Cancer-associated fi broblasts (CAF) are major players in the progression and drug resistance of pancreatic ductal adenocarcinoma (PDAC). CAFs constitute a diverse cell population consisting of several recently described subtypes, although the extent of CAF heterogeneity has remained undefi ned. Here we use single-cell RNA sequencing to thoroughly characterize the neoplastic and tumor microenvironment content of human and mouse PDAC tumors. We corroborate the presence of myofi broblastic CAFs and infl ammatory CAFs and defi ne their unique gene signatures in vivo. Moreover, we describe a new population of CAFs that express MHC class II and CD74, but do not express classic costimulatory molecules. We term this cell population "antigenpresenting CAFs" and fi nd that they activate CD4 + T cells in an antigen-specifi c fashion in a model system, confi rming their putative immune-modulatory capacity. Our cross-species analysis paves the way for investigating distinct functions of CAF subtypes in PDAC immunity and progression. SIGNIFICANCE : Appreciating the full spectrum of fi broblast heterogeneity in pancreatic ductal adenocarcinoma is crucial to developing therapies that specifi cally target tumor-promoting CAFs. This work identifi es MHC class II-expressing CAFs with a capacity to present antigens to CD4 + T cells, and potentially to modulate the immune response in pancreatic tumors.

Background: Smooth muscle cells (SMC) play significant roles in atherosclerosis via phenotypic switching, a pathological process in which SMC dedifferentiation, migration and transdifferentiation into other cell types. Yet, how SMC contribute to pathophysiology of atherosclerosis remains elusive. Methods: To reveal the trajectories of SMC transdifferentiation during atherosclerosis and to identify molecular targets for disease therapy, we combined SMC fate mapping and single-cell RNA sequencing of both mouse and human atherosclerotic plaques. We also performed cell biology experiments on isolated SMC-derived cells, conducted integrative human genomics, and employed pharmacological studies targeting SMC-derived cells both in vivo and in vitro . Results: We found that SMC transitioned to an intermediate cell state during atherosclerosis, which was also found in human atherosclerotic plaques of carotid and coronary arteries. SMC-derived intermediate cells, termed "SEM" cells, were multipotent and could differentiate into macrophage-like and fibrochondrocyte-like cells, as well as return towards SMC phenotype. Retinoic acid (RA) signaling was identified as a regulator of SMC to SEM cell transition and RA signaling was dysregulated in symptomatic human atherosclerosis. Human genomics revealed enrichment of genome wide association study (GWAS) signals for coronary artery disease (CAD) in RA signaling target gene loci and correlation between CAD risk alleles and repressed expression of these genes. Activation of RA signaling by all-trans retinoic acid (ATRA), an anti-cancer drug for acute promyelocytic leukemia, blocked SMC transition to SEM cells, reduced atherosclerotic burden and promoted fibrous cap stability. Conclusions: Integration of cell-specific fate mapping, single-cell genomics and human genetics adds novel insights into the complexity of SMC biology and reveals regulatory pathways for therapeutic targeting of SMC transitions in atherosclerotic cardiovascular disease.

ObjectivePancreatic ductal adenocarcinoma (PDA) has among the highest stromal fractions of any cancer and this has complicated attempts at expression-based molecular classification. The goal of this work is to profile purified samples of human PDA epithelium and stroma and examine their respective contributions to gene expression in bulk PDA samples.DesignWe used laser capture microdissection (LCM) and RNA sequencing to profile the expression of 60 matched pairs of human PDA malignant epithelium and stroma samples. We then used these data to train a computational model that allowed us to infer tissue composition and generate virtual compartment-specific expression profiles from bulk gene expression cohorts.ResultsOur analysis found significant variation in the tissue composition of pancreatic tumours from different public cohorts. Computational removal of stromal gene expression resulted in the reclassification of some tumours, reconciling functional differences between different cohorts. Furthermore, we established a novel classification signature from a total of 110 purified human PDA stroma samples, finding two groups that differ in the extracellular matrix-associated and immune-associated processes. Lastly, a systematic evaluation of cross-compartment subtypes spanning four patient cohorts indicated partial dependence between epithelial and stromal molecular subtypes.ConclusionOur findings add clarity to the nature and number of molecular subtypes in PDA, expand our understanding of global transcriptional programmes in the stroma and harmonise the results of molecular subtyping efforts across independent cohorts.

Transcription factor (TF)–induced reprogramming of somatic cells into induced pluripotent stem cells (iPSC) is associated with genome-wide changes in chromatin modifications. Polycomb-mediated histone H3 lysine-27 trimethylation (H3K27me3) has been proposed as a defining mark that distinguishes the somatic from the iPSC epigenome. Here, we dissected the functional role of H3K27me3 in TF–induced reprogramming through the inactivation of the H3K27 methylase EZH2 at the onset of reprogramming. Our results demonstrate that surprisingly the establishment of functional iPSC proceeds despite global loss of H3K27me3. iPSC lacking EZH2 efficiently silenced the somatic transcriptome and differentiated into tissues derived from the three germ layers. Remarkably, the genome-wide analysis of H3K27me3 in Ezh2 mutant iPSC cells revealed the retention of this mark on a highly selected group of Polycomb targets enriched for developmental regulators controlling the expression of lineage specific genes. Erasure of H3K27me3 from these targets led to a striking impairment in TF–induced reprogramming. These results indicate that PRC2-mediated H3K27 trimethylation is required on a highly selective core of Polycomb targets whose repression enables TF–dependent cell reprogramming.

