Abstract
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
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