CRISPR-Cas9-based target validation for p53-reactivating model compounds

Abstract

Inactivation of the p53 tumor suppressor by Mdm2 is one of the most frequent events in cancer, so compounds targeting the p53-Mdm2 interaction are promising for cancer therapy. Mechanisms conferring resistance to p53-reactivating compounds are largely unknown. Here we show using CRISPR-Cas9-based target validation in lung and colorectal cancer that the activity of nutlin, which blocks the p53-binding pocket of Mdm2, strictly depends on functional p53. In contrast, sensitivity to the drug RITA, which binds the Mdm2-interacting N terminus of p53, correlates with induction of DNA damage. Cells with primary or acquired RITA resistance display cross-resistance to DNA crosslinking compounds such as cisplatin and show increased DNA cross-link repair. Inhibition of FancD2 by RNA interference or pharmacological mTOR inhibitors restores RITA sensitivity. The therapeutic response to p53-reactivating compounds is therefore limited by compound-specific resistance mechanisms that can be resolved by CRISPR-Cas9-based target validation and should be considered when allocating patients to p53-reactivating treatments.

Figure 1. p53-deficient cells are resistant to nutlin but not RITA.

(a) Schematic outline of CRISPR-Cas9–based p53 enrichment assay. WT, wild type. (b) Schematic representation of the TP53 gene locus. Cleavage sites (black triangles) of CRISPR-Cas9 nucleases and primers (arrows) used for PCR analysis are indicated. (c) Enrichment of cells with TP53 indel mutations. Shown are percentages of mutated sequencing reads in cells treated with 7 μM nutlin or 1 μM RITA for the indicated time points. Enrichment versus untreated cells at day 0 was tested for significance using the nonparametric Friedman′s test and corrected for multiple comparisons using Dunn′s test (*P < 0.05; **P < 0.01; NS, not significant). (d) Enrichment of cells with deletion of exons 2–9 in TP53. Cells cotransfected with a nuclease pair were treated as in c for 10 d and analyzed by qPCR for deletion of exons 2–9. Shown is the fold enrichment in treated versus untreated cells (mean ± s.d., n = 3). Enrichment versus untreated cells was tested for significance by nonparametric Friedman′s test corrected for multiple comparisons by Dunn′s test (**P < 0.01; NS, not significant). (e) Colony formation of HCT116 parental and HCT116 p53^−/− (ref. 17) cells under the indicated treatments compared to CRISPR-Cas9-generated single cell clones with small indels or large deletions in TP53. ‘GFP’ represents negative control cells transfected with a GFP-targeted nuclease. Western blots confirm the successful inactivation of p53. Relative clonogenicity is shown as mean ± s.d. (n = 4). MW, molecular weight; R, RITA treatment; N, nutlin treatment.

Figure 2. RITA sensitivity correlates with induction of DNA damage.

(a,b) Expression of p53 protein, p53-Ser15 phosphorylation and p21 mRNA of parental H460 cells and two RITA-resistant H460 cell pools treated as indicated for 24 h (R, 1 μM RITA; N, 10 μM nutlin). p21 expression is shown normalized to GAPDH expression and relative to untreated cells (mean ± s.d. n = 3; *, P < 0.0001, by two-way analysis of variance and Sidak′s multiple comparisons test) by western blotting (a) and RTqPCR( b). MW, molecular weight; R, RITA treatment; N, nutlin treatment. (c,d) Expression of p53 and γH2A.X in parental and HCT116 derivate clones after a 16 h treatment with 1 μM RITA, as shown by western blotting (c) and immunofluorescence (d). Color bars indicate RITA sensitivity, as in Figure 1e.

Figure 3. RITA resistant cells are cross-resistant to cisplatin.

