DDDR-08. ARF4-MEDIATED RETROGRADE TRAFFICKING AS A MECHANISM OF CHEMORESISTANCE IN GLIOBLASTOMA

Abstract

Abstract Glioblastoma (GBM) remains a deadly disease with an extremely poor prognosis, owing largely to its high rate of resistance to conventional temozolomide (TMZ) chemotherapy. To identify the unknown drivers of this chemoresistance, we performed a genome-wide CRISPR knockout sensitivity screen. Results showed significant enrichment of ~200 novel genes, including ARF4, a previously unstudied gene involved in retrograde trafficking—a well-regulated process by which cargo is transported internally from endosomes to ultimately the nucleus. Initial investigation showed that ARF4-knockdowns resulted in significantly heightened susceptibility to TMZ in multiple GBM patient-derived xenograft lines and extended survival compared to the controls (p< 0.01) in vivo in both primary and recurrent lines. Live-cell imaging further revealed that ARF4-knockdowns significantly inhibited retrograde trafficking, while ARF4-overexpressions resulted in an untenable increase in retrograde trafficking (p < 0.001) in vitro. Enhanced trafficking was also observed in TMZ-treated cells (p < 0.001), suggesting that ARF4 may drive dysregulation of retrograde trafficking to promote chemoresistance.We then performed an unbiased proteomics screen in control and ARF4-knockdown GBM cells with and without therapy to identify which proteins were being uniquely transported to the nucleus during therapy as a result of ARF4-mediated retrograde trafficking. Results showed the greatest enrichment of EGFR signaling, as validated by an increase in EGFR trafficking and nuclear EGFR expression in ARF4-overexpression and TMZ-treated conditions and a decrease of this phenomenon in ARF4-knockdown conditions.We also show that DNA-PK, a DNA repair protein that is transcriptionally activated by nuclear EGFR, is similarly downregulated in ARF4-knockdown conditions and elevated in ARF4-overexpression and TMZ-treated conditions. Treatment with DNA-PK-inhibitor, KU57788, resulted in extended survival compared to the controls (p < 0.01) in vivo in a recurrent line, indicating potential clinical benefit from targeting this pathway. Overall, we present a novel understanding of how ARF4 acts during therapy to increase nuclear localization of chemoresistance-promoting proteins.