TP53 loss enables blood cancer cells to rapidly escape suboptimal BCL-2 inhibition. (A) Western blot confirming CRISPR/Cas9-induced loss of TP-53 expression in independent clones of the RS4;11 human ALL-derived cell line, expressing sgRNAs 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) TP53 wild-type (WT) or TP53-deficient (KO) clones of RS4;11 were treated with 0 to 10 μM venetoclax and their viability determined 24 hours later; the IC₅₀ values are indicated in parentheses. (C) The growth of WT or TP53 KO RS4;11 cells treated continuously with a suboptimal (IC₂₀) dose (supplemental Table 4) of venetoclax (or control treated) was monitored. (D) The outgrowth of TP53 KO RS4;11 cells seeded in a 5 TP53 KO:95 TP53 WT ratio treated continuously with an IC₅₀ dose of venetoclax (or control treated) was monitored by flow cytometric analysis. Data are means ± SD of ≥3 independent experiments. (E) The viability of aliquots of TP53 KO RS4;11 cells or their TP53 WT controls (EV⁻ empty vector) maintained continuously in an IC₂₀ dose of venetoclax for 0, 7, 14, 21, and 28 days (see panel C) treated for 24 hours with 0 to 10 μM venetoclax was determined as in panel B; the IC₅₀ values are indicated in supplemental Table 5. (F) Experiments similar to those with RS4;11 ALL cells (A-B, D) were undertaken with OCI-LY19 cells, a human DLBCL-derived cell line. Data from cell viability (Cell-Titer Glo) or cell competition (fluorescence-activated cell sorting [FACS]) assays represent means ± SD of ≥3 independent experiments.