Neural G0:a quiescent-like state found in neuroepithelial-derived cells and glioma

Single‐cell RNA sequencing has emerged as a powerful tool for resolving cellular states associated with normal and maligned developmental processes. Here, we used scRNA‐seq to examine the cell cycle states of expanding human neural stem cells (hNSCs). From these data, we constructed a cell cycle classifier that identifies traditional cell cycle phases and a putative quiescent‐like state in neuroepithelial‐derived cell types during mammalian neurogenesis and in gliomas. The Neural G0 markers are enriched with quiescent NSC genes and other neurodevelopmental markers found in non‐dividing neural progenitors. Putative glioblastoma stem‐like cells were significantly enriched in the Neural G0 cell population. Neural G0 cell populations and gene expression are significantly associated with less aggressive tumors and extended patient survival for gliomas. Genetic screens to identify modulators of Neural G0 revealed that knockout of genes associated with the Hippo/Yap and p53 pathways diminished Neural G0 in vitro, resulting in faster G1 transit, down‐regulation of quiescence‐associated markers, and loss of Neural G0 gene expression. Thus, Neural G0 represents a dynamic quiescent‐like state found in neuroepithelial‐derived cells and gliomas

Mechanism of acquired TMZ resistance in GBM

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2014.Cataloged from PDF version of thesis.Includes bibliographical references.Glioblastoma (GBM) is the most common and malignant form of brain cancer. After aggressive treatment, therapy resistant tumors inevitably recur. However, the molecular mechanisms underlying such resistance remain unclear. We isolated GBM cells resistant to temozolomide (TMZ), the frontline chemotherapy agent for GBM, and observed modest decreases in the mismatch repair (MMR) components MSH2 and MSH6. The modest decrease in MSH2, and relatively modest decrease in MSH6, did not seem sufficient to account for the very large increase in TMZ resistance. However, shRNA-mediated modulation of MSH2 and MSH6 levels in vitro confirmed that such decreases in MSH2 and MSH6 provide a potent mechanism for TMZ resistance. We demonstrate in an in vivo GBM mouse model that minor changes in MSH2 suppress TMZ-induced tumor regression, and moreover, show that even minor decreases in MSH2 transcript levels correlate with decreased survival in TMZ treated GBM patients. These modest changes in MMR are unlikely to alter classical markers of MMR deficiency, namely microsatellite instability and a mutator phenotype. Our results suggest that the involvement of MMR deregulation in mediating TMZ resistance is likely to be much more prevalent than previously appreciated. Additionally, we have employed phosphoproteomic network analysis to identify changes at the signaling network level that accompany the acquisition of TMZ resistance. Through mathematical and computational approaches, we identified changes that suggest increased PDGFR and integrin/FAK1 signaling in response to repeated TMZ exposure. Additionally, kinase motif analysis identified widespread alterations in phosphorylation of peptides containing motifs associated with the CDK/MAPK kinase family. Currently, we are applying molecular biology techniques to investigate the effects of these altered cellular signals on MMR activity and the sensitivity of GBM cells to TMZ.by José L. McFaline-Figueroa.Ph. D

High-capacity sample multiplexing for single cell chromatin accessibility profiling

Abstract Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first demonstrate our method with a chemical epigenomics screen, in which we identify drug-altered distal regulatory sites predictive of compound- and dose-dependent effects on transcription. We then analyze cell type-specific chromatin changes in PBMCs from multiple donors responding to synthetic and allogeneic immune stimulation. We quantify stimulation-altered immune cell compositions and isolate the unique effects of allogeneic stimulation on chromatin accessibility specific to T-lymphocytes. Finally, we observe that impaired global chromatin decondensation often coincides with chemical inhibition of allogeneic T-cell activation

