retroviral insertional mutagenesis and cancer in animal models linda wolff, ph.d. chief,...
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Retroviral Insertional Mutagenesis and Cancer in Animal Models
Linda Wolff, Ph.D.
Chief, Leukemogenesis Section, Laboratory of Cellular Oncology
Center for Cancer Research National Cancer Institute, NIH
FDA Center for Biologics Evaluation and ResearchBiological Response Modifiers Advisory Committee
Meeting #33, October 10, 2002
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•Brief historical overview
•Example of a model where inflammation promotes leukemia progression in conjunction with retroviral mutagenesis
•Collaboration of two genetic events: examples from our studies of retroviral insertional mutagenesis in transgenic and knockout mice.
Retrovirus Integration in DNA and Cancer
•Cancer caused by non-replicating retrovirus vector
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Retroviruses were first discovered in association with cancer around the turn of the century
cell-free extract
cell-free extract
Leukemia Leukemia
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Many cancer causing retrovirus isolateswere composed of two different viruses
LTRLTR LTR LTRONC gag pol env
Defective genome Replication competent genome“helper-virus”
Rapid diseaseDisease caused by
Insertional mutagenesis
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Integration into genomic DNA
Nucleus Integration is essentially random throughout the genome
Cell division required for efficient integration
provirus
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LTR LTRgag pol env
Proto-oncogene
mRNA
protein
Provirus
Cellular Genome
Proto-oncogene = stimulates accumulation of cells in normal processes
Oncogene = activated proto-oncogene having increased capacity to cause continued inappropriate growth.
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Most Common Mechanisms of Transcriptional Activation
Virus integrated at the 3’ end of gene---enhancer activation
Virus integrated at the 5’ end of gene---promoter and or enhancer activation
provirus
proviruspromoter
provirus
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Insertional Mutagenesis
Type of genes growth factors growth factor receptors cytoplasmic kinases transcription factors
Species- virus avian ALV rodent MuLV, MMTV, IAP feline FeLV
Disease myeloid leukemia lymphoid leukemia erythroleukemia mammary carcinomas
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Normal progenitorblood cells
Preleukemic phasewith progression
Leukemia- malignant transformation
Insertional mutagenesis
Additional oncogenic event(s)
Rapid Expansion
How Insertional Mutagenesis Leads to Leukemia
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Types of Cooperating events
Inflammation (immunological response)
Activation of a another oncogeneTranslocation, mutation, deletion(transgenic mouse expressing an oncogene)
Inactivation of a tumor suppressor (TS)Deletion, mutation, hypermethylation(mouse with a targeted deletion of a TS)
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How these events affect cells
Loss of cell cycle control
Block in terminal differentiation which is normally associated with growth arrest
Inhibition of apoptosis
Altered adhesion to stromal cells-allowing metastasis
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Model Involving Insertional Mutagenesis and InflammationThat Leads to Acute Myeloid Leukemia
Wolff et al, J Immunol. 141:688,1988
Wolff and Nason-Burchenal, Curr. Topics in Immunol. 149:79,1989
100 % of mice
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Effects of Provirus Into an Oncogenic Locus Is Not Observed Without Chronic Inflammation
Pristane, week after virus Incidence (%)
Latency after
Virus (days)
Latency after pristane (days)
None 0
-3 63 109
1 58 103 96
3 43 116 95
16 25 207 95
Nason-Burchenal and Wolff. PNAS 90:1619, 1993
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Lessons learned about insertional mutagenesis from this study
Provirus integrated next to the oncogene (c-Myb) can be detected in the bone marrow of 83% of the mice as early as 3 weeks following virus inoculation using a sensitive nested RT-PCR. This was way before any sign of disease (approx. 3 mo). (Nason-Burchenal and Wolff. PNAS 90:1619, 1993)
A minumum of one provirus can be found in many neoplasms (Wolff et al., J. Virology 65:3607, 1991)
(Koller et al. Virology 224:224,1996)
Effects of provirus at site of an oncogene can remain “dormant” until these cells are effected by other cancer promoting events such as an inflammatory response (stimulates cells to proliferate).
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Southern Analysis Showing Single Proviruses in Genome
BK
BK
BK
Proviruses in the Mml locus or unknown locus
Proviruses inThe Myb locus
EcoRI / Viral LTR probe
Mm
l1
Mm
l1
Mm
l1
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Collaboration of two genetic oncogenic events: use of the retrovirus to provide a second hit in genetically
engineered mice.
