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. 2022 Oct 14;141(6):634–644. doi: 10.1182/blood.2022016090

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Figure 4. — BAX deficiency confers resistance to combined BCL-2 and MCL1 targeting in vitro. (A) BAX expression in CRISPR-Cas9–edited OCI-AML3 cells. Immunoblot demonstrating CRISPR-Cas9–induced BAX depletion in OCI-AML3 cells using guide RNAs (gRNAs) targeting BAX (gRNA-1.1 or gRNA-2.1) or a nontargeted (empty vector [EV]) control. Cells were treated with 5 μg/mL doxycycline for 72 hours to induce BAX loss. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Drug sensitivity of CRISPR-Cas9–edited OCI-AML3 cells. OCI-AML3 cells transduced with gRNA targeting BAX or a nontargeted control were treated with indicated drugs (0.001-10 μM for BH3-mimetics, 0.01-100 μM for cytarabine, and 0.001 to 1μM for idarubicin) and the LC₅₀ determined by flow cytometry after 48-hour exposure. Error bars are SD of 2 independent experiments. ∗P < .05, ∗∗P <.01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. (C) Survival of mice engrafted with BAX knockout cells in response to BH3-mimetic therapy. Irradiated NSG mice were transplanted with 10⁵ OCI-AML3 nontargeted EV control or OCI-AML3 BAX knockout cells (gRNA 2.1). Dosing commenced on day 4 posttransplant and mice were randomly divided into cohorts of 6 mice and treated with vehicle or a combination of venetoclax (75 mg/kg, weekdays by gavage) and S63845 (25 mg/kg, IV weekly) for 4 weeks. Kaplan-Meier survival analysis (ethical end point) shows that combined treatment with venetoclax/S63845 significantly prolongs survival in OCI-AML3 EV but not OCI-AML3 BAX knockout cohorts (black bar indicates duration of treatment). (D) Survival of mice engrafted with BAK knockout cells in response to BH3-mimetic therapy. Irradiated NSG mice were transplanted with 10⁵ OCI-AML3 nontargeted EV control or OCI-AML3 BAK knockout cells (gRNA 2.1). Dosing commenced on day 4 posttransplant and mice were randomly divided into cohorts of 6 mice and treated with vehicle or a combination of venetoclax (75 mg/kg, weekdays by gavage) and S63845 (25 mg/kg, IV weekly) for 4 weeks. Kaplan-Meier survival analysis (ethical end point) shows that combined treatment with venetoclax/S63845 significantly prolongs survival in both the OCI-AML3 EV and OCI-AML3 BAK knockout cohorts (black bar indicates duration of treatment). ns, not significant.