RBIO-09. Preclinical evaluation of the synergistic effect of heat shock protein 90 inhibitors with temozolomide and external beam radiotherapy in glioblastoma models

Abstract INTRODUCTION Heat shock protein 90 (Hsp90) is a molecular chaperone that interacts with other co-chaperones to stabilize, mature and (re)fold proteins1. Its overexpression in cancer cells makes it attractive as a chemotherapeutic target. However, due to insufficient efficacy or toxicity, only TAS116 (pimitespib) was approved for clinical use in advanced gastrointestinal tumors in Japan2. A synergistic anti-cancer effect of Onalespib with external beam radiotherapy (EBRT) was preclinically observed in malignant gliomas3. This study evaluates the synergistic effect of Hsp90 inhibitor treatment combined with EBRT and temozolomide (TMZ) in glioblastoma models. Methodology: The Hsp90 inhibitors Geldanamycin, Onalespib, BIIB021, NVP-HSP990 and NMS-E973 were evaluated in U-87 MG-WT/IDH1 mutant glioblastoma cell models. IC50 values were determined via sulforhodamine B assay. Western blot was performed for Hsp90 expression evaluation following TMZ treatment. Clonogenic assays were conducted 10 days following 96-hour treatment with monotherapy or dual therapy using Geldanamycin, TMZ, and/or EBRT. RESULTS In U-87 MG-WT cells, IC50 values were determined for Geldanamycin (53 nM), Onalespib (135 nM), BIIB021 (97 nM), NVP-HSP990 (35 nM), and NMS-E973 (193 nM). For U-87 MG-IDH1 mutant cells, IC50 values were slightly elevated. Preliminary clonogenic assays showed reduced colony formation following EBRT, with enhanced effects in combination with Geldanamycin or TMZ. CONCLUSION U-87 MG cell viability assays showed nanomolar-range IC50 values for all Hsp90 inhibitors. A trend towards reduced colony formation was observed following monotherapy and combination therapy. Ongoing in vitro experiments include assays for viability, clonogenic survival, and DNA damage following monotherapy, dual, and triple combination treatments using Hsp90 inhibitors, TMZ and EBRT in additional cell lines. The best performing compounds will be tested in subcutaneous, patient-derived xenografts and orthotopic mouse models, focusing on tumor growth and survival. [1]Mol Pharmacol 95, 468-474 (2019). [2]Journal of Clinical Oncology 39, 11524-11524 (2021). [3]Uffenorde et al. Front Oncol 15 (2025).