Fig. 7: MRE11 and RAD51 cooperation contributes to RSR and mitosis and is essential in PARP1-upregulating CRC-SCs.

a, b Cell viability (assessed by CellTiter-Glo^® assay) and clonogenic survival of neoR-CRC-SCs left untreated or exposed to inhibitors of MRE11 (mirin, MRE11i) and/or RAD51 (B02, RAD51i) for 72 h (b) or 96 h (a) as indicated. Data are reported as means ± SEM from 5 (a) or 3 (b) independent experiments. In b, representative images and individual data points are also shown (dose range: #19neoR: 15 µM MRE11i, 2.5 µM RAD51i). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 (one-way ANOVA and Bonferroni post-hoc test), as indicated. See also Supplementary Fig. S7a–c. c Cell viability (assessed by CellTiter-Glo^® assay) of innR-CRC-SCs left untreated or exposed to inhibitors of MRE11 (mirin, MRE11i) and/or RAD51 (B02, RAD51i) or 96 h as indicated. Data are reported as means ± SEM from 9 independent experiments for all conditions with the exception of RAD51i 2.5 µM in which 5 independent experiments were performed. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 (one-way ANOVA and Bonferroni or Dunnett T3 post-hoc test), as indicated. See also Supplementary Fig. S7d, e. d–f Flow cytometry and western-blot analyses of RSR functionality in representative neoR-CRC-SCs either treated with 1 mM hydroxyurea (HU) and, after drug washout, with 1 µM nocodazole (N) alone or together with MRE11i and/or RAD51i (the HU + N assay, see Fig. 2a) (d; #1neoR), or treated 24 h only with MRE11i and/or RAD51i (e; #1neoR; f; #19neoR). In d and f, cell cycle profiles (mitotic (pH3⁺) cells are in red) and quantification of S-phase or G₂/M-phase or mitotic cells are represented. Results are reported as means ± SEM from 5 independent experiments (d) or as means ± SEM and individual data points from 4 independent experiments (f). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 (one-way ANOVA and Bonferroni or Dunnett T3 post-hoc test) as depicted (d) or as compared to control condition (f). In e, antibodies recognizing γH2AX, pRPA32 and/or RPA32 and/or nucleolin (as loading control) were used. See also Supplementary Fig. S7f, g. g Immunofluorescence analysis in neoR-CRC-SCs treated with MRE11i and/or RAD51i for 24 h and then costained with antibodies against the spindle assembly checkpoint (SAC) activation marker BUBR1 (in red) and the mitotic marker pH3 (in green). Representative aberrant metaphases activating the SAC and the anaphase ratio (i.e., of the fraction of anaphases on 100 prophases+metaphases+anaphases, see Materials and Methods) are reported. Results are means ± SEM from 6 independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 (one-way ANOVA and Dunnett T3 post-hoc test) as compared to control condition. h Immunofluorescence analysis in neoR-CRC-SCs treated with MRE11i and/or RAD51i for 48 h and then stained with an anti-cleaved caspase-3 (CASP3A) antibody. Representative images and the quantification of CASP3A⁺ cells are shown. Results are expressed as means ± SEM and individual data points from 3 independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 (one-way ANOVA and Bonferroni post-hoc test) as indicated. i Proposed model. CRC-SCs resistant to therapeutically-relevant replication poisons or ATR-CHK1 inhibitors display low levels of RS and PARP1 upregulation and can be efficiently targeted by the combined inhibition of (i) CHK1 + PARP1, which provokes fork degradation and severe RS, resulting in lethal replication catastrophe, or (ii) RAD51 + MRE11, which deregulates RSR and mitosis, resulting in cell death via mitotic catastrophe. Dose range in d–g: #1neoR: 25 µM MRE11i, 7.5 µM RAD51i; #19neoR: 20 µM MRE11i, 5–7.5 µM RAD51i. All significant P values are shown in Supplementary Table S4. Supplementary figures associated: Supplementary Figs. S7 and S8.