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•  JoeGozgit• RibonTherapeutics

PARP7NegativelyRegulatestheTypeIInterferonResponseinCancerCellsanditsInhibitionLeadstoTumorRegression

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Disclosure Statement

•  IamanemployeeandshareholderofRibonTherapeutics

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Not All PARPs Are Alike – Outside of PolyPARPs the PARP Family Is Unexplored for Therapeutic Development

•  PARPfamilyconsistsof17members•  Threesubfamiliesbasedoncatalyticactivity(polyPARPs,monoPARPsandinactive)

•  PARPsregulatetheircellularfunctionbymodifyingtargetproteinswithADP-ribose

•  monoPARPstransferasingleunitofADP-riboseontotheirsubstrateswherepolyPARPsattachpolymersofADP-riboseunits

•  PARP7isamonoPARP•  Targetgeneofthearylhydrocarbonreceptor(AHR)thatcanbeinducedbycancerrelevantstresses(e.g.chemicalsincigarettesmoke)

•  Genelocusisamplifiedincancerswithstrongsmokingassociation(e.g.,squamouscellcarcinomaofthelung(SCCL),esophagealandheadandnecksquamouscancers)

Vyas,Changet.al.NatureComm.2013

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PARP7isfrequentlyamplifiedincancer

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AberrantnucleicacidscanbedetectedbyvarioussensingmechanismsincludingcGAS/STINGandRIG-I

PARP7 Acts as a Brake in Cytosolic Nucleic Acid Sensing and the Type I Interferon (IFN) Response

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TypeIIFNsignalingplaysakeyroleinantitumorimmunitybyinducingbothinnateandadaptive

immunemechanisms

Zitvogel-NatureRevCancer-2015

•  Hypothesis:HighlevelsofPARP7intumorsblocksinterferonproductionresultinginanimmunosuppressiveenvironment

•  Targetinganegativeregulator“brake”ofTypeIIFNsignalingisanoveltherapeuticstrategyincancer

PARP7hasbeenreportedtonegativelyregulatetheTypeIresponsebyinteractingwithTBK1duringviralinfection

(Yamada-NatImmunol-2016)

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RBN-2397 is a Potent and Selective Inhibitor of PARP7

•  RBN-2397isapotentinhibitorofPARP7•  BindstoPARP7intheNAD+bindingpocketwithkeyinteractionsinadenosinesub-pocketdrivingpotencyandselectivity

•  Sub-nanomolarbiochemicalactivity•  PotentinhibitionofcellularMARylation

•  RBN-2397displaysselectivitytoPARP7•  >50-foldselectivevs.PARPfamily•  Noinhibitioninkinasepanel(1µM)

•  Drug-likepropertiessupportoraldosinginhumans

•  FirstinhumanPhaseImulti-centerclinicaltrialisunderway(NCT04053673)

StructureofRBN-2397

Co-crystalstructureofRBN-2397boundtoPARP12/7

PARP12wasusedasasurrogateforPARP7.FourlabeledresiduesweremutatedfromPARP12tomatchthePARP7sequence.

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RBN-2397 Restores Cytosolic Nucleic Acid Sensing in the Mouse CT26 Cancer Cell Line

0.1 1 10 100 10000

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InductionofCXCL10mRNA

RBN-2397restoresTypeIIFNresponseinCT26cells

InductionofpSTAT1

•  RestorationofTypeIIFNresponseismeasuredbyanincreaseinSTAT1phosphorylationandinterferonstimulatedgenes(ISGs)

PARP7inhibition“releasesthebrake”oncytosolicnucleicacidsensingandinducesTypeIIFNs

e.g.CXCL10

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RBN-2397 Induces Tumor-Specific Adaptive Immune Memory in CT26 Syngeneic Model with Durable Complete Responses

CT26re-challenge 4T1re-challenge

•  OncedailyoraldosingofRBN-2397inCT26tumor-bearingBALB/cmice

•  Tumor-freemiceweremonitoredfor60days

•  Tumor-freemicere-challengedwithCT26andsubsequently4T1cells

•  Alltumor-freemicerejectedCT26cellsbutnot4T1,demonstratinginductionoftumor-specificadaptiveimmunememory

