Summary We leveraged IDH wild-type glioblastomas, derivative neurospheres, and single cell gene expression profiles to define three tumor-intrinsic transcriptional subtypes designated as proneural, mesenchymal, and classical. Transcriptomic subtype multiplicity correlated with increased intratumoral heterogeneity and presence of tumor microenvironment. In silico cell sorting identified macrophages/microglia, CD4+ T lymphocytes, and neutrophils in the glioma microenvironment. NF1 deficiency resulted in increased tumor-associated macrophages/microglia infiltration. Longitudinal transcriptome analysis showed that expression subtype is retained in 55% of cases. Gene signature-based tumor microenvironment inference revealed a decrease in invading monocytes and a subtype-dependent increase in macrophages/microglia cells upon disease recurrence. Hypermutation at diagnosis or at recurrence associated with CD8+ T cell enrichment. Frequency of M2 macrophages detection associated with short-term relapse after radiation therapy.

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Graphical abstract Epigenetic activation of WNT5A expression contributes to glioblastoma tumor recurrence by promoting differentiation of glioma-derived stem cells into endothelial cells.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas without effective therapeutics. Bioinformatics was used to identify potential therapeutic targets. Paired Box (PAX), Eyes Absent (EYA), Dachsund (DACH), and Sine Oculis (SIX) genes, which form a regulatory interactive network in drosophila, were found to be dysregulated in human MPNST cell lines and solid tumors. We identified a decrease in DACH1 expression, and increases in expression of PAX6, EYA1, EYA2, EYA4, and SIX1- 4. Consistent with the observation that half of MPNSTs develop in neurofibromatosis type 1 patients, subsequent to NF1 mutation, we found that exogenous expression of the NF1-GAP related domain (GRD) normalized DACH1 expression. EYA4 mRNA was elevated more than 100-fold as estimated by quantitative real time PCR in most MPSNT cell lines. In vitro, suppression of EYA4 expression using shRNA reduced cell adhesion and migration and caused cellular necrosis without affecting cell proliferation or apoptotic cell death. MPNST cells expressing sh-EYA4 either failed to form tumors in nude mice or formed very small tumors, with extensive necrosis but similar levels of proliferation and apoptosis as control cells. Our findings identify a role for EYA4 and possibly interacting SIX and DACH proteins in MPNSTs and suggest the EYA4 pathway as a rational therapeutic target.