Inhibition of RAF dimers: it takes two to tango

Abstract

The RAS-regulated RAF–MEK1/2–ERK1/2 pathway promotes cell proliferation and survival and RAS and BRAF proteins are commonly mutated in cancer. This has fuelled the development of small molecule kinase inhibitors including ATP-competitive RAF inhibitors. Type I and type I½ ATP-competitive RAF inhibitors are effective in BRAFV600E/K-mutant cancer cells. However, in RAS-mutant cells these compounds instead promote RAS-dependent dimerisation and paradoxical activation of wild-type RAF proteins. RAF dimerisation is mediated by two key regions within each RAF protein; the RKTR motif of the αC-helix and the NtA-region of the dimer partner. Dimer formation requires the adoption of a closed, active kinase conformation which can be induced by RAS-dependent activation of RAF or by the binding of type I and I½ RAF inhibitors. Binding of type I or I½ RAF inhibitors to one dimer partner reduces the binding affinity of the other, thereby leaving a single dimer partner uninhibited and able to activate MEK. To overcome this paradox two classes of drug are currently under development; type II pan-RAF inhibitors that induce RAF dimer formation but bind both dimer partners thus allowing effective inhibition of both wild-type RAF dimer partners and monomeric active class I mutant RAF, and the recently developed “paradox breakers” which interrupt BRAF dimerisation through disruption of the αC-helix. Here we review the regulation of RAF proteins, including RAF dimers, and the progress towards effective targeting of the wild-type RAF proteins

Inhibitory feedback control of NF-κB signalling in health and disease

“…This is seen most obviously with the negative feedback loops that operate in RAS-RAF-MEK-ERK1/2 signalling, which give the pathway the properties of a negative feedback amplifier [ 358 ]. For example, ERK1/2 feedback phosphorylation of RAF proteins impairs RAS-dependent RAF dimerisation, which is critical for activation of wild type RAF proteins and downstream signalling [ 359 ]. Consequently, whilst inhibitors of MEK or ERK1/2 will disrupt downstream signalling, they will also prevent ERK1/2-mediated feedback phosphorylation of RAF, resulting in further RAF activation; this drives the rapid ‘rebound’ reactivation of ERK1/2 signalling that is observed in RAS-mutant tumour cells with wild type RAF proteins.…”

Hidden Targets in RAF Signalling Pathways to Block Oncogenic RAS Signalling

“…For instance, we do not know whether and how current RAF kinase-inhibitors affect RAF1's antiapoptotic kinase-independent functions. This is becoming important as current drug development is shifting from BRAF selective to pan-RAF inhibitors in the hope to block BRAF-RAF1 heterodimer signalling [8]. However, it will be equally important to assess how such inhibitors impact on the kinase-independent functions of RAF1 due to allosteric changes in protein conformation that could influence binding to ASK1 or MST2.…”

“…A main reason is that RAS induces homo-and heterodimerization of RAF kinases, and that the dimer is resistant to drug inhibition. The drug resistance is caused by the ability of a drug-bound, catalytically inhibited RAF protomer to allosterically transactivate the kinase activity of the other protomer [7,8]. This mechanism leads to a paradoxical activation of ERK in RAS-mutated cells in response to RAF inhibitors [9][10][11].…”

Hidden Targets in RAF Signalling Pathways to Block Oncogenic RAS Signalling

“…For instance, we do not know whether and how current RAF kinase inhibitors affect RAF1's antiapoptotic kinase independent functions. This is becoming important as current drug development is shifting from BRAF selective to pan-RAF inhibitors in the hope to block BRAF-RAF1 heterodimer signalling [69]. However, it will be equally important to assess how such inhibitors impact on the kinase independent functions of RAF1 due to allosteric changes in protein conformation that could influence binding to ASK1 or MST2.…”