Alteration of the Signal Transduction Pathway in RASopathies as a

Basis of Targeted Therapeutic Drug Development

Made Ananda Krisna

, Yulia Ariani Aswin

Master’s programme in Biomedical Science, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No,6,Central

Jakarta Indonesia

Medical Biology Department, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

Keywords: Ras protein, Mitogen-activated protein kinase, ERK, RASopathy, molecular targeted therapy

Abstract: Ras/mitogen-activated protein kinase (MAPK) pathway is one of the most critical intracellular signalling

cascades, relaying the extracellular signal in the form of growth factor into specific responses. The primary

responses of Ras/MAPK pathway activation are cellular proliferation and differentiation. Therefore, a

mutation in genes encoding one of its components or regulators causes a severe developmental disorder.

RASopathy is a group of genetic syndromes originating from a germline mutation in the Ras/MAPK

pathway's regulators' genes encoding components. More than 20 genes are associated with and seven

syndromes included in RASopathy: Noonan, LEOPARD, neurofibromatosis type 1, CM-AVM, Costello,

cardio-facio-cutaneous, and Legius syndrome. Genotype-phenotype associations in RASopathy are

complicated, the mutation in one gene could result in different syndromes, while a mutation in different genes

could cause one syndrome. Molecular diagnostic at the genomic level is crucial in establishing the definitive

diagnosis and as the basis for targeted therapy. Several therapeutic agents target the MAPK pathway, but they

have been mainly utilized in malignancy cases in which aberrant MAPK pathway was detected. Research in

targeted therapeutic drug development in RASopathy is still limited, yet it is eminently needed for further

elaboration.

1 INTRODUCTION

RASopathy is a group of syndromic genetic diseases

due to germline mutation in genes encoding

components or regulators of the Ras/mitogen-

activated protein kinase (MAPK) signalling pathway.

(Rauen, 2013; Romano et al., 2010) This pathway

mediates the effects of growth factors and,

consequently, plays an essential role in the growth of

many cells and tissues. Ras is a GTPase protein

encoded by the RAS gene that works following

growth factor receptors activation, usually in tyrosine

kinase receptor (TKR). The MAPK pathway is one of

the most vital downstream signalling cascades of Ras.

Regulatory nuclear proteins are the most common

final target of this pathway most of which are

transcription factors, histones, and other proteins

having a role in the cell cycle, proliferation,

differentiation, and cellular apoptosis and

https://orcid.org/0000-0002-7681-7135

https://orcid.org/0000-0002-6640-8530

senescence. (Alberts et al., 2008; Morrison, 2012;

Plotnikov, Zehorai, Procaccia, & Seger, 2011)

Disruptions in the Ras/MAPK pathway will

predictably result in a severe developmental disorder,

either localized or systemic, such as what is found in

RASopathy syndromes. Each type of RASopathy has

distinctive characteristics, although there are still

overlapping pathologies amongst them. Some

common clinical manifestations observed in almost

all RASopathy syndromes are abnormalities in the

craniofacial region; malformation of the heart; skin,

muscles, and ocular findings; neurologic and

cognitive disorder; hypotonia; and a higher risk of

developing malignancy. (Rauen, 2013)

The cumulative incidence of all RASopathy

syndromes is 1 case in every 1000 live births. More

than 20 mutated genes had been identified in

RASopathies, and these genes encode proteins

directly involved in the Ras/MAPK signalling

318

Krisna, M. and Aswin, Y.

Alteration of the Signal Transduction Pathway in RASopathies as a Basis of Targeted Therapeutic Drug Development.

DOI: 10.5220/0010492003180329

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 318-329

ISBN: 978-989-758-499-2

pathway or their regulators. The relative position of

these proteins in the pathway affects the degree of

clinical severity: the more upstream its involvement

in the pathway, the more severe the phenotypes.

However, this correlation is not clear-cut as clinical

heterogeneity is commonly observed in one specific

RASopathy caused by q disruption in the same gene.

