HK40032701B - Diagnostic and therapeutic methods for cancer - Google Patents

Description

Diagnosis and treatment of cancer

sequence list

This application contains a sequence list, which has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on August 9, 2018, is named 50474-171WO2_Sequence_Listing_8.09.18_ST25 and is 1,738 bytes in size.

Invention Field

This invention relates to diagnostic and therapeutic methods for treating proliferative cell disorders (such as cancer) using pan-RAF dimer inhibitors and MEK or PI3K inhibitors. Related kits and compositions are also provided.

Background of the Invention

Cancer remains one of the deadliest threats to human health. Some cancers can grow and metastasize rapidly and uncontrollably, making timely detection and treatment extremely difficult. In the United States, cancer afflicts nearly 1.3 million new patients each year and is the second leading cause of death after heart disease, accounting for approximately one in four deaths. The mitogen-activated protein kinase (MAPK) signaling pathway is activated in more than 30% of human cancers, most commonly via oncogenic mutations in the MEK/ERK branch of this pathway.

Therefore, there is still a need to develop improved alternatives for the diagnosis and treatment of patient populations with MAPK-dysregulated tumors who may be best suited for treatment targeting the MAPK signaling pathway.

Invention Overview

This invention provides diagnostic and treatment methods, kits, and compositions for treating proliferative cell disorders (such as cancer).

In a first aspect, the invention is characterized by a method for identifying an individual with cancer who may benefit from treatment including pan-RAF dimer inhibitors and MEK inhibitors, the method comprising screening a sample from the individual for KRAS-G13D mutations, wherein the presence of a KRAS-G13D mutation in the sample identifies the individual as an individual who may benefit from treatment including pan-RAF dimer inhibitors and MEK inhibitors.

In a second aspect, the invention is characterized by a method for selecting a treatment for an individual with cancer, the method comprising screening a sample from the individual for a KRAS-G13D mutation, wherein the presence of a KRAS-G13D mutation in the sample identifies the individual as potentially eligible for treatment, including pan-RAF dimer inhibitors and MEK inhibitors.

In some embodiments of the first or second aspect, the individual has a KRAS-G13D mutation and the method further includes administering a therapeutically effective amount of a pan-RAF dimer inhibitor and a MEK inhibitor to the individual.

In a third aspect, the invention is characterized by a method for treating an individual with cancer, the method comprising: (a) screening a sample from the individual for a KRAS-G13D mutation, wherein the individual has been identified as having a KRAS-G13D mutation, and (b) administering a therapeutically effective amount of a pan-RAF dimer inhibitor and a MEK inhibitor to the individual based on the presence of the KRAS-G13D mutation identified in step (a).

In a fourth aspect, the invention is characterized by a method for treating an individual with cancer, the method comprising administering to the individual a therapeutically effective amount of a pan-RAF dimer inhibitor and a MEK inhibitor, wherein a sample from the individual has been screened for KRAS-G13D mutations prior to treatment and the presence of KRAS-G13D mutations in the sample has been determined.

In some embodiments of any of the foregoing aspects, the screening includes amplification and sequencing of the entire or a portion of the KRAS gene. In some embodiments, the portion of the KRAS gene is exon 2 of the KRAS gene. In some embodiments, a KRAS c.38G>A nucleotide substitution mutation at codon 13 of exon 2 of the KRAS gene indicates a KRAS-G13D mutation.

In a fifth aspect, the invention is characterized by a composition comprising a pan-RAF dimer inhibitor and a MEK inhibitor for use in the therapeutic treatment of cancers characterized by KRAS-G13D mutations.

In a sixth aspect, the invention is characterized by the use of a composition comprising a pan-RAF dimer inhibitor and a MEK inhibitor in the preparation of a medicament for the therapeutic treatment of cancers characterized by KRAS-G13D mutations.

In a seventh aspect, the invention is characterized by a method for identifying an individual with cancer who may benefit from treatment including pan-RAF dimer inhibitors and MEK inhibitors, the method comprising screening a sample from the individual for NRAS activating mutations, wherein the presence of NRAS activating mutations in the sample identifies the individual as an individual who may benefit from treatment including pan-RAF dimer inhibitors and MEK inhibitors.

In an eighth aspect, the invention is characterized by a method for selecting a treatment for an individual with cancer, the method comprising screening a sample from the individual for NRAS activating mutations, wherein the presence of NRAS activating mutations in the sample identifies the individual as potentially eligible for treatment, including pan-RAF dimer inhibitors and MEK inhibitors.

In some embodiments of the seventh or eighth aspect, the individual has an activating mutation in NRAS and the method further includes administering a therapeutically effective amount of a pan-RAF dimer inhibitor and a MEK inhibitor to the individual.

In a ninth aspect, the invention is characterized by a method for treating an individual with cancer, the method comprising: (a) screening a sample from the individual for NRAS activating mutations, wherein the individual has been identified as having NRAS activating mutations, and (b) administering therapeutically effective amounts of a pan-RAF dimer inhibitor and a MEK inhibitor to the individual based on the presence of the NRAS activating mutation identified in step (a).

In a tenth aspect, the invention is characterized by a method for treating an individual with cancer, the method comprising administering to the individual a therapeutically effective amount of a pan-RAF dimer inhibitor and a MEK inhibitor, wherein prior to treatment, a sample from the individual has been screened for NRAS activating mutations and the presence of NRAS activating mutations in the sample has been determined.

In some embodiments of any of the seventh, eighth, ninth, and tenth aspects, the screening includes amplification and sequencing of the entire or a portion of the NRAS gene.

In some embodiments of any of the first, second, third, fourth, seventh, eighth, ninth, and tenth aspects, the MEK inhibitor is a small molecule inhibitor. In some embodiments, the small molecule inhibitor is selected from the group consisting of cobimetinib (GDC-0973), selumetinib (AZD6244), pimasetinib (AS-703026), PD0325901, remetinib (BAY86-9766), binimetinib (MEK162), BI-847325, trametinib, GDC-0623, G-573, and CH5126766 (RO5126766), or pharmaceutically acceptable salts thereof. In some embodiments, the small molecule inhibitor is cobimetinib (GDC-0973), selmetinib (AZD6244), pimecrolimus (AS-703026), PD0325901, remectinib (BAY86-9766), or bimetinib (MEK162), or a pharmaceutically acceptable salt thereof. In some embodiments, the small molecule inhibitor is cobimetinib (GDC-0973), or a pharmaceutically acceptable salt thereof.

In an eleventh aspect, the invention features a method for treating an individual with cancer including a KRAS activating mutation, the method comprising administering to the individual a therapeutically effective amount of a pan-RAF dimer inhibitor and a PI3K inhibitor. In some embodiments, the PI3K inhibitor is a small molecule inhibitor. In some embodiments, the small molecule inhibitor is selected from the group consisting of pictilisib (GDC-0941), taselisib (GDC-0032), and alpelisib (BYL719), or a pharmaceutically acceptable salt thereof. In some embodiments, the PI3K inhibitor is a pan-PI3K inhibitor. In some embodiments, the pan-PI3K inhibitor is pictilisib (GDC-0941) or taselisib (GDC-0032), or a pharmaceutically acceptable salt thereof. In some embodiments, the individual does not have a BRAF activating mutation.

In some embodiments of any of the first, second, third, fourth, seventh, eighth, ninth, tenth, and eleventh aspects, the method further includes administering an additional therapeutic agent to the individual. In some embodiments, the additional therapeutic agent is selected from the group consisting of immunotherapeutic agents, cytotoxic agents, growth inhibitors, radiotherapy agents, and anti-angiogenic agents.

In a twelfth aspect, the invention is characterized by a composition comprising a pan-RAF dimer inhibitor and a PI3K inhibitor for use in the therapeutic treatment of cancers characterized by KRAS activating mutations.

In a thirteenth aspect, the invention is characterized by the use of a composition comprising a pan-RAF dimer inhibitor and a PI3K inhibitor in the preparation of a medicament for the therapeutic treatment of cancers characterized by KRAS activating mutations.

In a fourteenth aspect, the invention features a composition comprising a pan-RAF dimer inhibitor and a pan-PI3K inhibitor. In some embodiments, the pan-PI3K inhibitor is picotelicoxib (GDC-0941) or tasericoxib (GDC-0032), or a pharmaceutically acceptable salt thereof. In some embodiments, the pan-RAF dimer inhibitor is selected from the group consisting of HM95573, LY-3009120, AZ-628, LXH-254, MLN2480, BeiGene-283, RXDX-105, BAL3833, regorafenib, and sorafenib, or a pharmaceutically acceptable salt thereof. In some embodiments, the composition comprises selected from HM95573 and picolixib (GDC-0941), LY-3009120 and picolixib (GDC-0941), AZ-628 and picolixib (GDC-0941), LXH-254 and picolixib (GDC-0941), MLN2480 and picolixib (GDC-0941), BeiGene-283 and picolixib (GDC-0941), RXDX-105 and picolixib (GDC-0941), BAL3833 and picolixib (GDC-0941), regorafenib and picolixib (GDC-0941), sorafenib and picolixib (GDC-0941). Combinations of the following groups: 0941), HM95573 and tacelicoxib (GDC-0032), LY-3009120 and tacelicoxib (GDC-0032), AZ-628 and tacelicoxib (GDC-0032), LXH-254 and tacelicoxib (GDC-0032), MLN2480 and tacelicoxib (GDC-0032), BeiGene-283 and tacelicoxib (GDC-0032), RXDX-105 and tacelicoxib (GDC-0032), BAL3833 and tacelicoxib (GDC-0032), regorafenib and tacelicoxib (GDC-0032), and sorafenib and tacelicoxib (GDC-0032), or pharmaceutically acceptable salts thereof. In some embodiments, the composition is intended for use in the therapeutic treatment of cancer. In some implementations, the cancer is selected from the group consisting of colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, breast cancer, stomach cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and hematologic malignancies.

In a fifteenth aspect, the invention is characterized by a pharmaceutical composition comprising the composition according to the fourteenth aspect.

In a sixteenth aspect, the invention is characterized by the use of the composition according to the fourteenth aspect for the therapeutic treatment of cancer. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, breast cancer, gastric cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and hematologic malignancies.

In a seventeenth aspect, the invention features a kit for identifying individuals with cancer who may benefit from treatment including pan-RAF dimer inhibitors and MEK inhibitors, the kit comprising: (a) a reagent for determining the presence of a KRAS-G13D mutation in a sample from the individual, and, optionally, (b) instructions for use of the reagent to identify individuals with cancer who may benefit from treatment including pan-RAF dimer inhibitors and MEK inhibitors. In some embodiments, the reagent comprises a first oligonucleotide and a second oligonucleotide for use in amplifying the whole or a portion of the KRAS gene.

In an eighteenth aspect, the invention features a kit for identifying individuals with cancer who may benefit from treatment including pan-RAF dimer inhibitors and MEK inhibitors, the kit comprising: (a) reagents for determining the presence of NRAS activating mutations in a sample from the individual, and, optionally, (b) instructions for use of the reagents to identify individuals with cancer who may benefit from treatment including pan-RAF dimer inhibitors and MEK inhibitors. In some embodiments, the reagents comprise a first oligonucleotide and a second oligonucleotide for amplifying the entire or a portion of the NRAS gene.

In some embodiments of the seventeenth or eighteenth aspect, the MEK inhibitor is a small molecule inhibitor. In some embodiments, the small molecule inhibitor is selected from the group consisting of cobimetinib (GDC-0973), selmetinib (AZD6244), pimecrolimus (AS-703026), PD0325901, remetinib (BAY86-9766), bimemetinib (MEK162), BI-847325, trametinib, GDC-0623, G-573, and CH5126766 (RO5126766), or pharmaceutically acceptable salts thereof. In some embodiments, the small molecule inhibitor is cobimetinib (GDC-0973), selmetinib (AZD6244), pimecrolimus (AS-703026), PD0325901, remectinib (BAY86-9766), or bimetinib (MEK162), or a pharmaceutically acceptable salt thereof. In some embodiments, the small molecule inhibitor is cobimetinib (GDC-0973), or a pharmaceutically acceptable salt thereof.

In a nineteenth aspect, the invention features a kit for identifying individuals with cancer who may benefit from treatment including pan-RAF dimer inhibitors and PI3K inhibitors, the kit comprising: (a) reagents for determining the presence of KRAS activating mutations in a sample from the individual, and optionally, (b) instructions for use of the reagents to identify individuals with cancer who may benefit from treatment including pan-RAF dimer inhibitors and PI3K inhibitors. In some embodiments, the reagents comprise a first oligonucleotide and a second oligonucleotide for amplifying the whole or a portion of the KRAS gene. In some embodiments, the PI3K inhibitor is a small molecule inhibitor. In some embodiments, the small molecule inhibitor is selected from the group consisting of picolixib (GDC-0941), taxerixib (GDC-0032), and apirilxib (BYL719), or pharmaceutically acceptable salts thereof. In some embodiments, the PI3K inhibitor is a pan-PI3K inhibitor. In some embodiments, the pan-PI3K inhibitor is picotelixicob (GDC-0941) or taxerixicob (GDC-0032), or a pharmaceutically acceptable salt thereof. In some embodiments, the individual does not have a BRAF activating mutation.

In some embodiments of any of the foregoing aspects, the panRAF dimer inhibitor is selected from HM95573, LY-3009120, AZ-628, LXH-254, MLN2480, BeiGene-283, RXDX-105, BAL3833, regorafenib, and sorafenib, or pharmaceutically acceptable salts thereof.

In some embodiments of any of the foregoing aspects, the sample is a tissue sample, a cell sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some embodiments, the sample is a tissue sample. In some embodiments, the tissue sample is a tumor tissue sample. In some embodiments, the tumor tissue sample is a formalin-fixed and paraffin-embedded (FFPE) sample, an archived sample, a fresh sample, or a frozen sample.

In some embodiments of any of the foregoing aspects, the cancer is selected from the group consisting of colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, breast cancer, stomach cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, or hematologic malignancies. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is skin cancer.

In some implementations of any of the foregoing aspects, the individual is a person.

Brief description of the attached diagram

Figure 1A is a series of graphs showing a profile analysis of the KRAS mutant cell line (red) and the BRAF ^V600E cell line (black) against the indicated RAF inhibitor in a 3-day viability assay. The curves were determined using four-parameter sigmoid curve fitting.

Figure 1B is a series of graphs showing the ratio of P-MEK to total MEK for different concentrations of type 1, 5 and type 2 RAF inhibitors.

Figure 1C is a series of plots showing a profile analysis of the sensitivity of colon, lung, and skin cancer cell lines to three type II pan-RAF inhibitors (AZ-628, LY-3009120, and MLN-2480) and the type 1.5 “abnormal disruptor” RAF inhibitor, PLX-8394, in a 3-day cell viability study. _IC50 values (μM) were determined using a four-parameter fit with nonlinear regression analysis.

Figure 1D is a plot showing the mean viability differences in the A549 KRAS-G12S mutant system after treatment with tool compounds from a small molecule library of 430 tool compounds using 1 μM AZ-628+/-. Cell viability in the A549 KRAS-G12S mutant system was measured 3 days after treatment with 1 μM AZ-628+/- tool compounds. Mean viability was calculated for each treatment, and the mean viability differences were normalized for DMSO-treated cells. Colored vertical bars indicate inhibitor categories.

Figure 1E shows a series of representative dose-response curves for the MEK inhibitors shown, selected from compounds screened in A549 cells, with or without 1 μM AZ-628.

Figure 1F is an immunoblot showing total BRAF and CRAF, and phosphate and total MEK, relative to the control (actin) after A549 cells were treated with the indicated molecules at 1 μM for 24 hours.

Figure 1G is a set of images showing the calculated Bliss overdose scores from 12x12 matrix titrations of AZ-628 and the MEK inhibitors indicated. A549 cells were treated for 3 days with 12x12 matrix titrations of AZ-628 and multiple MEK inhibitors. Cell viability was assessed and Bliss overdose scores were calculated.

Figure 2A is a set of images showing the calculated Bliss overdose scores of 12x12 matrix titrations of cobimetinib and the RAF inhibitor shown. Cell viability was assessed in A549 cells treated with cobimetinib and the RAF inhibitor shown for 3 days.

Figure 2B is an immunoblot showing the phosphate and total CRAF, MEK, ERK, RSK and AKT levels of A549 cells relative to the control (actin) 24 hours after treatment with 50 nM cobimetinib and/or the RAF inhibitor shown.

Figure 2C is a set of graphs showing the RNA-seq fold change expression data of DUSP6 and SPRY4 in four KRAS mutant lung cancer cell lines treated with 0.1 μM cobitinib, 0.1 μM AZ-628, or both for 6 hours relative to the DMSO control.

Figure 2D is an immunoblot showing the increase in cleaved PARP relative to the control (actin) in A549 cells treated with AZ-628 in combination with cobimetinib at the indicated concentration for 48 hours compared to vemurafenib with or without cobimetinib.

Figure 2E is a set of graphs showing flow cytometry cell cycle/apoptosis analysis of A549 cells treated with the indicated concentrations of AZ-628, cobimetinib, or both (propidium iodide and annexin V).

Figure 2F shows a set of images from the colony growth assay, demonstrating that type II RAF inhibitors exhibit synergistic activity with cobimetinib and enhance cell death even at sub-effective single-agent concentrations. A549 cells were treated with the RAF inhibitor or cobimetinib at the indicated concentrations, and NCI-H2122 cells were treated with the RAF inhibitor LY-3009120 or cobimetinib at the indicated concentrations. Medium containing the appropriate compound was added every 72 hours. Cells were cultured for 8 days and then stained with crystal violet.

Figure 3A is a set of figures showing the tumor volume over time in the NCI-H2122 and HCT 116 xenograft tumor models of the MEK inhibitor cobimetinib (5 mg/kg, oral (PO), daily (QD)) and the pan-RAF inhibitor AZ-628 (25 mg/kg, intraperitoneal (IP), QD) or LY-3009120 (25 mg/kg, IP, QD) (n = 10/group, mean tumor volume ± SEM).

Figure 3B is a set of graphs showing mouse body weight data (n = 10/group, mean tumor volume ± SEM) in NCI-H2122 and HCT-116 xenograft mice for single-agent MEK inhibitor cobimetinib (GDC-0973), pan-RAF inhibitor AZ-628 or LY-3009120, or combinations thereof.

Figure 3C is a set of bar charts showing the tumor pharmacodynamics in NCI-H2122 and HCT 116 tumor-bearing mice at time points (2, 8, 16, and 24 hours) after the last dose, 4 days after treatment. The bars indicate the quantitative values (n = 4/group, mean ± SD) of the proteins shown (cleaved PARP (Western blot), pMEK/total MEK (ECLA), pRSK/total RSK (Western blot), or downstream RNA transcript DUSP6 (RT-PCR)) normalized against the media controls.

Figure 3D is a set of bar charts showing the plasma concentrations of the compounds after the indicated time on the x-axis.

Figure 4A shows the immunoblotting results in KRAS mutant cell lines where MEK inhibition induces greater pathway feedback reactivation, leading to elevated RAS-GTP levels. The cancer cell lines shown were treated with either DMSO or 250 nM cobimethinib for 24 hours, and protein lysates were analyzed by Western blotting relative to a control (actin) for phosphate or total BRAF, CRAF, or MEK. CRAF, RBD-RAF1, and BRAF were immunoprecipitated (IP) from the same lysates and analyzed by Western blotting. Kinase activity was analyzed using inactive MEK as a substrate for CRAF immunoprecipitate.

Figure 4B shows a series of immunoblots illustrating MEK inhibition induced by cobimetinib in cooperative and non-cooperative KRAS mutant and wild-type colon cancer cell lines. The cancer cell lines shown were treated with either DMSO or 250 nM cobimetinib for 24 hours, and the protein lysates were analyzed by Western blotting relative to the control (actin) for phosphate or total MEK.

Figure 4C shows a series of immunoblots illustrating MEK inhibition induced by cobimetinib in cooperitoneal and non-cooperitoneal KRAS mutant and wild-type lung cancer cell lines. The cancer cell lines shown were treated with either DMSO or 250 nM cobimetinib for 24 hours, and the protein lysates were analyzed by Western blotting relative to the control (actin) for phosphate or total MEK.

Figure 4D is a plot showing the relative fold increase in cobimetinib-induced pMEK levels in cooperative and non-cooperative KRAS mutant and wild-type lung cancer cell lines compared to the normalized DMSO-treated control cell lines, as seen in Figures 4B and 4C. P-values were determined using the Wilcoxon rank-sum test.

Figure 4E shows the results of the immunoblotting, illustrating the CRAF and BRAF IP assays and subsequent kinase activity assays. A549 cells were treated with progressively increasing concentrations of the MEK inhibitor GDC-0973, and CRAF and BRAF were immunoprecipitated in the same lysate and analyzed by Western blotting. Kinase activity of the CRAF immunoprecipitate was analyzed using inactive MEK as a substrate.

Figure 4F shows the immunoblotting results, indicating that the type II pan-RAF inhibitor, rather than the residual type 1.5 RAF inhibitor, reversed MEK inhibitor-induced MEK hyperphosphorylation in KRAS mutant cells. MEK phosphorylation was assessed by Western blotting in A549 cells treated with 250 nM cobimethinib for 24 hours, followed by 24 hours of incremental addition of the indicated RAF inhibitor.

Figure 4G is an immunoblot showing MAPK reactivation after KRAS knockdown weakens MEK inhibition. Cell lines were transfected in reverse with 20 nM si-KRAS (L-005069-00-0020) or si-NTC (D-001810-10-20) (Dharmacon Collection) in the presence of lipofectamine reagent (Life Technologies) according to the manufacturer's instructions. The culture medium was changed on the day of transfection, and the knockdown efficiency was assessed by Western blotting on day 4. 250 nM cobimethinib was added to the cells for 24 hours.

Figure 5A is a plot showing that the combination of pan-RAF and MEK inhibitors resulted in greater synergy compared to BRAF-V600 cell lines in a subset of RAS mutant (G12/G13/Q61) cell lines and RAS/RAF wild-type cell lines. Cell viability was assessed using a cohort of 322 colon, lung, and pancreatic cancer cell lines treated with 9-step 3-fold serial dilutions of 10 μM AZ-628 and 1 μM cobimethinib, or their co-dilutions, for 3 days. The synergy score was the sum of positive Bliss excesses measured on the combination of concentrations. The bar chart shows four states of synergy: no synergy (light gray), low synergy (dark gray), moderate synergy (blue), and high synergy (orange). Synergy levels were determined by mixed modeling of positive Bliss excesses on multiple tissues and the distribution of these states.

Figure 5B is a set of immunoblots showing KRAS mutant and wild-type cancer cell lines representing the indicated synergistic state and demonstrating the relationship between the response and basal levels of MAPK signaling and feedback reactivation via KRAS, RAF dimer, and kinase activity. Cells were reverse-transfected in the presence of Lipofectamine reagent (Life Technologies) with 20 nM si-KRAS (L-005069-00-0020) or si-NTC (D-001810-10-20) (Dharmacon set) according to the manufacturer's instructions. The medium was changed on the day after transfection, and the knockdown efficiency was assessed on day 4. 250 nM cobimethinib was added to the cells for up to 24 hours. Phosphate and/or total KRAS, BRAF, CRAF, MEK, ERK, and RSK were analyzed from the protein lysates relative to the control (actin) by Western blotting. CRAF and RAF1-RBD were immunoprecipitated from the same lysates and analyzed by Western blotting. We used inactive MEK as a substrate to analyze kinase activity in CRAF immunoprecipitates.

