ATRX INACTIVATION AND IDH1-R132H DRIVE PREFERENTIAL SENSITIVITY TO PROTON VS. X-RAY RADIOTHERAPY IN GLIOMA STEM CELLS
Author ORCID Identifier
Date of Graduation
Masters of Science (MS)
David Grosshans, M.D., Ph.D.
Krishna P.L. Bhat, Ph.D.
Ahsan Farooqi, M.D., Ph.D.
Steven Lin, M.D., Ph.D.
Gabriel Sawakuchi, Ph.D.
Background: Glioma Stem Cells (GSCs) are self-renewable, treatment resistant cells in the glioma tumor mass known to promote tumor development. In contrast to traditional photon-based radiation therapy (XRT), proton radiation therapy (PRT) may induce more complex DNA damage and therefore might have the potential to eliminate GSCs. Although previous studies have individually linked IDH mutations, specifically IDH1R132H, and ATRX inactivating mutations to improved patient outcomes and suppressed DNA damage repair compared to their respective wild-types, the mechanisms by which these two genetic alterations interact in GSCs treated with PRT compared to XRT are currently unknown. We hypothesize that ATRXLoss and IDH1R132H both drive preferential sensitivity to PRT compared to XRT.
Methods: Isogenic human GSC lines TS543-ATRXWT, TS543-ATRXLoss, MGG18-IDH1WT, and MGG18-IDH1R132H were subjected to either XRT or PRT. Human GSC lines TS603-ATRXWT/IDH1R132H and GS522-ATRXLoss/IDH1R132H were subjected to a combination of Ivosidenib, a reversible selective IDH1R132H inhibitor, and either XRT or PRT. Extreme limiting dilution analysis (ELDA) was used to calculate the active cell frequency, a measure of GSC self-renewal. Post-radiation GSC viability was quantified using the CellTiterGlo 3D assay at 14 days after XRT or PRT. The primary mechanisms of radiation-induced cell death were determined using the RealTime-Glo Annexin V apoptosis and necrosis assay at 0-72 hours after irradiation.
Results: Using isogenic TS543 GSCs, ATRXLoss diminished cell viability and self-renewal primarily by inducing cell death via apoptosis and secondary necrosis compared to ATRXWT. Isogenic MGG18-IDH1R132H GSCs treated with PRT consistently exhibited increased apoptotic and necrotic cell death compared to XRT. MGG18-IDH1WT demonstrated increased apoptotic cell death after PRT compared to XRT. Finally, combining Ivosidenib with either XRT or PRT diminished survival by upregulating apoptotic and necrotic cell death in TS603-ATRXWT/IDH1R132H GSCs. However, the opposite effects were observed in GS522-ATRXLoss/IDH1R132H GSCs.
Conclusions: PRT was more effective than XRT in inducing GSC death across several cell lines. ATRX inactivation increased the efficacy of PRT via apoptotic and necrotic cell death. IDH1R132H does not significantly improve radiation induced cell death in ATRXWT GSCs. Combining IDH1R132H inhibitors with PRT in ATRXWT/IDH1R132H GSCs may represent a novel treatment strategy to overcome radioresistance.
Glioblastoma, Glioma Stem Cells, Proton Radiotherapy, Necroptosis.