ERK pathway agonism for cancer therapy: evidence, insights, and a target discovery framework

Abstract

At least 40% of human cancers are associated with aberrant ERK pathway activity (ERKp). Inhibitors targeting various effectors within the ERKp have been developed and explored for over two decades. Conversely, a substantial body of evidence suggests that both normal human cells and, notably to a greater extent, cancer cells exhibit susceptibility to hyperactivation of ERKp. However, this vulnerability of cancer cells remains relatively unexplored. In this review, we reexamine the evidence on the selective lethality of highly elevated ERKp activity in human cancer cells of varying backgrounds. We synthesize the insights proposed for harnessing this vulnerability of ERK-associated cancers for therapeutical approaches and contextualize these insights within established pharmacological cancer-targeting models. Moreover, we compile the intriguing preclinical findings of ERK pathway agonism in diverse cancer models. Lastly, we present a conceptual framework for target discovery regarding ERKp agonism, emphasizing the utilization of mutual exclusivity among oncogenes to develop novel targeted therapies for precision oncology.

Full text

npj | precision oncology Review article
Published in partnership with The Hormel Institute, University of Minnesota
https://doi.org/10.1038/s41698-024-00554-5
ERK pathway agonism for cancer therapy:
evidence, insights, and a target discovery
framework
Check for updates
Oleg Timofeev1,PhilippeGiron
2, Steffen Lawo 3, Martin Pichler4& Maxim Noeparast 4
At least 40% of human cancers are associated with aberrant ERK pathway activity (ERKp). Inhibitors
targeting various effectors within the ERKp have been developed and explored for over two decades.
Conversely, a substantial body of evidence suggests that both normal human cells and, notably to a
greater extent, cancer cells exhibit susceptibility to hyperactivation of ERKp. However, this
vulnerability of cancer cells remains relatively unexplored. In this review, we reexamine the evidence on
the selective lethality of highly elevated ERKp activity in human cancer cells of varying backgrounds.
We synthesize the insights proposed for harnessing this vulnerability of ERK-associated cancers for
therapeutical approaches and contextualize these insights within established pharmacological
cancer-targeting models. Moreover, we compile the intriguing preclinical findings of ERK pathway
agonism in diverse cancer models. Lastly, we present a conceptual framework for target discovery
regarding ERKp agonism, emphasizing the utilization of mutual exclusivity among oncogenes to
develop novel targeted therapies for precision oncology.
Background
Cancer targeting: from chemotherapy to precision oncology
In 1904, after a series of studies on different model organisms, Paul
Erlich, a highly meritorious German medical scientist, coined che-
motherapy to address his approach when chemicals were used to target
infectious diseases1,2(see Fig. 1). Ehrlich humbly stated: Indeed, from the
very origin of the art of healing, chemotherapy has existed since almost all
the medicaments we employ are chemicals1. However, Ehrlich’s concept
of chemotherapy unprecedently linked the chemical structure and
chemoreceptors on the target cells to make sense of the pharmacological
activity3,4. He address ed chemicals that selectively exert their detrimental
effect on the target cells (e.g., parasites) and not on the treated organism
cells as Zauberkugeln or magic bullets1,2. Erlich had tested compounds,
such as alkylating agents, to target cancer, which, in his hands, showed
limited effects, far from being considered Zauberkugeln. Still, Ehrlich
remained optimistic that effective chemotherapeutics could be explored
in the future1. He explained this lack of effect by the high similarity
between cancer and non-cancer cells1.
During World War II, in a mouse xenograft lymphoma model, two
American pharmacologists, Alfred Gilman, and Louis Goodman, showed
that nitrogen mustard, a warfare gas, had (chemo)therapeutic effects5,6.
Their work immediately justified the treatment of a non–Hodgkin’slym-
phoma patient and other cancers with nitrogen mustard5,7.Despitefurther
knowledge about its limited efficacy and proneness to resistance, nitrogen
mustard’s ephemerous success laid the foundation for establishing cancer
chemotherapy as a valid field. Since then, several cancer chemotherapeutics
such as Alkylating, Antimicrotubular agents, and Antimetabolites have been
developed8. Cancer chemotherapy is a term whose definition was progres-
sively elucidated long after it was coined, considering the expanding
knowledge about chemotherapeutics’mechanism of action and limitations.
Compared to more selective therapeutics, Chemotherapeutics are con-
sidered one-size-fits-all treatments targeting different cancer types carrying
distinct driver oncogenes8. Despite their narrow therapeutic window and the
possibility of targeting non-cancerous dividing cells, chemotherapeutics
have saved lives in some cancer populations, such as pediatric hematologic
cancers, or among adult malignancies, such as testicular cancer9.Until
1Institute of Molecular Oncology, Member of the German Center for Lung Research (DZL), Philipps University, 35043 Marburg, Germany. 2Vrije Universiteit Brussel
(VUB), Universitair Ziekenhuis Brussel (UZ Brussel), Clinical Sciences, Research group Genetics, Reproduction and Development, Centre for Medical Genetics,
Laarbeeklaan 101, 1090 Brussels, Belgium. 3CRISPR Screening Core Facility, Max Planck Institute for Biology of
Ageing, 50931 Cologne, Germany. 4Translational Oncology, II. Med Clinics Hematology and Oncology, 86156
Augsburg, Germany. e-mail: maxim.noeparast@gmail.com
npj Precision Oncology | (2024) 8:70 1
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today, chemotherapy, surgery, and radiotherapy are on the list of available
neoadjuvant, adjuvant, or combined cancer treatments8,10.
Followed by the burst of knowledge about cancer-related gene muta-
tions in the 1990s, targeting oncogenes laid the foundation for targeted
therapy in cancer11. Indeed, since the New Era of Personalized Medicine in
199911, cancer treatment has been revolutionized. We have seen the main-
stream shift from “one‐size‐fits‐all”approaches, such as classical che-
motherapy, to individualized treatments according to patients’tumor
genetic profiles12,13. A milestone in personalized cancer therapy was the
success of Imatinib, a small molecule inhibitor of the BCR‐ABL protein
tyrosine kinase. This inhibitor was initially conceptualized and profiled by
Nicholas Lydon and colleagues14. Imatinib demonstrated remarkable clin-
ical advantages during phase II trials, particularly among patients with
chronic myeloid leukemia (CML)15,16.
By 2002, to explain mechanisms behind the sensitivity of some cancers
to mutation-specific targeting, Bernard Weinstein coined the term oncogene
addiction to describe a phenomenon that, despite extensive genetic altera-
tions, cancer cells can depend on a single oncogene activity17–23.Onecan
analogize eliminating that very oncogene activity in the addicted cell to
surpassing the cell’s system biology threshold, ultimately leading to a lethal
trade-off favoring proapoptotic vs. the prosurvival signals. Various ther-
apeutics have been developed against other oncogenes to which different
cancers are addicted. EGFR- or ALK-altered lung cancers and BRAF mutant
melanoma are some success stories of personalized cancer treatments24–26.
All these therapeutics work by inhibiting a driver oncogene or its down-
stream effector, based on the rationale that withdrawal from oncogene
addiction leads to deleterious oncogenic shock27. Indeed, target inhibition is a
shared feature of all these treatments. Although the targets are often kinase,
non-kinase protooncogenes, such as KRASG12C, are also actionable28.Sooner
or later, however, all these treatments are doomed to the rise of resistance
mechanisms, leading to the relapse of the treated cancer25,29–33.
