1. Retroviral Insertional Mutagenesis and Cancer in Animal Models
FDA Center for Biologics Evaluation and Research
Biological Response Modifiers Advisory Committee
Meeting #33, October 10, 2002
Linda Wolff, Ph.D.
Chief, Leukemogenesis Section,
Laboratory of Cellular Oncology
Center for Cancer Research
National Cancer Institute, NIH

2. Outline of talk Retrovirus Integration in DNA and Cancer
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.
Cancer caused by non-replicating retrovirus
vector

3. Retroviruses were first discovered in association with cancer around the turn of the century
cell-free extract
Leukemia
Leukemia
cell-free extract

4. Many cancer causing retrovirus isolates were composed of two different viruses
LTR
ONC
LTR
LTR
gag pol env
LTR
Defective genome
Replication competent genome
“helper-virus”
Rapid disease
Disease caused by
Insertional mutagenesis

5. During replication:integrate in DNA
Integration
into genomic DNA
Nucleus
Integration is essentially random
throughout the genome
Cell division required for efficient
integration
provirus

6. Cellular Genome Provirus Proto-oncogene mRNA protein
LTR
gag pol env
LTR
mRNA
protein
Proto-oncogene = stimulates accumulation of cells in normal processes
Oncogene = activated proto-oncogene having increased capacity
to cause continued inappropriate growth.

7. Most Common Mechanisms of Transcriptional Activation
Virus integrated at the 5’ end of gene---promoter and or enhancer activation
provirus
promoter
Virus integrated at the 3’ end of gene---enhancer activation
provirus

8. Onocogenes Activated

9. 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

10. How Insertional Mutagenesis Leads to Leukemia
Clonal progression
How Insertional Mutagenesis Leads to Leukemia
Normal progenitor
blood cells
Insertional mutagenesis
Additional oncogenic event(s)
Preleukemic phase
with progression
Rapid Expansion
Leukemia- malignant
transformation

11. Types of Cooperating events
Inflammation (immunological response)
Activation of a another oncogene
Translocation, mutation, deletion
(transgenic mouse expressing an oncogene)
Inactivation of a tumor suppressor (TS)
Deletion, mutation, hypermethylation
(mouse with a targeted deletion of a TS)

12. 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

13. Model Involving Insertional Mutagenesis and Inflammation That 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

14. 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 -3 63
109 1 58
103 96 3 43
116 95 16 25
207
Nason-Burchenal and Wolff. PNAS 90:1619, 1993

15. Lessons learned about insertional mutagenesis from this study
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).
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)

16. Southern Analysis Showing Single Proviruses in Genome
Mml1
Mml1
Mml1 BK
Proviruses in the
Mml locus
or unknown locus BK BK
Proviruses in
The Myb locus
EcoRI / Viral LTR probe

17. Collaboration of two genetic oncogenic events: use of the retrovirus to provide a second hit in genetically engineered mice. x
virus
virus
Tumor
suppressor
Human
Oncogene
Transgenic mouse expressing an activated oncogene
Knockout mouse with deleted
tumor suppressor
Provides proof that the genetic alteration in the mouse is indeed
oncogenic in the case that it has no effect by itself.
Used to identify cooperating genetic events. Provirus tags the site of
integration

18. 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
Human MYH11 sequence
knockin at the mouse Cbf locus
Paul Liu, NHGRI, NIH
Castilla et al. Cell 87:687, 1996

19. Use of retroviruses in acceleration of AML in mice expressing Cbf-MYH11
Retrovirus 4070A
Cbf-MYH11 alone
Cbf-MYH11
+ Retrovirus 4070A
Collaboration between the
Paul Liu and Linda Wolff labs
(unpublished)

20. p15INK4b is Hypermethylation in 80% Human AML
Retrovirus Provides Second Hit in Validation of a Proposed Human Tumor Suppressor (p15INK4b) in Leukemia
p15INK4b is Hypermethylation in 80% Human AML
EVENTS
Myeloid Leukemia
Reference
p15Ink4b -/-
NONE
(Extramedulary myelopoiesis, lymphoid hyperplasia)
Latres et al. EMBO J 19:3496, (M. Barbacid lab)
Wild-type +/+ mice and retrovirus
Wolff lab
p15INK4b +/- and retrovirus
18%
same
p15INK4b -/- and retrovirus
15%

21. Can non-replicating virus such as a vector cause leukemia through the process of insertional mutagenesis?

22. Scan paper titles 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.
2. Later evidence that malignant transformation due to
Retroviral insertional mutagenesis

23. Expansion of erythroblasts
Friend SFFV disease
Erythroleukemia Induced by Friend Virus in Mice
env: Recombination
deletion, insertion
Fr-SFFV
Fr-MuLV (replication competent
helper-virus)
LTR
gag pol env
LTR
LTR
gag pol env
LTR
Expansion of erythroblasts
in spleen due to gp52
Malignant transformation of erythroblasts
-block in differentiation (due to helper-virus?)
1st Stage
2nd Stage
gp52
2nd stage transformation
Demonstrated by:
1.transplantation to other mice
2.growth outside of the spleen
in the omentum-autonomy

24. Production of helper-free virus
Test for lack of
helper virus
Transfect SFFV
DNA
SFFV
SFFV
pMov-
SFFV
NIH3T3 cells
5 days
SFFV
-2
NIH3T3 with supes
SFFV
SFFV
-2
-2
-2
Packaging cell line
Mann, Mulligan, Baltimore
Cell 33:153, 1983
SFFV
SFFV
gp85env
gp52
gp52
Wolff and Ruscetti, Science 228: 1549, 1985

25. Injection of help-free SFFV into mice
Tests for lack of replicating virus
Spleen
Cell free extracts
No disease
NIH3T3 cells
No replicating
virus
SFFV
Enlarged spleen
Erythroblast hyperplasia
And malignant transformation

26. Tranplantation and growth in the omentum
Wolff, Tambourin, Ruscetti, Virology 152: 272, 1986.

27. Cell lines derived from tranplantable neoplasms were free of replication competent helper virus
Wolff, Tambourin, Ruscetti, Virology 152: 272, 1986.

28. Moreau-Gachelin, et al. Spi-1 is a putative oncogene in virally
induced murine erythroleukemias. Nature 331: 277,1988

29. Malignant Transformation by Helper-free SFFV Is Associated With Retrovirus Integration into Spi-1/PU.1

30. Summary
Retroviruses are capable of activating oncogenes by integrating next
to or near these genes and activating them transcriptionally so that they
are expressed.
These activating events can collaborate with previous or future
oncogenic events in the cell to induce lymphoid, myeloid, or erythroid
leukemia.
Chronic inflammation in a mouse model was shown to promote
neoplastic progression in conjunction with retroviral mutagenesis.
Evidence was provided in a mouse model that replication defective
viruses can integrate into DNA, activating an oncogene leading to
overt leukemia.