(Rauen, 2013; Tajan, Paccoud, Branka, Edouard, &

Yart, 2018) Several syndromes which belong to the

RASopathy group are 1) Neurofibromatosis type 1

(NF1); 2) Noonan syndrome (NS); 3) NS with

multiple lentigo or LEOPARD syndrome; 4)

Capillary malformation-arteriovenous malformation

(CM-AVM) syndrome; 5) Costello syndrome (CS);

6) Cardio-facio-cutaneous (CFC) syndrome; and 7)

Legius syndrome. These syndromes overlap with

each other in terms of clinical manifestations and

their causal gene. On the other hand, one specific

syndrome could also be caused by a mutation in

different genes. This occurs partly due to the high

degree of cross-linking in the MAPK pathway, and it

has a potential clinical impact, especially for targeted

therapy development. (Rauen, 2013; Wu-Chou et al.,

2018)

As with other syndromic diseases, each

RASopathy type is suspected based on the

constellation of signs and symptoms. However,

etiologic diagnosis to elucidate which gene is

affected, and the type of the mutation affecting it is

paramount. RASopathy syndromes caused by

different genes or different mutation in the same gene

might have distinct hereditary patterns and clinical

courses. This information needs to be addressed in a

genetic counselling session with the patients and their

families. Moreover, to know the exact genetic defect

is the first step in building the precise formula for

personalized medicine through targeted therapy or

even gene therapy. (Bai et al., 2019)

In this review, biomedical aspects of RASopathy,

starting from the Ras/MAPK pathway's role in

healthy to various genetic defects underlying each

syndrome, and how it is related to the latest and future

potential drug development in targeted therapy

RASopathy were assessed.

2 RAS AND MAPK SIGNALING

PATHWAY

The MAPK cascade comprises of four families, all of

which are well-characterized, including classical

MAPK known as ERK1/2, C-Jun N-terminal

kinase/stress-activated protein kinase (JNK/SAPK),

p38 kinase, and ERK 5. (Belov & Mohammadi, 2012;

Plotnikov et al., 2011) In each cascade three main

kinases are sequentially activated, including MAPK

kinase kinase (MAPKKK), MAPK kinase (MAPKK),

and MAP kinase (MAPK). To this date, there are at

least 17 MAPKKK, 8 MAPK, and 10 MAPK

identified in mammalian cells. (Morrison, 2012;

Zhang & Liu, 2002) As kinase is an enzyme catalyzing

phosphorylation reaction, this sequential activation

leads to phosphorylation of the regulatory proteins that

are the signalling pathway's final target. This protein

can be located in different subcellular locations, such

as cytoplasm, mitochondria, Golgi apparatus,

endoplasmic reticulum, and nucleus. However, the

most common and important target protein in the

MAPK signalling cascade is located in the nucleus and

functions as gene expression regulators, whether it is

a transcription factor, transcription activator/

suppressor, or protein modulating chromatin

remodelling. (Plotnikov et al., 2011)

2.1 ERK Pathway

The first and foremost identified MAPK pathway is

the ERK1/2 cascade, and therefore it has been the

benchmark for all other kinase cascades. This cascade

has an important role in transducing extracellular

signals mediated through various receptors,

especially the RTK. Phosphorylation of receptor upon

binding with its ligand provides a docking site for

other proteins. Usually, the docked protein is an

intermediary which promotes binding and subsequent

interaction of other proteins in its vicinity. Such

protein is known as an adaptor, and the most

important adaptor in the MAPK pathway is growth

factor receptor-bound protein 2 (Grb2). The Grb2

protein enables interaction between Ras protein and

its activator, the sevenless (SOS) protein which

functions as a guanine nucleotide exchange factor

(GEF). Ras protein superfamily was named after the

tissue and species from which it was first identified:

Rat Sarcoma factor. It is found in the plasma

membrane's cytoplasmic surface, anchored through a

covalent bond to its lipid moiety. The protein has an

intrinsic GTPase activity which hydrolyzes GTP into

GDP so that the activated form has a very short half-

life. Another protein serving as Ras regulator called

GTPase-activating protein (GAP) promotes Ras

GTPase activity. (Belov & Mohammadi, 2012;

Nandan & Yang, 2011; Zenonos & Kyprianou, 2013)

The activated Ras protein has its GDP dissociated

and prefers binding to GTP. This GTP-binding Ras

can recruit and activate MAPKKK proteins such as

Raf-1 and B-Raf. The exact mechanism for

Alteration of the Signal Transduction Pathway in RASopathies as a Basis of Targeted Therapeutic Drug Development

319

MAPKKK activation has not been defined yet.

