Cancer is characterized as a complex disease caused by coordinated alterations of multiple signaling pathways. The Ras/RAF/MEK/ERK (MAPK) signaling is one of the best-defined pathways in cancer biology, and its hyperactivation is responsible for over 40% human cancer cases. To drive carcinogenesis, this signaling promotes cellular overgrowth by turning on proliferative genes, and simultaneously enables cells to overcome metabolic stress by inhibiting AMPK signaling, a key singular node of cellular metabolism. Recent studies have shown that AMPK signaling can also reversibly regulate hyperactive MAPK signaling in cancer cells by phosphorylating its key components, RAF/KSR family kinases, which affects not only carcinogenesis but also the outcomes of targeted cancer therapies against the MAPK signaling. In this review, we will summarize the current proceedings of how MAPK-AMPK signalings interplay with each other in cancer biology, as well as its implications in clinic cancer treatment with MAPK inhibition and AMPK modulators, and discuss the exploitation of combinatory therapies targeting both MAPK and AMPK as a novel therapeutic intervention.

Although extensively studied for three decades, the molecular mechanisms that regulate the RAF/MEK/ERK kinase cascade remain ambiguous. Recent studies identified the dimerization of RAF as a key event in the activation of this cascade. Here, we show that in-frame deletions in the β3-αC loop activate ARAF as well as BRAF and other oncogenic kinases by enforcing homodimerization. By characterizing these RAF mutants, we find that ARAF has less allosteric and catalytic activity than the other two RAF isoforms, which arises from its non-canonical APE motif. Further, these RAF mutants exhibit a strong oncogenic potential, and a differential inhibitor resistance that correlates with their dimer affinity. Using these unique mutants, we demonstrate that active RAFs, including the BRAF(V600E) mutant, phosphorylate MEK in a dimer-dependent manner. This study characterizes a special category of oncogenic kinase mutations, and elucidates the molecular basis that underlies the differential ability of RAF isoforms to stimulate MEK-ERK pathway. Further, this study reveals a unique catalytic feature of RAF family kinases that can be exploited to control their activities for cancer therapies.

RAS-RAF-MEK-ERK signaling has a well-defined role in cancer biology. Although aberrant pathway activation occurs mostly upstream of the kinase MEK, mutations in MEK are prevalent in some cancer subsets. Here, we found that cancer-related, activating mutations in MEK can be classified into two groups: those that relieve inhibitory interactions with the helix A region and those that are in-frame deletions of the β3-αC loop, which enhance MEK1 homodimerization. The former, helix A–associated mutants, are inhibited by traditional MEK inhibitors. However, we found that the increased homodimerization associated with the loop-deletion mutants promoted intradimer cross-phosphorylation of the activation loop and conferred differential resistance to MEK inhibitors both in vitro and in vivo. MEK1 dimerization was required both for its activation by the kinase RAF and for its catalytic activity toward the kinase ERK. Our findings not only identify a previously unknown group of MEK mutants and provide insight into some key steps in RAF-MEK-ERK activation but also have implications for the design of therapies targeting RAS-ERK signaling in cancers.

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.

Although targeting BRAF mutants with RAF inhibitors has achieved promising outcomes in cancer therapy, drug resistance remains a remarkable challenge, and underlying molecular mechanisms are not fully understood. Here, we characterized a previously unknown group of oncogenic BRAF mutants with in-frame insertions (LLRins506 or VLRins506) of αC-β4 loop. Using structure modeling and molecular dynamics simulation, we found that these insertions formed a large hydrophobic network that stabilizes R-spine and thus triggers the catalytic activity of BRAF. Furthermore, these insertions disrupted BRAF dimer interface and impaired dimerization. Unlike BRAF(V600E), these BRAF mutants with low dimer affinity were strongly resistant to all RAF inhibitors in clinic or clinical trials, which arises from their stabilized R-spines. As predicted by molecular docking, the stabilized R-spines in other BRAF mutants also conferred drug resistance. Together, our data indicated that the stability of R-spine but not dimer affinity determines the RAF inhibitor resistance of oncogenic BRAF mutants.