Preclinical modeling of drug resistantacute lymphoblastic leukemia

Jean-PierreBourquin,MD-PhDDivisionof Oncology

Children‘s HospitalZürich

CRPPHumanHemato-Lymphatic Diseases

Refractory

Firstdiagnosis

DrugdevelopmentExp.TherapyInnovativeconceptse.g‚basket‘trials

Acute lymphoblastic leukemia - clinical context

Relapse

PredictivemarkersMolecularclassificationFunctionalunderstandingFunctionalclassification

15%

55%

100%

Needs

MechanismsAdaptivetreatments

Research

IntegrationofMulti-OmicsFunctionalProfiling

ModelempiryModeldefinedmoleculartypes

GeneticlandscapeofALL

Hematopoieticstemcell

Bprogenitor MatureBcell

Initiatinglesions(translocations)transcriptionfactorsKinases

Developmentalarrest(deletions)

IKZF1,PAX5,TCF3,EBF1

Cooperatinglesions(SNV,indels,others)CellcycleSignalingEpigeneticmechanisms

Leukemia

AnthonyMoorman,UKALL

MAINTENANCEINTENSIFICATIONINDUCTION

0

FirstlineALLchemo

Functionalapplicationofgenomicsstilllimitedintheclinic

Entitiesforexperimentaltreatment

day33 78 154

Very highriskMRD highatday 78MRDpos d33and IKZF1-plushypodiploidyMLL/AF4,TCF3/HLF

Genomics MinimalResidualDisease(MRD)

Patient-derived models

MSCLive ALL

DeadALL

In vivo testing

Small molecule library

Automated microscopy

MSC Primary ALL

Time(h): 0 24 48 120

- Customized drug libraries- Precise dose response curves- Combinatorials

Drugs Image analysis

ALL sample direct on MSC

Drug

resp

onse

pro

filin

gH

igh

risk

ALL

- Drug testing- Functional genetics- Microenvironment

Relevant cohorts

- First line trial- Relapse trial- Phase I setting - Refractory disease

Patient derived xenograft (PDX)

biobank

NSG mice

Frismantas etalBlood2017,inpress

Number of events (patient samples)

Num

ber o

f eve

nts

(PD

X sa

mpl

es)

BCP-ALL T-ALL

% match0255075100

0.02.55.07.510.00.02.55.07.5 10.0

2.5

5.0

7.5

10.0

Leukemia PDXare genetically stabletargeted sequencing 52genes,SNVsand indels

Frismantas etalBlood2017PamDobay,bioinformatics,IntReALL panel,C.Eckert,AMoorman,J.IrvingFP7

Drugresponse profiling identifies phenotypes

T-ALLsensitiveto Dasatinib

BCP-ALL

T-ALL

Frismantas etalBlood2017

IC50

log[

nM]

0

1

2

3

4***

BCR-ABL1

NUP214-ABL1TCF3-PBX1

BCP-ALL T-ALLinvitro

Ahighly dasatinib sensitiveT-ALLsubset

SR VHR Relapse SR VHR RelapseFrismantas etalBlood2017,inpress

dasatinib inT-ALL:validationIC

50 lo

g[nM

]

0

1

2

3

Dasatinib

40%

Day 0 Day 90

RefractoryT-ALLadultpatientDasatinib +Asparaginase

IndependentcohortJeffreyTyner,OSHU

Frismantas etalBlood2017,inpress

0 1 2 30

20

40

60

80

100

120

Control

0 1 2 30

20

40

60

80

100

120

Control

BCPALL

T-ALL

Concentrationlog[nM]

%Livingcells

Pre-T-ALL

TCF3-HLF

0 1 2 30

20

40

60

80

100

120

Control 0 1 2 30

20

40

60

80

100

120

Control

Cortical-T-ALL Mature-T-ALL

0 1 2 30

20

40

60

80

100

120

Control

MLL-AF4

AsubsetofALLrespondstoBCL2inhibitionwithvenetoclax

ABBVIEPediatric PhaseIstudy,Protocol+PIPin2017single agent and combination

Celldeathsignal

BH3-only

Apoptosis

BCL-2

BCL-XL

ABT-199venetoclax

bHLHTransactivationdomainTCF3Chr19

DNAbindingdomain

breakpoint

TCF3-HLFt(17;19)HLFbZIP

notcurable

PBX1HD

TCF3-PBX1t(1;19) favorable

GeneticlandscapestableinPDX

TCF3-HLF:aparadigmofresistantdisease

U.FischerA.Rinaldi etal,NatureGenetics 2015

Cooperativelesions(SNV)RASpathway

Hematopoieticstemcell

MatureBcell

TCF3-HLFDELPAX5,BTG1,VPREB1

Leukemia

Bprogenitor

−1 0 1 2 3 4

CYTARABINEGEMCITABINE

MERCAPTOPURINEMETHOTREXATE

THIOGUANINEDEXAMETHASONE

VINCRISTINEAT9283

BARASERTIBBI−2536

BX795TEMSIROLIMUS

TORIN−1DASATINIB

LESTAURTINIBXL228

DOXORUBICINETOPOSIDEIDARUBICIN

MITOXANTRONETOPOTECAN

BORTEZOMIBABT−199

Drugs

log IC50

E2A−HLF_T(17;19)

E2A−PBX1_ T(1;19)

