Resistant Glioblastoma Research Articles

A Metal-Phenolic Network Nanoresensitizer Overcoming Glioblastoma Drug Resistance through the Metabolic Adaptive Strategy and Targeting Drug-Tolerant Cells.

Glioblastoma (GBM) is the most lethal primary brain tumor with limited therapeutic efficiency because of resistance to Temozolomide (TMZ), which is the standard chemotherapy drug. Here, we developed the metabolic adaptive strategy based on the complex TMZ resistance mechanisms, and engineered metal-phenolic networks (TBFP-MT MPNs) by self-assembly of PEG-polyphenol encapsulating FeIII, TMZ, and dihydroorotate dehydrogenase (DHODH) inhibitor, modifying T10 and cMBP for blood-brain barrier (BBB) penetration and targeting resistant cells. TBFP-MT suppressed drug efflux by inhibiting mesenchymal epithelial transition (MET) signaling and reduced DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT) by blocking pyrimidine synthesis via DHODH inhibition. Additionally, it triggered ferroptosis by disrupting the DHODH/GPX4 defense systems, overcoming the tumor cell survival mechanisms. In vitro and in vivo studies confirmed its ability to suppress resistant GBM growth and extend survival. This study reveals drug-resistant cell vulnerabilities and provides a new pathway to overcome chemoresistance by disrupting multiple resistance mechanisms in GBM.

Emerging Insights into the PI3K/AKT/mTOR Signaling Pathway and Non-Coding RNA-mediated Drug Resistance in Glioblastoma.

Glioblastoma multiforme [GBM] is a highly aggressive grade IV central nervous system tumor with a dismal prognosis. Factors such as late detection, treatment limitations due to its aggressive nature, and, notably, drug resistance significantly affect clinical outcomes. Despite the effectiveness of Temozolomide [TMZ], a potent chemotherapy agent, the development of drug resistance remains a major challenge. Given the poor survival rates and chemoresistance, there is an urgent need for novel treatment strategies. Non-coding RNAs, particularly microRNAs [miRNAs], offer a promising approach to GBM diagnosis and treatment. These small non-coding RNAs play crucial roles in tumor progression, either suppressing or promoting oncogenic characteristics. The phosphoinositide-3 kinase [PI3K]/AKT/ mTOR pathway, which regulates essential biological processes like proliferation and survival, is a key target of miRNAs in cancer. Studies have underscored the significance of PI3K/AKT/mTOR signaling in drug resistance development and its interplay with non-coding RNAs as mediators of tumorigenesis. This review aims to outline the involvement of PI3K/AKT/mTOR signaling in miRNA modulation and strategies to overcome chemoresistance in GBM.

Role of extracellular vesicles in immune modulation, angiogenesis, progression and therapeutic resistance of glioblastoma

Role of extracellular vesicles in immune modulation, angiogenesis, progression and therapeutic resistance of glioblastoma

Transglutaminase 2 nuclear localization enhances glioblastoma radiation resistance

Radiotherapy remains the cornerstone of treatment for glioblastoma (GBM). However, the frequent occurrence of radiation resistance presents a significant therapeutic challenge. A comprehensive understanding of the mechanisms underlying this resistance is essential for improving GBM treatment strategies. In the present study, live-dead cell staining and immunofluorescence staining were employed, and irradiation-resistant cell lines were established. It was observed that transglutaminase 2 (TGM2) plays a pivotal role in enhancing radiation resistance in GBM, facilitating cell proliferation, and promoting DNA damage repair following irradiation. Moreover, immunofluorescence and nucleoplasmic protein extraction assays revealed that TGM2 in GBM rapidly translocates into the nucleus upon irradiation. Through co-immunoprecipitation assays, TGM2 was identified as binding to an increased amount of p53 proteins, thereby promoting p53 degradation post-irradiation. Notably, inhibition of this interaction resulted in a reduction of radiation resistance in GBM. In summary, this study underscores the significance of TGM2 nuclear translocation in radiation resistance and suggests that disrupting TGM2 binding to p53 may offer novel therapeutic insights for overcoming radiation resistance in GBM.

