DNA methylation changes induced by hypomethylating agent, Azacitidine (AZA); IDH1 inhibitor, BAY1436032 (BAY) and sequential and simultaneous combination of BAY and AZA in in vitro treated human (CD45+) cells isolated from the bone marrow of IDH1mut

Mutations in the enzymes IDH1 and IDH2 have been identified in a wide variety of tumors like glioma, chondrosarcoma, thyroid cancer, lymphoma, melanoma, and in acute myeloid leukemia. Mutated IDH1/2 produces the metabolite 2-hydroxyglutarate (2HG), which interferes with epigenetic regulation of gene expression, and thus may promote tumorigenesis. Data for IDH1 inhibitors show that 30-40% of AML patients respond to monotherapy with a median duration of response of 8 months, suggesting that IDH1 inhibitors should be combined with other agents to improve efficacy. BAY 1436032 (BAY) is an oral pan-mutant IDH1 inhibitor currently undergoing phase 1 clinical trials. 5-Azacitidine (AZA) is a hypomethylating agent and can activate key epigenetically silenced pathways in AML cells, leading to an arrest of AML cell proliferation. Human AML cells isolated from the spleen and bone marrow of IDH1 R132C (PDX1) mice were cultured in IMDM medium (Stemcell Technology Inc. Vancouver, Canada) supplemented with 10% FBS (Sigma-Aldrich, Munich, Germany), 2 mM L-Glutamine (Life Technologies, Karlsruhe, Germany), 1% Penicillin/Streptomycin (Gibco, ThermoFischer Scientific, Braunschweig, Germany) , 20 ng/ml human GMCSF, 20 ng/ml human GCSF, 20 ng/ml human IL6, 50 ng/μl, human SCF and 20 ng/μl human IL-3 (all from PeproTech, Hamburg, Germany) and were incubated at 37 °C with 5% CO2 in humidified atmosphere. Treatment was carried out with DMSO (vehicle), 500nm of azacitidine for day 1 to day 4, 50nm of BAY1436032 for day 1 to day 5, sequential combination (azacitidine for day 1 to day 4 and BAY1436032 for day 5 to day 9) and simultaneous combination (azacitidine for day 1 to day 4 and BAY1436032 for day 1 to day 5). RNA was isolated on day 6 for cells treated with vehicle, azacitidine, BAY1436032 and simultaneous combination and on day 10 for sequential combination using AllPrep DNA/RNA mini kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions.