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Conroy et al. Cancer Drug Resist 2021;4:543-58 https://dx.doi.org/10.20517/cdr.2021.07 Page 11

to bind effectors in RAS-binding domains. Mutations in RAS that are relevant to cancer usually lead to RAS
permanently in the GTP bound active state. Efforts have been made to block this nucleotide exchange in an
attempt to stop RAS transitioning to the active GTP bound form.

An alternative to blocking the direct nucleotide site is to inhibit the proteins that regulate the nucleotide
exchange process. Normal RAS activation requires nucleotide exchange, processing, membrane localization
and effector binding. Targeting any of these steps can be used to indirectly inhibit RAS. GEFs are
responsible for releasing GDP from RAS allowing it to be replaced by GTP and ultimately leading to
activation of RAS. In mammals, three families of RAS-specific GEFs exist: SOS, RASGRF1/Cdc25Mm and
GRP/Cal-DAG-GEF . Of these, SOS is the best known and studied of the RAS-specific GEFs and this has
[84]
led to the development of a number of strategies and attempts to block it and ultimately inhibit RAS. SOS1
binds RAS at its catalytic binding site and thereby promotes exchange of GDP for GTP. RAS-GTP can also
[85]
bind at an allosteric site on SOS1 to enhance GEF activity . Genetic inactivation of SOS1 has been shown
to decrease the survival of RAS-mutant tumor cells, but not in RAS wild type cells that are not reliant on
RAS signaling . Inhibition of SOS1 has been thought of as an attractive mechanism of RAS inhibition over
[86]
direct RAS inhibitors as it does not appear to depend on targeting specific mutations.

BI-3406 is a potent and selective SOS1:K-RAS interaction inhibitor that potently decreased the formation of
GTP-bound RAS and reduced cell proliferation of RAS-driven cancers both in vitro and in vivo. This orally
bioavailable agent appears to reduce RAS-GTP level and inhibits ERK pathway signaling, thereby limiting
the growth of tumor cells driven by RAS. Whilst most RAS variants appear to show reduction in cell
proliferation when exposed to BI-3406, certain variants appear less sensitive. Mutations in codon 61 due to
the resultant molecular conformation appear to have low intrinsic GTPase activity and are subsequently less
[87]
sensitive to SOS1 inhibition . Initial studies of SOS1 inhibitor BI-3406 suggest that it may benefit as many
as 80%-90% of RAS-driven cancers .
[88]
SOS1 is phosphorylated by ERK, a kinase in the ERK pathway downstream of RAS, ultimately leading to the
reduction of its GEF activity. It is thought that efforts to treat RAS-driven cancers with MEK inhibitors have
failed in part as inhibition of MEK reduces the activity of ERK1/2, resulting in the release of a negative
feedback loop, thus increasing the activity of SOS1-dependent formation of GTP-bound RAS. Combination
therapy of a MEK inhibitor with BI-3406 blocks this negative feedback by reducing levels of phospho-MEK
and phospho-ERK leading to sustained pathway inhibition and potentiating the benefit of SOS1 inhibition.
A Phase I clinical trial of this combination to assess safety, tolerability and preliminary efficacy, as well as
another SOS1 inhibitor, BI-1701963 in combination with Trametinib is ongoing (NCT040111458).

Although much less defined than SOS, another protein involved in the nucleotide exchange process is
SHP2. SHP2 is a non-receptor protein tyrosine phosphatase that is required for the full activation of the
ERK pathway . Mutations in PTPN11, which encodes SHP2, cause “rasopathies” and are found in about
[89]
50% of patients with Noonan syndrome . Although not fully defined, SHP2 appears to act as a scaffold
[90]
protein, binding GRB2 and SOS1 in close proximity to RAS and ultimately thereby increasing RAS
nucleotide exchange . Research has explored the role of SHP2 inhibitors in the treatment of various
[91]
cancers. In the preclinical setting, the allosteric SHP2 inhibitor SHP099 has been shown to inhibit myeloid
leukaemia cell lines , and to elicit a response in colorectal cancer cell lines . Of note, in the colorectal
[93]
[92]
cancer cells, these responses seemed limited to cells that were sensitive to lapatinib, and therefore dependent
on EGFR signalling. By contrast, RAS- or BRAF-mutant cells were generally resistant to SHP099.