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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.

Entry of mammalian cells into DNA synthesis (S-phase) represents a key event in cell division(1). According to the current cell-cycle models, the Cdc7 kinase constitutes an essential and rate-limiting trigger of DNA replication, acting together with cyclin-dependent kinase Cdk2. Here we show, using chemical-genetic systems which allow an acute shutdown of Cdc7 in in vitro cultured cells as well as in vivo in a living mouse, that Cdc7 is dispensable for cell division of many different cell types. 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 play 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, while Cdc7 plays a redundant function in DNA replication.

Figure S4

Supplementary Figure 1 from PARC and CUL7 Form Atypical Cullin RING Ligase Complexes

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PDF file - 4178K, Supplemental Figure 3. Imatinib treatment does not induce a senescence phenotype.

Supplementary Figure Legends. Legends for Supplementary Figures S1-S2.

PDF file - 1731K, Supplemental Figure 1. SKP2 expression levels do not predict p27Kip1 levels after imatinib therapy.

PDF file - 3386K, Supplemental Figure 2. GIST cell growth completely recovers after imatinib washout.

PDF file - 1110K, Supplemental Figure 4. Dual siRNA transfection of E2F4/LIN54 leads to an increased apoptotic response after imatinib while single knock-down of DREAM complex subunits does not.

Supplementary Figure S1. Histology and MCM7 expression in the stratified epithelium of the whisker pad, footpad, and dorsal skin of ROSA26-LSL-MCPyV168 and K14Cre-MCPyV168 neonatal and adult tissues.

CUL7 and the p53-associated, PARkin-like cytoplasmic protein (PARC) were previously reported to form homodimers and heterodimers, the first demonstration of cullin dimerization. Although a CUL7-based SKP1/CUL1/F-box (SCF)–like complex has been observed, little is known about the existence of a PARC-based SCF-like complex and how PARC interacts with CUL7-based complexes. To further characterize PARC-containing complexes, we examined the ability of PARC to form an SCF-like complex. PARC binds RBX1 and is covalently modified by NEDD8, defining PARC as a true cullin. However, PARC fails to bind SKP1 or F-box proteins, including the CUL7-associated FBXW8. To examine the assembly of PARC- and CUL7-containing complexes, tandem affinity purification followed by multidimensional protein identification technology were used. Multidimensional protein identification technology analysis revealed that the CUL7 interaction with FBXW8 was mutually exclusive of CUL7 binding to PARC or p53. Notably, although heterodimers of CUL7 and PARC bind p53, p53 is not required for the dimerization of CUL7 and PARC. The observed physical separation of FBXW8 and PARC is supported functionally by the generation of Parc−/−, Fbxw8−/− mice, which do not show exacerbation of the Fbxw8−/− phenotype. Finally, all of the PARC and CUL7 subcomplexes examined exhibit E3 ubiquitin ligase activity in vitro. Together, these findings indicate that the intricate assembly of PARC- and CUL7-containing complexes is highly regulated, and multiple subcomplexes may exhibit ubiquitin ligase activity. [Cancer Res 2007;67(5):2006–14]

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.

Background The use of targeted therapy remains a treatment consideration for some patients with advanced Merkel cell carcinoma (MCC). However, supportive data on the use of targeted therapy approaches are limited. Thus, we sought to evaluate the responsiveness of targeted agents in patients with advanced MCC. Methods An institutional MCC database identified patients who were treated with targeted therapy. For the purpose of this study, targeted therapy was defined as any multi‐targeted tyrosine kinase inhibitor or inhibitor of the PI3K‐pathway. Clinical benefit was defined as complete response, partial response, or stable disease (SD) ≥6 months. A subset of patient samples underwent next‐generation sequencing (NGS), Merkel cell polyomavirus testing, and PD‐L1/PD‐1 expression testing. Results Nineteen patients with MCC treated with targeted therapy were identified, 21 targeted therapy regimens were evaluable for response in 18 patients. Four of twenty‐one (19%) of evaluable regimens were associated with clinical benefit with the best overall response of SD. The durations of SD were 13.6 months (59 weeks), 9.7 months (42 weeks), 7.6 months (33 weeks), and 7.2 months (31 weeks). Of the four patients who derived clinical benefit, three were treated with pazopanib alone and one was treated with pazopanib plus everolimus. No difference in the rate of clinical benefit between molecular disease subtypes was detected nor was associated with any specific genomic alteration. Conclusion In our series, targeted agents elicited a disease control rate of 19% in patients with advanced MCC, with a best overall response of SD. Pazopanib alone or in combination exhibited a rate of disease control of 36% (4 of 11 with SD ≥6 months)., The use of targeted therapy remains a treatment consideration for some patients with advanced Merkel cell carcinoma (MCC); however, supportive data are limited. In this retrospective analysis, we find that treatment with pazopanib led to clinical benefit in 4/11 (36%) of patients with heavily pretreated advanced MCC. Outside of pazopanib, we were unable to find anecdotal evidence of any meaningful clinical activity for several other kinase inhibitors, although the use was limited to small patient numbers for each.

