ABSTRACT
Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high mortality rate. MC polyomavirus (MCPyV) causes 80% of MCCs, encoding the viral oncogenes small T (sT) and truncated large T antigens (tLT). These proteins impair the Rb1-dependent G1/S checkpoint blockade and subvert the host cell epigenome to promote cancer. Whole proteome analysis and proximal interactomics identified a tLT-dependent deregulation of DNA damage response (DDR). Our investigation revealed a previously unreported interaction between tLT and the histone methyltransferase EHMT2, to our knowledge. T Antigens knockdown reduced DDR protein levels and increased levels of the DNA damage marker γH2Ax. EHMT2 normally promotes H3K9 methylation and DDR signaling. Given that inhibition of EHMT2 did not significantly change the MCC cells proteome, tLT-EHMT2 interaction could affect the DDR. With tLT, we report that EHMT2 gained DNA damage repair proximal interactors. EHMT2 inhibition rescued proliferation in MCC cells depleted for their T antigens, suggesting impaired DDR and/or lack of checkpoint efficiency. Combined tLT and EHMT2 inhibition led to altered DDR, evidenced by multiple signaling alterations. Here we show that tLT hijacks multiple components of the DNA damage machinery to enhance tolerance to DNA damage in MCC cells, which could explain the genetic stability of these cancers.
ABSTRACT
Merkel cell carcinoma (MCC) is an aggressive skin cancer for which Merkel cell polyomavirus integration and expression of viral oncogenes small T and Large T have been identified as major oncogenic determinants. Recently, a component of the PRC2 complex, the histone methyltransferase enhancer of zeste homolog 2 (EZH2) that induces H3K27 trimethylation as a repressive mark has been proposed as a potential therapeutic target in MCC. Because divergent results have been reported for the levels of EZH2 and trimethylation of lysine 27 on histone 3, we analyzed these factors in a large MCC cohort to identify the molecular determinants of EZH2 activity in MCC and to establish MCC cell lines' sensitivity to EZH2 inhibitors. Immunohistochemical expression of EZH2 was observed in 92% of MCC tumors (156 of 170), with higher expression levels in virus-positive than virus-negative tumors (P = 0.026). For the latter, we showed overexpression of EZHIP, a negative regulator of the PRC2 complex. In vitro, ectopic expression of the large T antigen in fibroblasts led to the induction of EZH2 expression, whereas the knockdown of T antigens in MCC cell lines resulted in decreased EZH2 expression. EZH2 inhibition led to selective cytotoxicity on virus-positive MCC cell lines. This study highlights the distinct mechanisms of EZH2 induction between virus-negative and -positive MCC.
