Receptor Tyrosine Kinase AXL: A Potential Strategy to Counter Immune Suppression and Dormancy in Multiple Myeloma

Introduction

The AXL receptor tyrosine kinase (AXL) has emerged as a promising therapeutic target for cancer therapy. Recent studies revealed a crucial role of AXL signaling in proliferation, survival, dormancy and therapy resistance in different cancers including lung cancer, hepatocellular cancer and AML. In this study, we aimed to investigate the role of AXL in Multiple Myeloma (MM), focusing on myeloma cell dormancy and AXL expression in different cellular components of the bone marrow microenvironment.

Material & Methods

To investigate dormancy, we used the syngeneic murine 5TGM1 MM model. 5TGM1-GFP⁺cells were DiD-labeled and injected intravenously in naïve C57BL/KaLwRij mice. At end-stage, GFP⁺DiD⁺('dormant', non-proliferating) and GFP⁺DiD^-('proliferating') MM cells were analyzed by flow cytometry for AXL expression. In addition, AXL expression was also analyzed in CD11b+ myeloid cells and in in vitrogenerated macrophages from the 5TMM model. The effects of AXL inhibition by R428 (BGB324|Bemcentinib, Sigma-Aldrich), a highly potent and AXL-specific small molecular inhibitor, on viability and induced apoptosis of MM cells was determined by Cell Titer Glo and AnnexinV/7AAD staining respectively. AXL expression in human myeloma cell lines (HMCL) (JJN3, U266 and LP-1) and murine 5TGM1 cells was analyzed by qRT-PCR and cytospin stainings. Patient cohorts (TT2/TT3) were used to correlate AXL expression and overall survival. Plasma of healthy donors and MM patients was analyzed by ELISA (R&D).

Results

Using the in vivo5TGM1 dormancy model, we demonstrated an increased expression of AXL (4x higher) in dormant MM cells compared to proliferating MM cells (n=3, p<0,05). Myeloma cell lines (JJN3, U266, 5TGM1) had a very low AXL expression, however, treatment with melphalan induced a more than twofold increase in AXL expression (n=3, p<0.05). The combination of melphalan and R428 significantly increased apoptosis of JJN3 (>10%), U266 (>20%) and LP-1 (>10%) cells compared to single agent therapy (n=6) (p<0.01). Using patient cohorts, we observed that AXL expression correlated with a good overall survival (p=0.006). In addition, plasma samples of patients (n=31) showed a decreased expression of AXL compared to samples of healthy controls (n=9) (p<0.001). This confirms our hypothesis that AXL is associated with dormancy and therefore correlates with a better overall survival. In a second part, we investigated AXL expression in 5TMM-derived myeloid cells and macrophages (n=3). We observed a high expression of AXL in myeloid derived suppressor cells and tumor associated macrophages compared to myeloma cells. In addition, we observed that myeloid cells were much more sensitive to R428 compared to MM cells (n=5, p>0.01).

Conclusion

We observed that AXL is highly expressed in dormant MM cells and environmental myeloid cells. Despite its association with a good prognosis in MM, AXL serves as an interesting target to eradicate dormant myeloma cells as AXL inhibitors affect viability and induce apoptosis of myeloma cells, especially in combination with melphalan. Therefore, AXL can be considered as a new therapeutic strategy, to target both the immunosuppressive myeloid cells and the residual cancer cells in MM patients.