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.

Asbstract: Despite aggressive therapy, glioblastoma (GBM) recurs in almost all patients and treatment options are very limited. Despite our growing understanding of how cellular transitions associate with relapse in GBM, critical gaps remain in our ability to block these molecular changes and treat recurrent disease. In this study we combine computational biology, forward-thinking understanding of miRNA biology and cutting-edge nucleic acid delivery vehicles to advance targeted therapeutics for GBM. Computational analysis of RNA sequencing from clinical GBM specimens identified TGFβ type II receptor (TGFBR2) as a key player in the mesenchymal transition associated with worse outcome in GBM. Mechanistically, we show that elevated levels of TGFBR2 is conducive to reduced temozolomide (TMZ) sensitivity. This effect is, at least partially, induced by stem-cell driving events coordinated by the reprogramming transcription factors Oct4 and Sox2 that lead to open chromatin states. We show that blocking TGFBR2 via molecular and pharmacological approaches decreases stem cell capacity and sensitivity of clinical recurrent GBM (rGBM) isolates to TMZ in vitro. Network analysis uncovered miR-590-3p as a tumor suppressor that simultaneously inhibits multiple oncogenic nodes downstream of TGFBR2. We also developed novel biodegradable lipophilic poly(β-amino ester) nanoparticles (LiPBAEs) for in vivo microRNA (miRNAs) delivery. Following direct intra-tumoral infusion, these nanomiRs efficiently distribute through the tumors. Importantly, miR-590-3p nanomiRs inhibited the growth and extended survival of mice bearing orthotopic human rGBM xenografts, with an apparent 30% cure rate. These results show that miRNA-based targeted therapeutics provide new opportunities to treat rGBM and bypass the resistance to standard of care therapy.

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 molecular and pharmacologic manipulation of patient-derived cells identified GSCs expressing immunosuppressive effectors mimicking regulatory T cells (Tregs). We show that this I mmunosuppressive T reg- L ike (ITL) GSC state is specific to the mesenchymal GSC subset and is associated with and driven specifically by TGF-β type II receptor (TGFBR2) in contrast to TGFBR1. Transgenic TGFBR2 expression in patient-derived GBM neurospheres promoted a mesenchymal transition and induced a 6-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.

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.

Glioblastoma (GBM) is the most lethal and aggressive primary brain malignancy with high recurrence rates and virtually no long-term survival. Despite our expanding knowledge regarding GBM at the genomic/molecular level, we still don’t fully comprehend the molecular mechanisms responsible for GBM recurrence. Current knowledge implicates tumor-propagating Glioma Stem Cells (GSCs) in the evolution of treatment resistance and tumor recurrence. Understanding these cellular/molecular events will inform strategies for preventing and/or treating recurrent GBM (rGBM). Gene set enrichment analysis (GSEA) of RNA sequencing datasets obtained from GSCs and clinical rGBM specimens identified TGFBR2 signaling as a high frequency and potentially targetable pathway in rGBM. We show that SMAD2/3 signaling, a downstream effector of TGFBR2, is enriched in rGBM and that patients with elevated TGFBR2 have statistically shorter long-term survival outlooks. The functional relevance of these findings was supported using ITD1, a selective TGFBR2 inhibitor, which decreased the GSC phenotype and cell viability of cells derived from clinical rGBM (rGBM cells), while simultaneously re-sensitizing them to temozolomide (TMZ) in-vitro. Since GBM is known to efficiently develop resistance to receptor-targeting monotherapies, we sought to simultaneously target multiple putative oncogenic drivers downstream of TGFBR2. To achieve this, we applied an unbiased miRNA-based network analysis that identified miR-590-3p as an inhibitor of SMAD2/3 signaling at multiple nodes and thus a potential tumor suppressor in rGBM. Transgenic miR-590-3p robustly decreased the expression of >30 putative oncogenes, reduced the GSC phenotype and cell viability, as well as re-sensitized rGBM cells to TMZ. These results predict that blocking TGFBR2 signaling will negatively impact the tumor phenotype of rGBM as well as its GSC capacity. Ongoing directions for this research include comparing the efficacy of ITD1 monotherapy with miR-590-3p therapy in rGBM models in-vivo. These results identify a significant role of TGFBR2 signaling in both TMZ resistance and the GSC phenotype in rGBM and provide a miRNA-based strategy for simultaneously targeting multiple oncogenic nodes within the TGFBR2 and potentially other oncogenic signaling pathways. Citation Format: Sophie Sall, Jack Korleski, Amanda Johnson, Maya Johnson, Hernando Lopez-Bertoni. miR-590-3p inhibits multiple oncogenic nodes within the TGFBR2:SMAD2/3 pathway in recurrent GBM cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3750.