Others

ZH89

ZH96

Risk

HR

MR

Others

SRZH89

ZH96

ZH96TCF3-HLF

TCF3-HLF

TCF3-PBX1

primary refractory ALL

Celldeathsignal

BH3-only

Apoptosis

BCL-2

BCL-XL

ABT-199venetoclax

A.Rinaldi,Fischeretal,NatureGenetics 2015

TCF3-HLF ALLTreatment

VEN+Dexa +VincristineFollow up leukemia

ControlVenetoclax

Treatment blocks

DEX+VCR

DEX+VCR+Venetoclax

0 20 40 60 80 100 1200

20

40

60

80

100

230

0 20 40 60 80 100 1200

20

40

60

80

100

2300 20 40 60 80 100 1200

20

40

60

80

100

230

0 20 40 60 80 100 1200

20

40

60

80

100

230hCD4

5+hC

D19+

inPB[%

]

Timeposttransplantation[days]

Survival[%

]

V.Frismantas and A.Rinaldi,Fischeretal,NatureGenetics 2015,Blood2017

3Drugminiinduction+VENcontrolsTCF3-HLFALL

SR03SR06

SR04

MNR-B03

VHR1016

959SR13

VHR03

MNR-B01

MNR-B10SR19

VHR27

MNR-B14

VHR02HR05

SR22B17SR02

SR01SR10

HR1215

00

VHR07HR03

VHR25SR08

SR12 5a

MNR-B09SR11

HR11SR14

MNR-B04 2a 7a

VHR28SR0911

c13

a

VHR06

VHR12 6a

VHR04

VHR0117

505

VHR23

MNR-B17SR17

SR21

VHR150

500

1000

1500

2000

SM IC

50 (n

M)

~20%IC50<100nM

SelectiveactivityoftheSMACmimeticbirinapant

VHR10

0 20 40 60 80 1000

20

40

60

80 ControlTreated

Day

% h

CD45

/hCD

19+

Denovo resistant (highMRDload)

B-cell precursor ALL

SMACmimeticripoptosomeapoptosis

necroptosomenecroptosis

RIP1

McCombS,Aguadé-GorgorióJ,etal.Science Translational Medicine,2016

Functional genomics:lentiviral CRISPRto target RIP1

McCombS,Aguadé-GorgorióJ,etal.Science Translational Medicine,2016

Selection with SMbirinapant

McCombS,Aguadé-GorgorióJ,etal.Science Translational Medicine,2016

SM:simultaneousapoptosisandnecroptosis

Foldchangeknockout

Biri

napa

ntLC

L161

Velc

ade

Vinc

ristin

eD

oxor

ubic

inA

BT1

99O

bato

clax

Topo

isom

eras

eEt

opos

ide

JQ1

Dex

amet

haso

neC

ytar

abin

e6-

thio

guan

ine

6-m

erca

ptop

urin

e

1

10

100

1000

10000 VHR10 ConVHR10 LC-Rip1MNRB03 ConMNRB03 LC-Rip1

IC50

(nM

)

Detection Limit

RIP1dependentdeathisnotexploitedbycurrentchemotherapeuticagents

Pat1controlPat1RIP1KOPat2controlPat2RIP1KO

McCombS,Aguadé-GorgorióJ,etal.Science Translational Medicine,2016

Preclinical modeling with PDX:

• Modelingand definition of clinically relevantcohorts

• Mechanistic studies inthe realcellular context

• Exvivoscreening,drug activity profiling,functional omics

• Proofof concept for new combination invivo

• Correlation with clinical activity of new agents

Leukemia Research GroupSeniorScientist: BeatBornhauser (CH)Biofinformatics: MariaPamelaDobay (CH)Postdocs LenaHarder(DE)

YunHuang(China)JuliaAguadé(SP)

PhD-Students:Danaelle Capron(F)Viktoras Frismantas (Lituania)VirginiaRodriguezMartinez(SP)BriceMouttet (CH)MD-PhDProgramCatarinaMezzatesta (IT)

MasterBiologie/MD: Seok YunLee(CH)GamzeAlteplin(TK)

: FabianRietmann (CH)Technician: Blerim Marovca (CH)

SilviaJenni(CH)Biobank/Labmanagement IrinaBanzola (IT)Alumni: LauraBonapace,AnnaRinaldi,PaulinaMirkowska,JeannetteBoutter,Maike Schmitz,ScottMcComb,OrrinPailKinderspitalJoelleTchinda,BeatSchaefer,FelixNiggli,NicoleBodmer

InternationalBFM-SGMartinStanulla,MartinSchrappeArnt Borkhardt,UteFischerArendvonStackelberg,ConnyEckert,AngelikaEggertVaskar Saha,AnthonyMoormannAndishe Atterbachi,RenatePanzerShaiIzraeli,RenatePfanzerBeppe Basso,Truus TeKronnieAndreaBiondi,GianniCazzanigaHeleneCavé,JeanSoulier,AndreBaruchel,UZH:MarkusManzSwissInstituteof bioinformaticsMariaPamelaDobayEPFLausanneFreddyRadtke,RajLehal,BartDeplanckeETHZurichPeterHorvath,BerndWollscheidMPIBerlinMLYaspo,MMPU/EMBL Andreas Kulozik, Paulina Richter-Pechańska, Joachim Kunz, Elena OrlovaCaroline von Knoebel Doeberitz,Tobias RauschJan Korbel, Sebastian Uhrig, G P Balasubramanian