slides - pdf - Blog Science Connections

G-CSF – a wonderful molecule
Prof. Dr. med. Karl Welte

1964
Donald Metcalf
The growth of mouse bone marrow
cells in vitro. Aust J Exp Bio Med Sci
1966
Leo Sachs
The cloning of normal mast cells
in tissue cultures. J.Cell.Physiol.
1965
The early 1960s

Metcalf, D., Nature Review Cancer, 2010

Purification of G-CSF
(40 L supernatant of the 5637 cell line: 5 µg G-CSF)
hu G-CSF
Welte, K., et al., PNAS, 1985

MSKCC New York, G-CSF-Laboratory, 1984
Pluripoietin ?
No,
G-CSF !

Effects of G-CSF on Myelopoiesis
CFU-
GM
Myeloblast Promyelocyt Myelocyt Metamyelocyt Band Segmented
Neutrophil
Proliferation
Anti-apoptosis
Differentiation
Hannover Medical School

Gibbs KD, ..., Weissman, IL, et al., Blood 2011
HSC
Engraftment of G-CSFR (CD114)
positive cells in NSG-mice

Protein
cDNA
Souza et al., Science, 1986

Welte, K., et al., PNAS 1985
Souza, L., et al., Science 1986
Biochemical Characteristics of G-CSF:
174 amino acids
Glycosylation: yes
Molecular weight: 19.6 kD
Filgrastim (E.Coli):
175 amino acids
Glycosylation: no
MW18.7 kD,
Nagata, S., et al., Nature 1986,
G-CSF splice form : 177 amino acids

Poster in Wilsede 1984
Modern Trends in Human Leukemia VI
R Neth, RC Gallo, MF Greaves, et al.
Biological activities of a human pluripotent hemopoietic colonystimulating
factor.
Platzer E, Welte K, Lu L, Gabrilove JL, Yung YP, Nathan CF,
Mertelsmann R, Moore MA.
Haematol Blood Transfus. 1985;29:418-22.
Hannover Medical School

Hannover Medical School

G-CSF in Primates

G-CSF in Primates
G-CSF in Primates
G-CSF (µg/kg/d)
100
G-CSF (10 µg/kg/d)
WBC
ANC
ALC
10
1
Welte, et al., J. Exp. Med. 165, 941, 1987
Hannover Medical School

G-CSF in Primates
Welte, K., et al., J Exp Med 1987

G-CSF in Childhood ALL (BFM2000):
Does administration of more chemotherapy in time
lead to increased survival ?
Welte, K., et al. Blood 1996

al95_end.tab 16OCT07P
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
ALL-BFM 90 Survival (10 years)
HRG-2 (with G-CSF) (N= 29, 16 events)
0.45, SE=0.09
0.37, SE=0.09
HRG-1 (without G-CSF) (N= 32, 20 events)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
years
Hannover Medical School

Souza and Welte, Science 1986

Ch. Schiffer, N Engl J Med 349, 727, 2003

Priming with G-CSF in AML
Löwenberg, et al NEJM 2003

AML-BFM 98
Ind. 1 Ind. 2
Consolidation
A I E
12 mg
H A M
nicht M 3
und DS
Rand II
HAE
Rand III
12 Gy
vs. 18 Gy
A I
haM
Maintenance
1 year +
i.th. Ara-C**
Rand
I
+ G-CSF* +
G-CSF*
Day 1 15 21-28 42-56 112 140 360 540
MRD MRD MRD MRD MRD MRD BM
* G-CSF 5-8 days or until ANC >500/mm³ except patients with > 5% Blasts on day 15
Hannover Medical School
G-CSF

fm1004.tab 21OCT04
AML-BFM 98 Standard risk patients (without Down-S.): EFS (3 years)
1.0
0.9
0.8
.71, SE=.06
0.7
P
0.6
0.5
.57, SE=.07
0.4
0.3
0.2
0.1
Log-Rank p = .07
0.0
0 1 2 3 4 5 years 6
No G-CSF (N= 66, 19 events)
G-CSF (N= 58, 25 events)

Ehlers S., et al., J Clin Oncol 2010

G-CSFR isoform I G-CSFR isoform IV
The isoform IV replaces the carboxy-terminal 87 aa
with 34 aa of novel sequence
G-CSF
G-CSF
JAK2
Y
Y
Y
Y
STAT5
STAT3
Shp2
SOCS3
normal ratio 200 : 1
Y
Y
JAK2
Y
STAT3
Shp2
STAT5
SOCS3

Mobilization of hematopoietic stem cells by
G-CSF
Vose et al., 09

2000
1973
1800
1739
1847
1600
1400
1614
1200
1225
1000
1055 1051
1100 1093
800
600
400
200
559
559
896
521
383
329 316 314 288 289
266 264
314
0
1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008
Knochenmark (KM)
Peripheres Blut (PB)
Paradigm shift of SCT by G-CSF mobilization
(Germany 1998 – 2008)
Hannover Medical School

Transplantation`s balancing act !
Martin, PJ, Nature Medicine 2009
Morris, ES, Nature Medicine 2009
Hannover Medical School

pluripotent
stem cell
CFU-GEMM CFU-GM myeloblast promyelocyte maturation
arrest
G-CSF
Severe congenital neutropenia
Hannover Medical School

