Inhibiting elements of the proteasome recovery pathway sensitizes glioblastoma to proteasome inhibition

Abstract

Glioblastoma (GBM) is the most aggressive brain cancer in adults. Due to the complexities and dynamic nature inherent to this tumor, effective treatments have been hard to come by. Thus, there is a dire need to discover newer therapies to help manage tumor progression and prevent recurrence. One type of therapy that has shown promise in many cancers including acute myeloid leukemia and mantle cell lymphoma are proteasome inhibitors (PIs). These drugs block the degradation of proteins that regulate cell growth and survival leading to the accumulation of pro-apoptotic factors and the induction of cancer cell death. Despite promising preliminary results to support the inclusion of PIs for the treatment of GBM; many patients develop resistance and eventually succumb to the disease. 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. Previous research has unveiled the ubiquitin-proteasome pathway (UPP) to be upregulated and essential for GBM survival and proliferation, thus serving as an amenable vulnerability. Studies have shown that UPP related proteolysis remains constitutively active in cancer cells leading to the degradation of proteins that regulate tumor suppressor genes and oncogenes. Despite robust preclinical evaluations for the usage of PIs against GBM, most tested PIs have shown clinical inefficacy in phase II and III trials, indicating resistance. We conducted an unbiased genome wide CRISPR-Cas9 screen in human HAP1 cells treated with the PI, Bortezomib (BTZ), to identify genes that may lead to a BTZ- resistant phenotype. We identified several genes that when perturbed, enhanced cytotoxicity of cells treated with BTZ, namely N-glycanase-1 (NGLY-1), Nuclear factor Erythroid 2-Like-1 (NFE2L1), and DNA damage inducible 1 homolog 2 (DDI2). Currently, literature points to a proteasome rescue pathway involving these genes, wherein NGLY-1 and DDI2 post-translationally modify the transcription factor NFE2L1. Upon successful modification, NFE2L1 is activated and traffics from the endoplasmic reticulum to the nucleus and binds to the cis-regulated antioxidant response element to upregulate proteasome expression. This is contingent on the accumulation of NFE2L1 in the cytosol which can result from either an endogenous or exogenous block on proteolytic activity leading to proteotoxic stress. This mechanism, although established in mouse fibroblasts, has yet to be validated in GBM. In this thesis, we validated these findings in our patient derived GBM cell lines, wherein the genetic perturbation of NGLY-1, DDI2 and NFE2L1 using CRISPR/Cas9 technology led to the sensitization of GBM to the PI, Marizomib in vitro. Ongoing efforts aim to recapitulate these findings in our patient-derived orthotopic murine models of GBM. We also aim to develop a novel NGLY-1 inhibitor in collaboration with Dr. Gunning’s lab to leverage a combination technique in vitro and in vivo that can ultimately potentiate the cytotoxic effects of proteasome inhibition and manage tumor progression.