A theoretical alternative of oncogene inhibition as a therapeutic con-
cept is the re-introduction or restoration of a lost tumor suppressor (TS)
activity, as cancer cells might be sensitive to that lost function34,35. However,
this strategy has been proven challenging as often, if not always (e.g., some
p53 variants), TS genes are lost due to various genetic and epigenetic
alterations and, therefore, non-targetable in cancer cells. The p53 targeted
therapies, conceptualized according to the p53 status of the cancer cell, are
being explored36. These approaches can involve different strategies, from
stabilizing the wild-type p53 and unleashing its wide range of TS functions
in cells with existing wild-type copies to restoring the wild-type con-
formation among responsive mutant p53 variants36. In the bargain,
numerous efforts have been made to re-express tumor suppressors by viral
and non-viral gene therapy in tumor cells37.Viraltumorsuppressorgene
transfer has even been combined with the TS-loss-targeted-oncolytic
capacity of therapeutic viruses or synergistic effects of TS re-expression with
immunotherapeutics to double-punch the cancer cells37–40.Despiteallthese
cumbersome efforts, none has yet borne fruit in the clinic among large
patient populations. Moreover, in the late stages of cancer, due to the highly
divergent genetic build-up of late-stage tumor cells, it is to be further
elucidated whether cells have already circumvented their native sensitivity
and response to the lost tumor suppressors.
By 1945, Theodosius Dobzhansky, a Russian-American geneticist, had
coined the term synthetic lethality, describing a scenario when two dis-
tinctive chromosomes in Drosophila pseudoobscura were tolerated if exist-
ing alone but became lethal when they co-existed synthetically, as being
imposed in the lab, in cross-over flies41. In 2009, Nobel Prize laureate
William Kaelin Jr. put synthetic lethality forward as a conceptual framework
for exploring novel anticancer treatments42. The cancer research field fur-
ther translates this as the genetic build-up in certain cancers conferring
sensitivity to specific chemical or genetic perturbations, opening novel
avenues for indirectly targeting previously non-druggable targets43,44.Today,
synthetic lethality is described as the cellular lethality of at least two co-
occurring contextual (genetic) or chemical perturbations targeting at least
two separate genes. In contrast, these perturbations are tolerated at
variance45. As a result, within the scope of personalized medicine, treatments
have been developed by targeting specific non-oncogenes based on the
concept of synthetic lethality43,44. An example of such an approach in the
clinic is the adjuvant as well as palliative PARP inhibitor treatment of
BRCA1/2-mutant HER2-negative breast cancer46.Anexcitingfeatureof
synthetic lethality is that it can be applied to both an oncogene and a tumor
suppressor as long as they confer sensitivity to a synthetically lethal target44.
An interesting reconceptualization of synthetic lethality as a cancer-
targeting approach, namely the one-two punch47 model, is being pioneered
and explored by René Bernards and colleagues. In this model, the first-line
treatment is designed to induce senescence-associated vulnerabilities in
cancer cells that are to be targeted with the second-line senolytic
compounds47.
Notably, in recent years, targeting cancer-related immune cells or their
interaction with cancer cells has opened a new front in the battle against
cancer48–50. For instance, in BRAF mutant melanoma, in which targeted
inhibitors are known to be at their highest performance, immune check-
point inhibitors showed a 20% benefit over targeted compounds in terms of
overall survival among therapy-naïve and metastatic patients51.Assuch,
oncogene-targeted therapy might seem on the verge oflosing momentum in
personalized medicine. However, despite the striking responses to different
cancer immunotherapeutics in a group of patients, many patients remain
non-responsive or fast-resistant to such therapies48–50,52.
While it is essential to work towards enhancing existing therapeutic
strategies, it is also highly justified to put forward and explore innovative and
unconsumed concepts.
By 2015, two significant studies showed that mutual exclusivity among
BRAF/KRAS and EGFR/KRAS oncogenes can result from synthetic leth-
ality or senescence43,53.
The British fairy tale “Three Bears”(Eleanor Mure, 1831) revolves
around a greedy individual who, in the absence of the three bachelor bears,
each of varying sizes, enters their cottage54. She samples their trio of distinct
milk portions, chairs, and beds. Repeatedly,amidstthesetriads,shedis-
covers contentment solely when the element is precisely balanced –not too
much, not too little, but just right54. In later biological and medical contexts,
Fig. 1 | Timeline of cancer-targeting discoveries and concepts relevant tothis writi ng. The focus of the timeline is mainly on therapeutics targeting the cancer cells. Figure
generated in Biorender.
https://doi.org/10.1038/s41698-024-00554-5 Review article
npj Precision Oncology | (2024) 8:70 2

the widely spread but modified version of the original story “Goldilocks and
the Three Bears,”in which a young girl replaces the elderly woman, has
inspired the Goldilocks principle. This principle signifies that, for a biological
system to function optimally, its components should possess the correct
levels of abundance and activity and be temporally right –avoiding both
excess and deficiency55.
Amit Dipak Amin and Jonathan Schatz, inspired by the Goldilocks
principle in Biology, introduced the term and the concept of “Oncogene
Overdose.”This concept compares cancer cells to drug addicts, as both can
die due to an overwhelming excess of what they are addicted to55.
In 2016, Nobel laureate Harold Varmus introduced the concept of
harnessing mechanisms elicited by the co-induction of mutually exclusive
genes as a therapeutic model56.Furthermore,Xuetal.fromDeng’s
laboratory have explored a striking KRAS agonist for targeting KRAS
mutant cancers57. More recently, Sugiura et al. presented the therapeutic
concept of ERKp activators58–60. Concurrently, René Bernards and collea-
gues described Paradoxical Intervention, signifying the simultaneous acti-
vation of mitogenic signals and the employment of stress-inducing
compounds in cells with pre-existing ERK pathway-activating
alterations61,62. Remarkably, they have demonstrated that such treatment
enforces unprecedented tumor-suppressive resistance mechanisms62.
Dostoevsky once remarked, ‘We all come out from Gogol’s‘Overcoat,”
paying homage to Nikolai Gogol as the iconic predecessor of Russian lit-
erature preceding his own generation. In doing so, he employed wordplay by
referring to one of Gogol’s renowned short stories, namely, ‘The Overcoat.’“
Based on chronological order and logical reasoning, all the above concepts
revolve around over-activating the already activated pathway as a ther-
apeutic approach, akin to stepping out from oncogene overdose overcoat.
In this writing, we review the evidence and insights that reinforced ERK
pathway effectors activity in cells with aberrantly increased ERK activity is
damaging to these cells. The pathway is also known as mitogen-activated
protein kinase (MAPK) or Ras-Raf-MEK-ERK pathway. In this writing, for
simplicity, we will mention the pathway by its major effector ERK without
distinguishing between the two human ERK genes, ERK1 and ERK2. Fur-
thermore, we will present a conceptual framework for therapeutic targeting
of such vulnerabilities.
The double-life of ERKp in human cancer and the curious case of
the ERK proteins
The ERK pathway is one of the extensively studied signal transduction
cascades. Essential effectors of this signaling cascade in humans fall into
three types of eukaryotic protein kinases: (1) HER family Receptor Tyrosine
Kinases (RTKs, EGFR, HER2-4), RAF family serine/threonine kinases (A/
B/CRAF), and (3) dual specificity protein kinase families MEK (MEK1/2)
and ERK (ERK1/2)63–65. In humans, the RAS family, consisting of three
essential members, H/N/KRAS, serve as the principal non-kinase effectors
within the ERK pathway66.
Feedback loops regulate ERK signaling during development and in
normal physiologic conditions for cell proliferation, survival, and
homeostasis63–66.
