Abstract NG04: Clinical acquired resistance to RAF inhibitor combinations in BRAF mutant colorectal cancer through MAPK pathway alterations
Authors: Corcoran Ryan B. , Ahronian Leanne G. , Van Allen Eliezer , Coffee Erin M. , Wagle Nikhil , Kwak Eunice L. , Faris Jason E. , Iafrate A. John , Garraway Levi A. , Engelman Jeffrey A. 
Published: August 2015

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 patient's 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