RASopathies...The RASopathies Noonan 1:1000 to 2500 BRAF CBL KRAS LZTR1 MAP2K1 (MEK1) NRAS PTPN11 RAF1 RASA2 RIT1 SOS1 SOS2 NSML/ LEOPARD BRAF PTPN11 RAF1 Legius SPRED1 NF1 1:3000

RASopathies

2ndAnnualRAS-TargetedDrugDevelopmentSummit

WorkshopF

9/14/2020

BethStronach,PhD,RASopathiesNet

BruceD.Gelb,MD,IcahnSchoolofMedicineatMountSinai

PhotosbyRickGuidottiofPOSITIVEEXPOSURE“Changehowyousee,Seehowyouchange”

W

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Hi,I’mSam

Introductions

BethStronach,LisaSchoyer,ElisabethParker,LisaSchill

Iusedtostudytissueclosureinfruitflies!

MyRoleswithRASNet:

Co-organizingRASopathiessymposia

Writingcontent-website,newsletters,grantapplications

Curatingresearchpublicationsandopportunities

TweetingforRASNet@rasopathiesnet

AdvocatingforRASopathiesresearchandtreatmentsatmeetings

co-presenter

Dr.BruceGelb

MoreaboutBruce

S  Physician-Scientist

S  TrainedinPediatricCardiology

S  MolecularGenetics

S  IcahnSchoolofMedicineatMountSinai(NYC)

S  Director,MindichChildHealthandDevelopmentInstitute

S  Co-Direct,CardiovascularGeneticsProgram

Topicsfortoday

S  RASopathysyndromes,prevalence,manifestations

S  RASopathymutationscomparedwithRAScancermutations

S  GettingtotreatmentsforRASopathies

S  Targetsandtreatmentconsiderations

S  Preclinicalmodels

S  Endpoints

S  Whathaveearly/smallclinicaltrialstoldus?

Topic1RASopathysyndromes

Prevalenceandmanifestations

InmemoryofDr.JacquelineNoonan

Pediatriccardiologistwhodescribedapediatriccohort,inthe1960’s,withcongenitalheartdefectsandothersharedtraits

Thisdisorderwaslatercoined“Noonansyndrome”

2021marksthe20thanniversaryofthepublicationofthefirstNSgene,PTPN11(SHP2)

AssociationofCostellosyndromewithHRAS,Cardio-facio-cutaneoussyndromewithBRAF,andNeurofibromatosis-1withaRAS-Gap,ledtotheinsightthatgermlinealterationofRAS-MAPKsignalingunderliesagroupofneurodevelopmentaldisorders…

TheRASopathies

Noonan1:1000to2500

BRAFCBL

KRASLZTR1MAP2K1(MEK1)

NRASPTPN11

RAF1RASA2RIT1

SOS1SOS2

NSML/LEOPARDBRAF

PTPN11RAF1

LegiusSPRED1

NF11:3000

NF1

Costello1:390,000

HRAS

CFC1:150,000

KRASBRAF

MAP2K1(MEK1)MAP2K2(MEK2)

MolecularGeneticsandPrevalence

inactive

Nucleus

MEK

ERK

Ras-GTP

RTK

SHP2(PTPN11)CBL

SHCGRB2 SOS

SHOC2

PPP1CB

Ras-GDP

RAF

Neurofibromin

SPRED1

CM-AVMRASA1

active

RASA1

NSLHSHOC2

PPP1CB

LZTR1

RASA2

Syndromes

CostelloCS

Cardio-facio-cutaneousCFC

Legius/NF1-likeLS

Neurofibromatosis1NF1

NoonanandNoonan-likeNS

NSwithmultiplelentiginesNSML

NSwithlooseanagenhairNSLH

Capillarymalformation-arteriovenousmalformationCM-AVM

Aokietal,2015,JHumGen

Castel,Rauen,McCormick,2020,NatReviews

CausalMechanisms

Howdothesyndromesmanifest?

S  Shortstature

S  Characteristicfacialfeatures

S  Developmentaldelay

S  Congenitalheartandvalvedefects

S  Gastrointestinaldysfunction

S  Neurocognitiveissues,ADHD

S  Bleedingandlymphaticabnormalities

S  Lowmuscletone

S  Pain

S  Hypertrophiccardiomyopathy

S  Skinandhairanomalies

S  Seizures

S  Cancers

Jindaletal.,2015,DiseaseModels&Mechanisms

HypertrophicCardiomyopathyOutcomes

Wilkinsonetal.,AmHeartJ2012

Discussion

Topic2RASopathyvs.Cancer

mutations

AnalysisofcancerandRASopathygeneticdatabasesrevealsthat

~19%ofallcancercases&~4%ofdevelopmentaldisorders

containRasmutations.

