Gene expression 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. Overall design: 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.