Abstract LB-055: Clinical acquired resistance to RAF inhibitor combinations in BRAF-mutant colorectal cancer through MAPK pathway alterations
Authors: Ahronian Leanne G. , Sennott Erin M. , Van Allen Eliezer M. , Wagle Nikhil , Kwak Eunice L. , Faris Jason E. , Godfrey Jason T. , Nishimura Koki , Lynch Kerry D. , Mermel Craig H. , Lockerman Elizabeth L. , Kalsy Anuj , Gurski Joseph M. , Bahl Samira , Anderka Kristin , Green Lisa M. , Lennon Niall J. , Huynh Tiffany G. , Mino-Kenudson Mari , Getz Gad , Dias-Santagata Dora , Iafrate A. John , Engelman Jeffrey A. , Garraway Levi A. , Corcoran Ryan B. 
Published: August 2015

BRAF V600E mutations occur in ∼10% of colorectal cancer (CRC), and are associated with poor prognosis. RAF inhibition alone has not been an effective treatment in BRAF-mutant (BRAFm) CRC patients, with response rates of only 5%, due to persistence of MAPK signaling. Combined RAF/EGFR, RAF/MEK, or RAF/MEK/EGFR inhibitors have produced improved efficacy in BRAFm CRC patients, yet ultimately resistance develops after an initial treatment response. Understanding the mechanisms of clinical acquired resistance that arise to RAF inhibitor combinations in BRAFm CRC patients may lead to valuable opportunities to overcome resistance and prolong clinical response.

We sought to identify clinically relevant mechanisms of acquired resistance to RAF inhibitor combinations by obtaining tumor biopsies from BRAFm CRC patients upon disease progression, after initial response to RAF/EGFR or RAF/MEK inhibitor combinations. Matched pre-treatment, post-progression, and normal DNA were analyzed by whole exome sequencing (WES) and RNA-seq.

In one BRAFm CRC patient with prolonged stable disease on a RAF/EGFR combination, WES identified KRAS amplification in a progressing lesion. FISH confirmed the presence of KRAS amplification in the post-progression biopsy, and RNA-seq revealed KRAS transcript overexpression. Interestingly, in resistant clones generated from BRAFm CRC cell lines selected with either RAF/EGFR or RAF/MEK inhibitors, 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.

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. BRAF amplification led to cross-resistance to the BRAF/MEK inhibitor combination.

In a third 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 MEK1 F53L seemed to function as the primary driver of acquired resistance in these resistant cells. MEK1 F53L expression markedly abrogated the ability of RAF/MEK and RAF/EGFR inhibitor combinations to suppress MAPK signaling.

Despite developing resistance to upstream MAPK pathway inhibitors, we found that each of the acquired resistance mechanisms we detected remained sensitive to ERK inhibition, which could effectively suppress MAPK signaling. Our findings demonstrate the central importance of MAPK pathway activity in BRAFm CRC, and highlight the critical need for MAPK pathway inhibition in the prevention of disease progression. Additionally, our work indicates ERK inhibitors may be valuable additions to future therapeutic combinations for BRAFm CRC patients. 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: Leanne G. Ahronian, Erin M. Sennott, Eliezer M. Van Allen, Nikhil Wagle, Eunice L. Kwak, Jason E. Faris, Jason T. Godfrey, Koki Nishimura, Kerry D. Lynch, Craig H. Mermel, Elizabeth L. Lockerman, Anuj Kalsy, Joseph M. Gurski, Samira Bahl, Kristin Anderka, Lisa M. Green, Niall J. Lennon, Tiffany G. Huynh, Mari Mino-Kenudson, Gad Getz, Dora Dias-Santagata, A. John Iafrate, Jeffrey A. Engelman, Levi A. Garraway, Ryan B. Corcoran. 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 LB-055. doi:10.1158/1538-7445.AM2015-LB-055