American Society of Hematology

Figure 5.

$The BAX P168A variant impairs BAX translocation and confers resistance to venetoclax in vitro and in vivo. (A) Expression of BAX P168A in BAX/BAK DKO MOLM-13 cells. Western blot showing BAX expression levels in MOLM-13 WT, BAX/BAK DKO (−) cells, or BAX/BAK DKO engineered to express BAX WT or BAX P168A upon transduction with retroviral expression constructs. (B) Sensitivity of BAX/BAK DKO plus BAX P168A cells to venetoclax. The indicated MOLM-13 cell lines (BAX/BAX DKO, BAX/BAX DKO + BAX WT, BAX/BAX DKO + BAX P168A, and parental MOLM13 WT) were treated with increasing concentrations of venetoclax (0.05-5 μM) for 24 hours and cell viability determined by annexin V/4′,6-diamidino-2-phenylindole staining and flow cytometry. Data are means ± standard error of the mean of 3 independent experiments. (C) BAX P168A does not have dominant-negative activity. BAX WT or BAX P168A was retrovirally expressed in MOLM-13 WT cells and cells treated with 500 nM venetoclax for 24 hours. Cell viability was determined by annexin V/4′,6-diamidino-2-phenylindole staining and flow cytometry. Data are means ± standard error of the mean of 2 independent experiments. (D) The BAX P168A mutation reduces MOM translocation and integration of BAX. MOLM-13 DKO cells expressing BAX WT or BAX P168A were pre-incubated with 25 μM Q-VD-OPh for 1 hour and then treated with 500 nM venetoclax. After 5 hours, cells were subjected to carbonate extraction and fractions run on sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted for BAX. The results are representative of 3 independent experiments. (E-F) MOLM-13 BAX P168A cells are resistant to venetoclax in vivo. Irradiated NSG mice were transplanted with 5 × 105 MOLM13 WT or MOLM13 BAX/BAK DKO cells expressing BAX P168A. On day 4 posttransplant, cohorts of 3 mice were treated with vehicle or venetoclax 75 mg/kg gavage weekdays for 2 weeks. Cohorts were euthanized and human CD45+ cells in BM and peripheral blood enumerated by flow cytometry. Error bars represent SD of 3 mice per treatment arm.$

The BAX P168A variant impairs BAX translocation and confers resistance to venetoclax in vitro and in vivo. (A) Expression of BAX P168A in BAX/BAK DKO MOLM-13 cells. Western blot showing BAX expression levels in MOLM-13 WT, BAX/BAK DKO (−) cells, or BAX/BAK DKO engineered to express BAX WT or BAX P168A upon transduction with retroviral expression constructs. (B) Sensitivity of BAX/BAK DKO plus BAX P168A cells to venetoclax. The indicated MOLM-13 cell lines (BAX/BAX DKO, BAX/BAX DKO + BAX WT, BAX/BAX DKO + BAX P168A, and parental MOLM13 WT) were treated with increasing concentrations of venetoclax (0.05-5 μM) for 24 hours and cell viability determined by annexin V/4′,6-diamidino-2-phenylindole staining and flow cytometry. Data are means ± standard error of the mean of 3 independent experiments. (C) BAX P168A does not have dominant-negative activity. BAX WT or BAX P168A was retrovirally expressed in MOLM-13 WT cells and cells treated with 500 nM venetoclax for 24 hours. Cell viability was determined by annexin V/4′,6-diamidino-2-phenylindole staining and flow cytometry. Data are means ± standard error of the mean of 2 independent experiments. (D) The BAX P168A mutation reduces MOM translocation and integration of BAX. MOLM-13 DKO cells expressing BAX WT or BAX P168A were pre-incubated with 25 μM Q-VD-OPh for 1 hour and then treated with 500 nM venetoclax. After 5 hours, cells were subjected to carbonate extraction and fractions run on sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted for BAX. The results are representative of 3 independent experiments. (E-F) MOLM-13 BAX P168A cells are resistant to venetoclax in vivo. Irradiated NSG mice were transplanted with 5 × 10⁵ MOLM13 WT or MOLM13 BAX/BAK DKO cells expressing BAX P168A. On day 4 posttransplant, cohorts of 3 mice were treated with vehicle or venetoclax 75 mg/kg gavage weekdays for 2 weeks. Cohorts were euthanized and human CD45+ cells in BM and peripheral blood enumerated by flow cytometry. Error bars represent SD of 3 mice per treatment arm.

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