In vivo CRISPR screens reveal SCAF1 and USP15 as drivers of pancreatic cancer

Abstract

Functionally characterizing the genetic alterations that drive pancreatic cancer is a prerequisite for precision medicine. Here, we perform somatic CRISPR/Cas9 mutagenesis screens to assess the transforming potential of 125 recurrently mutated pancreatic cancer genes, which revealed USP15 and SCAF1 as pancreatic tumor suppressors. Mechanistically, we find that USP15 functions in a haploinsufficient manner and that loss of USP15 or SCAF1 leads to reduced inflammatory TNFα, TGF-β and IL6 responses and increased sensitivity to PARP inhibition and Gemcitabine. Furthermore, we find that loss of SCAF1 leads to the formation of a truncated, inactive USP15 isoform at the expense of full-length USP15, functionally coupling SCAF1 and USP15. Notably, USP15 and SCAF1 alterations are observed in 31% of pancreatic cancer patients. Our results highlight the utility of in vivo CRISPR screens to integrate human cancer genomics and mouse modeling for the discovery of cancer driver genes with potential prognostic and therapeutic implications.

Fig. 1. In vivo CRISPR screen reveals pancreatic cancer tumors suppressors.

A Experimental design of the in vivo PDAC CRISPR screen, showing gene selection from long-tail mutations, pancreatic injection of AAV libraries and tumor sequencing. B Tumor-free survival of Pdx1-Cre;LSL-Kras^G12D;LSL-Cas9-GFP mice transduced with a sgRNA library targeting putative pancreatic cancer genes (n = 23) or a control sgRNA library (n = 13) C Representative whole-mount, H&E and immunofluorescent images of an H2B-RFP+ pancreatic PDAC-library tumor: Scale bar 2 mm. H&E image: scale bar 250 µm. Representative immunofluorescence image shows H2B-RFP and CK19 expression. Scale bar 50 µm. Similar results were observed in all collected tumor. D Representative pie charts showing tumor suppressor genes with enriched sgRNAs in tumor DNA obtained from three different pancreatic tumors and a control-transduced pancreas with multifocal PanINs. E Bar graph showing putative tumor suppressor genes with enriched sgRNAs in tumor DNA obtained from the PDAC mouse model (sgRNA enriched per tumors are indicated by color).

Fig. 2. Usp15 functions as PDAC tumor suppressor.

A Tumor-free survival of Pdx1-Cre;LSL-Kras^G12D;LSL-Cas9-GFP mice injected with CRISPR AAV-sgRNAs targeting the indicated gene or non-targeting control sgRNA (sgCtrl, n = 6). Two independent sgRNAs were used (sgUsp15_1 n = 9, sgUsp15_2 n = 9). Log-Rank test (Mantel-Cox). B Representative H&E images showing multifocal PanINs in sgCtrl transduced pancreas and PADC tumors in sgUsp15 transduced pancreas. Scale bar 100 µm. C Tumor-free survival of Pdx1-Cre;LSL-Kras^G12D mice with the indicated Usp15 genotype where ‘+’ indicates the wildtype allele and ‘Δ’ indicates a conditionally deleted allele. KrasG12D +/- Usp15 +/+ (n = 12); KrasG12D +/- Usp15 +/- (n = 11); KrasG12D +/- Usp15 -/- (n = 13). Log-Rank test (Mantel-Cox). D Representative H&E images of mice with the indicated genotype showing multifocal PanINs and PADC tumors. Scale bar 100 µm. E Cell proliferation curves of KC cells transduced with the indicated sgRNA obtained using the IncuCyte live-cell imaging. Cells were grown for five days and data are expressed as cell confluence percentage (%; mean ± SD, n = 3 independent experiments, Two-way ANOVA (sgUsp15_1 p = 3.43e-8: sgUsp15_2 p = 4.89e-9), Dunnett’s multiple comparison. F Cell proliferation curves of KC cells expressing ubiquitin variants inhibiting Usp15 (Ubv15.1a and Ubv15.1/d) or wildtype ubiquitin (Ub^wt) as control. (%; mean ± SD, n = 3 independent experiments, Two-way ANOVA (Ubv15.1a p = 2.74e-6: Ubv15.1/d p = 2.06e-7), Dunnett’s multiple comparison. G Tumor-free survival of NSG (NOD Scid Gamma) mice after orthotopic injection sgCtrl (n = 5) or sgUsp15_1/2 (n = 5; n = 5) KC cells. Two independent sgRNAs were used. Log-Rank test (Mantel-Cox). H Dose-response curves for KPC sgCtrl or sgUsp15 cells treated with the indicated concentration of Olaparib (mean ± SD, n = 3 independent experiments). Two-way ANOVA (sgUsp15_1 p = 0.0349: sgUsp15_2 p = 0.0431), Dunnett’s multiple comparison.