However, either dimerization or phosphorylation may

be involved in the process. The MAPKKK protein

phosphorylates MAPKK, the MEK1/2 and it turns

phosphorylates ERK1/2 as the last kinase tier, the

MAPK. The phosphorylated ERK is translocated to

the nucleus and can bind either transcription factor

affecting gene expression or the DNA itself. The

affected genes usually encode for proteins promoting

cellular proliferation, differentiation and survival,

and preventing apoptosis. (Morrison, 2012; Plotnikov

et al., 2011)

2.2 JNK and p38 Pathway

These pathways are functionally different from the

ERK pathway because they operate when intra- or

extracellular stressors are present. Although both JNK

and p38 pathway has its particular protein in each

kinase tier, there is a substantial cross-talk between

them. This cross-talk is kept in check by other

proteins, the scaffold-like JNK-interacting proteins

(JIP) so those specific substrates for each pathway are

concentrated and well-compartmentalized.

Because the JNK pathway is the first to be

known as responding to cellular stress, it is also named

a stress-activated protein kinase (SAPK) pathway.

There are several notable distinctions between the

JNK and classical ERK pathway. First, its MAPKKK

can be activated by proteins having an intrinsic

GTPase activity other than Ras, such as Rac1 and

CDC42. Second, the MAPKKK activation can also be

achieved without those proteins' involvement but

rather directly stimulated by an adaptor (e.g. TRAF).

Third, all proteins at the MAPK level belong to the

JNK protein family, which has a threonine-proline-

tyrosine (TPY) motif in their active domains. The JNK

pathway is mainly detected in cells that respond

rapidly to stress, such as neurons, cells of the immune

system, and cells whose activity under the influence of

insulin. (Morrison, 2012; Plotnikov et al., 2011;

Zhang & Liu, 2002) The p38 pathway has its

particularity: as a MAPK, the p38 protein can undergo

autophosphorylation when it is near other molecules,

for example, certain adaptor protein (Tab1 and ZAP-

70) and an analogue of phosphatidylinositol.

(Morrison, 2012; Plotnikov et al., 2011)/

3 RASOPATHY SYNDROMES

Each syndromic disease classified as RASopathy

results from a defect of at least one gene encoding

signal transduction components in the MAPK

pathway or its regulators. The relationship between

the mutated gene and the resulting clinical syndrome

is depicted in Figure 1 as a dashed blue line.

3.1 Noonan Syndrome

Noonan syndrome is a congenital genetic disease with

a relatively high prevalence, the incidence of which is

1 in 1000 to 2500 live births. This syndrome has an

autosomal dominant inheritance pattern with

complete penetrance, but variable expressivity.

Several clinical characteristics can be found in

patients with NS, including the distinctive facial and

musculoskeletal features which include a large skull

with a narrow facial area, wide eyes, prominent

epicanthal folds, ptosis, down-slanting palpebral

fissure, low-set ear, as well as a short nose with a

depressed nasal bridge; thoracic deformity; and

congenital heart disease. (Romano et al., 2010)

Noonan syndrome is a type of RASopathy with

the most heterogeneous genetic defects. The mutated

gene and the type of mutation influence the

phenotype. Mutations in PTPN11, encoding the

enzyme tyrosine phosphatase, consistently show a

significant association with the incidence of thoracic

deformity, mild bleeding disorders, and distinctive

facial features and stature. The nature of the mutation

in that gene also influences the type of heart defect:

missense mutations are associated with a higher

likelihood of pulmonary stenosis and ASD, while a

lower likelihood of cardiomyopathy. Meanwhile,

mutations in SOS1, the gene coding for sons of

sevenless (SOS) protein, have phenotypic

characteristics of the integumentary system that

overlap with CFC syndrome, and mutations in the

SHOC2 gene are associated with different

phenotypes (i.e. mitral valve disorders, growth

hormone deficiency, hyperpigmented skin,

ichthyosis, and developmental disorders). The

Noonan syndrome caused by a KRAS mutation tends

to have a more severe phenotype with more

significant developmental and learning disorders.1

However, not a single phenotype is unique to either a

specific gene or type of mutation. The illustrates the

complexity of interactions between genes in the RAS

/ MAPK pathway. Other than the genes mentioned

above, there are several other causative genes for

Noonan syndrome (Table 1). However, there was no

identified mutation in any genes associated with the

RAS/MAPK pathway in a small number of cases.

