Active Notch Protects Mapk Activated Melanoma Cell Lines From Mek Inhibitor Cobimetinib

The crosstalk between Notch and MAPK pathway plays a role in MEK inhibitor resistance in BRAF(V600E) metastatic melanoma (MM) and promotes migration in GNAQ(Q209L) uveal melanoma (UM) cells. We determined the cytotoxicity of combinatorial inhibition of MEK and Notch by cobimetinib and gamma-secretase inhibitor (GSI) nirogacestat, in BRAF(V600E) and BRAF wt MM and GNAQ(Q209L) UM cells displaying different Erk1/2 and Notch activation status, with the aim to elucidate the impact of Notch signaling in the response to MEK inhibitor. Overall the combination was synergic in BRAF(V600E) MM and GNAQ(Q209L) UM cells and antagonistic in BRAF wt one. Focusing on UM cells, we found that cobimetinib resulted in G0/G1 phase arrest and apoptosis induction, whereas the combination with GSI increased treatment efficacy by inducing a senescent-like state of cells and by blocking migration towards liver cancer cells. Mechanistically, this was reflected in a strong reduction of cyclin D1, in the inactivation of retinoblastoma protein and in the increase of p27(KIP1) expression levels. Of note, each drug alone prevented Notch signaling activation resulting in inhibition of c-jun(Ser63) and Hes-1 expression. The combination achieved the strongest inhibition on Notch signaling and on both c-jun(Ser63) and Erk1/2 activation level. In conclusion we unveiled a coordinate action of MAPK and Notch signaling in promoting proliferation of BRAF(V600E) MM and GNAQ(Q209L) UM cells. Remarkably, the simultaneous inhibition of MEK and Notch signaling highlighted a role for the second pathway in protecting cells against senescence in GNAQ(Q209L) UM cells treated with the MEK inhibitor.