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 a highly aggressive neuroendocrine cutaneous malignancy arising from either Ultraviolet-induced mutagenesis or Merkel cell polyomavirus (MCPyV) integration. Pyrvinium Pamoate (PP), an FDA-approved anthelminthic drug, has been reported to exhibit anti-tumor abilities in multiple cancers. By integrating transcriptomic, network, and molecular analyses, we discovered that PP is broadly effective against Merkel cell carcinoma cell lines, and that PP targets multiple proposed MCC tumorigenesis pathways. Methods: We performed time-series RNA-seq profiling on a MCPyV inducible cell model and on PP-treated MCC cell lines, and inferred gene regulatory networks associated with PP response. We then integrated the network models with publicly available MCC tumor profiles and the LINCS L1000 database to explore the targets of PP in MCC. In addition, we performed protein, RNA, growth rate, metabolic, and apoptosis assays on multiple MCC cell lines exposed to PP. Results: Analyzing the effect of PP in the L1000 cancer cell line panel suggested that PP could inhibit key drivers of MCC tumorigenesis. We therefore tested the anti-proliferation and pro-apoptosis effects of PP in MCC cell lines. PP effectively inhibits proliferation of MCC at concentrations as low as 100 nM and in a dose and time-dependent manner. RNA-seq analysis of PP treated MCC cells suggested that PP triggers the p53-mediated intrinsic apoptotic signaling pathway, impairs the mitochondria’s oxidative phosphorylation function and induces endoplasmic reticulum (ER) stress. Immunoblotting intrinsic apoptotic pathway protein markers and measuring the IC50 of PP and the MDM2 inhibitor Nutlin-3a in p53WT and p53mut or p53−/− MCC cell lines, we found that PP does not induce cell apoptosis only through p53 activation, but also through other mechanisms such as increasing Unfolded Protein Response (UPR) stress and decreasing expression of the anti-apoptotic protein survivin (BIRC5). Extracellular flux analysis revealed decreased oxidative phosphorylation rate in PP treated MCC cells. Western blot analysis suggests that PP directly inhibits mitochondria complexes genes ATP5A, MTCO1 and NDUF58 and induces UPR-ER stress. PP has also been reported to inhibit the canonical Wnt pathway. We found that WNT5B, the ligand for the non-canonical Wnt pathway, was significantly decreased in MCC tumors and in our MCPyV inducible cell model, and that PP can upregulate WNT5B significantly. MCC cells treated with WNT5B recombinant protein showed a retarded MCC phenotype. Conclusion: Our results suggest that PP exerts anti-tumor activity through both non-specific growth inhibition - via the modulation of mitochondrial function, ER stress, and apoptotic signaling - and more specifically by perturbing the non-canonical Wnt pathway. Citation Format: Jiawen Yang, James T. Lim, Paul Victor, Rick G. Schnellmann, James A. DeCaprio, Megha Padi. Multi-omics analysis of mechanisms underlying sensitivity of Merkel cell carcinoma to pyrvinium pamoate. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4882.

Background SARS-CoV-2, a positive-sense RNA virus in the family Coronaviridae, has caused a worldwide pandemic of coronavirus disease 2019 or COVID-19. Coronaviruses generate a tiered series of subgenomic RNAs (sgRNAs) through a process involving homology between transcriptional regulatory sequences (TRS) located after the leader sequence in the 5′ UTR (the TRS-L) and TRS located near the start of ORFs encoding structural and accessory proteins (TRS-B) near the 3′ end of the genome. In addition to the canonical sgRNAs generated by SARS-CoV-2, non-canonical sgRNAs (nc-sgRNAs) have been reported. However, the consistency of these nc-sgRNAs across viral isolates and infection conditions is unknown. The comprehensive definition of SARS-CoV-2 RNA products is a key step in understanding SARS-CoV-2 pathogenesis. Methods Here, we report an integrative analysis of eight independent SARS-CoV-2 transcriptomes generated using three sequencing strategies, five host systems, and seven viral isolates. Read-mapping to the SARS-CoV-2 genome was used to determine the 5′ and 3′ coordinates of all junctions in viral RNAs identified in these samples. Results Using junctional abundances, we show nc-sgRNAs make up as much as 33% of total sgRNAs in cell culture models of infection, are largely consistent in abundance across independent transcriptomes, and increase in abundance over time during infection. By assessing the homology between sequences flanking the 5′ and 3′ junction points, we show that nc-sgRNAs are not associated with TRS-like homology. By incorporating read coverage information, we find strong evidence for subgenomic RNAs that contain only 5′ regions of ORF1a. Finally, we show that non-canonical junctions change the landscape of viral open reading frames. Conclusions We identify canonical and non-canonical junctions in SARS-CoV-2 sgRNAs and show that these RNA products are consistently generated by many independent viral isolates and sequencing approaches. These analyses highlight the diverse transcriptional activity of SARS-CoV-2 and offer important insights into SARS-CoV-2 biology.

