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Conroy et al. Cancer Drug Resist 2021;4:543-58 https://dx.doi.org/10.20517/cdr.2021.07 Page 7

[31]
EGFR: There is substantial crosstalk between EGFR tyrosine kinase and RAS . With EGFR upstream of
RAS, inactivation of these receptor tyrosine kinases can in theory reduce RAS activation, and this linear
model explains the lack of clinical activity for EGFR inhibitors in colorectal cancer in the setting of K-RAS
or N-RAS-mutant tumours [32,33] . This has also been demonstrated experimentally, through activation of the
RAS signalling pathway by introduction of an activated K-RAS allele, confirming that as mutated RAS is
constitutively active, disruption of signalling from EGFR impairs the therapeutic effect of anti-EGFR
monoclonal antibodies . Similarly in advanced non-small cell lung cancer (NSCLC), EGFR inhibition is
[34]
[35]
insufficient to produce a response in the setting of RAS-mutant disease . It is possible, however, that the
specific RAS mutation plays a significant role in determining whether upstream inhibition of EGFR may be
effective. We have seen in K-RAS G12D mutation-specific advanced solid tumours that a response can be
achieved with pan-ERBB/EGFR inhibitors Afatinib and neratinib [36,37] . EGFR and pan-ERBB inhibitors have
shown promise in preclinical studies in combination with both direct covalent RAS inhibitors and MEK
inhibitors . This synergistic effect between EGFR inhibitors and covalent RAS inhibitors appears to be the
[37]
result of EGFR inhibition leading to a reduced amount of GTP-bound RAS, therefore leaving RAS in the
unbound GDP state open to targeting by direct inhibition.

ERK pathway: Directly downstream of RAS signaling are the ERK and PI3K signaling pathways . Active
[38]
GTP-bound RAS results in RAF dimerization and phosphorylation, RAF kinase activity and, ultimately,
phosphorylation of its substrates MEK1 and MEK2. The terminal kinases of this pathway, ERK1 and ERK2,
act as growth promoting transcription factors. The RAS-RAF-MEK-ERK cascade is targeted with RAF
kinase inhibitors, ERK inhibitors or MEK inhibitors. Eleven RAF kinase inhibitors have reached clinical
evaluation and four are approved for use by the US Food and Drug Administration (FDA). Vemurafenib
and dabrafenib are two ATP-competitive RAF inhibitors that are approved for use in BRAF-mutant
metastatic melanoma. Further clinical evidence in both lung cancer and colorectal cancer has shown benefit
for these agents when BRAF is mutated [39,40] . The current agents used in clinical practice, dabrafenib and
vemurafenib, act on RAF monomers. However, in RAS-mutant cancer, RAF inhibition has been
unsuccessful and the clinical experience has been very negative. The reason for this is that clinically used
RAF inhibitors enhance RAF kinase homo-and heterodimerization, leading to the paradoxical activation of
[41]
ERK signalling . Homo- and heterodimerization of the RAF kinases BRAF and CRAF significantly
increases their catalytic activities. The binding of RAF molecules to active RAS drives RAF dimerization by
inducing conformational changes, dephosphorylation of inhibitory residues, and brings RAF molecules into
proximity of each other . Due to allosteric interactions between protomers in the RAF dimer, inhibitor
[42]
binding to the first protomer in a dimer strongly decreases the affinity of the second protomer to the
inhibitor. In this constellation the drug-bound RAF protomer allosterically activates the drug-free protomer
causing paradoxical pathway activation and drug resistance . As oncogenic RAS proteins are effective
[42]
drivers of RAF kinase dimerization, RAS mutations lead to intrinsic or acquired resistance to RAF
inhibitors. Overcoming dimerization-induced resistance to RAF inhibitors could lead to effective anti-RAS
therapy .
[43]

Another interesting drawback to targeting wildtype RAF in RAS-mutant disease is that inhibition of
wildtype RAF can paradoxically upregulate the ERK pathway in the setting of RAS mutations leading to
[44]
downstream phosphorylation of MEK and ERK . There are a number of newer agents that target RAF
dimers rather than the monomer isoform all of which appear to demonstrate less paradoxical upregulation
of the ERK pathway [45,46] . Belvarafenib and LXH-254 are pan-RAF inhibitors, effective against the monomer
and dimer isoform, which are under clinical evaluation both as monotherapy and in combinations for RAS-
mutant advanced solid tumours.