Bo Hu

Background: Recurrent isocitrate dehydrogenase (IDH)-mutant gliomas frequently acquire increased radioresistance, leading to poorer outcomes. Their underlying mechanisms, however, remain largely unknown. We hypothesize that dysregulated RNA alternative splicing (AS) during IDH-mutant glioma recurrence contributes to the enhanced radioresistance by influencing critical cellular pathways. Methods: RNA sequencing of paired primary and recurrent IDH-mutant gliomas were analyzed to identify recurrence-associated AS events. Functional effects were assessed in patient-derived glioma stem cells using RNA interference and CRISPR-dCas13-mediated isoform switching. Candidate upstream RNA-binding proteins and antisense oligonucleotide (ASO)-based therapeutic strategies were evaluated in vitro and in vivo. Results: We identified differentially spliced MutS homolog 5 (MSH5) isoforms between primary and recurrent IDH-mutant gliomas. Primary gliomas predominantly expressed an exon 11/12-skipped MSH5 transcript, whereas recurrent tumors largely retained the full-length isoform. Exon 11/12 skipping introduced a premature termination codon, leading to nonsense-mediated decay and diminished MSH5 expression in primary tumors. Further analyses identified elongation factor Tu GTP binding domain containing 2 (EFTUD2) as an upstream splicing regulator that was upregulated in recurrent tumors and promoted exon 11/12 inclusion, thereby maintaining MSH5 expression and enhancing the repair of radiation-induced DNA double-strand breaks. Inducing MSH5 exon 11/12 skipping with CRISPR-dCas13 or inhibiting EFTUD2 with ASOs reduced MSH5 expression, impaired DNA repair, and sensitized recurrent IDH-mutant glioma to radiotherapy in vitro and in vivo. Conclusions: These findings identify an EFTUD2-MSH5 splicing axis that contributes to radioresistance in recurrent IDH-mutant glioma. Therapeutic disruption of this splicing program may represent a strategy to enhance the radiation response in recurrent IDH-mutant glioma.

Background: In glioblastoma (GBM), epidermal growth factor receptor (EGFR) amplification, one of the most prevalent genetic alterations, often occurs on extrachromosomal DNA (ecDNA) elements that contain amplified oncogenes and regulatory elements, driving tumor progression. Despite the central oncogenic role of EGFR amplification, therapeutic strategies targeting EGFR have demonstrated limited clinical efficacy, suggesting that additional mechanisms may underlie EGFR-driven GBM malignancy and treatment resistance. Long non-coding RNAs (lncRNAs) are critical regulators in cancer; however, the roles of EGFR-associated lncRNAs—particularly those localized on ecDNA—in GBM tumorigenicity and therapeutic resistance remain poorly understood. Methods: Transcriptomic and genomic analyses were performed to identify lncRNAs co-amplified with EGFR. Biochemical and molecular biological studies were carried out to reveal the mechanisms. In vivo xenograft models were used to evaluate the tumorigenicity and the therapeutic efficacy of combination treatment strategies. Results: The lncRNA EGFR long non-coding downstream RNA (ELDR) was co-amplified with EGFR on ecDNA and chromosomes and was associated with poor prognosis in GBM. ELDR promoted GBM tumorigenicity through a BMI1-dependent epigenetic mechanism operating in parallel with canonical EGFR signaling. Mechanistically, ELDR interacted with purine-rich element-binding protein A (PURA), disrupted the inhibitory PURA-BMI1 interaction, and thereby enhanced the activity of BMI1, a core component of Polycomb repressive complex 1 (PRC1). Therapeutically, combining a BMI1 inhibitor or ELDR-targeting antisense oligonucleotides (ASOs) with an EGFR inhibitor erlotinib significantly enhanced antitumor efficacy in preclinical models of EGFR-amplified GBM with high ELDR expression. Conclusion: EGFR co-amplified ELDR promotes GBM tumorigenicity by enhancing BMI1 activity. Targeting the ELDR-BMI1 axis in combination with EGFR inhibition represents a promising therapeutic strategy for a subset of EGFR-amplified GBMs with high ELDR expression.

EGFR amplification frequently occurs within extrachromosomal DNAs (ecDNAs) and is the most prevalent mutation in glioblastoma (GBM). However, targeting EGFR for GBM treatments has been unsuccessful. Here we show a long non-coding RNA (lncRNA) that is co-amplified with EGFR, which we name hidden EGFR long non-coding downstream RNA (HELDR). HELDR is a GBM-selective lncRNA that promotes tumorigenicity independent of EGFR signalling. HELDR exhibits widespread chromatin association and recruits the transcription co-activator p300 to the KAT7 promoter. p300-induced H3K27ac at the KAT7 promoter enlists other co-transcription factors, activating KAT7 transcription. KAT7 induces H3K14ac and H4K12ac that activate KAT7-driven gene programmes that are critical for GBM malignancy. Targeting KAT7 or HELDR markedly enhances therapeutic effects of anti-EGFR treatments for GBM. These results not only reveal the role of HELDR in EGFR-amplified GBM but also provide a strong rationale to characterize the role of lncRNAs co-amplified with driver oncogenes in human cancers.

