AXL/Mertk Inhibitor ONO-7475 Potently Synergizes with BCL2 Inhibitor ABT-199, Overcomes ABT-199 Resistance Mechanisms, and Kills FLT3 ITD AML Cells

Background: Internal tandem duplication (ITD) mutation in Fms-like kinase 3 (FLT3) occur in ~ 25% of newly diagnosed acute myeloid leukemia (AML) patients and is associated with poor survival outcome. We recently demonstrated that the highly specific AXL/MERTK inhibitor ONO-7475 (Ono Pharmaceutical Co, Osaka, Japan) was effective as a single agent against FLT3-ITD AML cells in both in vitro and in vivo models (Ruvolo et al Haematologica, 2017). The mechanism of ONO-7475 action involved diverse targets including inhibition of survival kinase Extracellular-signal Regulated Kinase 1/2 (ERK 1/2) and reduction of anti-apoptotic BCL2 family member Myeloid Cell Leukemia 1 (MCL-1). Considering that MCL-1 is a major factor in ABT-199 (Venetoclax) resistance, we studied efficacy of ONO-7475/ABT-199 combination using in vitro and in vivo models and the ability of ONO-7475 to overcome ABT-199 resistance.

Methods: FLT3-ITD AML cell lines Molm13 and MV4;11 as well as ABT-199 resistant MV4;11 cells and MV4;11 cells that overexpress MCL-1 or gain-of-function (GOF) MCL-1 mutant S159A were treated with varying doses of ONO-7475 in the presence and absence of ABT-199. The effect on cell viability and induction of apoptosis was assessed by flow cytometry. Effect of 24 hour treatment of DMSO vehicle, 10 nM ONO-7475, 30 nM ABT-199, or 10 nM ONO-7475/ 30 nM ABT-199 combination on various ONO-7475 targets was assessed by immunoblot analysis. The efficacy of ONO-7475 combination with ABT-199 in an in vivo FLT3 ITD AML xenograft model was tested using Molm13 cells expressing luciferase/GFP in NSG mice. Both drugs were given 5 days a week by oral gavage (ONO-7475 at 10 mg/kg and ABT-199 at 100 mg/kg). Leukemia burden was assessed by IVIS imaging and examination of H&E stained slides of bone marrow (BM), spleen, and liver.

Results: As single agent, a dose of 10 nM ONO-7475 or 30 nM ABT-199 reduced viable MV4;11 cells by ~ 60% after 72 hours. However, combination of ONO-7475 with ABT-199 at those doses potently induced apoptosis and eliminated > 98% of cells. Molm13 cells treated with 10 nM ONO-7475/ 30 nM ABT-199 combination for 48 hours displayed > 90% of cells killed. Immunoblot analysis of Molm13 and MV4;11 cells treated for 24 hours with vehicle, ONO-7475, ABT-199, or combination of both drugs revealed ONO-7475 alone and in combination with ABT-199 potently suppressed ERK phosphorylation in both cell lines but MCL-1 was only suppressed in MV4;11 cells treated with both drugs. Interestingly, ABT-199 resistant MV4;11 cells were equally as sensitive to ONO-7475 as parental cells. MV4;11 cells that overexpress WT MCL-1 or GOF mutant S159A MCL-1 provide a MCL-1-dependent ABT-199 resistance model. While MV4;11 cells overexpressing WT or GOF mutant MCL-1 are resistant to ABT-199 compared to control cells, all three cell variants are similarly sensitive to ONO-7475. Finally in the Molm13 in vivo model, ONO-7475/ABT-199 combination potently reduced leukemia burden and prolonged survival of mice compared to either single agent. The median survival of vehicle treated mice was 16 days, ABT-199 treated mice 18 days, ONO-7475 treated mice 22 days, and ONO-7475/ABT-199 treated mice 31 days (p = 0.002). ONO-7475/ABT-199 combination was superior to either single agent in reducing leukemia burden as determined by IVIS imaging and examination of BM, spleen and liver. Critically, the combination of these two agents had a synergistic effect and significantly increased survival compared to either ABT-199 or ONO-7475 alone. Together, these results provide evidence that AXL/MERTK inhibition overcomes ABT-199 resistance.

Conclusions: These results suggest that ONO-7475 in combination with ABT-199 is an effective therapeutic strategy for treating FLT3-ITD-dependent AML, including those that are resistant to the BCL2 specific inhibitors. Critically, these results indicate that inhibition of AXL/MERTK axis overcomes survival advantages conferred by MCL-1, a common mechanism of Venetoclax resistance. The ability of ONO-7475/ABT-199 combination to eliminate Molm13 cells without completely suppressing MCL-1 suggests that drug combination involves MCL-1 independent targets. That MCL-1 GOF mutant confers ABT-199 resistance but not resistance to ONO-7475 supports this hypothesis. In summary, the data presented suggest that ONO-7475 combination with Venetoclax may be an effective strategy for treating AML patients with FLT3 ITD mutation.