EACR26-1463

A Ras family member mediates reciprocal activation of ERK5 and ERK1/2 signaling in response to inhibition of either pathway

I. Tusa¹, C. Mazzei¹, D. Papini¹, A. Menconi¹, Y. Sfragano¹, A. Pandiella², A. Esparis-Ogando², E. Rovida¹

¹University of Florence, Department of Experimental and Clinical Biomedical Sciences "Mario Serio", Florence, Italy
²Instituto de Biología Molecular y Celular del Cáncer (IBMCC)-Consejo Superior de Investigaciones Científicas (CSIC); Instituto de Investigación Biomédica de Salamanca (IBSAL); Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Salamanca, Spain

Introduction:

Malignant melanoma is a highly aggressive cancer with a steadily rising incidence. The Mitogen Activated Protein Kinase (MAPK) family, in particular ERK1/2 and ERK5 pathways, plays a key role in tumor growth and progression, including in melanoma. In BRAFV600E-mutant patients, BRAF or MEK1/2 inhibitors have significantly improved outcomes, especially in combination with immunotherapy. However, resistance mechanisms frequently limit their efficacy. Preclinical evidence suggests that RAS-RAF-MEK1/2-ERK1/2 inhibition may induce compensatory ERK5 activation that supports tumor growth. Here, we investigated the role of a small GTPase (referred to as X-Ras) in the cross-regulation of ERK1/2 and ERK5 pathways in BRAFV600E-mutant melanoma.

Material and method:

BRAFV600E-mutated A375 and Sk-Mel-5 melanoma cells were silenced for ERK5 using specific shRNAs. X-RAS was silenced using siRNAs. Small-molecule inhibitors used included ERK5 inhibitors (JWG-071, XMD8-92), MEK1/2 inhibitor (Trametinib), BRAF inhibitor (Vemurafenib), ERK1/2 inhibitor (SCH-772984) and pan-RAS inhibitor (RMC-6236). HEK-293T cells were transfected with a constitutively active form of X-Ras. 3D speroid assays were performed in melanoma cells treated with RMC-6236 in combination with the ERK5 inhibitors or BRAF-MEK1/2-ERK1/2 inhibitors.

Result and discussion:

Genetic and pharmacological inhibition of ERK5 increased MEK1/2 and ERK1/2 phosphorylation and X-Ras expression in melanoma cells. X-Ras silencing prevented this activation and potentiated the anti-proliferative and pro-apoptotic effects of ERK5 inhibition, pointing to a compensatory effect. Notably, X-Ras also mediated ERK5 activation following inhibition of the BRAF–MEK1/2–ERK1/2 axis with vemurafenib/trametinib or SCH-772984. X-Ras knockdown prevented ERK5 phosphorylation induced by these treatments and enhanced their pro-apoptotic effects, pointing to a resistance/compensatory mechanism exerted by X-Ras upon ERK1/2 pathway inhibition. Consistently, overexpression of a constitutively active X-Ras mutant increased ERK5 phosphorylation, further supporting a role of X-Ras in regulating ERK5 activation. Treatment with the pan-RAS inhibitor RMC-6236 reduced melanoma cell viability in a dose-dependent manner, and its combination with ERK5 or BRAF–MEK1/2–ERK1/2 inhibitors more effectively impaired both cell viability and 3D tumor growth compared with single agents.

Conclusion:

X-Ras emerges as the tipping point for the reciprocal compensatory activation of ERK1/2 and ERK5 to overcome single pathway inhibition. Targeting X-Ras potentiates the effects of ERK1/2- and ERK5-directed therapies, highlighting its potential as a combinatorial strategy to overcome MAPK inhibitor resistance in BRAFV600E-mutant melanoma.