Azacitidine combined with the selective FLT3 kinase inhibitor crenolanib disrupts stromal protection and inhibits expansion of residual leukemia-initiating cells in FLT3-ITD AML with concurrent epigenetic mutations

// Anne-Kathrin Garz 1, 2 , Saskia Wolf 1 , Sonja Grath 3 , Verena Gaidzik 4 , Stefan Habringer 1, 2 , Binje Vick 2, 5 , Martina Rudelius 6 , Christoph Ziegenhain 3 , Sylvia Herold 2, 7 , Marie-Theresa Weickert 1 , Martha Smets 3 , Christian Peschel 1, 2 , Robert A.J. Oostendorp 1 , Sebastian Bultmann 3 , Irmela Jeremias 2, 5 , Christian Thiede 2, 7 , Konstanze Döhner 4 , Ulrich Keller 1, 2 and Katharina S. Götze 1, 2 1 Department of Medicine III, Klinikum rechts der Isar, Technische Universität München (TUM), Munich, Germany 2 German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany 3 Department of Biology II, Ludwig-Maximilians-University (LMU), Munich, Germany 4 Department of Internal Medicine III, University of Ulm, Ulm, Germany 5 Helmholtz Zentrum München, German Research Center for Environmental Health, Munich, Germany 6 Department of Pathology, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany 7 Department of Internal Medicine I, Gustav Carus University Dresden, Dresden, Germany Correspondence to: Katharina S. Götze, email: katharina.goetze@tum.de Keywords: FLT3-ITD, crenolanib, azacitidine, leukemia-initiating cell (LIC), TET2 Received: May 18, 2017      Accepted: September 20, 2017      Published: October 16, 2017 ABSTRACT Effectively targeting leukemia-initiating cells (LIC) in FLT3 -ITD-mutated acute myeloid leukemia (AML) is crucial for cure. Tyrosine kinase inhibitors (TKI) have limited impact as single agents, failing to eradicate LIC in the bone marrow. Using primary AML samples and a patient-derived xenograft model, we investigated whether combining the FLT3-selective TKI crenolanib with the hypomethylating agent azacitidine (AZA) eliminates FLT3 -ITD LIC and whether efficacy of this combination depends on co-existing mutations. Using multiparameter flow cytometry, we show FLT3 -ITD occurs within the most primitive Lin - /CD33 (+) /CD45 dim /CD34 + CD38 - LIC compartment. Crenolanib alone could not target FLT3 -ITD LIC in contact with niche cells while addition of AZA overcame stromal protection resulting in dramatically reduced clonogenic capacity of LIC in vitro and severely impaired engraftment in NSG mice. Strikingly, FLT3 -mutated samples harboring TET2 mutations were completely resistant to crenolanib whereas neither NPM1 nor DNMT3A mutations influenced response. Conversely, primary AML LIC harboring either TET2, DNMT3A or NPM1 mutations did not show increased sensitivity to AZA. In summary, resistance of FLT3 -ITD LIC to TKI depends on co-existing epigenetic mutations. However, AZA + crenolanib effectively abrogates stromal protection and inhibits survival of FLT3 -ITD LIC irrespective of mutations, providing evidence for this combination as a means to suppress residual LIC.