OS08.6.A Novel role of ARF4-mediated retrograde trafficking as a driver of chemoresistance in GBM

Abstract

Abstract Background Glioblastoma (GBM) is the most common type of adult malignant brain tumor, with a median survival of only 21 months. This is partly due to the high rate of resistance to conventional therapy, including temozolomide (TMZ), leading to recurrence rates close to 100%. To identify the unknown genes driving the development of this resistance, we performed a genome-wide CRISPR knockout screen comparing a DMSO-treated population with a TMZ-treated population over 14 days. Results showed substantial enrichment of ~200 novel genes, including a previously unstudied gene ARF4—involved in retrograde trafficking to the nucleus. Here, we set out to characterize the mechanism by which ARF4 may be acting to promote chemoresistance. Material and Methods A whole-genome CRISPR-Cas9 sensitivity screen as well as a variety of in vitro and in vivo experiments, such as live-cell imaging, cell viability assays, and western blotting were conducted. Results Initial investigation into ARF4 showed significant elevations in expression at RNA and protein levels (p<0.05) in recurrent patient tumors, as well as a significant survival benefit in patient datasets when downregulated (p<0.05). Knocking out ARF4 resulted in significantly heightened sensitivity to TMZ in multiple GBM patient-derived xenograft lines and extended survival compared to the controls (p<0.01) in vivo. Further investigation via live-cell imaging of transferrin receptors, a retrograde transport marker, revealed that ARF4-knockdowns significantly inhibited retrograde trafficking, while ARF4-overexpressions resulted in an untenable increase in retrograde trafficking in vitro. This effect was also seen in TMZ-treated cells, which displayed enhanced trafficking dynamics, suggesting that ARF4-mediated retrograde trafficking is elevated during therapy to drive nuclear localization of key chemoresistance-promoting factors. We then performed an unbiased proteomics screen to identify which genes were being uniquely transported to the nucleus as a product of ARF4-mediated retrograde trafficking, which revealed enrichment of the EGFR signaling pathway in particular. Validation experiments confirmed a decrease in EGFR trafficking and nuclear EGFR expression in ARF4-knockdowns and an increase in EGFR trafficking and nuclear EGFR expression in ARF4-overexpression and TMZ-treated GBM cells. Furthermore, nuclear DNA-PK, a DNA repair protein known to be transcriptionally activated by EGFR, was similarly found to be downregulated in ARF4-knockdown conditions and elevated in ARF4-overexpression and TMZ-treated conditions. Conclusion: Here, we show that ARF4 may be responsible for promoting chemoresistance through altered retrograde trafficking of EGFR specifically. Thus, our study has yielded a promising and novel therapeutic target for GBM, a disease desperately in need of new therapeutic strategies.