Figure 5C shows the immunoblot of the wild-type KRAS line without co-inhibition. Cells were transfected in reverse with 20 nM si-KRAS (L-005069-00-0020) or si-NTC (D-001810-10-20) (Dharmacon Collection) in the presence of Lipofectamine reagent (Life Technologies) according to the manufacturer's instructions. The medium was changed on the day of transfection, and the knockdown efficiency was assessed on day 4. 250 nM cobimethinib was added to the cells for 24 hours. Phosphate and/or total KRAS, BRAF, CRAF, MEK, ERK, and RSK were analyzed from protein lysates relative to a control (actin) by Western blotting. CRAF and RAF1-RBD were immunoprecipitated from the same lysates and analyzed by Western blotting. Kinase activity was analyzed from CRAF immunoprecipitates using inactive MEK as a substrate.

Figure 5D is a set of immunoblots showing the combined activity observed in the type II pan-RAF inhibitor HM95573 (GDC-5573) and the MEK inhibitor cobimetinib in the A549 KRAS mutant cell line, as assessed by the levels of MAPK pathway markers (pMEK, pERK, and pRSK) at the indicated time points after treatment with either 1 μM HM95573 (GDC-5573), 250 nM cobimetinib, or both 1 μM HM95573 (GDC-5573) and 250 nM cobimetinib.

Figure 5E is a set of images from a colony growth assay, showing that type II RAF inhibitors exhibit synergistic activity with cobimetinib and enhance cell death even at sub-effective single-agent concentrations. A549 cells (left) and HCT-116 cells (right) were treated with the RAF inhibitor HM95573 (GDC-5573), cobimetinib, or both HM95573 (GDC-5573) and cobimetinib at the indicated concentrations. Medium containing the appropriate compound was added every 72 hours. Cells were cultured for 8 days and then stained with crystal violet.

Figure 5F is a set of plots showing that treatment with HM95573 (GDC-5573) and cobimetinib resulted in improved tumor growth inhibition (TGI) in the KRAS mutant synergistic colorectal cancer (CRC) xenograft model CT26. Animals (n = 10/group) were treated for 21 days with: HM95573 (GDC-5573) at 5 mg/kg (orally, once daily); cobimetinib at 5 mg/kg (orally, once daily); or both agents. Tumor growth in individual animals is shown along the bold trend line for each group, represented by tumor volume ( ^mm³ ) over time (days).

Figure 5G is a graph showing the fitted tumor volume curves for each treatment group described in Figure 5F.

Figure 5H is a plot showing that the combination of pan-RAF and MEK inhibitors resulted in greater synergy compared to BRAF-V600 cell lines in a subset of RAS mutant cell lines and RAS/RAF wild-type cell lines. Cell viability was assessed using a co-dilution of HM95573 (GDC-5573), cobimetinib, or both HM95573 (GDC-5573) and cobimetinib for 3 days. Synergy score was the sum of positive Bliss excesses measured on the combination of concentrations. Significance was assessed by a two-tailed t-test.

Figure 5I is a set of immunoblotting results showing that the combined activity of pan-RAF and MEK inhibition is associated with MEK inhibitor-induced RAS-GTP levels in a group of colorectal cancer cell lines. RAS activity was immunoprecipitated using RAF1-RBD in the illustrated colorectal cell lines treated with DMSO or cobimetinib.

Figure 6A is a graph showing that the KRAS-G13D mutant cell line is more sensitive to the combination of RAF and MEK inhibition. It shows positive Bliss overdose of the AZ-628/cobimetinib combination for cell lines containing different RAS mutant codons (P value is a two-sided Wilcoxon rank-sum test).

Figure 6B is a graph showing that the KRAS- ^G13D mutant cell line is more sensitive to the combination of RAF and MEK inhibition. It shows positive Bliss overdose of the AZ-628/cobimetinib combination for cell lines containing different RAS mutant codons (P value is a two-sided Wilcoxon rank-sum test).

Figure 6C shows the bar and box plots, illustrating the excess Bliss score of the lung cell line (P-value is a two-sided Wilcoxon rank-sum test).

Figure 6D is a set of box plots showing the excess Bliss scores of KRAS-G13D colorectal (left) and lung (right) cancer cell lines after 3 days of co-dilution with HM95573 (GDC-5573) and cobimetinib, compared with other cell lines carrying non-KRAS-G13D mutations. The synergy score is the sum of positive Bliss excess at the measured concentration combinations. Significance was assessed using a two-tailed t-test.

Figure 6E is a set of immunoblots showing that the KRAS-G13D mutant colorectal cell line exhibits significantly enhanced pMEK induction compared to other KRAS mutants after MEK inhibition. The colorectal cell lines shown were treated with either DMSO or cobimetinib (250 nM) for 24 hours, followed by Western blotting to assess phosphate and total MEK, ERK, RSK, and AKT levels relative to a control (actin).

Figure 6F shows a set of images from a colony growth assay, demonstrating the synergistic activity of the type II RAF inhibitor and cobimetinib in KRAS-G13D mutant SW48 cells compared to KRAS-G12D or KRAS-G12C mutant SW48 cells. Syngeneic SW48 cells with KRAS-G13D (left), KRAS-G12D (middle), or KRAS-G12C (right) knock-in were treated with AZ-628, cobimetinib, or both AZ-628 and cobimetinib. Medium containing the appropriate compound was added every 72 hours. Cells were cultured for 8 days and then stained with crystal violet.

Figure 6G is a set of immunoblots showing the ERK and pERK levels in syngeneic SW48 cells with KRAS-G13D (left), KRAS-G12D (middle), or KRAS-G12C (right) knock-in at the concentrations shown, with AZ-628, cobimetinib, or both of these agents, relative to the control (actin).

Figure 6H is a graph showing the relative IC50 values (μM) of AZ-628 in 322 colon, lung, pancreas, ovary, blood, and skin cell lines, as determined by a 3-day assay. Nonlinear regression and four-parameter fit analysis were used to fit the IC50 curves.

Figure 6I shows the immunoblotting of active RAS in lysates from three syngeneic colorectal cell lines using RAF1-RBD immunoprecipitation: SW48 parent with wild-type KRAS, SW48 with an introduced heterozygous KRAS G12D mutation, and SW48 with an introduced heterozygous KRAS G13D mutation.

Figure 6J is a plot showing the higher KRAS-GTP levels in SW48 KRAS-G13D cells compared to SW48 KRAS-G12D and SW48 KRAS wild-type cells, as assessed using a nucleotide exchange reaction by time-resolved fluorescence energy transfer (TR-FRET). 6xHis-tagged KRAS wild-type, KRAS-G12D, and GDP-loaded KRAS-G13D cells were incubated with FLAG-tagged RAF1-RBD in the presence of anti-FLAG-Tb and anti-6xHis-d2 antibodies. GTP was added to initiate the nucleotide exchange reaction, and TR-FRET was measured.

Figure 6K is an immunoblot showing the MAPK pathway protein levels, including total EGFR and P-EGFR at the Y1068 locus, 24 hours after the cell lines were treated with either DMSO or 250 nM cobimethinib.

Figure 6L shows the levels of KRAS, SOS1, MEK, pMEK, and actin from cell lysates after reverse transfection of HCT 116 and DLD-1 cells with si-SOS1 or si-NTC in the presence of lipofectamine RNAiMAX reagent (Life Technologies) according to the manufacturer's instructions, followed by treatment with either DMSO or 250 nM cobimethinib for 24 hours. RAS-GTP was immunoprecipitated from RAF1-RBD autolysates and analyzed along with other proteins.

Figure 7A is a plot showing the synergistic effect of the pan-RAF inhibitor (AZ-628) and the pan-PI3K inhibitor picoteliximab (GDC-0941). Cell viability was assessed 3 days later in a cohort of 213 tumor cell lines treated with co-diluted doses of AZ-628/picoteliximab, cobimetinib/picoteliximab, or single-agent doses. Synergistic scores, representing the sum of positive Bliss overdoses on the measured concentration combinations, are plotted.

Figure 7B is an immunoblot showing that inhibition of KRAS-G12S A549 cells with multiple PI3K inhibitors led to the induction of MAPK pathway activity. A549 cells were treated with either a PI3K inhibitor or cobimetinib at the indicated concentrations for 24 hours and then processed for Western blot analysis targeting phosphate and total MEK levels relative to the control (actin). As previously described, CRAF was also assessed by immunoprecipitation (IP), and immunoimmunizations were performed targeting CRAF and BRAF, followed by processing for RAF kinase activity.

Figure 7C is an immunoblot of the proteins shown from the lysate, which was derived from HCT116 cells treated with the PI3K inhibitor or GDC-0973 at the concentration shown for 24 hours.

Figure 7D is an immunoblot showing that PI3K inhibition led to dose-dependent pMEK induction. A549 cells were treated with the indicated concentration of picotelixib for 24 hours and then processed for Western blot analysis targeting phosphate and total MEK, ERK, and RSK levels relative to the control (actin).

Figure 7E is an immunoblot showing that PI3K inhibition led to dose-dependent induction of RAS-GTP levels. A549 cells were treated with picotelixib and cobimetinib at the indicated concentrations for 24 hours and active RAS was immunoprecipitated using RAF1-RBD.

Figure 7F is a set of images showing the Bliss overdose scores calculated by 12x12 matrix titration of the combination of cobimetinib or AZ-628 pan-RAF inhibitors with PI3K/AKT inhibitors, demonstrating the synergistic effect of the type II pan-RAF inhibitor AZ-628 with multiple PI3K/AKT inhibitors in KRAS mutant cell lines. Cell viability was assessed by 12x12 matrix titration of A549 and HCT116 cells treated for 3 days with the combination of cobimetinib or AZ-628 pan-RAF inhibitors with PI3K/AKT inhibitors.

Figure 7G is an immunoblot showing that the combination of a type II pan-RAF inhibitor and a pan-PI3K inhibitor blocks the reactivation of the MAPK pathway mediated by PI3K inhibition. A549 cells were treated with picotelixib and/or AZ-628 at the indicated concentrations for 24 hours and then processed for Western blot analysis targeting phosphate and total MEK, ERK, RSK, and AKT levels relative to the control (actin).

Figures 8A and 8B are a series of figures showing a profile analysis of the sensitivity of NRAS mutant melanoma cell lines to three type II pan-RAF inhibitors (AZ-628, LY-3009120, and MLN-2480) and the type 1.5 “abnormal disruptor” RAF inhibitor PLX-8394, compared to BRAF-V600E and NRAS/BRAF wild-type cell lines in viability studies. The figures show the mean viability (Figure 8A) and the calculated _IC50 values (μM) determined using a four-parameter fit with nonlinear regression analysis (Figure 8B).

Figures 8C and 8D are a series of figures showing a profile analysis of the sensitivity of NRAS mutant melanoma cell lines to the type II pan-RAF inhibitor AZ-628 and the MEK inhibitor cobimetinib in viability studies compared to BRAF-V600E and NRAS/BRAF wild-type cell lines, showing the mean viability (Figure 8C) and the calculated _IC50 value (μM) determined using a four-parameter fit with nonlinear regression analysis (Figure 8D).

Invention Details

I. Introduction

This invention provides diagnostic methods, treatment methods, and compositions for the treatment of proliferative cell diseases, such as cancers (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancers (e.g., melanoma)). The invention is based, at least in part, on the discovery that MEK and PI3K inhibitors exhibit synergistic activity with pan-RAF dimer inhibitors via a RAS-GTP-dependent mechanism in cancers with NRAS activating mutations or KRAS activating mutations, particularly KRAS-G13D mutations. Therefore, KRAS-G13D and NRAS activating mutations can be used as biomarkers (e.g., predictive biomarkers) in methods of identifying individuals with cancer who may benefit from treatment including pan-RAF dimer inhibitors and MEK or PI3K inhibitors; selecting treatment including pan-RAF dimer inhibitors and MEK or PI3K inhibitors for individuals with cancer; and treating individuals with cancer with therapies including pan-RAF dimer inhibitors and MEK or PI3K inhibitors.

II. Definition

It should be understood that the various aspects and embodiments of the invention described herein include aspects and embodiments of “comprising,” “forming of,” and “essentially forming of.” As used herein, the singular forms “a,” “an,” and “the” include plural references unless otherwise specified.

As used herein, the term "about" refers to a typical range of error for a corresponding value that is readily known to those skilled in the art. References to "about" a value or parameter herein include (and describe) embodiments relating to that value or parameter itself. For example, a description of "about X" includes a description of X.

The term "MAPK signaling pathway" refers to mitogen-activated protein kinase signaling pathways (e.g., the RAS/RAF/MEK/ERK signaling pathway) and encompasses conserved serine/threonine protein kinase families (e.g., mitogen-activated protein kinases (MAPK)). Aberrant regulation of the MAPK pathway leads to uncontrolled proliferation, invasion, metastasis, angiogenesis, and reduced apoptosis. GTPases of the RAS family include KRAS, HRAS, and NRAS. Serine/threonine protein kinases of the RAF family include ARAF, BRAF, and CRAF (RAF1). Exemplary MAPKs include extracellular signal-regulated kinases 1 and 2 (i.e., ERK1 and ERK2), c-Jun N-terminal kinases 1–3 (i.e., JNK1, JNK2, and JNK3), p38 isoforms (i.e., p38α, p38β, p38γ, and p38δ), and Erk5. Other MAPKs include Nemo-like kinases (NLK), Erk3/4 (i.e., ERK3 and ERK4), and Erk7/8 (i.e., ERK7 and ERK8).

The term "MAPK signaling inhibitor" or "MAPK pathway signaling inhibitor" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction via the MAPK pathway (e.g., the RAS/RAF/MEK/ERK pathway). In some embodiments, MAPK signaling inhibitors can inhibit the activity of one or more proteins involved in MAPK signaling activation. In some embodiments, MAPK signaling inhibitors can increase the activity of one or more proteins involved in MAPK signaling inhibition. MAPK signaling inhibitors include, but are not limited to, MEK inhibitors (e.g., MEK1 inhibitors, MEK2 inhibitors, and inhibitors of both MEK1 and MEK2), RAF inhibitors (e.g., ARAF inhibitors, BRAF inhibitors, CRAF inhibitors, and pan-RAF inhibitors (i.e., RAF inhibitors that inhibit more than one member of the RAF family (i.e., two or all three of ARAF, BRAF, and CRAF), such as pan-RAF dimer inhibitors (i.e., pan-RAF inhibitors that bind to and inhibit RAF dimers (e.g., RAF heterodimers)), and ERK inhibitors (e.g., ERK1 inhibitors and ERK2 inhibitors).

The term "BRAF inhibitor" or "BRAF antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with BRAF activation or function. In one specific embodiment, the BRAF inhibitor has a binding affinity (dissociation constant) of about 1,000 nM or less for BRAF. In another embodiment, the BRAF inhibitor has a binding affinity of about 100 nM or less for BRAF. In another embodiment, the BRAF inhibitor has a binding affinity of about 50 nM or less for BRAF. In another embodiment, the BRAF inhibitor has a binding affinity of about 10 nM or less for BRAF. In another embodiment, the BRAF inhibitor has a binding affinity of about 1 nM or less for BRAF. In one specific embodiment, the BRAF inhibitor inhibits BRAF signaling with an IC50 of 1,000 nM or less. In another embodiment, the BRAF inhibitor inhibits BRAF signaling with an IC50 of 500 nM or less. In another embodiment, the BRAF inhibitor inhibits BRAF signaling with an IC50 of 50 nM or less. In another embodiment, the BRAF inhibitor inhibits BRAF signaling with an IC50 of 10 nM or less. In yet another embodiment, the BRAF inhibitor inhibits BRAF signaling with an IC50 of 1 nM or less. Examples of BRAF inhibitors that can be used according to the invention include, but are not limited to, vemurafenib, dabrafenib, encorafenib (LGX818), GDC-0879, XL281, ARQ736, PLX3603, RAF265, and sorafenib, or pharmaceutically acceptable salts thereof. The BRAF inhibitor may inhibit BRAF alone or may inhibit BRAF and one or more other targets. Preferred BRAF inhibitors are described in PCT application publications WO 2005/062795, WO 2007/002325, WO2007/002433, WO 2008/079903, and WO 2008/079906, each of which is incorporated herein by reference in its entirety.

The term "ERK inhibitor" or "ERK antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with the activation or function of ERK (e.g., ERK1 and/or ERK2). In one particular embodiment, the ERK inhibitor has a binding affinity (dissociation constant) of about 1,000 nM or less for ERK. In another embodiment, the ERK inhibitor has a binding affinity of about 100 nM or less for ERK. In another embodiment, the ERK inhibitor has a binding affinity of about 50 nM or less for ERK. In another embodiment, the ERK inhibitor has a binding affinity of about 10 nM or less for ERK. In another embodiment, the ERK inhibitor has a binding affinity of about 1 nM or less for ERK. In one particular embodiment, the ERK inhibitor inhibits ERK signaling with an IC50 of 1,000 nM or less. In another embodiment, the ERK inhibitor inhibits ERK signaling with an IC50 of 500 nM or less. In another embodiment, the ERK inhibitor inhibits ERK signaling with an IC50 of 50 nM or less. In another embodiment, the ERK inhibitor inhibits ERK signaling with an IC50 of 10 nM or less. In yet another embodiment, the ERK inhibitor inhibits ERK signaling with an IC50 of 1 nM or less. Examples of ERK inhibitors that can be used according to the invention include, but are not limited to, ravoxertinib (GDC-0994) and ulixertinib (BVD-523), or pharmaceutically acceptable salts thereof (e.g., benzenesulfonates (e.g., benzenesulfonates of ravoxertinib)). ERK inhibitors may inhibit ERK alone or may inhibit ERK and one or more other targets. Preferred ERK inhibitors are described in PCT application publications WO 2013/130976, WO 2012/118850, WO 2013/020062, WO 2015/154674, WO 2015/085007, WO 2015/032840, WO 2014/036015, WO 2014/060395, WO 2015/103137, and WO 2015/103133, each of which is incorporated herein by reference in its entirety.

The term "MEK inhibitor" or "MEK antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with the activation or function of MEK (e.g., MEK1 and/or MEK2). In one specific embodiment, the MEK inhibitor has a binding affinity (dissociation constant) of about 1,000 nM or less for MEK. In another embodiment, the MEK inhibitor has a binding affinity of about 100 nM or less for MEK. In another embodiment, the MEK inhibitor has a binding affinity of about 50 nM or less for MEK. In another embodiment, the MEK inhibitor has a binding affinity of about 10 nM or less for MEK. In another embodiment, the MEK inhibitor has a binding affinity of about 1 nM or less for MEK. In one specific embodiment, the MEK inhibitor inhibits MEK signaling with an IC50 of 1,000 nM or less. In another embodiment, the MEK inhibitor inhibits MEK signaling with an IC50 of 500 nM or less. In another embodiment, the MEK inhibitor inhibits MEK signaling with an IC50 of 50 nM or less. In another embodiment, the MEK inhibitor inhibits MEK signaling with an IC50 of 10 nM or less. In yet another embodiment, the MEK inhibitor inhibits MEK signaling with an IC50 of 1 nM or less. Examples of MEK inhibitors that can be used according to the invention include, but are not limited to, cobimetinib (e.g., cobimetinib hemifumarate), trametinib, bimetinib, selemetinib, pimozide, remexinib, GDC-0623, PD-0325901, and BI-847325, or pharmaceutically acceptable salts thereof. The MEK inhibitor may inhibit MEK alone or may inhibit MEK and one or more other targets. Preferred MEK inhibitors are described in PCT application publications WO 2007/044515, WO 2008/024725, WO 2008/024724, WO 2008/067481, WO 2008/157179, WO 2009/085983, WO 2009/085980, WO 2009/082687, WO 2010/003025, and WO 2010/003022, each of which is incorporated herein by reference in its entirety.

The term "CRAF inhibitor" or "CRAF antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with CRAF activation or function. In one specific embodiment, the CRAF inhibitor has a binding affinity (dissociation constant) of about 1,000 nM or less for CRAF. In another embodiment, the CRAF inhibitor has a binding affinity of about 100 nM or less for CRAF. In another embodiment, the CRAF inhibitor has a binding affinity of about 50 nM or less for CRAF. In another embodiment, the CRAF inhibitor has a binding affinity of about 10 nM or less for CRAF. In another embodiment, the CRAF inhibitor has a binding affinity of about 1 nM or less for CRAF. In one specific embodiment, the CRAF inhibitor inhibits CRAF signaling with an IC50 of 1,000 nM or less. In another embodiment, the CRAF inhibitor inhibits CRAF signaling with an IC50 of 500 nM or less. In another embodiment, the CRAF inhibitor inhibits CRAF signaling with an IC50 of 50 nM or less. In another embodiment, the CRAF inhibitor inhibits CRAF signaling with an IC50 of 10 nM or less. Examples of CRAF inhibitors that can be used according to the invention include, but are not limited to, sorafenib, or pharmaceutically acceptable salts thereof. CRAF inhibitors may inhibit CRAF alone or may inhibit CRAF and one or more other targets.

The term "pan-RAF inhibitor" or "pan-RAF antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with the activation or function of two or more RAF family members (e.g., two or more of ARAF, BRAF, and CRAF). In one embodiment, the pan-RAF inhibitor inhibits all three RAF family members (i.e., ARAF, BRAF, and CRAF) to a certain extent. In a particular embodiment, the pan-RAF inhibitor has a binding affinity (dissociation constant) of about 1,000 nM or less for one, two, or three of ARAF, BRAF, and/or CRAF. In another embodiment, the pan-RAF inhibitor has a binding affinity of about 100 nM or less for one, two, or three of ARAF, BRAF, and/or CRAF. In yet another embodiment, the pan-RAF inhibitor has a binding affinity of about 50 nM or less for one, two, or three of ARAF, BRAF, and/or CRAF. In another embodiment, the pan-RAF inhibitor has a binding affinity of about 10 nM or less for one, two, or three of ARAF, BRAF, and/or CRAF. In another embodiment, the pan-RAF inhibitor has a binding affinity of about 1 nM or less for one, two, or three of ARAF, BRAF, and/or CRAF. In a particular embodiment, the pan-RAF inhibitor inhibits ARAF, BRAF, and/or CRAF signaling with an IC50 of 1,000 nM or less. In another embodiment, the pan-RAF inhibitor inhibits ARAF, BRAF, and/or CRAF signaling with an IC50 of 500 nM or less. In another embodiment, the pan-RAF inhibitor inhibits ARAF, BRAF, and/or CRAF signaling with an IC50 of 50 nM or less. In yet another embodiment, the pan-RAF inhibitor inhibits ARAF, BRAF, and/or CRAF signaling with an IC50 of 10 nM or less. In another embodiment, the pan-RAF inhibitor inhibits ARAF, BRAF, and/or CRAF signaling with an IC50 of 1 nM or less. Examples of pan-RAF inhibitors that can be used according to the present invention include, but are not limited to, LY-3009120, HM95573 (GDC-5573), LXH-254, MLN2480, BeiGene-283, RXDX-105, BAL3833, regorafenib, and sorafenib, or pharmaceutically acceptable salts thereof. The pan-RAF inhibitor can inhibit ARAF, BRAF, and/or CRAF and one or more additional targets. Preferred pan-RAF inhibitors are described in PCT application publications WO2013/100632, WO2014/151616, and WO2015/075483, each of which is incorporated herein by reference in its entirety.

In some implementations, pan-RAF inhibitors are "pan-RAF dimer inhibitors" capable of binding to and inhibiting RAF dimers (e.g., RAF heterodimers, such as BRAF-CRAF heterodimers). In addition to RAF dimers (e.g., RAF heterodimers, such as BRAF-CRAF heterodimers), pan-RAF dimer inhibitors may also bind to and inhibit RAF monomers. Pan-RAF dimer inhibitors include, for example, type II RAF inhibitors capable of reducing, blocking, inhibiting, eliminating, or interfering with the activation or function of two or more RAF family members.

The term "PI3K inhibitor" refers to molecules that reduce, block, inhibit, eliminate, or interfere with the activation or function of one or more classes of phosphatidylinositol 3-kinases (PI3K), including classes I, II, III, and IV PI3K. The four classes of PI3K are classified based on structure and substrate specificity. Class I PI3Ks are most closely associated with human diseases such as cancer. Class I PI3Ks can be further divided into four distinct isoforms: PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ.

