Flow cytometric signal typingFlow cytometric signal typingfor therapy response predictionfor therapy response prediction

in pediatric myeloid leukemiain pediatric myeloid leukemia

Michael N. DworzakMichael N. DworzakVeronika SexlVeronika Sexl

Linking Research and Patients' Needs

Childhood cancers – Cure rates

still poor in acute myeloid leukemia (AML)

external stimuli

SCFFLIL3GCSFGMCSFTPOSDF-1IL6VEGFPDGFTGFβIFNγ

STAT1,3,5

JAK2,3

PI3K AKT

Bad Bcl-XL

SRCRas

Raf

MEK

c-Myc,Elk,CREB,c-Jun,FKHR,…

Rac

ERK

JNK (p38)

RS6K

S6NFκB

Bad Bcl-XL

mTOR

mutation

RTKs: FLT3, KIT, PDGFR, VEGFRCytokine-Receptors

proliferation differentiation

protein- synthesis

survival apoptosis

AML: New therapeutic options byAML: New therapeutic options by blocking blocking intracellular signal transductionintracellular signal transduction

external stimuli

SCFFLIL3GCSFGMCSFTPOSDF-1IL6VEGFPDGFTGFβIFNγ

STAT1,3,5

JAK2,3

PI3K AKT

Bad Bcl-XL

SRCRas

Raf

MEK

c-Myc,Elk,CREB,c-Jun,FKHR,…

Rac

ERK

JNK (p38)

RS6K

S6NFκB

Bad Bcl-XL

mTOR

mutation

RTKs: FLT3, KIT, PDGFR, VEGFRCytokine-Receptors

mutation

????

AML: New therapeutic options byAML: New therapeutic options by blocking blocking intracellular signal transductionintracellular signal transduction

Study premises and aimsWork package 2 “Flow cytometric assay development”Aim: Development and validation of a flow cytometric assay allowing for interrogation of the activation state of multiple signal pathways potentially relevant for the pathobiology of acute myeloid leukemia in children.

Work package 3 “Clinically relevant leukemic cell analysis”Aim: Assess and correlate the signaling profiles of pediatric AML samples with morphological, genetic, as well as clinical parameters, aiming at describing AML-subtype- or patient-specific patterns potentially relevant for the choice of signal transduction inhibitors.

Work package 4 “Transplantation of human AML and CML cells into NSG mice”Aim: Amplification of limiting human patient samples of AML and CML through the transplantation of human cells into NSG for assay development. Testing long term effects of inhibitor treatment in vivo. Leukemia arises in NSG animals only when viable stem cells are injected.

Divergent Divergent signal pathway activation signal pathway activation after after different cytokine stimulidifferent cytokine stimuli

74007400 signal activation read-outs from N=74 pediatric AML cases

MLL-normalFAB myeloid

MLL-rearrangedFAB monoblastic

HCA-analysis

Patient Patient classification classification and stratification and stratification upon upon patient-specific patient-specific signal-activation signal-activation bio-signaturesbio-signatures

“hot” cohort

relapsed 44% (11/25)

“cold” cohort

relapsed 15% (8/52)

FLT3-ITD t(8;21)constitutive

GCSFGMCSF

FLSCF

p38

STAT

3ER

KAK

TST

AT5

p38

STAT

3ER

KAK

TST

AT5

FLT3-ITD t(8;21)constitutive

GCSFGMCSF

FLSCF

p38

STAT

3ER

KAK

TST

AT5

p38

STAT

3ER

KAK

TST

AT5

p38

STAT

3ER

KAK

TST

AT5

p38

STAT

3ER

KAK

TST

AT5

log2 scale

-2.0

-1.0

back

grou

ndpo

sitiv

e+

1.0

+ 2.

0+

3.0

log2 scale

-2.0

-1.0

back

grou

ndpo

sitiv

e+

1.0

+ 2.

0+

3.0additional

effects over constitutive

STAT1,3,5

JAK2,3

PI3KAKT

SRCRas

Raf

MEK

Rac

ERK

JNK (p38)

RS6K

S6NFκB

mTOR

RTK

STAT1,3,5

JAK2,3

PI3KAKT

SRCRas

Raf

MEK

Rac

ERK

JNK (p38)

RS6K

S6NFκB

mTOR

RTK

Signal activation signaturesSignal activation signaturesin pediatric AML:in pediatric AML:- patient-specific „finger-prints“- differentiation-associated (FAB types)- genotype-related- outcome-associated

Cytokine-RRTKs, eg. FLT3, KIT, PDGFRb, VEGFR

STAT1,3,5

JAK2,3

PI3K AKT

Bad Bcl-XL

SRCRas

Raf

MEK

c-Myc,Elk,CREB,c-Jun,FKHR,…

Rac

ERK

JNK (p38)

