Targeted Axl Inhibition Primes Chronic Lymphocytic Leukemia B Cells to Apoptosis and Shows Synergistic/Additive Effects in Combination with BTK Inhibitors.

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. 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. CLL B-cells overexpress Tyro3, but not MER. Of interest, Tyro3 remains as constitutively phosphorylated and form a complex with Axl in CLL B-cells. TP-0903 induces massive apoptosis in CLL B-cells with LD50 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. Administration of TP-0903 either as a single agent or in combination with BTK inhibitors may be effective in treating CLL patients.

B细胞慢性淋巴细胞白血病（CLL）尽管有积极的治疗方法，但仍是一种无法治愈的疾病。我们先前发现Axl受体酪氨酸激酶（RTK）在CLL B细胞存活中起关键作用。在此，我们探讨了将一种高亲和力Axl抑制剂作为单一药物或与布鲁顿酪氨酸激酶（BTK）抑制剂联合用于未来临床试验以治疗CLL患者的可能性。 评估了RTKs的TAM（Tyro3、Axl、MER）家族其他成员在CLL B细胞中的表达/激活状态。在有或无CLL骨髓基质细胞（BMSCs）存在的情况下，用一种高亲和力、口服生物可利用的Axl抑制剂TP - 0903处理细胞。还评估了TP - 0903对CLL B细胞中Axl信号通路的抑制作用。最后，将细胞暴露于TP - 0903与BTK抑制剂联合的环境中，以确定这种联合的任何协同/相加效应。 CLL B细胞过度表达Tyro3，但不表达MER。有趣的是，在CLL B细胞中Tyro3持续磷酸化并与Axl形成复合物。TP - 0903在CLL B细胞中诱导大量凋亡，半数致死量（LD50）值在纳摩尔范围内。重要的是，CLL骨髓基质细胞无法保护白血病B细胞免受TP - 0903诱导的凋亡。由于Axl受到抑制，在CLL B细胞中检测到抗凋亡蛋白Mcl - 1、Bcl - 2、XIAP显著减少，促凋亡蛋白BIM上调。最后，TP - 0903与BTK抑制剂联合可增强CLL B细胞的凋亡。 TP - 0903无论是作为单一药物还是与BTK抑制剂联合使用，在治疗CLL患者方面可能都是有效的。