Elevated nonhomologous end-joining by AATF enables efficient DNA damage repair and therapeutic resistance in glioblastoma

Glioblastoma (GB) is a highly aggressive brain tumor resistant to chemoradiotherapy, largely due to glioma stem-like cells (GSCs) with robust DNA damage repair capabilities. Here we reveal that GSCs enhance their DNA repair capacity by activating non-homologous end-joining (NHEJ) through upregulation of the apoptosis antagonizing transcription factor (AATF), thereby promoting therapeutic resistance in GB. AATF interacts with XRCC4, a core NHEJ subunit, preventing its degradation via ubiquitin-mediated proteasomal processes. Upon DNA damage, AATF undergoes phosphorylation at Ser189 by ATM, leading to its dissociation from XRCC4 and rapid recruitment of XRCC4 to DNA break sites for efficient NHEJ repair. Moreover, AATF depletion or deficient AATF phosphorylation impedes NHEJ in GSCs, sensitizing GB xenografts to chemoradiotherapy. Additionally, elevated levels of AATF inform poor prognosis in GB patients. Collectively, our findings unveil a crucial role of AATF in XRCC4-mediated NHEJ repair, and underscore targeting AATF as a potential strategy to overcome GB resistance to chemoradiotherapy.

Polyamine acetylation mediates crosstalk between cancer cells and myeloid cells to promote mesenchymal/plurimetabolic states in glioblastoma.

Metabolic reprogramming in glioblastoma (GBM) is a putative determinant of GBM subtype, malignant cell state and tumor-immune crosstalk. In the present study, we investigated how polyamine metabolic rewiring contributes to the malignant cell-intrinsic and microenvironment-dependent biological processes underpinning GBM subtype classification. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) was used for polyamine quantification in human and murine GBM tumors and cell lines. Through single-cell RNA sequencing, metabolic profiling and additional functional experiments, we dissect the malignant cell-intrinsic and paracrine signaling processes regulated by SAT1 (spermidine/spermine-N1-acetyltransferase1) and its product, N1-acetylspermidine. We find that polyamine acetylation is elevated in human and murine GBM tumors and contributes to the classification of mesenchymal/plurimetabolic GBM through both regulation of tumor-cell intrinsic glucose metabolism and by facilitating metabolic crosstalk with tumor-associated macrophages/myeloid cells (TAMs). The impact of SAT1 on tumor cell metabolism is mediated, at least in part, by N1-acetylspermdine, the sole polyamine elevated in human and murine tumors. Furthermore, the relatively high levels of N1-acetylspermidine released by GBM is taken up by myeloid cells to promote intracellular polyamine flux, cellular respiration and migration. In vivo, both genetic disruption of polyamine acetylation and pharmacological inhibition of polyamine transport reduced myeloid cell infiltration and sensitized tumors to chemoradiation. Collectively, the findings highlight a previously unidentified role for SAT1 and its product, N1-acetylspermidine, in bridging the metabolic activity of tumor cells and tumor-associated macrophages/myeloid cells (TAMs), together promoting mesenchymal/plurimetabolic states and therapeutic resistance in GBM.

ATP11B triggers the infiltration of T cells into GBM and intensifies anti-GBM immunity by upregulating and externalizing S1PR1

BackgroundInsufficient T-cell infiltration in tumours causes immune checkpoint inhibitor (ICI) resistance in glioblastoma (GBM) patients. The aim of this study was to demonstrate a preferable way to facilitate T-cell infiltration and improve the therapeutic effects of ICIs in GBM.MethodsFlow cytometry, western blot and immunofluorescence staining were used to detect the effects of ATP11B upregulation on S1PR1 expression and distribution, T-cell infiltration and differentiation. A coculture system and an intracranial GBM model were established to explore the anti-GBM potential of ATP11B/S1PR1 signaling through systemic administration of CD3-DSPE-PEG2K-NHS/ATP11B nanoparticles to specifically deliver ATP11B overexpressing plasmids to T cells.ResultsS1PR1 deficiency in T cells caused T-cell lymphopenia and systemic immunosuppression in GBM, whereas ATP11B overexpression induced the upregulation and externalization of S1PR1 on T-cell membranes, thus increasing the ability of T cells to eliminate GBM cells. In intracranial GBM models, an ATP11B overexpression plasmid was specifically delivered to T cells in the peripheral blood, bone marrow and spleen, then triggering the infiltration of T cells deeply into the GBM and reversing systemic immunosuppression, ultimately enhancing the therapeutic outcomes of ICIs.ConclusionsThe upregulation and externalization of S1PR1 on T cells mediated by ATP11B overexpression may be promising immunotherapeutic alternatives for GBM treatment.

