Supplementary Information for: CRISPR/Cas9-based target validation. for p53-reactivating model compounds

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1 for: CRISPR/Cas9-based target validation for p53-reactivating model compounds Michael Wanzel 1,5, Jonas B. Vischedyk 1, Miriam P. Gittler 1, Niklas Gremke 1, Julia R. Seiz 1, Mirjam Hefter 1, Magdalena Noack 1, Rajkumar Savai 3,4,5, Marco Mernberger 1, Joël P. Charles 1, Jean Schneikert 1, Anne Catherine Bretz 1, Andrea Nist 1,2, Thorsten Stiewe 1,2,5 * Authors affiliation: 1 Institute of Molecular Oncology, 2 Genomics Core Facility, Philipps-University, Marburg, Germany. 3 Max-Planck-Institute for Heart and Lung Research, Department of Lung Development and Remodeling, Bad Nauheim, Germany. 4 Department of Internal Medicine, Justus-Liebig University, Giessen, Germany. 5 Universities of Giessen and Marburg Lung Center, member of the German Center for Lung Research (DZL), Germany. *Corresponding author: thorsten.stiewe@uni-marburg.de page 1 / 14

2 SUPPLEMENTARY RESULTS Supplementary Figure 1. T7 endonuclease assay reveals an enrichment of p53 mutated cells by nutlin but not RITA. Genomic DNA from HCT116 and H460 that was analyzed by next generation sequencing in Fig. 1c was PCR amplified using primers spanning the CRISPR nuclease target sites in TP53 exon 3 (ex3) or exon 5 (ex5). Ctrl, control cells not transfected with CRISPR nucleases; e3/e5.1/e5.2, cells transfected with CRISPR nucleases targeting the indicated TP53 sequences. Following denaturation, reannealing and incubation with T7 endonuclease PCR products were separated on agarose gels to visualize cleavage of heteroduplices as an indication for the presence of mutations. Supplementary Figure 2. Enrichment of cells depleted of p53 by RNAi. left: Western Blot of HCT116 cells transduced with indicated lentiviral constructs co-expressing shrnas together with a secreted luciferase (GLuc or CLuc) and treated as indicated (N, 10 µm nutlin; R, 1 µm RITA). nsh, non-targeting control shrna, shp53.1/5, p53-targeting shrnas. right: mean fold enrichment of p53-knockdown (p53-kd) cells from 1:1 mixtures of CLuc-positive nsh cells with GLuc-positive cells expressing p53-shrnas treated as indicated. Mean enrichment was calculated based on luciferase activities in the supernatant of triplicate cell mixtures (n=3) and tested for significant differences between treated and untreated groups by two-way ANOVA corrected for multiple comparisons by Dunnett's test (****, P<0.0001; n.s., not significant). page 2 / 14

3 Supplementary Figure 3. Colony formation of parental H460 under indicated treatment compared to CRISPR/Cas9-generated single cell clones with small TP53 indels. GFP: negative control H460 transfected with a GFP-targeted nuclease. Western Blots confirm the successful inactivation of p53. Relative clonogenicity is shown as mean ± s.d. (n=4). page 3 / 14

4 Supplementary Figure 4. Rapid adapation to RITA does not confer cross-resistance to nutlin. (a) Colony formation assay of H460 cells treated with 1 µm RITA for 2 weeks shows outgrowth of RITA resistant cell clones (H460 res ). (b) Resistance of indicated parental and RITA-adapted cell lines to 3 day treatment with 1 µm RITA (MCF7: 0.5 µm) or 10 µm nutlin. Shown is the relative survival to parental cells. Differences in survival between resistant and parental cells were tested for statistical significance by two-way ANOVA corrected with Sidak's multiple comparisons test (*, P<0.0001, n=3). No significant differences in relative survival of parental and RITA-adapted cells were observed under nutlin treatment. (c) Colony formation of parental H460, RITA-adapted H460 and intrinsically RITA-resistant A549 cells to 1 µm RITA and 10 µm nutlin. Supplementary Figure 5. RITA resistance is reversible. (a) Independent RITA-adapted H460 cell clones (H460 res #1-4) were continuously passaged for one year without RITA and tested for clonogenic growth in the absence or presence of 1 µm RITA (late) in comparison to the parental and original resistant cell clones (early). (b) Survival of parental and RITAresistant H460 cell clones treated with the indicated concentrations of RITA. Shown is the mean survival (n=2) relative to untreated as determined by CellTiter-Glo assays (Promega). Early, clones at early passages; late, clones one year after continuous passaging without RITA. page 4 / 14

