Chen Yaoyu, Favata Margaret, Pusey Michelle, Li Jun, Lo Yvonne, Ye Min, Wynn Richard, Wang Xiaozhao, Yao Wenqing, Chen Yingnan
Incyte Research Institute, 1801 Augustine Cut-off, Wilmington, DE 19803 USA.
Biomark Res. 2020 Feb 4;8:4. doi: 10.1186/s40364-020-0184-9. eCollection 2020.
The MER signaling pathway represents an attractive therapeutic target for human cancers. Growth arrest-specific protein 6 (GAS6)-induced MER phosphorylation is often unstable and difficult to detect without pervanadate pretreatment in human cancer cells, posing a challenge for the development of selective MER kinase inhibitors. Here, we identified phosphorylated AKT (pAKT) as a specific pharmacodynamic marker for MER kinase inhibitors in human melanoma G361 cells.
The expression of MER, TYRO3, and AXL were profiled among multiple human cancer cells. To determine whether they play a role in the activation of pAKT, MER and TYRO3 were selectively depleted by small, interfering RNA knockdown. In addition, using AKT phosphorylation as a readout, a high-throughput cell-based assay was established in G361 cells for evaluation of the potency of potential inhibitors of MER pathway activation.
We demonstrated that high levels of MER and TYRO3, but not AXL, were expressed in G361 cells. In these cells, pAKT was induced by GAS6 treatment, which could be reversed by AXL/MER inhibitors. We showed that GAS6-induced pAKT is only dependent on MER kinase, but not TYRO3, in G361 cells. Furthermore, we observed a correlation in potency between inhibition of pAKT in G361 cells and pMER in MER-overexpressing Ba/F3 cells by these inhibitors.
In summary, we have demonstrated that GAS6-induced pAKT is a possible pharmacodynamic marker for the inhibition of MER kinase, and we have successfully developed a cell-based functional assay for screening small-molecule inhibitors of MER kinase for potential therapeutic utility in treating GAS6/MER-deregulated human cancers.
MER信号通路是人类癌症颇具吸引力的治疗靶点。在人类癌细胞中，生长停滞特异性蛋白6（GAS6）诱导的MER磷酸化通常不稳定，且未经过钒酸盐预处理很难检测到，这给选择性MER激酶抑制剂的研发带来了挑战。在此，我们确定磷酸化的AKT（pAKT）是人黑色素瘤G361细胞中MER激酶抑制剂的一种特异性药效学标志物。
在多种人类癌细胞中分析了MER、TYRO3和AXL的表达情况。为确定它们是否在pAKT的激活中发挥作用，通过小干扰RNA敲低选择性去除了MER和TYRO3。此外，以AKT磷酸化作为读数，在G361细胞中建立了一种基于细胞的高通量分析方法，用于评估MER通路激活潜在抑制剂的效力。
我们证明G361细胞中表达高水平的MER和TYRO3，但不表达AXL。在这些细胞中，GAS6处理可诱导pAKT，而AXL/MER抑制剂可使其逆转。我们表明，在G361细胞中，GAS6诱导的pAKT仅依赖于MER激酶，而不依赖于TYRO3。此外，我们观察到这些抑制剂对G361细胞中pAKT的抑制作用与对MER过表达的Ba/F3细胞中pMER的抑制作用之间存在效力相关性。
总之，我们证明了GAS6诱导的pAKT是抑制MER激酶的一种可能的药效学标志物，并且我们成功开发了一种基于细胞的功能分析方法，用于筛选MER激酶的小分子抑制剂，以用于治疗GAS6/MER失调的人类癌症的潜在治疗用途。
