Abstract 4052: Effects of CRISPR/Cas9-mediated knockout of SIRT6 in human melanoma cells, in vitro and in vivo

Abstract

Abstract Melanoma is one of the most aggressive and fatal forms of skin cancer, with dismal 5-year survival if not diagnosed early. Although recent advances have led to better targeted- and immuno- therapeutic approaches, many patients develop resistance and recurrence. Therefore, it is crucial to develop newer druggable molecular targets that could be exploited towards the management of this deleterious neoplasm. Earlier studies from our laboratory have shown that SIRT6, a member of the sirtuin family of class III histone deacetylases, possesses a potential pro-proliferative role in melanoma. Extending on these novel findings, the objective of this study was to determine the effects of CRISPR/Cas9-mediated knockout (KO) of SIRT6 in melanoma cells, both in vitro and in vivo. First, we evaluated the effects of SIRT6 KO on growth, viability, and clonogenic survival in the A375 human melanoma cell line. We found that KO of SIRT6 resulted in a significant anti-proliferative response in melanoma cells, as measured by trypan blue and RealTime-Glo assays. Further, SIRT6 KO also resulted in a significant decrease in the long-term clonogenic survival of melanoma cells, as measured by a colony formation assay. Additionally, we used DNA cell cycle analysis by flow cytometry to assess the effect of SIRT6 KO on cell cycle perturbation in melanoma cells. Our data demonstrated that SIRT6 KO induced a G1-phase arrest in the A375 melanoma cells. Furthermore, we employed a PCR array (RT2 Profiler Cancer Pathway Array; with 84 genes) to understand the molecular mechanisms associated with the observed anti-proliferative response of SIRT6 KO. We found that SIRT6 KO caused statistically significant alteration in genes involved in important cellular pathways, including angiogenesis (CCL2, KDR, SERPINF1, and FLT1), hypoxia signaling (ADM and CA9), cellular senescence (IGFBP5 and TBX2), apoptosis (CFLAR), epithelial-to-mesenchymal transition (EMT) (SNAI2), and telomere maintenance (TEP1). Finally, to validate the in vitro findings to in vivo situation, we determined the growth of SIRT6 KO A375 human melanoma cells-implanted tumors in immunocompromised mice. We observed a significant slow growth of SIRT6 KO tumors compared to wild-type A375 tumors, suggesting that SIRT6 reduction decreases cell survival in melanoma both in vitro and in vivo. Overall, our results re-emphasize the tumor promoter role of SIRT6 in melanoma. Further detailed studies are required to determine the potential of SIRT6 inhibition as a novel treatment regimen against melanoma. Citation Format: Liz M. Garcia-Peterson, Mary A. Ndiaye, Glorimar Guzman-Perez, Chandra K. Singh, Gagan Chhabra, Nihal Ahmad. Effects of CRISPR/Cas9-mediated knockout of SIRT6 in human melanoma cells, in vitro and in vivo [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4052.