Tumor cell-intrinsic PD-1 promotes Merkel cell carcinoma growth by activating downstream mTOR-mitochondrial ROS signaling.

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer. Inhibitors targeting the programmed cell death 1 (PD-1) immune checkpoint have improved MCC patient outcomes by boosting antitumor T cell immunity. Here, we identify PD-1 as a growth-promoting receptor intrinsic to MCC cells. In human MCC lines and clinical tumors, RT-PCR-based sequencing, immunoblotting, flow cytometry, and immunofluorescence analyses demonstrated PD-1 gene and protein expression by MCC cells. MCC-PD-1 ligation enhanced, and its inhibition or silencing suppressed, in vitro proliferation and in vivo tumor xenograft growth. Consistently, MCC-PD-1 binding to PD-L1 or PD-L2 induced, while antibody-mediated PD-1 blockade inhibited, protumorigenic mTOR signaling, mitochondrial (mt) respiration, and ROS generation. Last, pharmacologic inhibition of mTOR or mtROS reversed MCC-PD-1:PD-L1-dependent proliferation and synergized with PD-1 checkpoint blockade in suppressing tumorigenesis. Our results identify an MCC-PD-1-mTOR-mtROS axis as a tumor growth-accelerating mechanism, the blockade of which might contribute to clinical response in patients with MCC.

Integrative analysis reveals therapeutic potential of pyrvinium pamoate in Merkel cell carcinoma.

Merkel Cell Carcinoma (MCC) is a highly aggressive neuroendocrine cutaneous malignancy arising from either ultraviolet-induced mutagenesis or Merkel cell polyomavirus (MCPyV) integration. It is the only known neuroendocrine tumor (NET) with a virus etiology. Despite extensive research, our understanding of the molecular mechanisms driving the transition from normal cells to MCC remains limited. To address this knowledge gap, we assessed the impact of inducible MCPyV T antigens into normal human fibroblasts by performing RNA sequencing. Our findings suggested that the WNT signaling pathway plays a critical role in the development of MCC. To test this model, we bioinformatically evaluated various perturbagens for their ability to reverse the MCC gene expression signature and identified pyrvinium pamoate, an FDA-approved anthelminthic drug known for its anti-tumor potential in multiple cancers. Leveraging transcriptomic, network, and molecular analyses, we found that pyrvinium effectively targets multiple MCC vulnerabilities. Specifically, pyrvinium not only reverses the neuroendocrine features of MCC by modulating canonical and non-canonical WNT signaling pathways but also inhibits cancer cell growth by activating the p53-mediated apoptosis pathway, disrupting mitochondrial function, and inducing endoplasmic reticulum (ER) stress. Pyrvinium also effectively inhibits tumor growth in an MCC mouse xenograft model. These findings offer new avenues for the development of therapeutic strategies for neuroendocrine cancer and highlight the utility of pyrvinium as a potential treatment for MCC.

Liquid biopsy epigenomic profiling for cancer subtyping.

Although circulating tumor DNA (ctDNA) assays are increasingly used to inform clinical decisions in cancer care, they have limited ability to identify the transcriptional programs that govern cancer phenotypes and their dynamic changes during the course of disease. To address these limitations, we developed a method for comprehensive epigenomic profiling of cancer from 1 ml of patient plasma. Using an immunoprecipitation-based approach targeting histone modifications and DNA methylation, we measured 1,268 epigenomic profiles in plasma from 433 individuals with one of 15 cancers. Our assay provided a robust proxy for transcriptional activity, allowing us to infer the expression levels of diagnostic markers and drug targets, measure the activity of therapeutically targetable transcription factors and detect epigenetic mechanisms of resistance. This proof-of-concept study in advanced cancers shows how plasma epigenomic profiling has the potential to unlock clinically actionable information that is currently accessible only via direct tissue sampling.

YAP1 and WWTR1 expression inversely correlates with neuroendocrine markers in Merkel cell carcinoma.

BackgroundMerkel 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, and the expression profile of MCC tumors is, to our knowledge, unexplored.MethodsHere, we leveraged bulk and single-cell RNA-Seq of patient-derived tumor biopsies and cell lines to explore the underlying transcriptional environment of MCC.ResultsStrikingly, 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 1 (WWTR1). This inverse correlation was broadly 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 TEA domain-dependent (TEAD-dependent) transcriptional repression of MCPyV LT.ConclusionThese findings identify what we believe to be a previously unrecognized heterogeneity in NE gene expression within MCC and support a model of YAP1/WWTR1 silencing as essential for the development of MCPyV-positive MCC.FundingUS Public Health Service grants R35CA232128, P01CA203655, and P30CA06516.

