Evaluation of Synergistic Anti-Leukemic Efficacy of PHI-101 in Preclinical Model of FLT3-ITD Acute Myeloid Leukemia

Background: AML is a heterogeneous disease with an overall cure rate of 40-50%. Some types of AML have even poorer prognosis with FLT3 mutant AML patients having only a 20-25% cure rate with chemotherapy alone. In order to improve the outcome for this high-risk AML, a number of tyrosine kinase inhibitors (TKI) have been developed and to date. Given the difficulty of identifying appropriate targets and treatment options for AML, combining drug options for AML treatment may produce clinical benefits. PHI-101 is a selective next-generation type I FLT3 inhibitor that shows potent anti-leukemic activity against AML samples harboring FLT3-ITD and/or TKD mutations. In a clinical trial, PHI-101 demonstrates significant clinical responses and tolerability in refractory or relapsed AML patients as monotherapy. To investigate the optimal way to combine PHI-101 with other therapies, a number of other drugs utilized in treating AML were tested both, in vitro and in vivo to determine synergistic or additive effects that might improve efficacy for relapsed/refractory/elderly FLT3 mutant AML patients. Study Design and Methods: Combination effects of simultaneous versus sequential treatment of PHI-101 with other agents, including daunorubicin, cytarabine, venetoclax (Ven), and azacytidine (Aza) were tested using human leukemia cell lines harboring FLT3-ITD mutations (MV4-11, Molm13, Molm14) or FLT3-wild type as controls. The cell growth inhibition was assessed by CCK-8, MTT and/or CellTiter-Glo assays as well as annexin V-binding by flow cytometry and the Chou-Talalay method was used to calculate the combination index (CI. using Compusyn software. The potential mechanisms of effect on combination therapy were explored by Western blotting of a variety of signal transduction pathways including FLT3 phosphorylation and downstream pathways BCL-2 family expression, and DNA damage. The MV4-11 xenograft mouse model was used to assess in vivo efficacy of PHI-101 combination therapy. Results: Among the substances tested for combination therapy with PHI-101 in FLT3-ITD cell lines MV4-11 and Molm14 cell lines, Aza and Ven treatment both showed synergy with both simultaneous and sequential treatment. Ven was highly synergistic when used simultaneously with PHI-101 in MV4-11 cells. Cell growth inhibition effect (GI 50) of PHI-101, Aza, and Ven used alone was measured first to calculate the CI in combination with increasing concentrations of each drug together of treatment for 72hrs. Th