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A genome-wide CRISPR-Cas9 knockout screen identifies novel PARP inhibitor resistance genes in prostate cancer

Abstract

DNA repair gene mutations are frequent in castration-resistant prostate cancer (CRPC), suggesting eligibility for poly(ADP-ribose) polymerase inhibitor (PARPi) treatment. However, therapy resistance is a major clinical challenge and genes contributing to PARPi resistance are poorly understood. Using a genome-wide CRISPR-Cas9 knockout screen, this study aimed at identifying genes involved in PARPi resistance in CRPC. Based on the screen, we identified PARP1, and six novel candidates associated with olaparib resistance upon knockout. For validation, we generated multiple knockout populations/clones per gene in C4 and/or LNCaP CRPC cells, which confirmed that loss of PARP1, ARH3, YWHAE, or UBR5 caused olaparib resistance. PARP1 or ARH3 knockout caused cross-resistance to other PARPis (veliparib and niraparib). Furthermore, PARP1 or ARH3 knockout led to reduced autophagy, while pharmacological induction of autophagy partially reverted their PARPi resistant phenotype. Tumor RNA sequencing of 126 prostate cancer patients identified low ARH3 expression as an independent predictor of recurrence. Our results advance the understanding of PARPi response by identifying four novel genes that contribute to PARPi sensitivity in CRPC and suggest a new model of PARPi resistance through decreased autophagy.

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Fig. 1: Genome-wide CRISPR-Cas9 knockout (KO) functional screen of olaparib resistance in CRPC cells.
Fig. 2: Generation and characterization of single-gene knockout (KO) populations and KO clones in C4-Cas9 CRPC cells.
Fig. 3: PARPi cross-resistance in olaparib resistant KO cell lines.
Fig. 4: ARH3 KO contributes to increased expression of oxidative phosphorylation genes.
Fig. 5: Clinical relevance of candidate genes.
Fig. 6: Model for the role of ARH3 in PARPi response.

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Acknowledgements

This work was supported by grants from The Novo Nordisk Foundation (KDS), Aase & Ejnar Danielsens Fond (MBI), Fabrikant Einar Willumsens Mindelegat (MBI), Tømrermester Jørgen Holm & Hustru Elisa F. Hansens Mindelegat (MBI), Direktør Emil C. Hertz og Hustru Inge Hertz Fond (MBI), Graduate School of Health (Aarhus University; MBI), Helge Peetz & Verner Peetz & Hustru Vilma Peetz Fond (MBI), Beckett Fonden (MBI), and NEYE Fonden (MBI). The Danish Cancer Biobank is acknowledged for providing patient samples and information on handling and storage.

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KDS, MBI, EMGS, JGM, and JH designed the study. MBI and EAT conducted the CRISPR-Cas9 screen. MBI and EMGS produced single gene knockout cell lines and performed all cell line experiments. MBI performed RNA-sequencing analyses for cell lines. AD performed western blots of LC3II/I. JP performed LC-MS experiments. PB supervised Seahorse assays. KDS, JF, MR, SK, MRJ, KB, BPU, and MB were responsible for collection and sequencing of patient samples. MBI and EMGS analyzed patient RNA-sequencing data. MBI, EMGS, SW, PB, JP, and KDS interpreted data. MBI, EMGS and KDS wrote the manuscript. KDS, SW, and JGM supervised the study. All authors read and approved the final manuscript.

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Correspondence to Karina Dalsgaard Sørensen.

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Ipsen, M.B., Sørensen, E.M.G., Thomsen, E.A. et al. A genome-wide CRISPR-Cas9 knockout screen identifies novel PARP inhibitor resistance genes in prostate cancer. Oncogene 41, 4271–4281 (2022). https://doi.org/10.1038/s41388-022-02427-2

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