Tumor viruses are reported to cause 20% of human cancer world-wide. Small DNA tumor viruses including polyomaviruses have provided advantages for oncologists to focus on a few viral proteins to dissect gene regulatory networks in cancer. In particular, Merkel cell polyomavirus (MCV) causes 80% of Merkel cell carcinoma (MCC). Given the oncogenic potential of this virus that is widespread in the general population, it is important for us to understand the functions of MCV viral proteins and their downstream targets that may serve as therapeutic targets for MCC.
We recently determined that the MCV ST antigen forms a stable complex with MYCL and the Tip60-p400 complex to specifically activate a group of genes including PRMT5. As a positive feedback loop, the methyltransferase activity of PRMT5 is required for full activity of Tip60. PRMT5 methylates splicing factors to promote exon inclusion of critical cellular factors including Tip60. Full length Tip60 alpha-alpha?splicing isoform is required for histone H4 acetylation that mediates ejection of 53BP1 from sites of DNA damage and promotes homologous recombination over error-prone non-homologous end joining (NHEJ). From CRISPR screens, we identified that knockout of writers and readers of m6A RNA methylation may confer MCC cells resistance to PRMT5 inhibitors. We hypothesize that PRMT5 methylates serine/arginine-rich splicing factors (SRSFs) while m6A RNA methylation recruits SRSF proteins complexed with m6A readers, therefore PRMT5 and m6A coordinate to promote proper splicing of transcripts associated with cancer cell proliferation.
We will perform knockdown and rescue experiments to confirm that the PRMT5-Tip60 alpha-alpha-H4K16ac axis promotes homologous recombination over NHEJ in MCC, which will lay the groundwork for combination therapies using PRMT5 inhibitors with DNA damaging agents. We will identify the correlation between PRMT5-promoted exon inclusion and m6A-mediated alternative splicing, which will help us further understand the essential role of PRMT5 in normal physiology and dose limiting of PRMT5 inhibitors in a therapeutic setting. We will perform SDMA immunoprecipitation followed by Mass Spectrometry to systematically identify PRMT5 substrates besides splicing factors that may contribute to PRMT5-promoted dedifferentiation of MCC. Completion of the proposed aims will help develop targeted therapeutics for highly aggressive MCC.
