AXL Inhibitor ONO-9330547 Suppresses PLK1 Gene and Protein Expression and Effectively Induces Growth Arrest and Apoptosis in FLT3 ITD Acute Myeloid Leukemia Cells
Authors: Ruvolo Peter , Ma Huaxian , Ruvolo Vivian , Mu Hong , Schober Wendy , Yasuhiro Tomoko , Yoshizawa Toshio , Gallardo Miguel , Zhang Xiaorui , Khoury Joseph D , Cortes Jorge , Andreeff Michael , Post Sean M. 
Published: October 2019

Abstract
Background: Internal tandem duplication (ITD) mutation in Fms-like kinase 3 (FLT3) are observed in approximately 25% of newly diagnosed acute myeloid leukemia (AML) patients. FLT3 ITD is generally associated with poor survival outcome. A recent study has demonstrated that the TAM (Tyro3/MER/AXL) tyrosine kinase AXL is required for FLT3 inhibitor resistance in FLT3 ITD AML cells (Park et al Leukemia 2014). AXL expression is prognostic for poor outcome in AML (Ben-Batalla et al Blood 2013). These findings suggest that AXL is an excellent candidate for AML therapy particularly in patients with FLT3 ITD. ONO-9330547 (Ono Pharmaceutical Co, Osaka, Japan) is a highly specific AXL/MER inhibitor being developed for AML therapy. Here we report on the in vitro and in vivo effects of ONO-9330547 in AML models.
Methods: FLT3 WT (HL60, OCI-AML3) and FLT3 ITD (Molm13, MV4;11) cells were treated with varying doses of ONO-9330547 and effect on cell viability and induction of apoptosis was assessed by flow cytometry using DAPI, Annexin V, and counting beads. Cell cycle was assessed by flow cytometry using EdU incorporation and FX Cycle Violet dye. Protein was isolated and reverse phase protein array (RPPA) analysis was performed on ONO-9330547 treated cells. RPPA results were validated by immunoblot analysis. RNA was collected and qRT-PCR performed. The drug was also tested on FLT3 ITD cells in an in vitro model of the AML microenvironment using co-culture with mesenchymal stromal cells (MSC). Finally, efficacy of ONO-9330547 in an in vivo AML xenograft model was tested using Molm13 cells expressing luciferase/GFP in NSG mice. Drug was given in feed at 0.013% (estimated ~ 20 mg/kg/day) and 0.004% (estimated ~ 6 mg/kg/day). Leukemia burden was assessed by IVIS imaging.
Results: FLT3 ITD cell lines were highly sensitive to ONO-9330547. A dose of 10 nM drug induces apoptosis after 48 hours and 50 nM eliminates > 95% after 72 hours. MSC protect leukemia cells from ONO-9330547, but killing was still effective with higher doses. ONO-9330547 was effective arresting cell growth of Molm13 and MV4;11 cells. After 24 hours, 5 nM ONO-9330547 resulted in accumulation of cells in G1/G0. RPPA analysis of cells treated with the AXL inhibitor revealed suppression of known AXL targets (e.g. p-S6RP likely via AKT). RPPA also revealed novel targets including CDK1, p-RB, Cyclin B1, and PLK1. Immunoblot analysis also demonstrated that the drug suppressed expression of MCL-1 (predicted due to block of ERK and AKT signaling by the drug). Analysis of PLK1 gene expression by qRT-PCR revealed potent suppression of PLK1 mRNA by ONO-9330547. These findings suggest that inhibition of AXL results in block of CDK1 activity with concomitant loss of RB phosphorylation leading to suppression of PLK1 expression (presumably via RB association with E2F). This model is consistent with the observed effect of ONO-9330547 blocking cell entry into S phase. Finally, both low dose (~ 6 mg/kg) and high dose (~ 20 mg/kg) ONO-9330547 was effective reducing leukemia burden and significantly enhancing survival of mice bearing Molm13 leukemia cells. Interestingly, while mice receiving control feed displayed leukemia infiltrate in the liver, ONO-9330547 greatly reduced or prevented Molm13 cell infiltration of the liver.
Conclusions: These results suggest that ONO-9330547 is effective killing AML cells with FLT3 ITD. The identification of p-RB and PLK1 as novel targets of the drug suggests for the first time that AXL regulates cell cycle in AML cells via CDK/RB/PLK1 axis. Finally, ONO-9330547 proved effective in in vivo AML xenograft models and the drug prevented AML infiltration of the liver. These results suggest that ONO-9330547 is a promising candidate for AML therapy particularly in AML patients with FLT3 ITD.

Disclosures
Yasuhiro: 3Ono Pharmaceutical Co. Ltd: Employment. Yoshizawa:3Ono Pharmaceutical Co. Ltd: Employment. Cortes:Ariad: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding; Arog: Research Funding; Astellas: Research Funding; Ambit: Research Funding.