In some embodiments, the PI3K inhibitor is a “pan-PI3K inhibitor,” meaning any compound capable of preferentially inhibiting class I PI3K over any other kinase. For example, a pan-I PI3K inhibitor is at least 2-fold, preferably at least 5-fold, and more preferably at least 10-fold more potent against class I PI3K than against other kinases, including the mammalian target of associated phosphatidylinositol 3-kinase-associated kinase (PIKK), and rapamycin (mTOR). In particular, the PI3K inhibitor can be a small molecule or a biological macromolecule. Typically, the PI3K inhibitor is a small molecule, preferably a synthetic compound, such as those described in U.S. Patent Application Publication No. 2010/0249126.

In other embodiments, the PI3K inhibitor may be specific to a particular PI3K isoform. For example, "PI3Kα-specific inhibitor" means any compound that preferentially inhibits PI3Kα over at least one (e.g., one, two, or three) other PI3K class I isoforms (PI3Kβ, PI3Kδ, and/or PI3Kγ). For example, the PI3Kα-specific inhibitor may be at least 2-fold, preferably at least 5-fold, and more preferably at least 10-fold more potent against PI3Kα than against PI3Kβ, but may not be at least 2-fold, at least 5-fold, or at least 10-fold more potent against PI3Kδ and/or PI3Kγ or other non-class I PI3Ks, such as class II PI3Ks (e.g., PI3K-C2α), class III PI3Ks (e.g., Vps34), or class IV PI3Ks (e.g., mTOR or DNA-PK). Similarly, "PI3Kδ specific inhibitor" means any compound that can preferentially inhibit PI3Kδ over at least one (e.g., one, two, or three) other PI3K I isoforms (PI3Kα, PI3Kβ, and/or PI3Kγ).

A “KRAS activating mutation” is any mutation in the KRAS gene (i.e., a nucleic acid mutation) or the KRAS protein (i.e., an amino acid mutation) that results in abnormal KRAS protein function associated with increased and/or constitutive activity (through a GTP-binding state favorable to the activity of the KRAS protein). The mutation can occur at conserved sites favorable to GTP binding and constitutive activity of the KRAS protein. In some cases, the mutation occurs at one or more of codons 12, 13, and 16 of the KRAS gene (e.g., the KRAS c.38G>A conversion mutation resulting in an aspartic acid (D) substitution for glycine (G) at amino acid position 13 of the KRAS protein, i.e., the KRAS-G13D mutant protein). Other illustrative KRAS activating mutations include, for example, KRAS-G12D, KRAS-G12C, KRAS-G12V, KRAS-G12A, KRAS-G12R, KRAS-G12S, KRAS-G13C, KRAS-G13A, KRAS-G13R, KRAS-G13S, KRAS-G13V, KRAS-Q61H, KRAS-Q61K, KRAS-Q61E, KRAS-Q61L, KRAS-Q61P, and KRAS-Q61R, as well as the corresponding nucleic acid mutations in the KRAS gene that encode the amino acid changes of the KRAS protein shown.

"NRAS activating mutations" are any mutations in the NRAS gene (i.e., nucleic acid mutations) or the Nras protein (i.e., amino acid mutations) that result in abnormal Nras protein function associated with increased and/or constitutive activity (through a GTP-binding state favorable to Nras protein activity). The mutation can occur at conserved sites favorable to GTP binding and constitutive activity of the Nras protein. In some cases, the mutation occurs at one or more of codons 12, 13, and 16 of the NRAS gene. Exemplary NRAS activating mutations include, for example, NRAS-Q61R, NRAS-Q61K, NRAS-G12D, NRAS-G13D, NRAS-G12S, NRAS-G12C, NRAS-G12V, NRAS-G12A, NRAS-G12R, NRAS-G13C, NRAS-G13A, NRAS-G13R, NRAS-G13S, NRAS-G13V, NRAS-Q61H, NRAS-Q61E, NRAS-Q61L, and NRAS-Q61P, as well as corresponding nucleic acid mutations in the NRAS gene that encode the Nras protein shown.

A “BRAF activating mutation” is any mutation in the BRAF gene (i.e., a nucleic acid mutation) or the B-Raf protein (i.e., an amino acid mutation) that results in abnormal B-Raf protein function associated with elevated and/or constitutive activity (through a state favorable to the active state of the B-Raf protein). The mutation can occur at conserved sites that favor RAS-GTP binding and constitutive activity of the B-Raf protein. In some cases, the mutation is at codon 600 of the BRAF gene. Exemplary BRAF activating mutations include, for example, BRAF-V600E, BRAF-V600K, BRAF-V600R, and BRAF-V600D, and the corresponding nucleic acid mutations in the BRAF gene encoding the amino acid alterations of the B-Raf protein described herein.

"Individual," "patient," or "subject" in this document refers to an animal (including, for example, mammals such as dogs, cats, horses, rabbits, zoo animals, cattle, pigs, sheep, non-human primates, and humans) who is experiencing, has experienced, or is at risk of developing one or more signs, symptoms, or other indicators of a proliferative disease or condition, such as cancer, or who has a family history of such a disease. Intended as a patient includes any patient who does not exhibit any clinical signs of disease and is participating in a clinical research trial, an epidemiological study, or was previously used as a control. A patient may have been previously treated with a MAPK signaling inhibitor, another drug (e.g., a PI3K inhibitor), or may not have been previously treated. Patients may be naive at the start of treatment, meaning they may have not previously been treated with any other medications used, i.e., at “baseline” (i.e., a predetermined point in time prior to administration of the first dose of one or more MAPK pathway and/or PI3K inhibitors (e.g., pan-RAF dimer inhibitors and MEK inhibitors or any PI3K inhibitor) as described in this article, such as the day subjects are screened prior to the start of treatment). Such naive patients or subjects are generally considered candidates for treatment with such other medications.

The term “antibody” in this article is used in the broadest sense and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

A “mutation” is the deletion, insertion, or substitution of one or more nucleotides or amino acids relative to a reference nucleotide sequence or a reference amino acid sequence, such as a wild-type sequence.

As used interchangeably herein, “polynucleotide” or “nucleic acid” refers to a polymer of nucleotides of any length and includes both DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be incorporated into the polymer by DNA or RNA polymerases, or through a synthetic reaction. Thus, for example, polynucleotides as defined herein include, but are not limited to, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, and hybrid molecules containing DNA and RNA, which may be single-stranded, or more typically double-stranded, or include single- and double-stranded regions. Additionally, as used herein, the term “polynucleotide” refers to a triple-stranded region containing RNA or DNA, or both RNA and DNA. The strands in such regions may originate from the same molecule or from different molecules. The region may include the entirety of one or more molecules, but more typically a region involving only some molecules. One of the molecules in a triple-stranded region is often an oligonucleotide. The term “polynucleotide” specifically includes cDNA.

Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogues. Modifications to the nucleotide structure, if present, can be given before or after polymer assembly. The sequence of the nucleotide can be interrupted by non-nucleotide components. Polynucleotides can be further modified after synthesis, such as by conjugation with tags. Other types of modifications include, for example, “capping”, replacing one or more naturally occurring nucleotides with analogs, internucleotide modifications such as those with uncharged linkages (e.g., methylphosphonates, triphosphates, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., thiophosphates, dithiophosphates, etc.), those containing pendant moiety, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalating agents (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those with modified linkages (e.g., α-terminal isomers of nucleic acids), and unmodified polynucleotides. Additionally, any hydroxyl groups typically present in sugars can be replaced with, for example, phosphonate groups, phosphate groups, protected with standard protecting groups, or activated to prepare for further linkage with other nucleotides, or conjugated to solid or semi-solid supports. The 5' and 3' terminal OH groups can be phosphorylated or replaced with amines or organic capping modules of 1 to 20 carbon atoms. Other hydroxyl groups can also be derived into standard protecting groups. Polynucleotides may also contain sugar analogs of ribose or deoxyribose known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl-, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-terminal isomers, epimeric sugars such as arabinose, xylose, or lythose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and non-base nucleoside analogs such as methylribonucleotides. One or more phosphodiester linkages can be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, the following embodiments, wherein the phosphate ester is replaced with P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR ₂ (“amidate”), P(O)R, P(O)OR', CO or CH ₂ (“formacetal”), wherein each R or R' is independently H or a substituted or unsubstituted alkyl group (1-20 Cs), optionally containing an ether (-O-) linker, aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl. All links in a polynucleotide do not necessarily have to be identical. A polynucleotide may contain one or more modifications of different types as described herein and/or multiple modifications of the same type. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

As used herein, "oligonucleotide" generally refers to a short, single-stranded polynucleotide, typically less than about 250 nucleotides in length, but this is not mandatory. Oligonucleotides can be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equivalent to and fully applicable to oligonucleotides.

The term "primer" refers to a single-stranded polynucleotide that can hybridize with nucleic acids and allows complementary nucleic acid polymerization (generally by providing a free 3'-OH group).

The term "small molecule" refers to any molecule having a molecular weight of about 2,000 Daltons or less, preferably about 500 Daltons or less.

The term "detection" includes any means of detection, including both direct and indirect detection.

As used herein, the term "biomarker" refers to an indicator molecule or set of molecules that can be detected in a sample (e.g., a predictive, diagnostic, and/or prognostic indicator) and includes, for example, KRAS-G13D mutations or NRAS activating mutations in proteins and/or corresponding activating mutations at nucleotide levels (e.g., nucleotide mutations in the KRAS or NRAS genes, such as the KRAS c.38G>A nucleotide substitution mutation). Biomarkers can be predictive biomarkers and serve as indicators of the likelihood of sensitivity or benefit to treatment with, for example, pan-RAF dimer inhibitors and MEK inhibitors or PI3K inhibitors in patients with a specific disease or condition (e.g., proliferative cell disorders (e.g., cancer)). Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and/or RNA (e.g., mRNA)), polynucleotide copy number alterations (e.g., DNA copy number), peptides, peptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and/or glycolipid-based molecular markers. In some embodiments, as mentioned above, the biomarker is a gene (e.g., the KRAS or NRAS gene).

As used herein, the “presence” of a biomarker is a detectable amount in a biological sample. These can be measured by methods known to those skilled in the art and also disclosed herein.

As used herein, “amplification” generally refers to the process of generating multiple copies of the desired sequence. “Multiple copies” means at least two copies. “Copy” does not necessarily mean complete sequence complementarity or identity with the template sequence. For example, copies may include nucleotide analogues such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced via primers containing sequences that hybridize with but are not complementary to the template), and/or sequence errors that occur during amplification.

The term "multiplex PCR" refers to a single PCR reaction performed on nucleic acids obtained from a single source (e.g., an individual) using more than one set of primers for the purpose of amplifying two or more DNA sequences in a single reaction.

As used herein, “polymerase chain reaction” or “PCR” generally refers to a procedure for amplifying small amounts of specific segments of nucleic acids, RNA, and/or DNA, as described, for example, in U.S. Patent No. 4,683,195. Generally, sequence information from the ends or beyond the region of interest needs to be available to allow the design of oligonucleotide primers; these primers will be identical or similar in sequence to the opposite strand of the template to be amplified. The 5’ terminal nucleotide of both primers can coincide with the ends of the material being amplified. PCR can amplify specific RNA sequences and specific DNA sequences from total genomic DNA and cDNA transcribed from total cellular RNA, bacteriophages, or plasmid sequences, etc. See Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263 (1987) and Erlich, ed., PCR Technology (Stockton Press, NY, 1989). As used herein, PCR is considered as a method for amplifying nucleic acid test samples, including one, but not the only, example of a nucleic acid polymerase reaction method that uses known nucleic acids (DNA or RNA) as primers and utilizes nucleic acid polymerases to amplify or generate specific nucleic acid segments or amplify or generate specific nucleic acid segments complementary to specific nucleic acids.

"Quantitative real-time polymerase chain reaction" or "qRT-PCR" refers to a form of PCR in which the amount of PCR product is measured at each step of the PCR reaction. This technique has been described in numerous publications, including, for example, Cronin et al., Am. J. Pathol. 164(1):35-42 (2004) and Ma et al., Cancer Cell 5:607-616 (2004).

The term "microarray" refers to hybridizable array elements, preferably an ordered arrangement of polynucleotide probes on a substrate.

As used herein, the term “sample” means a composition obtained from or derived from a subject (e.g., an individual of interest) that contains cells and/or other molecular entities to be characterized and/or identified, for example, based on physical, biochemical, chemical, and/or physiological characteristics. For example, the phrase “disease sample” and its variations refer to any sample obtained from a subject of interest that is expected or known to contain the cells and/or molecular entities to be characterized. Samples include, but are not limited to, tissue samples (e.g., tumor tissue samples), primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysates, and tissue culture media, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

“Tissue sample” means a collection of similar cells obtained from the tissue of a subject or individual. The source of a tissue sample can be solid tissue, such as fresh, frozen, and/or preserved organs, tissue samples, biopsies, and/or aspirates; blood or any blood component, such as plasma; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; and cells from any stage of pregnancy or development in the subject. Tissue samples can also be primary or cultured cells or cell lines. Optionally, tissue samples are obtained from diseased tissue/organ. For example, a “tumor tissue sample” is a tissue sample obtained from a tumor or other cancerous tissue. Tissue samples can contain a mixed population of multiple cell types, such as tumor cells and non-tumor cells, cancerous cells and non-cancer cells. Tissue samples may contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

As used herein, “reference sample,” “reference cell,” “reference tissue,” “control sample,” “control cell,” or “control tissue” refers to a sample, cell, tissue, standard, or level used for comparative purposes. In one embodiment, the reference level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or disease-free body part (e.g., tissue or cell) of the same subject or individual. For example, the reference level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be a healthy and/or disease-free cell or tissue adjacent to diseased cells or tissue (e.g., cells or tissue adjacent to a tumor). In another embodiment, the reference sample is obtained from untreated tissue and/or cells of the same subject or individual's body. In yet another embodiment, the reference level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or disease-free body part (e.g., tissue or cell) of an individual who is not the subject or individual. In even another implementation, the reference level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from untreated tissue and/or cells from the body of an individual who is not the subject or individual.

For the purposes of this invention, a “slice” of a tissue sample refers to a piece or sheet of tissue sample, such as a thin slice of tissue or cells cut from a tissue sample (e.g., a tumor sample). It is to be understood that multiple tissue sample slices can be prepared and analyzed, provided that the same slice of a tissue sample can be used for analysis at both the morphological and molecular levels, or for analysis of peptides (e.g., by immunohistochemistry) and/or polynucleotides (e.g., by in situ hybridization).

"Association" means comparing the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol in any way. For example, the results of the first analysis or protocol may be used in performing the second protocol and/or the results of the first analysis or protocol may be used to determine whether the second analysis or protocol should be implemented. With regard to the implementation of a peptide analysis or protocol, the results of a peptide expression analysis or protocol may be used to determine whether a specific treatment regimen should be implemented. With regard to the implementation of a polynucleotide analysis or protocol, the results of a polynucleotide expression analysis or protocol may be used to determine whether a specific treatment regimen should be implemented.

“Individual response/reaction” or “response/response” can be assessed using any endpoint indicating benefit to an individual, including but not limited to: (1) some degree of inhibition of disease progression (e.g., cancer progression), including slowing or completely blocking it; (2) reduction of tumor size; (3) inhibition (i.e., reduction, slowing, or complete cessation) of cancer cell infiltration into adjacent peripheral organs and/or tissues; (4) inhibition (i.e., reduction, slowing, or complete cessation) of metastasis; (5) some degree of relief of one or more symptoms associated with the disease or condition (e.g., cancer); (6) prolongation or extension of the length of survival (including overall survival and progression-free survival); and/or (7) a reduction in mortality at a given time point after treatment.

An individual’s “effective response” or “responsiveness” to treatment refers to a clinical or therapeutic benefit given to a patient who is at risk of or has a disease or condition (such as cancer). In one implementation, such benefit includes one or more of the following: prolonged survival (including overall survival and/or progression-free survival); resulting in an objective response (including a complete or partial response); or improvement of signs or symptoms of cancer. In one implementation, at least one biomarker (e.g., a KRAS-G13D mutation or an activating mutation in NRAS) is used to identify patients predicted to have an elevated likelihood of responding to treatment with a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor), either alone or in combination with a MEK inhibitor, a PI3K inhibitor (e.g., a pan-PI3K inhibitor), and/or other therapeutic agents (e.g., immunotherapeutic agents, cytotoxic agents, growth inhibitors, radiotherapy agents, and anti-angiogenic agents). In one implementation, at least one biomarker (e.g., KRAS-G13D mutation or NRAS activating mutation) is used to identify patients predicted to have an elevated likelihood of responding to treatment with a pan-RAF inhibitor (e.g., pan-RAF dimer inhibitor) in combination with MEK inhibitors, PI3K inhibitors (e.g., pan-PI3K inhibitors), and/or other therapeutic agents (e.g., immunotherapeutic agents, cytotoxic agents, growth inhibitors, radiotherapy agents, and anti-angiogenic agents).

"Objective response" refers to a measurable response, including complete response (CR) or partial response (PR). In some implementations, "objective response rate (ORR)" refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

"Complete response" or "CR" refers to the disappearance of all signs of a proliferative cell disease, such as cancer, in response to treatment (e.g., disappearance of all target damage). This does not always mean that the disease (e.g., cancer) has been cured.

"Durable response" refers to a lasting effect in reducing tumor growth after treatment has been stopped. For example, the tumor size may remain the same or smaller compared to its size at the start of the drug administration phase. In some embodiments, a durable response has a duration at least the same as, at least 1.5 times, 2.0 times, 2.5 times, or 3.0 times longer than the duration of treatment.

As used herein, “reducing or inhibiting cancer recurrence” means reducing or inhibiting tumor or cancer recurrence or tumor or cancer progression. As disclosed herein, cancer recurrence and/or cancer progression includes, but is not limited to, cancer metastasis.

As used herein, “partial response” or “PR” refers to a reduction in the size of one or more tumors or lesions or the extent of cancer in the body in response to treatment. For example, in some embodiments, PR refers to a reduction of at least 30% in the longest diameter (SLD) of the target lesion relative to a baseline SLD.

The term "survival" refers to a patient still being alive, and includes both overall survival and progression-free survival.

As used in this article, “progression-free survival” or “PFS” refers to the length of time during and after treatment when the treated disease (e.g., cancer) does not worsen. Progression-free survival can include the amount of time a patient has experienced a complete or partial response, and the amount of time a patient has experienced stable disease.

As used in this article, “overall survival” or “OS” refers to the percentage of individuals in a group who are likely to be alive after a specific duration.

"Prolonged survival" means extending overall or progression-free survival in treated patients relative to untreated patients (i.e., patients not treated with drugs), or patients who do not express biomarkers at specified levels, and/or patients treated with antitumor agents.

"Therapeutic effective dose" refers to the amount of a therapeutic agent that treats or prevents a disease or symptom in mammals. In the case of cancer, a therapeutically effective dose of a therapeutic agent can reduce the number of cancer cells; shrink the size of the primary tumor; inhibit (i.e., slow down, preferably stop) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down, preferably stop) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate one or more symptoms associated with the symptom to some extent. In terms of the extent to which a drug can prevent the growth of existing cancer cells and/or kill existing cancer cells, it can be cytosuppressive and/or cytotoxic. For cancer therapies, in vivo efficacy can be measured, for example, by assessing duration of survival, time to disease progression (TTP), response rate (e.g., CR and PR), duration of response, and/or quality of life.

“Symptom” is any condition that would benefit from treatment, including but not limited to chronic and acute symptom or disease, including those pathological conditions that make mammals susceptible to the symptom discussed.

The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals characterized by unregulated cell growth. This definition includes both benign and malignant cancers. Examples of cancers include, but are not limited to, carcinomas; lymphomas; blastomas (including medulloblastoma and retinoblastoma); sarcomas (including liposarcoma and synovial cell sarcoma); neuroendocrine tumors (including carcinoid tumors, gastrinomas, and islet cell carcinomas); mesotheliomas; Schwann cell tumors (including acoustic neuromas); meningiomas; adenocarcinomas; melanomas; and leukemias or lymphoid malignancies. More specific examples of this type of cancer include bladder cancer (e.g., urothelial bladder cancer (e.g., transitional cell or urothelial carcinoma, non-muscle-invasive bladder cancer, muscle-invasive bladder cancer, and metastatic bladder cancer) and non-urothelial bladder cancer); squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung, and squamous cell carcinoma of the lung; peritoneal cancer; hepatocellular carcinoma; stomach cancer or cancer of the stomach, including gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; hepatoma; breast cancer (including metastatic breast cancer); colon cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary gland cancer; kidney cancer or cancer of the kidney; prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; Merkel cell carcinoma; mycosis fungoides; testicular cancer; esophageal cancer; bile duct tumors; head and neck cancer; and hematopoietic malignancies. In some implementations, the cancer is triple-negative metastatic breast cancer, including any histologically confirmed triple-negative (ER-, PR-, HER2-) breast cancer with locally recurrent or metastatic disease (where the locally recurrent disease is not suitable for curative resection). In some implementations, the cancer is skin cancer, including melanoma, such as melanoma with locally recurrent or metastatic disease (where the locally recurrent disease is not suitable for curative resection). Any cancer can be in an early or late stage. “Early-stage cancer” or “early-stage tumor” means cancer that is not invasive or metastatic or classified as stage 0, 1, or 2 cancer.

As used herein, the term “tumor” refers to all proliferative cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” and “tumor” are not mutually exclusive when used herein.

The term "pharmaceutical formulation" refers to a formulation whose form allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any other ingredients that would have unacceptable toxicity to a subject who would administer the formulation.

"Pharmaceutical-acceptable excipients" refer to components in pharmaceutical formulations that are non-toxic to subjects, other than the active ingredient. Pharmaceutical-acceptable excipients include, but are not limited to, buffers, carriers, stabilizers, or preservatives.

The term "pharmaceutically acceptable salt" means a salt that is not biologically or otherwise undesirable. Pharmaceutically acceptable salts include both acid and base addition salts. The phrase "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with other components constituting the formulation and/or the mammals being treated with it.

The term "pharmaceutically acceptable acid addition salt" refers to pharmaceutically acceptable salts formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid) and organic acids (selected from aliphatic, cycloaliphatic, aromatic, aromatic aliphatic, heterocyclic, carboxyl, and sulfonic acid groups, such as formic acid, acetic acid, propionic acid, glycolic acid/glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid/maleic acid, malonic acid, succinic acid, fumaric acid/fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, aminoanilic acid, benzoic acid, cinnamic acid, mandelic acid, dihydroxynaphthyl acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid).

The term "pharmaceutically acceptable base addition salt" refers to those pharmaceutically acceptable salts formed with organic or inorganic bases. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and salts of base ion exchange resins (such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, aminobutanetriol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, halamine, choline, betaine, ethylenediamine, glucosamine, meglumine diatrizoate, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins).

As used herein, “treatment/management” refers to a clinical intervention that attempts to alter the natural course of the disease in the individual being treated, and may be implemented either for prevention or in the course of clinicopathology. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, relieving symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis. In some implementations, pan-RAF dimer inhibitors are used to delay the onset of disease or slow disease progression. In some implementations, pan-RAF dimer inhibitors are used in combination with MEK inhibitors or PI3K inhibitors (e.g., pan-PI3K inhibitors) to delay the onset of disease or slow disease progression.

