American Society of Hematology

Figure 2.

$Figure 2. Combinatorial anti-AML efficacy for Lip-C6 and vinblastine. (A) Eight human AML patient samples were exposed for 24 hours in vitro to 10 µM Lip-C6 alone or in combination with 2.5 nM vinblastine (VB), and the metabolism of C6-ceramide was evaluated by lipidomic analysis. Vinblastine cotreatment significantly shifted C6-ceramide metabolism to sphingosine and endogenous/physiological ceramides, both proapoptotic metabolites. Unpaired Student t test with Welch’s correction; *P ≤ .02 (specific values indicated). Data points are averages of biological replicates (n ≥ 3). AML-MRC (n = 15) (B) or DN-AML (n = 15) (C) human patient samples were exposed for 48 hours to 20 µM Lip-C6, Lip-Ghost, 5 nM VB, or the combination of Lip-C6 and VB, and apoptosis within the leukemia stem cell (CD34+CD38–) and bulk leukemia fractions was quantified by flow cytometry (1-way ANOVA with Tukey’s post hoc comparison; *P < .0001, #P < .0001, $P ≤ .0072, or &P ≤ .0375 compared with untreated and Lip-Ghost; **P = .0006 or ##P = .0036 compared with Lip-C6; $$P ≤ .0003 or &&P ≤ .0375 compared with untreated and Lip-Ghost; ***P < .0001 or ###P < .0001 compared with untreated, Lip-Ghost, and VB; $$$P ≤ .0001 or &&&P < .001 compared with all other treatments). (D) Survival of C57BL/6J mice engrafted with murine C1498 AML cells and then treated with intravenous injections of anionic formulations of Lip-Ghost control, Lip-C6, liposomal vinblastine (Lip-VB), or the combination of Lip-C6 and Lip-VB. Mantel-Cox log rank test *P = .008 (n ≥ 6). (E) Leukemia burden was quantified after bioluminescent imaging of NSG mice engrafted with MV4-11-LUC-YFP AML cells. Mice were treated with intravenous injections of either the Lip-Ghost control, Lip-C6, VB (not formulated in a liposome), or the combination of both Lip-C6 and VB. Shaded area corresponds to the treatment duration. ****P < .0001 comparing all the Lip-C6 + VB treatment mice with all those that received VB treatment alone at day 106 (2-way ANOVA with Tukey’s post hoc comparison). Each line represents the leukemia burden of an individual mouse. (F) Survival of NSG mice engrafted with human MV4-11-LUC-YFP AML cells that were treated with intravenous injections of Lip-Ghost control, Lip-C6, VB (not formulated in a liposome), or Lip-C6 and VB combined. Mantel-Cox log rank test *P = .0159 (n = 6).$

Combinatorial anti-AML efficacy for Lip-C6 and vinblastine. (A) Eight human AML patient samples were exposed for 24 hours in vitro to 10 µM Lip-C6 alone or in combination with 2.5 nM vinblastine (VB), and the metabolism of C6-ceramide was evaluated by lipidomic analysis. Vinblastine cotreatment significantly shifted C6-ceramide metabolism to sphingosine and endogenous/physiological ceramides, both proapoptotic metabolites. Unpaired Student t test with Welch’s correction; *P ≤ .02 (specific values indicated). Data points are averages of biological replicates (n ≥ 3). AML-MRC (n = 15) (B) or DN-AML (n = 15) (C) human patient samples were exposed for 48 hours to 20 µM Lip-C6, Lip-Ghost, 5 nM VB, or the combination of Lip-C6 and VB, and apoptosis within the leukemia stem cell (CD34⁺CD38^–) and bulk leukemia fractions was quantified by flow cytometry (1-way ANOVA with Tukey’s post hoc comparison; *P < .0001, ^#P < .0001, ^$P ≤ .0072, or ^&P ≤ .0375 compared with untreated and Lip-Ghost; **P = .0006 or ^##P = .0036 compared with Lip-C6; ^$$P ≤ .0003 or ^&&P ≤ .0375 compared with untreated and Lip-Ghost; ***P < .0001 or ^###P < .0001 compared with untreated, Lip-Ghost, and VB; ^$$$P ≤ .0001 or ^&&&P < .001 compared with all other treatments). (D) Survival of C57BL/6J mice engrafted with murine C1498 AML cells and then treated with intravenous injections of anionic formulations of Lip-Ghost control, Lip-C6, liposomal vinblastine (Lip-VB), or the combination of Lip-C6 and Lip-VB. Mantel-Cox log rank test *P = .008 (n ≥ 6). (E) Leukemia burden was quantified after bioluminescent imaging of NSG mice engrafted with MV4-11-LUC-YFP AML cells. Mice were treated with intravenous injections of either the Lip-Ghost control, Lip-C6, VB (not formulated in a liposome), or the combination of both Lip-C6 and VB. Shaded area corresponds to the treatment duration. ****P < .0001 comparing all the Lip-C6 + VB treatment mice with all those that received VB treatment alone at day 106 (2-way ANOVA with Tukey’s post hoc comparison). Each line represents the leukemia burden of an individual mouse. (F) Survival of NSG mice engrafted with human MV4-11-LUC-YFP AML cells that were treated with intravenous injections of Lip-Ghost control, Lip-C6, VB (not formulated in a liposome), or Lip-C6 and VB combined. Mantel-Cox log rank test *P = .0159 (n = 6).

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