Post-translational modifications in DNA damage repair: mechanisms underlying temozolomide resistance in glioblastoma.

Temozolomide (TMZ) resistance is one of the critical factors contributing to the poor prognosis of glioblastoma (GBM). As a first-line chemotherapeutic agent for GBM, TMZ exerts its cytotoxic effects through DNA alkylation. However, its therapeutic efficacy is significantly compromised by enhanced DNA damage repair (DDR) mechanisms in GBM cells. Although several DDR-targeting drugs have been developed, their clinical outcomes remain suboptimal. Post-translational modifications (PTMs) in GBM cells play a pivotal role in maintaining the genomic stability of DDR mechanisms, including methylguanine-DNA methyltransferase-mediated repair, DNA mismatch repair dysfunction, base excision repair, and double-strand break repair. This review focuses on elucidating the regulatory roles of PTMs in the intrinsic mechanisms underlying TMZ resistance in GBM. Furthermore, we explore the feasibility of enhancing TMZ-induced cytotoxicity by targeting PTM-related enzymatic to disrupt key steps in PTM-mediated DDR pathways. By integrating current preclinical insights and clinical challenges, this work highlights the potential of modulating PTM-driven networks as a novel therapeutic strategy to overcome TMZ resistance and improve treatment outcomes for GBM patients.

Epigenetic mechanisms of plasticity and resistance in glioblastoma: therapeutic targets and implications

Glioblastoma (GBM), a highly aggressive and malignant form of primary adult brain cancer, poses significant therapeutic challenges. Despite our improved understanding of the cellular and molecular mechanisms underlying tumorigenesis and the evolution of GBM, targeted molecular therapies have failed to improve patient survival outcomes. The failure of standard treatments and targeted therapies is mainly attributed to the acquisition of phenotypic plasticity of tumor cells and GBM stem-like cells. Epigenetic modifications and their mediators have emerged as crucial regulators of phenotypic plasticity, influencing tumor heterogeneity, therapy resistance and disease progression. Here, we summarize and provide insights into epigenetic regulation of GBM plasticity and specifically, focus on the roles played by DNA- and histone modifiers and non-coding RNAs in driving phenotypic plasticity and resistance. We also delve into their dynamics in response to standard therapies and the challenges for targeting them to overcome phenotypic plasticity and resistance in GBM.

Biomimetic Hybrid PROTAC Nanovesicles Block Multiple DNA Repair Pathways to Overcome Temozolomide Resistance Against Orthotopic Glioblastoma.

Glioblastoma (GBM) remains one of the deadliest forms of cancer due to its high rates of postoperative recurrence and resistance to treatment. Temozolomide (TMZ) is the standard chemotherapy for GBM. However, the therapeutic efficacy of TMZ is significantly compromised by the activation of various intracellular DNA repair mechanisms that facilitate TMZ resistance. Herein, the upregulation of bromodomain-containing protein 4 (BRD4) expression is demonstrated to be a key contributor to TMZ resistance in GBM. To address this challenge, a biomimetic hybrid PROteolysis TArgeting Chimeras (PROTAC) liposome delivery system (M@TP) is developed. This system efficiently penetrates the blood-brain barrier (BBB) and specifically targets GBM cells through homotypic recognition. Once within TMZ-resistant GBM cells, the released PROTAC from M@TP can specifically degrade BRD4, effectively inhibiting multiple DNA repair pathways and restoring sensitivity to TMZ treatment. In vivo, studies showed that M@TP demonstrated significant efficacy in suppressing tumor growth in both TMZ-resistant and postoperative GBM, with prolonged mouse survival times. These findings highlight the potential of M@TP as a promising strategy to overcome TMZ resistance and improve therapeutic outcomes in GBM.

Regulatory Mechanisms of STAT3 in GBM and its Impact on TMZ Resistance.

