Targeted BRAF inhibition in melanoma yields frequent and dramatic responses, which are unfortunately followed by almost universal development of therapeutic resistance. In contrast to MAP kinase pathway activation, (e.g., differential BRAF splicing, MEK1 mutations), direct gatekeeper mutations, seen with other targeted therapies, have not yet been reported in association with resistance to targeted BRAF inhibition. Through a combination of genomic profiling of clonal diversity and molecular modeling, we documented a new molecular mechanism of vemurafenib resistance in a melanoma patient, occurring by selection of a pre-existing clonal subpopulation harboring a novel mutation in the vemurafenib-binding site of BRAF. We assessed melanoma clonal diversity using a novel approach for studying tumor heterogeneity in solid tumors, based on separating cellular subpopulations by nuclear flow sorting coupled to next generation sequencing. This approach identified distinct, coexisting melanoma subpopulations prior to initiation of vemurafenib. While vemurafenib treatment suppressed multiple clonal populations, a distinct population was noted at the time of relapse, harboring both the common activating V600E and a novel L567V mutation in the vemurafenib-binding site. Molecular modeling predicted that L567V would interfere with drug binding leading to decreased therapeutic response. Using a lentiviral expression system we assessed the functional effects of the L567V mutation both alone, and in combination with V600E. Assessment of the kinase activity in an in vitro kinase assay using MEK as a substrate indicates that the L567V mutant retains kinase activity. However, expression in a panel of melanoma cell lines demonstrates that while both the V600E single mutant and the L567V single mutant respond to BRAF inhibition by decreasing the levels of phospho-Erk, the double mutant actually responds to treatment by increasing phospho-Erk levels. This is also reflected in the increased viability of cells expressing double mutant in presence of increasing drug levels (PLX4720), compared to either of the single mutants. Taken together, the data suggest that the BRAF L567V/V600E double mutant confers insensitivity to vemurafenib, and that patients harboring both mutations may demonstrate clinically significant resistance to vemurafenib. Further, this data illustrates the role of clonal heterogeneity in mediating key clinical events including response to therapy and development of resistance, a major challenge of targeted BRAF therapies.

Citation Format: Aleksandar Sekulic, Ashani T. Weeraratna, Jefferey L. MacKeigan, Amanpreet Kaur, Michael O'Connell, Megan L. Goodall, Michael Barrett, Elizabeth Lenkiewitz, Tara Holley, Victoria Zismann, Michael Gordon, Gideon Bollag, Chao Zhang, Jeffrey M. Trent. Discovery and molecular modeling of a novel mutation in the vemurafenib binding site of BRAF associated with therapeutic resistance in a patient with metastatic melanoma. [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 LB-213. doi:10.1158/1538-7445.AM2014-LB-213