Fig. 3. Usp15 regulates several pathways involved in PDAC development.

A Volcano Blot showing differential expressed genes between Usp15-knockout compared to sgCtrl control KC cells. Wald test and Benjamini-Hochberg (BH)-adjusted P-value. Two independent sgRNAs, two biological duplicates. B Bar graph showing gene set enrichment analysis (GSEA) of Usp15-knockout compared to sgCtrl control KC cells. GSEA nominal p-values. Two independent sgRNAs, two biological duplicates. C GSEA plots and Heatmaps of log2 counts per million for selected differentially expressed pathways and genes in sgUsp15 versus sgCtrl control KC cells. GSEA nominal p-values. Two independent sgRNAs, two biological duplicates. D Expression levels of genes related to TNFα signaling evaluated by RT-qPCR. Results were normalized with Gapdh and are expressed in fold change compared to Ctrl (mean ± SEM, n = 3 independent experiments). Cells were incubated with 10 ng/mL TNFα-for 30 min. Two-sided T-test, Rel-B p = 0.043; TRAF-1 p = 0.037/p = 0.034; NFKB1 p = 0.036; Rel-B p = 0.042; TRAF-1 p = 0.039; CXCL2 p = 0.028; CXCL3 p = 0.047/p = 0.043; NFKB1 p = 0.038/p = 0.040; NFKB2 p = 0.039/p = 0.043.

Fig. 4. Scaf1 functions as PDAC tumor suppressor.

A Tumor-free survival of Pdx1-Cre;LSL-Kras^G12D;LSL-Cas9-GFP mice injected with CRISPR AAV targeting the indicated gene or non-targeting control sgRNA (sgCtrl n = 6). Two independent sgRNAs were used (sgScaf1_1 n = 8, sgScaf1_2 n = 7). Log-Rank test (Mantel-Cox). B Representative H&E images showing multifocal PanINs in sgCtrl-transduced pancreas and PADC tumors in sgScaf1-transduced pancreas. Scale bar 100 µm. C Cell proliferation curves of KC sgCtrl and sgScaf1 cells were obtained using the IncuCyte live-cell imaging and data are expressed as cell confluence percentage (%; mean ± SD, n = 3 independent experiments). Two-way ANOVA (sgScaf1_1 p = 0.0039: sgScaf1_2 p = 0.00042), Dunnett’s multiple comparison. D Tumor-free survival of NSG mice orthotopically injected with sgCtrl (n = 5) or sgScaf1_1/2 (n = 5; n = 5) KC cells. Two independent sgRNAs were used. Log-Rank test (Mantel-Cox). E Dose-response curves for KPC sgCtrl or sgScaf1 cells treated with the indicated concentration of Olaparib (mean ± SD, n = 3 independent experiments). Two-way ANOVA (sgScaf1_1 p = 0.0084: sgScaf1_2 p = 0.0028), Dunnett’s multiple comparisons. F Representative Western Blot of Usp15 in KC cells transduced with the indicated sgRNAs and treated as indicated. This experiment was repeated independently two times with similar results. G Cell growth curves of KC sgCtrl and sgScaf1 cells expressing the listed isoform of USP15 or an empty vector (EV). Data are expressed as cell confluence percentage (%; mean ± SD, n = 3 independent experiments); two-way ANOVA (sgScaf1+EV p = 0.0342), Dunnett’s multiple comparison. Dose-response curves for KC sgCtrl and sgScaf1 cells expressing the listed isoforms of USP15 or EV and treated with Olaparib. (%; mean ± SD, n = 3 independent experiments; two-way ANOVA (sgScaf1+EV p = 0.00129), Sidak multiple comparison H Tumor-free survival of NSG mice orthotopically injected with sgCtrl (n = 5) or sgScaf1 KC cells expressing the listed isoforms of USP15 (n = 5: n = 5) or EV (n = 5; n = 5). Log-Rank test (Mantel-Cox).