Due to genetic alterations of the ERK pathway effectors and/or its
regulatory components, ERK signaling may become pathologically
deregulated. Aberrant down-regulation of the ERK pathway can be asso-
ciated with some neurodegenerative or autoimmune disorders, while its
abnormal upregulation is associated with human cancers, rasopathies, and
Erdheim-Chester disease66–69.
Under normal cellular conditions, upon ligands binding, HER recep-
tors can undergo conformational changes, transitioning to an active state
and forming homo- or hetero-dimers70. The induced conformational
alterations in the tyrosine kinase domain of dimerization partners facilitate
ATP binding in their ATP binding cleft and, as suggested, in the case of
EGFR, might lead to the relief of cis-autoinhibition and trans-
autophosphorylation of the tyrosine kinase domains70,71.
Under physiological circumstances, the mature RAS protein, when
residing in the inner surface of the plasma membrane, undergoes
conformational changes mediated by the upstream HER signaling or reg-
ulatory feedback signals66,72. RAS alternates between a guanosine tripho-
sphate (GTP)-bound state and a guanosine diphosphate (GDP)-bound
state28,66,73. The oscillation of RAS into its active conformation involves GTP
loading28,66,73. This process can be initiated by recruiting GRB2 proteins to
the plasma membrane, where the GRB2 is activated via its SH2 domain by
an active RTK66. Subsequently, SOS is recruited, forming a complex invol-
ving at least GRB2, SOS, and RAS66. This complex facilitates the displace-
ment of GDP and promotes the loading of GTP onto the RAS molecule66.
RAS can hydrolyze its GTP as a GTPase protein, converting it to GDP and
becoming inactive28,66,73. This process is facilitated by GTPase-activating
proteins (GAPs)28,66,73. Feedback loops play a regulatory role in the GTPase
activity of RAS66. The GTP-bound RAS triggers the activation of down-
stream RAF by engaging in a physical (allosteric) interaction with RAF or
mediating the release of its autoinhibition74. RAFs, in turn, phosphorylate
the MEK1/2 through phosphotransferase activity, and MEKs, in turn,
phosphorylate the ERKs, which, due to the Erks’vast array of network,
majorcellulareventssuchasproliferationandsurvivalcantakeplace
63–66.At
least 659 of ERKs’direct substrates are discovered75.
Approximately 40% of human cancers are linked to the abberant
upregulation of ERKp76. Notably, cancers that harbor genetic alterations in
an ERKp effector can be associated with worse clinical outcomes than
cancers where such alterations are absent, emphasizing the significance of
these alterations on cancer prognosis77. For over two decades, the effective
inhibition of the ERKp has remained a key focus in cancer research and
targeted therapy efforts12,24,26,28,77.
However, in an ironic twist, a wealth of evidence underscores the re ality
that human cells, including cancer cells, cannot tolerate excessive levels of
ERKp activity. While upstream effectors of the ERKp, such as RTKs, RAS,
and RAFs, are subject to activating and oncogenic mutations in humans, the
mutations in downstream effectors, such as MEK and even to a greater
extent ERK, are infrequent26,73,77,78. Before the ERK small molecule inhibi-
tors’development and clinical employment, ERK mutations were hardly
reported in human cancer79. These mutations can be associated with sec-
ondary resistance mechanisms that lead to loss-of the protein affinity to
ERKinhibitorsincellstreatedwiththesecompounds
79–81. As illustrated in
Fig. 2, among the conventional ERK signaling pathway effectors, the ERK
mutations still rank at the bottom of the list regarding the frequency of
mutations in human cancer.
The ERKs need two critical phosphorylation events before full con-
formational activation, none triggered by either cis- or trans-
autophosphorylation mechanisms82.AsopposedtoRTKs,RAFs,oreven
MEKs, ERKs protein conformations have evolved in such a way to be less
poised for autoactivation and instead rely on direct phosphotransferase
activity of MEKs for full activation82,83.
Several activating variants that cause increased kinase-dependent or
-independent activity of the ERK orthologues, such as Sevenmaker variants,
are studied in Yeast and Drosophila models83–85. However, how such
mutations can induce or contribute to human carcinogenesis remains
enigmatic. Several investigations have concentrated on characterizing a few
purportedly activating ERK variants by expressing them in mammalian
cells82,86,87. These studies have looked into activating variants of ERK that
render the ERK protein an autoactivation capacity82,86,87. In particular, the
activating variant ERK1R84H, reported twice in human cancer, was found to
transform NIH3T3 cells87. While introducing these presumably activating
ERK variants into non-cancerous mammalian cells was occasionally fea-
sible, accomplishing the same in human cancer cell lines appeared to pose
challenges. Markedly, Goetz et al. had shown in the past that overexpression
of wild-type ERK1/2 and not the kinase-dead or low-kinase ERK1/2 variants
in BRAFV600E mutant melanoma cell line A-375 leads to growth inhibitory
effects88. Interestingly, the ERK1/2 variants with increased kinase activity,
which were found to confer resistance against RAF inhibitors (RAFi) and
MEK inhibitors (MEKi) during a random mutation screen, even exerted
more potent growth inhibition when overexpressed in A-375 cells. The most
active ERK1/2 variants exhibited growth-suppressive effects in two other
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npj Precision Oncology | (2024) 8:70 3

BRAF mutant melanoma cell lines beyond A-375 cells(SKMEL-19, and
WM266.4)88. It was also shown that inducible overexpression of ERK2 in
A-375 cells was selectively detrimental to these cells vs. cells with wild-type
BRAF and caused anti-tumor effects in vitro and in vivo89. Such detrimental
effects could only be rescued uponERK2orBRAFknock-down
89.The
ERK2 overexpression in these cells was associated with the induction of ER
stress and DNA damage in addition to proapoptotic signals89.Thefactthat
some ERK-associated cancers, such as BRAF mutant melanoma, are sen-
sitive to ERK activation is well-established.
Interestingly, the negative effect of putative Gain-of-Function
(GOF) mutations on the proliferation of these cells has been utilized as a
model in an ERK saturation mutagenesis study90. Such a comprehensive
approach by Brenan et al. has shed light on the relevance of ERK
mutations in human cancer. It is worth noting that saturation muta-
genesis of ERK was unsuccessful in identifying the direct GOF impact of
activating variants, unless upon MEKi and BRAFi treatment. Indeed,
expression of GOF sevenmaker ERK mutations ERK2D321N and
ERK2E322K or other supposedly GOF variants in the A-375 cell line had a
robust anti-proliferative effect in these cells90. Interestingly, the seven-
maker variant and thos e that phenocopy its effect could only be tole rated
by the BRAFV600E cells under MEK or RAF inhibition90. These variants
are proposed to enhance ERK activity by disrupting ERK’s interaction
with inhibitory DUSP phosphatases90.
The rarity of ERK mutations in human cancer raises an intriguing
question, prompting consideration of at least two alternative scenarios to
explain this phenomenon.
One possibility is that the ERK protein, by default, exhibits low basal
activity and has a weak impact on its network. Consequently, even an
activating mutation might not be sufficient to manifest an effective GOF
phenotype leading to cell transformation. An illustrative example of such a
scenario is the case of CRAF mutations in human cancer, which are rarer
than mutations of another RAF isoform, BRAF91,92.Pastexplanations
attribute this rarity to the low basal activity of CRAF compared to BRAF93.
Conversely, the effectors, such as EGFR,whicharemoreupstreamandhave
a wider array of targets belonging to distinctive pathways, exhibit a higher
frequency of mutations in human cancer. From a vertical signaling cascade
standpoint, the three commonly mutated effectors of the ERKp in human
cancer are arranged as EGFR, KRAS, and BRAF. EGFR and BRAF muta-
tions exhibit a comparable frequency in human cancer (see Fig. 2). Notably,
KRAS mutations occur at a frequency equivalent to the combined occur-
rences of EGFR and BRAF oncogenes (Fig. 2). The higher mutational fre-
quency of KRAS over BRAF can be explained by its vertical rank along the
signaling cascade, a rationale not applicable to the other two effectors82.
