AXL is a novel ERK5/KLF4 target in MEK inhibitor-treated melanoma.

RAS/RAF/MEK/ERK1/2 mitogen-activated protein kinase (MAPK) pathway-based treatments, typically combination therapies with inhibitors for oncogenic BRAF V600 (BRAFi) and MEK1/2 (MEKi), are important treatment alternatives to immunotherapy in advanced BRAF-mutated melanoma. However, their benefit is limited by frequent therapy resistance, i.e. persistence and progression/metastasis of tumor cells under sustained treatment. Furthermore, approximately 50% of the patients, including the NRAS-mutated subset, lack targetable BRAF oncogenes and profit poorly from MEKi. Recent preclinical studies suggest co-inhibition of the MEK5/ERK5 MAPK pathway, which in different MAPK-activated tumors is compensatorily activated by MEKi, as promising strategy to overcome therapy resistance and trigger apoptosis and/or sustained cell cycle arrest. In NRAS-mutant melanoma, compensatory ERK5 activation is accompanied by the induction of the Krüppel-like factors KLF2 and KLF4 but their role in MEKi resistance remains unclear. Using RNA interference and CRISPR/Cas9, we examined their contribution to MEKi resistance through RNA sequencing and functional assays. Surprisingly, KLF2 and KLF4 were dispensable for the proliferative and anti-apoptotic effects of compensatory ERK5 activation in MEKi-exposed melanoma. Instead, we identified AXL, a key receptor tyrosine kinase associated with metastasis and phenotypic switching, as critical ERK5/KLF4 target induced during MEKi resistance. Genetic loss of KLF4 or AXL depletion reduced melanoma cell migration and invasion, suggesting a key role of KLF4 in the regulation of invasiveness. Our study describes a novel ERK5/KLF4/AXL signaling axis that drives MEKi resistance and metastatic potential in NRAS-mutant melanoma and highlights this axis as a potential target to improve MAPK-directed and potentially immune therapies.