Running title: Resistance mechanisms to MAPK inhibitors in melanoma

39 p.-7 fig.Combined treatment of metastatic melanoma with BRAF and MEK inhibitors has improved survival, but the emergence of resistance represents an important clinical challenge. Targeting ERK is a suitable strategy currently being investigated in melanoma and other cancers. To anticipate possible resistance to ERK inhibitors (ERKi), we used SCH772984 (SCH) as a model ERKi to characterize resistance mechanisms in two BRAF V600E melanoma cell lines. The ERKi-resistant cells were also resistant to vemurafenib (VMF), trametinib (TMT), and to combined treatment with either VMF and SCH or TMT and SCH. Resistance to SCH involved stimulation of the IGF-1R-MEK5-Erk5 signaling pathway, which counteracted inhibition of Erk1/2 activation and cell growth. Inhibition of IGF-1R with linsitinib blocked Erk5 activation in SCH-resistant cells and decreased their growth in 3D spheroid growth assays as well as in NOD scid gamma (NSG) mice. Cells doubly resistant to VMF and TMT or to VMF and SCH also exhibited downregulated Erk1/2 activation linked to stimulation of the IGF-1R-MEK5-Erk5 pathway, which accounted for resistance. In addition, we found that the decreased Erk1/2 activation in SCH-resistant cells involved reduced expression and function of TGF-alpha. These data reveal an escape signaling route that melanoma cells use to bypass Erk1/2 blockade during targeted melanoma treatment and offer several possible targets whose disruption may circumvent resistance.This work was supported by grants SAF2014-53059-R, SAF2017-85146-R and RD12/0036/0061 to J.T., and PI15/01180 to A.E-O.Peer reviewe