The term "anticancer therapy" refers to a therapy that is useful in treating cancer. Examples of anticancer therapeutic agents include, but are not limited to, cytotoxic agents, chemotherapeutic agents, growth inhibitors, agents used in radiotherapy, anti-angiogenic agents, apoptosis agents, anti-microtubule agents, and other agents for treating cancer, such as anti-CD20 antibodies, platelet-derived growth factor inhibitors (e.g., imatinib mesylate), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists that bind to one or more of the following targets (e.g., neutralizing antibodies): PDGFR-β, BlyS, APRIL, BCMA receptor, TRAIL/Apo2, other biologically active and organic chemical agents, etc. Combinations thereof are also included in this invention.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and/or causes cellular destruction. This term is intended to include radioactive isotopes (e.g., radioactive isotopes of At- ²¹¹ , I ^-131 , I- ¹²⁵ , Y ^-90 , Re ^-186 , Re ^-188 , Sm ^-153 , Bi ^-212 , P- ³² , and Lu), chemotherapeutic agents such as methotrexate, doxorubicin, vinblastine alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as lysozymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and/or variants thereof, and various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. Tumor-killing agents cause destruction of tumor cells.

Chemotherapy agents are chemical compounds used in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; and ethylenimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolamine. Melamine); acetogenins (especially bullatacin and bullatacinone); tetrahydrocannabinol (dronabinol); lapachone; lapachol; colchicines; betulinic acid Camptothecin (including synthetic analogues such as topotecan CPT-11 (irinotecan), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllinic acid acid); teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide Hydrochlorides, melphalan, novombhichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ω1I (see, for example, Nicolaou et al., Angew. Chem. Intl. Ed. Engl. 33:183-186 (1994)); anthracycline antibiotics, including dynemicin A; esperamicin; and neocarzinostatin chromophores and related chromogenic chromophores of alkenyne antibiotics, aclacinomycins, actinomycins, autramycins, azaserine, bleomycin, cactinomycin C, carrubicin, carminomycin, carzinophilin, chromomycin. Actinomycin D, daunorubicin, detorubicin, 6-diaza-5-oxo-L-leucine, doxorubicin (including morpholine doxorubicin, cyanomorpholine doxorubicin, 2-pyrrole doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid Folic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, methotrexate, and pteropterin. Trimetrexate; purine analogues, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues, such as ancitabine, azacitidine, azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluxuridine; and androgens, such as calusterone and dromostanolone. Propionate, epitiostanol, mepitiostane, testolactone; anti-adrenergics, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; eflornithine; elliptinium acetate); epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; ethylhydrazide; procarbazine; polysaccharide complex (JHS Natural) Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonicacid; triaziquone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urana Urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; piperoman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, such as taxanes, including paclitaxel (Bristol-Myers Squibb). Squibb Oncology, Princeton, NJ), ABRAXANE ^™ palitaxel formulations without cremophor, albumin-modified nanoparticles (American Pharmaceutical Partners, Schaumberg, Illinois) and docetaxel (Rorer, Antony, France); chlorambucil; gemcitabine, thioguanine; mecaptopurine; methotrexate; platinum or platinum-based chemotherapy agents and platinum analogues, such as cisplatin, carboplatin, oxaliplatin (ELOXATIN ^™) . Satraplatin, picoplatin, nedaplatin, triplatin, and lipoplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; oxaliplatin; leucovovin; vinorelbine; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; pharmaceutically acceptable salts or acids of any of the above substances; and combinations of two or more of the above substances, such as CHOP (an abbreviation for a combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone) and FOLFOX (an abbreviation for a treatment regimen of oxaliplatin (ELOXATIN ^™ ) combined with 5-FU and leucovorin). Other chemotherapeutic agents include cytotoxic agents useful as antibody drug conjugates, such as mayendime alkaloids (e.g., DM1) and auristatin (e.g., MMAE and MMAF).

Chemotherapy agents also include "anti-hormonal agents" or "endocrine therapy agents" that regulate, reduce, block, or inhibit the effects of hormones that promote cancer growth, and are often in the form of systemic or systemic treatment. They themselves can be hormones. Examples include anti-estrogens and selective estrogen receptor modulators (SERMs), including tamoxifen (including tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; anti-progesterones; estrogen receptor downregulators (ERDs); and agents that inhibit or shut down ovarian function, such as luteinizing hormone-releasing hormone (LHRH) agonists, such as leuprolide acetate and goserelin acetate. Buserelin acetate and tripterelin; other antiandrogens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit aromatase, an enzyme that regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole. Additionally, this definition of chemotherapeutic agents includes bisphosphonates such as clodronate (e.g., or), etidronate, NE-58095, zoledronic acid/zoledronate, alendronate, pamidronate, tiludronate, or risedronate; and troxacita. bine (1,3-dioxolane cytosine analogue); antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGFR); vaccines, such as vaccines and gene therapy vaccines, such as vaccines, vaccines, and vaccines; topoisomerase 1 inhibitors; lapatinib ditosylate (a small molecule inhibitor of dual tyrosine kinases of ErbB-2 and EGFR, also known as GW572016); and pharmaceutically acceptable salts or acids of any of the above substances.

Chemotherapy agents also include antibodies, such as alemtuzumab (bevacizumab) (Genentech); cetuximab (Imclone); panitumumab (Amgen); rituximab (Genentech/Biogen Idec); pertuzumab (2C4, Genentech); trastuzumab (Genentech); tositumomab (Corixa); and antibody-drug conjugates, gemtuzumab ozogamicin (Wyeth). Other humanized monoclonal antibodies with therapeutic potential as pharmaceutical agents in combination with the compounds of the present invention include: apolizumab, acelizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cituzumab, daclizumab, eculizumab, efalizumab, epratuzumab, and others. Erlizumab, Felvizumab, Fontolizumab, Gemtuzumab (Ozogamicin), Intozumab (Ozogamicin), Ipilimumab, Labetuzumab, Lintuzumab, Matuzumab, Mepolizumab, Motavizumab, Motavizumab, Natalizumab, Nimotuzumab, Nolovizumab, Nunavisumab b, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxe Tan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab, celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and anti-interleukin-12 (ABT-874/J695, Wyeth Research and Abbott Laboratories), which is a genetically modified recombinant proprietary sequence to recognize the interleukin-12p40 protein, a full-length IgG1λ antibody.

Chemotherapy agents also include “EGFR inhibitors,” which are compounds that bind to EGFR or otherwise directly interact with EGFR and block or reduce its signaling activity; they are also known as “EGFR antagonists.” Examples of such agents include antibodies and small molecules that bind EGFR. Examples of EGFR-binding antibodies include MAb 579 (ATCC CRL HB8506), MAb455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see U.S. Patent No. 4,943,533, Mendelsohn et al.) and their variants, such as chimeric 225 (C225 or cetuximab) and reconstructed human 225 (H225). 5)(See WO 96/40210, Imclone Systems Inc.); IMC-11F8, a fully human EGFR-targeting antibody (Imclone); an antibody binding to type II mutant EGFR (US Patent No. 5,212,290); humanized and chimeric antibodies binding EGFR, as described in US Patent No. 5,891,996; and human antibodies binding EGFR, such as ABX-EGF or panitumumab (see WO 98/50433, Abgenix/Amgen); EMD 55900 (Stragliotto et al., Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab), a humanized EGFR antibody targeting EGFR and competing with both EGF and TGF-α for EGFR binding (EMD/Merck); human EGFR antibody, HuM anti-EGFR (GenMab); known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E7.6.3 and described in US 6,235,883 as fully human antibodies; MDX-447 (Medarex Inc); and mAb 806 or humanized mAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384(2004)). Anti-EGFR antibodies can be conjugated with cytotoxic agents, thus forming immunoconjugates (see, for example, EP 659,439A2, Merck Patent GmbH). EGFR antagonists include small molecules, such as those described in U.S. Patent Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332,5. The compounds described in PCT publications WO 98/14451, WO 98/50038, WO 99/09016, and WO 99/24037. Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, Genentech/OSI Pharmaceuticals); PD183805 (CI 1033, 2-acrylamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (ge fitinib)4-(3’-chloro-4’-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); ZM 105180((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382(N8-(3-chloro-4-fluorophenyl)-N2-(1-methylpiperidin-4-yl)pyrimidino[5,4-d]pyrimidin-2,8-diamine, Boehringer Ingelhei m); PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol); (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidin); CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7- [Ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide (Wyeth); AG1478 (Pfizer); AG1571 (SU 5271; Pfizer); and dual EGFR/HER2 tyrosine kinase inhibitors, such as lapatinib (GSK572016 or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[2-methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamide).

Chemotherapy agents also include "tyrosine kinase inhibitors," including EGFR-targeted drugs mentioned earlier; small molecule HER2 tyrosine kinase inhibitors, such as TAK165 available from Takeda; CP-724, 714, an oral ErbB2 receptor tyrosine kinase selective inhibitor (Pfizer and OSI); dual HER inhibitors that preferentially bind to EGFR but inhibit both HER2 and EGFR-overexpressing cells, such as EKB-569 (available from Wyeth); lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors, such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors, such as those from ISIS Available from pharmaceuticals are the antisense agent ISIS-5132, which inhibits Raf-1 signaling; non-HER-targeting TK inhibitors, such as imatinib mesylate (available from Glaxo SmithKline); multi-targeting tyrosine kinase inhibitors, such as sunitinib (available from Pfizer); VEGF receptor tyrosine kinase inhibitors, such as vatalanib (PTK787/ZK222584, available from Novartis/Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035 and 4-(3-chloroaniline)quinazoline; pyridopyrimidine; pyrimidine; pyrrolopyrimidine, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidine, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferoylmethane, 4,5-bis(4-fluoroaniline)-phthalimide); tyrphostin containing a nitrothiophene module; PD-0183805 (Warner-Lamber); antisense molecules (e.g., those that bind to nucleic acids encoding HER); quinoxaline (US Patent No. 5,804,396); tyrphostin (US Patent No. 5,804,396); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors, such as CI-1033 (Pfizer); Affinitac ^™ (ISIS 3521; Isis/Lilly); imatinib mesylate PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone); rapamycin (sirolimus); or as described in any of the following patent publications: U.S. Patent No. 5,804,396; WO 1999/09016 (American Cyanamid); WO 1998/43960 (American Cyanamid); WO 1997/38983 (Warner Lambert); WO 1999/06378 (Warner Lambert); WO 1999/06396 (Warner Lambert); WO 1996/30347 (Pfizer, Inc); WO 1996/33978 (Zeneca); WO 1996/3397 (Zeneca) and WO 1996/33980 (Zeneca).

Chemotherapy agents also include dexamethasone, interferon, colchicine, metoprine, cyclosporine, amphotericin B, metronidazole, alemtuzumab, alitretinoin, allopurinol, aifostine, arsenic trioxide, and asparaginase. Live BCG, bevacizumab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon α-2a, interferon α-2 b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, PEG filgrastim, pemetrexed Disodium), plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, and their pharmaceutically acceptable salts.

Chemotherapy agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, and dexamethasone sodium phosphate. Phosphate), fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate, and fluprednidene acetate. Acetate); Immunoselective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and its D-isomer (feG) (IMULAN BioTherapeutics, LLC); Antirheumatic drugs, such as azathioprine, cyclosporin (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide, minocycline, sulfasalazine; Tumor necrosis factor α (TNFα) blockers, such as etanercept, infliximab, adalimumab, and certolizumab. pegolgolimumab, interleukin-1 (IL-1) blockers, such as anakinra; T-cell co-stimulation blockers, such as abatacept; interleukin-6 (IL-6) blockers, such as tocilizumab; interleukin-13 (IL-13) blockers, such as lebrikizumab; interferon-alpha (IFN) blockers, such as rontalizumab; β7 integrin blockers, such as rhuMAb Beta7; IgE pathway blockers, such as anti-M1 prime; secretory homotrimeric LTa3 and membrane-bound heterotrimeric LTa1/β2 blockers, such as anti-lymphotoxin alpha (LTa); mixed investigational agents, such as thioplatin, PS-341, phenyl butyrate, ET-18- _OCH3. And farnesyltransferase inhibitors (L-739749, L-744832); polyphenols, such as quercetin, resveratrol, piceatannol, epigallocatechinegallate, theaflavin, flavanol, procyanidins, betulinic acid; autophagy inhibitors, such as chloroquine; δ-9-tetrahydrocannabinol (dronabinol); β-lapachone; laparol; colchicine derivatives; betulinic acid (acid); acetylcamptothecin, scopolectin, and 9-aminocamptothecin; podophyllotoxin; tegafur; bexarotene; bisphosphonates, such as clodronate (e.g., or), etidronate NE-58095, zoledronic acid/zoledronic acid Alandronate, pamidronate, tiludronate, or risedronate; epidermal growth factor receptor (EGF-R); vaccines, such as vaccines; perifosine; COX-2 inhibitors (e.g., celecoxib or etoricoxib); protein body inhibitors (e.g., PS341); CCI-779; tipifarnib (R11577); sorafenib; ABT510; Bcl-2 inhibitors, such as oblimersensodiumpixantrone; farnesyltransferase inhibitors, such as lonafarnib (SCH6636, SARASAR ^™ ); and pharmaceutically acceptable salts or acids of any of the above substances; and combinations of two or more of the above substances.

As used herein, the term "prodrug" refers to a precursor form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and can be enzymatically activated or converted into a more active parent form. See, for example, Wilman, "Prodrugs in Cancer Chemotherapy," Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al. (ed.), pp. 247-267, Humana Press (1985). The prodrugs of this invention include, but are not limited to, phosphate/ester-containing prodrugs, thiophosphate/ester-containing prodrugs, sulfate/ester-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide prodrugs, or optionally substituted phenylacetamide prodrugs, and 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted into drugs with higher activity and no cytotoxicity. Examples of cytotoxic drugs that can be derived into prodrug forms for use in this invention include, but are not limited to, the chemotherapeutic agents described above.

As used herein, “growth inhibitor” refers to a compound or composition that inhibits the growth and/or proliferation of cells (e.g., cells whose growth depends on MAPK pathway signaling) either in vitro or in vivo. Thus, a growth inhibitor can be an agent that significantly reduces the percentage of cells in the S phase. Examples of growth inhibitors include agents that block cell cycle progression (at positions other than S phase), such as agents that induce G1 arrest and M phase arrest. Classical M-phase blockers include vincristine (vincristine and vinblastine), taxane, and topoisomerase II inhibitors such as the anthracycline antibiotic doxorubicin ((8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lythopyranoside)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthyldione, i.e., (8S-cis)-10 -[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione), epirubicin, daunorubicin, etoposide, and bleomycin. Agents that block G1 phase also overflow into S-phase arrest, such as DNA alkylating agents like tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in "The Molecular Basis of Cancer," edited by Mendelsohn and Israel, Chapter 1, entitled "Cell cycle regulation, oncogenes, and anti-neoplastic drugs," by Murakami et al. (WB Saunders: Philadelphia, 1995), especially page 13. Paclitaxel and docetaxel are both anticancer drugs derived from the yew tree. Rhone-Poulenc Rorrer, derived from the European yew, is a semi-synthetic analog of paclitaxel (Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of tubulin dimers into microtubules and stabilize microtubules by preventing depolymerization, leading to inhibition of mitosis in cells.

"Radiotherapy" means using targeted gamma or beta rays to induce sufficient damage to a cell, thereby limiting its ability to function normally or destroying the cell entirely. It will be understood that there are many ways known in the art to determine the dosage and duration of treatment. Typical treatment is given as a single administration and the typical dosage range is 10 to 200 units (Grays) per day.

As used herein, “administration” means the method of giving a dose of a compound (e.g., an inhibitor or antagonist) or a pharmaceutical composition (e.g., a pharmaceutical composition comprising an inhibitor or antagonist) to a subject (e.g., a patient). Administration can be by any suitable means, including parenteral, intrapulmonary, and intranasal, and intralesional administration if intended for local treatment. Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Depending in part on whether the administration is transient or prolonged, dose delivery can be via any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various dose delivery schedules are covered herein, including but not limited to single or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

The term "co-administration" is used herein to refer to the administration of two or more therapeutic agents, wherein at least some of the administration overlaps in time. Thus, concurrent administration includes dosage regimens in which one or more agents are administered after an interruption of the administration of one or more other agents.

"Reduction or inhibition" means the ability to cause an overall reduction of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. For example, reduction or inhibition can refer to the level of activity and/or function of proteins (e.g., RAF, MEK, and/or PI3K) in, for example, the MAPK signaling pathway/PI3K pathway. Additionally, reduction or inhibition can refer to, for example, the symptoms of the treated condition (e.g., cancer), the presence or size of metastases, or the size of the primary tumor.

The term "packaging insert" is used to refer to the instruction leaflet typically included in the commercial packaging of a therapeutic product, which contains information about the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings for the use of such therapeutic products.

"Article" is any manufactured item (e.g., package or container) or kit containing at least one reagent, such as a medicine for treating a disease or condition (e.g., cancer), or a probe for specifically detecting a biomarker described herein (e.g., a KRAS-G13D mutation or an NRAS activating mutation). In some embodiments, the manufactured item or kit is marketed, distributed, or sold in units for implementing the methods described herein.

When used in this article, the phrase “based on” means using information about one or more biomarkers to inform diagnostic decisions, treatment decisions, information provided in packaging inserts, or marketing/promotional guidance, etc.

III. Methods

A. Diagnostic methods

This invention provides a method for identifying individuals with cancer (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)) who may benefit from treatment including a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor or a PI3K inhibitor. The method includes screening samples from the individual (e.g., tissue samples, such as tumor tissue samples) for KRAS activating mutations (e.g., KRAS-G13D mutations), wherein the presence of a KRAS activating mutation (e.g., KRAS-G13D mutation) in the sample identifies the individual as potentially benefiting from treatment including a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK or PI3K inhibitor. A method for selecting treatment for individuals with cancer is also provided. These methods similarly include the step of screening samples from individuals for KRAS activating mutations (e.g., KRAS-G13D mutations), wherein the presence of a KRAS activating mutation (e.g., KRAS-G13D mutation) in the sample identifies the individual as potentially eligible to benefit from treatment comprising a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK or PI3K inhibitor. Methods are also provided for optimizing the therapeutic efficacy of treatments for individuals with cancer, wherein the treatment comprises a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK or PI3K inhibitor. Further provided herein are methods for predicting the responsiveness of individuals with cancer to treatment comprising a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK or PI3K inhibitor. Any method may further include administering a therapeutically effective amount of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK or PI3K inhibitor to the individual, for example based on the presence of a KRAS activating mutation (e.g., KRAS-G13D mutation). In addition, any method may further include administering a therapeutically effective amount of another therapeutic agent (e.g., immunotherapeutic agents, cytotoxic agents, growth inhibitors, radiotherapy agents, and anti-angiogenic agents) to the individual.

In some cases, the present invention provides a method for identifying individuals with cancer (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)) who may benefit from treatment including pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK inhibitors based on screening samples (e.g., tissue samples, such as tumor tissue samples) from the individual for KRAS-G13D mutations, wherein the presence of a KRAS-G13D mutation in the sample indicates an elevated likelihood that the individual will benefit from treatment including pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK inhibitors. Screening may include amplification and sequencing of the entire or a portion of the KRAS gene, for example, to determine a specific genotype of the individual. Thus, screening may include specific amplification and sequencing of the entire or a portion of exon 2 of the KRAS gene, wherein a KRASc.38G>A nucleotide substitution mutation at codon 13 of exon 2 of the KRAS gene indicates a KRAS-G13D mutation. In other cases, screening may include sequencing of the whole or a portion of the KRAS protein, for example, to determine whether an individual has a KRAS-G13D amino acid mutation. In cases where an individual has a KRAS-G13D mutation, the method may further include administering a therapeutically effective amount of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor to the individual, optionally in combination with one or more other therapeutic agents (e.g., immunotherapeutic agents, cytotoxic agents, growth inhibitors, radiotherapy agents, and anti-angiogenic agents).

In some cases, the present invention also provides a method for selecting a treatment for an individual with cancer (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)), wherein the method includes screening samples from the individual (e.g., tissue samples (e.g., tumor tissue samples)) for KRAS-G13D mutations, wherein the presence of a KRAS-G13D mutation in the sample identifies the individual as potentially eligible for treatment, including pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK inhibitors. Screening may include amplification and sequencing of the entire or a portion of the KRAS gene, for example, to determine a specific genotype of the individual. Thus, screening may include specific amplification and sequencing of exon 2 of the entire or a portion of the KRAS gene, wherein a KRAS c.38G>A nucleotide substitution mutation at codon 13 of exon 2 of the KRAS gene indicates a KRAS-G13D mutation. In other cases, screening may include sequencing of the entire or a portion of the KRAS protein, for example, to determine whether an individual has a KRAS-G13D amino acid mutation. In cases where an individual has a KRAS-G13D mutation, the method may further include the step of administering a therapeutically effective amount of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor to the individual, optionally in combination with one or more other therapeutic agents (e.g., immunotherapeutic agents, cytotoxic agents, growth inhibitors, radiotherapy agents, and anti-angiogenic agents).

In some cases, the present invention provides a method for screening for KRAS activating mutations (e.g., KRAS-G12D, KRAS-G12C, KRAS-G12V, KRAS-G13D, KRAS-G12A, KRAS-G12R, KRAS-G12S, KRAS-G13C, KRAS-G13A, KRAS-G13R, KRAS-G13S, KRAS-G13V, KRAS-Q61H, KRAS-Q61K, KRAS-Q61E, KRAS-G12D ...D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12D, KRAS-G12 A method for identifying individuals with cancer (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)) who may benefit from treatment including pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and PI3K inhibitors (e.g., pan-PI3K inhibitors), wherein the presence of a KRAS activating mutation in the sample indicates an elevated likelihood of benefiting from treatment including pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and PI3K inhibitors (e.g., pan-PI3K inhibitors). Screening may include amplification and sequencing of the entire or a portion of the KRAS gene, for example, to determine a specific genotype of the individual. Thus, screening may include specific amplification and sequencing of KRAS gene codons 12, 13, and/or 61. In other cases, screening may include sequencing of the entire or a portion of the KRAS protein, for example, to determine whether an individual has a KRAS activating amino acid mutation. In cases where the individual has a KRAS activating mutation, the method may further include administering a therapeutically effective amount of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a PI3K inhibitor (e.g., a pan-PI3K inhibitor) to the individual, optionally in combination with one or more other therapeutic agents (e.g., immunotherapeutic agents, cytotoxic agents, growth inhibitors, radiotherapy agents, and anti-angiogenic agents). In some cases, the individual does not have a BRAF activating mutation.

In some cases, the present invention also provides a method for selecting a treatment for an individual with cancer (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)), wherein the method includes screening samples from the individual (e.g., tissue samples (e.g., tumor tissue samples)) for KRAS activating mutations (e.g., KRAS-G12D, KRAS-G12C, KRAS-G12V, KRAS-G13D, KRAS-G12A, KRAS-G12R, KRAS-G12S, KRAS-G12C, KRAS-G12D, KRAS-G12D, KRAS-G12A, KRAS-G12R, KRAS-G12S, KRAS-G12D ... The KRAS gene mutations (G13C, KRAS-G13A, KRAS-G13R, KRAS-G13S, KRAS-G13V, KRAS-Q61H, KRAS-Q61K, KRAS-Q61E, KRAS-Q61L, KRAS-Q61P, or KRAS-Q61R) in a sample identify individuals who may benefit from treatments including pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and PI3K inhibitors (e.g., pan-PI3K inhibitors). Screening may include amplification and sequencing of the entire or partial KRAS gene, for example, to determine a specific genotype of an individual. Thus, screening may include specific amplification and sequencing of KRAS gene codons 12, 13, and/or 61. In other cases, screening may include sequencing of the entire or partial KRAS protein, for example, to determine whether an individual has a KRAS activating amino acid mutation. In cases where the individual has a KRAS activating mutation, the method may further include administering a therapeutically effective amount of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a PI3K inhibitor (e.g., a pan-PI3K inhibitor) to the individual, optionally in combination with one or more other therapeutic agents (e.g., immunotherapeutic agents, cytotoxic agents, growth inhibitors, radiotherapy agents, and anti-angiogenic agents). In some cases, the individual does not have a BRAF activating mutation.

