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Synergy between IDH1 inhibitor and hypomethylating agent
observed in the proportion of G0/G1 cells between the combination and monotherapy groups, while the subG1 population was increased in the combination treatment group indicating increased cell death. The percentage of Annexin V+ cells did not differ between any of the treat- ment groups in IDH1 wild-type or IDH1 mutant AML cells (Online Supplementary Figure S2). In order to further investi- gate the synergy between azacitidine and BAY1436032, we treated IDH1 mutant AML cells from six patients with seri- al dilutions of azacitidine and BAY1436032, either alone or in combination and performed colony forming unit assays. The combination of azacitidine with BAY14360932 signifi- cantly decreased the colony formation of cells treated with both agents (Figure 1D). In order to determine whether the combined effects of azacitidine and BAY1436032 are addi- tive or synergistic, we performed an isobologram analysis from an average of six patients. The combination of azaci- tidine with BAY1436032 proved highly synergistic with a combination index (CI) of <0.68 at an effective dose (ED) of 95, aCIof<0.65atED75,andaCIof<0.63atED50, respectively (Figure 1E; a CI of 1 reflects an additive effect, a CI <1 a synergistic effect and a CI >1 indicates antago- nism). This data suggest that BAY1436032 in combination with azacitidine more effectively inhibits proliferation of primary IDH1 mutant AML cells ex vivo than single agent treatment.
BAY1436032 synergizes with azacitidine to exert potent anti-leukemic activity in the patient-derived IDH1 mutant acute myeloid leukemia xenograft
models in vivo
We have previously developed and characterized an IDH1 mutant PDX mouse model (AML-PDX1) using pri- mary AML cells from a patient harboring IDH1 R132C, FLT3-TKD (p.D835del), an atypical NPM1 (p.S254LfsTer4), and a NRAS (p.Q61R) mutation.10 After 28 days of trans- plantation, having confirmed engraftment between 2-5% in peripheral blood, IDH1 mutant PDX mice were treated with either vehicle, BAY1436032, two cycles of azacitidine as monotherapy or in sequential or simultaneous combina- tion of both drugs for a total duration of 84 days (Figure 2A). We evaluated the simultaneous and sequential combi- nation treatment to derive mechanistic insight into the combination treatment and to instruct the clinical use of this combination, as mutation analysis usually takes some time and leaves a time window in which treatment with azacitidine may be started before the mutation status is known, followed by sequential treatment with a mIDH1 inhibitor.
While the engraftment of human leukemic cells increased in vehicle and azacitidine treated mice at week 8 after the start of treatment, the percentage of leukemic cells decreased in BAY1436032 treated mice as well as in the groups receiving the sequential and simultaneous combina- tion treatment (Figure 2B). However, after the stop of treat- ment at week 12 the percentage of leukemic cells increased after week 16 in the treatment groups receiving BAY1436032 or the sequential combination of BAY1436032 and azacitidine (Figure 2B). Interestingly, the percentage of leukemic cells in mice treated with the simultaneous com- bination of BAY1436032 and azacitidine showed a delayed increase of blasts and slower kinetics (Figure 2B) and 2 of 6 mice from this cohort remained negative in peripheral blood until the end of the study at 36 weeks (Figure 2C). The mean IDH1 mutant allele fraction in peripheral blood
at 8 weeks after the stop of the treatment was 48.8% in vehicle, 40.56% in the azacitidine, 46.43% in the BAY1436032, and 43.2% in the sequential treatment cohorts. However, median IDH1 mutant allele burden was 10.89% in the simultaneous treatment cohort, and below the detection limit in 3 of 5 analysed mice (Online Supplementary Figure S3A). While the white blood cell counts constantly increased and platelet counts, as well as hemoglobin, decreased in mice receiving vehicle, azaciti- dine or BAY1436032 monotherapy or the sequential combi- nation of BAY1436032 and azacitidine, all mice treated simultaneously had normal blood counts until week 36 (Figure 2D-E, Online Supplementary Figure S3B). In order to compare the effects of the combination therapy with single agents on the induction of myelomonocytic differentiation the expression of CD14 and CD15 in human CD45+ cells was monitored. At day 30 after initiation of treatment there was no significant difference in the expression of the myelomonocytic maturation markers CD14 and CD15 in the vehicle (CD14 0.05±0.05; CD15 0.86±0.3) and azaciti- dine treated groups (CD14 0; CD15 1.39±0.36). In contrast, mice treated with BAY1436032 had a significantly higher proportion of cells expressing CD14 (4.21±0.62) and CD15 (7.67±0.68). Mice treated in parallel with the combination of BAY1436032 and azacitidine had additive effects on the expression of these markers (CD14 7.44±1.27; CD15 11±0.79) (Online Supplementary Figure S4). While azacitidine treated mice survived longer with a median survival of 188 days compared to vehicle-treated mice with a median sur- vival of 139 days, BAY1436032 treated mice had significant- ly longer latency with a median survival of 220 days. However, mice treated sequentially with the combination of BAY1436032 and azacitidine survived longer than with BAY1436032 monotherapy with a median survival of 299 days. Strikingly, 5 of 6 mice treated simultaneously with BAY1436032 and azacitidine survived until the end of the study at 300 days and the median survival was not reached (Figure 2F).
In an independent, second PDX model (AML-PDX2), which harbored IDH1 p.R132H, DNMT3A p.R882H, PTPN11 p.A72T, and NPM1 p.T288CfsTer12 mutations (Online Supplementary Figure S5A), the percentage of human CD45+ cells increased in vehicle, azacitidine, BAY1436032 and sequential combination treatment groups albeit with different kinetics (Online Supplementary Figure S5B). However, even after the stop of treatment at week 12, the percentage of leukemic cells in mice treated with the simul- taneous combination of BAY1436032 and azacitidine remained low and 2 of 6 mice from this cohort had less than 10% human CD45+ cells in the peripheral blood until the end of the study at 28 weeks (Online Supplementary Figure S5C). In contrast to the other groups, all mice treated simul- taneously had normal blood counts until week 16 after the start of treatment (Online Supplementary Figure S5D-F). At day 90 after initiation of treatment mice treated simultane- ously with the combination of BAY1436032 and azacitidine had significantly higher expression of CD15 than mice treated with BAY1436032 alone or with the sequential combination of BAY1436032 and azacitidine (Online Supplementary Figure S5G). The mice treated simultaneously with BAY1436032 and azacitidine survived significantly longer with a median survival of 250 days than sequentially treated mice with a median median survival of 186 days (Online Supplementary Figure S5G).
In summary, the simultaneous combination of
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