Reversal of viral and epigenetic HLA class I repression in Merkel cell carcinoma.

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.

Milademetan is a highly potent MDM2 inhibitor in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin with 2 etiologies. Merkel cell polyomavirus (MCPyV) integration is present in about 80% of all MCC. Virus-positive MCC (MCCP) tumors have few somatic mutations and usually express WT p53 (TP53). By contrast, virus-negative MCC (MCCN) tumors present with a high tumor mutational burden and predominantly UV mutational signature. MCCN tumors typically contain mutated TP53. MCCP tumors express 2 viral proteins: MCPyV small T antigen and a truncated form of large T antigen. MCPyV ST specifically activates expression of MDM2, an E3 ubiquitin ligase of p53, to inhibit p53-mediated tumor suppression. In this study, we assessed the efficacy of milademetan, a potent, selective, and orally available MDM2 inhibitor in several MCC models. Milademetan reduced cell viability of WT p53 MCC cell lines and triggered a rapid and sustained p53 response. Milademetan showed a dose-dependent inhibition of tumor growth in MKL-1 xenograft and patient-derived xenograft models. Here, along with preclinical data for the efficacy of milademetan in WT p53 MCC tumors, we report several in vitro and in vivo models useful for future MCC studies.

An international report on bacterial communities in esophageal squamous cell carcinoma.

The incidence of esophageal squamous cell carcinoma (ESCC) is disproportionately high in the eastern corridor of Africa and parts of Asia. Emerging research has identified a potential association between poor oral health and ESCC. One possible link between poor oral health and ESCC involves the alteration of the microbiome. We performed an integrated analysis of four independent sequencing efforts of ESCC tumors from patients from high- and low-incidence regions of the world. Using whole genome sequencing (WGS) and RNA sequencing (RNAseq) of ESCC tumors from 61 patients in Tanzania, we identified a community of bacteria, including members of the genera Fusobacterium, Selenomonas, Prevotella, Streptococcus, Porphyromonas, Veillonella and Campylobacter, present at high abundance in ESCC tumors. We then characterized the microbiome of 238 ESCC tumor specimens collected in two additional independent sequencing efforts consisting of patients from other high-ESCC incidence regions (Tanzania, Malawi, Kenya, Iran, China). This analysis revealed similar ESCC-associated bacterial communities in these cancers. Because these genera are traditionally considered members of the oral microbiota, we next explored whether there was a relationship between the synchronous saliva and tumor microbiomes of ESCC patients in Tanzania. Comparative analyses revealed that paired saliva and tumor microbiomes were significantly similar with a specific enrichment of Fusobacterium and Prevotella in the tumor microbiome. Together, these data indicate that cancer-associated oral bacteria are associated with ESCC tumors at the time of diagnosis and support a model in which oral bacteria are present in high abundance in both saliva and tumors of some ESCC patients.

Coordinating gene expression during the cell cycle.

Cell cycle-dependent gene transcription is tightly controlled by the retinoblastoma (RB):E2F and DREAM complexes, which repress all cell cycle genes during quiescence. Cyclin-dependent kinase (CDK) phosphorylation of RB and DREAM allows for the expression of two gene sets. The first set of genes, with peak expression in G1/S, is activated by E2F transcription factors (TFs) and is required for DNA synthesis. The second set, with maximum expression during G2/M, is required for mitosis and is coordinated by the MuvB complex, together with B-MYB and Forkhead box M1 (FOXM1). In this review, we summarize the key findings that established the distinct control mechanisms regulating G1/S and G2/M gene expression in mammals and discuss recent advances in the understanding of the temporal control of these genes.

Disrupting the DREAM complex enables proliferation of adult human pancreatic ß cells.

Resistance to regeneration of insulin-producing pancreatic ß cells is a fundamental challenge for type 1 and type 2 diabetes. Recently, small molecule inhibitors of the kinase DYRK1A have proven effective in inducing adult human ß cells to proliferate, but their detailed mechanism of action is incompletely understood. We interrogated our human insulinoma and ß cell transcriptomic databases seeking to understand why ß cells in insulinomas proliferate, while normal ß cells do not. This search reveals the DREAM complex as a central regulator of quiescence in human ß cells. The DREAM complex consists of a module of transcriptionally repressive proteins that assemble in response to DYRK1A kinase activity, thereby inducing and maintaining cellular quiescence. In the absence of DYRK1A, DREAM subunits reassemble into the pro-proliferative MMB complex. Here, we demonstrate that small molecule DYRK1A inhibitors induce human ß cells to replicate by converting the repressive DREAM complex to its pro-proliferative MMB conformation.

