Identification of pAKT as a pharmacodynamic marker for MER kinase in human melanoma G361 cells

Abstract

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

A Potent and Selective Dual Inhibitor of AXL and MERTK Possesses Both Immunomodulatory and Tumor-Targeted Activity

“…A comparison between the nonresponsive CTG-1339 and responsive CTG-2041 sarcoma models demonstrated that while INCB081776 inhibited pAXL and pMERTK in both models, only pAKT was inhibited in CTG-2041. Our results are consistent with a prior report demonstrating that inhibition of pAKT is a biomarker of MERTK inhibition, although it is likely that inhibition of both pAXL and pMERTK contributed to the reduction of pAKT in the CTG-2041 model (36). These data indicate that AXL and MERTK are strong drivers of tumor growth in CTG-2041, and suggest that in the CTG-1339 model, there are other activated pathways that lead to AKT activation that are independent of AXL and MERTK.…”