Another explanation could be that while ERK mutations capable of
generating an effective GOF phenotype may exist synthetically, they are
negatively selected in the real world due to being poorly tolerated by human
Fig. 2 | ERK1/2 mutations are relatively rare. In July 2023, the query of
69223 samples belonging to 65853 patients in 213 curated and non-redundant
studies in cBiportal for only mutations among conventional effectors of the ERKp
yielded the approximate frequencies as displayed. Note that this oversimplified
schematic is not meant to communicate complex signaling and structures of the
effectors. Unlike typical oncogenes, ERK1/2 mutations are distributed along the
ERK protein’s conserved protein kinase and non-conserved regions, with no hotspot
mutational site. Figure generated in Biorender.
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cells. In contrast to, for instance, KRAS, which oscillates between active and
inactive states under normal physiological conditions and may become
constitutively active in human cancer73, human cells cannot tolerate ERKs in
a constitutively active state. It is crucial to distinguish between two types of
ERK mutations. The first type comprises mutations that occur in the real
world. As the above evidence suggests, these mutations exhibit low trans-
forming capacity. Then comes the synthetic ERK mutations, which do not
occurintherealworld.Indeed,asmentionedintheaboveparagraphs,such
synthetic variants have been studied in the past, and it has been shown that
they are not easily tolerated by human cells, particularly cancer
cells79,81,82,84–86,88–90. More interestingly, in addition to some ERK mutants,
even wild-type ERK overexpression is not tolerated in such human cellular
models88,90. Indeed, one of the vital pieces of evidence is ERK saturation
mutagenesis by Brenan et al., as they observed in human melanoma cell line
model with BRAFV600E, expression of wild-type ERK and activating ERK
variants could be tolerated only in the presence of ERK pathway inhibitors90.
This evidence, at least, can rule out the possibility that ERK, by nature,
cannot render gain-of-function. The scenario that ERK-activating muta-
tions are not easily tolerated by human cells aligns with one aspect of ERK
protein activity autoregulation, namely its reduced potential for auto-
activation, in contrast to other ERK pathway effectors like EGFR82,83.The
ERK protein is shown to tolerate synthetic mutations that can render it
increased autophosphorylation and kinase activity to levels comparable to
ERK582.
The RTK/RAS/RAF pathway and its inhibitors exhibit a Janus-faced
immunomodulatory effect in cancer (reviewed here94). In brief, inhibition of
the ERKp can enhance the activity of associated T-helper cells and cytotoxic
T-cells, thereby potentially enhancing the immune response against cancer
cells94. Additionally, it can enhance dendritic cell activity, which plays a
crucial role in antigen presentation and immune activation. Conversely, this
inhibition may hamper tumor infiltration of immunosuppressive regulatory
T-cells, monocytes, and macrophages, potentially limiting their immuno-
suppressive functions94.Ontheotherhand,long-termERKpinhibition
might eventually have an immunosuppressive effect95. Of note, oncogenic
RAS can contribute to immune evasion by stabilizing the PD-L1 mRNA and
subsequently favoring the PD-L1 expression on tumor cells96.
ERK pathway hyperactivation can be lethal to cells in
various ways
The ERKp activity can lead to cell toxicity and death (see Fig. 3). Numerous
studies have unraveled such a role in different model organisms60,97,98.The
ERK-induced cell apoptosis can involve extrinsic or intrinsic apoptotic
pathways60,97,99. ERK activity affects cell death by influencing multiple cel-
lular processes, including mitochondrial dysfunction100–102,DNADamage
Response103, Endoplasmic Reticulum stress104,Autophagymodulation
60,105,
Metabolic imbalance106, and accumulation of Reactive Oxygen
Species(ROS)97,103. ERKp activity can trigger senescence in vitro and
in vivo97. An interesting example of such a phenomenon is the existence of
benign human naevi with BRAFV600E mutation107,108. In cases where this
mutation occurs in isolation and without the concurrent loss-of the p53
tumor suppressor, it has been proposed to result in irreversible cellular
senescence rather than malignancy108. More recently, an alternative
mechanism involving non-coding RNAs has come to light, linking
BRAFV600E-carrying benign naevi to occurrences of mitotic failure and
reversible proliferation arrest107.
ERKp activity is crucial in determining divergent cellular outcomes,
including cell proliferation, growth arrest, or cell death. Notably, Hong et al.
from Park’s lab have elucidated an exclusive threshold for ERK activity109.
Previously, it was known that excessive activity of ERKs, like other effectors
such as MEKs, CRAF, and BRAF, can cause growth arrest110–113. However,
Hong et al. show that very high activity levels of ERKp, exceeding those that
cause growth arrest, can lead to apoptosis109. These ultra-high ERKp levels in
HEK293 and U251 cells could only be achieved by combining ERKs
overexpression and the presence of tamoxifen-inducible active CR3 catalytic
domain of CRAF109. As proposed by Hong et al., this cell death-inducing
characteristic is unique to the ERKs compared to the upstream kinase
effectors of the ERKp109.
Overall, the evidence is ample that excessive ERK activity is toxic to
cancer cells with different tissue backgrounds. First, we highlight a few
critical early studies among such evidence. Early evidence of ERK’s
proapoptotic activity was observed in the human breast cancer cell line
(MCF-7), as RAF depletion could desensitize these cells to Paclitaxel114.
During the characterizationof Phenethyl Isothiocyanate anti-proliferative
effects against p53-deficient prostate cancer cell line PC-3, ERKp pro-
longed activation was responsible for the compound’s growth inhibitory
effects115. Moreover, as shown in mouse embryonic fibroblasts and MCF-
7, the DNA Damage (DD) caused by different stimuli, including Etopo-
side, could trigger ERKp activationindependent of p53 but rest on Ataxia-
Telangiectasia Mutant (ATM)116. The ERK activity was essential for DD-
triggered growth arrest and apoptosis116. Later, it was shown that growth
inhibitory and proapoptotic effects of Asiatic acid against breast cancer
cell lines (MCF-7 and MDA-MB-231) were associated with the induction
of ERKp117. In another study investigating Lauryl-gallate’s growth-
suppressive and proapoptotic effect on three breast cancer cell lines MCF-
7, MCF-7 ADR, and MDA-MB-231, the observed phenotype was
accompanied by ERKp activity and p21-induced Cell Cycle (CC) arrest118.
As one of the investigated cell lines was p53 mutant (MDA-MB-231) and
the other possessed a multidrug-resistant phenotype (MCF-7 ADR),
authors conclude that none of these conditions affects the sensitivity of
cells to the tested compound118. Interestingly, MEK inhibition and the
resulting ERKp suppression were associated with rescuing the drug
effect118. In another study, sustained ERKp activity due to exogenous
expression of active MEK1/2 led to G1 CC arrest, marked by p21WAF
expression119. ProlongedERK activity was associated with the activation of
cellular protein biosynthesis regulator p70S6K, accompanied by an
increased translation of p21 protein120. Nonetheless, none of these studies
has fully elucidated the mechanisms elicited by ERK activation to lead to
the observed phenotypes. Several other studies demonstrate that rein-
forced ERK activity in cancer cells potentiates the cytotoxic effects of
various chemotherapeutical agents (reviewed here60,97). In many of these
studies, the MEK inhibitor compounds, if among other means, were
employed to investigate the rescue effects60,97. Moreover, as recently
highlighted by Sugiura et al.60 and taking into account subsequent reve-
lations regarding MEK inhibitors off-targets and mechanisms of action,
these factors could potentially undermine the precision of the above-
mentioned findings.
