Merkel cell polyomavirus recruits MYCL to the EP400 complex to promote oncogenesis

Abstract

Merkel cell carcinoma (MCC) frequently contains integrated copies of Merkel cell polyomavirus DNA that express a truncated form of Large T antigen (LT) and an intact Small T antigen (ST). While LT binds RB and inactivates its tumor suppressor function, it is less clear how ST contributes to MCC tumorigenesis. Here we show that ST binds specifically to the MYC homolog MYCL (L-MYC) and recruits it to the 15-component EP400 histone acetyltransferase and chromatin remodeling complex. We performed a large-scale immunoprecipitation for ST and identified co-precipitating proteins by mass spectrometry. In addition to protein phosphatase 2A (PP2A) subunits, we identified MYCL and its heterodimeric partner MAX plus the EP400 complex. Immunoprecipitation for MAX and EP400 complex components confirmed their association with ST. We determined that the ST-MYCL-EP400 complex binds together to specific gene promoters and activates their expression by integrating chromatin immunoprecipitation with sequencing (ChIP-seq) and RNA-seq. MYCL and EP400 were required for maintenance of cell viability and cooperated with ST to promote gene expression in MCC cell lines. A genome-wide CRISPR-Cas9 screen confirmed the requirement for MYCL and EP400 in MCPyV-positive MCC cell lines. We demonstrate that ST can activate gene expression in a EP400 and MYCL dependent manner and this activity contributes to cellular transformation and generation of induced pluripotent stem cells.

Characterization of six Merkel cell polyomavirus‐positive Merkel cell carcinoma cell lines: Integration pattern suggest that large T antigen truncating events occur before or during integration

“…Thus, we analyzed LT expression and its size by immunoblotting for all nine MCC cell lines, i.e., our six cell lines as well as MKL‐1, MKL‐2 and MS‐1. This analysis was performed with a monoclonal antibody which recognizes the common protein sequence of sT and LT (clone AB5;). All cell lines demonstrated expression of truncated LT (tLT) (Fig.…”

“…n.d.: not done. 10 Indicates whether complete amplification of integrated MCPyV was possible. 11 Accession numbers for the cells lines established by us were uploaded to NCBI.…”

“…Meanwhile, many studies confirmed the presence of Merkel cell polyomavirus (MCPyV) in about 75% of MCC cases, and given the high seropositivity observed in the general population MCPyV can trigger cancer in its natural host. Besides the monoclonal integration of the MCPyV genome into the cellular genomes, several other observations established MCPyV as etiological factor for MCC: ( i ) Expression of viral T antigen (TA) proteins, i.e., small and large T (sT and LT), in MCC tumors and MCPyV‐positive MCC cell lines depend on TA expression for their growth; ( ii ) MCPyV‐positive tumors harbor LT sequences encoding truncated proteins in which the RB binding domain is preserved but the DNA binding, helicase and cell growth inhibitory domains are lost resulting in a protein able to induce cellular proliferation but incapable of promoting virus replication; ( iii ) high levels of MCPyV vesicular protein 1 antibodies in patients sera are associated with MCC; ( iv ) sT has transforming activity both in vitro and in vivo and ( v ) MCPyV‐associated cancers are characterized by a low mutational load without any recurring mutations . In contrast, MCPyV‐negative MCC are associated with an exceptional high mutational burden displaying an ultraviolet (UV)‐signature .…”

STRIPAK directs PP2A activity toward MAP4K4 to promote oncogenic transformation

“…Notably, mutations in Aa occur at high frequency in premalignant endometrial lesions (Anglesio et al 2017). PP2A is also a target of the Small T antigens (ST) of SV40 and other polyomaviruses including the human oncogenic Merkel cell polyomavirus (Pallas et al 1990, Chen et al 2004, Cheng et al 2017) and this interaction contributes to cell transformation (Hahn et al 2002). Structural studies have shown that ST disrupts formation of a functional PP2A holoenzyme by displacing or hindering B subunit access to the PP2A core-enzyme (Chen et al 2007, Cho et al 2007).…”

Molecular and immune targets for Merkel cell carcinoma therapy and prevention

“…Current understanding suggests that LT and sT are the major oncoproteins mediating MCPyV‐driven tumorigenesis. Truncated MCPyV‐LT binds and inactivates pRB, while sT stabilizes and augments the activity of key oncoproteins including MCPyV‐LT and MYCL, among others . Further clarification of the mechanisms by which these viral oncoproteins promote tumorigenesis will suggest future targets for therapy.…”