flow cytometric signal typing for therapy response prediction in pediatric myeloid leukemia
DESCRIPTION
Flow cytometric signal typing for therapy response prediction in pediatric myeloid leukemia Michael N. Dworzak Veronika Sexl. Linking Research and Patients' Needs. Childhood cancers – Cure rates still poor in acute myeloid leukemia (AML). AML: New therapeutic options by - PowerPoint PPT PresentationTRANSCRIPT
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