RS6K

S6NFκB

Bad Bcl-XL

mTOR

mutation

SunitinibSorafenib PKC412

Dasatinib

AG490

PD98059Sorafenib

LY294002

Everolimus

Bortezomib

AML: New therapeutic options byAML: New therapeutic options by blocking blocking intracellular signal transductionintracellular signal transduction

Signal inhibition-assay in pediatric AML

correlates well with in-vitro drug activity - cell death

100 101 102 103 104p-Stat5 Ax 647

100 101 102 103 104p-S6 AX488

pSTAT5 pS6

100 101 102 103 104p-ERK Ax 647

100 101 102 103 104p-AKT AX647 (9DE)pERK pAKT

PKC412Sorafenib

Rapamycincontrol

100 101 102 103 104p-Stat5 Ax 647

100 101 102 103 104p-S6 AX488

pSTAT5 pS6

100 101 102 103 104p-ERK Ax 647

100 101 102 103 104p-AKT AX647 (9DE)pERK pAKT

100 101 102 103 104p-ERK Ax 647

100 101 102 103 104p-AKT AX647 (9DE)pERK pAKT

PKC412Sorafenib

Rapamycincontrol

control

PKC412

Sorafenib

Rapamycin

1.1.

2.2.

3.3.

BM orspleen of1st TP

Incubation: 24h supplementedDMSOSorafenibPKC412RapamycinRuxolitinib

Preliminary data:One representative experimentout of 2

In vitro drug activity in pediatric AML

correlates well with outcome in-vivo in the xenograft model

Signals in FLOW in pediatric AML – the international networkM. van Heuvel, M. Zwaan Erasmus MU Rotterdam

mutational screeningcross-platform validationCytokine-R

RTKs, eg. FLT3, KIT, PDGFRb, VEGF

External signal factor

SCF

FL

IL3

GCSF

GMCSF

TPO

SDF-1

IL6

VEGF

PDGF

TGFβ

IFNγ

Adhesion molecules

External signal factor

SCF

FL

IL3

GCSF

GMCSF

TPO

SDF-1

IL6

VEGF

PDGF

TGFβ

IFNγ

Adhesion molecules

STAT1,3,5

JAK2,3

PI3KAKT

Bad Bcl-XL

SRCRas

Raf

MEK

c-Myc,Elk,CREB,c-Jun,FKHR,…c-Myc,Elk,CREB,c-Jun,FKHR,…

Rac

ERK

JNK(p38)

RS6K

S6NFκB

Bad Bcl-XL

mTOR

External signal factor

SCF

FL

IL3

GCSF

GMCSF

TPO

SDF-1

IL6

VEGF

PDGF

TGFβ

IFNγ

adhesion molecules

External signal factor

SCF

FL

IL3

GCSF

GMCSF

TPO

SDF-1

IL6

VEGF

PDGF

TGFβ

IFNγ

adhesion molecules

signal transduction profilingpharmacodynamic monitoringM. Dworzak, CCRI Viennaxenograft models

V. Sexl, Veterinary University Vienna

D. Reinhardt, MH HannoverAML-BFM study headquarter

sample recruitmentclinical data repositorygenetic data repositorymutational screeningresearch data website

M. Dworzak, STAK ViennaAML-BFM-A study headquarter

Output and ImpactNew options

Output and ImpactPublications (preliminary)

Enhanced Ratio of Activated STAT5/STAT3 after G‑CSF Stimulation in

vitro is Associated with Favorable Prognosis in Pediatric Acute Myeloid

Leukemia 

Maibach S, Herbst C, Zimmermann M, Reinhardt K, Böhmer K, Dworzak M, Creutzig U, Reinhardt D, Ehlers S

submitted submitted Leukemia 2013

Output and ImpactClinical application

Leukemia – identification of vulnerable nodes

Andrea Hölbl-KovacicVeronika Sexl

Michael Dworzak

Leukemia – Aims of the Study

To find Achilles` heels within leukemic cells via identification of

essential signal transduction pathways

Tumor promoters: JAK2, STAT3, STAT5

Tumor suppressors:

STAT1

oncoproteins

A candidate approach:The JAK/STAT Signalling Pathway

Key findings of the STAT5 Study

STAT5

B-ALL maintenance

Leukemic Stem Cells

Imatinib-responsiveness

Mechanism:Serine

phosphorylation of Stat5 is critical

STAT5 is critical for B-ALL maintenance

delete Stat5in leukemic cells

44 0.2

CD19

bone marrow

B220

Stat5 present Stat5 absent

Hoelbl, Schuster et al, EMBO Mol Med 2010

STAT5 is critical for leukemic stem cells

STAT5 deletion ex vivo

GFP

SS

C

Sca-1

c-ki

t

leukemic mouse bone marrow

0

Sca-1

c-ki

t

GFP

SS

C

2nd transplant

Hoelbl, Schuster et al, EMBO Mol Med 2010

Imatinib treatment selects STAT5high cells

Days pdays of

treatment:

