FGFR1 but not S6K1/2 drives intrinsic BRAF inhibitor resistance in melanoma

A decrease in phosphorylation of S6 has been identified by many studies as one of the top markers of response to several kinase inhibitors in cancer, especially BRAF inhibitors (BRAFi) in melanoma. However, it is unknown whether it is merely a marker or whether it plays a functional role in resistance. We therefore sought to comprehensively assess the role of the primary S6 kinases S6K1 and S6K2 in intrinsic BRAFi resistance, an understudied topic compared to acquired BRAFi resistance. Surprisingly, we found that overexpression or double knockout of S6K1/2 had no effect on BRAFi sensitivity or resistance in multiple melanoma cell lines. Double S6K1/2 knockout left a small amount of residual S6 phosphorylation, which we determined was at least partially maintained by CK1a. To determine whether pS6 itself affects BRAFi resistance, we overexpressed a phosphomimetic S6 construct in sensitive cell lines, but this also had no impact on BRAFi sensitivity. Instead, a pooled CRISPR kinome knockout screen uncovered FGFR1 as a key resistance driver, and indeed intrinsic BRAFi resistance was partially to fully reversible in 6 cell lines by the FDA-approved pan-FGFR inhibitor pemigatinib. Overall, our results suggest that although changes in pS6 are an excellent marker of intrinsic BRAFi resistance, the S6K1/2-S6 axis itself is not primarily responsible, and instead that FGFR1 is worth further translational study in the context of intrinsic BRAFi resistance.