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. The GI 50 of PHI-101 in FLT3-mutant cell lines (MV4-11, Molm13, Molm14) were 7.0, 3.8, and 3.4 nM, respectively. The GI 50 of the gilteritinib was 5.7, 10.2, and 10.8 nM, respectively. On the other hand, the GI 50 of Aza were 198.9, 140.4, and 85.4 nM, that of Ven were 38.2, 8.4, and 56.5 nM. PHI-101 was the most efficient antitumor agent compared to other drugs tested in this study. The CI of Aza and PHI-101 was 0.01216 (EC 50), 0.05981 (EC 75), 0.29468 (EC 90) in MV4-11 cells. The observed synergistic effects were confirmed in the other FLT3-ITD cell lines as well. PHI-101 and venetoclax combined therapy in MV4-11 showed synergism (CI<0.1), and the CI was 0.00002 (EC 50), 0.00104 (EC 75), 0.01243 (EC 90). By Western blotting, the combination of Aza or Ven with PHI-101 resulted in a significant induction of p-H2AX and PARP cleavage in the FLT3-ITD cell lines (MV4-11), but not in FLT3-WT cell lines (THP-1). To further validate the synergistic anti-leukemic activity of PHI-101 with these agents, MV4-11-derived mouse xenograft models were generated and treated at various doses. In the MV4-11-xenograft model, PHI-101 alone or combined with Ven or Aza significantly decreased tumor volume. In line with the cell viability data, Ven and Aza combined with PHI-101 induced significant cell-killing in vivo. Conclusion: Treatment of AML cells with the combination of Ven or Aza with PHI-101 in vitro induced rapid induction of apoptosis and inhibition of cell growth that showed significant synergy. In vivo data showed improved efficacy for the combination treatment with these agents. In vitro and in vivo data from the preclinical AML models provides a rationale to evaluate the activity of PHI-101 in combination for patients who are ineligible for more intensive chemotherapeutic induction therapy. Improved anti-leukemic efficacy by combined therapy with PHI-101 represent a promising therapeutic strategy for newly diagnosed or R/R FLT3-mutant AML patients