R-Ras coordinates reciprocal activation of ERK5 and ERK1/2 under single pathway inhibition in melanoma

Malignant melanoma is frequently driven by constitutive activation of the RAS-RAF-MEK1/2-ERK1/2 pathway, yet adaptive signaling limits the long-term efficacy of MAPK-targeted therapies. Although activation of the MEK5-ERK5 pathway has emerged as a mechanism of resistance to RAF-MEK1/2-ERK1/2 inhibition, whether ERK5 inhibition reciprocally activates the canonical MAPK cascade and the molecular basis of this crosstalk remain unknown. Here, we show that genetic and pharmacological inhibition of ERK5 induces further activation of the MEK1/2-ERK1/2 pathway in BRAFV600E melanoma cells. Based on our previous transcriptomic analyses, we investigated the role of the small GTPase R-Ras, identified among the genes upregulated following ERK5 silencing. Accordingly, R-Ras mRNA and protein levels increased upon both genetic and pharmacological ERK5 inhibition, whereas R-Ras silencing abolished ERK1/2 hyperactivation and potentiated the anti-proliferative and pro-apoptotic effects of ERK5 targeting. Conversely, inhibition of the RAF-MEK1/2-ERK1/2 pathway increased R-Ras expression and ERK5 activation, both of which were prevented by R-Ras depletion. Besides ERK1/2, overexpression of a constitutively active mutant of R-Ras promoted ERK5 activation, placing R-Ras upstream of both signaling cascades. Finally, the pan-Ras inhibitor RMC-6236 potentiated the antitumor activity of either ERK5- or RAF-MEK1/2-ERK1/2-targeted therapies in either two-dimensional cultures or melanoma spheroids. Collectively, these findings identify R-Ras as a central regulator of reciprocal rewiring between ERK1/2 and ERK5 pathways under targeted MAPK inhibition. Functional disruption of this signaling circuit enhances melanoma cell death, providing a mechanistic rationale for co-targeting R-Ras together with MAPK signaling to limit adaptive responses to targeted therapy in BRAFV600E melanoma.