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Venetoclax enhances FLT3-ITD inhibition in AML
A
B
Figure 2. Venetoclax combined with quizartinib has greater anti-tumor efficacy against primary FLT3-ITD+ acute myeloid leukemia models. (A) Primary patient samples were treated with 20 nM quizartinib, 50 nM venetoclax or the combination in methylcellulose in triplicate for 14 days and colony forming units were counted. Data were normalized to the median colony number for the vehicle-treated cells for each sample and plotted as mean + standard deviation. (B) NOD/SCID/IL-2Rγnull (NSG) mice were engrafted with primary samples from Fms-like tyrosine kinase 3 wild-type (FLT3-WT) or FLT3 internal tandem duplication (FLT3-ITD+) patients and treated orally with 5 mg/kg quizartinib, 100 mg/kg venetoclax or the combination once daily for 28 days and efficacy was assessed. Kaplan-Meier survival curve for FLT3-WT and FLT3-ITD+ models. N=7-9 animals/group and median survival and statistical significance were determined by log-rank test: *P<0.0001 for vene- toclax vs. vehicle or quizartinib in FLT3-WT model. **P=0.0004 for quizartinib vs. vehicle or venetoclax and ***P=0.0003 for quizartinib + venetoclax vs. quizar- tinib in the FLT3-ITD+ model.
sublethal irradiation (250 cGY) 24 hours prior to cell inoculation. FLT3 WT or FLT3-ITD+ primary patient samples (1x106 cells) were suspended in Phosphate-buffered saline (PBS) and inoculated through tail vein injection. Engraftment and disease burden were determined by co-staining for human and murine anti-CD45 (BioLegend, San Diego, CA) in peripheral blood samples. Mice were grouped out based upon engraftment (WT average 2.2% engraftment; ITD average 0.8% engraftment) and treated as described for 28 continuous days. Animals were monitored for signs of disease progression and euthanized at first measurement of greater than 20% weight loss or when reaching any humane endpoint.
BLISS analysis
Details are provided in the Online Supplementary Appendix. BH3 profiling
Details are provided in the Online Supplementary Appendix.
Statistical analysis
Statistical comparisons included unpaired t-test or one-way ANOVA with Tukey post hoc test or Dunnett post-test, as indicated with P<0.05 considered statistically significant. Kaplan-Meier sur- vival analysis was used for in vivo studies and significance was determined by log-rank test with P<0.05 considered significant.
All analyses were completed using Prism software package (ver- sion 7; Graphpad Software, La Jolla, CA, USA).
Results
Venetoclax combined with FLT3-ITD inhibition prolonged survival of FLT3-ITD+ leukemic mice in vivo
In order to explore the impact FLT3-ITD on venetoclax activity, we assessed venetoclax treatment in combination with the selective FLT3 inhibitor quizartinib (AC220)32 in FLT3-ITD+ cell line xenograft models (MV4;11 and Molm13). Mice with established leukemia were treated orally with vehicle, venetoclax (100 mg/kg), quizartinib (2.5 or 5 mg/kg), or the combination for 21 continuous days. Consistent with clinical data, venetoclax alone demonstrated little to no efficacy based on survival with marginal improvement in the MV4;11 model (Figure 1A- C) and no anti-tumor activity in the Molm13 model (Figure 1D-F), while quizartinib significantly reduced dis- ease burden and increased survival compared to vehicle in both models (Figure 1A-F). Venetoclax combined with quizartinib led to further improvement in survival and reduction in tumor burden compared to quizartinib alone (Figure 1A-F). Additionally, in the MV4;11 model a subset
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