The present invention also provides a method for identifying individuals with cancer (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)) who may benefit from treatment including pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK inhibitors, wherein the method includes screening samples from the individual (e.g., tissue samples (e.g., tumor tissue samples)) for NRAS activating mutations (e.g., NRAS-Q61R, NRAS-Q61K, NRAS-G12D, NRAS-G13D, NRAS-G12S ... -G12C, NRAS-G12V, NRAS-G12A, NRAS-G12R, NRAS-G13C, NRAS-G13A, NRAS-G13R, NRAS-G13S, NRAS-G13V, NRAS-Q61H, NRAS-Q61E, NRAS-Q61L, or NRAS-Q61P), wherein the presence of NRAS activating mutations in a sample identifies the individual as potentially benefiting from treatment comprising a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor. Methods for selecting treatment for individuals with cancer are also provided. These methods similarly include the step of screening samples from individuals for NRAS activating mutations, wherein the presence of NRAS activating mutations in a sample identifies the individual as potentially benefiting from treatment comprising a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor. Also provided are methods for optimizing the therapeutic efficacy of treatments for individuals with cancer, wherein the treatment includes pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK inhibitors. Further provided herein are methods for predicting the response of individuals with cancer to treatments including pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK inhibitors. Any method may further include administering a therapeutically effective amount of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor to the individual, for example based on the presence of NRAS activating mutations. Additionally, any method may further include administering a therapeutically effective amount of another therapeutic agent (e.g., an immunotherapy agent, a cytotoxic agent, a growth inhibitor, a radiotherapy agent, and an anti-angiogenic agent) to the individual.

In some cases, the present invention provides a method for screening NRAS activating mutations (e.g., NRAS-Q61R, NRAS-Q61K, NRAS-G12D, NRAS-G13D, NRAS-G12S, NRAS-G12C, NRAS-G12V, NRAS-G12A, NRAS-G12R, NRAS-G13C, NRAS-G13A, NRAS-G13R, NRAS-G13S, NRAS-G13V) based on samples from individuals (e.g., tissue samples, such as tumor tissue samples). NRAS-Q61H, NRAS-Q61E, NRAS-Q61L, or NRAS-Q61P are methods used to identify individuals with cancer (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)) who may benefit from treatment including pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK inhibitors, wherein the presence of NRAS activating mutations in the sample indicates an elevated likelihood of benefiting from treatment including pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK inhibitors. Screening may include amplification and sequencing of the entire or partial NRAS gene, for example, to determine a specific genotype of the individual. Thus, screening may include specific amplification and sequencing of NRAS gene codons 12, 13, and/or 61. In other cases, screening may include sequencing of the entire or partial NRAS protein, for example, to determine whether an individual has NRAS activating amino acid mutations. In cases where an individual has an activating mutation in NRAS, the method may further include the step of administering a therapeutically effective amount of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor to the individual, optionally in combination with one or more other therapeutic agents (e.g., immunotherapeutic agents, cytotoxic agents, growth inhibitors, radiotherapy agents, and anti-angiogenic agents).

In some cases, the present invention also provides a method for selecting a treatment for an individual with cancer (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)), wherein the method includes screening samples from the individual (e.g., tissue samples (e.g., tumor tissue samples)) for NRAS activating mutations (e.g., NRAS-Q61R, NRAS-Q61K, NRAS-G12D, NRAS-G13D, NRAS-G12S, NRAS-G12C, NRAS-G12V). The screening criteria include NRAS-G12A, NRAS-G12R, NRAS-G13C, NRAS-G13A, NRAS-G13R, NRAS-G13S, NRAS-G13V, NRAS-Q61H, NRAS-Q61E, NRAS-Q61L, or NRAS-Q61P), where the presence of NRAS activating mutations in the sample identifies the individual as potentially eligible for treatment, including pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK inhibitors. Screening may include amplification and sequencing of the entire or partial NRAS gene, for example, to determine a specific genotype of the individual. Thus, screening may include specific amplification and sequencing of NRAS gene codons 12, 13, and/or 61. In other cases, screening may include sequencing of the entire or partial NRAS protein, for example, to determine whether an individual has an NRAS activating mutation. In cases where an individual has an activating mutation in NRAS, the method may further include the step of administering a therapeutically effective amount of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor to the individual, optionally in combination with one or more other therapeutic agents (e.g., immunotherapeutic agents, cytotoxic agents, growth inhibitors, radiotherapy agents, and anti-angiogenic agents).

In any of the aforementioned methods in which a MEK inhibitor is a component of the treatment, the MEK inhibitor may be a small molecule inhibitor, which may be a prodrug or a biologically active form. For example, a MEK inhibitor may be a small molecule inhibitor selected from the group consisting of cobimetinib (GDC-0973), selmetinib (AZD6244), pimecrolimus (AS-703026), PD0325901, remetinib (BAY86-9766), bimetinib (MEK162), BI-847325, trametinib, GDC-0623, G-573, and CH5126766 (RO5126766), or a pharmaceutically acceptable salt thereof. In specific cases, small molecule inhibitors are cobimetinib (GDC-0973), selmetinib (AZD6244), pimecrolimus (AS-703026), PD0325901, remetinib (BAY86-9766), or bimemetinib (MEK162), or pharmaceutically acceptable salts thereof.

In any of the aforementioned methods in which a PI3K inhibitor is a component of the treatment, the PI3K inhibitor may be a small molecule inhibitor, which may be a prodrug or a biologically active form. For example, a small molecule inhibitor may be selected from the group consisting of picotelixicob (GDC-0941), taserixicob (GDC-0032), and apirilixicob (BYL719), or pharmaceutically acceptable salts thereof. The PI3K inhibitor may be a pan-PI3K inhibitor (e.g., picotelixicob (GDC-0941) or taserixicob (GDC-0032), or pharmaceutically acceptable salts thereof), or a PI3Kα-specific inhibitor and/or a PI3Kδ-specific inhibitor.

In any of the foregoing methods, the pan-RAF inhibitor may be a pan-RAF dimer inhibitor. In some cases, the pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) may be a small molecule inhibitor, which may be a prodrug or a biologically active form. In some cases, the pan-RAF dimer inhibitor may be HM95573, LY-3009120, AZ-628, LXH-254, MLN2480, BeiGene-283, RXDX-105, BAL3833, regorafenib, or sorafenib, or a pharmaceutically acceptable salt thereof.

The disclosed methods and assays provide a convenient, efficient, and potentially cost-effective means of obtaining data and information useful in evaluating appropriate or effective therapies for treating patients. For example, patients can provide tissue samples (e.g., tumor biopsies or blood samples) before and/or after treatment with pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors, and these samples can be examined via various in vitro assays to determine whether the patient's cells are sensitive to pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors.

In any of the foregoing methods, a specific mutational state of the KRAS and/or NRAS genes, mRNA, or protein products can be identified in a sample obtained from an individual by any of a variety of methods known to those skilled in the art. For example, mutations can be identified by cloning the KRAS and/or NRAS genes or portions thereof using techniques known in the art and sequencing them. Alternatively, gene sequences can be amplified from genomic DNA, for example using PCR, and the products can be sequenced. Several non-limiting methods for analyzing mutations at a given locus in a patient's DNA are described below.

DNA microarray technology can be used, such as DNA chip devices for high-throughput screening applications and high-density and low-density microarrays. Methods for microarray fabrication are known in the art and include various inkjet and microjet deposition or spotting techniques and methods, in-situ or on-chip photolithography oligonucleotide synthesis methods, and electronic DNA probe addressing methods. DNA microarray hybridization applications have been successfully applied in gene expression analysis and genotyping of point mutations, single nucleotide polymorphisms (SNPs), and short tandem repeats (STRs). Other methods include interfering RNA microarrays and combinations of microarrays with other methods such as laser capture microdissection (LCM), comparative genomic hybridization (CGH), and chromatin immunoprecipitation (ChiP). See, for example, Heet et al. (2007) Adv. Exp. Med. Biol. 593: 117-133 and Heller (2002) Annu. Rev. Biomed. Eng. 4: 129-153. Other methods include PCR, xMAP, invader assay, mass spectrometry, and pyrosequencing (Wang et al. (2007) 593: 105-106).

Another detection method involves allele-specific hybridization using probes with overlapping polymorphic sites and approximately 5, or alternatively 10, 20, 25, or 30 nucleotides around the polymorphic region. For example, several probes capable of specifically hybridizing to a particular mutant variant (e.g., KRAS-G13D, corresponding to the KRAS c.38G>A nucleotide substitution mutation) are attached to a solid support, such as a “chip.” Oligonucleotides can be bound to the solid support using various methods, including lithography. Mutation detection analysis using these oligonucleotide-containing chips (also known as “DNA probe arrays”) is documented, for example, in Cronin et al. (1996) Human Mutation 7:244.

In other detection methods, it is necessary to first amplify at least a portion of the gene before identifying the mutant variant. This amplification can be performed, for example, by PCR and/or LCR or other methods known in the art.

In some cases, restriction enzyme analysis can be used to reveal the presence of specific mutations in a subject's DNA. For example, a particular mutation can result in a nucleotide sequence lacking a restriction site that is present in another mutant variant of the gene or the wild-type nucleotide sequence.

In another embodiment, the detection of mismatched bases in RNA/RNA, DNA/DNA, or RNA/DNA heteroduplexes can be performed using protection from cleavage agents (such as nucleases, hydroxylamine, or osmium tetroxide and piperidine) (see, for example, Myers et al. (1985) Science 230:1242). Generally, the “mismatch cleavage” technique is initiated by providing a heteroduplex formed by hybridizing a control nucleic acid (e.g., RNA or DNA) containing a nucleotide sequence of a gene allele optionally labeled with that of the sample nucleic acid (e.g., RNA or DNA) with a self-organized sample. The duplex is treated with a reagent that cleaves the single-stranded region of the duplex, such as a duplex formed based on base pair mismatches between the control and sample strands. For example, RNA/DNA duplexes can be treated with RNases, and DNA/DNA hybrids can be treated with S1 nucleases to enzymatically digest the mismatched regions. Alternatively, DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and piperidine to digest the mismatched regions. After digesting the mismatched regions, the resulting material is then separated by size on a denaturing polyacrylamide gel to determine whether the control and sample nucleic acids have the same nucleotide sequence or in which nucleotides they differ. See, for example, U.S. Patent No. 6,455,249; Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) Meth. Enzymol. 217:286-295.

Changes in electrophoretic mobility can also be used to identify specific allelic variants. For example, single-strand conformational polymorphism (SSCP) can be used to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766; Cotton (1993) Mutat. Res. 285:125-144 and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of the sample and control nucleic acids are denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies with sequence, and the resulting changes in electrophoretic mobility allow for the detection of even single-base changes. The DNA fragments can be labeled or detected using labeled probes. The sensitivity of the assay can be enhanced by using RNA (instead of DNA) (where secondary structure is more sensitive to sequence changes). In another implementation, the subject method utilizes heteroduplex analysis to separate double-stranded heteroduplex molecules, based on changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

The identity of mutant variants can also be determined by analyzing the migration of nucleic acids containing polymorphic regions in a polyacrylamide gel containing a denaturing agent gradient, using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When using DGGE as an analytical method, the DNA is modified to ensure that it is not completely denatured, for example by adding a GC clip containing approximately 40 bp of high-melting-point GC-rich DNA via PCR. In yet another embodiment, a temperature gradient is used instead of a denaturing agent gradient to identify differences in migration rates between control and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chem. 265:1275).

Examples of techniques for detecting differences of at least one nucleotide between two nucleic acid molecules (e.g., DNA or RNA molecules) include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide probes can be prepared in which a known polymorphic nucleotide is placed in the center (allelic-specific probe), and then hybridized with target DNA under conditions where hybridization is only permitted when a perfect match is found (Saiki et al. (1986) Nature 324:163; Saiki et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allelic-specific oligonucleotide hybridization techniques can be used to detect nucleotide variations in polymorphic regions of genes. For example, oligonucleotides with nucleotide sequences of specific allelic variants are attached to a hybridization membrane, which is then hybridized with labeled sample nucleic acids. Analysis of the hybridization signal then reveals the identity of the nucleotides in the sample nucleic acid.

Alternatively, allelic-specific amplification techniques relying on selective PCR amplification may be useful in conjunction with this invention. The oligonucleotides used as primers for specific amplification can carry the allele of interest at the center of the molecule (making amplification dependent on differential hybridization) (Gibbs et al. (1989) Nucl. Acids Res. 17:2437-2448) or at the 3' end of a primer (where, under suitable conditions, mismatches can prevent or reduce polymerase extension) (Prossner (1993) Tibtech 11:238 and Newton et al. (1989) Nucl. Acids Res. 17:2503). This technique is also known as "PROBE," which stands for Probe Oligo Base Extension. Additionally, it is desirable to introduce novel restriction sites in the mutant region to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell. Probes 6:1).

In another embodiment, oligonucleotide ligation assay (OLA) is used to identify mutant variants, as described, for example, in U.S. Patent No. 4,998,617 and Laridegren, U. et al. Science 241:1077-1080 (1988). The OLA protocol uses two oligonucleotides designed to hybridize to adjacent sequences on a single strand of the target. One oligonucleotide is ligated to a separation marker, such as a biotinylated one, while the other is a detectable label. If a precise complementary sequence is found in the target molecule, the oligonucleotides hybridize, resulting in terminal adjacency and creating a ligation substrate. The ligation then allows for the recovery of the labeled oligonucleotide using avidin, or another biotinylate ligand. Nickerson et al. have described a nucleic acid detection assay combining PCR and OLA properties (Nickerson, D.A. et al. (1990) Proc. Natl. Acad. Sci. USA 87:8923-8927). In this method, PCR is used to achieve exponential amplification of the target DNA, and then OLA is used for detection.

This describes a method for detecting single nucleotide mutations in a given gene, such as the RAS gene, or genes like the KRAS or NRAS genes. Because single nucleotide variations are flanked by invariant sequence regions, the analysis only requires identifying the individual variant nucleotide and does not need to determine the complete gene sequence for each patient. Several methods have been developed to facilitate the analysis of such mutations.

Single-base mutations can be detected using specialized exonuclease-resistant nucleotides, as disclosed, for example, in U.S. Patent No. 4,656,127. According to this method, a primer complementary to the mutation sequence at the 3' of the mutation site is allowed to hybridize with a target molecule obtained from a specific animal or human. If the polymorphic site on the target molecule contains a nucleotide complementary to a specific exonuclease-resistant nucleotide derivative present, this derivative is incorporated into the end of the hybridization primer. This incorporation makes the primer resistant to the exonuclease, thereby allowing its detection. Because the identity of the exonuclease-resistant derivative of the sample is known, the discovery that the primer has become resistant to the exonuclease reveals that the nucleotide present at the mutation site of the target molecule is complementary to the nucleotide derivative used in the reaction. This method has the advantage that it does not require the determination of large amounts of irrelevant sequence data.

Solution-based methods can also be used to determine the identity of the nucleotide at the mutation site (WO 91/02087). As described above, primers complementary to the mutation sequence at the 3' of the mutation site are used. This method uses a labeled dideoxynucleotide derivative to determine the identity of the nucleotide at the site; if complementary to the nucleotide at the mutation site, the labeled dideoxynucleotide derivative is incorporated into the end of the primer.

An alternative method is described in WO 92/15712. This method uses a mixture of a labeled terminator and primers, the primers being complementary to the 3' sequence of the mutation or polymorphism site. Thus, the incorporated labeled terminator is identified by the nucleotide present at the mutation site of the target molecule being evaluated and is complementary to said nucleotide. This method is typically a heterophase assay in which the primers or target molecule are immobilized on a solid phase.

Several other primer-guided nucleotide incorporation protocols for determining mutation sites in DNA have been described (Komher, J.S. et al. (1989) Nucl. Acids. Res. 17:7779-7784; Sokolov, B.P. (1990) Nucl. Acids Res. 18:3671; Syvanen, A.-C., et al. (1990) Genomics 8:684-692; Kuppuswa My, M.N. et al. (1991) Proc. Natl. Acad. Sci. USA 88:1143-1147; Prezant, T.R. et al. (1992) Hum. Mutat. 1:159-164; Ugozzoli, L. et al. (1992) GATA 9:107-112; Nyren, P. et al. (1993) Anal. Biochem. 208:171-175). These methods all rely on the incorporation of labeled deoxynucleotides to distinguish the bases at the mutation site.

Generally, the presence and/or quantity of biomarker genes described herein can be analyzed using a variety of methodologies, including those described above, as well as many other methodologies known in the art and understood by those skilled in the art, such as Southern blotting, Northern blotting, whole-genome sequencing, polymerase chain reaction (PCR) (including quantitative real-time PCR (qRT-PCR) and other amplification assays, such as, for example, branched DNA, SISBA, TMA, etc.), RNA-Seq, microarray analysis, nanostrings, gene expression profiling, and/or gene expression serial analysis (“SAGE”), and any of a great variety of assays that can be performed using protein, gene, and/or tissue array analysis. Typical protocols for assessing the status of genes and gene products are found, for example, in Ausubel et al., eds., 1995, Current Protocols in Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting), and 18 (PCR Analysis). Multiplex immunoassays, such as those available from Rules Based Medicine or MesoScale Discovery (“MSD”), can also be used.

In addition, the presence and/or quantity of the biomarker proteins (i.e., gene products) described herein can be analyzed using a variety of methodologies, including immunohistochemistry (“IHC”), Western blot analysis, immunoprecipitation, spectroscopy, molecular binding assays, HPLC, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofiltration assay (ELIFA), fluorescence activated cell sorting (“FACS”), MassARRAY, proteomics, blood-based quantitative assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization, fluorescence in situ hybridization (FISH), and protein sequencing. In some cases, the method involves contacting a biological sample from an individual with an antibody that specifically binds to the protein biomarker described herein under conditions that allow biomarker binding, and detecting whether a complex is formed between the antibody and the biomarker. Such methods can be in vitro or in vivo. In some cases, the protein expression level of the biomarker (e.g., KRAS-G13D protein or NRAS protein with an activating mutation, such as those described above herein) is measured in tumor cells (e.g., from biopsies).

Furthermore, it should be understood that any of the aforementioned methods used to detect mutations in genes or gene products may also be used to monitor treatment or therapeutic processes (e.g., treatments involving pan-RAF inhibitors, such as pan-RAF dimer inhibitors, and MEK or PI3K inhibitors).

The methods described herein can be implemented, for example, by utilizing pre-packaged diagnostic kits, such as those described below, which contain at least one probe or primer nucleic acid that can be readily used, for example, to determine whether a subject is likely to benefit from treatment including panRAF inhibitors (e.g., panRAF dimer inhibitors) and MEK or PI3K inhibitors.

The nucleic acid samples used in the diagnostic methods described above can be obtained from any cell type or tissue of an individual (including tumor tissue or blood).

In all the screening methods described above or mentioned herein, mutations in one or more RAS genes (e.g., KRAS or NRAS) or their protein products are generally identified by determining the nucleic acid sequence (e.g., DNA or RNA sequence) or protein sequence (i.e., amino acid sequence) in a sample obtained from an individual and comparing that sequence with a reference sequence (e.g., wild-type sequence). In some cases, a reference level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is one or more samples from the same subject or individual obtained at one or more time points different from when the test sample was obtained. For example, a reference level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be obtained from the same subject or individual at a time point earlier than when the test sample was obtained. Such a reference level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be useful if the reference sample was obtained during the initial diagnosis of cancer while the test sample was obtained later, when the cancer has become metastatic.

B. Treatment methods

This invention provides a method for treating an individual with cancer (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)) by administering a therapeutically effective amount of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor or PI3K inhibitor to the individual based on the presence of a KRAS activating mutation (e.g., a KRAS-G13D mutation). In some cases, the method includes a step of screening samples from the individual (e.g., tissue samples (e.g., tumor tissue samples)) for KRAS activating mutations, wherein the individual has been identified as having a KRAS activating mutation. In other cases, samples from the individual (e.g., tissue samples (e.g., tumor tissue samples)) have been screened for KRAS activating mutations prior to treatment, and the presence of a KRAS activating mutation has been identified.

In some cases, the present invention provides a method for treating an individual with cancer (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)), comprising (a) screening a sample (e.g., a tissue sample (e.g., a tumor tissue sample)) from the individual for a KRAS-G13D mutation, wherein the individual has been identified as having a KRAS-G13D mutation, and (b) administering a therapeutically effective amount of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor to the individual based on the presence of the KRAS-G13D mutation identified by the screening step.

In some cases, the present invention provides a method for treating an individual with cancer (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)), comprising administering to the individual a therapeutically effective amount of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor, wherein samples from the individual (e.g., tissue samples (e.g., tumor tissue samples)) have been screened for KRAS-G13D mutations prior to treatment and the presence of KRAS-G13D mutations has been determined.

In some cases, the present invention provides a method for treating an individual with cancer (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)), comprising (a) screening samples from the individual (e.g., tissue samples (e.g., tumor tissue samples)) for KRAS activating mutations (e.g., KRAS-G12D, KRAS-G12C, KRAS-G12V, KRAS-G13D, KRAS-G12A, KRAS-G12R, KRAS-G12S, KRAS-G13C, KRAS-G12 ... (a) KRAS-G13A, KRAS-G13R, KRAS-G13S, KRAS-G13V, KRAS-Q61H, KRAS-Q61K, KRAS-Q61E, KRAS-Q61L, KRAS-Q61P, or KRAS-Q61R), wherein the individual has been identified as having a KRAS activating mutation, and (b) the individual is given a therapeutically effective amount of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a PI3K inhibitor based on the presence of a KRAS activating mutation identified through a screening step. In some cases, the individual does not have a BRAF activating mutation.

In some cases, the present invention provides a method for treating an individual with cancer (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)), comprising administering to the individual a therapeutically effective amount of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a PI3K inhibitor, wherein samples from the individual (e.g., tissue samples (e.g., tumor tissue samples)) have been screened for KRAS activating mutations (e.g., KRAS-G12D, KRAS-G12C, KRAS-G12D) prior to treatment. -G12V, KRAS-G13D, KRAS-G12A, KRAS-G12R, KRAS-G12S, KRAS-G13C, KRAS-G13A, KRAS-G13R, KRAS-G13S, KRAS-G13V, KRAS-Q61H, KRAS-Q61K, KRAS-Q61E, KRAS-Q61L, KRAS-Q61P, or KRAS-Q61R) and the presence of a KRAS activating mutation has been confirmed. In some cases, the individual does not have a BRAF activating mutation.

This invention also provides a method for treating an individual with cancer (e.g., skin cancer (e.g., melanoma), colorectal cancer, ovarian cancer, lung cancer, and pancreatic cancer) by administering a therapeutically effective amount of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor to the individual based on the presence of an NRAS activating mutation. In some cases, the method includes the step of screening samples from the individual (e.g., tissue samples (e.g., tumor tissue samples)) for NRAS activating mutations, wherein the individual has been identified as having an NRAS activating mutation. In other cases, samples from the individual (e.g., tissue samples (e.g., tumor tissue samples)) have been screened for NRAS activating mutations prior to treatment, and the presence of an NRAS activating mutation has been determined.

In some cases, the present invention provides a method for treating an individual with cancer (e.g., skin cancer (e.g., melanoma), colorectal cancer, ovarian cancer, lung cancer, and pancreatic cancer), comprising (a) screening samples from the individual (e.g., tissue samples (e.g., tumor tissue samples)) for NRAS activating mutations (e.g., NRAS-Q61R, NRAS-Q61K, NRAS-G12D, NRAS-G13D, NRAS-G12S, NRAS-G12C, NRAS-G12V, NRAS-G12A, NRA). (a) The individual has been identified as having an NRAS activating mutation (S-G12R, NRAS-G13C, NRAS-G13A, NRAS-G13R, NRAS-G13S, NRAS-G13V, NRAS-Q61H, NRAS-Q61E, NRAS-Q61L, or NRAS-Q61P), and (b) the individual is given a therapeutically effective amount of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor based on the presence of the NRAS activating mutation identified by the screening step.

