The Anti-Leprosy Agent Clofazimine Inhibits FLT3-ITD Mutant AML Stem Cells and Potently Enhances Effects of TKI

Introduction: The FMS-like tyrosine kinase-3 (FLT3) internal tandem duplication (ITD) mutation is associated with poor prognosis in acute myeloid leukemia (AML). FLT3 tyrosine kinase inhibitors (TKIs) are clinically effective but are limited by frequent relapse and resistance. Thus, new approaches to enhance TKI mediated targeting of FLT3-ITD AML stem cells are required to improve outcomes. In previous studies the FDA-approved anti-leprosy agent Clofazimine (CFZ) was identified via a drug screen to specifically target quiescent CML stem cells by inhibiting STAT5 and inducing ROS-mediated apoptosis and differentiation. Given that these mechanisms are also relevant in FLT3-ITD AML, we investigated the efficacy of CFZ in targeting FLT3-ITD mutant AML cells by itself and in combination with TKI.

Method: FLT3-ITD mutant MV4-11 and MOLM13 cell lines and primary AML CD34+ cells were treated with CFZ as a single agent or in combination with FLT3 TKIs, Quizartinib (AC220) and Giltertinib followed by determination of cell cytotoxicity using CellTiter-Glo, apoptosis using Annexin V labeling, and differentiation using cell surface markers. Synergy between CFZ and TKIs was analyzed by Calcusyn software based on Chou-Talalay's combination index (CI) theorem. We also analyzed the effects of CFZ and FLT3 kinase inhibition in vivo using a FLT3-ITD+ TET2-deleted genetic mouse model of FLT3-ITD AML.

Results: Evaluation of the effect of CFZ with and without FLT3 TKIs Quizartinib or Giltertinib TKI in FLT3-ITD mutant MV4-11 and MOLM13 cell lines showed that that CFZ treatment resulted in potent inhibition of cell viability, and that the combination of the CFZ with TKI synergistically enhanced inhibition of FLT3-ITD+AML cells, compared to TKI or CFZ alone. We next studied the effects of CFZ alone or in combination with TKIs on CD34+ progenitor cells from FLT3-ITD mutated AML patients (n=8). CFZ treatment for 7 days effectively inhibited growth of CD34+ cells. The combination of CFZ and Quizartinib or Giltertinib resulted in significantly enhanced inhibition compared to either agent alone. Similar effects were observed in the most primitive CD34+CD38- cell population. We further show that CFZ enhanced apoptosis in CD34+ cells by itself and the combination of CFZ and TKI resulted in significantly enhanced apoptosis compared to CFZ or TKI alone. Finally, we studied the effect of CFZ in a genetic mouse model of FLT3-ITD AML. Mice were treated with vehicle (control), Giltertinib, CFZ or the combination of Giltertinib and CFZ for 3 weeks. We found significant inhibition of mature AML cells and AML stem and progenitor cells in the bone marrow and spleen with CFZ and significantly enhanced inhibition with combination of CFZ and Giltertinib. These results indicate that combination of CFZ with FLT3 TKIs leads to effective targeting of FLT3-ITD AML stem cells. We are currently investigating the effect of CFZ alone and with TKI in a large panel of FLT3-ITD AML samples and analyzing the role of STAT5 inhibition and increased oxidative stress in mediating the observed effects.

Conclusion: Our studies indicate that the FDA-approved anti-leprosy agent CFZ demonstrates activity against FLT3-ITD+ AML stem and progenitor cells and significantly enhances FLT3 TKI targeting of these populations. These results support further investigation of CFZ as a potential therapeutic agent in FLT3-ITD+ AML.

No relevant conflicts of interest to declare.