Background: Glioblastoma (GBM) is a highly aggressive brain tumor with limited treatment options. Tumor-associated astrocytes (TAAs) are crucial components of the GBM microenvironment, yet the contribution of alternative splicing (AS) in TAAs to tumor progression remains unclear. Methods: Transcriptomic and molecular analyses of GBM-associated astrocytes revealed a GBM-induced isoform switch in the RNA-binding protein Quaking (QKI) from the QKI-6 isoform to QKI-5 isoform. The biological role of QKI-5 was examined through gain- and loss-of-function approaches in human astrocytes and coculture systems with patient-derived glioma stem-like cells (GSCs). In vitro proliferation and sphere-formation assays, along with in vivo orthotopic xenograft models, were used to evaluate tumor growth. Immunoprecipitation and AlphaFold3 structural prediction were performed to investigate the mechanistic interaction between QKI-5 and sterol regulatory element-binding protein 2 (SREBP2). Results: GBM-induced QKI-5 interacts with SREBP2 to transcriptionally activate cholesterol metabolic enzymes, enhancing astrocyte-derived cholesterol production and promoting GBM growth. Knockdown of QKI-5 or inhibitors for SREBP2-driven signaling suppressed astrocyte-mediated tumor-supportive effects in vitro and in vivo. Conclusion: QKI-5 drives astrocytic metabolic reprogramming via the QKI-5-SREBP2 axis, fostering a cholesterol-rich tumor microenvironment that supports GBM progression. Targeting this pathway offers a potential therapeutic strategy.

Background: Cysteine is a multifunctional amino acid that can be oxidized affecting disulfide bond formation, redox signaling, and protein function. Reactive oxygen species (ROS) and the metabolic environment dictate cysteine uptake and oxidation status—especially in redox sensitive pathways. As many chemotherapeutic agents increase ROS, including the standard care for glioblastoma (GBM), temozolomide (TMZ), we hypothesized that TMZ-resistant (TMZ-R) GBM would have increased ROS affecting cysteine reactivity that could be therapeutically targeted. Methods: Here, to study the metabolic state within drug sensitive and resistant GBM, we used metabolite tracing with13 C-Cyst(e)ine, specialized cysteine reactivity proteomics and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) screening with drug treatments to determine the efficacy of targeting cysteine metabolic pathways with our designer selenium drug in both patient-derived cell lines and patient-derived xenograft GBM ­orthotopic models. Results: We show that TMZ-R have increased cyst(e)ine uptake, cysteine reactivity, and sensitivity to selenium (Se)-containing compounds—which can bind cysteine—in vitro and in vivo. We show that in TMZ-R models selenium compound treatment increases the need for thioredoxin reductases where co-treatment of Se compounds and the thioredoxin inhibitor auranofin significantly improves overall survival in mouse models. Conclusions: Overall, our findings show a unique metabolic environment in TMZ-R models where designer brain penetrant Se-containing compounds target cysteine reactivity within proteins necessary for cancer cell survival and hold therapeutic potential.

EGFR amplification frequently happens within extrachromosome DNAs (ecDNAs) and is a major mutation in glioblastoma (GBM). However, targeting EGFR for GBM treatments has been unsuccessful. Here we characterized a long non-coding RNA (lncRNA) that is co-amplified with EGFR within ecDNAs that we name hidden EGFR long non-coding downstream RNA (HELDR). HELDR is a GBM-specific lncRNA that promotes tumorigenicity independent of EGFR signaling. HELDR globally binds genomic DNA and recruits the transcription co-activator p300 to the KAT7 promoter. p300-induced H3K27ac at the KAT7 promoter enlists other co-transcription factors, activating KAT7 transcription. KAT7 induces H3K14ac and H4K12ac that activate KAT7-driven gene programs that are critical for GBM malignancy. Targeting KAT7 or HELDR markedly enhances therapeutic effects of anti-EGFR treatments for GBM. These results not only reveal the role of HELDR in EGFR-driven GBM but also provide a strong rationale to characterize the role of lncRNAs co-amplified with driver oncogenes in human cancers.

Background: Isocitrate dehydrogenase mutant (IDH-mut) gliomas represent a distinct subtype of glioma, characterized by a relatively better prognosis compared to IDH wildtype (wt) glioblastoma (GBM). Despite this advantage, these tumors remain incurable due to the limited availability of effective treatments. Targeting SHP2, a non-receptor protein tyrosine phosphatase, is a promising therapeutic strategy for several types of human cancers. In this study, we aim to determine the efficacy of SHP inhibition in IDH-mut gliomas. Methods: Bioinformatic and biological analyses revealed increased expression and activation of the PDGFRA-SHP2-ERK pathway in clinical IDH-mut gliomas and patient-derived IDH-mut glioma stem-like cells (GSCs). The effects of SHP2 inhibition, alone or with radiation therapy (RT), were assessed through assays including cell growth, sphere formation, cell differentiation markers, flow cytometry, immunoblotting, immunohistochemistry, and orthotopic brain tumor xenografts. Results: PDGFRA expression was elevated in IDH-mut gliomas and GSCs, activating the SHP2-ERK pathway. SHP099, a SHP2 inhibitor, reduced GSC tumorigenicity in vitro and in vivo by disrupting SHP2-ERK signaling and promoting differentiation. SHP099 also enhanced cytotoxicity of RT, the standard treatment for IDH-mut glioma, in IDH-mut GSCs and orthotopic glioma models. Mechanistically, the PDGFRA-SHP2-ERK axis is activated in IDH-mut gliomas and RT further activates this pathway. Targeting SHP2 suppressed ERK signaling thereby enhancing the therapeutic effect of RT. Conclusion: Combining SHP2 inhibition with RT is a promising therapeutic avenue for IDH-mut glioma by suppressing the activated SHP2-ERK axis.