Erin M. Coffee

EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition with vemurafenib

UNLABELLED BRAF mutations occur in 10-15% of colorectal cancers (CRCs) and confer adverse outcome. While RAF inhibitors such as vemurafenib (PLX4032) have proven effective in BRAF mutant melanoma, they are surprisingly ineffective in BRAF mutant CRCs, and the reason for this disparity remains unclear. Compared to BRAF mutant melanoma cells, BRAF mutant CRC cells were less sensitive to vemurafenib, and P-ERK suppression was not sustained in response to treatment. Although transient inhibition of phospho-ERK by vemurafenib was observed in CRC, rapid ERK re-activation occurred through EGFR-mediated activation of RAS and CRAF. BRAF mutant CRCs expressed higher levels of phospho-EGFR than BRAF mutant melanomas, suggesting that CRCs are specifically poised for EGFR-mediated resistance. Combined RAF and EGFR inhibition blocked reactivation of MAPK signaling in BRAF mutant CRC cells and markedly improved efficacy in vitro and in vivo. These findings support evaluation of combined RAF and EGFR inhibition in BRAF mutant CRC patients. SIGNIFICANCE BRAF valine 600 (V600) mutations occur in 10% to 15% of colorectal cancers, yet these tumors show a surprisingly low clinical response rate (~5%) to selective RAF inhibitors such as vemurafenib, which have produced dramatic response rates (60%–80%) in melanomas harboring the identical BRAF V600 mutation. We found that EGFR-mediated MAPK pathway reactivation leads to resistance to vemurafenib in BRAF-mutant colorectal cancers and that combined RAF and EGFR inhibition can lead to sustained MAPK pathway suppression and improved efficacy in vitro and in tumor xenografts.

Synthetic Lethal Interaction of Combined BCL-XL and MEK Inhibition Promotes Tumor Regressions in KRAS Mutant Cancer Models

KRAS is the most commonly mutated oncogene, yet no effective targeted therapies exist for KRAS mutant cancers. We developed a pooled shRNA-drug screen strategy to identify genes that, when inhibited, cooperate with MEK inhibitors to effectively treat KRAS mutant cancer cells. The anti-apoptotic BH3 family gene BCL-XL emerged as a top hit through this approach. ABT-263 (navitoclax), a chemical inhibitor that blocks the ability of BCL-XL to bind and inhibit pro-apoptotic proteins, in combination with a MEK inhibitor led to dramatic apoptosis in many KRAS mutant cell lines from different tissue types. This combination caused marked in vivo tumor regressions in KRAS mutant xenografts and in a genetically engineered KRAS-driven lung cancer mouse model, supporting combined BCL-XL/MEK inhibition as a potential therapeutic approach for KRAS mutant cancers.

The dual PI3K/mTOR inhibitor NVP-BEZ235 induces tumor regression in a genetically engineered mouse model of PIK3CA wild-type colorectal cancer.

Purpose To examine the in vitro and in vivo efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type colorectal cancer (CRC). Experimental Design PIK3CA mutant and wild-type human CRC cell lines were treated in vitro with NVP-BEZ235, and the resulting effects on proliferation, apoptosis, and signaling were assessed. Colonic tumors from a genetically engineered mouse (GEM) model for sporadic wild-type PIK3CA CRC were treated in vivo with NVP-BEZ235. The resulting effects on macroscopic tumor growth/regression, proliferation, apoptosis, angiogenesis, and signaling were examined. Results In vitro treatment of CRC cell lines with NVP-BEZ235 resulted in transient PI3K blockade, sustained decreases in mTORC1/mTORC2 signaling, and a corresponding decrease in cell viability (median IC50 = 9.0–14.3 nM). Similar effects were seen in paired isogenic CRC cell lines that differed only in the presence or absence of an activating PIK3CA mutant allele. In vivo treatment of colonic tumor-bearing mice with NVP-BEZ235 resulted in transient PI3K inhibition and sustained blockade of mTORC1/mTORC2 signaling. Longitudinal tumor surveillance by optical colonoscopy demonstrated a 97% increase in tumor size in control mice (p = 0.01) vs. a 43% decrease (p = 0.008) in treated mice. Ex vivo analysis of the NVP-BEZ235-treated tumors demonstrated a 56% decrease in proliferation (p = 0.003), no effects on apoptosis, and a 75% reduction in angiogenesis (p = 0.013). Conclusions These studies provide the preclinical rationale for studies examining the efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type CRC.

