AML in mice after retroviral cell marking

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AML in mice after retroviral cell marking
Heinrich-Pette-Institute, Hamburg Bernd
Schiedlmeier, Martin Forster, Carol Stocking,
Anke Wahlers, Oliver Frank, Wolfram
Ostertag University Hospital Eppendorf,
Hamburg Jochen Duellmann, Axel Zander, Boris
Fehse University Freiburg Manfred Schmidt,
Christof von Kalle EUFETS AG Klaus Kuehlcke,
Hans-Georg Eckert Hannover Medical
School Zhixiong Li, Johann Meyer, Christopher
Baum
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Oncogenic progression related to insertional
mutagenesis
Risk 10-7 per insertion in human TF-1 leukemia
cells (Stocking et al., 1993) Insertional
mutagenesis promotes tumor formation in
numerous animal models, but single insertion
never sufficient to explain malignancy No
disease induction reported using
replication-defective vectors designed for
gene therapy in numerous preclinical and
clinical trials, probably involving manipulation
of gt1012 hematopoietic or lymphoid cells Side
effects of transgene or active replication
required for pathogenesis
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Toxicity Assessment of Gene Transfer Technologies
dLNGFR
EGFP
tCD34
flCD34
SF
SF
SF
SF
SF
SF
SF
SF




Animal experiments with long-term follow-up
At least 5 recipients for each condition
One group of 5 recipients for each vector
MACS unselected
2d
ana- lysis
7mo
5mo
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• dLNGFR group, 2 recipients (n10)
• AML M5 n6
• Overt dysplasia n3
• Microscopic lesions n1

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AML after Retroviral Gene Marking in Mice
Long latency No overt disease in first cohort (7
mo) 10/10 secondary recipients developed
dysplasia or AML M5 (5 mo) Leukemia is
transplantable to 3 cohort (lethal) Monoclonal
origin, heterogenous kinetics, however
identical entity with reproducible
phenotype Aberrant clone has single vector
integration Vector is intact and continues to
express dLNGFR Insertional activation of
Evi-1 RCR and activation of endogenous MLV
excluded
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Vector integration in Evi-1
A
E1 LTR - dLNGFR - LTR E1
E2 E3
681 955
1
131
132
AUG
PCR
B
M P1 P2 P3 P4 P5 S6 S8 S9 S10 S3 S4
S5 S1 H M
PCR confirms integration and origin in primary
recipient P2
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Evi-1
Transcription factor, known oncogene Endogenous
expression in primitive stem cells Ectopic
expression blocks granulocytic and erythroid
differentiation promotes megakaryocytic
hematopoiesis Activation implicated in MDS and
AML (usually immature phenotype) Tg mice at
increased risk for leukemia (dysplastic
hematopoiesis) Not sufficient to explain AML M5
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dLNGFR variant of p75NTR
p75NTR
dLNGFR
Ligand binding domain
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dLNGFR structurally related to antiapoptotic
decoy receptors
DcR1
DcR2
p75NTR
dLNGFR
Ligand binding domain
Juxtamembrane domain Death domain
Differentiation Apoptosis
Shedding of dLNGFR may generate soluble decoy
receptor (see osteoprotegerin, OPG)
TRAIL family
Marsters et al., Curr Biol 1997
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p75NTR and Trk receptors A two-receptor-system
for neurotrophins
Trk
p75NTR
NT
p75NTR NGF BDNF NT-4
NT-3 TrkA TrkB TrkC
Differentiation Apoptosis
Survival Proliferation
Balanced growth
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The combination of dLNGFR, Trk and NT transforms
fibroblasts
Hantzopoulos et al., Neuron 1994, 13187
dLNGFR
Trk
p75NTR
NT
Trk
NT
No signal (?)
Differentiation Apoptosis
Survival Proliferation
Survival Proliferation
Balanced growth
Transformation
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AML cells express dLNGFR and TrkA and proliferate
in response to NGF
dLNGFR
TrkA
NGF
Enhancement
Loss of balance
Survival Proliferation
Expansion or Transformation ?
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Expression of Neurotrophins and their Receptors
in Human Hematopoiesis(Labouyrie et al., AJP
1999, 154411)
Progenitors Mature Cells
p75NTR absent B cells (mouse mast cells) TrkA
erythroblasts mono, baso, mast, B
cells TrkB eo TrkBi erythroblasts meg TrkC
myeloblasts eo, meg, granulo TrkCi myeloblasts g
ranulo NGF BDNF NT-3 NT-4/5
bone marrow stroma cells, monocytic
cells osteoblasts, osteoclasts, mast cells, B
cells (T cells ?)
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Trk receptors and human leukemia

• TrkA was detected in some leukemic cell lines,
  such as UT-7(acute megakaryoblastic leukemia),
  K562 and TF1 (erythroleukemia), and myeloid cell
  lines HEL, HL60 and KG1, but not in myeloid cell
  lines U937 and THP-1 (Chevalier et al., 1994,
  Auffray et al.,1996, Kaebisch et al., 1996).
• So far, there are only 3 reports on expression of
  p75NTR and Trk receptors in primary leukemia
• 44 TrkA gene expression in patients with AML
  (Kaebisch et al., 1996).
• A translocation t(1215) (p13q25) was found in
  an AML patient, which resulted in a fusion RNA
  ETV6-TrkC (Eguchi et al., 1999).
• A deleted form of TrkA, DTrkA, was identified in
  AML patients. 75-aa deletion in the extracellular
  domain resulted in constitutive tyrosine
  phosphorylation of the protein, which also
  transforms fibroblasts (Reuther et al., 2000).
• These data suggest a possible role of Trk
  receptors and their mutant forms in leukemia
  development (however, so far no evidence for
  transformation of lymphatic cells).

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AML after Retroviral Gene Transfer into Murine HSC
Integration site causal role likely, but not
sufficient Role of transgene causal
contribution suggested Role of vector
architecture no splice acceptor 5-FU exposure of
donor not a strong mutagen, common
procedure Forced expansion in serial BMT
possibly promoting, but not cause Difference
rodent vs. human cells ? Implications for other
cell types ?
SD Y
U3
R
U5
U3
R
U5
dLNGFR
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