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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

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AML in mice after retroviral cell marking

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  1. 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

  2. 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

  3. 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

  4. dLNGFR group, 2° recipients (n=10) • AML M5: n=6 • Overt dysplasia: n=3 • Microscopic lesions: n=1 CB 02

  5. 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

  6. 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

  7. 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

  8. Juxtamembrane domain Death domain Differentiation Apoptosis dLNGFR: variant of p75NTR p75NTR dLNGFR Ligand binding domain CB 02

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

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