EstimatingRASmutationfrequency

Prior,2020,inpress

Prior,Hood,Hartley,2020,CancerResearch

RASopathyvs.RAScancermutations

IanA.Prior,RasVariantBiologyandContributionstoHumanDisease,inpressSource:NSEuroNetdatabase

RASfamilyalignment

Khan,Rhett,O’Bryan,2020,BBAMolCellRes

Rasopathymutations

RASopathyassociatedcancers

S  Rhabdomyosarcoma

S  HematologicJMML,AML,ALL

S  Neuroblastoma

S  Glioma

S  Neurofibroma

S  Malignantperipheralnervesheathtumor

S  Bladdercancer

SomaticRAScancers

S  HeadandneckSCC

S  Lungadenocarcinoma

S  Cutaneousmelanoma

S  Thyroidcarcinoma

S  Pancreaticductaladenocarcinoma

S  Colorectaladenocarcinoma

S  Bladderurothelialadenocarcinoma

RASopathyvs.cancermutations

PTP

C-SH2

N-SH2

SHP2

PTP

C-SH2

N-SH2

SubstitutionsinNoonansyndrome

NoonanSyndromevs.Cancer

NoonanSyndromevs.Cancer

ImmunocomplexPhosphataseAssays

NoonanSyndromevs.Cancer

K76 SHP-2

D308 SHP-2

RTK

Growth Factor

Ras-GDP

Ras-GTP

Phenotype Embryonic Lethal

K76 SHP-2 Ras-GTP

GM-CFS

GMRb

GMRa

Proliferation

D308 SHP-2Ras-GDP

NoonanSyndrome Cancer

RASopathyvs.cancermutations

Jindaletal,2017,PNAS

Castel,Rauen,McCormick,2020,NatReviews

TheHapplehypothesis

RASopathyvs.Cancermutations

Ras:BeatingheartofcancerbyDarrylMcConnell

Discussion

Break

Topic3GettingtoTreatments:

Targetsandtreatmentconsiderations

TargetsandTreatment

S  Manymoleculartargets

S  Commonfeature:HyperactiveRAS/MAPKsignaling(autosomaldominantGOF,haploinsufficientorLOF)

S  Multi-systempathology

S  Nocurrenttreatmentsorcuresbeyondsymptommanagement

Treatmentconsiderations

S  Turnvolumedown,notoff

S  Multi-system,multi-organ

S  Timingandduration

S  Minimalchanceofescapethroughmutationselection

S  Somepotentialindicationsnotlife-threatening

S  Applicabilityofsideeffectprofilefrompatientswithcancer?

TreatmentconsiderationsIntegrins

ESR

P

GgGb

BAD

DAG

GTP

EPAC

B-Raf

Raf1

A-Raf

SOS

MEKK1

P

IkBa

NF-kB

IkBDegradation

NF-kB

P

P

P

Rac

FAKSHC

GRB2

EGFRSignaling

P

P

P

STAT1/3c-Myc

c-MaxP P P

PP

P

P

P P

P P

P CREB

c-Fos

c-Jun

MLCKFAK

P

P

P

P

P

PHistone

H3PHMG14

P

KSR

SHCPYK2

Src

UBF

p53Elk1

Pax6

PKA

MNK1/2

Ca2+

PLCb

PAK

MKP

VHR

cAMP

Rap1

PI3K

PP1/PP2

BRF1 GeneExpression

Cell Survival

Translation

Translation

Proliferation

Tumorigenesis

Differentiation

Development

Cell Survival

Migration

Raf

EGFR

Hormones,

Neurotransmitters

SOS

ZAP70

PKC

SOS

P

Gene

Expression

SRFP

EtsP

Ga

Gen

eE

xp

ressio

n

MEK1/2

MP1

ERK 1/2

MSK1/2

SAP1/2

G

P

C

R

N

C

p90RSK

IKKa

TGR-CD3Complex

AgECM

CaC

n

Ca2+CytokinesGrowth

Factors

RTK

AC

LAT

ATF1

Calpain

c-Raf

GRB2

PLCg

RasGTP

2009ProteinLounge.comC

Treatmentconsiderations

Goyal,NatGenetics2017

Treatmentconsiderations

Goyal,NatGenetics2017

Treatmentoptions

S  Smallmoleculeinhibitors[RAS,RAF,MEK,ERK,SHP2,SOS1]