Background Immunotherapy has revolutionised the treatment of advanced cutaneous squamous cell carcinoma (cSCC). It is important to understand both safety and efficacy in a real-world and trial-ineligible cSCC population. We aimed to evaluate safety, efficacy and molecular insights among a broader cSCC population, including immunosuppressed patients, treated with immune checkpoint inhibitors (CPI). Methods We present a cohort of advanced cSCC patients (n = 61) treated from 2015 to 2020 evaluating the best overall response (BOR) (RECISTv1.1) to CPI therapy, immune-related adverse events (irAEs) and tumour mutational burden (TMB) to correlate with outcomes. A validated geriatric scoring index (CIRS-G) was utilised to assess comorbidities among patients ≥75. These data were compared with published clinical trial results among the broader cSCC population. Results BOR to CPI was lower among the entire cohort when compared with trial data (31.5 vs. 48%, P P P = 0.02), while pre-treatment lymphocyte count and TMB predicted response (P = 0.02). Conclusions We demonstrate comparatively lower response rates to CPI among real-world cSCC patients not explained by older age or immunosuppression history alone. Immune-related toxicity, absolute lymphocyte count and TMB predicted CPI response.

Merkel cell carcinoma (MCC)-a neuroendocrine cancer of the skin-is caused by the integration of Merkel cell polyomavirus and persistent expression of large T antigen and small T antigen. We report that small T antigen in complex with MYCL and the EP400 complex activates the expression of LSD1 (KDM1A), RCOR2 and INSM1 to repress gene expression by the lineage transcription factor ATOH1. LSD1 inhibition reduces the growth of MCC in vitro and in vivo. Through a forward-genetics CRISPR-Cas9 screen, we identified an antagonistic relationship between LSD1 and the non-canonical BAF (ncBAF) chromatin remodelling complex. Changes in gene expression and chromatin accessibility caused by LSD1 inhibition were partially rescued by BRD9 inhibition, revealing that LSD1 and ncBAF antagonistically regulate an overlapping set of genes. Our work provides mechanistic insight into the dependence of MCC on LSD1 and a tumour suppressor role for ncBAF in cancer.

Precision cancer medicine aims to classify tumors by site, histology, and molecular testing to determine an individualized profile of cancer alterations. Viruses are a major contributor to oncogenesis, causing 12% to 20% of all human cancers. Several viruses are causal of specific types of cancer, promoting dysregulation of signaling pathways and resulting in carcinogenesis. In addition, integration of viral DNA into the host (human) genome is a hallmark of some viral species. Tests for the presence of viral infection used in the clinical setting most often use quantitative PCR or immunohistochemical staining. Both approaches have limitations and need to be interpreted/scored appropriately. In some cases, results are not binary (virus present/absent), and it is unclear what to do with a weakly or partially positive result. In addition, viral testing of cancers is performed separately from tests to detect human genomic alterations and can thus be time-consuming and use limited valuable specimen. We present a hybrid-capture and massively parallel sequencing approach to detect viral infection that is integrated with targeted genomic analysis to provide a more complete tumor profile from a single sample.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a hematologic malignancy believed to originate from plasmacytoid dendritic cells (pDCs), the immune cells responsible for producing type 1 interferons during infection. Nearly all patients with BPDCN have prominent skin involvement, with cutaneous infiltration occupying the dermis and subcutis. One half of patients present with BPDCN cells only in the skin, with no evidence of disease elsewhere. Because normal pDCs are rare or absent in cutaneous sites, and they only traffic to the skin after activation by pathogen or inflammation, our aim was to determine if a microorganism is associated with BPDCN. We performed RNA sequencing in BPDCN skin and bone marrow, with cutaneous T-cell lymphoma (CTCL) and normal skin as controls. GATK-PathSeq was used to identify known microbial sequences. Bacterial reads in BPDCN skin were components of normal flora and did not distinguish BPDCN from controls. We then developed a new computational tool, virID (Viral Identification and Discovery; https://github.com/jnoms/virID), for identification of microbial-associated reads remaining unassigned after GATK-PathSeq. We found no evidence for a known or novel virus in BPDCN skin or bone marrow, despite confirming that virID could identify Merkel cell polyomavirus in Merkel cell carcinoma, human papillomavirus in head and neck squamous cell carcinoma, and Kaposi’s sarcoma herpesvirus in Kaposi’s sarcoma in a blinded fashion. Thus, at the level of sensitivity used here, we found no clear pathogen linked to BPDCN.