5 Supplementary Figure 6. Accumulation of RITA in parental and resistant H460 cells. Intracellular accumulation of RITA was measured by flow cytometry following a 2 day treatment of parental and RITA resistant H460 cells with the depicted autofluorescent RITA derivative (RITA*) at a final concentration of 1 µm. RITA* has the same cytotoxicity and resistance profile as RITA. Supplementary Figure 7. Pharmacological inhibitors of ABC transporters fail to overcome RITA resistance. RITA-resistant H460 cell clones were treated with indicated concentrations of RITA together with multi-abc transporter inhibitors verapamil (25 µm) and reserpine (5 µm) or without (untreated). Shown is the survival relative to cells cultured without RITA as determined by CellTiter-Glo assays (Promega). Supplementary Figure 8. Depletion of individual ABC transporters fails to overcome RITA resistance. RITA-resistant H460 cells were transfected with sirna pools (Dharmacon SMARTpools) targeting the indicated genes at a final concentration of 25 nm. Cells were cultured with or without 1 µm RITA for 3 days. Cell viability was determined by CellTiter-Glo assays (Promega). Shown is the mean viability ± s.d. (n=4) relative to untransfected and untreated control cells. Blue: ABC transporter sirnas; red: cytotoxic sirnas (positive control); grey/black: non-targeting sirnas (negative control). page 5 / 14

6 Supplementary Figure 9. Induction of γh2a.x after a 16 hour treatment with 1 µm RITA in sensitive but not RITA resistant H460 or primary resistant A549 cells. (a) Western Blot; (b) immunofluorescence. Color bars indicate RITA sensitivity (as shown in Supplementary Figure 3; sens: sensitive; res: resistant). page 6 / 14

7 Supplementary Figure 10. Immunofluorescence analysis of parental (H460), RITA-resistant (H460 res ) and CDDP-resistant (H460 CDDPres ) cells for γh2a.x 6 hours following a 2 hour treatment with 16 µm CDDP indicates similar levels of DNA damage irrespective of sensitivity. Supplementary Figure 11. Western Blot of parental and RITA resistant H460 cells transduced with lentiviral vectors expressing secreted luciferases (GLuc or CLuc) coupled to 3 different shrnas targeting FancD2. nsh, non-targeting control shrna. page 7 / 14

8 Supplementary Figure 12. FancD2 depletion sensitizes multiple cell lines with acquired (U2OS res ) or intrinsic resistance (H1299, A549, H661) to 1 µm RITA. Supplementary Figure 13. (a) Colony formation assay: FancD2 depletion sensitizes a p53- negative, RITA-resistant HCT116 clone (i5#2, Figure 1e) with a CRISPR-generated small TP53 indel mutation to 1 µm RITA. (b) Western Blot demonstrates knockdown efficiency. page 8 / 14

9 Supplementary Figure 14. (a) Colony formation of RITA resistant H460 cells treated with 2 µm RITA following knockdown of FancA, FancL and Rad18. (b) Western Blots demonstrate knockdown efficiency. Supplementary Figure 15. Western blot for Rad18 and FancD2 in RITA resistant H460 cells transfected with two independent Rad18-targeting sirna. nsi, non-targeting control sirna. Supplementary Figure 16. Western blot for FancD2 and Rad18 in 4 in dependent CDDP resistant H460 cell clones (H460 CDDPres ) compared to parental H460 cells. page 9 / 14

10 Supplementary Figure 17. Chemical structures of compounds used in this study. page 10 / 14

11 Supplementary Figure 18. The full blots used for figures 1e, 2a and 2c. The specific bands shown in the main figures are highlighted. page 11 / 14

12 Supplementary Figure 19. The full blots used for figures 4d, 5b, 6a, 6b, 6c, 6e and 6h. The specific bands shown in the main figures are highlighted. page 12 / 14

13 Supplementary Figure 20. The full blots used for supplementary figures 2a, 3, 9a and 11. The specific bands shown in the main figures are highlighted. page 13 / 14

14 Supplementary Figure 21. The full blots used for supplementary figures 13, 14b, 15 and 16. The specific bands shown in the main figures are highlighted. page 14 / 14