mTOR Inhibition Specifically Sensitizes Colorectal Cancers with KRAS or BRAF Mutations to BCL-2/BCL-XL Inhibition by Suppressing MCL-1

Colorectal cancers harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that targets the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS- and BRAF-mutant but not wild-type (WT) colorectal cancer cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT, colorectal cancers, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS-mutant colorectal cancer xenograft and genetically engineered mouse models of colorectal cancer, but not in the corresponding KRAS-WT colorectal cancer models. These data suggest that the combination of BCL-2/BCL-XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers.

Concomitant BRAF and PI3K/mTOR blockade is required for effective treatment of BRAF(V600E) colorectal cancer.

Purpose: BRAFV600E mutations are associated with poor clinical prognosis in colorectal cancer (CRC). Although selective BRAF inhibitors are effective for treatment of melanoma, comparable efforts in CRC have been disappointing. Here, we investigated potential mechanisms underlying this resistance to BRAF inhibitors in BRAFV600E CRC. Experimental Design: We examined phosphoinositide 3-kinase (PI3K)/mTOR signaling in BRAFV600E CRC cell lines after BRAF inhibition and cell viability and apoptosis after combined BRAF and PI3K/mTOR inhibition. We assessed the efficacy of in vivo combination treatment using a novel genetically engineered mouse model (GEMM) for BRAFV600E CRC. Results: Western blot analysis revealed sustained PI3K/mTOR signaling upon BRAF inhibition. Our BRAFV600E GEMM presented with sessile serrated adenomas/polyps, as seen in humans. Combination treatment in vivo resulted in induction of apoptosis and tumor regression. Conclusions: We have established a novel GEMM to interrogate BRAFV600E CRC biology and identify more efficacious treatment strategies. Combination BRAF and PI3K/mTOR inhibitor treatment should be explored in clinical trials. Clin Cancer Res; 19(10); 2688–98. ©2013 AACR.

Combination PI3K/MEK inhibition promotes tumor apoptosis and regression in PIK3CA wild-type, KRAS mutant colorectal cancer

PI3K inhibition in combination with other agents has not been studied in the context of PIK3CA wild-type, KRAS mutant cancer. In a screen of phospho-kinases, PI3K inhibition of KRAS mutant colorectal cancer cells activated the MAPK pathway. Combination PI3K/MEK inhibition with NVP-BKM120 and PD-0325901 induced tumor regression in a mouse model of PIK3CA wild-type, KRAS mutant colorectal cancer, which was mediated by inhibition of mTORC1, inhibition of MCL-1, and activation of BIM. These findings implicate mitochondrial-dependent apoptotic mechanisms as determinants for the efficacy of PI3K/MEK inhibition in the treatment of PIK3CA wild-type, KRAS mutant cancer.

Cross-species analysis of genetically engineered mouse models of MAPK-driven colorectal cancer identifies hallmarks of the human disease

Effective treatment options for advanced colorectal cancer (CRC) are limited, survival rates are poor and this disease continues to be a leading cause of cancer-related deaths worldwide. Despite being a highly heterogeneous disease, a large subset of individuals with sporadic CRC typically harbor relatively few established ‘driver’ lesions. Here, we describe a collection of genetically engineered mouse models (GEMMs) of sporadic CRC that combine lesions frequently altered in human patients, including well-characterized tumor suppressors and activators of MAPK signaling. Primary tumors from these models were profiled, and individual GEMM tumors segregated into groups based on their genotypes. Unique allelic and genotypic expression signatures were generated from these GEMMs and applied to clinically annotated human CRC patient samples. We provide evidence that a Kras signature derived from these GEMMs is capable of distinguishing human tumors harboring KRAS mutation, and tracks with poor prognosis in two independent human patient cohorts. Furthermore, the analysis of a panel of human CRC cell lines suggests that high expression of the GEMM Kras signature correlates with sensitivity to targeted pathway inhibitors. Together, these findings implicate GEMMs as powerful preclinical tools with the capacity to recapitulate relevant human disease biology, and support the use of genetic signatures generated in these models to facilitate future drug discovery and validation efforts.

