A novel CRISPR-engineered prostate cancer cell line defines the AR-V transcriptome and identifies PARP inhibitor sensitivities

Abstract Resistance to androgen receptor (AR)-targeted therapies in prostate cancer (PC) is a major clinical problem. A key mechanism of treatment resistance in advanced PC is the generation of alternatively spliced forms of the AR termed AR variants (AR-Vs) that are refractory to targeted agents and drive tumour progression. Our understanding of how AR-Vs function is limited due to difficulties in distinguishing their discriminate activities from full-length AR (FL-AR). Here we report the development of a novel CRISPR-derived cell line which is a derivative of CWR22Rv1 cells, called CWR22Rv1-AR-EK, that has lost expression of FL-AR, but retains all endogenous AR-Vs. From this, we show that AR-Vs act unhindered by loss of FL-AR to drive cell growth and expression of androgenic genes. Global transcriptomics demonstrate that AR-Vs drive expression of a cohort of DNA damage response genes and depletion of AR-Vs sensitises cells to ionising radiation. Moreover, we demonstrate that AR-Vs interact with PARP1 and PARP2 and are dependent upon their catalytic function for transcriptional activation. Importantly, PARP blockade compromises expression of AR-V-target genes and reduces growth of CRPC cell lines suggesting a synthetic lethality relationship between AR-Vs and PARP, advocating the use of PARP inhibitors in AR-V positive PC.


Supplementary Figure S2. Validating loss of FL-AR in CWR22Rv1-AR-EK cells. CWR22Rv1-and
CWR22Rv1-AR-EK-derived lysates were subject to immunoblotting using four distinct AR N-terminaltargeting antibodies: AR N20 (Santa Cruz Biotechnology -discontinued), AR-BD (BD Pharmingen), AR ab74272 (Abcam) and AR-441 (Santa Cruz Biotechnology). Short and long exposure were performed to enable detection of FL-AR in CWR22Rv1 parental cells. α-tubulin was used as loading control.

Supplementary Figure S3. Validating genomic integrity of CWR22Rv1-AR-EK cells. a. Bright field
images of CWR22Rv1 and CWR22Rv1-AR-EK cells were taken at a 10x magnification to demonstrate similar morphology of the two cell lines. b. CWR22Rv1 and CWR22Rv1-AR-EK share equivalent genomic SNP markers as determined using conventional cell line authentication. c. Predicted potential CRISPR off-target sites were amplified using specific primers and resultant amplicons were analysed by TIDE. Sequencing chromatograms of the three highest ranked off-target loci within COL1A2, KMT2B and SLC7A8 genes are shown for parental and CWR22Rv1-AR-EK cells (left panel) and analysed by TIDE (right panel).

Supplementary Figure S4. AR-V depletion in CWR22Rv1-AR-EK down-regulates androgenic gene
expression. CWR22Rv1-AR-EK cells grown in steroid-depleted media were subject to control (siScr) or AR-V (siAR-V) depletion for 48 hours with either vehicle, 10 nM DHT or 10 µM enzalutamide (Enz) treatment for the final 24 hours before quantitative RT-PCR to assess UBE2C, ATAD2 and KLK2 expression. Data represents the average of three independent experiments +/-SD.

Supplementary Figure S5. AR target gene expression in CWR22Rv1-AR-EK is driven exclusively by
AR-Vs. a. CWR22Rv1-AR-EK cells grown in steroid-depleted media were subject to transfection with control (siScr) or FL-AR-targeting siRNAs (siARex4 and siARex7) for 48 hours with either vehicle or 10 nM DHT treatment for the final 24 hours before quantitative RT-PCR to assess PSA, KLK2 and UBE2C expression. Data represents the average of three independent experiments +/-SD. b. Cell lysates harvested from the parallel experiment were subject to western blot analysis using anti-AR and αtubulin antibodies to demonstrate that unlike siARex1, both siARex4 and siARex7 have no impact on AR-V levels in CWR22Rv1-AR-EK cells (left panel). Using CWR22Rv1 parental cells, siARex4 and siARex7 were able to successfully deplete FL-AR levels (right panel).

Supplementary Figure S6. AR-Vs associate with AR-target genes in CWR22Rv1-AR-EK cells.
CWR22Rv1-AR-EK cells were subject to control (siScr) or AR (siARex1) knockdown for 48 hours before ChIP experiments, incorporating either N-terminal AR-binding or control (IgG) antibodies. Data represents the average of three independent experiments +/-SD (** represents p<0.01 as determined using one-way ANOVA). Figure S7. Validating samples for RNA sequencing analysis. a. Triplicate CWR22Rv1-AR-EK samples transfected with either control (siScr) or AR-V-targeting (siARex1) siRNAs were subject to quantitative RT-PCR and immunoblotting to assess effect on AR-target gene expression (left panel) and validate AR-V depletion (right panel). Data represents the average of three independent experiments +/-SD (** represents p<0.01 as determined using a two-tailed student T-test). b. MA plot of RNA sequencing data from three experimental replicates showing statistically significant gene expression changes (p<0.01) above and below the 0 y-intercept representing up-and down-regulated genes, respectively, shown in red. Figure S8. Reduced CWR22Rv1-AR-EK cell growth in response to AR-V depletion.