(Bai et al., 2019)

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Table 1. Mutated genes and its prevalence in Noonan syndrome.

o Gene/protein Subcellular

effects

Characteristics of mutation Prevalence (%)

1 PTPN11/SHP-

Cytosolic

phosphatase

cutting

phosphotyrosyl

bond on activated

RTK and

indirectly

activating SOS-

activato

Conformational change causing

catalytic site exposure  ↑

phosphatase  ↑ MAPK activation

42 (Bertola et al., 2006)

58,3 (Chinton et al.,

2019)

60 (F. R. Lepri et al.,

2014)

2 SOS1/Sos As a Ras-

activating GEF

Mutation in autoinhibition sites

 ↑ GEF activity  ↑ RAS/MAPK

pathway activation

11 (Cessans et al., 2016)

18 (F. Lepri et al., 2011)

20 (Roberts et al., 2007)

3 KRAS and

NRAS/Ras

One of Ras

subtypes which

activates

MAPKKK tier

KRAS: ↑ GDP/GTP dissociation

rate  ↑ RAS GEF-independent

RAS activation* (Carta et al., 2006)

NRAS: de novo somatic

mutation  ↑ GAP-resistant RAS

activation (Cirstea et al., 2010)

1 (Cirstea et al., 2010)

4 RIT1/Ras

subfamily

idem ↑ ELK1 activity, a TF which

activates MAPK and activated by the

MAPK pathway(Aoki et al., 2013)

(positive c

cle feedback)

3.8

9 (Aoki et al., 2013)

5 RAF1 dan

BRAF/Raf

family

A type of

MAPKKK

RAF1: ↑ Raf activity

BRAF: ↑ Raf activity

15 (Kobayashi et al.,

2010)

1.7

AP2K1/MEK idem ↑ MEK kinase activit

4.2

*Based on structural analysis study digitally (Carta et al., 2006)

3.2 LEOPARD Syndrome

LEOPARD syndrome is also known as Noonan

syndrome with multiple lentigos. The name

"LEOPARD" is an acronym for its main

manifestations: multiple Lentigos,

Electrocardiographic abnormalities, Ocular

hypertelorism, Pulmonary stenosis, Abnormal

genitalia, developmental Retardation, and

sensorineural Deafness. The prevalence of

LEOPARD syndrome in the general population is

unknown. However, it is thought to be significantly

rare because to date, the total number of cases

reported in the publications is no more than 300

patients. (Martinez-Quintana & Rodriguez-Gonzalez,

2012; Sarkozy, Digilio, & Dallapiccola, 2008) This

syndrome is either has an autosomal dominant

inheritance pattern with complete penetrance or

occurred sporadically due to de novo germline

mutation. The main clinical manifestations based on

which the clinical diagnosis is made 1) characteristic

facial features such as hypertelorism, ear

malformations, and low ear with a folded helix; 2)

hypertrophic cardiomyopathy; and 3) café-au-lait

macules which are usually found on the face, neck

and upper part of the torso. Also, several other signs

and symptoms could be encountered in a person with

LEOPARD syndrome, such as thoracic deformity,

cryptorchidism, mild learning, and psychomotor skill

development disorders, and malignancy in the form

of juvenile myelomonocytic leukaemia. (Martinez-

Quintana & Rodriguez-Gonzalez, 2012)

Although the Leopard syndrome is genetically

heterogeneous, 95% of cases have mutations in one

of the following genes: PTPN11, RAF1, and BRAF.

However, as depicted in Table 1, these genes are the

causative genes for more than 50% of NS cases, while

mutations in BRAF are also found in a significant

number of CFC syndrome cases. (Martinez-Quintana

& Rodriguez-Gonzalez, 2012) (Table 2)

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Table 2. Mutated genes and its prevalence in LEOPARD syndrome

No Gene/protein Subcellular effects Characteristics of

mutation

Prevalence (%)

1 PTPN11/SHP-

Cytosolic phosphatase

cutting phosphotyrosyl

bond on activated RTK

and indirectly activating

SOS-activator

Mutation inactive catalytic

site  ↓ phosphatase  but

↑ MAPK activation

42 (Bertola et al.,

2006)

