MNT deletion enhances the survival of NSG mice engrafted with MV4;11 human AML cells. (A) Western blot showing the in vitro MNT deletion after DOX induction of sgRNA expression in the batch of MV4;11 human AML cells used for transplantation into NSG mice. (B) DOX-induced MNT loss in MV4;11 human AML cells in vivo enhanced the survival of NSG mice that received transplants. Transplant recipients treated with DOX (pink curve) survived significantly longer than the untreated mice (compare pink and pale blue curves) and those treated with VEN (compare pink and dark blue curves). Combining VEN with MNT loss (green curve) did not statistically improve the survival when compared with MNT loss alone (pink curve). NSG mice were transplanted with 5 × 105 MV4;11 human AML cells that coexpressed Cas9 and 2 independent DOX-inducible sgRNAs that targeted human MNT (MNT2 and MNT4). Recipient mice were either left untreated, treated with DOX to delete MNT, treated with the BH3 mimetic drug VEN to inhibit BCL-2, or treated with both DOX and VEN (6 mice per treatment arm). Dosing with DOX to induce expression of MNT sgRNAs commenced on day 4 after transplantation (600 mg/kg DOX hyclate in base rodent chow, fed orally ad libitum). Dosing with VEN (50 mg/kg, by oral gavage every day except weekends, for 4 weeks) commenced on day 10 after transplantation. Statistical significance was determined using a log-rank (Mantle-Cox) test with Bonferroni correction (ns, P > .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001). (C) MNT deletion and VEN treatment significantly reduced the tumor burden in the BM of mice transplanted with MV4;11 cells. Flow cytometric analysis of hCD45+ cells in BM isolated by intrafemoral sampling of mice in panel B, 28 days after transplantation. Reduction of AML cells was most efficient for the combination of MNT deletion plus VEN treatment (compare green to pale blue column); the differences between the various treatment groups did not reach statistical significance. The data are presented as the mean ± SEM of 3 to 6 mice per treatment arm. A 1-way ANOVA with Dunnett multiple comparison test was used to determine statistical significance (∗∗P ≤ .01; ∗∗∗ P ≤ .001). (D) A comparison of MNT messenger RNA expression in AMLs that had MLL (KMT2A) or other indicated fusion genes, other types of AMLs, and CD34+ HSCs from healthy individuals, determined by the analysis of BEAT-AML 1.0 (accessed on 16 May 2025; https://registry.opendata.aws/beataml). hCD45, human CD45; sgh, short guide human.
Figure 6.

MNT deletion enhances the survival of NSG mice engrafted with MV4;11 human AML cells. (A) Western blot showing the in vitro MNT deletion after DOX induction of sgRNA expression in the batch of MV4;11 human AML cells used for transplantation into NSG mice. (B) DOX-induced MNT loss in MV4;11 human AML cells in vivo enhanced the survival of NSG mice that received transplants. Transplant recipients treated with DOX (pink curve) survived significantly longer than the untreated mice (compare pink and pale blue curves) and those treated with VEN (compare pink and dark blue curves). Combining VEN with MNT loss (green curve) did not statistically improve the survival when compared with MNT loss alone (pink curve). NSG mice were transplanted with 5 × 105 MV4;11 human AML cells that coexpressed Cas9 and 2 independent DOX-inducible sgRNAs that targeted human MNT (MNT2 and MNT4). Recipient mice were either left untreated, treated with DOX to delete MNT, treated with the BH3 mimetic drug VEN to inhibit BCL-2, or treated with both DOX and VEN (6 mice per treatment arm). Dosing with DOX to induce expression of MNT sgRNAs commenced on day 4 after transplantation (600 mg/kg DOX hyclate in base rodent chow, fed orally ad libitum). Dosing with VEN (50 mg/kg, by oral gavage every day except weekends, for 4 weeks) commenced on day 10 after transplantation. Statistical significance was determined using a log-rank (Mantle-Cox) test with Bonferroni correction (ns, P > .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001). (C) MNT deletion and VEN treatment significantly reduced the tumor burden in the BM of mice transplanted with MV4;11 cells. Flow cytometric analysis of hCD45+ cells in BM isolated by intrafemoral sampling of mice in panel B, 28 days after transplantation. Reduction of AML cells was most efficient for the combination of MNT deletion plus VEN treatment (compare green to pale blue column); the differences between the various treatment groups did not reach statistical significance. The data are presented as the mean ± SEM of 3 to 6 mice per treatment arm. A 1-way ANOVA with Dunnett multiple comparison test was used to determine statistical significance (∗∗P ≤ .01; ∗∗∗ P ≤ .001). (D) A comparison of MNT messenger RNA expression in AMLs that had MLL (KMT2A) or other indicated fusion genes, other types of AMLs, and CD34+ HSCs from healthy individuals, determined by the analysis of BEAT-AML 1.0 (accessed on 16 May 2025; https://registry.opendata.aws/beataml). hCD45, human CD45; sgh, short guide human.

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