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.

Predictors of immunotherapy benefit in Merkel cell carcinoma.

Merkel cell carcinoma is a rare cancer for which immune checkpoint blockade is standard-of-care for recurrent/metastatic disease. However, not all patients benefit from immunotherapy. A greater understanding of molecular mechanisms and predictive biomarkers are unmet needs. We retrospectively analyzed electronic health records and next-generation sequencing data of 45 patients treated at our institution from 2013 to 2020 to understand clinical and genomic correlates of benefit from immunotherapy. Our cohort predominantly included individuals with stage III disease at primary disease diagnosis and individuals with stage IV disease at recurrent/metastatic disease diagnosis. Most received immunotherapy as first-line treatment. 43% experienced objective response (median duration of response 24.2 months, 95% confidence interval 8.8-not reached). Median overall survival was 15.5 months (95% confidence interval 9.0-28.7) (median follow-up 25.2 months). Less advanced stage at primary disease diagnosis and shorter disease-free interval between completion of initial treatment and recurrence were each associated with greater odds of response (odds ratio of 0.06, p = 0.04 for stage; odds ratio 0.75, p = 0.05 for disease-free interval). Single-nucleotide variants in ARID2 and NTRK1 were associated with response (p = 0.05, without Bonferroni correction), while none of Merkel cell polyomavirus status, total mutational burden, ultraviolet mutational signatures, and copy-number alterations predicted outcomes. Patients with shorter disease-free interval may be particularly suitable immunotherapy candidates. Our molecular findings point to ARID2 and NTRK1 as potential predictive markers and/or therapeutic targets (e.g., with Trk inhibitors), although this association needs to be confirmed in a larger sample.

Endothelial pyruvate kinase M2 maintains vascular integrity.

The M2 isoform of pyruvate kinase (PKM2) is highly expressed in most cancer cells, and has been studied extensively as a driver of oncogenic metabolism. In contrast, the role of PKM2 in nontransformed cells is little studied, and nearly nothing is known of its role, if any, in quiescent cells. We show here that endothelial cells express PKM2 almost exclusively over PKM1. In proliferating endothelial cells, PKM2 is required to suppress p53 and maintain cell cycle progression. In sharp contrast, PKM2 has a strikingly different role in quiescent endothelial cells, where inhibition of PKM2 leads to degeneration of tight junctions and barrier function. Mechanistically, PKM2 regulates barrier function independently of its canonical activity as a pyruvate kinase. Instead, PKM2 suppresses NF-kB and its downstream target, the vascular permeability factor angiopoietin 2. As a consequence, loss of endothelial cell PKM2 in vivo predisposes mice to VEGF-induced vascular leak, and to severe bacteremia and death in response to sepsis. Together, these data demonstrate new roles of PKM2 in quiescent cells, and highlight the need for caution in developing cancer therapies that target PKM2.

Identification of extant vertebrate Myxine glutinosa VWF: evolutionary conservation of primary hemostasis.

Hemostasis in vertebrates involves both a cellular and a protein component. Previous studies in jawless vertebrates (cyclostomes) suggest that the protein response, which involves thrombin-catalyzed conversion of a soluble plasma protein, fibrinogen, into a polymeric fibrin clot, is conserved in all vertebrates. However, similar data are lacking for the cellular response, which in gnathostomes is regulated by von Willebrand factor (VWF), a glycoprotein that mediates the adhesion of platelets to the subendothelial matrix of injured blood vessels. To gain evolutionary insights into the cellular phase of coagulation, we asked whether a functional vwf gene is present in the Atlantic hagfish, Myxine glutinosa We found a single vwf transcript that encodes a simpler protein compared with higher vertebrates, the most striking difference being the absence of an A3 domain, which otherwise binds collagen under high-flow conditions. Immunohistochemical analyses of hagfish tissues and blood revealed Vwf expression in endothelial cells and thrombocytes. Electron microscopic studies of hagfish tissues demonstrated the presence of Weibel-Palade bodies in the endothelium. Hagfish Vwf formed high-molecular-weight multimers in hagfish plasma and in stably transfected CHO cells. In functional assays, botrocetin promoted VWF-dependent thrombocyte aggregation. A search for vwf sequences in the genome of sea squirts, the closest invertebrate relatives of hagfish, failed to reveal evidence of an intact vwf gene. Together, our findings suggest that VWF evolved in the ancestral vertebrate following the divergence of the urochordates some 500 million years ago and that it acquired increasing complexity though sequential insertion of functional modules.

A role of stochastic phenotype switching in generating mosaic endothelial cell heterogeneity.

Previous studies have shown that biological noise may drive dynamic phenotypic mosaicism in isogenic unicellular organisms. However, there is no evidence for a similar mechanism operating in metazoans. Here we show that the endothelial-restricted gene, von Willebrand factor (VWF), is expressed in a mosaic pattern in the capillaries of many vascular beds and in the aorta. In capillaries, the mosaicism is dynamically regulated, with VWF switching between ON and OFF states during the lifetime of the animal. Clonal analysis of cultured endothelial cells reveals that dynamic mosaic heterogeneity is controlled by a low-barrier, noise-sensitive bistable switch that involves random transitions in the DNA methylation status of the VWF promoter. Finally, the hearts of VWF-null mice demonstrate an abnormal endothelial phenotype as well as cardiac dysfunction. Together, these findings suggest a novel stochastic phenotype switching strategy for adaptive homoeostasis in the adult vasculature.

FOXO1-mediated activation of Akt plays a critical role in vascular homeostasis.

RATIONALE: Forkhead box-O transcription factors (FoxOs) transduce a wide range of extracellular signals, resulting in changes in cell survival, cell cycle progression, and several cell type-specific responses. FoxO1 is expressed in many cell types, including endothelial cells (ECs). Previous studies have shown that Foxo1 knockout in mice results in embryonic lethality at E11 because of impaired vascular development. In contrast, somatic deletion of Foxo1 is associated with hyperproliferation of ECs. Thus, the precise role of FoxO1 in the endothelium remains enigmatic. OBJECTIVE: To determine the effect of endothelial-specific knockout and overexpression of FoxO1 on vascular homeostasis. METHODS AND RESULTS: We show that EC-specific disruption of Foxo1 in mice phenocopies the full knockout. Although endothelial expression of FoxO1 rescued otherwise Foxo1-null animals, overexpression of constitutively active FoxO1 resulted in increased EC size, occlusion of capillaries, elevated peripheral resistance, heart failure, and death. Knockdown of FoxO1 in ECs resulted in marked inhibition of basal and vascular endothelial growth factor-induced Akt-mammalian target of rapamycin complex 1 (mTORC1) signaling. CONCLUSIONS: Our findings suggest that in mice, endothelial expression of FoxO1 is both necessary and sufficient for embryonic development. Moreover, FoxO1-mediated feedback activation of Akt maintains growth factor responsive Akt/mTORC1 activity within a homeostatic range.