DDDR-41. A GENOME-WIDE CRISPR/CAS9 SCREEN IN GLIOBLASTOMA STEM CELLS UNVEILS SYNTHETIC LETHAL INTERACTIONS AND RESISTANCE MECHANISMS OF JIN001, A BLOOD-BRAIN BARRIER-PENETRANT HSP90 INHIBITOR

Abstract

Abstract Glioblastomas (GBMs) are aggressive brain tumors characterized by extensive genetic heterogeneity and therapeutic resistance. Heat shock protein 90 (Hsp90) is a molecular chaperone that stabilizes and facilitates the proper folding of numerous proteins, including oncogenic drivers and is overexpressed in cancers. Hsp90 inhibitors can disrupt multiple oncogenic signaling pathways by degrading various client proteins essential for tumor growth and survival, making them attractive therapeutic candidates for GBMs. We have previously reported on the efficacy of Hsp90 inhibitors against gliomas. However, cancer cells develop resistance to Hsp90 inhibitors by upregulating compensatory chaperones (e.g., Hsp70), mutating client proteins, or activating alternative survival pathways, reducing the long-term efficacy of these agents. Combination approaches overcoming such resistance mechanisms are needed. JIN001, a novel Hsp90 inhibitor, demonstrates superior blood-brain barrier (BBB) penetration, and high potential for clinical translation. we performed a genome-wide CRISPR/Cas9 knockout screen using the Toronto Knockout Library v3 (TKOv3) with screen selection pressure from JIN001 in GBM stem cells (GSC272) to identify synthetic lethal interactions and resistance mechanisms associated with JIN001 treatment. Experiments were performed in triplicate with two time points, T22 and T37. Quality control analysis of the NGS data indicated excellent quality and consistency between different replicates. DrugZ analysis revealed a set of sensitive and resistant genes, informing potential combination therapies and further elucidating JIN001’s mechanism of action. Validation studies for the top synthetic lethal targets are underway to confirm the identified interactions and will be presented. This CRISPR-Cas9 screen offers valuable insights into JIN001’s therapeutic potential, reveals new combination strategies to overcome resistance to Hsp90 inhibitors and for use of these agents in other cancers, and paves the way for the development of more effective GBM combination treatment strategies.