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

•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

Retroviruses were first discovered in association with cancer around the turn of the century

cell-free extract

cell-free extract

Leukemia Leukemia

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

Integration into genomic DNA

Nucleus Integration is essentially random throughout the genome

Cell division required for efficient integration

provirus

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.

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

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

Normal progenitorblood cells

Preleukemic phasewith progression

Leukemia- malignant transformation

Insertional mutagenesis

Additional oncogenic event(s)

Rapid Expansion

How Insertional Mutagenesis Leads to Leukemia

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)

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

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

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

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).

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

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

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

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

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

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

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.

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

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

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

Tranplantation and growth in the omentum

Wolff, Tambourin, Ruscetti, Virology 152: 272, 1986.

Cell lines derived from tranplantable neoplasms were free of replication competent helper virus

Wolff, Tambourin, Ruscetti, Virology 152: 272, 1986.

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

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

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.