Abstract 3170: Identification of novel genetic alterations driving melanoma tumorigenesis.
Authors: Kim Minjung , Ding Li , Dees Nathan , Kanchi Krishna L. , Sung Hyeran , Fenstermacher David , Griffith Malachi , Linette Gerry , Cornelius Lynn , Sondak Vernon K. , Mulé James J. , Wilson Richard K. , Weber Jeffrey S. 
Published: August 2014

Abstract
Recent high-throughput sequencing efforts have provided a comprehensive view of cancer genomes, revealing their complexity and heterogeneity. However, most of these studies remain descriptive without further functional and clinical validation of the candidate alterations, mainly due to the overwhelming number of somatic alterations present. To discover novel driver mutations of melanoma, massively parallel whole genome sequencing (WGS) was used to characterize 15 metastatic melanomas derived from 13 patients. A large number of somatic alterations were discovered in these tumors and hybridization capture-based validation confirmed 17,361 tier 1 point mutations, 84 tier 1 indels, and 411 somatic structural variants. As a pilot study to exploit this genomic data in order to identify novel genetic alterations driving melanoma tumorigenesis, we performed mutation proximity analysis to select candidates for further analysis. In this study, we addressed possible roles of DBC1 (Deleted in Bladder Cancer 1) and RASA1 (RAS p21 protein activator 1), which showed previously undocumented neighboring mutations. RASA1 is a GTPase activation protein that acts as a suppressor of RAS function. RASA1 has been implicated in actin filament polymerization, vascular development, cellular apoptosis, and cell motility. Our whole-genome analyses of melanomas identified two somatic missense mutations, targeting highly conserved neighboring Y472 and L481 in or around the PH domain in RASA1 in two samples. DBC1, also called BRINP, DBCCR1, and FAM5A, was previously shown to undergo loss of heterozygosity at 9q32-q33 in bladder cancer, and methylation silencing in bladder, breast, and lung cancers. Ectopic expression of DBC1 in bladder and lung cancer cells was reported to cause cell death and to inhibit cell proliferation, respectively. We observed six DBC1 missense mutations by whole genome analyses in four patients, including 2 neighboring mutations targeting S688 and S690. The shRNA-mediated knock down of DBC1 and RASA1 in melanocyte derived from Ink4a/Arf deletion/BRAF mutation background promoted proliferation, soft agar colony formation, and invasion. Ectopic expression of wild type DBC1 and RASA1 in human melanoma cell lines SKmel28 and WM983C (all with BRAFV600E), respectively, decreased soft agar colony formation, supporting their tumor suppressive roles. Various mutant forms of RASA1 and DBC1 were addressed for their roles. Interestingly, loss of RASA1 conferred decreased sensitivity to BRAF inhibitor Vemurafenib. In order to address the mutation frequency of DBC1 and RASA1, we analyzed additional melanoma samples and observed mutation rates of 21% for DBC1 (20/96 patients) and 9% for RASA1 (20/221). Therefore, our findings support that DBC1 and RASA1 play roles in melanoma suppression and the utility of genomic data for the identification of novel genes involved in tumorigenesis.
Citation Format: Minjung Kim, Li Ding, Nathan Dees, Krishna L. Kanchi, Hyeran Sung, David Fenstermacher, Malachi Griffith, Gerry Linette, Lynn Cornelius, Vernon K. Sondak, James J. Mulé, Richard K. Wilson, Jeffrey S. Weber. Identification of novel genetic alterations driving melanoma tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3170. doi:10.1158/1538-7445.AM2013-3170