Supplementary
CWR22Rv1-AR-EK and CWR22Rv1 cells grown in steroid-depleted media were subject to control (siScr), total AR (siARex1) or AR-V only (siAR-V) knockdown for 96 hours before 10 x magnification bright field images were taken. Figure S9. CWR22Rv1-AR-EK RNA sequencing demonstrates down-regulation of DDR-associated genes in response to AR-V depletion. Expression of the 41 DDR-associated genes identified to be consistently and significantly down-regulated (* p<0.01) in response to AR-V knockdown. Figure S10. Validation of DDR-associated gene regulation by AR-Vs. a. CWR22Rv1-AR-EK cells depleted of AR-Vs (siARex1) for 48 hours were subject to quantitative RT-PCR to assess expression of several DDR-associated genes. Data represents the average of three independent experiments +/-SD (* p< 0.05 as determined using a two-tailed student T-test). genes. CWR22Rv1 cells grown in steroid-depleted medium were transfected for 24 hours with control (siScr) or AR-targeting siRNAs (siARex1) before 24 hour treatment with and without 10 µM enzalutamide (Enz) were subject to quantitative RT-PCR to assess 'BRCAness' gene expression. Data is presented as a heatmap (upper panel) and in graphical form; both represent the mean +/-SD of three independent experiments (* p<0.05 as determined using a two-tailed student T-test). Figure S17. AR-V depletion in CWR22Rv1-AR-EK cells reduces expression of 'BRCAness' genes. CWR22Rv1-AR-EK cells grown in steroid-depleted medium were transfected with control (siScr) or AR-V-targeting siRNAs (siAR-V) for 48 hours and were subject to quantitative RT-PCR to assess gene expression. Data represent the mean +/-SD of three independent experiments (*p<0.05 as determined using a two-tailed student T-test). Parallel samples were subject to immunoblotting using AR and α-tubulin antibodies. Figure S18. PARP inhibition using talazoparib reduces AR-V activity. CWR22Rv1-AR-EK cells were treated with 1 µM talazoparib (Talaz) for 24 hours before quantitative RT-PCR to assess AR-V target gene expression. Data represents three independent experiments +/-SD (***p<0.001 as determined using a two-tailed student T-test). Parallel samples were subject to immunoblotting using AR and α-tubulin antibodies.

Supplementary Figure S19. PARP inhibition using rucaparib reduces AR-V activity. CWR22Rv1-AR-EK
and CWR22Rv1 cells were treated with 0.5 and 1 µM olaparib (Olap) for 24 hours before quantitative RT-PCR to assess AR-V target gene expression. Data represents three independent experiments +/-SD (***p<0.001 as determined using a two-tailed student T-test). Figure S20. PARP inhibition reduces AR enrichment at target genes. CWR22Rv1-AR-EK (a.) or CWR22Rv1 cells (b.) treated for 4 (and 8) hours with 1 µM talazoparib (Talaz) were subject to ChIP using either anti-AR or control (IgG) antibodies to assess AR enrichment at AR target gene promoters PSA and KLK2. Data represents two independent experiments +/-SD (*, ** p<0.05, 0.01, respectively, as determined using a two-tailed student T-test). Immunoblotting of resultant CWR22Rv1 cell lysates using AR and α-tubulin antibodies is shown in the right panel. Figure S21. PARP inhibition reduces AR enrichment at target genes. CWR22Rv1-AR-EK cells were treated with and without 1 µM talazoparib (Talaz) for 4 hours before ChIP using PARP1/2 and control (IgG) antibodies to assess protein enrichment at AR target gene promoters PSA and KLK2, and the TMPRSS2 enhancer. Data represents two independent experiments +/-SD (** p<0.01 as determined using a two-tailed student T-test). Figure S22. Effect of ectopic AR-V7 and PARP1/2 inhibition on DDR-associated gene expression in LNCaP cells. LNCaP cells transiently transduced with control or AR-V7-expressing lentivirus for 24 hours and then treated with 1 µM talazoparib (Talaz) for an additional 24 hours were subject to quantitative RT-PCR to assess expression of DDR-associated genes. Data represents three independent experiments +/-SD (** p<0.01 as determined using a two-tailed student T-test). Genes were segregated into those up-regulated by ectopic AR-V expression and sensitive to PARP inhibitor (top left panel); those enhanced by AR-V7, but insensitive to PARP blockade (top right panel); and those AR-V independent (bottom panel).