A revisit of the precedent evidence reveals an intriguing aspect of
ERKp-induced cell death and senescence, as it can happe nin both p53 wild-
type and p53-altered backgrounds114–120. Numerous studies have indicated
crosstalk and interactions between the p53 and ERKp, suggesting that they
can influence each other’s functions and responses under specific cellular
conditions. As a major tumor suppressor known as “the guardian of the
genome121,”p53 governs safeguard mechanisms that prevent the accumu-
lation of DNA damage and the proliferation of damaged cells122.Thep53
protein primarily functions as a transcription factor, regulating a plethora of
genes that control cell proliferation, apoptosis, DNA repair, and other cel-
lular processes123. Additionally, p53 possesses multiple transcription-
independent activities in cell death124–126, metabolism127,128,autophagy
129,
DNA replication130, and repair131, all of which contribute to tumor sup-
pression and the maintenance of genomic integrity. The aberrant activation
of the ERKp leads to the induction of the p53 response, which can trigger
senescence108,132–136 or cell death109,137. The classical mechanism of p53 acti-
vation by oncogenes is mediated by the p14ARF protein, which sequesters
the p53 inhibitory ubiquitin ligase Mdm2 (HDM2 in humans), allowing for
the accumulation of the p53 protein138. Additionally, studies have shown
that ERK can phosphorylate p53 on Ser15 under stress conditions, inhi-
biting its interaction with Mdm2 and promoting the stabilization of the p53
protein139–141. The phosphorylation of p53 by ERK2 on Thr55 was reported
to induce p53 stabilization and activation in doxorubicin-treated MCF-7
cells142. However, it remains unclear to what extent the direct
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phosphorylation of p53 by ERK contributes to the induction of senescence
and apoptosis in tumor cells without additional stress stimuli.
In the process of malignant transformation, cells with hyperactive ERK
signaling experience strong negative selective pressure from p53132,143–149
which they can counteract by blunting p53 activity, often through the
acquisition of TP53 mutations that entirely or partially inactivate the tumor
suppressor150. Large-scale genomic studies have demonstrated that TP53
mutations are found in all types of cancers151. The frequency of TP53 genetic
alterations, estimated at an average of 50%, varies significantly depending on
the tumor entity, ranging from 5% in neuroblastoma to over 90% in ovarian
and small-cell lung cancer151–156.
Somatic mutations that inactivate TP53 are predominantly observed in
the later stages of tumorigenesis, indicating its role in advanced cancer
progression157. It is estimated that 80% of genetic alterations in the TP53
gene are missense mutations158,159 that lead to the expression of mutant p53
proteins. These mutants can possess oncogenic properties, driving cancer
progression and conferring therapy resistance160,161. However, the functional
impact of the majority of cancer-associated TP53 missense mutations
(approximately 70%) remains poorly characterized, and their role in
tumorigenesis and therapy response remains unclear162.
TP53 mutations oftenco-occur with oncogenicEGFR, BRAF,and RAS
mutations, but the extent of cooperation between TP53 and oncogenes may
vary even within the same tumor type. For instance, in lung adenocarci-
noma (LUAD), TP53 mutations are prevalent in EGFR mutant tumors but
are strongly underrepresented in KRAS-driven LUAD151,163, indicating
different paths of tumor evolution driven by different oncogenes. The
intricate interplay between various oncogenes within the ERKp and its
impact on the residual activity of wild-type or partial Loss-of-Function
(LOF) p53, retained in tumors, which can potentially lead to tumor-
suppressive effects, remains an area of ongoing investigation162,164.More-
over, different TP53 variants are suggested to cause distinctive LOF,
dominant negative, or even GOF phenotypes, which needs to be acknowl-
edged in further exploration of ERKp-induced lethality in different cellular
contexts with distinctive TP53 status165–168.
ERK pathway in light of emerging knowledge about vulnerability
of cancers to replication stress targeting
In eukaryotes, transmitting genetic material to daughter cells is a crucial
event and is thus tightly regulated. Cyclin-dependent kinases (CDKs) are
pivotal in advancing the CC during interphase and M phases169–171.TheCC
represents a series of sequential decisions and commitments169–171.EachCC
stage is intricately governed by a complex interplay involving CDKs coupled
with E3 ubiquitin ligases like APC/C and their activating proteins169–171.
Varied levels of CC-related proteins can establish decision windows that
impact entry, prevention of re-entry, or even the deceleration of CCs169–171.
Although tightly controlled, the CC can face undesired events due to DD,
Replication Stress (RS), or spindle assembly malfunctions169–171. Eukaryotic
cells rely on distinct checkpoints to tackle each scenario169–171.RSemerges
when diverse factors slowdown or stall the replication fork. DD and RS
differ, but the latter can trigger DD as the collapse of the stalled fork can lead
to double-strand breaks169–172. RS checkpoints function to avoid RS-induced
DNA damage response, while DD checkpoints are meant to prevent the
accumulation of DNA damage and thereby protect cells from subsequent
complications170. A cancer’s hallmark is uncontrolled proliferation, ham-
pering apoptosis and avoiding long-term CC exits34. Oncogenes like RAS
and MYC can negatively affect DNA replication licensing and firing,
Fig. 3 | Intertwining mechanisms by which excessive ERK activity leads to cell damage. Figure generated in Biorender.
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npj Precision Oncology | (2024) 8:70 6

inciting RS169,170,172. Deregulation of DD and growth pathways due to cancer-
related mutations causes excessive S phase entry and subsequent RS. While a
temporary check-out from the Mitosis/entry is possible, leaving the CC is
not favored169–172. Cancer cells hold a higher basal level of RS vs. normal
cells170. The severity of the damage and the context determines the cell fate,
which can be apoptosis, quiescence, or senescence. It deserves to be men-
tioned that DD checkpoint responses can lead to a temporary exit or, as
opposed to RS, an eternal exit of the CC169–172. However, DD responses and
RS are so intertwined that, ultimately, RS can lead to such events169–172.
Curiously, cancer cells highlight a distinct approach to DD and RS check-
points. DD checkpoints are often surrogated in cancer; cancer cells tolerate
defects in DD response and perhaps even benefit from such a compromise
in favor of increased selection pressure, all while sidestepping unfavorable
exits from the cell cycle169–172. Conversely, RS checkpoints are somehow
intact169–172. Indeed, cancer cells are more sensitive to DD responses than RS
responses170. Cancer cells tolerate and even favor some Chromosomal
instability (CIN) levels170. In contrast, they cannot tolerate excessive CIN,
which can be caused by excessive RS and DD, leading to catastrophic mitotic
defects, loss-of-essential genes, and cell death170. Therefore, cancer relies on
RS checkpoints to avoid too much CIN. Consequently, targeting RS toler-
ance in cancer holds promise as a viable cancer therapy approach169–173.Of
note, studies have demonstrated that elevated oncogenic RAS activity can
trigger RS by ubiquitously enhancing cellular transcription events174–176.This
augmented transcription activity increases the likelihood of collisions
between replication and transcription processes174,176.InadditiontoRS
induction, oncogenic RAS (HRASG12V) can circumvent the p53 activity,
thereby sensitizing cancer cells to RS-inducing compounds176.
Withdrawing ERK pathway inhibitors from addicted cells: lethal
consequences of excessive pathway activity
There are several pieces of evidence that ERK-related cancer cells can tol-
erate upregulation of the ERKp only in the presence of ERKp inhibitors.
