant (for example, where the implant is of a porous, non-porous, or gelatinous material, including membranes, such as silastic membranes, or fibers). Formulations for injection can be presented in unit dosage form, such as in ampoules or in multi-dose containers, with an added preservative.

The present disclosure also provides dual RAF/MEK degraders for use in treating human diseases that require RAF-MEK. The dual RAF/MEK degraders can be any of the dual RAF/MEK degraders described herein. The human diseases that require RAF-MEK can be any of the human diseases that require RAF-MEK described herein.

The present disclosure also provides dual RAF/MEK degraders for use in the preparation of a medicament for treating human diseases that require RAF-MEK. The dual RAF/MEK degraders can be any of the dual RAF/MEK degraders described herein. The human diseases that require RAF-MEK can be any of the human diseases that require RAF- MEK described herein.

In order that the subject matter disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the claimed subject matter in any manner. Throughout these examples, molecular cloning reactions, and other standard recombinant DNA techniques, were carried out according to methods described in Maniatis et al., Molecular Cloning - A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989), using commercially available reagents, except where otherwise noted.

Examples

Example 1: General Methodology

Cell culture and drug treatment. For short-term growth assays, 1000-5000 cells were plated per well in 96-well plates and allowed to adhere and equilibrate overnight. Drug was added the following morning and after 120 hours of drug treatment, cell viability was assessed using the CellTiter-Glo Luminescent cell viability' assay according to the manufacturer's instructions (Promega). Samples were run in biological triplicates (N=3) and Student’s t-tests were performed for statistical analyses and p values < 0.05 were considered significant. For long term colony formation assays, cells were plated in 24-well dishes (1000- 5000 cells per well) and incubated overnight before continuous drug treatment for 2 weeks, with drug and medium replenished twice weekly. Following the final treatment, cells were rinsed with PBS and fixed with chilled methanol for 10 minutes at -20°C. Methanol was removed by aspiration, and cells were stained with 0.5% crystal violet in 20% methanol for 1 hour at room temperature. Samples were run in biological triplicates (N=3).

Immunoblotting/Quantitation. Samples w ere harvested in lysis buffer (50 mM HEPES (pH 7.5), 0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM sodium fluoride, 2.5 mM sodium orthovanadate, IX protease inhibitor cocktail (Roche), and 1% each of phosphatase inhibitor cocktails 2 and 3 (Sigma)). Particulate was removed by centrifugation of lysates at 21,000 rpm for 15 minutes at 4°C. Lysates w ere subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) chromatography and transferred to PVDF membranes before western blotting with primary antibodies. Secondary HRP-anti-rabbit and HRP-anti-mouse were obtained from ThermoFisher Scientific. SuperSignal West Pico and Femto Chemiluminescent Substrates (Thermo) were used to visualize blots. Western blot images were quantified using the Analyze>Gels function in Image J open-source software (National Institutes of Health). Optical density values for total protein levels were normalized by GAPDH. DC50 (the concentration where 50% of protein has been degraded) plots and values were calculated using PRISM software. Samples were run in biological triplicates (N=3) and Student’s t-tests were performed for statistical analyses and P values < 0.05 were considered significant.

VHL/MEK1/MEK2 knockdown. Small interfering RNAs (siRNA) transfections were performed using 25 nM siRNA duplex and the reverse transfection protocol. 3000-5000 cells per well were added to 96-well plates with media containing the VHL, MEK1, or MEK2 siRNA and transfection reagent (Lipofectamine RNAiMax) according to the manufacturer’s instructions. Cells were allowed to grow for 120 hours after transfection prior to CellTiter- Glo (Promega) analysis. Two to three independent experiments were performed with each cell line and siRNA. Samples were run in biological triplicates (N=3) and Student’s t-tests were performed for statistical analyses and P values < 0.05 w ere considered significant. For Western blot (immunoblotting) studies, the same procedure was performed with volumes and cell numbers proportionally scaled to a 60 mm or 10 cm dish, and cells were collected 72 hours after transfection.

