AML in mice after retroviral cell marking

Heinrich-Pette-Institute, HamburgBernd Schiedlmeier, Martin Forster, Carol Stocking, Anke Wahlers, Oliver Frank, Wolfram Ostertag

University Hospital Eppendorf, HamburgJochen Duellmann, Axel Zander, Boris Fehse

University FreiburgManfred Schmidt, Christof von Kalle

EUFETS AGKlaus Kuehlcke, Hans-Georg Eckert

Hannover Medical SchoolZhixiong 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 >1012

hematopoietic or lymphoid cells

Side effects of transgene or active replication required for

pathogenesisCB 02

Toxicity Assessment of Gene Transfer Technologies

Animal experiments with long-term follow-up

2d 7moMACS

unselected

5mo ana-lysis

dLNGFRSF SF

EGFPSF SF

tCD34SF SF

flCD34SF SF

One group of 5 recipients

for each vector

At least5 recipients

for each condition

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dLNGFR group, 2° recipients (n=10)

– AML M5: n=6

– Overt dysplasia: n=3

– Microscopic lesions: n=1

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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 excludedCB 02

Vector integration in Evi-1

SD

681 9551 131 132

U3 R U5 U3 R U5

E1 LTR - dLNGFR - LTR E1 E2 E3

M P1 P2 P3 P4 P5 S6 S8 S9 S10 S3 S4 S5 S1 H M

PCR confirms integrationand origin in primary recipient P2

PCR

A

B

AUG

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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

DifferentiationApoptosis

Juxtamembrane domainDeath domain

Ligand binding domain

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dLNGFR: structurally related to antiapoptotic decoy receptors

Marsters et al., Curr Biol 1997

DifferentiationApoptosis

p75NTR dLNGFR

Juxtamembrane domainDeath domain

Ligand binding domain

DcR1

TRAIL family

DcR2

Shedding of dLNGFR maygenerate soluble decoy receptor

(see osteoprotegerin, OPG)CB 02

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

SurvivalProliferation

DifferentiationApoptosis

Trk p75NTRNT

Balanced growth

p75NTR

NGF BDNF NT-4 NT-3

TrkA TrkB TrkC

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The combination of dLNGFR, Trk and NT transforms fibroblasts

Hantzopoulos et al., Neuron 1994, 13:187

SurvivalProliferation

DifferentiationApoptosis

Trk p75NTRNT

Balanced growth

SurvivalProliferation

No signal (?)

Trk dLNGFRNT

Transformation

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

N L K S

TrkA4.4 kb

GAPDH

SurvivalProliferation

Enhancement

TrkA dLNGFRNGF

Expansion or Transformation ?

CD11b

77 %

dLNGFR Loss of balance

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Expression of Neurotrophins and their Receptors in Human Hematopoiesis

(Labouyrie et al., AJP 1999, 154:411)

p75NTR absent B cells (mouse mast cells)

TrkA erythroblasts mono, baso, mast, B cells

TrkB eoTrkBi erythroblasts meg

TrkC myeloblasts eo, meg, granuloTrkCi myeloblasts granulo

NGFBDNFNT-3NT-4/5

Progenitors Mature Cells

bone marrow stroma cells, monocytic cellsosteoblasts, 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(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, TrkA, 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 ?

U3 R U5 U3 R U5 SD

dLNGFRCB 02