DNA methylation is a reversible process catalyzed by the ten–eleven translocation (TET) family of enzymes (TET1, TET2, TET3) that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Altered patterns of 5hmC and 5mC are widely reported in human cancers and loss of 5hmC correlates with poor prognosis. Understanding the mechanisms leading to 5hmC loss and its role in oncogenesis will advance the development of epigenetic-based therapeutics. We show that TET2 loss associates with glioblastoma (GBM) stem cells and correlates with poor survival of GBM patients. We further identify a SOX2:miR-10b-5p:TET2 axis that represses TET2 expression, represses 5hmC, increases 5mC levels, and induces GBM cell stemness and tumor-propagating potential. In vivo delivery of a miR-10b-5p inhibitor that normalizes TET2 expression and 5hmC levels inhibits tumor growth and prolongs survival of animals bearing pre-established orthotopic GBM xenografts. These findings highlight the importance of TET2 and 5hmC loss in Sox2-driven oncogenesis and their potential for therapeutic targeting.

Purpose: Glioblastoma (GBM) is the most common brain malignancy with median survival <2 years. Standard-of-care temozolomide has marginal efficacy in approximately 70% of patients due to MGMT expression. LP-184 is an acylfulvene-derived prodrug activated by the oxidoreductase PTGR1 that alkylates at N3-adenine, not reported to be repaired by MGMT. This article examines LP-184 efficacy against preclinical GBM models and identifies molecular predictors of LP-184 efficacy in clinical GBM. Experimental Design: LP-184 effects on GBM cell viability and DNA damage were determined using cell lines, primary PDX-derived cells and patient-derived neurospheres. GBM cell sensitivities to LP-184 relative to temozolomide and MGMT expression were examined. Pharmacokinetics and CNS bioavailability were evaluated in mice with GBM xenografts. LP-184 effects on GBM xenograft growth and animal survival were determined. Machine learning, bioinformatic tools, and clinical databases identified molecular predictors of GBM cells and tumors to LP-184 responsiveness. Results: LP-184 inhibited viability of multiple GBM cell isolates including temozolomide-resistant and MGMT-expressing cells at IC50 = approximately 22–310 nmol/L. Pharmacokinetics showed favorable AUCbrain/plasma and AUCtumor/plasma ratios of 0.11 (brain Cmax = 839 nmol/L) and 0.2 (tumor Cmax = 2,530 nmol/L), respectively. LP-184 induced regression of GBM xenografts and prolonged survival of mice bearing orthotopic xenografts. Bioinformatic analyses identified PTGR1 elevation in clinical GBM subtypes and associated LP-184 sensitivity with EGFR signaling, low nucleotide excision repair (NER), and low ERCC3 expression. Spironolactone, which induces ERCC3 degradation, decreased LP-184 IC50 3 to 6 fold and enhanced GBM xenograft antitumor responses. Conclusions: These results establish LP-184 as a promising chemotherapeutic for GBM with enhanced efficacy in intrinsic or spironolactone-induced TC-NER–deficient tumors.

Attempts to activate an anti-tumor immune response in glioblastoma (GBM) have been met with many challenges due to its inherently immunosuppressive tumor microenvironment. The degree and mechanisms by which molecularly and phenotypically diverse tumor-propagating glioma stem cells (GSCs) contribute to this state are poorly defined. In this study, our multifaceted approach combining bioinformatics analyses of clinical and experimental datasets, single-cell sequencing, and the molecular and pharmacologic manipulation of patient-derived cells identified GSCs expressing immunosuppressive effectors mimicking regulatory T cells (Tregs). We showed that this immunosuppressive Treg-like (ITL) GSC state is specific to the mesenchymal GSC subset and is associated with and driven specifically by TGFβ type II receptor (TGFBR2) in contrast to TGFBR1. Transgenic TGFBR2 expression in patient-derived GBM neurospheres promoted a mesenchymal transition and induced a six-gene ITL signature consisting of CD274 (PD-L1), NT5E (CD73), ENTPD1 (CD39), LGALS1 (galectin-1), PDCD1LG2 (PD-L2), and TGFB1. This TGFBR2-driven ITL signature was identified in clinical GBM specimens, patient-derived GSCs, and systemic mesenchymal malignancies. TGFBR2^high GSCs inhibited CD4+ and CD8+ T cell viability and their capacity to kill GBM cells, effects reversed by pharmacologic and shRNA-based TGFBR2 inhibition. Collectively, our data identify an immunosuppressive GSC state that is TGFBR2-dependent and susceptible to TGFBR2-targeted therapeutics.

