Abstract 475: Identification of SHANK2 as a tumor suppressor disrupted by recurrent somatic structural variation (SV) in neuroblastoma

Background. Neuroblastoma is a malignancy of the developing sympathetic nervous system which exacts significant morbidity and mortality in children. Large sequencing efforts have revealed a relative paucity of somatic point mutations in this and other childhood cancers. Structural variations (SVs) including translocations, inversions, deletions, duplications, and other complex events can occur in germline DNA or be acquired somatically in tumors. Recurrent somatic SVs affecting known genes are likely to be functional and may elucidate novel tumor suppressors or oncogenes. Methods. We are performing whole-genome sequencing (WGS) of 187 matched tumor-normal pairs from high-risk patients as part of the NCI-TARGET project (http://www.ocg.cancer.gov/programs/target) using Complete Genomics technology. High confidence somatic SVs were identified and a subset subsequently validated with Sanger sequencing; in vitro functional studies were performed using human derived neuroblastoma cell line models. Results. To date, we have analyzed WGS data from 106 matched stage 4 tumor-normal pairs. Among 2,770 high-confidence somatic SVs (average 26.1 per tumor, range 1-158), we observed recurrent focal deletions in ZFHX3 (n = 5) and EZH2 (n = 2), in addition to ATRX (n = 2), ARID1B (n = 2), and PTPRD (n = 5), each of the latter genes previously reported in neuroblastoma. A total of 210 inter-chromosomal translocations disrupting known genes were detected (average 2.0 per tumor, range 0-12). Notably, 14.4% of MYCN non-amplified stage 4 tumors (11/76) harbored SV breakpoints within SHANK2 at 11q13; all SHANK2 breakpoints were validated by Sanger sequencing. With the exception of a single deletion at the c-terminus, all breakpoints mapped within the long isoform of SHANK2 (transcript variant 1: NM_012309), and clustered within the ankyrin repeat domain which is involved in protein-protein interactions. Low SHANK2 expression in primary tumors obtained at diagnosis was associated with worse overall survival (p = 6.3 × 10−5), suggesting SHANK2, known to regulate neuronal differentiation, may function as a tumor suppressor. Consistent with this hypothesis, mRNA expression of SHANK2 was low in a panel of 27 neuroblastoma cell lines and further silencing with siRNA did not produce an observable phenotype. In contrast, forced expression of the long isoform of SHANK2 in three independent neuroblastoma cell lines with low endogenous SHANK2 levels resulted in profound reduction in cell g