STAT3, a key member of the Signal Transducer and Activator of Transcription (STAT) family, plays a vital role in the development and progression of glioblastoma (GBM), as well as in the resistance to the chemotherapy drug temozolomide (TMZ). This review outlines the dysregulation of STAT3 in GBM, focusing on its activation mechanisms and its contribution to TMZ resistance. STAT3 can be activated by cytokines, like IL-6, growth factors, and membrane receptors, like EGFR. In GBM, constitutively active STAT3 enhances tumor growth and therapy resistance. Specifically, resistance to TMZ, a standard chemotherapeutic agent for GBM, is facilitated by STAT3-induced expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase and anti-apoptotic proteins like Bcl-2, as well as through the regulation of microRNAs. To combat TMZ resistance in GBM, strategies that inhibit STAT3 activity have been explored. Recent advancements, such as the use of small molecule inhibitors targeting STAT3 and its upstream or downstream regulators, RNA-based therapies, as well as the development of nanocarriers for targeted delivery of STAT3-targeting small interfering RNA across the blood-brain barrier, have demonstrated significant potential in enhancing the sensitivity of GBM to TMZ. These targeted therapies hold promise for improving the treatment outcomes of patients with GBM.

Centromere protein U mediates the ubiquitination and degradation of RPS3 to facilitate temozolomide resistance in glioblastoma.

Centromere protein U mediates the ubiquitination and degradation of RPS3 to facilitate temozolomide resistance in glioblastoma.

Tumoroid Model Reveals Synergistic Impairment of Metabolism by Iron Chelators and Temozolomide in Chemo-Resistant Patient-derived Glioblastoma Cells.

Chemoresistance poses a significant clinical challenge in managing glioblastoma (GBM), limiting the long-term success of traditional treatments. Here, a 3D tumoroid model is used to investigate the metabolic sensitivity of temozolomide (TMZ)-resistant GBM cells to iron chelation by deferoxamine (DFO) and deferiprone (DFP). This work shows that TMZ-resistant GBM cells acquire stem-like characteristics, higher intracellular iron levels, higher expression of aconitase, and elevated reliance on oxidative phosphorylation and proteins associated with iron metabolism. Using a microphysiological model of GBM-on-a-chip consisting of extracellular matrix (ECM)-incorporated tumoroids, this work demonstrates that the combination of iron chelators with TMZ induces a synergistic effect on an in vitro tumoroid model of newly diagnosed and recurrent chemo-resistant patient-derived GBM and reduced their size and invasion. Investigating downstream metabolic variations reveal reduced intracellular iron, increased reactive oxygen species (ROS), upregulated hypoxia-inducible factor-1α, reduced viability, increased autophagy, upregulated ribonucleotide reductase (RRM2), arrested proliferation, and induced cell death in normoxic TMZ-resistant cells. Hypoxic cells, while showing similar results, display reduced responses to iron deficiency, less blebbing, and an induced autophagic flux, suggesting an adaptive mechanism associated with hypoxia. These findings show that co-treatment with iron chelators and TMZ induces a synergistic effect, making this combination a promising GBM therapy.

TLK1 as a therapeutic target in TMZ resistant glioblastoma using small molecule inhibitor

The acquired resistance to existing therapies poses a grave concern in achieving successful therapeutic outcomes. Temozolomide (TMZ), a widely used alkylating chemotherapeutic in Glioblastoma therapy, often encounters resistance, necessitating the investigation of the underlying mechanisms of TMZ-acquired resistance. To study TMZ resistance, a cell-based model system was generated by intermittently exposing glioblastoma cells to increasing concentrations and time of TMZ over six months. The survival response of cells at higher concentrations confirmed TMZ-resistant cells, which exhibited a phenotypic shift toward a mesenchymal-like state, with decreased epithelial traits, indicating mesenchymal-epithelial transition (MET). This transition likely facilitates the stabilization and clonal growth of TMZ-resistant cells. Subsequent analysis revealed elevated expression of TLK1, a DNA repair protein, thus reinforcing its potential involvement in mechanisms associated with acquired resistance. To explore the therapeutic aspect of TLK1 inhibition, we utilized an in-house developed TLK1 inhibitor, J54. The inhibition of TLK1 in TMZ-resistant cells enhanced cytotoxicity, indicating TLK1 as a potential target to combat TMZ resistance. Moreover, TLK1 inhibition reduced cell migration and invasion, implying its role in promoting metastasis. In conclusion, our study sheds light on the role of TLK1 in the context of TMZ resistance, highlighting its potential as a valuable target for therapeutic intervention.

