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Cordover et al. J Cancer Metastasis Treat 2020;6:45 I http://dx.doi.org/10.20517/2394-4722.2020.101 Page 5 of 19

abnormal Ras signaling that is seen in many cancers. While more research needs to be done to determine
the mechanism behind Ras dimerization, current data suggests that disruption of Ras dimerization has the
[19]
capacity to suppress Ras-Raf signaling in cancer .
Raf and MEK inhibitors
The Raf family of protein kinases are central components of the ERK cascade as they interact with Ras
and initiate the activation of the kinase cascade. When Raf is dysregulated, it can exert oncogenic effects,
[20]
including metastasis, invasion, and uncontrolled cellular proliferation . Researchers have discovered
mutations that affect the catalytic activity of Raf to be present in several human tumors. Of the three
different isoforms of Raf, A-, B-, and C-Raf, B-Raf was found to be commonly mutated in numerous
[21]
cancers, specifically melanoma. In fact, B-Raf mutations are found in 66% of all melanomas . In wild type
cells, Ras prompts the dimerization of B-Raf and its subsequent activation. In cancer cells with mutated
B-RAF, activating mutations cause B-RAF to be expressed constitutively, which promotes cell growth and
inhibits apoptosis .
[22]

In particular, the B-Raf V600E mutation, present in melanoma and colon cancer, is shown to drive cancer
[23]
development, and serves as an important diagnostic and prognostic biomarker . Inhibitors that target
B-RAF V600E have been clinically proven to prolong the survival of advanced stage melanoma patients.
Nonetheless, these agents display adverse effects, and patients often relapse due to acquired resistance.
A major problem with first generation B-RAF inhibitors is that drug treatment provokes increased wild-
type Raf membrane localization and B-/C-Raf dimerization. This means that when the inhibitor is under
non-saturating conditions, drug bound B-Raf can dimerize with drug-free Raf, resulting in the activation
of MEK and ERK in the presence of active Ras. This contributes to the limited activity of these first-
generation inhibitors in cancers with upregulated Ras despite having the V600E B-Raf mutation. As such,
this poses a challenge for next generation B-RAF V600E inhibitors. One way in which researchers are looking
to combat the dilemma of Raf dimerization and drug resistance is by creating inhibitors that target both
monomeric and dimeric Raf with equal affinity. These agents, known as pan-Raf inhibitors, have been
shown to inhibit Raf signaling and paradoxical ERK activation in colorectal cancer and melanomas.
Another approach that is being studied to combat paradoxical ERK activation is through an inhibitor that
[20]
binds to the ATP binding pocket of B-Raf and impedes Raf dimerization .
In addition to Ras and Raf, MEK is another kinase that has an important role in the ERK pathway and
serves as a promising drug target. Once it is activated, MEK phosphorylates ERK. ERK in turn plays critical
roles in the regulation of cellular proliferation and apoptosis. As such, the inhibition of both isoforms
of MEK, MEK1 and MEK2, have the potential to cause growth inhibition in tumors where the ERK
pathway is activated. Interestingly, trametinib, the FDA approved MEK1 and MEK2 inhibitor, is shown
to inhibit tumor proliferation in advanced stage melanomas with the B-RAF V600E mutation. Compared to
standard chemotherapy, trametinib given as a single agent showed statistically significant improvement in
[24]
terms of disease progression-free survival during treatment . The other FDA-approved MEK inhibitor,
cobimetinib, proved successful in blocking ERK/MAPK signaling in B-RAF and K-RAS mutated cell
lines. Clinically, cobimetinib is well tolerated in patients with advanced solid tumors with the B-RAF V600E
[25]
mutation . Further, several preclinical and clinical studies show that the use of a MEK inhibitor
alongside a BRAF inhibitor is a promising form of treatment for advanced stage melanomas. As previously
mentioned, melanoma patients treated with first generation BRAF inhibitors often relapsed due to acquired
drug resistance. The dual inhibition of both BRAF and MEK leads to more significant tumor response than
BRAF therapy alone .
[26]

ERK inhibitors
ERKs are important effectors in the Ras/ERK pathway. There are two structurally similar isoforms of
ERK, ERK1 and ERK2, and they belong to the MAPK family of protein kinases that are central to signal