Proteomics /pathway analysis. Single Run Total Proteomics and Nano LC MS/MS: Parental or PROT AC -resistant cells were lysed in a buffer containing 50 mM HEPES pH 8.0 + 4% SDS, and 100 ig of protein was digested using LysC for 3 hours and try psin overnight. Digested peptides were isolated using C-18 and PGC columns, then dried and cleaned with ethyl acetate. Three pg of proteolytic peptides were resuspended in 0.1% formic acid and separated with a Thermo Scientific RSTC nano Ultimate 3000 LC on a Thermo Scientific Easy-Spray C-18 PepMap 75 pm x 50 cm C-18 2 pm column. A 305 minute gradient of 2- 20% (180 minute) 20%-28% (45 minute) 28%-48% (20 minute) acetonitrile with 0.1% formic acid was run at 300 nL/min at 50°C. Eluted peptides were analyzed by Thermo Scientific Q Exactive or Q Exactive plus mass spectrometers utilizing a top 15 methodology in which the 15 most intense peptide precursor ions were subjected to fragmentation. The AGC for MSI was set to 3xl0⁶ with a max injection time of 120 ms, the AGC for MS2 ions was set to 1x10⁵ with a max injection time of 150 ms, and the dynamic exclusion was set to 90 s.

Proteomics Data Processing: Raw data analysis of LFQ experiments was performed using MaxQuant software 1.6.0. 1 and searched using Andromeda 1.5.6.0 against the Swiss- Prot human protein database (downloaded on April 24. 2019, 20402 entries). The search was set up for full tryptic peptides with a maximum of two missed cleavage sites. All settings were default and searched using acetylation of protein N-terminus and oxidized methionine as variable modifications. Carbamidomethylation of cysteine was set as fixed modification. The precursor mass tolerance threshold was set at 10 ppm and maximum fragment mass error was 0.02 Da. LFQ quantitation was performed using MaxQuant with the following parameters; LFQ minimum ratio count: Global parameters for protein quantitation were as follows: label minimum ratio count: 1, peptides used for quantitation: unique, only use modified proteins selected and with normalized average ratio estimation selected. Match between runs was employed for LFQ quantitation and the significance threshold of the ion score was calculated based on a false discovery rate of < 1%. MaxQuant normalized LFQ values were imported into Perseus software (1.6.2.3) and filtered in the following manner: Proteins identified by site only were removed, reverse, or potential contaminant were removed then filtered for proteins identified by >1 unique peptide. Protein LFQ values were log2 transformed, filtered for a minimum percent in runs (100%), annotated, and subjected to a Student's /-test with comparing PROT AC -resistant cells vs. parental cells. Samples were run in biological triplicates (N=3). Parameters for the Student's /-test were the following: S0=2, two-sided using Permutation-based FDR <0.05. Volcano plots depicting differences in protein abundance were generated using R studio software and Prism graphics. Proteins repressed by MS934 or PD0325901 treatment (FDR<0.05) were imported into Metascape for pathway analysis.

Example 2: MS934-Treatment Reduces CRAF, MEK1 and MEK2 Protein Levels in Cancer Cell Lines

To explore the effect of MEK1 and MEK2 degradation on KRAS mutant cells, a KRAS mutant pancreatic cancer cell line. PANC-1, was treated with increasing concentrations of PD0325901 or MS934 for 24 hours and MEK1 and MEK2 protein levels were assessed, as well as other MAPK signaling components, by immunoblotting. A reduction in MEK1 and MEK2 protein levels was observed, as well as active phosphorylated MEK1/2(S217/S223) form with increasing doses of MS934 but not PD0325901 (see, FIGS. 1 A and IB). Tn contrast, elevated levels of p-MEKl/2(S217/S223) were observed with increasing doses of PD0325901, consistent with feedback-induced activation of CRAF following MEK-ERK pathway blockade. Surprisingly, a reduction in active phosphory lated CRAF(S338) and total CRAF protein levels in response to MS934 treatment in PANC-1 cells was observed that was not observed with PD0325901 (see, FIGS. 1A and IB). Quantitation of immunoblots revealed that the DC50 (the concentration at which 50% of the target protein is degraded) of MS934 for MEK1 and MEK2 was 0.01 pM, while phosphory lated CRAF(S338) and MEK1/2(S217/S223) bands were reduced by half at 0.15 pM and 0.2 pM MS934. respectively, while the CRAF total proteins were reduced by 50% at a concentration of 0.4 pM MS934 (see, FIG. 1C). Notably, ARAF and BRAF protein levels were not reduced by MS934-treatment for 24 hours but rather showed an increase in protein levels at lower concentrations of MS934.