PrimaryEfficacy:RBN-2397inducesdurableregressions Re-challengeoftumor-freemice:RejectionofCT26cells

TF:TumorfreemiceAllgroupsco-dosedwithABT

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RBN-2397 Induces Type I IFN Signaling and Enhances Immune Markers in CT26 Tumors

Increase in GrzB on CD8 T cells

RBN-2397showsexposure-dependenteffectsonPDmarkersinCT26tumors

RBN-2397enhancesantigenpresentationandTcellactivationintumor-infiltratingimmunecells

IncreaseinMHCIIonDC1cells IncreaseinCD86onDC1cells

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RBN-2397increasestumorantigen-specificTcells:IncreaseinthenumberofsplenicTcellsproducingIFN-γin

responsetotheCT26antigen

CT26tumor-bearingmiceadministeredassingleoraldose.

CT26tumor-bearingmicedosedwithRBN-2397100mg/kg+ABTfor15Days.

CT26tumor-bearingmicedosedwithRBN-2397500mg/kg.Tumorscollectedondays3,6&12.

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Combination of Anti-PD1 with RBN-2397 Increases the Number of Tumor-Free Mice in the CT26 Syngeneic Model

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IncreaseinmarkersofimmunefeedbackregulationfollowingtreatmentwithRBN-2397inCT26

Enhancedantitumorimmunitywithcombinationwithanti-PD1inCT26

IncreaseinPD-L1onmacrophages

IncreaseinLAG3onCD8Tcells

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CRISPR-Cas9 Used to Ablate either TBK1 or IFNAR1 in CT26 Cells to Investigate the Mechanism of Action of RBN-2397

ModelofPARP7insuppressingTypeIIFNsignaling

•  TBK1knockout(KO)preventsbothIRF3&STAT1phosphorylationbyRBN-2397•  IFNAR1KOpreventsSTAT1phosphorylationbyRBN-2397

TBK1KO

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IFNAR1KO

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Tumorcells

CT26WTandKOcellstreatedwithRBN-2397

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Tumor-derived Interferon Is Key for Antitumor Activity of RBN-2397

•  AblationoftumorTBK1nearlyeliminatestheantitumoractivityofRBN-2397intheCT26tumormodel

•  Tumor-derivedIFN-βisthesourceoftheinnateimmuneactivationandcrucialforRBN-2397mediatedantitumorresponse

Allgroupsco-dosedwithABTDONOTPOST

•  NoPARP7i-mediatedIFN-βrelease•  Noeffectsoncancerorimmunecells

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Tumorcell

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•  IFNAR1KOinitiallyattenuatesantitumoractivityofRBN-2397,butasubsetoftumorsstartrespondingafterDay12

•  IFNAR1KOdoesnotpreventIRF3phosphorylationbyRBN-2397•  SuggestsonsetofantitumorimmunityaroundDay12,induced

byeffectsoftumor-derivedIFN-βonimmunecells

IFNAR1 Knockout in CT26 Tumor Cells Partially Attenuates Antitumor Activity of RBN-2397 in the CT26 Tumor Model

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•  NoISGs•  Notumorintrinsiceffect

•  Immuneresponse

Tumorcells

Immunecell

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•  Dosingofanti-IFNAR1ABonthebackgroundofCT26IFNAR1KOtumorspreventstheantitumoractivityofRBN-2397

•  SuggestscontributionofimmunesystemthroughactivationofIFN-βsignalinginimmunecells

•  Tumor-producedTypeIIFNbyRBN-2397playsamajorroleinthedevelopmentofdurableantitumorimmunity

Allgroupsco-dosedwithABT

IFNAR1 Blockade on Tumor and Immune Cells Is Necessary to Prevent Antitumor Activity of RBN-2397 in the CT26 Tumor Model

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•  NoISGs•  Notumorintrinsiceffect

•  Immuneresponse

Tumorcells

Immunecell

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RBN-2397 Shows Cancer Cell Autonomous Effects and Restores Type I IFN Signaling in Human Cancer Cell Lines

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Eachdotrepresentsacancercellline6dayassay

