Visualizing genomic characteristics across an RNA-Seq based reference landscape of normal and neoplastic brain

Abstract In order to better understand the relationship between normal and neoplastic brain, we combined five publicly available large-scale datasets, correcting for batch effects and applying Uniform Manifold Approximation and Projection (UMAP) to RNA-Seq data. We assembled a reference Brain-UMAP including 702 adult gliomas, 802 pediatric tumors and 1409 healthy normal brain samples, which can be utilized to investigate the wealth of information obtained from combining several publicly available datasets to study a single organ site. Normal brain regions and tumor types create distinct clusters and because the landscape is generated by RNA-Seq, comparative gene expression profiles and gene ontology patterns are readily evident. To our knowledge, this is the first meta-analysis that allows for comparison of gene expression and pathways of interest across adult gliomas, pediatric brain tumors, and normal brain regions. We provide access to this resource via the open source, interactive online tool Oncoscape, where the scientific community can readily visualize clinical metadata, gene expression patterns, gene fusions, mutations, and copy number patterns for individual genes and pathway over this reference landscape

Computational modelling of perivascular-niche dynamics for the optimization of treatment schedules for glioblastoma

Glioblastoma stem-like cells dynamically transition between a chemoradiation-resistant state and a chemoradiation-sensitive state. However, physical barriers in the tumour microenvironment restrict the delivery of chemotherapy to tumour compartments that are distant from blood vessels. Here, we show that a massively parallel computational model of the spatiotemporal dynamics of the perivascular niche that incorporates glioblastoma stem-like cells and differentiated tumour cells as well as relevant tissue-level phenomena can be used to optimize the administration schedules of concurrent radiation and temozolomide—the standard-of-care treatment for glioblastoma. In mice with platelet-derived growth factor (PDGF)-driven glioblastoma, the model-optimized treatment schedule increased the survival of the animals. For standard radiation fractionation in patients, the model predicts that chemotherapy may be optimally administered about one hour before radiation treatment. Computational models of the spatiotemporal dynamics of the tumour microenvironment could be used to predict tumour responses to a broader range of treatments and to optimize treatment regimens

Multimodal single-cell analysis reveals distinct radioresistant stem-like and progenitor cell populations in murine glioma

Radiation therapy is part of the standard of care for gliomas and kills a subset of tumor cells, while also altering the tumor microenvironment. Tumor cells with stem-like properties preferentially survive radiation and give rise to glioma recurrence. Various techniques for enriching and quantifying cells with stem-like properties have been used, including the fluorescence activated cell sorting (FACS)-based side population (SP) assay, which is a functional assay that enriches for stem-like tumor cells. In these analyses, mouse models of glioma have been used to understand the biology of this disease and therapeutic responses, including the radiation response. We present combined SP analysis and single-cell RNA sequencing of genetically-engineered mouse models of glioma to show a time course of cellular response to radiation. We identify and characterize two distinct tumor cell populations that are inherently radioresistant and also distinct effects of radiation on immune cell populations within the tumor microenvironment

A kinase-deficient NTRK2 splice variant predominates in glioma and amplifies several oncogenic signaling pathways.

Independent scientific achievements have led to the discovery of aberrant splicing patterns in oncogenesis, while more recent advances have uncovered novel gene fusions involving neurotrophic tyrosine receptor kinases (NTRKs) in gliomas. The exploration of NTRK splice variants in normal and neoplastic brain provides an intersection of these two rapidly evolving fields. Tropomyosin receptor kinase B (TrkB), encoded NTRK2, is known for critical roles in neuronal survival, differentiation, molecular properties associated with memory, and exhibits intricate splicing patterns and post-translational modifications. Here, we show a role for a truncated NTRK2 splice variant, TrkB.T1, in human glioma. TrkB.T1 enhances PDGF-driven gliomas in vivo, augments PDGF-induced Akt and STAT3 signaling in vitro, while next generation sequencing broadly implicates TrkB.T1 in the PI3K signaling cascades in a ligand-independent fashion. These TrkB.T1 findings highlight the importance of expanding upon whole gene and gene fusion analyses to include splice variants in basic and translational neuro-oncology research.We thank James Yan, Jenny Zhang, Deby Kumasaka, and Denis Adair for continued technical and administrative assistance and support throughout these experiments. Eero Castren at University of Helsinki provided pEF-BOS-TrkB plasmids used for RCAS-TrkB.T1 generation. We thank Francis S. Lee and Lino Tessarollo for providing breeding pairs of TrkB.T1-/- mice for antibody production. Luis Chiriboga at New York University School of Medicine provided helpful insight into histology protocols. We thank William A. Johnsen and Midori Clarke for assistance with antibody sequencing and protein purification. We thank the Tracy A. Goodpaster and Julie Randolph-Habecker at the Fred Hutchinson Experimental Histopathology Core for histology assistance during the antibody validation phase and Elizabeth Jensen at the Fred Hutchinson Genomics Core for help with all DNA sequencing. We thank Jeongwu Lee Do-Hyun Nam and Steven M. Pollard for providing cell isolates. Funding was provided by National Institutes of Health R01 CA195718 (E.C.H.), U54 CA193461 (E.C.H., F.S.), R01 CA100688 (E.C.H.), T32 CA965725 (S.S.P.), U54 DK106829 (S.S.P.), R21 CA223531 (S.S.P., E.C.H.), T32 CA080416 (P.H.), R01 CA190957 (P.J.P.; T.B.); Jacobs Foundation Research Fellowship (S.S.P.); American Cancer Society ACS-RSG-14-056-01 (P.J.P.); National Research Agency RTI2018-102035-B-I00 (M.S.); Seve Ballesteros Foundation (M.S.). Autopsy materials used in this study were obtained from the University of Washington Neuropathology Core, which is supported by the Alzheimer's Disease Research Center (AG05136), the Adult Changes in Thought Study (AG006781), and Morris K Udall Center of Excellence for Parkinson's Disease Research (NS062684)S

Therapeutic HDAC inhibition in hypermutant diffuse intrinsic pontine glioma

Constitutional mismatch repair deficiency (CMMRD) is a cancer predisposition syndrome associated with the development of hypermutant pediatric high-grade glioma, and confers a poor prognosis. While therapeutic histone deacetylase (HDAC) inhibition of diffuse intrinsic pontine glioma (DIPG) has been reported; here, we use a clinically relevant biopsy-derived hypermutant DIPG model (PBT-24FH) and a CRISPR-Cas9 induced genetic model to evaluate the efficacy of HDAC inhibition against hypermutant DIPG. We screened PBT-24FH cells for sensitivity to a panel of HDAC inhibitors (HDACis) in vitro, identifying two HDACis associated with low nanomolar IC50s, quisinostat (27 nM) and romidepsin (2 nM). In vivo, quisinostat proved more efficacious, inducing near-complete tumor regression in a PBT-24FH flank model. RNA sequencing revealed significant quisinostat-driven changes in gene expression, including upregulation of neural and pro-inflammatory genes. To validate the observed potency of quisinostat in vivo against additional hypermutant DIPG models, we tested quisinostat in genetically-induced mismatch repair (MMR)-deficient DIPG flank tumors, demonstrating that loss of MMR function increases sensitivity to quisinostat in vivo. Here, we establish the preclinical efficacy of quisinostat against hypermutant DIPG, supporting further investigation of epigenetic targeting of hypermutant pediatric cancers with the potential for clinical translation. These findings support further investigation of HDAC inhibitors against pontine high-grade gliomas, beyond only those with histone mutations, as well as against other hypermutant central nervous system tumors