+ imatinib Warsch et al, Blood 2011

Serine phosphorylation of STAT5 is critical for leukemia

Friedbichler et al, Blood 2011

STAT5 serine phosphorylation – a potential therapeutic target

Another Candidate Approach:Switching gears from JAK/STAT signalling

to cell cycle regulation

Friedbichler et al, Blood 2011

The cell-cycle dependent kinase 6 (CDK6) comes into a privileged role in

angiogenesis…

CDK6 regulates angiogenesis in lymphoma

Cdk6-/-Cdk6+/+

B-ALL tumors NPM-ALK+ tumors

0 10 200

20

40

60

80

100Cdk6+/+ Cdk6-/-

time (days)

% s

urvi

val

Nu/NuCdk6+/+Cdk6-/-

0.00

0.10

0.20

0.30

0.40

0.50*

tum

or w

eigh

t (g)

Cdk6-/-Cdk6+/+

Kollmann et al, Cancer Cell 2013

Output and ImpactFollow-up publication

Laimer, Dolznig, Kollmann, Vesely et al, Nature Medicine 2012

Imatinib treatment of a human ALCL patient

NPM-ALK+ tumors+ imatinib

Lab of Lukas Kenner, LBI-CR

Output and ImpactFollow-up projects

STAT5 serine phosphorylation

in leukemia (FWF-Grant to

A.H.)

CDK6 in stem-cell-derived leukemia

(FWF-Grant to V.S.)

Output and Impact - Publications Hoelbl A, Schuster C, Kovacic B, Hoelzl M, Fajmann S, Grebien F, Warsch W, Stengl G, Hennighausen L, Beug H, Moriggl R, Sexl V. Stat5 is

indispensable for the maintenance of bcr/abl positive leukemia (2010). EMBO Mol Med; 2: 98-110.

Friedbichler K, Kerenyi MA, Kovacic B, Li G, Hoelbl A, Yahiaoui S, Sexl V, Müllner E, Fajmann S, Cerny-Reiterer S, Valent P, Beug H, Gouilleux F, Bunting KD,

Moriggl R. Stat5a serine 725 and 779 phosphorylation is a prerequisite for hematopoietic transformation (2010). Blood. 116 1548.

Warsch W, Kollmann K, Eckelhart E, Fajmann S, Cerny-Reiterer S, Hoelbl A,

Gleixner K, Dworzak M, Mayerhofer M, Hoermann G, Hermann H, SillaberC, Egger G, Valent P, Moriggl R, Sexl V. High Stat5 levels mediate

imatinib –resistance and indicate disease progression in chronicmyeloid leukemia (2011). Blood. 117:3409

Hantschel O, Warsch W, Eckelhart E, Grebien F, Superti-Furga G, Sexl V.Bcr/Abl directly activates Stat5 independent of Jak2. (2012) Nat. Chem

Biol 8(3):285-93

Kovacic, B; Hoelbl, A; Litos, G; Alacakaptan, M; Schuster, C; Fischhuber, K;Kerenyi, M; Stengl, G; Moriggl, R; Sexl, V; Beug, H: Diverging fates of

cells of origin in acute and chronic leukemia. EMBO Mol Med,Apr;4(4):283-97

Kollmann K, Heller G, Schneckenleithner C, Warsch W, Scheicher R, Ott RG, Schäfer M, Fajmann S, Schlederer M, Schiefer AI, Reichart U, Mayerhofer M, Hoeller C, Zöchbauer-Müller S, Kerjaschki D, Bock C, Kenner L, Hoefler G, Freissmuth M, Green AR, Moriggl R, Busslinger M,

Malumbres M, Sexl V. A Kinase-Independent Function of CDK6 Links the Cell Cycle to Tumor Angiogenesis.Cancer Cell. 2013 Aug 12;24(2):167-81

Output and ImpactCareer Development

Veronika Sexl: 2010: full professorship at the VetmedUni Vienna (VUV); head of the institute2013: member of the EHA Fellowship Grant Committee2013: Supervisory Board Member of the Medical University Vienna (MUV)

Andrea Hölbl-Kovacic:2009: University Assistant at the MUV2011: FWF-Project Leader „STAT5 Serine Phosphorylation in Leukemia“2011: Award „Forschungspreis der Stadt Wien für innovative Krebsforschung“2012: University Assistant at the VUV

Wolfgang Warsch:2012: Award „Forschungspreis der Stadt Wien für innovative Krebsforschung“2012: PostDoc at the „Cambridge Institute of Medical Research“, Lab of Tony Green

Thanks to…

…you for your attention

Christine Schneckenleithner

Wolfgang Warsch

Sabine FajmannMichael

Dworzak

and Angela Schumich

MichaelaPrchal