Targeting the GAS6/AXL Pathway with a Newly Developed AXL-Selective Inhibitor SLC-0211 for the Treatment of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by rapid clonal growth of myeloid lineage cells, with accumulation of immature blasts. Overall survival of AML patients is low and resistance to frontline chemotherapy is a major cause of treatment failure, highlighting the need for new therapies. It has been reported that AXL, a TAM family receptor tyrosine kinase, along with its ligand growth arrest-specific gene 6 (GAS6), mediates proliferation and survival of many types of cancer cells. An increasing body of evidence suggests that the GAS6/AXL pathway plays a major role in resistance to targeted therapies and conventional cytotoxic drugs. Particularly, increased expression levels of AXL and GAS6 have been reported in some AML and chronic myeloid leukemia (CML) patients, which are associated with poor prognosis. Several AXL inhibitors have recently been developed and some of them are in clinical trials. However, many of these small molecules are multi-kinase inhibitors and off-target effects and/or toxicity on healthy hematopoietic cells remain challenging. Moreover, it is still not known if the GAS6/AXL pathway is specifically activated in subgroups of AML patients carrying specific chromosomal abnormalities or mutations, and if AXL inhibitors can sensitize AML stem/progenitor cells to chemotherapy drugs and targeted therapeutics, since these cells are highly resistant to current anti-cancer therapies. We have therefore investigated expression changes in AXL and GAS6 in 15 AML cell lines and CD34⁺ stem/progenitor cells obtained from 11 primary AML patient samples. Interestingly, the transcript levels of AXL and GAS6 were found to be significantly increased in several AML cell lines carrying MLL fusion genes as compared to cells without MLL fusions (~6-fold and 8-fold). This result was further confirmed in these cells by Western blot analysis. Moreover, AXL transcripts were highly increased in primary CD34⁺ stem/progenitor cells as compared to more mature CD34^- cells from nine AML patient samples studied (~7-fold). Transcript levels of GAS6 were slightly increased in CD34⁺ cells compared to CD34^- cells from the same patient samples. These results indicate that both AXL and GAS6 are highly expressed in AML cell lines harboring MLL fusion genes, as well as in patient stem/progenitor cells.

Interestingly, we have further demonstrated that AML cells with high expression of GAS6/AXL were more sensitive to the highly selective and novel AXL inhibitor SLC-0211, as compared to AML cells with low expression of GAS6 and/or AXL, as assessed by viability and apoptosis assays (2-3 fold). Notably, AML cells with high expression of both GAS6 and AXL, but without FLT3-ITD mutation, were highly sensitive to SLC-0211 treatment compared to other AXL inhibitors (>10-fold). Additive effects of SLC-0211 with chemotherapeutic drugs cytarabine or daunorubicin were also observed. In addition, SLC-0211 modestly reduced the colony forming ability of CD34⁺ AML cells, but the inhibitory effect of SLC-0211 was strikingly enhanced in re-plating assays, resulting in a significant reduction in re-plating efficiency of more primitive AML cells (75-97% inhibition). Importantly, the drug concentrations used to inhibit AML CFCs were not toxic to normal human CD34⁺ stem/progenitor cells. At the molecular level, treatment with SLC-0211 greatly reduced phosphorylation of AXL^Y779, AKT^S473 and ERK^T202/Y204 in AML cells in a dose-dependent manner as compared to control cells, as demonstrated by Western blot analysis. Taken together, these results indicate that inhibition of AXL by SLC-0211 specifically targets primitive AML cells where the AXL/GAS6 pathway is highly activated. Thus, targeting AXL may provide improved therapies for specific subgroups of AML.