A Genome-wide Framework for Mapping Gene Regulation via Cellular Genetic Screens

Over one million candidate regulatory elements have been identified across the human genome, but nearly all are unvalidated and their target genes uncertain. Approaches based on human genetics are limited in scope to common variants and in resolution by linkage disequilibrium. We present a multiplex, expression quantitative trait locus (eQTL)-inspired framework for mapping enhancer-gene pairs by introducing random combinations of CRISPR/Cas9-mediated perturbations to each of many cells, followed by single-cell RNA sequencing (RNA-seq). Across two experiments, we used dCas9-KRAB to perturb 5,920 candidate enhancers with no strong a priori hypothesis as to their target gene(s), measuring effects by profiling 254,974 single-cell transcriptomes. We identified 664 (470 high-confidence) cis enhancer-gene pairs, which were enriched for specific transcription factors, non-housekeeping status, and genomic and 3D conformational proximity to their target genes. This framework will facilitate the large-scale mapping of enhancer-gene regulatory interactions, a critical yet largely uncharted component of the cis-regulatory landscape of the human genome

Targeting TWIST1 through loss of function inhibits tumorigenicity of human glioblastoma

TWIST1 (TW) is a bHLH transcription factor (TF) and master regulator of the epithelial‐to‐mesenchymal transition (EMT). In vitro, TW promotes mesenchymal change, invasion, and self‐renewal in glioblastoma (GBM) cells. However, the potential therapeutic relevance of TW has not been established through loss‐of‐function studies in human GBM cell xenograft models. The effects of TW loss of function (gene editing and knockdown) on inhibition of tumorigenicity of U87MG and GBM4 glioma stem cells were tested in orthotopic xenograft models and conditional knockdown in established flank xenograft tumors. RNAseq and the analysis of tumors investigated putative TW‐associated mechanisms. Multiple bioinformatic tools revealed significant alteration of ECM, membrane receptors, signaling transduction kinases, and cytoskeleton dynamics leading to identification of PI3K/AKT signaling. We experimentally show alteration of AKT activity and periostin (POSTN) expression in vivo and/or in vitro. For the first time, we show that effect of TW knockout inhibits AKT activity in U87MG cells in vivo independent of PTEN mutation. The clinical relevance of TW and candidate mechanisms was established by analysis of the TCGA and ENCODE databases. TW expression was associated with decreased patient survival and LASSO regression analysis identified POSTN as one of top targets of TW in human GBM. While we previously demonstrated the role of TW in promoting EMT and invasion of glioma cells, these studies provide direct experimental evidence supporting protumorigenic role of TW independent of invasion in vivo and the therapeutic relevance of targeting TW in human GBM. Further, the role of TW driving POSTN expression and AKT signaling suggests actionable targets, which could be leveraged to mitigate the oncogenic effects of TW in GBM

Minor Changes in Expression of the Mismatch Repair Protein MSH2 Exert a Major Impact on Glioblastoma Response to Temozolomide

Glioblastoma (GBM) is often treated with the cytotoxic drug temozolomide, but the disease inevitably recurs in a drug-resistant form after initial treatment. Here, we report that in GBM cells, even a modest decrease in the mismatch repair (MMR) components MSH2 and MSH6 have profound effects on temozolomide sensitivity. RNAi-mediated attenuation of MSH2 and MSH6 showed that such modest decreases provided an unexpectedly strong mechanism of temozolomide resistance. In a mouse xenograft model of human GBM, small changes in MSH2 were sufficient to suppress temozolomide-induced tumor regression. Using The Cancer Genome Atlas to analyze mRNA expression patterns in tumors from temozolomide-treated GBM patients, we found that MSH2 transcripts in primary GBM could predict patient responses to initial temozolomide therapy. In recurrent disease, the absence of microsatellite instability (the standard marker for MMR deficiency) suggests a lack of involvement of MMR in the resistant phenotype of recurrent disease. However, more recent studies reveal that decreased MMR protein levels occur often in recurrent GBM. In accordance with our findings, these reported decreases may constitute a mechanism by which GBM evades temozolomide sensitivity while maintaining microsatellite stability. Overall, our results highlight the powerful effects of MSH2 attenuation as a potent mediator of temozolomide resistance and argue that MMR activity offers a predictive marker for initial therapeutic response to temozolomide treatment.National Cancer Institute (U.S.). Integrative Cancer Biology Program (Grant U54-CA112967)National Institutes of Health (U.S.) (Grants R01-ES022872, P30-CA014051, P30-ES002109, T32GM007287, T32-GM081081 and DP1-ES022576)German Cancer Aid (Mildred-Scheel Fellowship)National Cancer Institute (U.S.) (Ruth L. Kirschstein National Research Service Award 5F31CA165735