Abstract 4333: Identification of glucose-6-phosphate dehydrogenase dependency in IDH1 mutant glioma cells using functional genomics

Abstract Astrocytoma and oligodendroglioma are most commonly initiated by missense mutations at the arginine 132 codon of isocitrate dehydrogenase 1 (IDH1). Biochemically the mutant IDH1 protein acquires neomorphic activity: reductive NADPH-dependent catalysis of α-ketoglutarate (αKG) to (R)-2-hydroxyglutarate [(R)-2-HG]. (R)-2-HG interferes with the function of αKG-dependent epigenetic modifiers, resulting in extensive epigenetic remodeling that impairs cellular differentiation and promotes gliomagenesis. Strategies for selectively targeting IDH1-mutant cells offer promise for improved treatment over the standard modalities of surgery, chemotherapy and radiotherapy. Whole-genome CRISPR/Cas9 knockout screens were conducted to identify gene dependencies in isogenic IDH1-mutant and wild-type (WT) U-87 MG cells. Cells were transduced with the Brunello single guide RNA (sgRNA) library, grown for 21 days, then sgRNA distributions were assessed by amplicon sequencing. Gene dependencies were identified using CRISPRcleanR and BAGELR pipelines. We discovered that glucose-6-phosphate dehydrogenase (G6PD), a pentose phosphate pathway (PPP) enzyme, was needed for viability of IDH1-mutant cells, but was dispensable for U-87 MG WT cells. The PPP is the main producer of cytosolic NADPH that is required for de novo lipogenesis, maintenance of antioxidants and production of (R)-2-HG by mutant IDH1. Previous reports demonstrated that IDH1-mutant cells increase PPP flux to support these processes. We have validated this dependency using G6PDi-1, a cell-active inhibitor of G6PD. Growth inhibition (IC50) assays demonstrated that IDH1-mutant U-87 MG cells were threefold more sensitive to G6PDi-1 with IC50 of 23 μM and enhanced cell death (increased propidium iodide uptake), compared with WT U-87 MG cells with IC50 of 67 μM. G6PD knockout was then conducted to further validate this dependency. Nucleofection of IDH1 WT U-87 MG cells with G6PD multi guide RNA-Cas9 ribonucleoproteins resulted in highly efficient knockout of G6PD at day 5 (99-100%), which was maintained at day 21 (96-98%). In contrast, efficient G6PD knockout in IDH1-mutant U-87 MG cells at day 5 (98-100%) was lost following 21 days culture (0-5%), indicating death of almost all edited cells within the culture. Finally, Kaplan-Meier analysis of IDH1-mutant patients in the TCGA lower grade glioma dataset demonstrated that higher G6PD expression correlated with worse median overall survival (7.3 versus 9.5 years; log-rank p=0.01), but was not prognostic in the IDH1 WT TCGA glioblastoma multiforme dataset. These findings demonstrate that a cell line model of IDH1-mutant glioma is highly dependent on G6PD for survival. This observation builds on the recognized importance of the PPP in the metabolic biology of IDH1-mutant malignancy by establishing G6PD as a targetable dependency in this disease. Citation Format: Kim M. Paras, Pooja Patel, Paul Choi, William R. Wilson, Tet-Woo Lee, Stephen M. Jamieson, Dean C. Singleton. Identification of glucose-6-phosphate dehydrogenase dependency in IDH1 mutant glioma cells using functional genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4333.