Understanding the requirement of PRMT5 splicing regulation in Merkel cell carcinoma.

Abstract

Merkel cell carcinoma (MCC) is an aggressive, highly metastatic neuroendocrine skin cancer. Immune checkpoint therapies have greatly improved the overall survival rate of patients with late-stage disease, however patients with resistant tumors have very limited alternative treatment options. In this dissertation, we investigate the molecular mechanisms of MCC growth and survival to identify new avenues of targeted therapeutics for MCC. Previous efforts to characterize the molecular features of MCC have identified two distinct subtypes: polyomavirus-associated MCC (MCCP) and non-viral MCC (MCCN). MCCP is caused from integrated Merkel cell polyomavirus (MCV) DNA while MCCN tumors are caused from UV damage. Recent work showed that MCV small tumor antigen (ST) regulates transcription by binding with the proto-oncogene L-MYC and the TIP60/P400 chromatin remodeling complex (the SLaP complex). In this dissertation, we extend these findings by showing that multiple TIP60 isoforms bind to the SLaP complex and that chromatin binding of SLaP is correlated with H2A.Z acetylation, a histone variant mark associated with gene activation. These findings provide further support for a model where ST recruits the TIP60/P400 complex to activate specific gene expression via chromatin remodeling at the transcriptional start site of L-MYC target genes. Next, we show that PRMT5, a transcriptional target of the SLaP complex, is required for growth of MCC cells. PRMT5 is an arginine methyltransferase with diverse cellular targets, including the well-described tumor suppressor p53. We found that PRMT5 loss induces p53 activity, cell cycle arrest, and apoptosis; however, we show that p53 activity is not required for cell viability loss. Using unbiased approaches, we identify mRNA processing as a critical pathway dysregulated by PRMT5 inhibition in MCC. We show that PRMT5 is required for splicing of detained introns in cell cycle and DNA damage genes. We further investigate the role of PRMT5 in regulating the DNA damage response and find that PRMT5 is required for the ATR/CHK1 response and genome stability. Overall, we take a molecular approach to identify PRMT5 as a potential therapeutic target for MCC and characterize the role of PRMT5 in regulating both mRNA splicing and the response to DNA damage.