Resistance mechanisms against ERKp inhibitors predominantly involve
ERKp effectors and regulators30,31,81,177–181. Different RAF and MEK inhibi-
tors can trigger clonal expansion of drug-tolerant cells, which maintain a
proliferative advantage, perhaps preferably in the presence of inhibitors by
virtue of enhanced ERKp activity182,183. This elevated activity could be lethal
upon inhibitor cessation, potentially resulting in ERK-related cellular
toxicities183. It is suggested ERKp inhibitors can create a window upon drug
removal, in which cells lose their fitness advantage gained during drug
treatment and may even experience growth disadvantage due to excessive
ERKp activity and adaptive switching182,184. In an elegant study by Kong et al.
conducted in Peeper lab185, a CRISPR knock-out screen of melanoma cells
resistant and addicted to BRAFi revealed a phenotypic switch dependent on
ERK2 kinase and JUNB and FRA1 transcription factors accompanied by
suppression of microphthalmia-associated transcription factor (MITF)185.
The ERK2 dependency of the observed phenotype was supported by in vivo
and clinical findings185. This dependency was also observed in lung cancer
cells resistant and addicted to EGFRi185. The addicted Melanoma cells
experienced grave cell death upon withdrawal from BRAFi, which could be
rescued by ERK2 targeting or restoring MITF activity185. Another intriguing
aspect of this study was that the authors opted to investigate the intermittent
drug treatment with the chemotherapeutic agent dacarbazine, and they
showed BRAFi-addicted melanoma cells were sensitized to this compound
accompanied with MITF inhibition185.
Aissa et al. elegantly showed at the single-cell level that drug-resistant
EGFR mutant lung cancer cell clusters exhibited markers indicative of
activated ERKp180. Recent work by Nuria Gutierrez-Prat et al.186 reported
similar findings concerning ERK and MITF dependency on drug with-
drawal toxicity. Moreover, the authors find that the knock-down of DUSP4,
an ERK phosphatase and negative regulator, was lethal by causing excessive
ERK activity186. This effect was observed not only in melanoma cells
addicted to inhibitors of the ERKp but also, intriguingly, in drug-naïve
cells186.Xueetal.inPiroLito’s lab found the link between oncogenic BRAF
protein dosage in cells, the depth of ERKp inhibition and the related
resistance mechanisms. In their patient-derived xenograft lung cancer and
melanoma models, they discovered the more robust the ERK inhibition is,
the higher the oncogene dosage required for cells to retain proliferation
advantage in the presence of inhibitors183. They proposed a fitness threshold
model, suggesting that cells treated with regimens with a higher threshold,
such as upon combination of RAFi, MEKi, and ERKi vs. ERKi mono-
therapy, might face a disadvantageous outcome due to sustained and
excessive ERKp activation when the drug treatment is stopped183.
Some preclinical and early clinical evidence suggested the benefits of
intermittent treatment with RAFi and MEKi vs. sequential treatments, in
particular in melanoma182–184,187–197. This evidence laid the foundation for
clinical trials examining intermittent treatment regimens in individuals with
BRAF mutant melanoma198,199. Contrary to the expectations, these trials did
not show any overall survival benefits from the intermittent therapies, and
even worse progression-free survival outcomes were reported upon inter-
mittent treatments198,199. Nevertheless, it is still uncertain whether those
intermittent therapies meant to produce that high fitness threshold as
recommended by Xue et al. to cause selection disadvantages in tumor cells
during drug removal effectively.
Mutual exclusivity of highly activating variants of BRAF, KRAS
and EGFR oncogenes in cancer: induction of synthetic lethalityor
senescence
In 2006, Carlotta Petti et al.200 showed that synthetic expression of NRASQ61R
oncogene in a metastatic melanoma clone, which natively harbored the
mutually exclusive BRAFV600E oncogene, resulted in senescence. Later,
Cisowski et al. discovered that co-expression of BRAFV600E and KRASG12D
under their endogenous promotors provides a selective disadvantage
compared to single oncogene expression in mouse lung cells53. The decrease
in tumor burden in double oncogene-expressing tumors was associated with
hyperactivated ERK and AKT signaling and a decrease in proliferating
cells53. Further analysis demonstrated enhanced β-galactosidase expression
and increased p15, p16, and p19 levels upon oncogenes co-expression,
suggesting that double oncogene-expressing cells become senescent53.
Meanwhile, Unni et al. exogenously induced the expression of KRASG12V or
EGFRL858R in EGFREX19Del and KRASG12C LUAD cell lines, respectively43.
They observed decreased cell viability, indicating that mutant KRAS and
EGFR co-expression are not tolerated in cells43. Additionally, they generated
genetically engineered mice with co-induction of KRAS and EGFR mutants
in lung epithelium. The established lung tumors did not grow faster than
those harboring only one of the oncogenic mutations did43. Further analysis
indicated that only one of these oncogenes could be activated in the tumor
cells43. Furthermore, Unni et al. revealed that DUSP6 prevents ERK activity
from exceeding critical thresholds in EGFR and KRAS mutant cell lines201.
They found that targeting DUSP6 reduced cell viability due to unleashing
the excessive and toxic levels of RAS-mediated ERK activity in cancer cells
harboring mutations in EGFR and KRAS201. Markedly, Ambrogio et al.
showed that conditional induction of an EGFRL858R allele in KRASG12V
knock-in mouse LUAD models led to decreased tumor burden, increased
mice survival, and reversible cell toxicity in remaining tumor cells202.The
latter could further be recovered through ERKp activity reduction202.Of
note, all these consequences were associated with hyperactivation of ERKp
signaling43,201,202.
Harold Varmus, a Nobel Prize laureate, and his colleagues, who played
key roles in some of the aforementioned studies, articulated and cham-
pioned the intriguing concept that, unlike how previously considered, not
the redundancy of functions but synthetic lethality or senescence could lie
behind mutual exclusivity of certain oncogenes in cancer56.
We argue that the pathway redundancy and synthetic lethality,
senescence, or any other constraining phenomenon should not necessarily
be seen as conflicting scenarios. One can consider that pathway redundancy
and synthetic lethality could both play roles in explaining mutual exclusivity.
When two proteins with overlapping functions in a pathway are excessively
active, the likelihood of both events being mutually exclusive within a cancer
cell is increased.
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On co-induction of activating EGFR and BRAF events in the same
cell, it has been shown that exogenous expression of wild-type EGFR in a
Melanoma cell line with native BRAFV600E leads to decreased proliferation
of these cells in a dose-dependent manner, in-vitro and in-vivo203.The
slowdown in proliferation was associated with cellular senescence as
suggested by hypophosphorylation of RB1 and induction of CDKN1A,
CDKN1B, and Beta-galactosidase203. Concerning mutant EGFR and
mutant BRAFs, two studies have been inspired so far by the emergence of
BRAFV600E in treatment-refractory EGFRL858R lung cancers as they become
resistant to EGFR-targeted therapies177,179. In one study, exogenous
expression of BRAFV600E in a polyclonal pool of EGFR mutant lung cancer
cells led to no differences in cellproliferation and cell death ratecompared
to empty vector control177. Of note, the BRAFV600E protein levels and
mRNA levels showed an indispensable increase only in the presence of an
ERKp inhibitor, suggesting that in the polyclonal pool, cells with
BRAFV600E induction are not clonally expanded unless the ERKp activity is
suppressed177. As such, further investigation is required to determine the
existence and the mechanism behind such clonal disfavor. Overall, the
findings of thesetwo studies align with the results of two independent GOF
CRISPRa screens in Vemurafenib-treated A-375 cells (BRAFV600E),
showing that overexpression of EGFR, among others, is a resistance
mechanism to BRAFi204,205.
