Glioblastoma (GBM) is the most lethal type of primary brain cancer. Standard care using chemo- and radio-therapy modestly increases the overall survival of patients; however, recurrence is inevitable, due to treatment resistance and lack of response to targeted therapies. GBM therapy resistance has been attributed to several extrinsic and intrinsic factors which affect the dynamics of tumor evolution and physiology thus creating clinical challenges. Tumor-intrinsic factors such as tumor heterogeneity, hypermutation, altered metabolomics and oncologically activated alternative splicing pathways change the tumor landscape to facilitate therapy failure and tumor progression. Moreover, tumor-extrinsic factors such as hypoxia and an immune-suppressive tumor microenvironment (TME) are the chief causes of immunotherapy failure in GBM. Amid the success of immunotherapy in other cancers, GBM has occurred as a model of resistance, thus focusing current efforts on not only alleviating the immunotolerance but also evading the escape mechanisms of tumor cells to therapy, caused by inter- and intra-tumoral heterogeneity. Here we review the various mechanisms of therapy resistance in GBM, caused by the continuously evolving tumor dynamics as well as the complex TME, which cumulatively contribute to GBM malignancy and therapy failure; in an attempt to understand and identify effective therapies for recurrent GBM.

For decades, research in cancer biology has been focused on the protein-coding fraction of the human genome. However, with the discovery of non-coding RNAs (ncRNAs), it has become known that these entities not only function in numerous fundamental life processes such as growth, differentiation, and development, but also play critical roles in a wide spectrum of human diseases, including cancer. Dysregulated ncRNA expression is found to affect cancer initiation, progression, and therapy resistance, through transcriptional, post-transcriptional, or epigenetic processes in the cell. In this review, we focus on the recent development and advances in ncRNA biology that are pertinent to their role in glioma tumorigenesis and therapy response. Gliomas are common, and are the most aggressive type of primary tumors, which account for ~30% of central nervous system (CNS) tumors. Of these, glioblastoma (GBM), which are grade IV tumors, are the most lethal brain tumors. Only 5% of GBM patients survive beyond five years upon diagnosis. Hence, a deeper understanding of the cellular non-coding transcriptome might help identify biomarkers and therapeutic agents for a better treatment of glioma. Here, we delve into the functional roles of microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA) in glioma tumorigenesis, discuss the function of their extracellular counterparts, and highlight their potential as biomarkers and therapeutic agents in glioma.

Widespread alterations in RNA alternative splicing (AS) have been identified in adult gliomas. However, their regulatory mechanism, biological significance, and therapeutic potential remain largely elusive. Here, using a computational approach with both bulk and single-cell RNA-Seq, we uncover a prognostic AS signature linked with neural developmental hierarchies. Using advanced iPSC glioma models driven by glioma driver mutations, we show that this AS signature could be enhanced by EGFRvIII and inhibited by in situ IDH1 mutation. Functional validations of 2 isoform switching events in CERS5 and MPZL1 show regulations of sphingolipid metabolism and SHP2 signaling, respectively. Analysis of upstream RNA binding proteins reveals PTBP1 as a key regulator of the AS signature where targeting of PTBP1 suppresses tumor growth and promotes the expression of a neuron marker TUJ1 in glioma stem-like cells. Overall, our data highlights the role of AS in affecting glioma malignancy and heterogeneity and its potential as a therapeutic vulnerability for treating adult gliomas.

Single-cell analysis has refined our understanding of cellular heterogeneity in glioma, yet RNA alternative splicing, a critical layer of transcriptome regulation, remains underexplored at single-cell resolution. Here, we present a pan-glioma single-cell alternative splicing analysis in both tumor and immune cells through integrating seven SMART-seq2 datasets of human gliomas to uncover overlooked isoform-level regulations shaping glioma progression and immune responses. Our analysis reveals lineage-specific alternative splicing regulation among glioma cellular states, with the most divergent alternative splicing landscapes observed between mesenchymal and neuronal-like glioma cells. Notably, this includes events in TCF12 exon 15 and PTBP2 exon 10, two key regulators of mesenchymal and neuronal transcriptomes. Inducing TCF12 exon 15 inclusion in glioma cells with a dCasRx-RBM25 system promotes neuronal gene expression, suppresses genes related to extracellular matrix organization, and enhances sensitivity to radiotherapy. Comparison of core and peripheral glioma cells highlights alternative splicing redox co-regulation of TPM1 and ACTN4, key genes involved in cytoskeletal organization. Further analysis of glioma-infiltrating immune cells reveals altered first exon usage of UGP2 in regulatory T cells and an association between MS4A7 alternative splicing in macrophages and clinical response to anti-PD-1 therapy. This study emphasizes the role of alternative splicing in glioma cellular heterogeneity, highlighting the importance of an isoform-centric approach to better understand the complex biological processes driving tumorigenesis.

Brain tumor-initiating cells (BTICs) and tumor cell plasticity promote glioblastoma (GBM) progression. Here, we demonstrate that clemastine, an over-the-counter drug for treating hay fever and allergy symptoms, effectively attenuated the stemness and suppressed the propagation of primary BTIC cultures bearing PDGFRA amplification. These effects on BTICs were accompanied by altered gene expression profiling indicative of their more differentiated states, resonating with the activity of clemastine in promoting the differentiation of normal oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes. Functional assays for pharmacological targets of clemastine revealed that the Emopamil Binding Protein (EBP), an enzyme in the cholesterol biosynthesis pathway, is essential for BTIC propagation and a target that mediates the suppressive effects of clemastine. Finally, we showed that a neural stem cell-derived mouse glioma model displaying predominantly proneural features was similarly susceptible to clemastine treatment. Collectively, these results identify pathways essential for maintaining the stemness and progenitor features of GBMs, uncover BTIC dependency on EBP, and suggest that non-oncology, low-toxicity drugs with OPC differentiation-promoting activity can be repurposed to target GBM stemness and aid in their treatment.