In some cases, the present invention provides a method for treating an individual with cancer (e.g., skin cancer (e.g., melanoma), colorectal cancer, ovarian cancer, lung cancer, and pancreatic cancer), comprising administering to the individual a therapeutically effective amount of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor, wherein samples from the individual (e.g., tissue samples (e.g., tumor tissue samples)) have been screened for NRAS activating mutations (e.g., NRAS-Q61R, NRAS-Q61K, NRAS...). -G12D, NRAS-G13D, NRAS-G12S, NRAS-G12C, NRAS-G12V, NRAS-G12A, NRAS-G12R, NRAS-G13C, NRAS-G13A, NRAS-G13R, NRAS-G13S, NRAS-G13V, NRAS-Q61H, NRAS-Q61E, NRAS-Q61L, or NRAS-Q61P) and the presence of NRAS activating mutations has been confirmed.

For any of the treatment methods described above, one or more of the methods described in Section III, Part A above can be used to screen samples or samples from individuals.

As described above, the administered MEK inhibitor can be a small molecule inhibitor, which can be a prodrug or a biologically active form. For example, MEK inhibitors can be small molecule inhibitors selected from cobimetinib (GDC-0973), selmetinib (AZD6244), pimecrolimus (AS-703026), PD0325901, remetinib (BAY86-9766), bimetinib (MEK162), BI-847325, trametinib, GDC-0623, G-573, and CH5126766 (RO5126766), or groups thereof that are pharmaceutically acceptable salts of the same. In specific cases, the small molecule inhibitor is cobimetinib (GDC-0973), selmetinib (AZD6244), pimecrolimus (AS-703026), PD0325901, remexinib (BAY86-9766), or bimetinib (MEK162), or a pharmaceutically acceptable salt thereof. The administered PI3K inhibitor can be a small molecule inhibitor, which can be a prodrug or a biologically active form. For example, the small molecule inhibitor is selected from the group consisting of picotinib (GDC-0941), taseriecoxib (GDC-0032), and apiricoxib (BYL719), or a pharmaceutically acceptable salt thereof. The PI3K inhibitor can be a pan-PI3K inhibitor (e.g., picotinib (GDC-0941) or taseriecoxib (GDC-0032), or a pharmaceutically acceptable salt thereof), or a PI3Kα-specific inhibitor and/or a PI3Kδ-specific inhibitor. Furthermore, the administered pan-RAF inhibitor can be a pan-RAF dimer inhibitor. In some cases, the pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) can be a small molecule inhibitor, which can be a prodrug or a biologically active form. In some cases, the pan-RAF dimer inhibitor can be HM95573, LY-3009120, AZ-628, LXH-254, MLN2480, BeiGene-283, RXDX-105, BAL3833, regorafenib, or sorafenib, or a pharmaceutically acceptable salt thereof.

In some implementations, the treatment method also includes administering one or more additional therapeutic agents to the individual (e.g., immunotherapeutic agents, cytotoxic agents, growth inhibitors, radiotherapy agents, and anti-angiogenic agents).

In any of the above methods, administration of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK or PI3K inhibitor may result in a cellular or biological response in the patient as a result of treatment with a pan-RAF inhibitor in combination with either a MEK inhibitor or a PI3K inhibitor, including a complete response, a partial response, stable disease (without progression or relapse), or a response with a later relapse (i.e., benefit). For example, an effective response could be a KRAS activating mutation (e.g., KRAS-G12D, KRAS-G12C, KRAS-G12V, KRAS-G13D, KRAS-G12A, KRAS-G12R, KRAS-G12S, KRAS-G13C, KRAS-G13A, KRAS-G13R, KRAS-G13S, KRAS-G13V, KRAS-Q61H, KRAS-Q61K, KRAS-Q61E, KRAS-Q61L, KRAS-Q61P, or KRAS-Q61R) or an NRAS activating mutation (e.g., NRAS-Q61R, NRAS-Q61R). The tumor size (volume), progression-free survival (PFS), and/or overall survival (OS) of individuals with KRAS-Q12, NRAS-G12D, NRAS-G13D, NRAS-G12S, NRAS-G12C, NRAS-G12V, NRAS-G12A, NRAS-G12R, NRAS-G13C, NRAS-G13A, NRAS-G13R, NRAS-G13S, NRAS-G13V, NRAS-Q61H, NRAS-Q61E, NRAS-Q61L, or NRAS-Q61P were reduced compared to individuals without KRAS-activating mutations or NRAS-activating mutations. In some cases, administration of pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors has resulted in a reduction in tumor size (volume) of 1% or more (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more). The presence of one or more KRAS activating mutations (e.g., KRAS-G13D) or NRAS activating mutations predicts such therapeutic efficacy. In some cases, administration of pan-RAF inhibitors (such as pan-RAF dimer inhibitors) and MEK or PI3K inhibitors has a therapeutic effect that prolongs progression-free survival (PFS) by 1 day or longer (e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or longer).

Dosage and administration

Once a patient is identified as responding to or sensitive to treatment with a pan-RAF inhibitor in combination with either a MEK inhibitor or a PI3K inhibitor, treatment can be initiated either alone or in combination with other therapeutic agents. As mentioned above, such treatment can lead to, for example, tumor size reduction or increased progression-free survival (PFS) and/or overall survival (OS). Furthermore, treatment with a pan-RAF inhibitor in combination with either a MEK inhibitor or a PI3K inhibitor preferably results in a synergistic (or greater than additive) therapeutic benefit for the patient. Preferably, in such a combination approach, the timing between at least one administration of the pan-RAF inhibitor in combination with either a MEK inhibitor or a PI3K inhibitor is about one month or less, more preferably about two weeks or less.

Those skilled in the art will appreciate that the exact manner in which a therapeutically effective dose of this combination therapy is administered to a patient after a diagnosis of their likely response to a pan-RAF inhibitor in combination with either a MEK inhibitor or a PI3K inhibitor will be determined by the attending physician. The mode of administration (including dosage, combination with other agents, timing and frequency of administration, etc.) can be influenced by the diagnosis of the patient's likely response to such combination therapy, as well as the patient's condition and history. Thus, even individuals with cancer who are predicted to be relatively insensitive to pan-RAF inhibitors in combination with either a MEK inhibitor or a PI3K inhibitor may still benefit from their treatment, particularly in combination with other agents (including those that can alter a patient's response to one or both inhibitors).

Compositions containing a pan-RAF inhibitor and either a MEK inhibitor or a PI3K inhibitor are formulated, dosed, and administered in accordance with good medical practice. Factors considered in this context include the specific type of cancer being treated (e.g., lung cancer, breast cancer, skin cancer, colorectal cancer, gastric cancer, lymphoid carcinoma, pancreatic cancer, ovarian cancer, and cervical cancer), the specific mammal being treated (e.g., human), the individual patient's clinical condition, the cause of the cancer, the site of drug delivery, possible side effects, the type of inhibitor, the method of administration, the schedule of administration, and other factors known to the medical practitioner. The effective amount of the pan-RAF inhibitor and MEK or PI3K inhibitor to be administered will depend on these considerations.

Depending on factors such as the specific type of antagonist, an internist with ordinary skills in the field can easily determine the effective amount of the required pharmaceutical composition and prescribe such a formula. For example, an internist can begin with multiple doses of a pan-RAF inhibitor in combination with either a MEK inhibitor or a PI3K inhibitor, administered at a level in the pharmaceutical composition lower than required to achieve the desired therapeutic effect, and gradually increasing the dose until the desired effect is achieved. The effectiveness of a given dose of the antagonist or treatment regimen can be determined, for example, by assessing the patient's signs and symptoms using standardized efficacy measures.

In some cases, a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) may be the only agent administered to the subject (i.e., as a monotherapy), such as in cases where the subject has an activating mutation in NRAS. In other cases, a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK or PI3K inhibitor may be administered to the subject (i.e., as a combination therapy).

In some cases, patients are treated with the same therapy at least twice. Thus, the initial and second exposures are preferably treated with the same inhibitor or the same combination of inhibitors, and more preferably all exposures are treated with the same inhibitor or the same combination of inhibitors, i.e., for the first two exposures, all exposures are preferably treated with the same inhibitor (e.g., the same pan-RAF dimer inhibitor) or the same combination of inhibitors (e.g., the same pan-RAF dimer inhibitor and the same MEK or PI3K inhibitor).

Treatment with MAPK signaling inhibitors or PI3K inhibitors, or their pharmaceutically acceptable salts, may be performed according to standard methods. For example, an illustrative method for administering the MEK inhibitor cobimetinib (e.g., cobimetinib fumarate) is described in the prescribing information for cobimetinib fumarate from Genentech, Inc., USA (November 10, 2015), which is incorporated herein by reference in its entirety. An illustrative method for administering vemurafenib is described in the prescribing information for vemurafenib from Hoffmann La Roche, Inc., USA (August 11, 2015), which is incorporated herein by reference in its entirety.

If multiple exposures to a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK or PI3K inhibitor are provided, each exposure may be delivered using the same or different methods of administration. In one embodiment, each exposure is administered orally. In another embodiment, each exposure is administered intravenously. In yet another embodiment, each exposure is administered subcutaneously.

The duration of treatment can continue for as long as directed by the physician or until the desired therapeutic effect is achieved (as described herein). In some implementations, treatment continues for periods of 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 1 year, 2 years, 3 years, 4 years, 5 years, or many years until the subject's lifetime.

However, as mentioned above, these recommended dosages of pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors require considerable therapeutic consideration. The key factor in selecting the appropriate dosage and schedule is the outcome, as indicated above. In some implementations, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors are administered as close as possible to the first sign, diagnosis, presentation, or onset of a proliferative cellular condition (e.g., cancer).

Application route

Pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) in combination with either a MEK inhibitor or a PI3K inhibitor and optionally any other therapeutic agent can be formulated, dosed, and administered in accordance with good medical practice. Factors considered in this context include the specific condition being treated (e.g., cancer), the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to the medical practitioner. For example, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) do not need to be formulated and/or administered in parallel with either a MEK inhibitor or a PI3K inhibitor, and optionally in combination with one or more agents currently used for the prevention or treatment of conditions (e.g., cancer).

For the prevention or treatment of cancer, the appropriate dosage of the pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors (when used alone or in combination with one or more other additional therapeutic agents) described herein will depend on the type, severity, and course of the disease (e.g., cancer), whether the inhibitor is administered for preventative or therapeutic purposes, prior therapy, the patient's clinical history and response to the inhibitor, and the attending physician's discretion. The pan-RAF inhibitor (e.g., pan-RAF dimer inhibitor) and MEK or PI3K inhibitor may be administered appropriately to the patient in a single dose or in a series of treatments. For repeated administration over several days or longer, depending on the condition, treatment generally continues until the desired disease symptom control is achieved. Such doses may be administered intermittently, such as weekly or every three weeks (e.g., so that the patient receives, for example, about 2 to about 20, or, for example, about 6 doses of the pan-RAF inhibitor (e.g., pan-RAF dimer inhibitor) and MEK or PI3K inhibitor). A higher initial loading dose may be administered, followed by a lower one or more doses. However, other dosing regimens may be useful. Progress in this therapy is easily monitored using routine techniques and assays.

Pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be administered by any suitable means, including oral, parenteral, topical, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and/or intra-injury administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Intrathecal administration is also covered. Additionally, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be appropriately administered by pulsatile infusion, for example, with a decreasing dose of one or both of the inhibitors. Optionally, dosage administration is given orally.

If multiple exposures to a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) in combination with a MEK or PI3K inhibitor are provided, each exposure may be delivered using the same or different methods of administration. In another embodiment, each exposure is administered intravenously (i.v.). In another embodiment, each exposure is administered subcutaneously (s.c.). In yet another embodiment, the exposure is administered by both intravenous and subcutaneous administration.

Combination therapy

The treatments described in this article generally involve the administration of more than one therapeutic agent (e.g., panRAF inhibitors in combination with MEK or PI3K inhibitors, such as panRAF dimer inhibitors).

Combination therapy can provide “synergistic effects” and is demonstrated to be “synergistic,” meaning that the effect achieved when the active components are used together is greater than the sum of the effects produced when the compounds are used separately. Synergistic effects can be achieved when the active components (i.e., pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors) are: (1) co-formulated and administered or delivered simultaneously in unit dose formulations of the combination; (2) delivered alternately or in parallel as separate formulations; or (3) through some other regimen. When delivered in alternating therapy, synergistic effects can be achieved when the compounds are administered or delivered sequentially. Generally, during alternating therapy, each active component is administered sequentially (i.e., continuously), while in combination therapy, two or more active components are administered together in effective doses.

In some cases, this method includes further administration of anticancer agents, such as chemotherapy agents, growth inhibitors, biotherapies, immunotherapies, or radiotherapy agents. Additionally, cytotoxic agents, anti-angiogenic agents, and antiproliferative agents can be used in combination with pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors. In some cases, the pan-RAF inhibitor (e.g., pan-RAF dimer inhibitor) and MEK or PI3K inhibitor are used in combination with anticancer therapies, such as surgery.

In other cases, treatment may include a combination of two or more (e.g., three or more) MAPK signaling inhibitors (e.g., two or more RAF, MEK, or ERK inhibitors) and/or PI3K inhibitors.

This method may also involve combining chemotherapy agents, such as docetaxel, doxorubicin, and cyclophosphamide, with an effective dose of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK or PI3K inhibitor.

In other cases, the method involves combining immunotherapy, such as the administration of a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK or PI3K inhibitor in combination with a therapeutic antibody. In one embodiment, the therapeutic antibody is an antibody that binds to a cancer cell surface marker or tumor-associated antigen (TAA). In one embodiment, the therapeutic antibody is an anti-HER2 antibody, such as trastuzumab. In another embodiment, the therapeutic antibody is an anti-HER2 antibody, such as pertuzumab (OMNITARG ^™ ). In yet another embodiment, the therapeutic antibody is either a naked antibody or an antibody-drug conjugate (ADC).

Unwilling to be bound by theory, some believe that enhancing T-cell stimulation by promoting activating co-stimulatory molecules or inhibiting negative co-stimulatory molecules can promote tumor cell death, thereby treating cancer or delaying cancer progression. Therefore, in some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be combined with agonists targeting activating co-stimulatory molecules. In some cases, activating co-stimulatory molecules may include CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some cases, the agonist targeting the activating co-stimulatory molecules is an agonistic antibody that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be combined with antagonists targeting inhibitory co-stimulatory molecules. In some cases, inhibitory co-stimulatory molecules may include CTLA-4 (also known as CD152), TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase. In some cases, antagonists against inhibitory co-stimulatory molecules are antagonistic antibodies that bind to CTLA-4, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase.

In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with antagonists against CTLA-4 (also known as CD152), such as blocking antibodies. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with ipilimumab (also known as MDX-010, MDX-101, or). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with tremelimumab (also known as ticilimumab or CP-675,206). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with antagonists against B7-H3 (also known as CD276), such as blocking antibodies. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with MGA271. In other cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with TGF-β antagonists, such as metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299.

In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with treatments involving adoptive transfer of T cells expressing chimeric antigen receptors (CARs), such as cytotoxic T cells or cytotoxic lymphocytes (CTLs). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with treatments involving adoptive transfer of T cells containing manifest-negative TGFβ receptors, such as manifest-negative TGFβ type II receptors. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with treatments including HERCREEM regimens (see, for example, ClinicalTrials.gov IdentifierNCT00889954).

In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be co-administered with agonists targeting CD137 (also known as TNFRSF9, 4-1BB, or ILA), such as activating antibodies. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be co-administered with urelumab (also known as BMS-663513). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be co-administered with agonists targeting CD40, such as activating antibodies. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be co-administered with CP-870893. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be co-administered with agonists targeting OX40 (also known as CD134), such as activating antibodies. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be co-administered with anti-OX40 antibodies (e.g., AgonOX). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be co-administered with agonists targeting CD27, such as activating antibodies. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be co-administered with CDX-1127. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be co-administered with antagonists targeting indoleamine-2,3-dioxygenase (IDO). In some cases, the IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be administered in combination with a PD-1 axis binding antagonist. In some cases, the PD-1 axis binding antagonist is a PD-L1 antibody.

In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be administered in combination with antibody-drug conjugates. In some cases, the antibody-drug conjugate contains mertansine or auristatin E (MMAE). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be administered in combination with anti-NaPi2b antibody-MMAE conjugates (also known as DNIB0600A or RG7599). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be administered in combination with trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine, or Genentech). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be administered in combination with DMUC5754A. In some cases, pan-RAF inhibitors (such as pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be co-administered with antibody-drug conjugates targeting the endothelin B receptor (EDNBR), such as antibodies against EDNBR conjugated with MMAE.

In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with anti-angiogenic agents. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with antibodies targeting VEGF, such as VEGF-A. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with bevacizumab (also known as Genentech). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with antibodies targeting angiopoietin 2 (also known as Ang2). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with MEDI3617. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with antitumor agents. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with agents targeting CSF-1R (also known as M-CSFR or CD115). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with anti-CSF-1R (also known as IMC-CS4). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with interferon, such as interferon α or interferon γ. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with Roferon-A (also known as recombinant interferon α-2a). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with GM-CSF (also known as recombinant human granulocyte-macrophage colony-stimulating factor, rhu GM-CSF, sargramostim, or). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with IL-2 (also known as aldesleukin, or). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with IL-12. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with antibodies targeting CD20. In some cases, the antibody targeting CD20 is obinutuzumab (also known as GA101, or) or rituximab. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be administered in combination with antibodies targeting GITR. In some cases, the antibody targeting GITR is TRX518.

In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be administered in combination with cancer vaccines. In some cases, the cancer vaccine is a peptide cancer vaccine, which in some cases is a personalized peptide vaccine. In some cases, the peptide cancer vaccine is a multivalent long peptide, multiple peptide, peptide mixture, hybrid peptide, or a dendritic cell vaccine via peptide pulses (see, for example, Yamada et al., Cancer Sci. 104:14-21, 2013). In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be administered in combination with adjuvants. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be administered in combination with treatments containing TLR agonists, such as Poly-ICLC (also known as), LPS, MPL, or CpG ODN. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with tumor necrosis factor (TNF) α. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with IL-1, such as IL-1β. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with HMGB1. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with IL-10 antagonists. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with IL-4 antagonists. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with IL-13 antagonists. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with HVEM antagonists. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with ICOS agonists, such as by administration of ICOS-L, or agonistic antibodies against ICOS. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with treatments targeting CX3CL1. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with treatments targeting CXCL9. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with treatments targeting CXCL10. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be used in combination with treatments targeting CCL5. In some cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be administered in combination with LFA-1 or ICAM1 agonists. In other cases, pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors can be administered in combination with selector protein agonists.

Generally, for the prevention or treatment of disease, a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK or PI3K inhibitor are appropriately administered to the patient in a single dose or in a series of treatments. Pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors are typically administered as listed above. Depending on the type and severity of the disease, an additional therapeutic agent, approximately 20 mg/ ^m² to 600 mg/ ^m², is the initial candidate dose administered to the patient, for example, by a single or multiple separate administration or by continuous infusion. A typical daily dose of any therapeutic agent in the treatment regimen can range from approximately 20 mg/ ^m² , 85 mg/ ^m² , 90 mg/ ^m² , 125 mg/ ^m² , 200 mg/ ^m² , 400 mg/ ^m² , 500 mg/ ^m² , or more, depending on the factors described above. For repeated administration over several days or longer, depending on the condition, treatment continues until the desired disease symptom control is achieved. Thus, one or more doses of approximately 20 mg/ ^m² , 85 mg/ ^m² , 90 mg/ ^m² , 125 mg/ ^m² , 200 mg/ ^m² , 400 mg/ ^m² , 500 mg/ ^m² , 600 mg/ ^m² (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, for example weekly or every two, three, four, five, or six weeks (e.g., so that the patient receives approximately 2 to approximately 20, or for example, approximately 6 additional doses). A higher initial loading dose can be administered, followed by one or more lower doses. However, other dosing regimens may be useful. Progression to this therapy is easily monitored using routine techniques and assays.

In one implementation, the subject has never previously received any medication to treat cancer. In another implementation, the subject or patient has previously received one or more medications to treat cancer. In yet another implementation, the subject or patient does not respond to one or more previously administered medications. Such medications that a subject may not respond to include, for example, antitumor agents, chemotherapeutic agents, cytotoxic agents, and/or growth inhibitors.

IV. Compositions and Their Uses

This invention is partly based on the finding that combinations of pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and MEK or PI3K inhibitors are effective in treating patients with KRAS activating mutations (e.g., KRAS-G12D, KRAS-G12C, KRAS-G12V, KRAS-G13D, KRAS-G12A, KRAS-G12R, KRAS-G12S, KRAS-G13C, KRAS-G13A, KRAS-G13R, KRAS-G13S, KRAS-G13V, KRAS-Q61H, KRAS-Q61K, KRAS-Q61E, KRAS-Q61L). This information is useful for individuals with cancer who have KRAS-Q61P or KRAS-Q61R mutations or NRAS activating mutations (e.g., NRAS-Q61R, NRAS-Q61K, NRAS-G12D, NRAS-G13D, NRAS-G12S, NRAS-G12C, NRAS-G12V, NRAS-G12A, NRAS-G12R, NRAS-G13C, NRAS-G13A, NRAS-G13R, NRAS-G13S, NRAS-G13V, NRAS-Q61H, NRAS-Q61E, NRAS-Q61L, or NRAS-Q61P).

In some cases, the present invention therefore provides a composition comprising a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor for use in methods of treating individuals with cancer (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)), wherein samples from the individual have been screened for KRAS-G13D and the presence of a KRAS-G13D mutation in the samples has been determined. In some cases, the present invention provides a composition comprising a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor for use in therapeutic treatment of cancers characterized by KRAS-G13D mutations (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)). In some cases, the present invention provides the use of a composition comprising a pan-RAF dimer inhibitor and a MEK inhibitor in the preparation of a medicament for therapeutic treatment of cancers characterized by KRAS-G13D mutations (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)).

In some cases, the present invention provides a composition comprising a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) and a MEK inhibitor for use in methods for treating individuals with cancer (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)), wherein samples from the individual have been screened for NRAS activating mutations and the presence of NRAS activating mutations in the samples has been determined. In some cases, the present invention provides a composition comprising a pan-RAF dimer inhibitor and a MEK inhibitor for use in therapeutic treatment of cancers characterized by NRAS activating mutations (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)). In some cases, the present invention provides the use of a composition comprising a pan-RAF dimer inhibitor and a MEK inhibitor in the preparation of a medicament for therapeutic treatment of cancers characterized by NRAS activating mutations (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)).

In other instances, the present invention provides compositions comprising pan-RAF inhibitors (e.g., pan-RAF dimer inhibitors) and PI3K inhibitors (e.g., pan-PI3K inhibitors such as picotelixicob (GDC-0941) or taserixicob (GDC-0032), or pharmaceutically acceptable salts thereof), or PI3Kα-specific inhibitors and/or PI3Kδ-specific inhibitors, for use in methods for treating individuals with cancers (e.g., colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancer (e.g., melanoma)), wherein samples from the individual have been screened for KRAS activating mutations. For example, KRAS-G12D, KRAS-G12C, KRAS-G12V, KRAS-G13D, KRAS-G12A, KRAS-G12R, KRAS-G12S, KRAS-G13C, KRAS-G13A, KRAS-G13R, KRAS-G13S, KRAS-G13V, KRAS-Q61H, KRAS-Q61K, KRAS-Q61E, KRAS-Q61L, KRAS-Q61P, or KRAS-Q61R mutations) and the presence of a KRAS activating mutation in the sample has been confirmed. In some cases, the present invention provides compositions comprising pan-RAF dimer inhibitors and PI3K inhibitors for use in the therapeutic treatment of cancers characterized by KRAS activating mutations. In some cases, the present invention provides the use of a composition comprising a pan-RAF dimer inhibitor and a PI3K inhibitor in the preparation of a medicament for the therapeutic treatment of cancers characterized by KRAS activating mutations.

