Laterra, John

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. TGFBR2high 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.

Abstract 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.

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.

Abstract Despite aggressive therapy consisting of surgery followed by radio/chemotherapy Glioblastoma (GBM) recurs in almost all patients and, currently, there are no proven therapies to treat recurrent GBM (rGBM). Recent developments in nanomedicine provide new and promising opportunities to develop new targeted therapeutics to treat brain tumors. In this study we combine bioinformatics, forward-thinking understanding of miRNA biology and cutting-edge nucleic acid delivery vehicles to advance targeted therapeutics for rGBM. Bioinformatic analysis of RNA sequencing from GSCs and clinical rGBM specimens identified TGF-beta receptor II (TGFBR2) signaling as a targetable pathway in rGBM. Mechanistically, we show that alterations in chromatin state driven by stem-cell driving events are conducive to a therapy-resistant state induced by TGFBR2. We show that blocking TGFBR2 via molecular and pharmacological approaches decreases the stem cell capacity, cell viability and re-sensitizes clinical rGBM isolates to temozolomide (TMZ) in vitro. Network analysis of SMAD2/3 targets enriched in rGBM predicted a subset of miRNAs that can target multiple genes within this set. Notably, miR-590-3p was predicted to inhibit 37 SMAD 2/3 gene targets that are also enriched in rGBMs. Bioinformatics analysis also showed that expression of 24 of those 37 miR-590-3p targets are positively correlated with TGFBR2 expression in rGBM clinical specimens. Consistent with these predictions, miR-590-3p inhibited expression of 25 of the 37 targets across 3 distinct therapy-resistant GSC isolates. To translate these in vitro finding, we developed novel bioreducible Lipophilic poly(β-amino ester) nanoparticles (LiPBAEs) for in vivo miRNA delivery. Following direct intratumoral infusion, these nanomiRs efficiently distribute through the tumors and spatial transcriptomic analysis (STA) found a decrease in 34 out of the 37 miR- 590-3p targets in animals that received the active nanomiR compared to control miRNA. Furthermore, STA identified 3 cell populations with distinct transcriptomic profiles reflecting a gradient pharmacodynamic response within tumors that received miR-590-3p nanomiRs with decreasing target expression towards the core of the tumor closest to catheter tip placement. To test the effect of 590-3p nanomiR therapy on survival, animals bearing pre-established intracranial rGBM xenografts received three infusions of either control or miR-590-3p nanomiRs every other day beginning on post-implantation day 7. All 10 animals treated with control nanomiRs were either dead or premorbid requiring euthanasia by post-implantation day 23. In contrast, 3 of 10 animals treated with miR-590-3p nanomiRs remained alive and healthy by post-implantation day 45, at which time the experiment was terminated. These results show that miRNA-based targeted therapeutics provide new opportunities to treat rGBM and bypass the resistance that is developed to standard of care. Citation Format: Hernando Lopez-Bertoni, Jack Korleski, Sophie Sall, Kathryn Luly, Maya K Johnson, Amanda L Johnson, Harmon Khela, Bachchu Lal, TC Taylor, Jean Micheal Ashby, Hector Alonso, Alice Li, Stephany Tzeng, John Laterra, Jordan J Green. Multipronged SMAD Pathway Targeting by LiPBAE miR-590-3p NanomiRs Inhibits Recurrent Glioblastoma Growth and Prolongs Survival [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR011.