Abstract NG04: Clinical acquired resistance to RAF inhibitor combinations in BRAF mutant colorectal cancer through MAPK pathway alterations

BRAFmutations occur in ∼10% of colorectal cancers (CRCs) and confer poor prognosis. While RAF inhibitor monotherapy leads to response rates of ∼60% in BRAF mutant melanoma, response rates in BRAF mutant CRC are disappointingly low (∼5%), suggesting a fundamental difference between these tumor types, despite the mutual presence of a BRAF V600 mutation. Previously, our group and others found that while RAF inhibitors lead to profound and sustained suppression of MAPK signaling in BRAF mutant melanoma cells, suppression of MAPK signaling by RAF inhibitors in BRAF mutant CRC cells is transient, and the MAPK pathway is rapidly reactivated despite the continued presence of drug. These data suggest that MAPK pathway suppression by RAF inhibitors alone may be inadequate in BRAF mutant CRC and that combinations of RAF inhibitors with other targeted agents might be required to achieve robust MAPK suppression and clinical responses. More recently, our group and others found that in many BRAF mutant CRCs, MAPK pathway reactivation is driven by feedback signaling through EGFR via RAS and CRAF. Importantly, we found that the combination of a RAF inhibitor and an EGFR inhibitor can lead to sustained MAPK suppression and improved efficacy, leading to marked tumor regressions in preclinical BRAF mutant CRC xenograft models. These data have led to clinical trials in BRAF mutant CRC patients evaluating RAF inhibitor combinations, including combinations of RAF+EGFR, RAF+MEK, and RAF+MEK+EGFR inhibitors. Initial data from these trials, reported at ASCO 2014, have shown encouraging efficacy, with some trials showing initial response rates of as much as 40%. However, as with all targeted therapies, clinical benefit is invariably limited by the emergence of acquired resistance to these therapies. In this study, we sought to identify clinically relevant mechanisms of acquired resistance to RAF inhibitor combinations in BRAF mutant CRC, in order to understand the signaling changes leading to resistance and to devise therapeutic strategies to overcome or prevent resistance. To do so, we obtained tumor biopsies from BRAF mutant CRC patients upon disease progression, after initial response or prolonged stable disease on RAF+EGFR, RAF+MEK, or RAF+MEK+EGFR inhibitor combinations. Matched pre-treatment, post-progression, and normal DNA were analyzed by whole exome sequencing (WES) and RNA sequencing. To supplement this approach, in vitro modeling of acquired resistance was performed in BRAF mutant CRC cell lines. In one BRAF mutant CRC patient with prolonged stable disease on a RAF+EGFR inhibitor combination, WES identified KRAS amplification in a progressing lesion. RNA sequencing confirmed KRAS transcript overexpression, and KRAS amplification (∼25-fold) was confirmed by FISH in the post-progression biopsy, but was absent in a pre-treatment biopsy. Interestingly, in resistant clones generated from BRAF mutant CRC cell lines selected with either RAF+EGFR or RAF+MEK inhibitor combinations, KRAS exon 2 mutations were identified. Either KRAS amplification or KRAS mutation led to sustained MAPK pathway activity and cross-resistance to either RAF+EGFR or RAF+MEK