Abstract 2587: RAC1P29S expression in melanocytes is associated with increased DNA damage and altered cell cycle progression

Abstract

Abstract Melanoma is the fifth most common cancer in the United States that has less than a 25% 5-year survival rate once the cancer has metastasized beyond the lymph nodes. Recent exome studies of cutaneous melanoma have identified RAC1P29S as the third most prevalent hotspot mutation in 4-9% of sun-exposed melanoma. RAC1 alteration in cancer is correlated to poor prognosis, resistance to standard chemotherapy, and insensitivity to targeted inhibitors. As a member of the Ras superfamily of small GTPases, RAC1 functions as a master regulator of actin cytoskeleton dynamics and influences proliferation, cell migration, motility, and polarity. Although RAC1P29S mutations in melanoma and RAC1 alterations in several other cancers are becoming increasingly evident, the RAC1-driven biological mechanisms contributing to melanomagenesis remain elusive. Lack of rigorous signaling analysis has prevented identification of alternative therapeutic targets for RAC1P29S melanomas. To investigate the RAC1P29S-driven effect on downstream molecular signaling pathways, we generated an inducible RAC1P29S expression melanocytic cell line and performed RNA-sequencing (RNA-seq) coupled with multiplexed kinase inhibitor beads and mass spectrometry (MIBs/MS) to establish enriched pathways from the genomic to proteomic level. Our proteogenomic analysis has identified the G2-M cell cycle phase transition as a hyperactive pathway in RAC1P29S-expressing melanocytes. RAC1P29S expression promotes increased kinase activity of the PAK-PLK1-AURORA A axis and increased transition into M-phase as indicated by FACS analysis. Interestingly, RAC1P29S-expressing melanocytes also possess increased DNA damage via γH2AX foci, suggesting a compromise in G2-M checkpoint integrity and an altered DNA damage response. Here, we assess the influence of RAC1P29S on the PAK-PLK1-AURORA A axis and its role in promoting G2-M transition. In addition, we investigate the DNA damage response and cell cycle regulation in the presence of cytotoxic agents. Collectively, these results suggest novel therapeutic pathways to target in RAC1P29S-driven melanoma. Citation Format: Alexa C. Cannon, Jonathan Chernoff. RAC1P29S expression in melanocytes is associated with increased DNA damage and altered cell cycle progression [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 2587.