Glioma-associated isocitrate dehydrogenase (IDH) mutations synthesize (R)-2-hydroxyglutarate (R2HG), which broadly inhibits 2-oxoglutarate-dependent enzymes. Given that mutant IDH inhibitors have limited efficacy in high-grade glioma subtypes, alternative treatment strategies are needed, such as therapies that exploit collateral vulnerabilities engendered by IDH mutations. In this vein, drugs that induce replication stress, such as Ataxia-telangiectasia and Rad3-related (ATR) inhibitors and dihydroorotate dehydrogenase (DHODH) inhibitors, have demonstrated efficacy in preclinical IDH-mutant glioma models. However, translation of this synthetic lethal framework to clinical testing has been limited in part by a lack of well-tolerated, effective, and on-target drugs that exploit this vulnerability. To address this need, we developed GLIO-1, a new small molecule inhibitor of DHODH. GLIO-1 preferentially killed IDH-mutant glioma cells in engineered, patient-derived, and orthotopic xenograft model systems. In a patient-derived orthotopic xenograft mouse model of IDH-mutant glioma, GLIO-1 displayed similar monotherapy antitumor efficacy and superior tolerability compared to a leading clinical-stage DHODH inhibitor (BAY 2402234). GLIO-1 also exhibited superior brain penetrance compared to BAY 2402234. The effects of GLIO-1 were on-target, as expression of a drug-resistant DHODH mutant (DHODH-A58T) completely rescued cell death induced by GLIO-1. To mechanistically understand how IDH-mutant gliomas are sensitized to DHODH inhibitors and replication stress, we performed forward genetic screens targeting all 2-oxoglutarate-dependent enzymes. Across six independent CRISPR screens using both DHODH and ATR inhibitors, we discovered that KDM6 histone demethylases play a vital role in protecting glioma cells from replication stress. Base editor screens directed against the KDM6A gene revealed that the demethylase activities of KDM6 enzymes are required for replication stress tolerance. Genetic or R2HG-dependent repression of KDM6 catalytic activity sensitized engineered, patient-derived, and xenograft models of glioma to both ATR and DHODH inhibitors. This liability was generalizable even to non-neural cells because KDM6A loss-of-function mutations commonly observed in urothelial carcinomas sensitized bladder cancer cells to DHODH inhibition including GLIO-1, thereby phenocopying IDH mutations in glioma. We developed an selective, on-target, and well-tolerated DHODH inhibitor, GLIO-1, that is effective in IDH-mutant gliomas. Moreover, we identify KDM6 enzymes as the mechanistic targets of R2HG that link IDH oncogenes with sensitivity to GLIO-1 and reveal KDM6 activity as a fundamental determinant of replication stress. Collectively, these results nominate a new pan-cancer biomarker and targeted therapy pairing, KDM6A inactivation and GLIO-1, that is poised for clinical translation.
Alexander C-Y. Tsai, Mathew D. Lin, Vinesh T. Puliyappadamba, Dorothy M. Junginger, Victoria G. Donovan, Eleanor G. Kaplan, Laura M. Drepanos, Hiroaki Wakimoto, Daniel P. Cahill, Julie A. Losman, Kent W. Mouw, Kalil G. Abdullah, John G. Doench, Duane Nash, Daniel Vitt, Christian Gege, Hella Kohlhof, Samuel K. McBrayer, William G. Kaelin Jr., Diana D. Shi. GLIO-1 is a selective DHODH inhibitor that is effective in IDH-mutant gliomas and KDM6-mutated cancers [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr PR012.
Alexander C-Y. Tsai, Mathew D. Lin, V. Puliyappadamba et al.· Clinical Cancer Research· 0 citations
Glioma-associated isocitrate dehydrogenase (IDH) mutations synthesize (R)-2-hydroxyglutarate (R2HG), which broadly inhibits 2-oxoglutarate-dependent enzymes. Given that mutant IDH inhibitors have limited efficacy in high-grade glioma subtypes, alternative treatment strategies are needed, such as therapies that exploit collateral vulnerabilities engendered by IDH mutations. In this vein, drugs that induce replication stress, such as Ataxia-telangiectasia and Rad3-related (ATR) inhibitors and dihydroorotate dehydrogenase (DHODH) inhibitors, have demonstrated efficacy in preclinical IDH-mutant glioma models. However, translation of this synthetic lethal framework to clinical testing has been limited in part by a lack of well-tolerated, effective, and on-target drugs that exploit this vulnerability. To address this need, we developed GLIO-1, a new small molecule inhibitor of DHODH. GLIO-1 preferentially killed IDH-mutant glioma cells in engineered, patient-derived, and orthotopic xenograft model systems. In a patient-derived orthotopic xenograft mouse model of IDH-mutant glioma, GLIO-1 displayed similar monotherapy antitumor efficacy and superior tolerability compared to a leading clinical-stage DHODH inhibitor (BAY 2402234). GLIO-1 also exhibited superior brain penetrance compared to BAY 2402234. The effects of GLIO-1 were on-target, as expression of a drug-resistant DHODH mutant (DHODH-A58T) completely rescued cell death induced by GLIO-1. To mechanistically understand how IDH-mutant gliomas are sensitized to DHODH inhibitors and replication stress, we performed forward genetic screens targeting all 2-oxoglutarate-dependent enzymes. Across six independent CRISPR screens using both DHODH and ATR inhibitors, we discovered that KDM6 histone demethylases play a vital role in protecting glioma cells from replication stress. Base editor screens directed against the KDM6A gene revealed that the demethylase activities of KDM6 enzymes are required for replication stress tolerance. Genetic or R2HG-dependent repression of KDM6 catalytic activity sensitized engineered, patient-derived, and xenograft models of glioma to both ATR and DHODH inhibitors. This liability was generalizable even to non-neural cells because KDM6A loss-of-function mutations commonly observed in urothelial carcinomas sensitized bladder cancer cells to DHODH inhibition including GLIO-1, thereby phenocopying IDH mutations in glioma. We developed an selective, on-target, and well-tolerated DHODH inhibitor, GLIO-1, that is effective in IDH-mutant gliomas. Moreover, we identify KDM6 enzymes as the mechanistic targets of R2HG that link IDH oncogenes with sensitivity to GLIO-1 and reveal KDM6 activity as a fundamental determinant of replication stress. Collectively, these results nominate a new pan-cancer biomarker and targeted therapy pairing, KDM6A inactivation and GLIO-1, that is poised for clinical translation.
Alexander C-Y. Tsai, Mathew D. Lin, Vinesh T. Puliyappadamba, Dorothy M. Junginger, Victoria G. Donovan, Eleanor G. Kaplan, Laura M. Drepanos, Hiroaki Wakimoto, Daniel P. Cahill, Julie A. Losman, Kent W. Mouw, Kalil G. Abdullah, John G. Doench, Duane Nash, Daniel Vitt, Christian Gege, Hella Kohlhof, Samuel K. McBrayer, William G. Kaelin Jr., Diana D. Shi. GLIO-1 is a selective DHODH inhibitor that is effective in IDH-mutant gliomas and KDM6-mutated cancers [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr B083.
Alexander C-Y. Tsai, Mathew D. Lin, V. Puliyappadamba et al.· Clinical Cancer Research· 0 citations
