Transcript Presentation Michael Dworzak and Andrea Hölbl

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
blocking intracellular signal transduction
external
stimuli
SCF
FL
IL3
GCSF
RTKs: FLT3, KIT, PDGFR, VEGFR
Cytokine-Receptors
PI3K
Ras
JAK2,3 SRC
Raf
GMCSF
Rac
MEK
TPO
SDF-1
IL6
VEGF
STAT1,3,5
survival
apoptosis
AKT
mutation
ERK
mTOR
RS6K
JNK
(p38)
NFκB
Bad Bcl-XL
c-Myc,Elk,CREB,c-Jun,FKHR,…
PDGF
TGFβ
IFNγ
S6
proliferation
differentiation
Bad Bcl-XL
proteinsynthesis
AML: New therapeutic options by
blocking intracellular signal transduction
external
stimuli
SCF
FL
IL3
GCSF
RTKs: FLT3, KIT, PDGFR, VEGFR
Cytokine-Receptors
PI3K
mutation
Ras
JAK2,3 SRC
Raf
GMCSF
IL6
VEGF
PDGF
TGFβ
IFNγ
Rac
STAT1,3,5
Bad Bcl-XL
mTOR
??
MEK
TPO
SDF-1
AKT
mutation
ERK
JNK
(p38)
RS6K
NFκB
S6
c-Myc,Elk,CREB,c-Jun,FKHR,…
Bad Bcl-XL
Study premises and aims
Work 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 AMLsubtype- 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 signal pathway activation
after different cytokine stimuli
7400 signal activation read-outs from N=74 pediatric AML cases
MLL-normal
MLL-rearranged
FAB myeloid
FAB monoblastic
HCAanalysis
Patient classification
and stratification
upon patient-specific
signal-activation
bio-signatures
“hot” cohort
relapsed 44%
(11/25)
“cold” cohort
relapsed 15%
(8/52)
RTK
AKT
Rac
STAT1,3,5
ERK
JNK
(p38)
RS6K
NFκB
S6
Signal activation signatures
in pediatric AML:
-
patient-specific „finger-prints“
differentiation-associated (FAB types)
genotype-related
outcome-associated
FLT3-ITD
t(8;21)
constitutive
GCSF
GMCSF
FL
SCF
p38
STAT3
ERK
AKT
STAT5
additional
effects over
constitutive
log2 scale
- 2.0
- 1.0
background
positive
+ 1.0
+ 2.0
+ 3.0
MEK
mTOR
p38
STAT3
ERK
AKT
STAT5
PI3K
Ras
JAK2,3 SRC
Raf
AML: New therapeutic options by
blocking intracellular signal transduction
Cytokine-R
RTKs, eg. FLT3, KIT, PDGFRb, VEGFR
LY294002
AKT
mutation
Sunitinib
Sorafenib
PKC412
PI3K
Ras
JAK2,3 SRC
Raf
Rac
MEK
AG490
STAT1,3,5
ERK
Everolimus
mTOR
JNK
(p38)
RS6K
NFκB
S6
Bad Bcl-XL
c-Myc,Elk,CREB,c-Jun,FKHR,…
Dasatinib
Sorafenib
PD98059
Bad Bcl-XL
Bortezomib
2.
Signal inhibition-assay
in pediatric AML
correlates well with in-vitro
drug activity - cell death
100
101
102
p-Stat5 Ax 647
103
pSTAT5
104
100
101
102
p-S6 AX4 88
103
104
pS6
PKC412
Sorafenib
Rapamycin
control
control
100
1.
101
102
p-ERK Ax 647
103
pERK
Sorafenib
Rapamycin
100
101
102
p-AKT AX647 (9DE)
pAKT
3.
