Abstract 5846: Inhibiting elements of the proteasome recovery pathway sensitizes glioblastoma to proteasome inhibitors

Abstract Bacground: Glioblastoma (GBM) is the most common primary brain tumor, accounting for 15% of all central nervous system related tumors. Despite the aggressive standard of care treatment including chemotherapy, radiotherapy, and maximally safe surgical resection, patient outcomes are abysmal: with 95% of patients relapsing and a median overall survival of 15 months. Thus, this necessitates the rapid query for personalized therapeutics and agents that can potentiate the clinical effects of currently approved treatments. One such pathway that demonstrates aberrant functioning in cancer and is a targetable mechanism is the ubiquitin-proteosome pathway (UPP). Studies have shown that UPP related proteolysis remains constitutively active in cancer cells leading to the rapid degradation of proteins that regulate tumor suppressor genes and oncogenes. Despite robust preclinical evaluations for the usage of proteosome inhibitors against GBM, most tested proteosome inhibitors failed in phase II and III trials, indicating resistance. Methods: We conducted an unbiased genome wide CRISPR-Cas9 screen in human HAP1 cells treated with the proteosome inhibitor, Bortezomib (BTZ), to identify genes that may lead to a BTZ-resistant phenotype. We identified several genes that when perturbed, sensitized cells to BTZ including N-glycanase-1 (NGLY-1), Nuclear factor Erythroid 2-Like-1 (NFE2L1), and DNA damage inducible 1 homolog 2 (DDI2). Herein we sought to evaluate the effects of perturbing components of the proteosome rescue pathway in our Singh lab patient derived GBM cell lines by utilizing CRISPR/Cas9 knockout technology in vitro and in vivo. Results: The generation of NGLY-1, DDI2, and NFE2L1 KO cell lines demonstrated functional sensitivity to the proteosome inhibitor Marizomib (MZB) as observed through a significant reduction of the IC50 in the KO cell lines compared to a control. Functional evaluation also revealed a reduction in proliferation capacity as well as sphere formation in these genetically modified GBM lines. Ongoing in vivo work will aim to evaluate the mitigation of this resistant pathway in our NSG mouse models orthotopically transplanted with these patient derived KO cell lines. In an ongoing collaborative effort, we are working to identify and functionally assess a novel small molecule NGLY-1 inhibitor to evaluate preclinical sensitivity to MZB in our in vitro and in vivo models. Citation Format: Alisha Anand, Muhammad Vaseem Shaikh, Chirayu Chokshi, Benjamin Brakel, William Maich, Shan Grewal, Chitra Venugopal, Sheila Singh. Inhibiting elements of the proteasome recovery pathway sensitizes glioblastoma to proteasome inhibitors 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 (6Suppl): Abstract nr 5846.