The AMPK inhibitor overcomes the paradoxical effect of RAF inhibitors through blocking phospho–Ser-621 in the C terminus of CRAF

Abstract

The dimerization-driven paradoxical activation of RAF proto-oncogene Ser/Thr kinase (RAF) is the predominant cause of drug resistance and toxicity in cancer therapies with RAF inhibitors. The scaffold protein 14-3-3, which binds to the RAF C terminus, is essential for RAF activation under physiological conditions, but the molecular basis is unclear. Here we investigated whether and how 14-3-3 regulates the dimerization-driven transactivation of the RAF isoform CRAF by RAF inhibitors and affects drug resistance and toxicity by virtue of the dominant role of CRAF in these processes. We demonstrated that 14-3-3 enhances the dimerization-driven transactivation of CRAF by stabilizing CRAF dimers. Further, we identified AMP-activated protein kinase (AMPK) and CRAF itself as two putative kinases that redundantly phosphorylate CRAF's C terminus and thereby control its association with 14-3-3. Next, we determined whether the combinatory inhibition of AMPK and CRAF could overcome the paradoxical effect of RAF inhibitors. We found that the AMPK inhibitor (AMPKi) not only blocked the RAF inhibitor–driven paradoxical activation of ERK signaling and cellular overgrowth in Ras-mutated cancer cells by blocking phosphorylation of Ser-621 in CRAF but also reduced the formation of drug-resistant clones of BRAFV600E-mutated cancer cells. Last, we investigated whether 14-3-3 binding to the C terminus of CRAF is required for CRAF catalytic activity and observed that it was dispensable in vivo. Altogether, our study unravels the molecular mechanism by which 14-3-3 regulates dimerization-driven RAF activation and identified AMPKi as a potential agent to counteract drug resistance and adverse effects of RAF inhibitors in cancer therapies.

Targeting Aberrant RAS/RAF/MEK/ERK Signaling for Cancer Therapy

“…On the other hand, if a dimeric 14-3-3 binds to the N-and C-terminus of a single RAF, respectively, it can stabilize autoinhibitory conformation [211,213]. The serine residues in conserved 14-3-3 binding motifs can be phosphorylated by Protein Kinase A (PKA) [214][215][216], Akt [216][217][218], AMPK [219,220], or by LATS1 from the MST2-Hippo pathway [221]. Regulation of phosphorylation events at two 14-3-3 binding sites can change the response drastically due to their opposite activity.…”

“…For example, in Ras-mutated cancer cells, the CRAF S621 is phosphorylated redundantly by AMPK and itself. Combination of RAF inhibitors with AMPK inhibitor could reduce the paradoxical activation [219]. Recognition of the alternative kinases that could phosphorylate 14-3-3 binding sites and thereby alter RAF activity, could improve clinical success.…”

RAF conformational autoinhibition and 14-3-3 proteins promote paradoxical activation

“…14-3-3 proteins bind and stabilize the autoinhibited form of BRAF (33,34,43), and a recent cryo-EM structure is available for the complex between 14-3-3 and autoinhibited BRAF (35). However, 14-3-3 also stabilizes the RAF dimers (25,33,34) and a recent cryo-EM structure illuminates the structure of dimerized RAF bound to 14-3-3 (30). We therefore extended our model to include both of these sets of reactions ( Figure 4A) and we then mathematically investigated how they may impact PA.…”

“…Alternatively, there may also be negative cooperativity (also referred to as negative allostery) in which the binding of a RAF inhibitor to one protomer of a RAF dimer reduces the affinity of the second protomer for RAF inhibitor (Figure 1D) (18,19,23). As only one protomer in a RAF dimer need be signaling competent for RAF signaling to propagate (15,24,25), negative cooperativity can result in a reduced ability to fully inhibit RAF signaling. However, the regulation of RAF kinase activation is complex with multiple regulatory steps (18,24,26,27), and other processes have been described to play a role in PA (15,(28)(29)(30)(31).…”

Overcoming Resistance to Therapies Targeting the MAPK Pathway in BRAF-Mutated Tumours

“…By contrast, mutating serine 729 in cells with intermediate BRAF kinase activity led to increased MEK activation [187]. While more investigation into the mechanism of AMPK regulation of BRAF V600E -mutated cells is necessary, AMPK inhibitors have shown efficacy in combination with BRAFi therapy [188]. Melanoma and CRC studies indicate that cancer cells increase their tolerance to MAPK pathway inhibition by activating AMPK-mediated autophagy [63,189].…”