(a) Colony formation of parental and RITA-resistant H460 cells 7 d following a 3-d treatment with 3 μΜ CDDP, 8 μg ml⁻¹ oxaliplatin, 50 μg ml⁻¹ carmustin and 0.05 μg ml⁻¹ mitomycin C (MMC) or treated continuously with 0.5 μg ml⁻¹ cytarabin (AraC) and 0.5 mM hydroxyurea. (b) Colony formation of parental and CDDP-resistant H460 cells continuously treated with 3 μM CDDP, 1 μM RITA and 10 μM nutlin. (c) Increased DNA damage repair in RITA- and CDDP-resistant H460 cells. Immunofluorescence analysis of γH2A.X at indicated time points following a 2-h treatment with 16 μM CDDP is shown.

Figure 4. RITA resistance is mediated by FancD2.

(a) Parental and RITA-resistant H460 cells transduced with lentiviral vectors expressing secreted luciferases (GLuc or CLuc) coupled to shRNA targeting FancD2. FancD2 knockdown sensitizes parental (left) and RITA-resistant (right) H460 cells to RITA in a competitive co-culture setting. Shown is the relative mean survival of GLuc⁺ cells expressing the indicated shRNAs in 1:1 cocultures with CLuc⁺ NSH cells assayed in triplicate (n = 3) in the absence (blue) or presence (red) of RITA (parental: 0.125 μM, resistant: 2 μM). *P < 0.0001; statistical significance between treated and untreated cell mixtures was assessed by two-way analysis of variance with Tukey's multiple comparisons test. (b) Colony formation of parental and RITA-resistant H460 cells transduced with the indicated shRNAs treated continuously with 1 μM RITA or for 3 d with 8 μg ml⁻¹ oxaliplatin and 3 μM CDDP. (c,d) FancD2 knockdown sensitizes parental HCT116 and HCT116 p53^−/− cells to RITA but not nutlin, as shown by a colony formation assay (c) and western blotting (d). MW, molecular weight; NSH, non-targeting negative control shRNA; NSI, non-targeting negative control siRNA; si6 and si8, FancD2-targeting siRNAs.

Figure 5. RAD18 depletion sensitizes to RITA.

(a) Colony formation of RITA-resistant H460 cells treated with 2 μM RITA following knockdown of Rad18. NSI, non-targeting negative control siRNA; si1 and si3, Rad18-targeting siRNAs. (b) Western blotting reveals high-level expression of FancD2 and Rad18 in multiple different RITA-resistant H460 clones compared to parental H460 cells. MW, molecular weight. (c) Immunofluorescence analysis indicates induction of DNA damage markers γH2A.X and RPA32 phosphorylated at Ser33 (RPA32^pS33) in FancD2-depleted H460 cells following 3-d treatment with 2 μM RITA.

Figure 6. RITA resistance is overcome with mTOR inhibitors.

(a) Elevated mTOR signaling in RITA-resistant H460 cells. Western blot of parental H460 and RITA-resistant H460^res cells in the presence and absence mTOR kinase inhibitor AZD8055, respectively. MW, molecular weight. (b) mTOR inhibition results in progressive downregulation of FancD2 in RITA-resistant H460^res cells treated with AZD8055 for the indicated time points. (c) FancD2 downregulation by 3-d treatment of RITA-resistant H460^res cells with AZD8055 is reversible upon removal of AZD8055 within 24–48 h. (d) AZD8055 reduces clonogenic growth of RITA-treated parental and RITA-resistant H460^res cells. Cells were grown in the absence or presence of AZD8055 for 3 d, treated with a combination of AZD8055 and RITA for 3 d, and finally treated with RITA only for a further 7 d. H460, 1 μΜ RITA; H460^res, 2 μΜ RITA. (e) AZD8055 treatment enhances γH2A.X induction by RITA. (f) AZD8055 reduces clonogenic growth of RITA-resistant H460^res cells treated for 3 d with 3.3 μΜ CDDP and of CDDP-resistant H460^CDDP treated continuously with 1 μΜ RITA. (g,h) mTOR knockdown reduces clonogenic growth of parental H460 and RITA-resistant H460^res cells treated continuously with 1 μΜ RITA or for 3 d with 3.3 μΜ CDDP. Western blotting confirms mTOR knockdown efficiency. In all experiments, AZD8055 was used at 1 μΜ for H460 and at 2 μΜ for H460^res and H460^CDDPres. si8-11, mTOR-targeting siRNAs.