TP53 mutations confer increased resistance to hypomethylating agents as well as BCL-2 inhibition in vitro. (A) Graphical representation of the experimental workflow for generating MOLM13-TP53 isogenic cell lines and MV4-11 and OCI-AML3 TP53^KO cell lines. (B) MOLM13-TP53 isogenic AML cell lines were treated with DMSO, decitabine, azacitidine, or venetoclax at increasing concentrations for 72 hours, after which cell viability was assessed using a CellTiter-Glo luminescent assay (symbols represent averages from 3 independent experiments; error bars indicate standard error of the mean). (C) MOLM13-TP53 isogenic AML cell lines were treated with HMAs in combination with venetoclax at increasing concentrations for 72 hours, after which cell viability was assessed using a CellTiter-Glo luminescent assay, and viabilities were plotted within a drug synergy matrix (data points represent averages of results from 2 independent experiments). Average zero interaction potency (ZIP) scores were calculated to assess potential synergism. (D) MOLM13-TP53 isogenic AML cell lines were treated with DMSO, decitabine (Dec), azacitidine (Aza), venetoclax (Ven), or a combination thereof at IC₂₅ and IC₅₀ for 48 hours. At this point, cells were stained with annexin V and analyzed by flow cytometry to assess total apoptotic cells (bar graphs represent averages of 3 independent experiments; error bars indicate standard error of the mean. (E) Experimental workflow for in vitro competition assays in MOLM13-TP53 isogenic AML cell lines. MOLM13-TP53^mutant RFP657⁺ cells were mixed with MOLM13-TP53^WT GFP⁺ cells at a 1:1 ratio and cultured in the presence of DMSO or the indicated drugs for 10 days, during which repetitive flow cytometric measurements were performed. (F) MOLM13-TP53 isogenic AML cell lines with TP53^WT, TP53^KO, or TP53^R248Q/− were treated with DMSO, Dec, or Aza at IC₂₅ or IC₅₀ for 24 hours, after which whole-cell protein lysates were collected, run on a polyacrylamide gel, and immunoblotted for p53, DNA methyltransferase 1 (DNMT1), and vinculin (3 independent experiments; 1 representative image is shown). (G) Heat maps depicting results from in vitro competition assays in MOLM13-TP53 isogenic AML cell lines. Equivalent (1:1) numbers of MOLM13-TP53^mutant (RFP657⁺) and MOLM13-TP53^WT (GFP⁺) cells were seeded and cocultured in the continued presence of the indicated doses of decitabine (D), azacitidine (A), or venetoclax (V) alone or HMAs + venetoclax (in this case at IC₂₅). Cell survival was monitored by flow cytometry to track RFP657⁺ and GFP⁺ cells (average results from 2-4 independent experiments are shown). (H) Outgrowth of TP53^mutant MOLM13-TP53 isogenic cell lines seeded in a 1:1 ratio with TP53^WT cells and treated with 2 repetitive cycles of DMSO, Dec, or Aza at IC₂₅ and IC₅₀ (symbols represent averages from 3-6 independent experiments). ***P < .001, 1-way ANOVA. CRISPR-HDR, CRISPR-Cas9–mediated homology directed repair; CRISPR-KO, CRISPR-Cas9–mediated gene knockout; d0, day 0; d2, day 2; d4, day 4; d6, day 6; d8, day 8; d10, day 10; ns, not significant; #, not applicable because of cell death.