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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

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aml in mice after retroviral cell marking
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

CB 02

o ncogenic progression related to insertional mutagenesis
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 >1012

hematopoietic or lymphoid cells

Side effects of transgene or active replication required for pathogenesis

CB 02

toxicity assessment of gene transfer technologies
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

ana-

lysis

2d

7mo

5mo

CB 02

slide4

dLNGFR group, 2° recipients (n=10)

  • AML M5: n=6
  • Overt dysplasia: n=3
  • Microscopic lesions: n=1

CB 02

aml after retroviral gene marking in mice
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

CB 02

vector integration in evi 1

U3 R U5

SD

U3 R U5

Vector integration in Evi-1

A

E1LTR - dLNGFR - LTR E1 E2 E3

1

131

132

681 955

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

CB 02

evi 1
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

CB 02

dlngfr variant of p75ntr

Juxtamembrane domain

Death domain

Differentiation

Apoptosis

dLNGFR: variant of p75NTR

p75NTR

dLNGFR

Ligand binding domain

CB 02

dlngfr structurally related to antiapoptotic decoy receptors
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

CB 02

p75ntr and trk receptors a two receptor system for neurotrophins
p75NTR and Trk receptors: A two-receptor-system for neurotrophins

p75NTR

Trk

NT

p75NTR

NGF BDNF NT-4 NT-3

TrkA TrkB TrkC

Differentiation

Apoptosis

Survival

Proliferation

Balanced growth

CB 02

the combination of dlngfr trk and nt transforms fibroblasts
The combination of dLNGFR, Trk and NT transforms fibroblasts

Hantzopoulos et al., Neuron 1994, 13:187

dLNGFR

p75NTR

Trk

Trk

NT

NT

No signal (?)

Differentiation

Apoptosis

Survival

Proliferation

Survival

Proliferation

Balanced growth

Transformation

CB 02

aml cells express dlngfr and trka and proliferate in response to ngf

N L K S

TrkA

4.4 kb

GAPDH

77 %

CD11b

dLNGFR

AML cells express dLNGFR and TrkA and proliferate in response to NGF

dLNGFR

TrkA

NGF

Enhancement

Loss of balance

Survival

Proliferation

Expansion or

Transformation ?

CB 02

slide13
Expression of Neurotrophins and their Receptors in Human Hematopoiesis(Labouyrie et al., AJP 1999, 154:411)

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 granulo

NGF

BDNF

NT-3

NT-4/5

bone marrow stroma cells, monocytic cells

osteoblasts, osteoclasts, mast cells, B cells

(T cells ?)

CB 02

trk receptors and human leukemia
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(12;15) (p13;q25) 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).

CB 02

aml after retroviral gene transfer into murine hsc
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

dLNGFR

U3

R

U5

CB 02