RNA sequencing (RNAseq) has become the method of choice for transcriptome analysis, yet no consensus exists as to the most appropriate pipeline for its analysis, with current benchmarks suffering important limitations. Here, we address these challenges through a rich benchmarking resource harnessing (i) two RNAseq datasets including ERCC ExFold spike-ins; (ii) Nanostring measurements of a panel of 150 genes on the same samples; (iii) a set of internal, genetically-determined controls; (iv) a reanalysis of the SEQC dataset; and (v) a focus on relative quantification (i.e. across-samples). We use this resource to compare different approaches to each step of RNAseq analysis, from alignment to differential expression testing. We show that methods providing the best absolute quantification do not necessarily provide good relative quantification across samples, that count-based methods are superior for gene-level relative quantification, and that the new generation of pseudo-alignment-based software performs as well as established methods, at a fraction of the computing time. We also assess the impact of library type and size on quantification and differential expression analysis. Finally, we have created a R package and a web platform to enable the simple and streamlined application of this resource to the benchmarking of future methods.

Malignant gliomas constitute one of the most significant areas of unmet medical need, owing to the invariable failure of surgical eradication and their marked molecular heterogeneity. Accumulating evidence has revealed a critical contribution by the Polycomb axis of epigenetic repression. However, a coherent understanding of the regulatory networks affected by Polycomb during gliomagenesis is still lacking. Here we integrate transcriptomic and epigenomic analyses to define Polycomb-dependent networks that promote gliomagenesis, validating them both in two independent mouse models and in a large cohort of human samples. We find that Polycomb dysregulation in gliomagenesis affects transcriptional networks associated with invasiveness and de-differentiation. The dissection of these networks uncovers Zfp423 as a critical Polycomb-dependent transcription factor whose silencing negatively impacts survival. The anti-gliomagenic activity of Zfp423 requires interaction with the SMAD proteins within the BMP signalling pathway, pointing to a novel synergic circuit through which Polycomb inhibits BMP signalling.

Most antiviral agents are designed to target virus-specific proteins and mechanisms rather than the host cell proteins that are critically dysregulated following virus-mediated reprogramming of the host cell transcriptional state. To overcome these limitations, we propose that elucidation and pharmacologic targeting of host cell Master Regulator proteins-whose aberrant activities govern the reprogramed state of coronavirusinfected cells-presents unique opportunities to develop novel mechanism-based therapeutic approaches to antiviral therapy, either as monotherapy or as a complement to established treatments. Specifically, we propose that a small module of host cell Master Regulator proteins (ViroCheckpoint) is hijacked by the virus to support its efficient replication and release. Conventional methodologies are not well suited to elucidate these potentially targetable proteins. By using the VIPER network-based algorithm, we successfully interrogated 12h, 24h, and 48h signatures from Calu-3 lung adenocarcinoma cells infected with SARS-CoV, to elucidate the time-dependent reprogramming of host cells and associated Master Regulator proteins. We used the NYS CLIA-certified Darwin OncoTreat algorithm, with an existing database of RNASeq profiles following cell perturbation with 133 FDA-approved and 195 late-stage experimental compounds, to identify drugs capable of virtually abrogating the virus-induced Master Regulator signature. This approach to drug prioritization and repurposing can be trivially extended to other viral pathogens, including SARS-CoV-2, as soon as the relevant infection signature becomes available.

Despite extensive efforts to characterize the transcriptional landscape of pancreatic ductal adenocarcinoma (PDA), reproducible assessment of subtypes with actionable dependencies remains challenging. Systematic, network-based analysis of regulatory protein activity stratified PDA tumours into novel functional subtypes that were highly conserved across multiple cohorts, including at the single cell level and in laser capture microdissected (LCM) samples. Identified subtypes were characterized by activation of master regulator proteins representing either gastrointestinal lineage markers or transcriptional effectors of morphogen pathways. Single cell analysis confirmed the existence of Lineage and Morphogenic states but also revealed a dominant population of more differentiated Oncogenic Precursor (OP) cells , present in all sampled patients, yet not apparent from bulk tumor analysis. Master regulators were validated by pooled, CRISPR/Cas9 screens, demonstrating both subtype-specific and universal dependencies. Conversely, ectopic expression of Lineage MRs, such as OVOL2, was sufficient to reprogram Morphogenic cells, thus providing a roadmap for the future targeting of patient-specific dependencies in PDA.