Purine metabolism rewiring improves glioblastoma susceptibility to temozolomide treatment

Glioblastoma (GBM) is among the deadliest cancers, characterized by poor prognosis and median survival of 12–15 months post-diagnosis. Despite aggressive therapeutic regimens, GBM treatment is still an unmet clinical need due to heterogeneity, recurrencies, and resistance. Metabolic reshaping is emerging as a critical mechanism supporting cell proliferation and sustaining chemoresistance. In this study, we explored metabolic changes induced by chemotherapy in temozolomide (TMZ)-sensitive and TMZ-resistant GBM cell lines. We found that purine levels were altered in sensitive versus resistant GBM cells, highlighting a critical role of guanosine and inosine metabolism. By using a mesenchymal-like GBM zebrafish model, we uncovered dysregulated pathways involved in purine metabolism, with a downregulation of catabolic processes. Our data indicate that combined treatment with TMZ plus guanosine and inosine increased cytotoxicity, enhancing chemotherapy effectiveness in TMZ-resistant cells. These effects correlated with alterations in mitochondrial dynamics and activity. Specifically, the combinatorial effectiveness of TMZ with guanosine and inosine was linked to Mitofusin-2 overexpression, enhancing mitochondrial fusion, typically associated with a better prognosis. Therefore, our findings suggest that purine metabolism is involved in the metabolic rewiring of TMZ-resistant cells, suggesting guanosine and inosine as potential adjuvant treatments to improve the cytotoxicity effects of chemotherapy in resistant GBM.

Mapping the spatial architecture of glioblastoma from core to edge delineates niche-specific tumor cell states and intercellular interactions.

Treatment resistance in glioblastoma (GBM) is largely driven by the extensive multi-level heterogeneity that typifies this disease. Despite significant progress toward elucidating GBM's genomic and transcriptional heterogeneity, a critical knowledge gap remains in defining this heterogeneity at the spatial level. To address this, we employed spatial transcriptomics to map the architecture of the GBM ecosystem. This revealed tumor cell states that are jointly defined by gene expression and spatial localization, and multicellular niches whose composition varies along the tumor core-edge axis. Ligand-receptor interaction analysis uncovered a complex network of intercellular communication, including niche- and region-specific interactions. Finally, we found that CD8 positive GZMK positive T cells colocalize with LYVE1 positive CD163 positive myeloid cells in vascular regions, suggesting a potential mechanism for immune evasion. These findings provide novel insights into the GBM tumor microenvironment, highlighting previously unrecognized patterns of spatial organization and intercellular interactions, and novel therapeutic avenues to disrupt tumor-promoting interactions and overcome immune resistance.

Abstract 3085: Upregulation of TAGLN2-PI3K-AKT signaling promotes therapeutic resistance in glioblastoma through a positive feedback mechanism

Abstract Objective: Despite aggressive treatment with surgery, radiation and temozolomide chemotherapy glioblastoma (GBM) remains the most lethal tumor of the human brain, which is largely due to inevitable recurrence of treatment refractory tumor. Studies have shown that aberrant activation of AKT significantly contributes to the development of treatment resistance in GBM. Molecular profiling and in vitro studies from our group identified TAGLN2, an actin binding cytoskeletal protein, as a top upregulated gene in GBM. In other cancer types, TAGLN2 has been shown to activate the PI3K-AKT pathway resulting in treatment resistance. This study aims to explore how TAGLN2 drives treatment resistance in GBM. Methods: PDX primary human GBM cell lines (08-387 and GBM 3359) were used. Co-immunoprecipitation (Co-IP) assays were performed to study the interaction of PTEN with TAGLN2 in vitro. Biacore/Surface plasmon resonance (SPR) analyses were performed to study binding affinity and kinetics of their interaction. Protein-protein interactions were validated by in silico (AlphaFold2) analysis. Protein expression was quantified by WB analysis. Cell viability was assessed by MTS assays. Results: We have previously shown that RNAi-mediated knockdown (KD) of TAGLN2 significantly reduced cell proliferation, survival, clonal expansion and the invasive potential of GBM PDX cell lines in vitro and reduced tumor growth using preclinical in vivo models. We found that KD of TAGLN2 reduced the levels of activated/phosphorylated AKT (pAKT) in GBM cells resulting in downregulation of this pathway. Subsequently, we found that TAGLN2 physically associated with PTEN as evidenced by Co-IP, SPR and in silico analysis. Binding of TAGLN2 to PTEN may attenuate PTEN function resulting in the activation of downstream oncogenic pathways. mTORC2 and DNA-PK are known to phosphorylate and activate AKT resulting in the upregulation of downstream signaling. Here, we show that inhibition of these kinases either individually or in combination reduced TAGLN2 protein levels in GBM cells in vitro. Importantly, direct inhibition of Akt phosphorylation also resulted in the reduction of TAGLN2 protein levels indicating that activated Akt stabilized TAGLN2 (likely by phosphorylation), which in turn promoted oncogenic pathways via a positive feedback mechanism. Also, KD of TAGLN2 increased the sensitivity of GBM cells to both TMZ and RT as assessed by multiple in vitro models. Conclusion: Taken together, our data suggest that Transgelin-2 promotes chemo-radiation treatment resistance in GBM through a novel positive feedback mechanism that involves the TAGLN2-PTEN-PI3K-AKT axis. Therefore, TAGLN2 may serve as potential therapeutic target in GBM and to this end, development of small molecule inhibitors of TAGLN2 is actively underway in our laboratory. Citation Format: Ashok Kumar, Sasha Beyer, Bryan Pérez-Soto, Anca-Ligia Grosu, Steffen Lindert, Heather R. Manring, Saikh Jaharul Haque, Arnab Chakravarti. Upregulation of TAGLN2-PI3K-AKT signaling promotes therapeutic resistance in glioblastoma through a positive feedback mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3085.