58,3 (Chinton,

Huckstadt, Moresco,

Gravina, & Obregon,

2019)

60 (F. R. Lepri et al.,

2014)

RAF1/Raf

family

A type of MAPKKK ↑ Raf activity

<5-10 (Carcavilla et

al., 2013)

BRAF/Raf

family

idem Limited ↑ Raf activity

<1-5 (Carcavilla et

al., 2013)

3.3 Neurofibromatosis Type 1

Neurofibromatosis-1 (NF-1) or Von

Recklinghausen's disease is one of the RASopathy

syndromes inherited with an autosomal dominant

inheritance pattern. (Upadhyaya & Cooper, 2012)

The prevalence of NF-1 reaches 1 every 3000 to 4000

individuals in the general population, based on

various studies from Europe and America States.

(Uusitalo et al., 2015) The diagnosis of NF-1 is made

based on clinical manifestations. Some of the most

commonly reported symptoms of NF-1 patients are

café-au-lait macules, neurofibromas of the skin or

oral mucosa, brownish (freckling) spot in the axillary

region, Lisch nodules on the iris, bone abnormalities,

and malignancy, particularly optic nerve gliomas,

astrocytoma, and schwannoma. (Upadhyaya &

Cooper, 2012) The cause of NF-1 is a mutation in the

NF1 gene that encodes the neurofibromin protein.

This protein functions as an activator of GTP-ase

which works on Ras: GTP-ase hydrolyzes GTP bound

to Ras for Ras to reinstate its inactive form. If there is

either a reduction in its level of expression or its

dysfunction, there will be overactivity of the Ras

signalling pathway and subsequent cell growth and

proliferation. (Bennett, Thomas, & Upadhyaya,

2009) Based on previous studies, 100% of patients

with NF-1 have mutations in the NF1 gene even with

heterogeneous types of mutations: it can be small

deletions, missense or nonsense mutations, as well as

splicing mutations. Mutations can accompany

mutations in NF1 in other genes, either the ones

which also play a role in the RAS/MAPK pathway

such as PTPN11 and BRAF, or those which are not,

such as P53. (Arima et al., 2017) Concurrent

mutations in PTPN11 and NF1 genes may indicate a

possibly different clinical entity known as

Neurofibromatosis-Noonan Syndrome (NFNS).

(Thiel et al., 2009; Wu-Chou et al., 2018) Meanwhile,

additional mutations, particularly in other oncogenes

(e.g. P53), increase a patient's probability with NF-1

developing a tumor. (Gottfried, Viskochil, &

Couldwell, 2010)

3.4 Capillary Malformation-arteriovenous

Malformation Syndrome

Capillary malformation-arteriovenous malformation

(CM-AVM) syndrome is a RASopathy syndrome

characterized by multifocal capillary malformations

that are typically found on the face and extremities.

This condition can also be accompanied by

arteriovenous malformations (AVM) and

arteriovenous fistulas. The AVM may occur in

various tissues, including the skin, muscle, bone, and

various internal organs, for instance, the heart and

brain. Consequently, if the AVM ruptures and

bleeding occurs, there will be life-threatening

complications. In general, this syndrome is inherited

with an autosomal dominant pattern. However, in 20-

30% of cases, pathogenic mutations occur de novo.

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Table 3. Mutated genes and its prevalence in CV-AVM syndrome

No Gene/protein Subcellular effects Characteristics of

mutation

Prevalence (%)

1 RASA1/RASA

Ras inactivation ↓ GTPase activity  ↑ Ras

activit

50 (Amyere et al.,

2017)

2 EPHB4/EphB4 RTK family which signals

through MAPK pathway to

exerts its effects

↑ MAPK pathway activation

3 Undetected - - 40

The mutated gene in CM-AVM syndrome can be

either the RASA1 gene or the EPHB4 gene (Table 3).

