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

Effectively targeting leukemia-initiating cells (LIC) in -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 -ITD LIC and whether efficacy of this combination depends on co-existing mutations. Using multiparameter flow cytometry, we show -ITD occurs within the most primitive Lin/CD33/CD45/CD34CD38 LIC compartment. Crenolanib alone could not target -ITD LIC in contact with niche cells while addition of AZA overcame stromal protection resulting in dramatically reduced clonogenic capacity of LIC and severely impaired engraftment in NSG mice. Strikingly, -mutated samples harboring mutations were completely resistant to crenolanib whereas neither nor mutations influenced response. Conversely, primary AML LIC harboring either or mutations did not show increased sensitivity to AZA. In summary, resistance of -ITD LIC to TKI depends on co-existing epigenetic mutations. However, AZA + crenolanib effectively abrogates stromal protection and inhibits survival of -ITD LIC irrespective of mutations, providing evidence for this combination as a means to suppress residual LIC.