Abstract 5395: CDK12 regulates cellular metabolism to promote therapy resistance in glioblastoma

Abstract Glioblastoma IDH-wildtype, the most common primary brain cancer in adults, has an exceedingly poor prognosis of only one to two years despite standard treatment involving surgery, temozolomide, and radiation. We evaluated patient-derived xenograft GBM cultures to determine their sensitivity to disrupting the function of CDK12, which was achieved through genetic manipulation, shRNA, or the novel inhibitor SR-4835. GBM metabolism was assessed using extracellular flux analysis and LC/MS-based carbon tracing. The in vivo efficacy was evaluated in orthotopic GBM mouse models. Comprehensive screening approaches identified CDK12 as essential for GBM growth. Consistently, inhibiting CDK12 function led to reduced growth in GBM PDX lines in vitro, but not in human astrocytes. Notably, SR-4835 inhibited GBM growth at low nanomolar concentrations and synergistically enhanced the potency of temozolomide. In vivo, SR-4835 activated cell death machinery related to intrinsic apoptosis. Metabolic analyses demonstrated that the loss of CDK12 function substantially impaired cellular respiration, coupled with the suppression of key metabolic transcriptional regulators, PGC1A and PPARD. This resulted in energy depletion and apoptotic cell death, partially mediated by the pro-apoptotic protein Noxa. The inactivation of CDK12 significantly extended the overall survival time without any harmful side effects in orthotopic mouse models of GBM. In some subjects, it also led to the complete elimination of brain tumors. When used together with temozolomide, blocking CDK12 eradicated brain tumors in all animals studied, indicating that CDK12 is a highly promising treatment target for GBM and providing a solid foundation for future clinical trials involving patients with this devastating disease. Citation Format: Jeong-Yeon Mun, Chang Shu, Qiuqiang Gao, Hasan O. Akman, Mike-Andrew Westhoff, Georg Karpel-Massler, Markus D. Siegelin. CDK12 regulates cellular metabolism to promote therapy resistance in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5395.

Abstract 6945: Targeting Chek2-YBX1&3 axis to enhance response to immune checkpoint blockade therapy in gliomas