S  RNAsilencing

S  Degradation

S  Genetherapy[NF]

S  Geneediting/Baseediting

S  Othercellularvulnerabilities

Topic3GettingtoTreatments:

PreclinicalModels

PreclinicalModels

S  Cell-based

S  Inducedpluripotentstemcell-derivedcells(e.g.,cardiomyocytes)

S  Animal

S  Fruitfly

S  Zebrafish

S  Mouse

S  Worm

PreclinicalRaf1Mouse

Wuetal.,JClinInvest2011

PreclinicalRaf1Mouse

Wuetal.,JClinInvest2011

S  MAPKsignaling

S  IncreasedErkactivation

S  Nochangeinp38orJnk

S  Mekinhibitor(PD0325901)

S  6-weektreatmentsfrom4weeksofage

S  Rescuedthehypertrophiccardiomyopathy

BirthDefectsResearch,2020

Methods:RNAormorpholinoinjectionNf1KO,transgeneinsertionHighthroughputscreening

Phenotypicassays:

Jindaletal.DiseaseModels&Mechanisms(2015)8,769-782

Topic3GettingtoTreatments:

Endpoints

Naturalhistorystudy

Howdothesyndromesmanifest?

S  Shortstature

S  Developmentaldelay

S  Neurocognitiveissues,ADHD

S  Congenitalheartandvalvedefects

S  Gastrointestinaldysfunction

S  Cancers

S  Bleedingandlymphaticabnormalities

S  Lowmuscletone

S  Pain

S  Hypertrophiccardiomyopathy

S  Skinandhairanomalies

S  Seizures

NeurocognitiveImpairment

S  Treatable?

S  AnimalPTPN11modelsS  Fruitflies

S  NotdevelopmentalS  NormalizedwithSHP-2inhibitor

S  MouseS  NeurobehavioraldeficitsS  AmelioratedwithMEKinhibition

S  HumangeneticdataS  SOS1

S  NoonansyndromewithnormalneurodevelopmentS  Onlyexpressedinfetalbrain

Topic3GettingtoTreatments:

ProofofConceptTrials

Proofofconcepttrials

S  MEKinhibitorSelumetinibforNF1–associatedinoperableplexiformneurofibromas

S  Mosthaddurabletumorshrinkageandclinicalbenefit

S  RecentFDAapprovalforpediatriccases

S  A.Grossetal.2020NEJM[ph2trial]

S  Off-labeluseofMEKinhibitorTrametinibforRIT1-associatedHCMin2infants(13and14wksofage)

S  AssociatedwithreversalofHCMandvalvularobstructionover17monthsoftherapy

S  G.Andelfingeretal.2019JACC

TrametinibforHCM

LVMass Pro-BNP

TrametinibforLymphaticDisease

S  Lietal.,NatureMedicine2019S  Centralconductinglymphaticanomaly(CCLA)

S  ARAFgain-of-functionmissensevariantS  ComparableallelesinRAF1forNoonansyndrome

S  ZebrafishmodelS  RecapitulatedlymphaticphenotypeS  Treatedwithcobemitinib

TrametinibforLymphaticDisease

Before After

TrametinibforLymphaticDisease

Before After

Engagedadvocacygroups

Socialmediagroups

HOWCANWEHELP?

Discussion

MovingForward-WhatisfeasiblefortheRASopathies?

END

Moore,Rosenberg,McCormick,Malek,2020,NatRevDrugDiscov

●  Noonansyndrome(NS)[PTPN11,SOS1,RAF1,BRAF,KRAS,NRAS,(SHOC2),CBL,RRAS,

RIT1,(RASA2),SOS2,MAP3K8,SPRY1,MYST4,LZTR1,(A2ML1)]

●  Noonansyndromewithmultiplelentigines(NSML)[PTPN11,RAF1]>formerlyLEOPARDsyndrome

●  Noonan-likesyndromewithlooseanagenhair(NS-LH)[SHOC2,PPP1CB]>SHOC2(NS-LH1)

>PPP1CB(NS-LH2)

●  Cardio-facio-cutaneoussyndrome(CFC)[BRAF,MAP2K1,MAP2K2,KRAS]

●  Costellosyndrome(CS)[HRAS]

●  Neurofibromatosistype1(NF1)[NF1-neurofibromin]

●  Legiussyndrome/NF1-like(LS)[SPRED1]

●  Capillarymalformation-arteriovenousmalformationsyndrome(CM-AVM1)[RASA1]

●  Centralconductinglymphaticanomaly(CCLA)[ARAF]

RASopathySyndromeGenes