Compositions are also provided comprising RAF inhibitors (e.g., pan-RAF dimer inhibitors) and PI3K inhibitors (e.g., pan-PI3K inhibitors such as picotelicoxib (GDC-0941) or tasericoxib (GDC-0032), or pharmaceutically acceptable salts thereof), or PI3Kα-specific inhibitors and/or PI3Kδ-specific inhibitors). In some cases, the present invention provides a composition wherein the pan-PI3K inhibitor is picotelicoxib (GDC-0941) or tasericoxib (GDC-0032), or a pharmaceutically acceptable salt thereof. In some cases, the present invention provides a composition wherein the pan-RAF dimer inhibitor is selected from the group consisting of HM95573, LY-3009120, AZ-628, LXH-254, MLN2480, BeiGene-283, RXDX-105, BAL3833, regorafenib, and sorafenib, or pharmaceutically acceptable salts thereof. In some cases, the composition comprises selected from HM95573 and picolixib (GDC-0941), LY-3009120 and picolixib (GDC-0941), AZ-628 and picolixib (GDC-0941), LXH-254 and picolixib (GDC-0941), MLN2480 and picolixib (GDC-0941), BeiGene-283 and picolixib (GDC-0941), RXDX-105 and picolixib (GDC-0941), BAL3833 and picolixib (GDC-0941), regorafenib and picolixib (GDC-0941), sorafenib and picolixib (GDC-0941). 41), HM95573 and tacelicoxib (GDC-0032), LY-3009120 and tacelicoxib (GDC-0032), AZ-628 and tacelicoxib (GDC-0032), LXH-254 and tacelicoxib (GDC-0032), MLN2480 and tacelicoxib (GDC-0032), BeiGene-283 and tacelicoxib (GDC-0032), RXDX-105 and tacelicoxib (GDC-0032), BAL3833 and tacelicoxib (GDC-0032), regorafenib and tacelicoxib (GDC-0032), and sorafenib and tacelicoxib (GDC-0032), or combinations thereof consisting of pharmaceutically acceptable salts.

In some cases, the present invention provides pharmaceutical compositions comprising the compositions described above herein.

In some cases, the present invention provides compositions for use in therapeutic treatments of cancer, such as those described above herein.

In some cases, the present invention provides the use of the compositions described above in the preparation of a medicament for the therapeutic treatment of cancer.

In any of the above cases, cancer can be selected from the group consisting of colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, breast cancer, stomach cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma, and hematologic malignancies.

V. Diagnostic kits

This article provides a diagnostic kit that includes biomarkers (e.g., KRAS activating mutations such as KRAS-G12D, KRAS-G12C, KRAS-G12V, KRAS-G13D, KRAS-G12A, KRAS-G12R, KRAS-G12S, KRAS-G13C, KRAS-G13A, KRAS-G13R, KRAS-G13S, KRAS-G13V, KRAS-Q61H, KRAS-Q61K, KRAS-Q61H) in samples from individuals or patients with diseases or conditions (e.g., proliferative cell diseases such as cancers such as colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, and skin cancers such as melanoma)). The presence of one or more reagents (e.g., peptides or polynucleotides) of NRAS activating mutations (e.g., NRAS-Q61R, NRAS-Q61K, NRAS-G12D, NRAS-G13D, NRAS-G12S, NRAS-G12C, NRAS-G12V, NRAS-G12A, NRAS-G12R, NRAS-G13C, NRAS-G13A, NRAS-G13R, NRAS-G13S, NRAS-G13V, NRAS-Q61H, NRAS-Q61E, NRAS-Q61L, or NRAS-Q61P).

In some cases, the presence of biomarkers in a sample indicates a higher probability of efficacy when an individual is treated with a pan-RAF inhibitor (e.g., a pan-RAF dimer inhibitor) in combination with a MEK inhibitor or a PI3K inhibitor. Optionally, the kit may further include instructions for use on using the reagent to identify individuals with cancer who may benefit from treatment comprising a pan-RAF dimer inhibitor and a MEK or PI3K inhibitor.

In some cases, for example, the invention is characterized by a kit for identifying individuals with cancer who may benefit from treatment comprising a pan-RAF dimer inhibitor and a MEK inhibitor, the kit comprising reagents for determining the presence of the KRAS-G13D mutation in a sample from the individual, and, optionally, instructions for use of the reagents for identifying individuals with cancer who may benefit from treatment comprising a pan-RAF dimer inhibitor and a MEK inhibitor. In some cases, the reagent comprises a first oligonucleotide and a second oligonucleotide for use in the amplification of the whole or part of the KRAS gene.

In some cases, for example, the invention is characterized by a kit for identifying individuals with cancer who may benefit from treatment comprising a pan-RAF dimer inhibitor and a MEK inhibitor, the kit comprising reagents for determining the presence of NRAS activating mutations in a sample from the individual, and, optionally, instructions for use of the reagents for identifying individuals with cancer who may benefit from treatment comprising a pan-RAF dimer inhibitor and a MEK inhibitor. In some cases, the reagent comprises a first oligonucleotide and a second oligonucleotide for use in the amplification of the entire or a portion of the NRAS gene.

In some cases, for example, the invention is characterized by a kit for identifying individuals with cancer who may benefit from treatment comprising a pan-RAF dimer inhibitor and a PI3K inhibitor, the kit comprising reagents for determining the presence of KRAS activating mutations in samples from the individual, and, optionally, instructions for use of the reagents for identifying individuals with cancer who may benefit from treatment comprising a pan-RAF dimer inhibitor and a PI3K inhibitor. In some cases, the reagent comprises a first oligonucleotide and a second oligonucleotide for use in the amplification of the whole or part of the KRAS gene.

Example

The following embodiments are provided to illustrate, and not to limit, the invention currently claimed.

Example 1: Materials and Methods

In vitro methods

Cell lines and reagents

Anti-BRAF (sc-5284) and anti-CRAF (sc-133) antibodies were purchased from Santa Cruz Biotechnology. Anti-MEK1 (610122) and anti-CRAF (610152) antibodies were purchased from BD Biosciences. Anti-pMEK (S217/S221) (9121), anti-ERK (9107), anti-pERK (T202/Y204) (9101), anti-pCRAF (S338) (9427), anti-pEGFR (Y1068) (3777), AKT (9272), pAKT (T308) (13038), cleaved PARP (9521), and anti-β-actin (4970) were purchased from Cell Signaling Technology. IR-conjugated secondary antibodies, goat anti-mouse 680LT (926-68020), goat anti-human 680LT (926-68032), and goat anti-rabbit 800CW (926-32211), were purchased from Li-Cor. All Western blots were scanned on Li-Cor CLX using dual IR-conjugated secondary antibodies. All cell lines were obtained from the American Type Culture Collection (ATCC) and maintained in recommended medium supplemented with 10% heat-inactivated FBS (HyClone, SH3007003HI), 1X GlutaMAX (Gibco, 35050-061), and 1X Pen Strep (Gibco, 15140-122). A549 shCRAF and HCT116 shCRAF cell lines were generated at Genentech.

Stable cell line generation

HCT116 colon cancer cell line was purchased from ATCC (American Type Culture Collection, Manassas, VA). HCT116 cells were cultured in RPMI 1640 containing 10% fetal bovine serum. HCT116 cells stably expressing luciferase and CRAF shRNA were generated using hairpin oligonucleotides (luciferase shRNA: sense: 5’-GAT CCC CCT TAC GCT GAG TAC TTCGAT TCA AGA GAT CGA AGT ACT CAG CGT AAG TTT TTT GGA AA-3’ (SEQ ID NO: 1), antisense: 5’-AGC TTT TCC AAA AAA CTT ACG CTG AGT ACT TCG ATC TCT TGA ATC GAA GTA CTCAGC GTA AGG). GG-3’ (SEQ ID NO:2) and CRAF shRNA: sense: 5’-GAT CCC CGA CAT GAA ATCCAA CAA TAT TCA AGA GAT ATT GTT GGA TTT CAT GTC TTT TTT GGA AA-3’ (SEQ ID NO:3), antisense: 5’-AGC TTT TCC AAA AAA GAC ATG AAA TCC AAC AAT ATC TCT TGA ATATTG TTGGAT TTC ATG TCG GG-3’ (SEQ ID NO:4)). Lentiviral constructs carrying inducible shRNAs were generated by co-transfecting HEK293T cells with a pHUSH-Lenti-puro construct containing either luciferase or CRAF shRNA and plasmids expressing vesicular stomatitis virus (VSV-G) envelope glycoprotein and HIV-1 packaging protein (GAG-POL) using Lipofectamine (Invitrogen, Carlsbad, CA). These viruses were then transduced into target cells and selected using puromycin. As previously described (Jaiswal et al., PLoS One.4:e5717, 2009), shRNA was induced using a medium containing 500 ng/ml doxycycline (Clontech, CA), and Western blot analysis was performed on the knockdown-characterized cells.

A549 lung cancer cell line was obtained from ATCC. Cells were cultured in RPMI 1640 + 10% fetal bovine serum. A549 cells stably expressing non-targeted control (NTC) or CRAF shRNA were generated using hairpin oligonucleotides: NTC shRNA targeting sequence: 5’TCC TGC GTC TAG AGG TTC CCA 3’ (SEQ ID NO: 5) and CRAF shRNA targeting sequence: 5’TAGGAG TAG ACA TCC GAC TGG 3’ (SEQ ID NO: 6). The vector used was pINDUCER 10, which was modified to express optimized miR-30-based hairpins (Meerbrey et al., PNAS 108(9):3665-3670, 2011; Fellmann et al., Cell Rep. 5:1704-1713, 2013). Lentiviral constructs carrying inducible shRNAs were generated by co-transfecting pINDUCER10-miRE constructs containing either NTC or CRAF shRNAs with plasmids expressing vesicular stomatitis virus (VSV-G) envelope glycoproteins and HIV-1 packaging protein (GAG-POL) in 293T cells using lipofectamine 2000 (Invitrogen). Viral supernatants were concentrated using a Lenti-X concentrator (Clontech). Target cells were transduced with these viral supernatants and selected using puromycin (2 μg/ml). Cell knockdown was assessed by Western blot analysis after shRNA induction using medium containing 2 μg/ml doxycycline (Sigma).

Cell viability assay

RAS mutant, BRAF mutant cell screening

Seeding density was optimized for each cell line to achieve 70-80% confluence after 4 days. Cells were distributed into 384-well plates (Griener, 781091) and treated with the compound the following day at a final DMSO concentration of 0.1%. The relative number of surviving cells was measured by luminescence using CellTiter-Glo (Promega, G7573). Viability curves were generated using four-parameter fitting in GraphPad Prism 6.

Tool compound combination screening

A library of 480 compounds was screened in A549 cells with a fixed dose of either AZ-628 or DMSO, either absent or present, arranged in a 9-point dose-response pattern. A549 cells were seeded into 384-well plates, and compounds were added 24 hours later. Cell viability was measured 120 hours after compound addition (CellTiter Glo). Curves were fitted, and both IC50 and mean viability measures were calculated. IC50 is the dose at which inhibition occurs relative to 50% of untreated wells. Mean viability is the average of the fitted viability at each tested dose. Mean viability is equivalent to the area under the log dose/viability curve divided by the total number of tested doses. All data were fitted using Genedata Screener (GDS) software. The combined measure is the difference in mean viability between the AZ-628-treated and DMSO-treated branches for each compound.

High-throughput cell viability assay

Compounds were screened using a three-fold dilution with a nine-point dose response. Cells were seeded into 384-well plates 24 hours before adding the compound. Cells were then incubated with the compound for 72 or 120 hours before viability assays (CellTiter-Glo, Promega). Assays were performed in triplicate. Through-the-loop assays were performed by incubating cells (37°C, 5% _CO2 ) in RPMI-1640, 2.5% FBS (72-hour assay) or 5% FBS (120-hour assay), and 2 mM glutamine. IC50 and mean viability values were reported as follows: IC50 is the dose at which inhibition is estimated to be 50% relative to untreated wells (i.e., absolute IC50). Mean viability is equivalent to the area under the log dose/viability curve divided by the total number of doses tested.

Determination of Phosphoric Acid (Ser 217/221)/Total MEK1/2

Cells were split at a density of 20,000 cells per 96 wells and treated with the compound at a final concentration of 0.2% DMSO for 2 hours the following day. After 2 hours, cells were lysed according to the manufacturer's protocol (Meso Scale Discovery, K15129D), and the lysate was added to a BSA-blocked plate and captured overnight at 4°C. The following day, the assay plate was washed three times with TBST and the detection antibody was added, incubated at room temperature for 1 hour. The plate was then washed three times with TBST. 1X reading buffer was added to the plate and read immediately on a Meso Scale Discovery SECTOR 6000 imager. Curves were generated using a GraphPad Prism 6.

Drug combination assay

For combined synergistic studies, cells were seeded into 384-well plates (Corning) and treated with different concentrations of compounds (either alone or in combination) for 72 hours. Cell viability was determined using the CellTiter-Glo Luminescent cell viability assay (Promega, G7573). Synergistic effects were determined using Bliss independence analysis (Greco et al., Pharmacol. Rev. 47:331-385, 1995; Chou and Talalay, Adv. Enzyme Regul. 22:27-55, 1984; Chou, Cancer Res. 70:440-446, 2010; Bliss, Ann. Appl. Biol. 25:31, 1939).

RNA sequencing experiment

Cells were seeded overnight and treated with AZ-628 and cobimetinib (either alone (0.1 μM) or in combination (0.1 μM each)) for 6 hours. Control cells were treated with DMSO (0.2% final concentration). Total RNA was extracted using the RNEasy Mini Kit and on-column DNase digestion (Qiagen #74106 and #79254) according to the manufacturer's protocol. Sample quality control was performed to determine RNA quantity and quality before processing by RNA-seq. RNA concentration was determined using a NanoDrop 8000 (Thermo Scientific), and RNA integrity was determined using a Fragment Analyzer (Advanced Analytical Technologies). 0.5 μg of total RNA was used as input material for library preparation using the TruSeq RNA Sample Preparation Kit v2 (Illumina). Library sizes were confirmed using a 2200 TapeStation and high-sensitivity D1000 selection bands (Agilent Technologies), and their concentrations were determined using a qPCR-based method with a Library Quantification Kit (KAPA). The multiplexed libraries were then sequenced on an Illumina HiSeq2500 (Illumina) to generate 30M single-end 50-base-pair reads.

Readings were localized to the hg19 genome using the RefSeq gene model and the GSNAP alignment tool. Differential gene expression was assessed using per-gene counting with limma and edgeR software implemented in the R programming language, following the limma user manual. Linear models were fitted to gene expression with respect to cell line identity, AZ-628 and cobimetinib treatments, and treatment interactions. The significance of the linear model terms was determined using a moderated t-test performed in the limma software.

Clonogenesis assay

Cells were partitioned in duplicate in 6-well plates at 10,000 cells/well, allowed to adhere overnight, and treated with the indicated compound for 8 days. Medium containing the appropriate compound was added every 72 hours. At day 8, cells were washed once with PBS, fixed, stained with crystal violet solution (Sigma Aldrich, HT90132) for 20 minutes, and washed with water.

Immunoblotting

Cells were lysed in lysis buffer (0.5% NP40, 20 mM Tris, pH 7.5, 137 mM NaCl, 10% glycerol, 1 mM EDTA) with a protease inhibitor mixture – a complete mini (Roche Applied Science, 11836170001) and a phosphatase inhibitor mixture (Thermo, 78426). The lysates were centrifuged at 15,000 rpm for 10 min and protein concentration was determined using BCA (Thermo, 23227). Equal volumes of protein were submitted to SDS-PAGE NuPAGE 4–12% Bis-Tris gel (Novex, WG-1403) and transferred to nitrocellulose membranes (BioRad, 170–4159). After blocking in blocking buffer (Li-Cor, 927-40000), the membrane was incubated with the indicated primary antibody and analyzed by either the secondary antibody IRDye 680LT goat anti-mouse IgG (H+L) (Li-Cor, 926-68050) or IRDye 800CW goat anti-rabbit IgG (H+L) (Li-Cor, 926-32211). The membrane was visualized on a LiCor Odyssey CLx scanner.

In vitro kinase assay

Cells were allocated and lysed as described in the immunoblotting method. Cell lysates were incubated at 4°C for 2 hours with anti-CRAF (Millipore 07-396) or anti-BRAF antibody (Millipore 07-453) and 50 μl of protein A agarose beads (Millipore 16-125). After washing with a mixture of protease and phosphatase inhibitors in lysis buffer, protein A beads were incubated with 0.4 μg of inactive MEK1 (Millipore 14-420) in 40 μl of kinase buffer (20 mM MOPS, pH 7.2, 25 mM β-glycerophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, 120 μM ATP, 18 mM _MgCl2 ) for 30 minutes. The samples were then analyzed by Western blot.

Ras activity assay

The level of active GTP-loaded Ras was determined using the GST-Raf-RBD pull-down assay (Thermo Scientific, 16117). In short, the GST-Raf-RBD fusion protein was incubated with glutathione beads, and cells were collected in lysis buffer as recommended by the manufacturer. Cell lysates were incubated with GST-Raf-RBD immobilized beads at 4°C for 1.5 h. The bound protein was eluted with SDS sample buffer and analyzed by Western blotting.

siRNA transfection

Cells were reverse transfected with 20 nM siKRAS (L-005069-00-0020) or siNTC (D-001810-10-20) (Dharmacon Collection) in the presence of lipofectamine RNAiMAX reagent (Life Technologies) according to the manufacturer's instructions. The culture medium was changed on the day after transfection, and the knockdown efficiency was assessed on day 4.

In vivo methods

Tumor protein and RNA separation

GEM model tumor samples were collected and preserved in RNALater (Qiagen, Valencia, CA). Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) following the manufacturer's instructions. RNA counts were determined using Nanodrop (ThermoScientific, Waltham, MA).

RT-PCR analysis

Transcriptional readouts were assessed using Fluidigm instruments or standard RT-PCR assays as recommended by the manufacturer. RNA (100 ng) was submitted to the cDNA synthesis/pre-amplification reaction using the Applied Biosystems High Capacity cDNA RT Kit and TaqMan PreAmp Master mixture, following the manufacturer's protocol (Life Technologies, Carlsbad, CA). After amplification, qPCR was performed on a Fluidigm 96.96 Dynamic array using the BIOMARK ^™ HD system with TE buffer at dilutions one to four, following the manufacturer's protocol. The cycle threshold (Ct) was converted to a fold change in relative expression (2^-(ddCt)) by subtracting the mean of the three reference genes from the mean of each target gene, followed by subtracting the mean medium dCt from the mean sample dCt.

Immunoblotting

To prepare protein lysates, cells were washed once with ice-cold PBS and lysed in 1X cell extraction buffer (Invitrogen) supplemented with protease inhibitor tablets (Roche) and phosphatase inhibitors (Sigma). Protein concentrations were determined using the BCA protein assay (Pierce). Equal volumes of protein were eluted via a 10% Bis-Tris gel in 1X MOPS running buffer (Invitrogen) and transferred to a nitrocellulose membrane (Invitrogen). Antibodies against the following proteins were used: CRAF, p-ERK, ERK1/2, p-MEK-1/2, MEK-1/2, cleaved PARP (Cell Signaling), p-p90RSK, GAPDH (EMD Millipore), and p90RSK (Invitrogen). Antigen-antibody interactions were detected using enhanced chemiluminescence assays (Pierce) with HRP-conjugated goat anti-rabbit and goat anti-mouse antibodies (Jackson Immuno Research) or with Odyssey infrared imaging systems (LI-COR Biosciences) with affinity-purified anti-rabbit IgG conjugated with IRDye 800 and Alexa Fluor 680 goat anti-mouse IgG secondary antibodies (Life Technologies).

in vivo model

The establishment and monitoring of all tumor xenograft models were previously shown and described in Hoeflich et al., Cancer Res. 72(1):210-219, 2012.

Test materials

Cobimetinib (GDC-0973) was prepared at Genentech as a suspension in methylcellulose Tween (MCT) at various concentrations. AZ-628 and LY3009120 were synthesized at Genentech and prepared as MCT nanosuspensions. Cobimetinib, AZ-628, LY3009120, and a median control drug solution were prepared once weekly for three weeks. The formulations were thoroughly mixed by vortexing before administration. Test items were stored in a refrigerator maintained at a temperature range of 4°C–7°C.

Subcutaneous tumor model

Xenograft studies were performed as previously described in Hoeflich et al., Cancer Res. 72(1):210-219, 2012. In short, 5 x ^10⁶ HCT116 or ^10⁷ NCI-H2122 cells were subcutaneously implanted into the right side of female NCR nude mice (6-8 weeks old) with an average weight of 24-26 g, obtained from Taconic (Cambridge City, IN). Mice were housed in standard rodent miniature isolation cages at Genentech and acclimatized to the study conditions for at least 3 days prior to tumor cell implantation. Only animals exhibiting healthy behavior and without apparent abnormalities were used in each study. Tumor volume was determined using a digital diameterometer (Fred V. Fowler Company, Inc.) using the formula (L x W x W)/2. Tumor growth inhibition (%TGI) was calculated as a percentage of the area under the curve (AUC) of each dose group relative to the medium per day, such that %TGI = 100 x [1 - (AUC _treatment /day) / (AUC _medium /day)]. Curve fitting was applied to individual tumor volume data transformed by Log ₂ using the R package nlme, version 3.1-97 in R v2.12.0 (Pinheiro et al. R Package Version 3.1-89, 2008). Mice were weighed twice a week using a standard balance.

Example 2: RAF kinase inhibitors lack potent single-agent efficacy in KRAS mutant cell lines

To determine whether RAF kinase inhibitors could be therapeutically used in KRAS mutant cell lines, cell viability was assessed in a group of RAF inhibitors in KRAS mutants compared to BRAF-V600E cell lines for type 1.5 BRAF inhibitors, dabrafenib, vemurafenib, and the “anomalous disruptor” PLX-8394, and type II RAF inhibitors, AZ-628 and LY3009120. Clinically approved type 1.5 BRAF inhibitors, vemurafenib and dabrafenib, and PLX-8394 showed activity in BRAF-V600E mutant cell lines (as indicated in black), but not in KRAS mutant cell lines (as indicated in red) (Figure 1A). As expected, the type 1.5 inhibitors vemurafenib and dabrafenib, but not PLX-8394, led to anomalous activation of downstream pMEK in KRAS mutant tumors (Figure 1B). In contrast to type 1.5 inhibitors, type II pan-RAF inhibitors showed better inhibition of KRAS mutant cell lines; however, their potency in KRAS mutant cell lines was weaker than the activity observed in the BRAF-V600E mutant background (Fig. 1A). Consistent with this activity, type II RAF inhibitors, as well as the recently reported anomalous disruptor PLX-8394, did not induce anomalous activation (Fig. 1B). To extend these results, three type II RAF inhibitors (LY-3009120, MNL-2480, and AZ-628) and PLX-8394 were screened in a cohort of 161 lung, skin, and colorectal cell lines (Fig. 1C). Although these inhibitors showed moderately increased activity in RAS mutant systems compared to BRAF/RAS wild-type lines, the BRAF-V600 cell line was the most sensitive (Fig. 1C). These data suggest that the lack of anomalous activation is insufficient to predict sensitivity in a RAS mutant background, and that RAF kinase activity may not be necessary for mutant KRAS-mediated growth.

