Impact of CRISPR/Cas9-mediated MNT deletion on the sensitivity of human AML CLs to BH3 mimetic drugs. (A) The expression of MYC, MNT, and the indicated BCL-2 protein family members in the human AML CLs MV4;11, MOLM-13, THP-1, OCI-AML3 and, as a positive control, the multiple myeloma CL U266B1, as determined by western blot. The protein levels were quantified in each blot relative to the actin loading control by densitometric analysis using ImageJ software; the values are indicated. (B) Western blot analysis of CRISPR/Cas9-mediated MNT loss in human AML CLs. MV4;11, MOLM-13, THP-1, and OCI-AML3 cells were infected with 2 lentiviruses, 1 carrying Cas9 and the mCherry marker and the other carrying an sgRNA and GFP marker. Double-positive (mCherry⁺GFP⁺) cells were collected by flow cytometry and treated with DOX for 3 or 5 days to induce expression of the sgRNAs that target MNT (MNT2, MNT4) or mouse Bim (mBim). AH15 is a control Eμ-Myc lymphoma CL in which Mnt was deleted by CRISPR/Cas9. (C-F) The impact of MNT loss on the sensitivity to BH3 mimetics. MV4;11, MOLM-13, THP-1, and OCI-AML3 cells infected with Cas9 and sgRNA lentiviruses were treated with DOX for 3 days, followed by treatment with the BH3 mimetic drugs S63845 (MCL-1i) or ABT-199/VEN (BCL-2i) in the presence of DOX for 48 hours. BH3 mimetic drug concentrations used were based on predetermined individual IC₅₀ values. Live cells were identified as annexin V–negative/PI-negative by flow cytometry. The data are shown normalized to the DMSO-treated control samples, and significance is indicated relative to the control sgRNA mBim. All data are presented as mean ± SEM of 2 independent experiments. A 1-way ANOVA with Dunnett multiple comparison test was used to determine statistical significance (ns, P > .05; ∗P ≤ .05; ∗∗P ≤ .01). BCL-2i, BCL-2 inhibitor; DOX, doxycycline; KO, knockout; MCL-1i, MCL-1 inhibitor; WT, wild type.