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Synergy between IDH1 inhibitor and hypomethylating agent
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Figure 2. BAY1436032 synergizes with azacitidine to exert potent anti-leukemic activity in a patient-derived IDH1 mutant acute myeloid leukemia xenograft model in vivo. (A) Schematic representation of the treatment regimens; sim: simultaneous treatment with BAY1436032 and azacitidine (Aza); seq: sequential treatment with BAY1436032 and azacitidine. (B) Percentage of hCD45+ leukemic cells in peripheral blood of IDH1 R132C PDX1 mice at different time points after treatment start with vehicle, azacitidine (1 mg/kg, subcutaneoulsy, days 1-5 and days 29-34), BAY1436032 (150 mg/kg orally, continuously), or the sequential or simultaneous combination of BAY1436032 and azacitidine according to the treatment regimen shown in Figure 2A (mean ± standard error of the mean [SEM]). (C) Percentage of hCD45+ leukemic cells in peripheral blood of individual mice transplanted with human IDH1 mutant AML cells and simultaneously treated with BAY1436032 and azacitidine. (D) White blood cell counts after different time points of treatment (mean ± SEM). (E) Platelet counts after different time points of treatment (mean ± SEM). (F) Kaplan–Meier survival curves of IDH1 mutant PDX1 mice treated with vehicle, azacitidine (1 mg/kg, subcutaneoulsy), BAY1436032 (150 mg/kg, orally), or the sequential or simultaneous combination of BAY1436032 and azacitidine according to the treatment regimen shown in Figure 2A. *P<0.5; **P<0.01; ***P<0.001; PDX: patient-derived xenograft; wt: wild-type; mut: mutant.
BAY1436032 and azacitidine is highly effective in inhibiting IDH1 mutated human leukemia in vivo, suggesting a specific dependency on the simultaneous presence of both drugs in vivo.
Combination treatment with BAY1436032 and azacitidine strongly depletes leukemia stem cells
in vivo through inhibition of MAP-kinase signaling and activation of myeloid differentiation
In order to assess the effect of simultaneous and sequen- tial treatment with BAY1436032 and azacitidine on leukemia stem cell self-renewal we performed a limiting dilution transplantation experiment with IDH1 mutant AML cells. NSG mice transplanted with primary human IDH1R132C mutant AML cells were treated when leukemias were fully established (70-80% human AML cells in the peripheral blood) with either vehicle, azaciti- dine, BAY1436032, or the sequential or simultaneous com- bination of BAY1436032 and azacitidine for 4 weeks (Online Supplementary Figure S6A). Subsequently, we har- vested bone marrow cells from these treatment groups and transplanted defined numbers of human AML cells in recip- ient mice at limiting dilution. Cell numbers of 2,000,000, 200,000, 20,000, 2,000, 200, and 20 human CD45+ AML cells were injected into three NSG recipient mice per cell dose for each treatment group. After 8 weeks the mice were bled and scored positive if more than 0.1% human CD45+
cells were present in the peripheral blood (Figure 3A). The LSC frequency was 1 in 73 in vehicle-treated mice, 4.1-fold lower in azacitidine treated mice (1 in 304), and 117-fold lower in BAY1436032 treated mice (1 in 8,580). The sequen- tial combination treatment of BAY1436032 and azacitidine decreased the stem cell frequency by 470-fold (1 in 34,300) compared to control treated mice and by 4-fold compared to BAY1436032 monotherapy. However, in mice treated simultaneously with BAY1436032 and azacitidine, the leukemia stem cell frequency was 33,150-fold lower com- pared to control treated mice, 282-fold lower compared to BAY1436032 treated mice, and 70-fold lower compared to mice treated sequentially with BAY1436032 and azacitidine (1 in 2,420,000, Figure 3A, Online Supplementary Figure 6B).
Next, we performed gene expression profiling of hCD45+ cells from NSG mice treated with vehicle, azacitidine, BAY1436032 or simultaneous treatment with BAY1436032 and azacitidine for 4 weeks. In unsupervised hierarchical clustering, azacitidine and vehicle-treated groups clustered together and separated from BAY1436032 and BAY1436032 + azacitidine treated groups. However, many genes that were overexpressed in BAY1436032 treated cells were sup- pressed in the combination group (Figure 3B). Principal component analysis showed that BAY1436032 + azaciti- dine treated cells separated well from the control or single- agent treated cells (Figure 3C). Interestingly, 11 of 17 genes from the LSC17 gene signature were downregulated by the
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