Mnt deletion increases the susceptibility of MLL::AF9 AML CLs to BH3 mimetic drugs. (A) Expression of BCL-2 family members in primary Mntfl/flCreERT2/MLL::AF9 AML CLs and control CreERT2/MLL::AF9 AML CLs. MCL-1, BCL-2, and BCL-XL promote cell survival. BAX, BAK, and BH3-only protein promote apoptosis. The western blots used β-actin as a loading control. Molecular weight markers are indicated (kilodalton). The results are representative of independent experiments. (B) Mnt deletion enhances the killing of AML cells by BH3 mimetic drugs. Short-term AML CLs established from 5 independent Mntfl/flCreERT2/MLL::AF9 AML tumors (2205, circle; 2206, square; 2207, triangle up; 2233, triangle down; 2235, diamond) were treated for 24 hours in triplicate with 0.5 μM 4-OHT or vehicle (ethanol), followed by treatment with the BH3 mimetic drugs S63845 (MCL-1 inhibitor), ABT-199/venetoclax (VEN; BCL-2 inhibitor), or A-1331852 (BCL-XL inhibitor) for a further 24 hours at the indicated concentrations, or with vehicle (DMSO), in the presence of 4-OHT. The BH3 mimetic drug concentrations used were based on predetermined 50% inhibitory concentration (IC50) values (data not shown). Live cells were quantified using annexin V–negative PI-negative staining with flow cytometry (supplemental Figure 3), and cell viability is shown normalized to treatment with DMSO. The results are from 2 independent experiments; mean ± standard error of mean (SEM); ∗∗P ≤ .01; ∗∗∗∗P ≤ .0001 as determined by 1-way ANOVA with Dunnett multiple comparison test. (C) MLL::AF9 AML cell death after Mnt loss and treatment with BH3 mimetic drugs occurs by apoptosis. Mntfl/flCreERT2/MLL::AF9 AML CLs used in panel B were treated in triplicate with 4-OHT or vehicle (ethanol) for 24 hours, with or without the addition of a pan-caspase inhibitor (QVD-OPH; 25 μM), and then treated for 24 hours with BH3 mimetic drugs or vehicle (DMSO). Viable cells (annexin V–negative PI-negative) were quantified by flow cytometry. Results are from 3 independent experiments with 1 CL (2206) analyzed twice; mean ± SEM; ns, P > .05; ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001 as determined by 1-way ANOVA with Tukey multiple comparison test. (D) Apoptosis is enhanced by combining Mnt deletion with synergistic concentrations of BH3 mimetic drugs. The Mntfl/flCreERT2/MLL::AF9 AML CLs described in panel B were treated in triplicate with 0.5 μM 4-OHT or vehicle (ethanol), with or without QVD-OPH (25 μM), for 24 hours and then treated for 24 hours with either 1.5 μM S63845 + 1.5 μM ABT-199 or 1.5 μM S63845 + 5 μM A-1331852 or carrier alone (DMSO). Viable cells (Annexin V-negative PI-negative) were identified by flow cytometry. Results are from 3 independent experiments with 1 CL (2206) analyzed twice. The data are presented as mean ± SEM; ns, P > .05; ∗P ≤ .05; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001 as determined by 2-way ANOVA with Tukey multiple comparison test. (E) Proapoptotic BIM is elevated in MNT-deficient AML cells. Western blot analysis of 2235 Mntfl/flCreERT2/MLL::AF9 AML CL treated as in panel C, showing the protein levels of MNT, BCL-2 family members, and PARP1, with actin as the loading control. The indicated ratios show the BIM level in comparison with that of actin on the same blot, determined by densitometric analysis using ImageJ software. Cleavage of PARP1, a marker of apoptosis, is inhibited in the presence of QVD-OPH. DMSO, dimethyl sulfoxide; KO, knockout; ns, not significant; PARP, poly ADP ribose polymerase; WT, wild type.
Figure 2.