Addiction of Merkel cell carcinoma to MUC1-C identifies a potential new target for treatment.

Merkel cell carcinoma (MCC) is an aggressive malignancy with neuroendocrine (NE) features, limited treatment options, and a lack of druggable targets. There is no reported involvement of the MUC1-C oncogenic protein in MCC progression. We show here that MUC1-C is broadly expressed in MCCs and at higher levels in Merkel cell polyomavirus (MCPyV)-positive (MCCP) relative to MCPyV-negative (MCCN) tumors. Our results further demonstrate that MUC1-C is expressed in MCCP, as well as MCCN, cell lines and regulates common sets of signaling pathways related to RNA synthesis, processing, and transport in both subtypes. Mechanistically, MUC1-C (i) interacts with MYCL, which drives MCC progression, (ii) is necessary for expression of the OCT4, SOX2, KLF4, MYC, and NANOG pluripotency factors, and (iii) induces the NEUROD1, BRN2 and ATOH1 NE lineage dictating transcription factors. We show that MUC1-C is also necessary for MCCP and MCCN cell survival by suppressing DNA replication stress, the p53 pathway, and apoptosis. In concert with these results, targeting MUC1-C genetically and pharmacologically inhibits MCC self-renewal capacity and tumorigenicity. These findings demonstrate that MCCP and MCCN cells are addicted to MUC1-C and identify MUC1-C as a potential target for MCC treatment.

Merkel cell polyomavirus large T antigen binding to pRb promotes skin hyperplasia and tumor development.

Clear evidence supports a causal link between Merkel cell polyomavirus (MCPyV) and the highly aggressive human skin cancer called Merkel cell carcinoma (MCC). Integration of viral DNA into the human genome facilitates continued expression of the MCPyV small tumor (ST) and large tumor (LT) antigens in virus-positive MCCs. In MCC tumors, MCPyV LT is truncated in a manner that renders the virus unable to replicate yet preserves the LXCXE motif that facilitates its binding to and inactivation of the retinoblastoma tumor suppressor protein (pRb). We previously developed a MCPyV transgenic mouse model in which MCC tumor-derived ST and truncated LT expression were targeted to the stratified epithelium of the skin, causing epithelial hyperplasia, increased proliferation, and spontaneous tumorigenesis. We sought to determine if any of these phenotypes required the association between the truncated MCPyV LT and pRb. Mice were generated in which K14-driven MCPyV ST/LT were expressed in the context of a homozygous RbΔLXCXE knock-in allele that attenuates LT-pRb interactions through LT's LXCXE motif. We found that many of the phenotypes including tumorigenesis that develop in the K14-driven MCPyV transgenic mice were dependent upon LT's LXCXE-dependent interaction with pRb. These findings highlight the importance of the MCPyV LT-pRb interaction in an in vivo model for MCPyV-induced tumorigenesis.

CDC7-independent G1/S transition revealed by targeted protein degradation.

The entry of mammalian cells into the DNA synthesis phase (S phase) represents a key event in cell division1. According to current models of the cell cycle, the kinase CDC7 constitutes an essential and rate-limiting trigger of DNA replication, acting together with the cyclin-dependent kinase CDK2. Here we show that CDC7 is dispensable for cell division of many different cell types, as determined using chemical genetic systems that enable acute shutdown of CDC7 in cultured cells and in live mice. We demonstrate that another cell cycle kinase, CDK1, is also active during G1/S transition both in cycling cells and in cells exiting quiescence. We show that CDC7 and CDK1 perform functionally redundant roles during G1/S transition, and at least one of these kinases must be present to allow S-phase entry. These observations revise our understanding of cell cycle progression by demonstrating that CDK1 physiologically regulates two distinct transitions during cell division cycle, whereas CDC7 has a redundant function in DNA replication.

Long-read sequencing reveals complex patterns of wraparound transcription in polyomaviruses.

