Abstract 1364: Tip60 dependent DNA homologous recombination repair is impaired in VHL-deficient clear cell renal cell carcinoma
Authors: Zhou Lijun , Pilie Patrick G. , Peterson Christine B. , Liu Xian-de , Zhang Xuesong , Jonasch Eric 
Published: August 2018

Abstract
Introduction: Clear cell renal cell carcinoma (ccRCC) displays genomic instability across all tumor stages, indicative of increased replicative stress and defects in DNA damage response (DDR) pathways including homologous repair (HR); however ccRCC does not display mutations in canonical DDR genes. We hypothesized that biallelic VHL loss is sufficient to cause HR deficiency (HRD) in ccRCC via direct regulation of DDR pathways. Experimental Procedures: We performed whole-exome (WES) sequencing of 15 small ccRCC tumors. We performed in silico genomic, transcriptomic, and proteomic analysis of tumors in KIRC TCGA to assess for HRD, using published HRD signatures. We assessed HR efficiency as a product of biallelic VHL loss in engineered cell line models. We studied the VHL-dependent DNA damage repair molecular mechanisms with cell line models, by HR and nonhomologous end joining (NHEJ) using reporter gene assays. We then assessed the status of TIP60 activation in Vhl deficient murine embryo fibroblasts, isogenic VHL deficient and proficient cell lines by determining DSB induced tip60 acetylation and foci formation. DSB induced HR and NHEJ activation were analyzed by assessing the induction of marker proteins including γH2AX, RAD51, and 53BP1. To examine VHL-protein interaction, green-fluorescent protein (GFP)-trap with GFP-tagged VHL and co-immunoprecipitation with specific antibodies were used. Results: 15/15 early stage ccRCC had biallelic VHL mutations and approximately 100 additional mutations per tumor, but no driver mutations typically associated with HRD. Nonetheless, in silico analysis showed 67% of KIRC TCGA displayed an HRD gene signature, and this signature was significantly higher in stage I disease (p=2.21e-08). Patients with VHL-mutated tumors were more commonly HRD than HRI (p=0.03), with frameshift/nonsense variants in VHL more likely to result in HRD than missense variants (p=0.02). Multivariate analysis showed HRD predicted for better overall survival compared to an HR intact (HRI) state (p&lt;0.0001). Using Vhl knockout and VHL overexpressing cell models we demonstrated that VHL interacts with the acetyltransferase Tip60, and Tip60 is required for VHL-dependent HR but not for nonhomologous end joining (NHEJ). We also observed that during genotoxic stress, VHL-associated-Tip60 dissociated from histone H3 and increased its association with H4, consistent with VHL having a regulatory role on histone H4. Conclusions: Early stage, VHL-deficient ccRCC shows a strong HRD signature in the absence of additional driver mutations associated with DDR. Biallelic loss of VHL is sufficient to cause HRD by impairing Tip60 mediated DDR complex activation. Our findings provide a mechanistic explanation for the DDR defect in ccRCC, and provide a foundation for treatment strategies that target specific elements of the DDR pathway in VHL deficient ccRCC.
Citation Format: Lijun Zhou, Patrick G. Pilie, Christine B. Peterson, Xian-de Liu, Xuesong Zhang, Eric Jonasch. Tip60 dependent DNA homologous recombination repair is impaired in VHL-deficient clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1364.