TP53 loss enables AML cells to rapidly escape suboptimal BCL-2 inhibition. (A) Western blot analysis confirming CRISPR/Cas9-induced loss of TP-53 expression in 2 independent clones of the MOLM-13 human AML derived cell line, expressing guide RNAs to target TP53. In all cases, cells were cotreated with an MDM2 inhibitor for 16 hours to induce expression and activation of TP-53, as well as the broad-spectrum caspase inhibitor Q-VD-OpH to prevent late stages of apoptosis that are associated with protein degradation. (B) Wild-type (WT) or TP53-deficient (KO) clones of MOLM-13 were treated with 0 to 10 μM venetoclax, and their viability was determined 24 hours later. (C) Schematic of the in vitro cell competition assay used to evaluate the impact of suboptimal venetoclax treatment over several weeks on TP53 WT and isogenic KO clones. Equivalent (50:50) numbers of TP53 WT (GFP⁺) and TP53 KO (BFP⁺) cells were seeded and cocultured in the continued presence of the indicated doses of venetoclax. Cell survival was monitored by flow cytometry to track GFP⁺ and BFP⁺ cells in viable PI⁻ cells. (D) The growth of WT or TP53 KO MOLM-13 cells treated continuously with a suboptimal (IC₂₀) dose (supplemental Table 4) of venetoclax (or DMSO) was monitored, as in panel C. (E) The outgrowth of TP53 KO MOLM-13 cells seeded in a 5:95 TP53 KO/TP53 WT ratio treated continuously with an IC₅₀ dose of venetoclax (or control DMSO treated) was monitored by flow cytometric analysis. Data are means ± SD of ≥3 independent experiments. (F) The viability of aliquots of TP53 KO (right) MOLM-13 cells or their TP53 WT controls (EV⁻, empty vector; left) maintained continuously in IC₂₀ dose of venetoclax for 0, 7, 14, 21, and 28 days (see panel D) treated for 24 hours with 0 to 10 μM venetoclax was determined as in panel B; the IC₅₀ values are detailed in supplemental Table 5. (G) WT MOLM-13 and TP53 KO cells were transplanted at a 50:50 ratio into NOD SCID IL2Rγ^−/− (NSG) mice. Three days after transplantation, the mice were treated with vehicle, or venetoclax (75 mg/kg by oral gavage once daily [QD] on week days) for 2 weeks. (H) NSG mice were transplanted with equal numbers of TP53 WT and TP53 KO MOLM-13 cells. Three days afterward, mice were treated with vehicle or venetoclax as shown in panel G. The proportions of TP53 WT (GFP⁺) and KO (BFP⁺) human (CD45⁺) cells in the peripheral blood were determined by flow cytometry after the 2-week treatment course. ***P = 5.5 × 10⁻⁵ vehicle vs venetoclax treatment of TP53 KO cells. Data are means ± SD of 5 animals per group from a representative experiment (n = 3 independent experiments).