Holley, Tara

Abstract 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

Abstract Background: Breast and other solid tumors exhibit high and varying degrees of intra-tumor heterogeneity that make the management and treatment of these diseases rather difficult. In order to study clinically relevant tumor heterogeneity in detail we performed an in-depth analysis of the clonal architecture of primary chemoradiation-naive breast cancers and their lymph node metastases. We combined DNA content-based flow cytometry and ploidy analysis with high-definition array comparative genomic hybridization (aCGH) and next-generation sequencing technologies to interrogate the genomes and transcriptomes of multiple biopsies from the tumors and involved lymph nodes. Thus rather than inferring the presence of distinct tumor cell populations, our flow-sorting based approach of first identifying and defining the clonal populations and then interrogating their genomes provides a highly objective method of exploring the clinical significance of tumor heterogeneity in solid tumors. Material and methods: We used DAPI-based DNA content measurements and flow sorting to isolate nuclei from distinct clonal populations of diploid and aneuploid tumor cells in surgical tumor samples and distant lymph nodes from chemoradiation naive patients. In parallel, matching samples were processed for pathological, transcriptome, and IHC based assays. Each distinct sorted tumor cell population was then interrogated with aCGH and next generation sequencing. For each patient, the detailed profiles of ploidy, copy number aberrations and mutations were used to recreate and map the lineages present in the primary tumor and lymph. In one detailed case, we used 12 fresh frozen sections morphologically mapped from within a chemo-naïve primary invasive ductal carcinoma of histological grade 3 with lymph node involvement, 2-3 fresh frozen sections from 2 out of 5 lymph nodes with metastatic carcinoma, formalin-fixed paraffin-embedded (FFPE) tissue sections from 2 more of the involved lymph nodes and 5 morphologically mapped FFPE samples of normal tissue adjacent to the tumor. Results: We identified multiple coexisting aneuploid populations within biopsies from individual patients. Our clonal analysis determined the genomic aberrations arising in lineages that spread throughout the primary tumor and later bifurcated to distinct lymph nodes, revealing distinct therapeutic targets in the trunk and branches of the tumor. We identified driver events including high-level focal amplifications (e.g. SARC) and homozygous deletions (e.g. ROBO1 and ROBO2), that converge on distinct cellular signaling pathways. Conclusion: The approach highlighted here has broad implications in the study of tumor heterogeneity in breast and other solid tumors by providing a unique ultra-high resolution view of tumor heterogeneity that can advance more effective treatment and clinical management of patients with this disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD4-7.