Next, studies explored whether MS934-treatment reduced CRAF and MEK1/2 protein levels in additional cancer cell lines including KRAS mutant cell lines (SKCO1, NCI- H23, Capan-1, Calu-1, HPAF-II, HCT-116, OVCAR8), a BRAF(V600E) mutant cell line (HT29), as well as a KRAS/BRAF wild-type cell line (LIM1215) (see, FIGS. 2A, 2B, 2C. 2D, 2E, 2F, 2G, 2H, and 21). Notably, a dose dependent reduction in the active phosphorylated CRAF(S338) and MEK1/2(S217/S223) forms, as well as CRAF, MEK1 and MEK2 total protein levels was observed following MS934-treatment for 24 hours in all the cell lines tested. Quantitation of the concentration of MS934 required to reduce CRAF by half across the cancer cell lines ranged from 0. 1 to 0.8 pM of MS934 with a median of 0.5 pM of MS934 (see, FIG. 2J). Together, these findings showed that MS934-treatment reduced CRAF protein levels in addition to the intended degrader-targets MEK1 and MEK2 in KRAS mutant, BRAFV600E and KRAS/BRAF-wild-type cancer cell lines.

Example 3: MS934-Mediated CRAF Protein Degradation Occurs via a PROTAC- Mechanism

To determine if CRAF protein reduction by MS934-treatment was through a PROTAC -mechanism, VHL knockdown was performed, proteasome machinery was inhibited using bortezomib or cells were treated with excess PD0325901 in the presence or absence of MS934. Notably, siRNA-mediated knockdown of VHL rescued MEK1, MEK2 and CRAF protein levels, establishing that CRAF degradation required the VHL E3 ligase activity of MS934 (see, FIGS. 3 A and 3B). Moreover, inhibiting the proteasome machinery in the presence of MS934 w ith the proteasome inhibitor, bortezomib, restored MEK1 , MEK2 and CRAF protein levels (see, FIGS. 3C and 3D). In addition, treating cells with 100-fold excess of the warhead PD0325901 in the presence of MS934 outcompeted MS934-binding to targets, restoring MEK1, MEK2 and CRAF protein levels (see, FIGS. 3C and 3D). Furthermore, an increase in CRAF ubiquitination was observed in PANC-1 cells treated with MS934 and bortezomib, consistent with MS934-mediated degradation of CRAF occurring by a PROTAC mechanism (see, FIGS. 3E and 3F).

Example 4: Relief of Negative Feedback Induces CRAF-MEK Interactions Causing Collateral Degradation of CRAF by MS934

It has previously been shown that CRAF binds directly to MEK following PD0325901 -treatment in KRAS mutant cells forming a protein complex due to relief of a negative feedback loop that occurs in cancer cells following inhibition of MEK-ERK activity (Lito et al., Cancer Cell, 2014, 25, 697-710). Consistent with these findings, this study show ed that treatment of PANC-1 cells with the warhead of MS934, PD0325901, for 3 hours induced the CRAF-MEK1 interaction (see, FIGS. 4A and 4B). To test whether the interaction of CRAF and MEK1/2 was required for CRAF degradation by MS934, a knockdown of MEK1 and MEK2 in MS934-treated PANC-1 cells was performed. Importantly, the study show ed that knockdown of MEK1 and MEK2 in PANC-1 cells does not cause reduced CRAF protein levels, signifying that loss of MEK1/2 alone does not result in concurrent loss of CRAF proteins (see, FIGS. 4C and 4D). Moreover, the study showed that the degradation of CRAF protein by MS934 required MEK.1/2 proteins, whereby siRNA-mediated knockdown of MEK1/2 prevented the degradation of CRAF by MS934-treatment (see, FIGS. 4C and 4D). These findings also demonstrate that MS934 does not bind directly to CRAF. Finally, it was observed that inhibition of CRAF using the inhibitor, RAF709, restored CRAF protein levels but not MEK1 or MEK2 in MS934-treated PANC-1 cells, suggesting RAF709 prevents CRAF-MEK interactions blocking collateral degradation of CRAF by MS934 (see, FIGS. 4E, 4F, 4G, and 4H). Together, these findings establish that the collateral degradation of CRAF by MS934 requires the feedback induced CRAF-MEK complex formation that occurs following blockade of MEK-ERK activity.

