Targeted Axl Inhibition Primes Chronic Lymphocytic Leukemia B-Cells to Apoptosis and Show Synergistic/Additive Effects in Combination with BTK inhibitors

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Despite aggressive treatment regimen B-Cell Chronic Lymphocytic Leukemia (CLL) is still incurable. Previously we defined the role of a novel receptor tyrosine kinase (RTK) Axl in CLL B-cell survival. In this study we demonstrated the impact of Axl inhibition on CLL B-cell survival using a high-affinity Axl inhibitor, TP-0903. Results suggest that TP-0903 is highly effective in inducing apoptosis in primary CLL B-cells via inhibition of AKT/Src signaling pathways, downstream of Axl, with nanomolar range LD 50 doses which overcome bone marrow stromal cell protection of the leukemic B-cells. Finally, combined treatment of CLL B-cells with TP-0903 and a reversible Bruton's tyrosine kinase (BTK) inhibitor induces apoptosis more efficiently with additive/synergistic effects. These findings underscore the usage of the Axl inhibitor as a single agent or in combination with BTK inhibitors to more effectively treat CLL patients. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Purpose: B-cell chronic lymphocytic leukemia (CLL) is an incurable disease despite aggressive therapeutic approaches. We previously found that Axl receptor tyrosine kinase (RTK) plays a critical role in CLL B-cell survival. Here, we explored the possibility of using a high-affinity Axl inhibitor as a single agent or in combination with Bruton's tyrosine kinase (BTK) inhibitors for future clinical trial to treat CLL patients. Experimental Design: Expression/activation status of other members of the TAM (Tyro3, Axl, MER) family of RTKs in CLL B-cells was evaluated. Cells were treated with a high-affinity orally bioavailable Axl inhibitor TP-0903 with or without presence of CLL bone marrow stromal cells (BMSCs). Inhibitory effects of TP-0903 on Axl signaling pathway was also evaluated in CLL B-cells. Finally, cells were exposed to TP-0903 in combination with BTK inhibitors to determine any synergistic/additive effects of the combination. constitutively phosphorylated and form a complex with Axl in CLL B-cells. TP-0903 induces massive apoptosis in CLL B-cells with LD 50 values of nanomolar ranges. Importantly, CLL BMSCs could not protect the leukemic B-cells from TP-0903 induced apoptosis. A marked reduction of the anti-apoptotic proteins Mcl-1, Bcl-2, XIAP and upregulation of the pro-apoptotic protein BIM in CLL B-cells were detected as a result of Axl inhibition. Finally, combination of TP-0903 with BTK inhibitors augments CLL B-cell apoptosis. Conclusion: Administration of TP-0903 either as a single agent or in combination with BTK …