Treatment with FHD-286–based combinations exerted synergistic in vitro lethality in cultured and PD AML cells expressing MLL1r or mtNPM1 with or without mtFLT3 and reduced leukemia burden and significantly improved survival of NSG mice bearing MLL1r or mtNPM1-expressing AML xenografts. (A) MOLM13, MV4-11, MV4-11-MITR, OCI-AML3, OCI-AML3-MITR, and PD MLL1r or mtNPM1-expressing AML cells were treated with FHD-286 (dose range, 10-250 nM) and MI SNDX-50469 (dose range, 50-1000 nM), BETi OTX015 (dose range, 50-250 nM), or venetoclax (dose range, 10-100 nM) for 72 to 96 hours. At the end of treatment, the percentage of nonviable cells was determined by staining with TO-PRO-3 iodide and flow cytometry analysis. Delta synergy scores were determined by the ZIP method within the SynergyFinder web application. Synergy scores >1.0 indicate a synergistic interaction of the 2 agents in the combination. Panel shows the mean Delta Synergy Score for each FHD-286–based combination in the cell lines and PD AML cells. (B) Total photon counts (flux; determined by bioluminescent imaging) in NSG mice engrafted with luciferized MLL-AF9 + FLT3-TKD AML PDX cells (AML number 6 in the oncoplot) and treated for 3 weeks with FHD-286 and/or venetoclax, decitabine, or OTX015 at the indicated doses. (C) Kaplan-Meier survival plot of NSG mice engrafted with luciferized MLL-AF9 + FLT3-TKD AML PDX cells and treated with 1.5 mg/kg of FHD-286 (daily ×5 days, P.O.) and/or 30 mg/kg of venetoclax (daily ×5 days, P.O.) for 4 weeks. Significance was calculated by a Mantel-Cox log-rank test. (D) Kaplan-Meier survival plot of NSG mice engrafted with luciferized MLL-AF9 + FLT3-TKD AML PDX cells and treated with 1.5 mg/kg of FHD-286 (daily ×5 days, P.O.) and/or 1 mg/kg of DAC (days 1-5 only, IP) for 6 weeks. Significance was calculated by a Mantel-Cox log-rank test. (E) Kaplan-Meier survival plot of NSG mice engrafted with luciferized MLL-AF9 + FLT3-TKD AML PDX cells and treated with 1.5 mg/kg of FHD-286 (daily ×5 days, P.O.) and/or 30 mg/kg of OTX015 (daily ×5 days, P.O.) for 7 weeks. Significance was calculated by a Mantel-Cox log-rank test. (F) Total photon counts (flux; determined by bioluminescent imaging) in NSG mice engrafted with luciferized mtNPM1 + FLT3-ITD PDX cells and treated for 5 weeks with FHD-286 and/or SNDX-5613 or OTX015 at the indicated doses. (G) Representative bioluminescent images of mice from panel F. (H-I) Kaplan-Meier survival plot of NSG mice engrafted with luciferized mtNPM1 + FLT3-ITD PDX cells and treated with 1.5 mg/kg of FHD-286 (daily ×5 days, P.O.) and/or 30 mg/kg of OTX015 (daily ×5 days, P.O.) or SNDX-5613 (50 mg/kg, B.I.D. ×5 days, P.O) for 8 weeks. Significance between cohorts was determined by a Mantel-Cox log-rank test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005; ∗∗∗∗P < .001. B.I.D., twice a day; IP, intraperitoneal; ns, not significant; P.O., oral.

Treatment with FHD-286–based combinations exerted synergistic in vitro lethality in cultured and PD AML cells expressing MLL1r or mtNPM1 with or without mtFLT3 and reduced leukemia burden and significantly improved survival of NSG mice bearing MLL1r or mtNPM1-expressing AML xenografts. (A) MOLM13, MV4-11, MV4-11-MITR, OCI-AML3, OCI-AML3-MITR, and PD MLL1r or mtNPM1-expressing AML cells were treated with FHD-286 (dose range, 10-250 nM) and MI SNDX-50469 (dose range, 50-1000 nM), BETi OTX015 (dose range, 50-250 nM), or venetoclax (dose range, 10-100 nM) for 72 to 96 hours. At the end of treatment, the percentage of nonviable cells was determined by staining with TO-PRO-3 iodide and flow cytometry analysis. Delta synergy scores were determined by the ZIP method within the SynergyFinder web application. Synergy scores >1.0 indicate a synergistic interaction of the 2 agents in the combination. Panel shows the mean Delta Synergy Score for each FHD-286–based combination in the cell lines and PD AML cells. (B) Total photon counts (flux; determined by bioluminescent imaging) in NSG mice engrafted with luciferized MLL-AF9 + FLT3-TKD AML PDX cells (AML number 6 in the oncoplot) and treated for 3 weeks with FHD-286 and/or venetoclax, decitabine, or OTX015 at the indicated doses. (C) Kaplan-Meier survival plot of NSG mice engrafted with luciferized MLL-AF9 + FLT3-TKD AML PDX cells and treated with 1.5 mg/kg of FHD-286 (daily ×5 days, P.O.) and/or 30 mg/kg of venetoclax (daily ×5 days, P.O.) for 4 weeks. Significance was calculated by a Mantel-Cox log-rank test. (D) Kaplan-Meier survival plot of NSG mice engrafted with luciferized MLL-AF9 + FLT3-TKD AML PDX cells and treated with 1.5 mg/kg of FHD-286 (daily ×5 days, P.O.) and/or 1 mg/kg of DAC (days 1-5 only, IP) for 6 weeks. Significance was calculated by a Mantel-Cox log-rank test. (E) Kaplan-Meier survival plot of NSG mice engrafted with luciferized MLL-AF9 + FLT3-TKD AML PDX cells and treated with 1.5 mg/kg of FHD-286 (daily ×5 days, P.O.) and/or 30 mg/kg of OTX015 (daily ×5 days, P.O.) for 7 weeks. Significance was calculated by a Mantel-Cox log-rank test. (F) Total photon counts (flux; determined by bioluminescent imaging) in NSG mice engrafted with luciferized mtNPM1 + FLT3-ITD PDX cells and treated for 5 weeks with FHD-286 and/or SNDX-5613 or OTX015 at the indicated doses. (G) Representative bioluminescent images of mice from panel F. (H-I) Kaplan-Meier survival plot of NSG mice engrafted with luciferized mtNPM1 + FLT3-ITD PDX cells and treated with 1.5 mg/kg of FHD-286 (daily ×5 days, P.O.) and/or 30 mg/kg of OTX015 (daily ×5 days, P.O.) or SNDX-5613 (50 mg/kg, B.I.D. ×5 days, P.O) for 8 weeks. Significance between cohorts was determined by a Mantel-Cox log-rank test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005; ∗∗∗∗P < .001. B.I.D., twice a day; IP, intraperitoneal; ns, not significant; P.O., oral.

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