Merkel cell polyomavirus pan-T antigen knockdown reduces cancer cell stemness and promotes neural differentiation independent of RB1

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer associated with integration of Merkel cell polyomavirus (MCPyV). MCPyV-encoded T-antigens (TAs) are pivotal for sustaining MCC's oncogenic phenotype, such as repression of TAs resulting in reactivation of the RB pathway and subsequent cell cycle arrest. Notably, the MCC cell line LoKe, characterized by a homozygous loss of the RB1 gene, exhibits uninterrupted cell cycle progression after shRNA-mediated TA repression. This unique feature allows an in-depth analysis of the effects of TAs beyond the inhibition of the RB pathway. Upon inducible shRNA-mediated TA knockdown, we utilized single-cell RNA sequencing (scRNA-seq) to delve into the molecular landscape, which revealed a decrease in the expression of genes associated with stemness. Gene regulatory network analysis pinpointed members of the E2F family (E2F1, E2F8, TFDP1) as crucial transcriptional regulators maintaining stem cell properties. Similarly, minichromosome maintenance (MCM) genes encoding DNA-binding license proteins essential for stem cell maintenance, were found to be repressed upon TA knockdown. Interestingly, reduction in stemness coincided with neural differentiation, as evidenced by the upregulation of neurogenesis-related genes (neurexins, BTG2, MYT1L likely due to increased expression of PBX1 and BPTF, key regulators of neurogenesis pathways. To validate these findings, we reanalyzed scRNA-seq data for CVG-1 cells and proteomics data for WaGa cells after TA knockdown, confirming consistent expression patterns of key regulators, including TFDP1 and MCM2/MCM6. In summary, TAs are critical for maintaining the stemness of MCC cells and suppressing neural differentiation, irrespective of their impact on the RB-signaling pathway.