(Bayrak-Toydemir & Stevenson, 1993-2020) The

RASA1 protein is a negative regulator of Ras through

its activity as a GAP. (Kawasaki et al., 2014)

Meanwhile, the EphB4 protein is a member of the

RTK Eph family which plays a role in embryonic

capillaries morphogenesis. Physiologically, one of

the effects of binding between the EphB4 receptor

and its ligand is the suppression of VEGF-initiated

endothelial proliferation and migration through the

RAS/MAPK signalling cascade. (Kim et al., 2002;

Xiao et al., 2012)

3.5 Costello Syndrome

Costello syndrome's prevalence rate, one of the

RASopathy clinical syndromes, is 1 case per 300,000

live births. This syndrome generally occurs due to

heterogeneous de novo germline mutations in the

HRAS gene. In most cases, the HRAS mutation is

inherited from the father and correlates with an

increase in paternal age. However, in some cases,

somatic HRAS mutations were found. These

mutations lead to continuous activation of the Ras

protein, which causes dysregulation of cell growth

and development. The clinical diagnosis of Costello's

syndrome should be followed by genotyping that

shows the HRAS mutation. If no mutations are found,

the likely diagnosis is not Costello's syndrome, but

other RASopathy syndromes. (Gripp et al., 2019)

Phenotypically, Costello's syndrome has a wide

spectrum of clinical manifestations and may involve

multiple organ systems. In infancy, patients with this

syndrome will experience severe feeding difficulties

that cause growth and developmental problems

including stunting, intellectual disabilities, several

developmental disorders; distinctive facial features

(largemouth, thick lips, large nose tip), papillomas in

the face, and perianal area; generalized hypotonia;

excessive flexibility of the wrist and finger joints; and

involvement of the cardiovascular system in the form

of ventricular hypertrophy, pulmonary stenosis, and

arrhythmias. (Gripp et al., 2019)

3.6 Cardio-Facio-Cutaneous Syndrome

Cardio-facio-cutaneous (CFC) syndrome is

characterized by prominent heart abnormalities

(usually in the form of pulmonary stenosis with other

valves' dysplasia, septal defects, hypertrophic

cardiomyopathy, and arrhythmia), notable

craniofacial characteristics, and concomitant skin

abnormalities including xerosis, hyperkeratosis,

ichthyosis, keratosis pilaris, dermatitis, melanocytic

nevi, hemangioma, and palmoplantar hyperkeratosis.

Almost all patients with CFC syndrome suffered from

neurological and cognitive disorders. Besides, some

of them had significant abnormalities in one or

several other organ systems: musculoskeletal,

lymphatic, ocular, gastrointestinal, and endocrine

systems. Because the clinical manifestations of CFC

syndrome are very diverse, the definitive diagnosis is

established when there is clinical suspicion and

confirmed a pathogenic variant in one of the genes

associated with the syndrome, that is the BRAF,

MAP2K1/2, and KRAS genes. (Table 4) Mutations in

these genes are predominantly de novo and have an

autosomal dominant pattern of inheritance. (Rauen,

1993-2020)

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Table 4. Mutated genes and its prevalence in CFC syndrome. (Rauen, 1993-2020)

No Gene/protein Subcellular effects Characteristics of mutation Prevalence (%)

1 BRAF/BRAF Activates MAPKK

(MEK1/2)

↑ MAPKK phosphorylation 75

2 MAP2K1/2/M

EK1 dan 2

Activates MAPK

↑ MAPK phosphorylation

3 KRAS/KRAS Ras subtype ↓ intrinsic GTPase activity

 ↑ Ras activit

2-3

3.7 Legius Syndrome

The characteristic feature of Legius syndrome is

multiple café au lait macules but without the presence

of neurofibromas or any tumor manifestation as found

in NF-1. Other clinical manifestations that can be

encountered in patients with this syndrome are

intertriginous freckles, lipomas, macrocephaly, and

developmental or learning disorders. To date, only

less than 500 individuals with Legius syndrome have

been reported worldwide with confirmatory

molecular diagnostic laboratory examinations. The

inheritance pattern is autosomal dominant, and

children born to individuals with Legius syndrome

have a 50% chance of inheriting the pathogenic

variant. The only gene associated with the incidence

of this syndrome is the SPRED1 gene. The gene

encodes the Spred1 protein, which physiologically

functions as an inhibitor of the Raf1 kinase activity.

The pathogenic variant of SPRED1 in Legius

syndrome loses its physiological function resulting in

continuous activation of the Raf1 kinase and

increases downstream signalling from the MAPK

pathway. A similar pathophysiological mechanism is

Found in NF-1 with NF1 gene mutation, whose

product's function is very similar to Spred1 protein.