Three BRAF mutational classes26 include Class I variants, like
BRAFV600E, which often function independently of upstream effectors as
constitutively active monomers. Class II variants, exemplified by BRAFG469A,
activate the ERK pathway independently of RAS and CRAF as active
homodimers. Class III involves kinase-dead BRAF mutants activating the
ERK pathway through RAS-dependent allosteric transactivation of CRAF.
Cancer-relevant KRAS mutations are classified into three categories73
based on their impact on KRAS protein functions: Class I (Hydrolysis)
includes mutations leading to the loss-of the GTP-hydrolyzing feature of
KRAS, Class II (Exchange) involves mutations causing a gain in KRAS
Exchange function facilitated by Guanine Nucleotide Exchange Factors,and
Class III (Hybrid) encompasses mutations affecting both functions.
A recent classification of EGFR mutations24 considers both the struc-
tural effects of mutations on the EGFR protein, specifically its drug-binding
pocket (DBP), and the implications of mutations on drug response.
Accordingly, one of the EGFR mutational classes is Classical-like (relevant
to this writing), where mutations like L858R have minimal impact on DBP
and the affinity for corresponding Tyrosine Kinase Inhibitors.
Recently, the variant-specific landscape of mutual exclusivity among
BRAF, KRAS, and EGFR mutations in cancer has been unraveled206.We
learn which oncogenic variants can co-occur in the same cancer sample
whilecertain driver events aremutually exclusive. The authors concludethat
class I BRAF(in line with another recent report206,207), Hydrolysis KRAS206,
and classical-like EGFR206 class mutations are less likely to co-occur. When
they dissected the analyses into variants, they discovered novel instances of
mutual exclusivity involving unconventional yet common oncogenic var-
iants. They showed that specific classical-like EGFR and BRAF mutations,
often the most frequent ones, are mutually exclusive in human cancer206.
Leveragingoncogenes mutual exclusivity for precision oncology:
a target discovery framework for RTK/RAS/RAF pathway
agonism
Reinforced activation of the ERKp could serve as a therapeutic strategy for
ERK-associated cancers. As previously explained, these cancers exhibit
susceptibility to elevated ERKp activity beyond conventional oncogenic
levels. Importantly, this vulnerability can be selectively targeted because
normal cells and ERK-associated cancer cells differ in their baseline ERKp
activities. Thus, reinforcing ERK activation to levels intolerable for cancer
cells may not necessarily push normal cells beyond the tolerable threshold
(see Fig. 4).
At first glance, the primary concern withthisapproachisthepotential
undesired activation of the ERKp in non-cancerous tissues. Interestingly,
this potential adverse event is not unfamiliar in precision oncology. Patients
with BRAFV600E mutant cancers have been treated with various RAF inhi-
bitors for years, initially as single agents and later combined with MEK
inhibitors. Type I RAF inhibitors like Vemurafenib and Dabrafenib can
cause paradoxical ERKp activation in non-cancerous cells with wild-type B/
CRAF. Some patients develop benign teratomas like keratoacanthoma, but
most do not26,208,209. Adding MEK inhibitors to therapy can significantly
reduce the occurrence of such adverse events, although MEK inhibition is
also associated with toxic effects210. Furthermore, predictive markers and
signatures can help identify patients at risk of these adverse events. On the
other hand, developing selective ERKp activators with exclusive effects on
cancer cells could mitigate this challenge from the outset.
Our proposed model recognizes three non-detrimental and two det-
rimental stages and four thresholds of ERKp activity (Fig. 4). The “normal
low”occurs when cells are at rest. The “normal high”occurs when cells are in
proliferating status or are stressed due to intrinsic or extrinsic signals. ERKp
activity spatiotemporally surpassing “normal high”physiological levels
enters the “oncogenic window.”Levels below the “normal low”and above
the “oncogenic window”can be detrimental. For any potential therapy, it
will be necessary to set pathway activity exceeding the “oncogenic window”
and entering the detrimental stage in the target cancer cells. Ideally, the
therapy should not push pathway activity beyond “normal high”levels in
normal cells, adhering to the Goldilocks principle.
This discussion will not delve into the chemistry and structural aspects
of potential therapeutics, including small molecule activators, monoclonal
antibodies, monobodies, etc. We propose three strategies to reinforce the
ERKp or ERK-independent signals for detrimental effects (Fig. 5a, b).
Perturbation 1 (P1)-global activators. Potential therapeutics activate
the ERKp through various means, such as ligand-mimetic molecules
targeting EGFR, irrespective of the specific oncogenic mechanism of the
treated cell. While not selective towards their direct targets, these ther-
apeutics can exert selective detrimental effects on cancer cells. Recently,
Xu et al. from Deng’s lab have introduced and characterized the first-in-
class small molecule KRAS agonist that activates both the wild-type and
mutant KRAS molecules and has shown selective effects towards KRAS
mutant lung cancer in-vitro and in-vivo57. Another tactic involves
directly targeting the ERK1/2 proteins. Exciting research in Sugiura’s lab
and colleagues has explored ERKp agonists and lead compounds that
Detrimental
Normal high
Oncogenic
Normal low
Stages of ERK pathway activity in health and disease
Fig. 4 | Simplified schematic illustrating the stages of ERKp activity in health and
disease, and upon explored and suggested therapeutic targeting. This schematic is
oversimplified and does not acknowledge the spatiotemporal contextof ERK activity and
regulation. The figure was generated using a template provided in https://www.
slideegg.com.
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show selective activity against ERK-associated cancer cell lines compared
to normal cells58–60. Unsupervised screens involving perturbations to
activate different activating effectors of the ERKp can be applied to find
the most effective targets.
P2-Mutation-specific targeting. Targeting protooncogenes like
BRAFV600, EGFRL858R, or KRASG12V with mutation-specific agonists can
elevate basal ERK activity beyond the oncogenic window in cancer cells,
leading to events like apoptosis or senescence. These agonists should
ideally be selective (if they ever could be) against oncogenic variants of the
targeted- versus the wild-type proteins, aligning with Paul Ehrlich’s
magic bullet concept. It deserves to be mentioned that as oncogenes, like
any other protein, have a threshold of conformational stability; it cannot
be ruled out that oncogene agonists might end up destabilizing and
further orchestrating protooncogene degradation processes before even
exerting the envisioned effects.
P3-Unsupervised target discovery and targeting aided by the
variant-specific landscape of mutual exclusivity among BRAF,
EGFR, and KRAS oncogenes in human cancer. We discussed how
induction of mutually exclusive BRAF, EGFR, and KRAS oncogenes
could be detrimental in affected cells. Nature itself is presenting these
scenarios to us. While all the hypothesis-driven approaches mentioned
above show promise for further investigation, we would like to
underscore an unbiased target discovery approach guided by the
uncovered variant-specific landscape of mutual exclusivity among
BRAF, EGFR, and KRAS oncogenes. Oncogene overdose55,mimicking
the co-expression of EGFR and KRAS oncogenes56,paradoxical
intervention61,andERK-Dependent Apoptosis60 as therapeutic models
proposed by different research groups share fundamental similarities.
Nevertheless, they vary in certain specific details. The recent unveiling
of the variant-specific landscape of mutual exclusivity among ERKp
oncogenes introduces a new layer of complexity to existing concepts.
This newfound complexity offers insights into the hyperactivation of
specific signaling pathways that certain cancer cells circumvent to
avoid most hostile conditions.