Background/Objectives: CSCs are critical drivers of the tumor and stem cell phenotypes of glioblastoma (GBM) cells. Chromatin modifications play a fundamental role in driving a GBM CSC phenotype. The goal of this study is to further our understanding of how stem cell-driving events control changes in chromatin architecture that contribute to the tumor-propagating phenotype of GBM. Methods: We utilized computational analyses to identify a subset of clinically relevant genes that were predicted to be repressed in a Polycomb repressive complex 2 (PRC2)-dependent manner in GBM upon induction of stem cell-driving events. These associations were validated in patient-derived GBM neurosphere models using state-of-the-art molecular techniques to express, silence, and measure microRNA (miRNA) and gene expression changes. Advanced Poly(β-amino ester) nanoparticle formulations (PBAEs) were used to deliver miRNAs in vivo to orthotopic human GBM tumor models. Results: We show that glioma stem cell (GSC) formation and tumor propagation involve the crosstalk between multiple epigenetic mechanisms, resulting in the repression of the miRNAs that regulate PRC2 function and histone H3 lysine 27 tri-methylation (H3K27me3). We also identified miR-217-5p as an EZH2 regulator repressed in GSCs and showed that miR-217-5p reconstitution using advanced nanoparticle formulations re-activates the PRC2-repressed genes, inhibits GSC formation, impairs tumor growth, and enhances the effects of ionizing radiation in an orthotopic model of GBM. Conclusions: These findings suggest that inhibiting PRC2 function by targeting EZH2 with miR-217-5p advanced nanoparticle formulations could have a therapeutic benefit in GBM.

The advent of immunotherapy has transformed our approach to cancer treatment; however, it is virtually ineffective in glioblastoma (GBM), the most common & malignant brain cancer in adults, due in part to the inherently immunosuppressive tumor microenvironment (TME). Recent research has identified GBM cancer stem cells (GSCs) as key modulators of the TME, mainly through activation and recruitment of pro-tumor immune cells (e.g., TAMs, regulatory T-cells (Tregs), MDSCs). We identified a novel immunosuppressive phenotype whereby GSCs mimic Treg function via TGF-b type II receptor (TGFBR2) signaling to suppress the immune response with the potential to promote tumor propagation and resist immunotherapy. Preliminary experiments revealed that TGFBR2 drives GBM cells to a CD44-high, mesenchymal-like GSC state. Transgenic TGFBR2 was found to induce a Treg-like transcriptional profile defined by expression of canonical Treg effector genes (e.g., CD274, ENTPD1, NT5E, LGALS1, TGFB1, PDCD1LG2) in human GBM neurospheres. Single-cell RNA-Seq and bioinformatics analyses confirmed the presence of this TGFBR2-mediated transcriptional signature in both patient-derived GSCs and patient tumors. Notably, in silico deconvolution of bulk GBM sequencing predicts that high expression of a TGFBR2-induced transcriptome associates with an immunosuppressive TME characterized by high infiltration of pro-tumor immune cells (e.g., TAMs, neutrophils). To target this Treg-like, immunosuppressive GSC phenotype, we utilized ITD-1, a small molecule inhibitor of TGFBR2. In vitro ITD-1 treatment reduced GSC proliferation capacity and downregulated expression of CD44 and Treg effectors. In T-cell co-cultures, pre-treating GSCs with ITD-1 increased T-cell viability and T-cell-mediated tumor cell killing. Administration of ITD-1 in tumor-bearing, immunocompetent mice reduced tumor volume and increased anti-tumor immune cell infiltration relative to vehicle controls. In all, ITD-1-mediated TGFBR2 inhibition attenuates a unique, Treg-like immunosuppressive and potentially tumor-protective phenotype in GSCs. Future studies will combine ITD-1 and checkpoint inhibitor therapy in an effort to increase the efficacy of immunotherapy in GBM.

Glioblastoma (GBM) contains mesenchymal cancer stem cells that drive tumor aggressiveness and recurrence and exhibit aberrant glycosylation during proneural-to-mesenchymal transition. A comprehensive computational analysis of human GBM transcriptomic datasets revealed an up-regulation of 13 genes involved in glycan mannosylation compared to normal brain, and histopathological staining of a tissue array representing 35 GBM cases revealed elevated mannose levels that correlated with increased expression of the mesenchymal marker CD44. Hydroxyl proton transfer–weighted magnetic resonance imaging (HPTw MRI) detected elevated mannose levels in aggressive human mesenchymal GBM in vitro and in vivo but not in GBM with a less aggressive nonmesenchymal phenotype. To establish causation over correlation, inhibiting expression of the mannose-binding lectins LMAN1/2 that regulate intracellular processing of mannosylated proteins decreased the glioma cell HPTw MRI signal. Our findings indicate that HPTw MRI correlates with high mannose and possibly other saccharide levels in mesenchymal GBM cells, serving as a surrogate imaging biomarker for predicting tumor aggressiveness and recurrence.