This explains the substantial clinical overlap between

Legius syndrome and NF-1. (Stevenson, Viskochil, &

Mao, 1993)

4 POTENTIAL TARGETED

THERAPEUTIC DRUG

DEVELOPMENT FOR

RASOPATHY

Despite the diverse genetic variations in

RASopathies, the impact of gene mutations in all

syndromes belonging to RASopathy cause an

increase or activation of the RAS/MAPK pathway.

Therefore, targeted therapy to the RAS/MAPK

pathway is predicted to have a role in managing

RASopathy (Figure 1, straight red lines). This

targeted therapy focuses on the downstream part of

the pathway to cover the full spectrum of

pathophysiology in the RASopathy syndromes. Some

of the MAPK pathway blockers include MEK

inhibitors (MAPKK tier), BRAF and Raf inhibitors

(MAPKKK tier), and an antagonist of RAS, the

farnesyltransferase inhibitors. However, the clinical

trials' target population evaluating these drugs was

cancer patients with proven mutations and

dysregulations in the MAPK pathway.

Although some RASopathy syndromes are

associated with an increased risk of developing

malignancy, not all patients with RASopathy exhibit

these clinical manifestations. This has resulted in

excluding most patients with RASopathy syndromes

from many clinical trials evaluating targeted therapy

for the MAPK pathway. (Gross et al., 2020)

Furthermore, most of the studies assessing the use of

inhibitors against components of the MAPK pathway

in RASopathy syndromes were preclinical studies

with the primary objective of explaining disease's

pathophysiological mechanisms, not determining the

effectiveness or efficacy of the inhibitors. However,

all trials reviewed in this article focused on evaluating

the RAS/MAPK pathway inhibitors' effectiveness for

RASopathy, although the majority of which were still

in the preclinical phase.

Ascota et al studied lovastatin therapy in mice

with NF1 mutation, and it was observed that the

attention deficit and spatial aspects of learning were

improved. Lovastatin is a statin drug that inhibits the

HMG-CoA reductase enzyme and acts as an inhibitor

of Ras isoprenylation. This post-translational

modification process is essential for normal Ras

protein function. (Acosta et al., 2011) On the other

hand, Lee et al used a different type of NS animal

model in which the genetic basis of mutation was the

overexpression of the PTPN11 allele. The mice

harboring the mutation increased excitatory synapse

function, deficits in long-term potentiation, and

spatial learning. After the MEK inhibitors

administration, all neurologic manifestations were

alleviated. (Lee et al., 2014) Although not as

prevalent as PTPN11 mutation, SOS1 mutation has

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also been implicated in NS (Table 1), and an animal

model bearing the mutations were either unviable or

having severe cardiac hypertrophy. Chen et al showed

that prenatal administration of MEK inhibitors to the

mutated mice prevented embryonal lethality and the

cardiac abnormalities that should have occurred

otherwise. (Chen et al., 2010)

Figure 1. RAS/MAPK pathway in RASopathy and potential targeted therapy. Its components and regulators implicated in

RASopathy are shown in elliptical boxes, and the dashed blue lines represent the association between a component/regulator

(if mutated) and each RASopathy syndrome. The red rectangle boxes are potential therapies, and the straight red lines connect

them to their specific targets in RAS/MAPK pathway

Other than NF-1 and NS, there is a relative

scarcity of study on other RASopathy syndromes. A

study by Inoue et al utilized transgenic mice with

BRAF gain-of-function mutation which are most

commonly seen in CFC syndrome, and it showed

consistent phenotypes of embryonic skeletal

abnormalities, lymphatic defects, heart defects, and

liver necrosis. These mutations are lethal in the

embryonic period. However, prenatal administration

of MEK inhibitors causes early embryo development

to return to normal. (Inoue et al., 2014) The Costello

syndrome experimental animal model with gain-of-

Function mutations in HRAS have been shown to

have increased activation of the ERK pathway and

cognitive deficits. In this study, the lovastatin effect

on the syndrome was evaluated. Unlike in the NF1

and NS experimental animal models, its

administration did not restore ERK signalling activity

to baseline level, and the cognitive deficits persisted.

There might have been pathophysiological

differences underlying cognitive deficits among

different syndromes and/or different mutations.