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.

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.

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.

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.

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.

Neuroendocrine carcinomas (NEC) are tumors expressing markers of neuronal differentiation that can arise at different anatomic sites but have strong histological and clinical similarities. Here we report the chromatin landscapes of a range of human NECs and show convergence to the activation of a common epigenetic program. With a particular focus on treatment emergent neuroendocrine prostate cancer (NEPC), we analyze cell lines, patient-derived xenograft (PDX) models and human clinical samples to show the existence of two distinct NEPC subtypes based on the expression of the neuronal transcription factors ASCL1 and NEUROD1. While in cell lines and PDX models these subtypes are mutually exclusive, single-cell analysis of human clinical samples exhibits a more complex tumor structure with subtypes coexisting as separate sub-populations within the same tumor. These tumor sub-populations differ genetically and epigenetically contributing to intra- and inter-tumoral heterogeneity in human metastases. Overall, our results provide a deeper understanding of the shared clinicopathological characteristics shown by NECs. Furthermore, the intratumoral heterogeneity of human NEPCs suggests the requirement of simultaneous targeting of coexisting tumor populations as a therapeutic strategy., Neuroendocrine carcinomas (NECs) arise from different anatomic sites, but have similar histological and clinical features. Here, the authors show that the epigenetic landscape of a range of NECs converges towards a common epigenetic state, while distinct subtypes occur within neuroendocrine prostate cancer contributing to intratumor heterogeneity in clinical samples.

Merkel cell polyomavirus (MCV) is the causative agent for the majority of Merkel cell carcinoma (MCC) cases. Polyomavirus-associated MCC (MCCP) is characterized by the integration of MCV DNA into the tumor genome and a low tumor mutational burden. In contrast, nonviral MCC (MCCN) is characterized by a high tumor mutational burden induced by UV damage. Since the discovery of MCV, much work in the field has focused on understanding the molecular mechanisms of oncogenesis driven by the MCV tumor (T) antigens. Here, we review our current understanding of how the activities of large T (LT) and small T (ST) promote MCC oncogenesis in the absence of genomic instability. We highlight how both LT and ST inhibit tumor suppressors to evade growth suppression, an important cancer hallmark. We discuss ST interactions with cellular proteins, with an emphasis on those that contribute to sustaining proliferative signaling. Finally, we examine active areas of research into open questions in the field, including the origin of MCC and mechanisms of viral integration.

Resistance to regeneration of insulin-producing pancreatic β 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 β cells to proliferate, but their detailed mechanism of action is incompletely understood. We interrogated our human insulinoma and β cell transcriptomic databases seeking to understand why β cells in insulinomas proliferate, while normal β cells do not. This search reveals the DREAM complex as a central regulator of quiescence in human β 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 β cells to replicate by converting the repressive DREAM complex to its pro-proliferative MMB conformation.

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.

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.

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. Citation Format: James A. DeCaprio, Jason Nomburg, Matthew Meyerson. Long-read sequencing reveals complex patterns of wraparound transcription in polyomaviruses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY11-02.

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 proposed biological pathway linking poor oral health and ESCC involves the alteration of the microbiome. Thus, 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 and WGS of synchronous collections of saliva specimens 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 a similar tumor enrichment of the ESCC-associated bacterial community in these cancers. Because these genera are traditionally considered members of the oral microbiota, we explored if there is a relationship between the synchronous saliva and tumor microbiomes of ESCC patients in Tanzania. Comparative analyses revealed that paired saliva and tumor microbiomes are 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 ESCC patients. Longitudinal studies of the pre-diagnostic oral microbiome are needed to investigate whether these cross-sectional similarities reflect temporal associations.