Targeting Alterations in the RAF-MEK Pathway

Abstract

The MAPK pathway is one of the most commonly mutated oncogenic pathways in cancer. Although RAS mutations are the most frequent MAPK alterations, less frequent alterations in downstream components of the pathway, including the RAF and MEK genes, offer promising therapeutic opportunities. In addition to BRAF^V600 mutations, for which several approved therapeutic regimens exist, other alterations in the RAF and MEK genes may provide more rare, but tractable, targets. However, recent studies have illustrated the complexity of MAPK signaling and highlighted that distinct alterations in these genes may have strikingly different properties. Understanding the unique functional characteristics of specific RAF and MEK alterations, reviewed herein, will be critical for developing effective therapeutic approaches for these targets. SIGNIFICANCE: Alterations in the RAF and MEK genes represent promising therapeutic targets in multiple cancer types. However, given the unique and complex signaling biology of the MAPK pathway, the diverse array of RAF and MEK alterations observed in cancer can possess distinct functional characteristics. As outlined in this review, understanding the key functional properties of different RAF and MEK alterations is fundamental to selecting the optimal therapeutic approach.

Figure 1:. Function of activator and amplifier mutations in the MAPK signaling pathway.

Schema showing the effect of “activator” versus “amplifier” mutants in the MAPK pathway. Activator alterations lead to constitutive MAPK signaling through ERK activation that is independent of upstream pathway activity. Activator mutants strongly activate ERK and lead to negative feedback suppression of upstream signaling. Amplifier mutants augment the downstream signal to ERK and commonly co-occur with other activating mutations upstream in the pathway. They lead to modest activation of ERK and consequently cause minimal negative feedback inhibition of upstream signaling.

Figure 2:. Functional classes of BRAF mutations.

Class I BRAF mutants can signal as monomers, independent of RAS activation. They lead to high ERK activation, which causes negative feedback inhibition of upstream signaling. RAS-GTP levels are therefore low in tumors with class I BRAF mutants. Class II BRAF mutants signal as RAS independent, mutant-mutant BRAF dimers. They strongly activate ERK, causing negative feedback inhibition of upstream signaling and low levels of RAS-GTP. Class III BRAF mutants exhibit enhanced binding to RAS and CRAF to signal as mutant BRAF-wild-type CRAF dimers. They amplify the signaling downstream of RAS and thus require upstream activation to increase ERK signaling, either through genomic alterations (RAS mutations or NF1 loss, as shown on the left) or receptor tyrosine kinase (RTK) signaling (as shown on the right). Tumors with these mutations therefore exhibit high RAS-GTP levels.

Figure 3:. Functional classes of MEK mutations.

Schema showing physiologic MAPK signaling (left panel) or signaling in cells with MEK mutants. RAF-independent MEK mutants strongly activate ERK and induce negative feedback regulation of upstream signaling. These mutants are able to auto-phosphorylate the key regulatory sites S218 and S222 on MEK in cis. RAF-regulated MEK mutants exhibit some independent kinase activity, but this activity can be increased further in the presence of activated RAF, augmenting signaling from RAF. They do not activate ERK signaling to the same degree as the RAF-independent MEK mutants and therefore exhibit more modest feedback inhibition of upstream signaling. RAF-dependent mutants increase ERK activation only in the setting of active RAF; they bind more tightly to RAF, augmenting ERK activation. They modestly activate ERK and lead to minimal feedback inhibition of upstream signaling and often co-occur with upstream, activating alterations.

Figure 4:. Distinct functional properties of RAF inhibitors.

Schema showing effect of different RAF inhibitors in monomeric RAF kinases (i.e. BRAF^V600E) (top section) or dimeric RAF kinases (bottom section). ERK activation is strongly activated downstream of BRAF^V600E, even more so than seen for RAF dimeric kinase signaling. Monomer-selective RAF inhibitors bind to the ATP site in BRAF monomers and inhibit downstream signaling. In RAF dimeric kinases, binding of drug inhibits the bound RAF protomer, but leads to a conformational change in the other protomer in the dimer pair and strong transactivation of this protomer, leading to overall increased ERK activation. Drug binding to one site of the RAF dimer pair leads to a negative cooperativity for binding to the other site, and therefore at clinical doses, only one protomer in the dimer pair binds drug. RAF dimer inhibitors are able to bind to mutant RAF monomers and dimers at equipotent doses without negative cooperativity for the second site, and therefore can inhibit mutant RAF monomers and dimers at the same dose. RAF dimer breakers bind to and inhibit BRAF monomers. In RAF dimeric kinases, these drugs act by directly disrupting dimerization, rather than binding to both protomers in the dimer pair and exhibit negative cooperativity for binding a second protomer in the dimer pair. Dimer breakers disrupt BRAF-containing dimers, but do not disrupt CRAF homodimers, where they cause transactivation of the unbound CRAF protomer.

Figure 5:. Therapeutic strategies for different classes of BRAF and MEK mutations.

The efficacy of specific inhibitors (green “+”: active; red “-”, inactive) and a potential rational therapeutic approach (blue boxes) are shown for each class of BRAF or MEK mutation. In general, the therapeutic approach for each class of mutation is based on its classification as an “activator” or “amplifier”, with activators requiring targeting downstream of or at the level of the mutation, and with amplifiers requiring upstream inhibition in combination with downstream inhibition. The level at which the pathway is targeted in each scenario is marked in orange. For MEK mutants, upstream inhibition would include RAF inhibition in BRAF^V600 cancers or RTK inhibition in RAS/BRAF wild type cancers. In some cases, upstream inhibition may be helpful even when targeting activator mutations as a means of disrupting adaptive feedback reactivation of MAPK signaling, for example as when adding an RTK inhibitor (e.g., anti-EGFR antibodies) in BRAF^V600 colorectal cancers (CRC).