Consequently, replicating these mutually exclusive scenarios could
lead to highly effective yet personalized therapies tailored for each oncogenic
variant (BRAF, KRAS, and EGFR). Therefore, in the proposed model, the
toxic effects of two oncogenes’joint expression would be limited to specific
variants, or at least more likely in those mutually exclusive scenarios.
Consequently, in our proposed model, both target (Fig. 5b) and co-target
discovery (Fig. 6) can be a matter of precision. Considering this, our model
suggests that the initial point of target discovery and potentially resulting
therapy, for instance, for the BRAFG4 69V variant, may differ from that of
BRAFV600E. Unbiased approaches, such as bulk RNA sequencing and single-
cell RNA sequencing, can help explore differentially expressed genes and
altered pathways resulting from the induction of mutually exclusive BRAF,
EGFR, and KRAS oncogenes. Genetic screens such as CRISPR screens,
especially dual LOF and GOF screens (i.e., CRISPRi and CRISPRa), may
identify the signaling nodes that govern the response and dictate cell fate
when mutually exclusive oncogenes are co-induced. These screens are
intended to elucidate targeting approaches that phenotypically replicate the
co-induction of two synthetically lethal and mutually exclusive gene
mutations. Merely Droup-out screens like KO screens may fall short in
identifying negative regulators of the ERKp as potential signaling nodes.
Of note, this approach can ideally be tailored to each tumor. For
example, for target discovery of cancer cells with BRAFV600E, one can refer to
the variant-specific landscape of mutual exclusivity database207 and find the
most mutually exclusive scenario with BRAFV600E, and as such, design a
model with inducible expression of KRASG12D or EGFRL858R in BRAFV600E
background. In a suppressor CRISPR screen, those genes whose activation
b
a
Fig. 5 | Three Perturbations (P1-3) to reinforce the already activated ERK
pathway activity in related cancers as a therapeutic approach. a Three suggested
approaches to target the vulnerabilities to reinforced RTK/RAS/RAF pathway
activation in affected cancers. bFramework for P3 target discovery is displayed in a
more detailed manner. Figure generated in Biorender.
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npj Precision Oncology | (2024) 8:70 9

or inhibition will lead to the rescue of senescence or synthetic lethality would
be the governing nodes and potential targets whose antagonism (when
activation rescues) or agonism (when inhibition rescues) replicate the
synthetically lethal co-induction of these mutually exclusive oncogenes (Fig.
5b).Thisapproachwidensthetargeting possibilities and may fitboththe
Goldilocks principle and the magic bullet concept. Besides, this approach
may fit the concept of precision oncology.
It is crucial to emphasize that the RTK/RAS/RAF effectors, the inhi-
bitors designed to target them, and their regulators can demonstrate func-
tions and interactions independently of one another, their kinase activity, or
even the ERKp itself211–213. Further investigation exploring the RTK/RAS/
RAF agonism as a therapeutic approach must address whether these
functions can be leveraged to enhance oncogenic activity beyond tolerable
levels for potential therapeutic benefits or, conversely, whether they might
counteract such a strategy. Therefore, any proposed approach to exploit the
susceptibility of ERK-associated cancers to hyperactivation of these effectors
should consider this notion. If the desired detrimental effect would be ERK-
independent, the P1 might fail to address it.
Widespread and highly active oncogenic variants (gene 1), in mutually
exclusive relationships with other variants (gene 2), align with the third
approach for target discovery (Fig. 5a, b: P3). Conversely, cells harboring
oncogenes with co-occurring tendencies206 (variants of gene 1 and gene 2)
may align with P1 and P2 (Fig. 5a).
Combined targeting
Cancer targeting resembles a time loop: as monotherapies are developed,
resistance emerges, driving the search for more effective combinatorial
treatments that not only offer a stronger initial impact but may also delay the
development of further resistance. Therefore, it would be wishful thinking to
predict that P1-3 would be exempt from the rise of resistance. As such,
concurrent, sequential, or intermittent combinatorial treatments can be
explored depending on the ultimate phenotypes exerted by putative ther-
apeutics. Based on the evidence revisited in this writing, opportunities for
combinatorial treatments with chemotherapeutics can be studied, whether as
double-punch or one-two-punch with senolytics. Suppose ERK agonism
would lead to immune evading phenotypes. In that case, a one-two-punch
model can be explored to address whether, after ERK agonism, the remaining
tumor could be sensitized to immune checkpoint inhibitors. The impact of
ERK agonism on cancer stem cells and in-parallel or subsequent sensitivity of
this subpopulation to chemotherapy or stem cell-targeted therapies is also an
avenue worthy of exploration. Notably, P1-P3 hold promise to be explored in
combination with therapeutics targeting the RS tolerance.
For co-target discovery, we propose that the initial conditions and
ongoing perturbations will be pre-determined using one of the P1-P3, as we
discussed for single-target discovery (Figs. 5a, b and 6). In this context, dual
loss- and GOF screens (e.g., CRISPR screens) will prove valuable in
identifying co-targets when ERK-associated cancer cells undergo the
Fig. 6 | Suggested approach for co-target discovery to more profoundly target the vulnerabilities to reinforced hyperactivation in ERK-associated human cancer.
Figure generated in Biorender.
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pre-determined ERK-activating perturbation (P1-P3). This approach aims
to uncover synthetically lethal co-perturbations (SP) with P1-3, enhancing
the detrimental effects to the levels warranting higher fitness threshold191
and the emergence of tumor-suppressive drug resistance mechanisms62.In
this respect, our proposed model differs from the paradoxical intervention
model, where co-perturbations are identified within the context of
hypothesis-driven constant perturbation, such as stress-inducing agents61,62.
Consequently, we envision unbiased target discovery for both the discovery
of the ERK-activating signaling nodes that govern the detrimental effects of
excessive ERKp activation and the subsequent co-target discovery phase (P3
combined with SP, see Fig. 6). Therefore, our model may precisely recapi-
tulate the mosthostile conditions cancercells are avoiding,which are theco-
induction of specific oncogenic scenarios.
Conclusions
Despite ample evidence that cancer cells are sensitive to excessive RTK/
RAS/RAF pathway activity, this approach has not been widely explored.
Perhaps years of relentless efforts to develop efficient ERK inhibitory
treatments have established a psychological barrier within us, the scientific
community, which, if not overcome, could become a dogma.
Learning from the past, we humbly recommend that all the proposed
strategies in this writing undergo unbiased preclinical exploration without
any premature preference for a specific strategy over the others.
Despite the primary focus of this writing being exclusively on the
ERKp, the mutual exclusivity of cancer-related genetic events offers a wealth
of information regarding unexplored synthetic lethality scenarios. Har-
nessing these scenarios for therapeutic purposes could open new horiz ons in
targeting cancer-related vulnerabilities. Apart from targeting oncogenes,
this approach can also encompass events related to tumor suppressors, such
as p53, especially when cancer cells remain sensitive to restoring the related
functions. Exploring these approaches and identifying targets demands a
collaborative effort involving collective benchwork and shared intellectual
contributions.
Received: 3 November 2023; Accepted: 16 February 2024;
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Acknowledgements
M.N. is grateful to Dr. ErikTeugels for years of scientific discussion aboutthe
idea behind this writing and beyond.
Author contributions
M.N. conceived and conceptualized the idea for the review and the
conceptual framework and authored the manuscript with scientific
support, authoring contribution, and intellectual input of O.T., P.G., S.L.,
and M.P. All authors critically revised the text as well. M.N. and S.L.
generated figures.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Competing interests
All authors,includingM.N., O.T., P.G., S.L., and M.P., declare no competing
(financial or non-financial) interests.
Additional information
Correspondence and requests for materials should be addressed to
Maxim Noeparast.
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