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A. Chaturvedi et al.
oral pan-mutant IDH1 inhibitor, which has strong anti- leukemic activity in patient-derived xenograft (PDX) mod- els of IDH1 mutant leukemia in vivo by inducing differenti- ation of leukemic blasts as well as inhibition of leukemia stem cell proliferation and self-renewal.10 BAY1436032 is currently evaluated in phase I clinical trials in AML and glioma patients (NCT03127735 and NCT02746081).
In this study, we evaluated the therapeutic efficacy and elucidated the mechanism of action of the combined treat- ment of azacitidine and BAY1436032 in IDH1 mutant AML in vitro as well as in vivo in two independent patient-derived xenograft models of IDH1 mutant AML. From these exper- iments, we conclude that the simultaneous combination of a mIDH inhibitor with a hypomethylating agent synergisti- cally inhibits LSC through suppression of MAP kinase and RB/E2F signaling, which are involved in cell survival and proliferation.
Methods
Combination index
Drug synergy was evaluated using a combination index (CI) equation based on the multiple drug-effect equation of Chou- Talalay.11,12 Colony-forming cell units were assayed in methylcellu- lose after treatment with varying doses of either azacitidine ( 62.5, 125, 250, 500, 1,000 or 2,000 nm), BAY 1436032 ( 6.25, 12.5, 25, 50, 100 or 200 nm) or a fixed ratio of the combination of azaciti- dine/BAY1436032 (62.5/6.25, 125/12.5, 250/25, 500/50, 1,000/100 or 2,000/200 nm). The analysis was performed with CompuSyn software (ComboSyn Inc., Paramus, USA).
Patient samples
Diagnostic bone marrow or peripheral blood collected from AML patients at Hannover Medical School were analyzed for mutations in IDH1 and IDH2 by Sanger sequencing. Details of the type of IDH mutation are described in the figure legends. Mononuclear cells were isolated by ficoll density centrifugation, washed with PBS, and red blood cells were lysed using a red blood cell (RBC) lysis buffer (BD Pharm Lyse, BD Biosciences, Heidelberg, Germany). The bone marrow samples for the develop- ment of the PDX models were collected prior to the start of AML treatment. Written informed consent was obtained according to the Declaration of Helsinki, and the study was approved by the Institutional Review Board of Hannover Medical School, Hannover, Germany.
Transplantation and treatment
Six to eight-week old female NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were purchased from Hannover Medical School, Germany and kept in pathogen free conditions at the central ani- mal laboratory of Hannover Medical School. Experimental proce- dures were approved by the governmental authorities of Lower Saxony, Germany, and supervised by local animal welfare officials. The IDH1 mutant AML PDX model was developed as described.10 One million patient-derived AML cells (hCD45+) were collected from the bone marrow and spleen of leukemic mice and were injected intravenously in the tail vein of sublethally (3 Gy) irradiat- ed NSG mice. Neither randomization, nor blinding was used since all animal experiments were performed with a homogeneous strain, comparable age, and similar variance of the mice. Treatment was started 28 days after transplantation. The control groups were treated with either vehicle, BAY1436032 150 mg/kg once daily orally continuously, or azacitidine 1 mg/kg once daily subcutaneoulsy on days 1-5, repeated once after 28 days. The test
groups were treated with BAY1436032 and azacitidine in the doses mentioned above either starting both drugs on day 1 (simultane- ous group) or starting azacitidine on day 1 but BAY1436032 on day 6 (sequential group). In the second cycle in the sequential treat- ment group, BAY1436032 was stopped during azacitidine treat- ment to better model the biologic and therapeutic effects of the synergy. The treatment was stopped at day 84 or day 96. The pro- portion of leukemic cells in the peripheral blood of mice was meas- ured with the human-specific CD45 antibody every 4 weeks by tail vein bleeds and fluorescence-activated cell sorting (FACS) analysis.13 Blood counts were performed using an ABC Vet Automated Blood counter (Scil animal care company GmbH, Viernheim, Germany). All animal experiments were started with 10 animals, however, the animals which died before the start of treatment due to engraftment failure, during injections or during bleeding were excluded from the study.
Statistical analysis
Two-tailed unpaired comparisons were performed by Mann- Whitney test in Graph pad prism 8.2.1 (GraphPad Software, La Jolla, CA). Comparison of survival curves were performed using the log-rank test. Statistical analyses were performed with Microsoft Excel 2016 (Microsoft, Munich, Germany) or GraphPad Prism 8.2.1 (GraphPad Software, La Jolla, CA, USA). Graphical rep- resentation was prepared using Adobe Illustrator CS6 (Adobe sys- tems GmbH, Munich, Germany). The size of the animal cohorts was based on our previous study.5,6,10 All in vitro experiments were performed at least three times and all attempts of replication were successful.
Results
mIDH1 inhibitor BAY1436032 and azacitidine synergize to inhibit human IDH1 mutant acute myeloid leukemia cells ex vivo
In order to test the effect of the combination of BAY1436032 and azacitidine on survival and proliferation of primary human AML cells, IDH1 wild-type (n=6) and IDH1 mutant (n=6) cells from AML patients were seeded in semi-solid medium supplemented without or with BAY1436032 in combination with varying concentrations of azacitidine. BAY1436032 reduced colony formation specifically in human IDH1 mutant AML cells with an IC50 of 100 nM, while IDH1 wild-type cells were not affected (Online Supplementary Figure S1A). Azacitidine alone induced a 20% reduction in colony formation in both IDH1 wild-type and IDH1 wild-type AML patient cells at 100 nM (Online Supplementary Figure S1B), while the combination of BAY1436032 with azacitidine reduced colony formation in a dose-dependent manner in IDH1 mutant cells with reduction of colonies by 80% in IDH1 mutant AML but only by 20% in IDH1 wild-type AML cells at 100 nM azac- itidine, suggesting improved efficacy of an IDH1 inhibitor in combination with azacitidine (Figure 1A, normalized to cells treated with BAY1436032 at 100 nM). In order to test the effect on cell cycle progression and apoptosis, IDH1 mutant and IDH1 wild-type AML cells were cultured in suspension medium and treated with vehicle, 100 nM BAY1436032 or 100 nM azacitidine as single agents or in combination. The proportion of cells in S phase of the cell cycle was strongly decreased by the combination treatment compared with either monotherapy or vehicle in IDH1 mutant AML cells, however, remained largely unchanged in IDH1 wild-type cells (Figure 1B-C). No difference was
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haematologica | 2021; 106(2)