inhibitor combinations. However, an ERK inhibitor, either alone or in combination with a RAF inhibitor retained the ability to suppress the MAPK pathway and could overcome resistance. In a second patient with a minor response to a RAF+EGFR inhibitor combination, BRAF amplification was identified in a progressing lesion, which was confirmed by FISH and was not present in a pre-treatment biopsy of the same lesion. Previously, our laboratory found that amplification of mutant BRAF could cause resistance to RAF or MEK inhibitors in BRAF mutant CRC cells by abrogating the ability of these inhibitors to suppress MAPK signaling. Importantly, an ERK inhibitor, either alone or in combination with a RAF inhibitor could suppress MAPK signaling and overcome resistance in this setting. In one patient with a minor response to a RAF+MEK inhibitor combination, WES identified the presence of an ARAF Q489L mutation and a MEK1 F53L mutation in a single progressing lesion, suggesting possible intra-lesional heterogeneity of acquired resistance mechanisms. However, utilizing a cell line derived from the patient9s post-progression biopsy, we found that 30 out of 30 single cell clones harbored both the ARAF and MEK1 mutations, and that the MEK1 F53L seemed to function as the primary driver of acquired resistance in these resistant tumor cells. MEK1 F53L expression markedly abrogated the ability of RAF+MEK and RAF+EGFR inhibitor combinations to suppress MAPK signaling. Interestingly, an ERK inhibitor was able to effectively suppress MAPK signaling and overcome resistance. In a second patient with a minor response to RAF+MEK, WES identified a focal amplicon on chromosome 2 in a progressing lesion, encompassing the c-mer oncogene (MERTK) receptor tyrosine kinase and the MAPK-regulated checkpoint kinase BUB1. Post-progression biopsies from two patients who progressed after prolonged stable disease to the triple combination of a RAF+MEK+EGFR inhibitor combination are currently being analyzed by WES, and updated results will be presented. In summary, RAF inhibitor combinations are leading to promising initial response rates in recent clinical trials in BRAF mutant CRC patients. The frequent identification of alterations leading to reactivation of MAPK pathway signaling upon clinical acquired resistance to RAF+EGFR or RAF+MEK combinations underscores the MAPK pathway as a valid and critical target in BRAF mutant CRC. Importantly, we found that many of these resistance mechanisms could be overcome by an ERK inhibitor or ERK inhibitor-based combinations, suggesting that ERK inhibitors may be key components of therapeutic combinations to be explored in future clinical trials for BRAF mutant CRC. Further efforts to understand acquired resistance mechanisms will be vital to developing novel therapeutic strategies to overcome resistance and extend clinical benefit in this lethal CRC subtype. Citation Format: Ryan B. Corcoran, Leanne G. Ahronian, Eliezer Van Allen, Erin M. Coffee, Nikhil Wagle, Eunice L. Kwak, Jason E. Faris, A. John Iafrate, Levi A. Garraway, Jeffrey A. Engelman. Clinical acquired resistance to RAF inhibitor combinations in BRAF mutant colorectal cancer through MAPK pathway alterations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr NG04. doi:10.1158/1538-7445.AM2015-NG04

