Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I–low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.

BACKGROUND. Merkel cell carcinoma (MCC) is an aggressive neuroendocrine (NE) skin cancer caused by severe UV-induced mutations or expression of Merkel cell polyomavirus (MCPyV) large and small T antigens (LT and ST). Despite deep genetic differences between MCPyV-positive and -negative subtypes, current clinical diagnostic markers are indistinguishable between subtypes and the expression profile of MCC tumors is unexplored. METHODS. Here we leveraged bulk and single-cell RNA sequencing of patient-derived tumor biopsies and cell lines to explore the underlying transcriptional diversity of MCC. RESULTS. Strikingly, MCC samples could be separated into transcriptional subtypes that were independent of MCPyV status. Instead, we observed an inverse correlation between a NE gene signature and the Hippo pathway transcription factors Yes1-associated transcriptional regulator (YAP1) and WW domain containing transcriptional regulator (WWTR1). This inverse correlation was present at the transcript and protein levels in the tumor biopsies as well as in established and patient-derived cell lines. Mechanistically, expression of YAP1 or WWTR1 in a MCPyV-positive MCC cell line induced cell-cycle arrest at least in part through TEAD-dependent transcriptional repression of MCPyV LT. CONCLUSION. These findings describe previously unrecognized heterogeneity in NE gene expression within MCC and support the model that YAP1/WWTR1 silencing is essential for the development of MCPyV-positive MCC. FUNDING. US Public Health Service grants R35CA232128, P01CA203655, and P30CA06516.

Background: We conducted a national antimicrobial surveillance study of both gram-positive and gram-negative organisms isolated from hospitalized patients. This report presents data on antimicrobial susceptibility among 4998 organisms collected in China between 2012 and 2014. Method: The minimum inhibitory concentrations (MICs) and susceptibilities of ceftaroline/avibactam (CPA), ceftazidime/avibactam (CZA) and a range of comparative agents were determined according to guidelines established by the Clinical and Laboratory Standards Institute (CLSI). Results: The highest overall susceptibility levels for all Enterobacteriaceae during the study period were observed for CPA, CZA, doripenem (DOR), meropenem (MEM), and amikacin (AMK), which were all >90%. However, both CPT and CAZ alone and in combination with avibactam showed low activities for Acinetobacter spp., whereas CPA and CZA exhibited MIC90 values for Pseudomonas aeruginosa that were reduced by 4- and 8-fold, respectively, compared with those of CPT and CAZ. High susceptibilities of Acinetobacter spp. and P. aeruginosa to colistin and P. aeruginosa to AMK were observed. For the gram-positive strains, no significant activity changes were seen for Enterococcus, Staphylococcus, and viridans group streptococci to CPT or CAZ alone or in combination with avibactam, whereas Streptococcus pneumoniae and β-hemolytic Streptococcus showed almost 100% susceptibility to both CPT and CPA. Conclusion: The addition of 4 mg/L avibactam greatly increased the activities of CPT and CAZ against most Enterobacteriaceae and P. aeruginosa isolates, whereas no significant changes were observed in Acinetobacter spp. or any of the gram-positive strains.

Merkel cell polyomavirus (MCPyV) causes the majority of human Merkel cell carcinomas (MCC), a rare but highly aggressive form of skin cancer. We recently reported that constitutive expression of MCC tumor-derived MCPyV tumor (T) antigens in the skin of transgenic mice leads to hyperplasia, increased proliferation, and spontaneous epithelial tumor development. We sought to evaluate how the MCPyV T antigens contribute to tumor formation in vivo using a classical, multi-stage model for squamous cell carcinoma development. In this model, two chemical carcinogens, DMBA and TPA, contribute to two distinct phases of carcinogenesis—initiation and promotion, respectively—that are required for tumors to develop. By treating the MCPyV transgenic mice with each chemical carcinogen, we determined how the viral oncogenes contributed to carcinogenesis. We observed that the MCPyV T antigens synergized with the tumor initiator DMBA, but not with the tumor promoter TPA, cause tumors. Therefore, the MCPyV tumor antigens function primarily as tumor promoters, similar to that seen with human papillomavirus (HPV) oncoproteins. These studies provide insight into the role of MCPyV T antigen expression in tumor formation in vivo and contribute to our understanding of how MCPyV may function as a human DNA tumor virus.

Protein arginine methyltransferase 5 (PRMT5) is a key regulator of gene expression and RNA splicing, with therapeutic potential demonstrated in MTAP-deleted cancers. Emerging evidence suggests that MYC -driven tumors or tumors with wild-type TP53 may also be sensitive to PRMT5 inhibition, though the underlying mechanisms remain unclear. Virus-positive Merkel cell carcinoma serves as an ideal model to explore this, as it is driven by the MYC paralog MYCL and retains wild-type TP53 . In this study, we examined how PRMT5 regulates the Tip60-EP400 complex, which is recruited by MYCL in Merkel cell carcinoma. Using RNA-seq, we characterized PRMT5-mediated gene expression, whereas Iso-Seq enabled in-depth analysis of PRMT5-mediated alternative splicing. How PRMT5 deficiency selectively affects certain splice sites remains unresolved. Our findings suggest that PRMT5-mediated modification of SRSF1 (serine/arginine-rich splicing factor 1) enhances its recruitment to m6A-modified RNA, ensuring proper KAT5 (Tip60) splicing and Tip60-EP400 activity. PRMT5 inhibition disrupts this recruitment, leading to widespread splicing defects, including exon skipping and intron retention. These results provide new insights into PRMT5’s role in splicing regulation and may have broader implications for targeting splicing dysregulation in MYC -driven cancers.

PRMT5 catalyzes symmetric dimethyl arginine on numerous proteins, with PRMT5 inhibitors (PRMT5i) inducing anti-proliferative effects in preclinical studies. Several models have been proposed to explain sensitivity to PRMT5i including through p53 activation and DNA damage response (DDR) regulation. Here, we interrogate the mechanisms of PRMT5i sensitivity in Merkel cell carcinoma, a neuroendocrine skin cancer sensitive to p53 activation. To identify critical pathways altered by PRMT5i, we performed CRISPR/Cas9 screening, proteomic, and transcriptomic analyses. Our results indicate that PRMT5i sensitivity is independent of p53 activation and is characterized by an increase in detained introns (DIs) and dependency on mRNA processing factors. Sensitivity correlated with elevated basal DI levels and was associated with replication-associated DNA damage accompanied by impaired ATR/CHK1 signaling. These findings are consistent with a threshold model of sensitivity in which excessive DI accumulation is associated with replication-associated DNA damage and apoptosis following PRMT5 inhibition.