Fig. 2: Loss of TOPORS sensitizes MDS and AML cell lines to AZA.

A, B Dose-survival plots of FP⁺ cell counts following four daily applications of AZA to MDS-L cells polyclonally expressing Cas9 plus (A) single sgRNAs, or (B) single shRNAs which targeted TOPORS or a non-targeting control. Dots are means (n = 4 technical replicates per data point) normalized to the vehicle control, ±SD. Each experiment was performed once, with two independent targeting RNAs tested per experiment. C Clonogenic assays performed using TOPORS-edited MDS-L cells pre-treated with 0.3 µM AZA as in A before plating in methylcellulose medium. Colonies were counted two weeks after methylcellulose plating. 2-way ANOVA: ***P ≤ 0.001, ****P ≤ 0.0001. n = 3 biological replicates per treatment. D Dose-survival plots of tagRFP⁺ cell counts following 4 days of daily treatment with the indicated AZA concentrations in AML cell lines polyclonally expressing Cas9 plus single sgRNAs or a non-targeting control sgRNA. Dots are means (n = 4 technical replicates per data point) normalized to the vehicle control, ±SD. Each experiment was performed once, with two independent targeting RNAs tested per experiment. E The change in whole body luminescence flux in MISTRG mice engrafted with 10⁵ MOLM-13 cells which polyclonally express luciferase, Cas9 and the indicated sgRNAs, immediately following 1 cycle of treatment with AZA or vehicle i.p.- as described by the time-based x-axis in F. FDR q-values (threshold = 0.01) are reported for a Mann–Whitney multiple comparison test; n = 7 (sgControl 0.3 mg/kg AZA), n = 8 (sgControl 1.0 mg/kg AZA, sgTOPORS 0.3 mg/kg AZA), n = 9 (sgControl 0 mg/kg AZA, sgTOPORS 1.0 mg/kg AZA) or n = 10 (sgTOPORS 0 mg/kg AZA) mice per treatment group. F Kaplan-Meier plots for survival of the same MISTRG mice as E. Whole body luminescence (IVIS) was performed 8 days after engraftment to give a pre-AZA baseline (which was used to rank-randomize mice into treatment groups based on sex and relative engraftment- pre-AZA in E), and again on day 18 – two days after completion of the treatment cycle (post-AZA in E). Event-free survival was scored according to ethics guidelines. **Gehan-Breslow-Wilcoxon test P = 0.0029 (one degree of freedom) for sgCONTROL versus sgTOPORS-2 at 1.0 mg/kg AZA. Source data are provided as a Source Data file.