SubsetofcancercelllinesexhibitdependencyonPARP7forproliferation

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•  RespondercelllinesareenrichedforhighexpressionofgenesinvolvedinTypeIIFNresponse

crControl crPARP70.0

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crControlcrPARP7P < 0.0001

RBN-2397inductionofpSTAT1

DependencyonPARP7forproliferationandsuppressionofTypeIIFNsignalinginNSCLCNCI-H1373cells

Proliferation:RBN-2397GI50

Proliferation:PARP7KO

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TranscriptionalchangesbyRBN-2397:IncreaseinmultipleISGs

GenesenrichedforTypeIIFN&viralresponse

PARP7inhibitor

PARP1inhibitor

NonsignificantSignificant

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CRISPRi/a Screen Highlights Innate Immune Response Genes in Driving RBN-2397 Activity in NCI-H1373 Cells

SignificantenrichmentofgenesinvolvedininnateimmuneresponsethataffectRBN-2397activity

ComparisonofCRISPRi/aphenotypescoreshighlightsgeneswithopposingfunctionalityuponRBN-2397treatment

GeneticscreenusingwholegenomeCRISPRi/alibraries•  CRISPRi:interferencetosilencegeneexpression•  CRISPRa:activationtoincreasegeneexpression

EffectsofindividualgenesonRBN-2397activity•  Resistancewhensilenced•  Sensitivitywhenactivated

GSEAofgenesthataffectRBN-2397activity•  Resistancewhensilenced•  Sensitivitywhenactivated

GSEA:Genesetenrichmentanalysis

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RBN-2397 Shows Antitumor Activity in Human Cancer Cell Lines that Show Induction of ISGs

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TranscriptomicchangesbyRBN-2397mRNAlevels(log2foldchange)

RBN-2397cellproliferation

ReactivationoftumorTypeIIFNsignalingisamajordeterminantforRBN-2397antitumoractivity

NoincreaseinISGs

IncreaseinISGs

= =

Noantitumoractivity

Antitumoractivity(50-100%TGI)•  Lungcancer•  Pancreaticcancer•  Oralsquamouscancer

RBN-2397efficacyinCB17SCIDxenograftmousemodel(*)

RBN-2397causescompleteregressionsinNCI-H1373xenografts

•  OncedailyoraldosingofRBN-2397inCB17SCIDmicewithNCI-H1373xenografts

•  Dose-dependenteffectsontumorgrowth•  Tumorregressionatdoselevelsof≥30mg/kg

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RBN-2397 – A Novel Cancer Therapeutic Being Tested in Clinical Trials

•  DiscoveredfirstpotentandselectivePARP7inhibitor•  Novelfirst-in-classtherapy

•  PARP7isanoveltherapeutictargetanditsinhibitioninducesbothantitumorimmunityandcancercellautonomouseffects• Increasedsignalingtotheimmunesystem• Developmentofimmunememory• Arrestofcancercellproliferationandtumorregression• AntitumoractivityinmultiplecancertypeswherereactivationofTypeIIFNsignalingisobserved

•  IdentifiedPARP7asafundamentalregulatorofintrinsicstresssupportpathwaysandanoveltumorvulnerabilityincancercells

•  FirstinhumanPhaseImulti-centerclinicaltrialunderway(NCT04053673)

Completeregressionsandantitumorimmunityasasingleagent

IFNUncloaksthetumorcellsandrecruitsimmunecells

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Acknowledgements

VictoriaRichonAndySantospagoLaurieSchenkelRichardSchroederPrashantShambharkarJeffSongTadStewartKerrenSwingerLukeUtleyZachareniaVarsamisMelissaVasbinderTimWigleJodieWong

RyanAboEllenBambergDanielleBlackwellRichardBushellAnneCheungW.DavidChurchLisaClearyDavidCordoBryanDorseyJenniferDowningJosephGozgitLinetteGreyBinGuiHeikeKeilhackPeterKimDanielleKnightKaikoKunii

TeamRibon:KevinKuntzKristyKuplast-BarrJenkinsLemeraChangLiuAlvinLuAhmedMadyChristinaMajerKristenMcEachernMaeganMikulaElenaMinissaleJasonMoJenniferMolinaSunainaNayakMarioNiepelSudhaParasuramanNicholasPerlYueRen

PaulChangLeeKrausTimothyMitchison

JamesAudiaLarryLaskyPatriciaRao

FoundersandAdvisors:

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