Development of an Axl/Mer Dual Inhibitor, ONO-9330547: Promising Single Agent Activity in an Acute Myeloid Leukemia (AML) Model

Purpose: Axl and Mer belong to the TAM (Tyro3, Axl, Mer) family of receptor tyrosine kinases and play a role in regulating cell proliferation, survival, migration and cytokine production. Axl/Mer are over-expressed in various types of haematological and solid tumour cancers and appear to play a key role in epithelial-to-mesenchymal transitions (EMTs), known to be important in tumour metastases and drug resistance. AML is a clonal disease of haematopoietic progenitors characterised by acquired heterogeneous genetic changes that alter cell proliferation and differentiation and Axl is an independent prognostic marker and therapeutic target in AML. Recent studies revealed that AML cells induce expression and secretion of the Axl ligand growth arrest specific gene 6 (Gas6) by bone marrow derived stromal cells (BMDSCs). Gas6 mediates proliferation, survival and chemoresistance of Axl-expressing AML cells, giving rise to a chemoprotective tumour niche environment. Standard chemo-treatment for AML affects not only AML cells but healthy cells which cause severe side effects. In addition, only 40-45% of patients <65 years reach long-term survival with current treatment options and only 10% of patients >65 can be cured. Therefore, there is still a high, unmet medical need for new therapies. ONO-9330547 is a small molecule inhibitor that binds to Axl and Mer kinase with a potency (IC₅₀) of 2.2 and 0.4 nM respectively, therefore, the anti-tumour activity of ONO-9330547 in AML cells was evaluated.

Methods: AML cell lines were implanted subcutaneously into female SCID mice. Randomization of mice occurred when mean tumour volume was 100-200 mm³. ONO-9330547 was administered orally at doses up to 3 mg/kg twice daily (BD) for 24 days. Tumour volumes were measured twice a week after initiation of treatment, and tumor volumes were determined using the formula volume (=width²xlength)/2. Animals were euthanized when the tumours reached a maximum volume of 2,000 mm³. Phosphorylation of Axl (P-Axl) and Mer (P-Mer) were detected by Western blotting or Flow cytometry.

Results: In AML cell lines assays, ONO-9330547 strongly inhibited both P-Axl and P-Mer with an IC₅₀in the nM range, which resulted in growth inhibitions and inductions of apoptosis. In the MV-4-11 xenograft model, tumour growth inhibition at the final treatment day was 40% in the 0.3 mg/kg BD group and 78% in the 1 mg/kg BD and 100% in the 3 mg/kg BD groups respectively (All treatment groups: P<0.001 vs Vehicle). Specifically, the treatment with ONO-9330547 1 or 3 mg/kg BD resulted in higher and sustained P-Axl inhibition throughout the study, and treatment of 3 mg kg BD resulted in complete remission in all treated mice. Interestingly, up-regulation of Axl and Mer were observed in AML cell lines after the treatment of doxorubicin or Ara-C. The combination of ONO-9330547 with Ara-C resulted in significant anti-tumour effect compared with respective monotherapy.

Conclusion: ONO-9330547 is a highly potent dual Axl/Mer inhibitor with evidence of efficacy in AML cells. Moreover, up-regulation of Axl/Mer after chemotherapy indicates the potential combination of ONO-9330547 with standard therapy to overcome treatment resistance in AML. Additional work to investigate and clarify the involvement of Axl/Mer in other types of leukemia are currently underway.