Mnt deletion increases the susceptibility of MLL::AF9 AML CLs to BH3 mimetic drugs. (A) Expression of BCL-2 family members in primary Mntfl/flCreERT2/MLL::AF9 AML CLs and control CreERT2/MLL::AF9 AML CLs. MCL-1, BCL-2, and BCL-XL promote cell survival. BAX, BAK, and BH3-only protein promote apoptosis. The western blots used β-actin as a loading control. Molecular weight markers are indicated (kilodalton). The results are representative of independent experiments. (B) Mnt deletion enhances the killing of AML cells by BH3 mimetic drugs. Short-term AML CLs established from 5 independent Mntfl/flCreERT2/MLL::AF9 AML tumors (2205, circle; 2206, square; 2207, triangle up; 2233, triangle down; 2235, diamond) were treated for 24 hours in triplicate with 0.5 μM 4-OHT or vehicle (ethanol), followed by treatment with the BH3 mimetic drugs S63845 (MCL-1 inhibitor), ABT-199/venetoclax (VEN; BCL-2 inhibitor), or A-1331852 (BCL-XL inhibitor) for a further 24 hours at the indicated concentrations, or with vehicle (DMSO), in the presence of 4-OHT. The BH3 mimetic drug concentrations used were based on predetermined 50% inhibitory concentration (IC50) values (data not shown). Live cells were quantified using annexin V–negative PI-negative staining with flow cytometry (supplemental Figure 3), and cell viability is shown normalized to treatment with DMSO. The results are from 2 independent experiments; mean ± standard error of mean (SEM); ∗∗P ≤ .01; ∗∗∗∗P ≤ .0001 as determined by 1-way ANOVA with Dunnett multiple comparison test. (C) MLL::AF9 AML cell death after Mnt loss and treatment with BH3 mimetic drugs occurs by apoptosis. Mntfl/flCreERT2/MLL::AF9 AML CLs used in panel B were treated in triplicate with 4-OHT or vehicle (ethanol) for 24 hours, with or without the addition of a pan-caspase inhibitor (QVD-OPH; 25 μM), and then treated for 24 hours with BH3 mimetic drugs or vehicle (DMSO). Viable cells (annexin V–negative PI-negative) were quantified by flow cytometry. Results are from 3 independent experiments with 1 CL (2206) analyzed twice; mean ± SEM; ns, P > .05; ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001 as determined by 1-way ANOVA with Tukey multiple comparison test. (D) Apoptosis is enhanced by combining Mnt deletion with synergistic concentrations of BH3 mimetic drugs. The Mntfl/flCreERT2/MLL::AF9 AML CLs described in panel B were treated in triplicate with 0.5 μM 4-OHT or vehicle (ethanol), with or without QVD-OPH (25 μM), for 24 hours and then treated for 24 hours with either 1.5 μM S63845 + 1.5 μM ABT-199 or 1.5 μM S63845 + 5 μM A-1331852 or carrier alone (DMSO). Viable cells (Annexin V-negative PI-negative) were identified by flow cytometry. Results are from 3 independent experiments with 1 CL (2206) analyzed twice. The data are presented as mean ± SEM; ns, P > .05; ∗P ≤ .05; ∗∗∗P ≤ .001; ∗∗∗∗P ≤ .0001 as determined by 2-way ANOVA with Tukey multiple comparison test. (E) Proapoptotic BIM is elevated in MNT-deficient AML cells. Western blot analysis of 2235 Mntfl/flCreERT2/MLL::AF9 AML CL treated as in panel C, showing the protein levels of MNT, BCL-2 family members, and PARP1, with actin as the loading control. The indicated ratios show the BIM level in comparison with that of actin on the same blot, determined by densitometric analysis using ImageJ software. Cleavage of PARP1, a marker of apoptosis, is inhibited in the presence of QVD-OPH. DMSO, dimethyl sulfoxide; KO, knockout; ns, not significant; PARP, poly ADP ribose polymerase; WT, wild type.

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