Resistance to androgen receptor (AR)-targeted therapies represent a major challenge in prostate cancer (PC). A key mechanism of treatment resistance in patients who progress to castrate-resistant PC (CRPC) is the generation of alternatively spliced androgen receptor variants (AR-Vs). Unlike full-length AR (FL-AR) isoforms, AR-Vs are constitutively active and refractory to current receptor-targeting agents hence drive tumour progression. Identifying regulators of AR-V synthesis may therefore provide new therapeutic opportunities in combination with conventional AR-targeting agents. Our understanding of AR transcript splicing, and the factors that control the synthesis of AR-Vs, remains limited. While candidate-based approaches have identified a small number of AR-V splicing regulators, an unbiased analysis of splicing factors important for AR-V generation is required to fill an important knowledge gap and furnish the field with novel and tractable targets for PC treatment. To that end, we conducted a bespoke CRISPR screen to profile splicing factor requirements for AR-V synthesis. MFAP1 and CWC22 were shown to be required for the generation of AR-V mRNA transcripts and their depletion resulted in reduced AR-V protein abundance and cell proliferation in several CRPC models. Global transcriptomic analysis of MFAP1-depleted cells revealed both AR-dependent and -independent transcriptional impact, including genes associated with DDR. As such, MFAP1 downregulation sensitised PC cells to ionising radiation suggesting therapeutically targeting AR-V splicing could provide novel cellular vulnerabilities which can be exploited in CRPC. Implications: We have utilised a CRISPR screening approach to identify key regulators of pathogenic AR splicing in prostate cancer.

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Supplementary data

Supplementary Information

Supplementary methods and legends for supplementary figures

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Supplementary Figure S1

Figure S1. Development of the inducible Cas9 CWR22Rv1-AR-EK derivative cell line (CWR22Rv1-AR-EK-iCas9)

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Supplementary Figure S2

Figure S2. Validating inducible Cas9 activity in the CWR22Rv1-AR-EK-iCas9 cell line.

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Supplementary Figure S3

Figure S3. Optimization of CRISPR screening pipeline.

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Supplementary Figure S4

Figure S4. PLK1 sgRNAs reduce cell growth in the CWR22Rv1-AR-EK-iCas9 cell line.

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Supplementary Figure S5

Figure S5. Initial assessment of knockout hits from CRISPR screen highlights MFAP1 and CWC22 as regulators of AR-V abundance.

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Supplementary Figure S6

Figure S6. Comparing cell proliferation and AR abundance data from the CRISPR/Cas9 KO screen confirms MFAP1 and CWC22 as key targets to progress.

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Supplementary Figure S7

Figure S7. Validating gene editing with MFAP-1 and CWC22-targeting CRISPR/Cas9 complexes.

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Supplementary Figure S8

Figure S8. Knockdown of MFAP1 reduces expression of AR-Vs across a panel of FL-AR/AR-V positive CRPC cell line models.

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Supplementary Figure S9

Figure S9. Knockdown of CWC22 reduces expression of FL-AR and AR-Vs across a panel of FL-AR/AR-V positive CRPC cell line models.

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Supplementary Figure S10

Figure S10. Cell counts and live cell imaging confirm knockdown of MFAP1 and CWC22 reduces growth of FL-AR/AR-V positive CRPC cell lines.

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Supplementary Figure S11

Figure S11. RNA sequencing samples were confirmed to have depletion of MFAP1, total AR and AR-V7 by western blot and RT-qPCR.

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Supplementary Figure S12

Figure S12. Comparison of overlapping SDEGs in response to AR depletion in the CWR22Rv1 cell line.

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Supplementary Figure S13

Figure S13. Comparison of overlapping SDEGs in response to MFAP1 and AR-V depletion in the CWR22Rv1, and derivative, cell line models reveals considerable overlap between MFAP1 and AR-V transcriptomes.

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Supplementary Figure S14

Figure S14. Comparison of Hallmark gene-sets using GSEA shows considerable overlap in Hallmarks regulated by both MFAP1 and AR-V7.

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Supplementary Figure S15

Figure S15. Comparison of KEGG pathways using GSEA shows considerable overlap in pathways regulated by both MFAP1 and AR-V7.

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Supplementary Figure S16

Figure S16. MFAP1 knockdown diminishes DNA damage repair in PC cells.

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Supplementary Figure S17

Figure S17. MFAP1 knockdown diminishes DNA damage repair in PC cells.

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Supplementary Figure S18

Figure S18. Knockdown of MFAP1 resensitizes CWR22Rv1 cell lines to enzalutamide, and MFAP1 and CWC22 show no consistent change in expression in PC.

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Supplementary Table S1

CRISPR screen targets and normalized screen data N=1-4

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Supplementary Table S2

DEGs and SDEGs from MFAP1 and AR-V depletion in CWR22Rv1 cells

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Supplementary Table S3

Overlapping and non-overlapping SDEGs from MFAP1 and AR-V depletion in CWR22Rv1 and CWR22Rv1-AR-EK cells

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Supplementary Table S4

MFAP1 depletion downregulates AR hallmark gene sets

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Supplementary Table S5

Cellular function associated with MFAP1/AR-V-overlapping genes

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Supplementary Table S6

AR-V-independent MFAP1-regulated gene list analysed by Enrichr

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Supplementary Table S7

MFAP1-regulated splicing events

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Supplementary Table S8

NER and HR gene set expression changes upon MFAP1 and AR knockdown

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Supplementary Table S9

siRNA sequences and supplier

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Supplementary Table S10

sgRNA sequences and supplier

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Supplementary Table S11

qRT-PCR primer sequences

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Supplementary Table S12

Antibodies used in study

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