Example 5: MS934-Treatment Reduces Both CRAF Kinase-Dependent and Independent Signaling Providing Superior Growth Inhibition in KRAS Mutant Cells

CRAF has been shown to have kinase-independent growth and survival functions outside the MEK-ERK pathway in KRAS mutant cells, making CRAF degradation an attractive therapeutic avenue (McCormick et al., Cancer Cell, 2018, 33, 158-159). To explore the difference between MEK inhibition and CRAF/MEK degradation on KRAS signaling, PANC-1 cells were treated with PD0325901 or MS934 and proteomics followed by pathw ay analysis were completed. The studies revealed that CRAF/MEK degradation reduced the expression of 348 proteins, while PD0325901-treatement reduced 94 proteins (see, FIG. 5A). demonstrating MS934-treatment exhibited a more pronounced effect on protein abundance than MEK inhibition. Metascape pathway analysis of proteins reduced by MEK inhibitor or CRAF/MEK degradation showed MS934-treatment exhibited a more potent reduction in the PLK1 pathway, signaling by Rho GTPases, and S phase signaling compared to PD0325901- treated PANC-1 cells (see, FIG. 5B). These findings are consistent with established kinase independent functions of CRAF, where prior studies have showor CRAF to signaling through PLK1. ROCK2 and CHEK2 signaling (McCormick et al., Cancer Cell, 2018, 33, 158-159). Moreover. MS934-treatment uniquely reduced pathways associated with chromatin organization, protein localization to organelle, regulation of attachment of spindle microtubules to kinetochore, Fanconi pathway, establishment of organelle localization, Human T-cell leukemia virus 1 infection, Retinoblastoma gene in cancer, and mitochondrial gene expression (see, FIG. 5B). Finally, transcription factors predicted to be reduced by treatments showed that MS934-treatment but not PD0325901-treatment uniquely reduced MYC signaling, an established driver in KRAS mutant pancreatic cancer (see. FIG. 5C). Treatment of KRAS mutant cell lines with MS934 reduced colony formation, with the majority of colony grow th inhibited by 1 pM MS934-treatment (see, FIG. 6). Notably, PANC-1 cells have been previously shown to be intrinsically resistant to MEK inhibitor PD0325901 -treatment (Zhong et al., PLoS One, 2013, 8. e77243), signifying MS934 causes growth inhibition of both MEKi-sensitive and MEKi-resistant KRAS mutant cell lines.

Example 6: ARAF, BRAF and CRAF Protein Levels Reduced at Higher Concentrations ofMS934

To determine if MS934-treatment altered levels of the RAF family (ARAF, BRAF and CRAF), cancer cell lines were treated with increasing doses of MS934. It was observed that treatment of cancer cell lines showed reduced levels of all three RAF members, ARAF, BRAF and CRAF at higher concentrations (10 pM). Notably, CRAF protein levels were reduced by about 50% at concentrations < 1 pM. demonstrating that MS934 exhibits selectivity for degrading CRAF at concentrations < 1 pM. Interestingly, a reduction in BRAF(V600E) protein levels was observed in HT-29 cells, suggesting MS934 could be beneficial in melanoma and colorectal cancers driven by activated BRAF by degrading both MEK1/2 and BRAF.

Various modifications of the described subject matter, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference (including, but not limited to, journal articles, U.S. and non-U. S. patents, patent application publications, international patent application publications, gene bank accession numbers, and the like) cited in the present application is incorporated herein by reference in its entirety⁷.

Claims

CLAIMS What Is Claimed Is:

1. A method of treating a human disease that requires RAF -Mitogen- Activated Protein Kinase Kinase (RAF-MEK) in a subject in need thereof, the method comprising administering a dual RAF/MEK degrader to the subj ect.