Transgenic mouse expressing an activated oncogene
Knockout mouse with deletedtumor suppressor
HumanOncogene
Tumor suppressorx
virus virus
1. Provides proof that the genetic alteration in the mouse is indeed oncogenic in the case that it has no effect by itself.
2. Used to identify cooperating genetic events. Provirus tags the site of integration
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Acceleration of Acute Myeloid Leukemia (AML) in a Transgenic Mouse Expressing
Human Oncogene CBF-MYH11
CBF-MYH11 - gene encoding an aberrant transcription factor INV16 in acute myeloid leukemia in man (12% of AML)
Cbf-MYH11
Paul Liu, NHGRI, NIH Castilla et al. Cell 87:687, 1996
Human MYH11 sequenceknockin at the mouse Cbf locus
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30
40
50
60
70
80
90
100
0 5 10 15 20 25
WT with ENU or retrovirusKI with retrovirusKI untreated
% S
urv
iva
l
time after treatment (month)
Cbf-MYH11 alone
Cbf-MYH11 + Retrovirus 4070A
Retrovirus 4070A
Collaboration between thePaul Liu and Linda Wolff labs(unpublished)
Use of retroviruses in acceleration of AML in mice expressing Cbf-MYH11
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EVENTS Myeloid Leukemia Reference
p15Ink4b -/- NONE
(Extramedulary myelopoiesis, lymphoid hyperplasia)
Latres et al. EMBO J 19:3496, 2000 (M. Barbacid lab)
Wild-type +/+ mice and retrovirus NONE Wolff lab
p15INK4b +/- and retrovirus 18% same
p15INK4b -/- and retrovirus 15% same
Retrovirus Provides Second Hit in Validation of a Proposed Human Tumor Suppressor (p15INK4b) in Leukemia
p15INK4b is Hypermethylation in 80% Human AML
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Can non-replicating virus such as a vector cause leukemia through the process of insertional mutagenesis?
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2. Later evidence that malignant transformation due to Retroviral insertional mutagenesis
1. Erythroleukemia without replicating helper-virus
Wolff and Ruscetti, Malignant Transformation of Erythroid Cells in Vivo by Introduction of a non-replicating Retrovirus Vector. Science 228: 1549, 1985
Wolff, Tambourin, Ruscetti, Induction of the Autonomous Stage of Transformation in Erythroid Cells Infected with SFFV: Helper Virus is Not Required. Virology 152: 272, 1986.
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LTR LTRgag pol env LTR LTRgag pol env
env: Recombinationdeletion, insertion
Fr-SFFV Fr-MuLV (replication competent helper-virus)
Erythroleukemia Induced by Friend Virus in Mice
gp52
Expansion of erythroblasts in spleen due to gp52
Malignant transformation of erythroblasts-block in differentiation (due to helper-virus?)
1st Stage
2nd Stage
2nd stage transformationDemonstrated by:
1.transplantation to other mice2.growth outside of the spleen in the omentum-autonomy
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Production of helper-free virus
pMov-
-2Packaging cell line
Mann, Mulligan, BaltimoreCell 33:153, 1983
Transfect SFFV DNA
SFFV
SFFV SFFV
SFFV
NIH3T3 cells
Test for lack of helper virus
5 days
gp85env
gp52gp52
-2
SF
FV
SF
FV
NIH3T3 with supes -2-2SFFVSFFV
Wolff and Ruscetti, Science 228: 1549, 1985
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Injection of help-free SFFV into mice
SFFV
Enlarged spleenErythroblast hyperplasia And malignant transformation
Tests for lack of replicating virus
Spleen
Cell free extracts
No disease
NIH3T3 cells
No replicating virus
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Tranplantation and growth in the omentum
Wolff, Tambourin, Ruscetti, Virology 152: 272, 1986.
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Cell lines derived from tranplantable neoplasms were free of replication competent helper virus
Wolff, Tambourin, Ruscetti, Virology 152: 272, 1986.
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Moreau-Gachelin, et al. Spi-1 is a putative oncogene in virally induced murine erythroleukemias. Nature 331: 277,1988
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Malignant Transformation by Helper-free SFFV Is Associated With Retrovirus Integration into Spi-1/PU.1
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Summary
1. Retroviruses are capable of activating oncogenes by integrating next to or near these genes and activating them transcriptionally so that they
are expressed.
2. These activating events can collaborate with previous or future oncogenic events in the cell to induce lymphoid, myeloid, or erythroid leukemia.
3. Chronic inflammation in a mouse model was shown to promote neoplastic progression in conjunction with retroviral mutagenesis.
4. Evidence was provided in a mouse model that replication defective viruses can integrate into DNA, activating an oncogene leading to overt leukemia.