Genetic distribution in 122 patients with CN
Hannover Medical School

Severe Chronic Neutropenia Registry, Europe
Long Term Course of Median Absolute Neutrophil Count
in Patients with Severe Congenital Neutropenia
10.000
Absolute Neutrophil Count / µL
1.000
100
10
N = 168 119 98 97 86 78 72 76 66 54 43 39 34 26 22 18 11 7
Years of G-CSF Treatment
Hannover Medical School

G-CSF induces Nampt and NAD + in myeloid progenitors from
G-CSF
CN patients
Nampt
NA NAD + SIRT1
5 *
4
3
2
1
0
CN Mctrl
G-CSF + + +
iNAD + , mg/l
Regulation of
transcription
Nampt/PBEF protein
Ng/ml
80
60
40
20
0
CN CN MN ctrl ctrl
G-CSF - + + - +
110kDa
loading
control
*
MN + G-CSF
bone marrow
promyelocytes
CN + G-CSF
*
SIRT1
Skokowa, J., et al., Nat Med 2009; 15: 151- 8
Hannover Medical School

Nampt induces G-CSF and G-CSFR in CD34 cells via Sirt1/C/EBPs
ctrl
Nampt
targetgene/ß-actin
mRNAratio, AU
16
8
0
*
150
100
50
*
60
30
45
30
15
0 0
0
C/EBPα C/EBPβ G-CSFR G-CSF
*
control Nampt/PBEF
20% 84%
G-CSFR
G-CSF
ng/ml medium
0.6
0.4
0.2
0
**
Hannover Medical School

Nampt triggers myeloid differentiation of CD34 + cells
% of CD16 + cells
60
30
0
ctrl
Nampt
G-CSF
Nampt +G-CSF
0 7 10 14
Time (d)
% of CD15 + cells
60
30
0
0 7 10 14
Time (d)
- NAMPT + NAMPT
10 µm 10 µm
Hannover Medical School

Vitamin B3 treatment increases number of
neutrophilic granulocytes in healthy individuals
Peripheral blood
neutrophilic granulocyte
( x 10 3 µl -1 )
7
6
5
4
3
Day 1
P = 0.002
Day 3
P = 0.002
Day 7
Day 10
Vitamin B3 (200 mg/kg/d) treatment (day 1-7)
Skokowa, J., et al., Nat Med 2009; 15: 151- 8
Hannover Medical School

Vitamin B3 treatment of patient with cyclic neutropenia
Treatment with G-CSF (3 ug/kg/d s.c.)
Neutrophil granulocytes
in peripheral bloot
( x 10 3 µl -1 )
Neutrophil granulocytes
in peripheral bloot
( x 10 3 µl -1 )
Treatment with Vitamin B3 (200 mg/kg/d p.o.)
without G-CSF
Medizinische Hochschule
Hannover

Hazard rates and cumulative incidence of
MDS/AML and sepsis death in patients with SCN.
Cumulative incidence of MDS/AML (blue
curve) and sepsis death (black curve)
among patients who achieved an adequate
mean absolute neutrophil count (ANC) by
months 6–18 (≥2.188 cells × 109/l) at
doses of G-CSF less than or equal to
8 μg/kg per d).
Corresponding cumulative incidence curves
among patients who failed to achieve an
adequate ANC despite higher doses of G-
CSF.
Rosenberg, Zeidler, et al., BJH 2010
Zeidler EHA 2010

G-CSFR mutations in CN patients

Hannover Medical School
Adopted from Germeshausen et al., Blood 2006

Leukemogenesis
Nampt deacetylates p53
G-CSF
STAT3/Nampt
NA NAD + SIRT1
p53 acetyl Lys382
p53 total
Nampt
β-actin
ctrl lv
Nampt lv
Deactelylation of p53,
FOXO3A,
C/EBPs, etc.
Hannover Medical School

Leukemogenesis
SIRT1 deacetylates p53 and FOXO3a
functional
inactivation of p53 and FOXO3a
SIRT1 also deacetylates ß-catenin and by this may induce genomic
instability
Hannover Medical School

Model of the malignant transformation in CN
G-CSF treatment
Severe congenital
neutropenia
Leukemia
ELA2- and HAX-
1-
mutations,
genotoxic stress,
ER-stress,
genetic instability
G-CSF
Rezeptormutations:
Selective
pressure
Deletion
Chrom. 7
or/and
Trisomy 21
N-rasmutation
sustained STAT5 activation,
deacetylation of p53 and FOXO3 by SIRT1
Hannover Medical School

Nature Review Cancer, 2010

Acknowlegement
G-CSF signaling
Julia Skokowa
Basant Kumar Thakur
Lan Dan
Kshama Gupta
SCN Registry
Cornelia Zeidler
European LLPs
SCNIR David Dale
Leukemias
Martin Schrappe
Haig Riehm
Dirk Reinhardt,
Ursula Creutzig
ELA2
Inga Köllner
Carmela Beger
Lentiviral vectors
Axel Schambach
Christopher Baum
G-CSF receptor mutations,
Manuela Germeshausen
Matthias Ballmaier
HAX 1
Christoph Klein
and his Lab
Hannover Medical School

Elternverein
krebskranker Kinder
Hannover e.V.
Abteilung und/oder Titel
Abteilung und/oder Titel
Datum
Hannover Medical School