(Schreiber et al., 2017)

Although their numbers are limited, several

studies have demonstrated the pharmacological

potential of MEK inhibitors in human subjects.

Dombi et al. conducted a clinical trial on NF-1

patients with inoperable plexiform neurofibroma

(PN). This benign tumor originates from the myelin

sheath covering the nerves and causes chronic pain,

physical disability, and impaired motor function. The

MEK inhibitor selumetinib administration in this

group of patients significantly reduced tumor size,

reduced pain intensity, and improved motor function.

This study is the first breakthrough to demonstrate the

clinical usefulness of MAPK inhibitors in RASopathy

syndrome and provides preliminary evidence as a

basis for the treatment of other RASopathies (non-

NF-1) using these substances. The United States Food

and Drug Administration has granted permission to

use selumetinib as a PN therapy in NF-1 patients.

(Dombi et al., 2016)

For other RASopathies, as stated before, the

clinical trials were still limited to animal subjects. Wu

et al. used a mice model of NS with mutations in

RAF1 and administered MEK inhibitor PD032590

during the prenatal period. The administration of this

therapy prevented developmental disorders of the

heart. (Wu et al., 2011) Even though it has not been

approved for NS standard therapy, Andelfinger et al

Alteration of the Signal Transduction Pathway in RASopathies as a Basis of Targeted Therapeutic Drug Development

325

applied the MEK inhibitor trametinib to two

newborns with NS who developed heart failure due to

hypertrophic cardiomyopathy. In both of these

infants, myocardial hypertrophy experienced a

significant improvement (partial reversal) within 4

months after the first therapy. (Andelfinger et al.,

2019)

Clinical trials for MAPK-targeted therapy

pathways that have been carried out in the context of

cancer treatment show some substantial side effects

and resistance that results from the initiation of

negative feedback cycles. This can be a problem if

these targeted therapies are used in RASopathy

because the treatment will be continuous and given

for a lifetime. However, the therapeutic doses

required for RASopathy are predictably to be

significantly lower than the doses required to produce

cytotoxic effects in cancer cells. Hence, the likelihood

of side effects is also lower. An essential

consideration for RASopathy targeted therapy is that

most RASopathies result from germline mutations,

while most mutations in cancers are somatic.

Germline mutations underlying RASopathy cause a

homeostatic burden in the form of continuous and

stable tonic activation of the MAPK pathway since

early in life. Consequently, there is a possibility of

harmful regulatory mechanisms in each organ system

as an adaptive attempt to restore homeostasis.

Targeted therapy for the MAPK pathway can be

optimized if supplemented with therapy targeting

these specific adaptation mechanisms. Another

challenge in treating RASopathy using targeted

therapy is that the specific mutation underlying each

syndrome can modify sensitivity to MAPK pathway

inhibitors. For example, several types of mutations in

the PTPN11 gene in NS cause mutant SHP2 protein

production that is resistant to the allosteric inhibitor

of SHP2. Likewise, mutated RAF1 or MEK1 possibly

encode mutant protein resistant to target inhibitor

MEK therapy. (Gripp et al., 2020; Gross et al., 2020;

Tajan et al., 2018)

5 CONCLUSION

RASopathies are a group of syndromic genetic

disorders that result from germline mutations in genes

encoding components or regulators of the Ras /

MAPK signalling pathway. The syndromes included

in RASopathy are Noonan syndrome, LEOPARD

syndrome, neurofibromatosis type 1, CM-AVM

syndrome, Costello syndrome, CFC syndrome, and

Legius syndrome. Clinically, there are plenty of

overlapping signs and symptoms among the

syndromes so that diagnosis based solely on clinical

manifestations is exceptionally challenging. On the

other hand, except for Legius syndrome, genetic

mutations underlying each syndrome are vastly

overlapped to one and another. Therefore, targeted

therapy, especially those that work in the downstream

part of the RAS/MAPK signalling, could be a solution

in the clinical management of RASopathy. All

RASopathy syndromes have in common, both

genotypically and phenotypically, the MAPK

pathway's overactivity. However, treatment in the

form of an inhibitor of the MAPK pathway has been

more widely studied for cases of malignancies with

signalling aberrations in this pathway. Thus, there is

still an opportunity for research development in

specific treatment targeting components of the

MAPK pathway to treat RASopathy syndromes

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