ARF4-mediated retrograde trafficking as a driver of chemoresistance in GBM

Shreya Budhiraja; Graysen McManus; Shivani Baisiwala; Ella N Perrault; Sia Cho; Miranda Saathoff; Li Chen; Cheol H Park; Hasaan A Kazi; Crismita Dmello; Peiyu Lin; C David James; Adam M Sonabend; Dieter H Heiland; Atique U Ahmed

doi:10.1093/neuonc/noae059

ARF4-mediated retrograde trafficking as a driver of chemoresistance in GBM

Neuro Oncol. 2024 Mar 20:noae059. doi: 10.1093/neuonc/noae059. Online ahead of print.

Authors

Shreya Budhiraja^{1

2}, Graysen McManus¹, Shivani Baisiwala¹, Ella N Perrault¹, Sia Cho³, Miranda Saathoff¹, Li Chen¹, Cheol H Park¹, Hasaan A Kazi¹, Crismita Dmello¹, Peiyu Lin¹, C David James^{1

2}, Adam M Sonabend^{1

2}, Dieter H Heiland^{1

4

5

6

7}, Atique U Ahmed^{1

2}

Affiliations

¹ Department of Neurological Surgery, Feinberg School of Medicine, Northwestern University.
² Northwestern Medicine Malnati Brain Tumor Institute of the Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University.
³ Northwestern University, Evanston, IL.
⁴ Microenvironment and Immunology Research Laboratory, Medical Center - University of Freiburg, Freiburg, Germany.
⁵ Department of Neurosurgery, Medical Center - University of Freiburg, Freiburg, German.
⁶ Faculty of Medicine, University of Freiburg, Freiburg, Germany.
⁷ German Cancer Consortium (DKTK), partner site Freiburg.

PMID: 38506351
DOI: 10.1093/neuonc/noae059

Abstract

Background: Cellular functions hinge on the meticulous orchestration of protein transport, both spatially and temporally. Central to this process is retrograde trafficking, responsible for targeting proteins to the nucleus. Despite its link to many diseases, the implications of retrograde trafficking in glioblastoma (GBM) are still unclear.

Methods: To identify genetic drivers of TMZ resistance, we conducted comprehensive CRISPR-knockout screening, revealing ADP-ribosylation factor 4 (ARF4), a regulator of retrograde trafficking, as a major contributor.

Results: Suppressing ARF4 significantly enhanced TMZ sensitivity in GBM patient-derived xenograft (PDX) models, leading to improved survival rates (p<0.01) in both primary and recurrent lines. We also observed that TMZ exposure stimulates ARF4-mediated retrograde trafficking. Proteomics analysis of GBM cells with varying levels of ARF4 unveiled the influence of this pathway on EGFR signaling, with increased nuclear trafficking of EGFR observed in cells with ARF4 overexpression and TMZ treatment. Additionally, spatially-resolved RNA-sequencing of GBM patient tissues revealed substantial correlations between ARF4 and crucial nuclear EGFR (nEGFR) downstream targets, such as MYC, STAT1, and DNA-PK. Decreased activity of DNA-PK, a DNA repair protein downstream of nEGFR signaling that contributes to TMZ resistance, was observed in cells with suppressed ARF4 levels. Notably, treatment with DNA-PK inhibitor, KU57788, in mice with a recurrent PDX line resulted in prolonged survival (p<0.01), highlighting the promising therapeutic implications of targeting proteins reliant on ARF4-mediated retrograde trafficking.

Conclusion: Our findings demonstrate that ARF4-mediated retrograde trafficking contributes to the development of TMZ resistance, cementing this pathway as a viable strategy to overcome chemoresistance in GBM.

Keywords: ARF4; Glioblastoma; chemoresistance; retrograde trafficking; whole-genome CRISPR screen.

Abstract

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