Medical Student Northwestern University Feinberg School of Medicine Chicago, Illinois, United States
Disclosure(s):
Shreya Budhiraja: No financial relationships to disclose
Introduction: GBM is the most common and aggressive type of adult malignant brain tumor. 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-Cas9 knockout screen.
Methods: We conducted a whole-genome CRISPR-Cas9 knockout screen in a human GBM line, comparing a DMSO-treated population with a TMZ-treated population over 14 days. Experiments to analyze the effects of top hits on TMZ-resistance were conducted. In vivo models of GBM were used to check for survival benefit. Functional studies to understand the mechanism of action were pursued.
Results: A previously unstudied gene in GBM, ARF4—involved in retrograde trafficking of proteins to the nucleus—was identified as a top driver of TMZ-resistance. Knocking out ARF4 resulted in significantly heightened sensitivity to TMZ in multiple GBM patient-derived xenograft lines. Mice bearing intracranial primary GBM with ARF4-knockdown showed increased sensitivity to TMZ and significantly improved survival (p < 0.01). Furthermore, knocking out ARF4 resulted in significantly heightened sensitivity to TMZ and extended survival in a recurrent GBM line (p < 0.01). Additional investigation via live-cell imaging of transferrin receptors, a retrograde transport marker, revealed that cells undergoing therapy show enhanced trafficking dynamics, suggesting that ARF4-mediated retrograde trafficking is elevated during therapy to drive nuclear localization of key chemoresistance-promoting factors. An unbiased proteomics screen to identify which genes were being uniquely transported to the nucleus during therapy revealed a host of novel therapeutic targets and key pathways—including DNA repair and EGFR signaling—that are regulated by this critical process, thus cementing it as a promising avenue for treatment.
Conclusion : Here, we show that ARF4 may be promoting chemoresistance through a specific dysregulation of retrograde trafficking, thus yielding a promising and novel therapeutic target for GBM.