PKC412
104
103
104
In vitro drug activity in pediatric AML
BM or
spleen of
1st TP
correlates well with outcome in-vivo
in the xenograft model
Incubation: 24h supplemented
DMSO
Sorafenib
PKC412
Rapamycin
Ruxolitinib
Preliminary data:
One representative experiment
out of 2
Signals in FLOW in pediatric AML – the international network
M. van Heuvel, M. Zwaan
Erasmus MU Rotterdam
External
signal
factor
Cytokine-R
RTKs, eg. FLT3, KIT, PDGFRb, VEGF
SCF
FL
IL3
GCSF
GMCSF
PI3K
Ras
JAK2,3 SRC
Raf
TPO
D. Reinhardt, MH Hannover
AML-BFM study headquarter
AKT
Rac
MEK
mTOR
JNK
(p38)
RS6K
SDF-1
STAT1,3,5
IL6
VEGF
PDGF
ERK
mutational screening
cross-platform validation
NFκB
S6
Bad Bcl-XL
sample recruitment
clinical data repository
genetic data repository
mutational screening
research data website
Bad Bcl-XL
c-Myc,Elk,CREB,c-Jun,FKHR,…
TGFβ
M. Dworzak, STAK Vienna
AML-BFM-A study headquarter
IFNγ
Adhesion
adhesion
molecules
xenograft models
V. Sexl, Veterinary University Vienna
signal transduction profiling
pharmacodynamic monitoring
M. Dworzak, CCRI Vienna
Output and Impact
New options
Output and Impact
Publications (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 Leukemia 2013
Output and Impact
Clinical application
Leukemia –
identification of vulnerable nodes
Andrea Hölbl-Kovacic
Veronika Sexl
Michael Dworzak
Leukemia –
Aims of the Study
To find Achilles` heels within
leukemic cells via identification of
essential signal transduction
pathways
A candidate approach:
The JAK/STAT Signalling Pathway
oncoproteins
Tumor promoters:
Tumor suppressors:
JAK2, STAT3, STAT5
STAT1
Key findings of the STAT5 Study
B-ALL
maintenance
Leukemic
Stem Cells
STAT5
Imatinibresponsiveness
Mechanism:
Serine
phosphorylation of
Stat5 is critical
STAT5 is critical for B-ALL maintenance
Stat5 present
Stat5 absent
44
0.2
B220
bone marrow
CD19
delete Stat5
in leukemic cells
Hoelbl, Schuster et al, EMBO Mol Med 2010
STAT5 is critical for leukemic stem cells
bone marrow
c-kit
SSC
leukemic mouse
GFP
Sca-1
2nd transplant
0
c-kit
SSC
STAT5 deletion
ex vivo
GFP
Sca-1
Hoelbl, Schuster et al, EMBO Mol Med 2010
Imatinib treatment selects STAT5high cells
days of
Days p
treatment:
+ imatinib
Warsch et al, Blood 2011
Serine phosphorylation of STAT5 is
critical for leukemia
STAT5 serine phosphorylation – a potential therapeutic target
Friedbichler et al, Blood 2011
Another Candidate Approach:
Switching gears from JAK/STAT signalling
to cell cycle regulation
The cell-cycle dependent kinase 6 (CDK6)
comes into a privileged role in
angiogenesis…
Friedbichler et al, Blood 2011
CDK6 regulates angiogenesis
in lymphoma
NPM-ALK+ tumors
B-ALL tumors
Cdk6+/+
Cdk6-/0.50
0.40
0.30
0.20
60
40
20
0.00
0
Kollmann et al, Cancer Cell 2013
Cdk6+/+
Cdk6-/-
80
0.10
Cdk6+/+Cdk6-/-
Cdk6-/-
100
% survival
tumor weight (g)
*
Cdk6+/+
Nu/Nu
0
10
time (days)
20
Output and Impact
Follow-up publication
NPM-ALK+ tumors
+ imatinib
Imatinib treatment of a
human ALCL patient
Laimer, Dolznig, Kollmann, Vesely et al, Nature Medicine 2012
Lab of Lukas Kenner, LBI-CR
Output and Impact
Follow-up projects
STAT5 serine
phosphorylation
in leukemia
(FWF-Grant to A.H.)
CDK6 in stem-cellderived 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, Sillaber
C, Egger G, Valent P, Moriggl R, Sexl V. High Stat5 levels mediate
imatinib –resistance and indicate disease progression in chronic
myeloid 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 Impact
Career Development

Veronika Sexl:
2010: full professorship at the VetmedUni Vienna (VUV); head of the institute
2013: member of the EHA Fellowship Grant Committee
2013: Supervisory Board Member of the Medical University Vienna (MUV)
 Andrea Hölbl-Kovacic:
2009: University Assistant at the MUV
2011: 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…
Michael
Dworzak
and Angela Schumich
Christine
Wolfgang
Schneckenleithner Warsch
Sabine
Fajmann
…you for your
attention
Michaela
Prchal