To determine whether a second pharmacological agent could sensitize KRAS mutant cell lines to RAF kinase inhibition, a library of 480 small molecule tool compounds was screened in the A549KRAS mutant lung cancer cell line for combination with either DMSO or the 1 μM type II RAF inhibitor AZ-628. The top hit from this screening was the MEK inhibitor cobimetinib (GDC-0973). AZ-628 showed good combination potential with other MAPK pathway inhibitors, including four distinct MEK inhibitors and two ERK inhibitors among the top 20 hits (Figure 1D and Supplementary Table 1). Other notable hits included several microtubule inhibitors, as well as the PI3K inhibitors picolixib (GDC-0941) and taxerixib (GDC-0032). To validate the hits from the primary screening, a first-class hit was obtained in the HCT-116 (KRAS-G13D/PIK3CA mutant) cell line, which confirmed that the MEK inhibitor is the most potent agent in combination with the type II RAF inhibitor AZ-628.

Example 3: Conformation-specific RAF and MEK inhibitors exhibit synergistic effects in RAS mutant cell lines

While the top hits from the screening were MEK inhibitors, not all MEK inhibitors are equally synergistic. Although cobimetinib, pimasertib, remetinib, and PD901 scored highest among the compounds tested, trametinib and GDC-0623 did not show differences in mean viability, despite being potent MEK inhibitors (Figure 1E). Previous reports have described differential mechanisms of action for MEK inhibitors based on their ability to trap inactive RAF-MEK complexes (Hatzivassiliou et al., Nature 501:232-236 (2013)). This drug-stabilized complex prevents RAF from phosphorylating MEK. To determine whether the mechanism of action of MEK inhibitors affects synergy with AZ-628, several MEK inhibitors with different molecular mechanisms were tested. It was found that MEK inhibitors that trap inactive RAF-MEK complexes and do not induce pMEK (e.g., trametinib, GDC-0623, G-573, and CH-6766) (Figure 1F) also showed lower synergy with AZ-628 in full-dose Bliss matrix analysis (Figure 1G).

Based on the above observations, it is hypothesized that, just as not all MEK inhibitors are synergistically equivalent to RAF inhibitors, there will be differences in synergy between different RAF inhibitors combined with MEK inhibitors. Although type II RAF inhibitors were found to readily combine with cobimetinib, type 1.5 RAF inhibitors (including the anomalous disruptor PLX-8394) did not synergize with MEK inhibitors in KRAS mutant cell lines (Figure 2A). This suggests that anomalous activation is not the primary reason why type 1.5 BRAF inhibitors do not synergize with MEK inhibitors in this mutant setting.

To test whether these changes in sensitivity were related to the expected changes in pathway signaling, A549 and HCT 116 cells were treated with escalating concentrations of the RAF inhibitors vemurafenib and AZ-628 (0.1–10 μM) and a fixed dose of cobimetinib. While vemurafenib did not reduce pathway signaling in the A549 KRAS mutant system in the presence or absence of cobimetinib, AZ-628 only effectively inhibited MAPK pathway output at the levels of pMEK, pERK, and pRSK when combined with cobimetinib (Figure 2B). Pathway inhibition was limited to the MAPK pathway, as few effects were observed at the pAKT level. DUSP6 and SPRY4 were examined 6 hours after treatment with 0.1 μM AZ-628, 0.1 μM cobimetinib, or a combination of these drugs in four KRAS mutant lung cancer cell lines using RNA-Seq canonicalizing downstream transcriptional targets of MAPK signaling (Figure 2C). Significant interactions were observed between treatments (P<0.01, moderate T-test), where the downregulation of these genes was more profound than expected if the effects of the two single agents were merely additive. These data indicate that the compounds also induce synergistic transcriptional repression of the MAPK pathway. The combination also resulted in significant apoptosis induction 48 hours after treatment, as demonstrated by elevated staining of cleaved PARP by Western blotting (Fig. 2D), and significant increases in sub-G1 and G1 populations, as assessed by flow cytometry targeting annexin V and propidium iodide (Fig. 2E). To test whether these combination effects lead to durable cell growth repression, the long-term growth effects of the MEK/RAF inhibitor combination were also tested in a colony formation assay. Significant synergy was observed between cobimetinib and the type II inhibitors AZ-628 or LY3009120, but not with the type 1.5 inhibitors vemurafenib and PLX-8394 (Fig. 2F).

Example 4: Combined treatment with RAF and MEK inhibitors demonstrates efficacy in vivo.

To determine whether the combination effect observed in vitro translated into in vivo efficacy, the type II pan-RAF inhibitors LY3009120 and AZ-628 were tested in combination with cobimetinib in NCI-H2122 lung (AZ-628+GDC-0973) and HCT116 colon (LY3009120+GDC-0973) xenograft tumor models. Consistent with in vitro data, in vivo data indicated a robust combination effect of either type II RAF inhibitor plus a MEK inhibitor compared to the efficacy of either molecule alone (Figure 3A). While LY3009120 or cobimetinib as a single agent moderately inhibited tumor growth, the combination effectively regressed tumors. Importantly, the combination was well tolerated in mice, resulting in minimal or minimal changes in body weight after treatment (Figure 3B). To assess how well the combination affected MAPK signaling, tumor samples were collected at multiple time points 4 days post-treatment. At all time points tested, compared to either inhibitor alone, the combination of LY3009120 and GDC-0973 demonstrated better inhibition of pERK and pRSK signaling in the MAPK pathway, leading to deeper inhibition of downstream MAPK target genes, DUSP6 and SPRY4 (Figure 3C). Plasma and tumor drug concentrations confirmed that the improved inhibitory activity was not due to increased drug exposure, but rather to drug-drug interactions (Figure 3D). In conclusion, the combination of RAF and MEK inhibition exhibited significant combined efficacy in vivo.

Example 5: MEK inhibitor treatment induces RAF kinase activity in a RAS-dependent manner.

To investigate the mechanism of the synergy observed between cobimetinib and the type II RAF inhibitor AZ-628, a cohort of KRAS mutant and wild-type cell lines were treated with cobimetinib for 24 hours, and the effect on MAPK pathway signaling was examined. Increased pMEK levels were observed in KRAS mutant cells but not in KRAS wild-type cells after cobimetinib treatment, indicating increased flux through the MAPK pathway (Fig. 4A). In a separate set of experiments, where pMEK induction was examined in a cohort of KRAS mutant and wild-type colon and lung cancer cell lines after 24 hours of cobimetinib treatment, increased pMEK levels were observed in synergistic KRAS mutant and wild-type cells but not in non-synergistic KRAS mutant and wild-type cells (Figs. 4B-4D). To further understand how cobimetinib treatment alters the pathway, RAF dimerization was assessed via CRAF immunoprecipitation. In KRAS mutant cell lines (A549, H2122, and HCT116) treated with cobimetinib, BRAF and CRAF underwent strong immunoprecipitation (Fig. 4A). This was not observed in the tested KRAS wild-type (293T) or BRAFV600E (A375) cell lines. In vitro kinase activity of RAF dimers was subsequently measured via exogenous addition of MEK and ATP. These results indicated that the RAF dimers present in the KRAS mutant cell lines after cobimetinib addition were highly active (Fig. 4A) and capable of phosphorylating MEK at significantly higher levels than those treated with DMSO. To further investigate the mechanism, RAS-GTP levels in KRAS mutant and wild-type cell lines after treatment with GDC-0973 were assessed. The elevated RAS-GTP levels in the KRAS mutant system at baseline and after cobimetinib treatment likely explain the induction of RAF dimer and RAF kinase activation (Fig. 4A). Following the addition of cobimetinib at concentrations of 50–100 nM, both the formation of BRAF-CRAF heterodimers and the increase in heterodimer kinase activity were dose-dependent (Figure 4E). This result explains the synergistic effect observed in the 8-day colony formation assay, where the concentrations of 50–100 nM cobimetinib and 100 nM AZ-628 had minimal effect on cell growth on their own (Figure 2F).

Next, the ability of RAF inhibitors to inhibit cobimetinib-induced RAF dimers was examined. Cobimetinib-induced RAF dimers were immunoprecipitated from A549 cells and then treated with AZ-628, LY3009120, or vemurafenib. Only type II RAF inhibitors that bind to the RAF dimer were able to inhibit kinase activity, as demonstrated by a decrease in pMEK levels in an IP kinase assay (Fig. 4F), and consistent with the synergistic effect observed in this line (Fig. 2A).

Cobimetinib treatment led to the reactivation of both the dysfunctional feedback loop and the robust pathway, as demonstrated by elevated RAS-GTP levels, RAF dimer, and pMEK, an effect more pronounced in KRAS mutant cell lines. To test whether mutant KRAS is required for this activity, KRAS-targeting siRNAs were used, and their effects on MAPK signaling in the presence or absence of cobimetinib were observed. In each KRAS mutant cell line tested, KRAS knockdown attenuated cobimetinib-induced pMEK levels, as well as RAF dimer formation and in vitro kinase activity (Figure 4G). In summary, these data indicate that mutant KRAS plays a crucial role in mediating the reactivation of the MAPK pathway, due to the increased RAS-GTP levels following single-agent MEK inhibition.

Example 6: Synergistic effect of type II RAF inhibitors and MEK inhibitors in subsets of KRAS mutant and wild-type cell lines

To determine whether specific genotypes are more sensitive to the combination of a type II pan-RAF inhibitor and a MEK inhibitor, 322 cell lines were screened using two single agents, AZ-628 (starting at 20 μM) and cobimetinib (starting at 1 μM), and co-dilutions of both compounds. These included cell lines from which RAS mutations were significantly and frequently found (pancreas, lung, colorectal, skin, ovary, and blood). Positive Bliss overdose on the co-dilution series was calculated from these data as a measure of synergy. Significantly higher synergy scores were observed in KRAS and NRAS mutant cell lines compared to either BRAF-V600 mutant or wild-type (non-RAS/BRAF-V600 mutant) cell lines (Figure 5A) (p < 0.001, two-tailed t-test). Cell lines from each tissue type were then grouped into the following categories using a mixed model approach: no synergy, low synergy, moderate synergy, or high synergy. When examining the distribution of these categories across all metrics, a strong association was observed between moderate and high co-responsive individuals in the RAS mutant group compared to wild-type or BRAF-V600 cell lines (Figure 5A).

While strong synergism was observed in RAS mutant cell lines, no RAS mutant cell lines were found to be synergistically suppressed. Conversely, several RAS/BRAFV600 wild-type cell lines showed strong synergism. Colorectal cancer lines exhibiting RAS mutation status and the presence or absence of synergism were selected, and MAPK signaling after KRAS knockdown with or without cobimetinib treatment was evaluated (Fig. 5B). Interestingly, lower overall levels of MAPK signaling were observed in KRAS mutant systems where the combination was not synergistic, as evidenced by lower total protein levels and phosphorylation at multiple nodes in the MAPK pathway. Conversely, significantly higher levels of MAPK signaling were observed in either KRAS mutant or wild-type lines where the combination showed synergism. MAPK signaling in lines synergistically suppressed by the combination was eliminated by the combination of KRAS knockdown and cobimetinib, as indicated by lower levels of pMEK, pERK, and pRSK (Fig. 5B). Cell lines subjected to combined synergistic inhibition showed elevated BRAF/CRAF heterodimer formation, as demonstrated by CRAF immunoprecipitation and subsequent CRAF and BRAF staining, independent of RAS mutation status (Fig. 5B). A strong increase in pMEK levels was observed after cobimetinib treatment using an in vitro RAF kinase assay, primarily in lines exhibiting synergistic inhibition (Fig. 5B), but not in wild-type KRAS lines not subjected to synergistic inhibition (Fig. 5C).

In a separate set of experiments, the combined activity of HM95573 (GDC-5573), a distinct type II pan-RAF inhibitor, with cobimetinib was also observed in both KRAS-mutant non-small cell lung cancer (NSCLC) cells (A549) and KRAS-mutant colorectal cancer (CRC) cells (HCT-116), as assessed by pMEK, pERK, and pRSK levels after treatment with HM95573, cobimetinib, or either of these agents (Figs. 5D-5E). The combination of HM95573 (GDC-5573) and cobimetinib also improved tumor growth inhibition (TGI) in vivo in the KRAS-mutant syngeneic CRC xenograft model CT26 compared to treatment with either agent alone (Figs. 5F-5G).

To determine whether the combination of HM95573 (GDC-5573) and cobimetinib exhibited similar synergy across cell lines, 196 lung, colon, skin, and ovarian cancer cell lines were screened using HM95573 (GDC-5573), cobimetinib, or co-dilutions of both compounds. Positive Bliss excess on the co-dilution series was calculated from these data as a measure of synergy, and significance was assessed by a two-tailed t-test. Similar to the combination of AZ-628 and cobimetinib, significantly higher synergy scores were observed in RAS mutant cell lines compared to either BRAF-V600 mutant or wild-type (non-RAS/BRAF-V600 mutant) cell lines (Figure 5H).

Given the observation of increased RAF dimer formation in RAS mutant and wild-type cell lines after cobimetinib treatment, we inquired whether these cell lines also exhibited increased RAS-GTP levels after cobimetinib treatment. Only the cell lines showing synergistic response to cobimetinib treatment showed increased active RAS-GTP, regardless of RAS mutation status (Figure 5I), consistent with the KRAS-dependent effect observed in cell lines synergistically inhibited by a combination of RAF and MEK inhibitors.

Because of the known biochemical differences among different RAS mutation isotypes due to intrinsic nucleotide changes dependent on position 12 or 13 (Hunter et al., Molecular Cancer Research 13:1325-1335 (2015)), synergistic differences in cell lines with mutations at G12 or G13 were examined using at least five representative cell lines. Interestingly, a significant association was observed between sensitivity to the RAF plus MEK inhibitor combination and the presence of the G13D mutation (Fig. 6A). This trend was most pronounced in colorectal cancer lines where the KRAS-G13D mutation was most prevalent, and similar trends were observed in lung adenocarcinomas, which had a lower prevalence, for both combinations tested (i.e., AZ-628 and cobimetinib (Fig. 6B-6C) and HM95573 (GDC-5573) and cobimetinib (Fig. 6D)). Pathway signaling analysis of the KRAS-G13D mutant colorectal cell line compared to the non-KRAS-G13D line revealed stronger pMEK induction in the G13D cell line (Fig. 6E). In a separate set of experiments using SW48 syngeneic cell lines, KRAS-G13D knock-in cells treated with AZ-628 (starting at 0.1 μM) and cobimetinib (at 250 nM) exhibited greater synergy than KRAS-G12D and KRAS-G12C knock-in cells treated with the same combination (AZ-628 and cobimetinib), as assessed by crystal violet staining (Fig. 6F) and pERK-induced Western blot analysis (Fig. 6G). Another experiment, a profile analysis of 322 cell lines targeting AZ-628, showed increased single-agent activity in KRAS-G13D cells compared to KRAS-G12C, KRAS-G12D, and KRAS-G12V cells (Fig. 6H).

These effects can be directly attributed to the RAS-G13D isoform, as SW48 syngeneic cell lines carrying KRAS-WT, KRAS-G12V, or KRAS-G13D exhibit significantly higher RAS-GTP levels in KRAS-G13D clones (Fig. 6I). The elevated nucleotide exchange rate reported with KRAS-G13D (Hunter et al., Molecular Cancer Research 13:1325-1335 (2015)) induces greater dependence on RAF heterodimer signaling, which is effectively eliminated by combined treatment with MEK and type II RAF inhibitors. Greater intrinsic nucleotide exchange with KRAS-G13D was confirmed in SW48 KRAS-G13D cells, as assessed using nucleotide exchange reactions by time-resolved fluorescence energy transfer (TR-FRET) (Fig. 6J).

Another explanation for elevated RAS-GTP levels is induction due to adaptive reprogramming following MEK inhibitor treatment, or activation upstream of RAS via EGFR or downstream via SOS1. To directly test this, pEGFR levels were assessed after cobimetinib treatment, but little evidence of EGFR activation was observed under the condition of elevated RAS-GTP levels (Figure 6K). Similarly, targeted knockdown of SOS1 had no effect on RAS-GTP levels (Figure 6L), suggesting that these mechanisms are not the cause of elevated RAS-GTP levels.

Example 7: Pan-RAF inhibitors also synergize with pan-PI3K inhibitors

The data above strongly suggest a MEK kinase inhibition-induced feedback loop, which is particularly potent in KRAS-G13D mutant tumors. Given these results, the initial compound screening was re-examined, where A549 cells were screened with combinations of AZ-628 and a library of 430 different small molecule inhibitors. The presence of picotinib, a pan-PI3K inhibitor, was noted as the top hit in the screening. Since A549 cells are PIK3CA wild-type, it was hypothesized that wild-type PI3K inhibition in this background could drive RAS activation via an inhibitory negative feedback loop and subsequent RAF activation. To examine this more broadly, a cohort of 213 cell lines was screened with a combination of AZ-628 and picotinib (Figure 7A). Interestingly, a larger overall increase in synergy was observed in the AZ-628/picotinib combination compared to the cobimetinib/picotinib combination, independent of RAS/RAF mutation status. Next, A549 and HCT116 cells were treated with a set of PI3K inhibitors, and pMEK levels were examined after treatment. All the PI3K inhibitors tested were found to increase pMEK in both A549 (KRAS-G12S) and HCT116 (KRAS-G13D/PIK3CA-H1047R) cells (Figs. 7B and 7C), with picolixib and taxerixib achieving this at lower concentrations. This suggests that the increased flux via the MAPK pathway following PI3K inhibition is independent of PI3K mutation status. This increased flux leads to greater downstream signaling, as demonstrated by elevated pERK and pRSK levels, and is dose-dependent for the PI3K inhibitor picolixib (Fig. 7D). To investigate whether the effect of PI3K inhibition led to a RAS-dependent increase in RAS-GTP levels, RAF1-RBD pull-down of active RAS-GTP was performed in cells treated with either picolixib or cobimetinib. Picotinib was found to increase RAS-GTP levels (albeit at higher concentrations), similar to the induction by cobimetinib (Figure 7E).

Full-dose matrix Bliss analysis confirmed the synergistic effect of PI3K with pan-RAF inhibitors. A group of PI3K inhibitors showed synergy with the pan-RAF inhibitor AZ-628 (producing an overdose Bliss score similar to the AZ-628/cobimetinib combination) (Figure 7F), the highly specific PIK3CA inhibitor iperitixib (BYL-719), and the AKT inhibitor ipatasertib (GDC-0068) (showing 2-3 times lower synergy). This indicates that broad-based wild-type PI3K inhibition can drive RAS activation and may induce subsequent RAF inhibition sensitization. When examining the effects of AZ-628 or iperitixib alone or in combination on MAPK signaling, PI3K inhibitors were observed to robustly inhibit pAKT, while the combination of the two agents was observed to prevent pMEK accumulation and eliminate downstream signaling to ERK (Figure 7G).

Example 8: Type II RAF inhibitors are effective in NRAS mutant cell lines.

To investigate whether RAF inhibitors, particularly type II pan-RAF dimer inhibitors, are also effective in the context of other RAS gene mutations, cell viability was assessed in a group of RAF inhibitors in NRAS mutant melanoma cell lines compared to BRAF-V600E mutant and NRAS/BRAF wild-type cell lines for type II RAF inhibitors, AZ-628, LY-3009120, and MNL-2480, as well as the “abnormal disruptor” PLX-8394. In the tested NRAS mutant melanoma, the type II RAF inhibitors AZ-628 and LY-3009120 showed substantially equivalent effects on mean viability (Fig. 8A) and inhibitory efficacy as measured by IC50 (Fig. 8B) compared to the tested BRAF-V600E mutant cell lines. These results suggest that type II pan-RAF dimer inhibitors (such as AZ-628 and LY-3009120), alone or in combination with MEK inhibitors (like cobimetinib), may be highly effective in treating cancers with NRAS activating mutations (like melanoma). Further confirmation of the efficacy of type II pan-RAF dimer inhibitors (such as AZ-628 and LY-3009120) in treating NRAS mutant cancers was achieved through separate evaluations of cell viability and IC50 in NRAS mutant, BRAF-V600E, and NRAS/BRAF wild-type cell lines treated with AZ-628 or cobimetinib (GDC-0973). The results showed that AZ-628 had equivalent efficacy in both NRAS mutant and BRAF-V600E mutant cell lines compared to cobimetinib, which showed greater efficacy in the BRAF-V600E cell line relative to NRAS mutant cell lines (Figures 8C and 8D).

Other implementation plans

While the foregoing invention has been described in considerable detail by way of example for clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. All patent and scientific disclosures cited herein are explicitly and fully incorporated by reference.

Sequence List

<110> Genentech, Inc.

<120> Used in the diagnosis and treatment of cancer

<130> 50474-171WO2

<150> US 62/556,277

<151> 2017-09-08

<160> 6

<170> PatentIn version 3.5

<210>  1

<211> 65

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400>  1

gatccccctt acgctgagta cttcgattca agagatcgaa gtactcagcg taagtttttt 60

ggaaa                                                                                                                                    

<210>  2

<211> 65

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400>  2

agcttttcca aaaaacttac gctgagtact tcgatctctt gaatcgaagt actcagcgta 60

agggg                                                                                                       65

<210> 3

<211> 65

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 3

gatccccgac atgaaatcca acaatattca agagatattg ttggatttca tgtctttttt 60

ggaaa                                                                                                                                    

<210> 4

<211> 65

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400>  4

agcttttcca aaaaagacat gaaatccaac aatatctctt gaatattgtt ggatttcatg 60

tcggg

<210> 5

<211>  21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 5

tcctgcgtct agaggttccc a                     21

<210> 6

<211>  21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 6

taggagtaga catccgactg g                         21

Claims (15)

1. Preparation of reagents for determining the presence of KRAS-G13D mutations in a sample: This is intended for use in a method for identifying an individual with cancer who may benefit from treatment comprising a pan-RAF dimer inhibitor and a MEK inhibitor, the method comprising screening a sample from the individual for a KRAS-G13D mutation, wherein the presence of a KRAS-G13D mutation in the sample identifies the individual as potentially benefiting from treatment comprising a pan-RAF dimer inhibitor and a MEK inhibitor, wherein the pan-RAF dimer inhibitor is HM95573 or a pharmaceutically acceptable salt thereof, wherein the MEK inhibitor is cobimetinib (GDC-0973) or a pharmaceutically acceptable salt thereof, wherein the cancer is selected from the group consisting of colorectal cancer, lung cancer, and skin cancer. 2. The use of claim 1, wherein the individual has a KRAS-G13D mutation and the method further comprises administering to the individual a therapeutically effective amount of a pan-RAF dimer inhibitor and a MEK inhibitor. 3. The use of claim 1 or 2, wherein screening comprises amplification and sequencing of the entire or part of the KRAS gene. 4. The use of claim 3, wherein the portion of the KRAS gene is exon 2 of the KRAS gene. 5. The use of claim 4, wherein the KRAS c.38G>A nucleotide substitution mutation at codon 13 of exon 2 of the KRAS gene indicates the KRAS-G13D mutation. 6. The use according to any one of claims 1-5, wherein the method further comprises administering an additional therapeutic agent to the individual. 7. The use of claim 6, wherein the additional therapeutic agent is selected from the group consisting of free immunotherapy agents, cytotoxic agents, growth inhibitors, radiotherapy agents, and anti-angiogenic agents. 8. The use according to any one of claims 1-7, wherein the sample is a tissue sample, a cell sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof. 9. The use of claim 8, wherein the sample is a tissue sample. 10. The use of claim 9, wherein the tissue sample is a tumor tissue sample. 11. The use of claim 10, wherein the tumor tissue sample is a formalin-fixed and paraffin-embedded (FFPE) sample, an archived sample, a fresh sample, or a frozen sample. 12. The use according to any one of claims 1-11, wherein the cancer is colorectal cancer. 13. The use according to any one of claims 1-11, wherein the cancer is lung cancer. 14. The use according to any one of claims 1-11, wherein the cancer is skin cancer. 15. The use of any one of claims 1-14, wherein the individual is a person.