Polyomaviruses (PyV) are ubiquitous pathogens that can cause devastating human diseases. Due to the small size of their genomes, PyV utilize complex patterns of RNA splicing to maximize their coding capacity. Despite the importance of PyV to human disease, their transcriptome architecture is poorly characterized. Here, we compare short- and long-read RNA sequencing data from eight human and non-human PyV. We provide a detailed transcriptome atlas for BK polyomavirus (BKPyV), an important human pathogen, and the prototype PyV, simian virus 40 (SV40). We identify pervasive wraparound transcription in PyV, wherein transcription runs through the polyA site and circles the genome multiple times. Comparative analyses identify novel, conserved transcripts that increase PyV coding capacity. One of these conserved transcripts encodes superT, a T antigen containing two RB-binding LxCxE motifs. We find that superT-encoding transcripts are abundant in PyV-associated human cancers. Together, we show that comparative transcriptomic approaches can greatly expand known transcript and coding capacity in one of the simplest and most well-studied viral families.

TargetGeneReg 2.0: a comprehensive web-atlas for p53, p63, and cell cycle-dependent gene regulation.

In recent years, our web-atlas at www.TargetGeneReg.org has enabled many researchers to uncover new biological insights and to identify novel regulatory mechanisms that affect p53 and the cell cycle - signaling pathways that are frequently dysregulated in diseases like cancer. Here, we provide a substantial upgrade of the database that comprises an extension to include non-coding genes and the transcription factors ΔNp63 and RFX7. TargetGeneReg 2.0 combines gene expression profiling and transcription factor DNA binding data to determine, for each gene, the response to p53, ΔNp63, and cell cycle signaling. It can be used to dissect common, cell type and treatment-specific effects, identify the most promising candidates, and validate findings. We demonstrate the increased power and more intuitive layout of the resource using realistic examples.

Merkel Cell Carcinoma Sensitivity to EZH2 Inhibition Is Mediated by SIX1 Derepression.

Polycomb repressive complex 2 has a critical role in the maintenance of bivalent promoters and is often perturbed in cancer, including neuroendocrine tumors. In this study, we investigated the susceptibility of Merkel cell carcinoma (MCC), a neuroendocrine carcinoma of the skin, to inhibitors of the Polycomb repressive complex 2 catalytic subunit EZH2. We show that a subset of MCC cell lines is sensitive to EZH2 inhibitor-induced cell viability loss. We find that inhibitor treatment of susceptible cells derepresses the Polycomb repressive complex 2 target SIX1, a transcription factor in the PAX-SIX-EYA-DACH network normally involved in inner ear hair cell development, and that PAX-SIX-EYA-DACH network transcription factors are critical contributors to EZH2 inhibitor-induced MCC cell viability loss. Furthermore, we show the EZH2 inhibitor tazemetostat slows the growth of MCC xenografts and derepresses SIX1 and its downstream inner ear transcriptional target MYO6 in vivo. We propose that EZH2 inhibition in MCC leads to SIX1 derepression with dysregulation of hearing-related transcriptional programs and growth inhibition. This study provides evidence that MCC tumors may be specifically susceptible to EZH2 inhibitors, while giving mechanistic insight into the transcriptional programs these inhibitors perturb in MCC, and potentially in other neuroendocrine cancers.

Simultaneous expression of MMB-FOXM1 complex components enables efficient bypass of senescence.

Cellular senescence is a stable cell cycle arrest that normal cells undergo after a finite number of divisions, in response to a variety of intrinsic and extrinsic stimuli. Although senescence is largely established and maintained by the p53/p21WAF1/CIP1 and pRB/p16INK4A tumour suppressor pathways, the downstream targets responsible for the stability of the growth arrest are not known. We have employed a stable senescence bypass assay in conditionally immortalised human breast fibroblasts (CL3EcoR) to investigate the role of the DREAM complex and its associated components in senescence. DREAM is a multi-subunit complex comprised of the MuvB core, containing LIN9, LIN37, LIN52, LIN54, and RBBP4, that when bound to p130, an RB1 like protein, and E2F4 inhibits cell cycle-dependent gene expression thereby arresting cell division. Phosphorylation of LIN52 at Serine 28 is required for DREAM assembly. Re-entry into the cell cycle upon phosphorylation of p130 leads to disruption of the DREAM complex and the MuvB core, associating initially to B-MYB and later to FOXM1 to form MMB and MMB-FOXM1 complexes respectively. Here we report that simultaneous expression of MMB-FOXM1 complex components efficiently bypasses senescence with LIN52, B-MYB, and FOXM1 as the crucial components. Moreover, bypass of senescence requires non-phosphorylated LIN52 that disrupts the DREAM complex, thereby indicating a central role for assembly of the DREAM complex in senescence.