2. The method of claim 1, wherein the human disease is a cancer having an alteration in a receptor-tyrosine kinase.

3. The method of claim 1, wherein the human disease is a RAS-altered cancer.

4. The method of claim 3, wherein the RAS-altered cancer is a KRAS-altered cancer.

5. The method of claim 3, wherein the RAS-altered cancer is an NRAS-altered cancer.

6. The method of claim 3, wherein the RAS-altered cancer is an HRAS-altered cancer.

7. The method of claim 3, wherein the RAS-altered cancer is non-small lung cancer, breast cancer, prostate cancer, ovarian cancer, testicular cancer, colon cancer, colorectal cancer, renal cancer, bladder cancer, pancreatic cancer, glioblastoma, neuroblastoma, retinoblastoma, leukemia, melanoma, kidney cancer, or osteosarcoma.

8. The method of claim 3, wherein the RAS-altered cancer is colon cancer, colorectal cancer, non-small lung cancer, or pancreatic cancer.

9. The method of claim 1, wherein the human disease is a RAF-altered cancer.

10. The method of claim 9, wherein the RAF-altered cancer is a BRAF mutant cancer.

11. The method of claim 10. wherein the BRAF mutant cancer is BRAF(V600E) cancer.

12. The method of claim 11. wherein the BRAF(V600E) cancer is melanoma or colorectal cancer.

13. The method of claim 9, wherein the RAF-altered cancer is a CRAF-altered cancer.

14. The method of claim 1, wherein the human disease is an MEK-altered cancer.

15. The method of claim 1, wherein the human disease is an ERK-altered cancer.

16. The method of claim 1, wherein the human disease is a RASopathy.

17. The method of claim 16, wherein the RASopathy is Noonan syndrome.

18. The method of claim 1, wherein the human disease is NF1 syndrome.

19. The method of claim 1, wherein the human disease is NF2 syndrome.

20. The method of claim 1, wherein the human disease is syndrome neurofibromatosis

Type 1, Type 2, or Type 3 (Schwannomatosis).

21. The method of claim 1, wherein the human disease is CoQ-deficiency kidney disease.

22. The method of any one of claims 1 to 21, wherein the dual RAF/MEK degrader is an ARAF/MEK degrader.

23. The method of any one of claims 1 to 21, wherein the dual RAF/MEK degrader is a BRAF/MEK degrader.

24. The method of any one of claims 1 to 21, wherein the dual RAF/MEK degrader is a CRAF/MEK degrader.

25. The method of claim 24, wherein the CRAF/MEK degrader comprises MS934, MS432, MS910, or MS928, or any combination thereof.

26. The method of claim 24, wherein the CRAF/MEK degrader comprises MS934.

27. The method of any one of claims 1 to 26, wherein the subject is also administered an MEK inhibitor, a BRAF inhibitor, a P13K inhibitor, an MTOR inhibitor, an SHP2 inhibitor, a KRAS inhibitor, an AKT inhibitor, an ERK inhibitor, an ERBB inhibitor, an ABCBl(MDRl) inhibitor, a CXCR1/2 inhibitor, a PD-L1/PD-1 inhibitor, an RTK inhibitor, an SOS1 inhibitor, a CDK4/6 inhibitor, an AURKA inhibitor, a WEE1 inhibitor, or an mTORC l inhibitor, a BET bromodomain protein inhibitor, an HD AC inhibitor, or any combination thereof.

28. The method of claim T1 , wherein the MEK inhibitor comprises trametinib, cobimetinib, binimetinib, mirdametinib, TAK733, selumetinib, refametinib, pimasertib, PD98059. U0126. Ro 09-2210. CI-1040. PD0325901, R04987655, R05126766. GDC-0623, G-573, E6201, AZD8330, or WX-554, or any combination thereof.

29. The method of claim 28, wherein the MEK inhibitor comprises PD0325901.

30. The method of any one of claims 1 to 26, wherein the subject is also administered an siRNA or a cancer vaccine.

31. The method of any one of claims 1 to 30, wherein the subject is a non-responder or is resistant to treatment with an MEK inhibitor, a KRAS inhibitor, a RAF inhibitor, an ERK inhibitor, a PI3K inhibitor, an AKT inhibitor, an MTOR inhibitor, a BET bromodomain inhibitor, an SHP2 inhibitor, an SOS inhibitor, or an RTK inhibitor.

32. A dual RAF/Mitogen-Activated Protein Kinase Kinase (RAF/MEK) degrader for use in treating a human disease that requires RAF-Mitogen- Activated Protein Kinase Kinase (RAF-MEK).