Abstract A small population of self-renewing stem cells initiate tumors and maintain therapeutic resistance in glioblastoma (GBM). Given the limited treatment options and dismal prognosis for this disease, there is urgent need to identify drivers of stem cells that could be druggable targets. Previous work showed that the endosomal pH regulator NHE9 is upregulated in GBM and correlates with worse survival prognosis. Here, we probed for aberrant signaling pathways in patient-derived GBM cells and found that NHE9 increases cell surface expression and phosphorylation of multiple receptor tyrosine kinases (RTKs) by promoting their escape from lysosomal degradation. Downstream of NHE9-mediated receptor activation, oncogenic signaling pathways converged on the JAK2-STAT3 transduction axis to induce pluripotency genes Oct4 and Nanog and suppress markers of glial differentiation. We used both genetic and chemical approaches to query the role of endosomal pH in GBM phenotypes. Loss-of-function mutations in NHE9 that failed to alkalinize endosomal lumen did not increase self-renewal capacity of gliomaspheres in vitro. However, monensin, a chemical mimetic of Na+/H+ exchanger activity, and the H+ pump inhibitor bafilomycin bypassed NHE9 to directly alkalinize the endosomal lumen resulting in stabilization of RTKs and induction of Oct4 and Nanog. Using orthotopic models of primary GBM cells we found that NHE9 increased tumor initiation in vivo. We propose that NHE9 initiates inside-out signaling from the endosomal lumen, distinct from the established effects of cytosolic and extracellular pH on tumorigenesis. Endosomal pH may be an attractive therapeutic target that diminishes stemness in GBM, agnostic of specific receptor subtype.

Abstract A subset of stem-like cells in glioblastoma (GBM; GSC) underlies tumor propagation, therapeutic resistance, and tumor recurrence. Immune evasion is critical for GSCs to carry out these functions. However, the molecular mechanisms employed by GSCs to escape antitumor immunity remain largely unknown. The reprogramming transcription factors Oct4 and Sox2 function as core multipotency factors and play an essential role in the formation and maintenance of GSCs, but the roles of these transcription factors in GSC immune escape have not been well explored. Here we examine how Oct4/Sox2 coexpression contributes to the immunosuppressive phenotype of GSCs. Combined transcription profiling and functional studies of Oct4/Sox2 coexpressing GSCs and differentiated GBM cells demonstrated that Oct4 and Sox2 cooperatively induce an immunosuppressive transcriptome consisting of multiple immunosuppressive checkpoints (i.e., PD-L1, CD70, A2aR, TDO) and dysregulation of cytokines and chemokines that are associated with an immunosuppressive tumor microenvironment. Mechanistically, induction and function of BRD/H3k27Ac-dependent immunosuppressive genes played a role in the immunosuppressive phenotype of GSCs. Pan-BET bromodomain inhibitors (e.g., JQ1) and shBRD4 constructs significantly inhibited the immunosuppressive transcriptome and immunosuppressive biological responses induced by Oct4/Sox2. Our findings identify targetable mechanisms by which tumor-propagating GSCs contribute to the immunosuppressive microenvironment in GBM. Significance: This report identifies mechanisms by which the reprogramming transcription factors Oct4 and Sox2 function to drive the immunomodulatory transcriptome of GSCs and contribute to the immunosuppressive microenvironment in GBM.

Abstract Glioblastoma multiforme (GBM) and other solid malignancies are heterogeneous and contain subpopulations of tumor cells that exhibit stem-like features. Our recent findings point to a dedifferentiation mechanism by which reprogramming transcription factors Oct4 and Sox2 drive the stem-like phenotype in glioblastoma, in part, by differentially regulating subsets of miRNAs. Currently, the molecular mechanisms by which reprogramming transcription factors and miRNAs coordinate cancer stem cell tumor-propagating capacity are unclear. In this study, we identified miR-486-5p as a Sox2-induced miRNA that targets the tumor suppressor genes PTEN and FoxO1 and regulates the GBM stem-like cells. miR-486-5p associated with the GBM stem cell phenotype and Sox2 expression and was directly induced by Sox2 in glioma cell lines and patient-derived neurospheres. Forced expression of miR-486-5p enhanced the self-renewal capacity of GBM neurospheres, and inhibition of endogenous miR-486-5p activated PTEN and FoxO1 and induced cell death by upregulating proapoptotic protein BIM via a PTEN-dependent mechanism. Furthermore, delivery of miR-486-5p antagomirs to preestablished orthotopic GBM neurosphere-derived xenografts using advanced nanoparticle formulations reduced tumor sizes in vivo and enhanced the cytotoxic response to ionizing radiation. These results define a previously unrecognized and therapeutically targetable Sox2:miR-486-5p axis that enhances the survival of GBM stem cells by repressing tumor suppressor pathways. Significance: This study identifies a novel axis that links core transcriptional drivers of cancer cell stemness to miR-486-5p–dependent modulation of tumor suppressor genes that feeds back to regulate glioma stem cell survival.