Fig. 5. Scaf1 regulates TNFa and p53 signaling as well as hedgehog signaling in response to Olaparib.

A Volcano Blot showing differential expressed genes between Scaf1-knockout compared to control KC cells. Wald test and Benjamini-Hochberg (BH)-adjusted P-value. Two independent sgRNAs, two biological duplicates. B Bar graph showing gene set enrichment analysis of Scaf1-knockout compared to control KC cells. GSEA nominal p-values. Two independent sgRNAs, two biological duplicates. C Bar graph showing gene set enrichment analysis of Usp15-knockout and Scaf1-knockout compared to sgCtrl control KC cells treated with Olaparib (1 µM). GSEA nominal p-values. Two independent sgRNAs, two biological duplicates. D Expression levels of genes related to HH signaling evaluated by RT-qPCR in the indicated KC cell lines. Results were normalized with Gapdh and are expressed in fold change to CTRL (mean ± SEM, n = 3 independent experiments). Cells were treated with 100 nM Smoothened Agonist (SAG) and 1 µM Olaparib. Two-sided T-test, for sgUSP15: NRP2 p = 0.021/p = 0.018; PTCH1 p = 0.037/p = 0.033; GLI1 p = 0.033; NRP2 p = 0.042/p = 0.037; for sgScaf1: PTCH1 p = 0.046; PTCH2 p = 0.040/p = 0.042/p = 0.033; GLI1 p = 0.037/p = 0.031; NRP2 p = 0.042.

Fig. 6. USP15 and SCAF1 function as tumor suppressor in human pancreatic cancer.

A Oncoprint of the indicated genes in PDAC samples (n = 293, TCGA). B Kaplan-Meier survival analyses of PDAC patients with deep or shallow USP15 or SCAF1 deletion. (n = 293, TCGA) Log-Rank test (Mantel-Cox). C Tumor-free survival of NSG mice orthotopically injected with sgCtrl (n = 5), sgUsp15_1/2 (n = 5; n = 5) or sgSCAF1_1/2 (n = 5; n = 5) PANC-1 cells. Two independent sgRNAs were used. Log-Rank test (Mantel-Cox) D Dose-response curves for sgCtrl, sgUsp15 or sgSCAF1 PANC-1 cells treated with Olaparib. (%; mean ± SD, n = 3 independent experiments) two-way ANOVA (sgUsp15_1 p = 0.0242; sgUsp15_2 p = 0.0387; sgScaf1_1 p = 0.0281; sgScaf1_2 p = 0.0371), Dunnett’s multiple comparison. E sgUSP15 and sgOR2W5 PDO Competition Assay. sgUSP15 and control sgOR2W5 patient-derived organoids were disassociated into single cells and mixed in a 20:80% ratio. Organoid cultures were passaged, and a sample was collected every ~7 days. Percentage of DNA indels is tracked over time by sanger-sequencing and TIDE analysis.