428 Clinical acquired resistance to combined RAF/EGFR or RAF/MEK inhibition in BRAF mutant colorectal cancer (CRC) patients through MAPK pathway alterations

Background: BRAF mutations occur in ~10% of CRC and confer poor prognosis. While RAF inhibitor monotherapy leads to response rates of 60−80% in BRAF mutant (BRAFm) melanoma, response rates in BRAFm CRC are poor (~5%). Promising recent studies with RAF inhibitor-based combinations, including combined RAF/EGFR or RAF/MEK inhibition, have produced higher response rates in BRAFm CRC, but a better understanding of acquired resistance mechanisms will be critical to improving therapy. Methods: Mechanisms of acquired resistance to RAF/EGFR or RAF/MEK inhibition were evaluated in BRAFm CRC cell lines and in clinical biopsies obtained from BRAFm CRC patients following disease progression after initial response or prolonged stable disease to either therapeutic combination. BRAFm CRC cell lines were cultured in the presence of combined RAF/EGFR or RAF/MEK inhibitors until resistant clones emerged. Candidate resistance mutations were identified through exome sequencing. Matched pre-treament, post-progression, and normal DNA from BRAFm CRC patients treated with either RAF/EGFR or RAF/MEK combinations were analyzed by whole exome sequencing to identify clinical acquired resistance mechanisms. Results: In resistant clones generated from BRAFm CRC cell lines selected with either RAF/EGFR or RAF/MEK inhibitor combinations, KRAS exon 2 mutations were identified. KRAS mutation led to sustained MAPK pathway activity and cross-resistance to either RAF/EGFR or RAF/MEK inhibitor combinations. Interestingly, the triple combination of RAF/EGFR/MEK inhibition was able to suppress MAPK activity and overcome resistance. In a BRAFm CRC patient with prolonged stable disease on a RAF/EGFR inhibitor combination, whole exome sequencing identified the presence of KRAS amplification in a progressing lesion. RNA sequencing of the same lesion confirmed KRAS transcript overexpression, and ~25-fold amplification of KRAS in the progressing lesion and absence of amplification in the pre-treatment biopsy was confirmed by FISH. In one patient with a minor response to a RAF/MEK inhibitor combination, whole exome sequencing identified amplification of the c-mer oncogene (MERTK) receptor tyrosine kinase as the likely acquired resistance mechanism. In another patient with a minor response to a RAF/MEK inhibitor combination, whole exome sequencing identified subclonal ARAF and MEK1 mutations in the post-progression biopsy, suggesting the presence of heterogeneous acquired resistance mechanisms in this progressing lesion. Conclusions: The identification of alterations affecting the MAPK pathway in BRAFm CRC patients who have developed clinical acquired resistance to RAF/EGFR or RAF/MEK inhibitor regimens underscores the importance of the MAPK pathway in this cancer. Understanding the mechanisms of acquired resistance can lead to novel combination strategies to overcome or delay resistance. Friday 21 November 2014

Abstract 3135: Pre-treatment p-EGFR levels in tumors from a genetically engineered mouse model of BRAFV600E colorectal cancer predict response to combined BRAF/EGFR inhibition

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BRAFV600E colorectal cancer (CRC) patients do not exhibit responses to single-agent BRAF inhibition due to activation of EGFR. While combined BRAF and EGFR inhibition has shown some success in early clinical trials, there are still a subset of patients who do not respond to this combination. The purpose of this study is to identify which BRAFV600E tumors would be expected to respond to combined BRAF/EGFR inhibition based on the pre-treatment levels of pEGFR. A genetically engineered mouse model (GEMM) for Apc-/- BrafV600E/+ p53-/- CRC was used for the study. Tumors were induced in the colons using adenovirus expressing Cre recombinase. Tumors were visualized by optical colonoscopy and pre-treatment tissue biopsy samples were obtained. BRAF mutant mice were randomized to a combination treatment with a BRAF inhibitor (Dabrafenib), combined with either an EGFR inhibitor (Erlotinib) or a MEK inhibitor (Trametinib). Tumor growth or regression was followed for 21 days by colonoscopy. A retrospective analysis indicated that those tumors with high pre-treatment phosphorylated EGFR (p-EGFR) levels had significant responses to combined BRAF/EGFR therapy, while those tumors with little to no response to combined BRAF/EGFR therapy had low levels of p-EGFR prior to treatment. Furthermore, the majority of tumors that had a significant response to the combined BRAF/MEK therapy had the lowest levels of pre-treatment p-EGFR. These pre-clinical data suggest that BRAFV600E CRC patients could benefit from being stratified by p-EGFR levels prior to treatment. Those patients with high p-EGFR levels would be expected to respond best to combined BRAF/EGFR treatment, while those patients with low p-EGFR levels might benefit from a different combination therapy, such as combined BRAF/MEK inhibition. Citation Format: Erin M. Coffee, Ryan B. Corcoran, Jeffrey A. Engelman. Pre-treatment p-EGFR levels in tumors from a genetically engineered mouse model of BRAFV600E colorectal cancer predict response to combined BRAF/EGFR inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3135. doi:10.1158/1538-7445.AM2014-3135