Abstract Although the immunosuppressive microenvironment of glioblastoma (GBM) is well-established, the tumor-intrinsic mechanisms underlying resistance to CD8+ T cell-mediated killing remain incompletely understood. Our previous study identified Checkpoint Kinase 2 (Chek2) as the top contributor to CD8+ T cell resistance in GBM through an in vivo CRISPR screen. Therapeutic evaluation demonstrated that Chek2 inhibition, combined with PD-1/PD-L1 immune checkpoint blockade, significantly enhanced CD8+ T cell-mediated tumor killing and improved survival in preclinical glioma models. Here, we aimed to elucidate the immunosuppressive function of Chek2. Immunoprecipitation-mass spectrometry (IP-MS) and phosphoproteomics identified the transcriptional repressors and RNA-stabilizing proteins YBX1 and YBX3 as critical interactors of Chek2. Single-gene knockout and overexpression studies of Chek2, YBX1, and YBX3 in multiple glioma cell lines revealed that these proteins positively regulate each other’s expression at the protein level. RNA sequencing coupled with ChIP-seq analysis demonstrated that the Chek2-YBX1-YBX3 interaction represses the expression of inflammatory cytokines. Targeting Chek2, YBX1, and YBX3 with the small-molecule inhibitor SU056 enhanced antigen presentation and promoted CD8+ T cell proliferation. Moreover, combining SU056 with PD-1 blockade significantly improved survival in multiple glioma models. Collectively, these findings reveal an immunosuppressive mechanism mediated by the Chek2-YBX1-YBX3 axis and propose its inhibition as a promising therapeutic strategy to enhance responses to immune checkpoint blockade therapies in gliomas. Citation Format: Heba Ali, Ningjia Zhou, Li Chen, Vivekanudeep Karri, Mahalakshmi Harish, Yalu Zhou, Vasilios Kalas, Karl Habashy, Victor A. Arrieta, Kwang-Soo Kim, Si Wang, Joseph Duffy, Ragini Yeeravalli, Deanna M. Tiek, Xiao Song, Snehasis Mishra, Catalina Leechang, Atique Ahmed, Prasanna Venkatraman, Gabriel J. Rocklin, Adam M. Sonabend, Crismita Dmello. Targeting Chek2-YBX1&3 axis to enhance response to immune checkpoint blockade therapy in gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6945.

Abstract 2901: Targeting miR-19b/PPP2R5E regulatory axis to enhance alkylating agent efficacy: a new avenue to counteract temozolomide resistance in recurrent glioblastomas

Abstract Glioblastoma (GBM), the most aggressive primary brain tumor, notorious for its inevitable recurrence despite standard of care, presents a pressing need for novel treatment paradigms. Temozolomide (TMZ), a frontline DNA alkylating agent, induces extensive DNA damage. However, TMZ response is counteracted by intricate or acquired survival mechanisms. Through functional TMZ-resistance screens using microRNAs, we uncovered a novel mechanism involving the miR-19b target, PPP2R5E, a subunit of PP2A serine-threonine phosphatase, in TMZ response. Specifically, the induced expression of PPP2R5E, resulting from miR-19b attenuation, triggers genotoxic stress, priming GBM cell lines and primary GBM cells for heightened TMZ cytotoxicity. Elevated DNA damage in PPP2R5E-overexpressing cells is attributed to increased nuclear and mitochondrial ROS production, which is accompanied by enhanced ROS-mediated senescence and ferroptosis. Of note, all these phenotypes are reversed in GBM cells with concomitant miR-19b and PPP2R5E attenuation, indicating that miR-19b exerts its role in TMZ resistance by targeting PPP2R5E. In line with this finding, our orthotopic human-derived GBM stem cell xenograft model confirmed that PPP2R5E attenuation not only decreases TMZ cytotoxicity in the brain, but also at the spinal metastatic location. Consistently, treating cells with the PP2A-activating drug FTY720 or knocking down endogenous PP2A-inhibiting proteins mirrors the effects of miR-19b attenuation in enhancing TMZ cytotoxicity. This intricate interplay between miR-19b and PPP2R5E underscores a novel strategy to modulate the TMZ response, highlighting promising results in our pre-clinical models. Intriguingly, PP2A inhibiting proteins CIP2A and ANP32A (PHAP1) are upregulated in TMZ-resistant GBM cells and recurrent GBM tumors following TMZ treatment, suggesting a role of the PPP2R5E axis in TMZ resistance. In conclusion, our findings advocate for the therapeutic targeting of the miR-19b/PPP2R5E axis as a promising strategy to overcome TMZ resistance in GBM, indicating a potential novel strategy in the pursuit of effective therapeutic interventions for the formidable recurrent GBM tumors. Citation Format: Erik Vassella, Elham Kashani. Targeting miR-19b/PPP2R5E regulatory axis to enhance alkylating agent efficacy: a new avenue to counteract temozolomide resistance in recurrent glioblastomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2901.