# Presentation: 656905

## 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 Research**
**Biological Response Modifiers Advisory Committee**
**Meeting #33, October 10, 2002**

## Outline of talk

- 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 isolateswere composed of two different viruses

**LTR**

**LTR**

**LTR**

**LTR**

**ONC**

***gag*****       *****pol       env***

- Defective genome

- Replication competent genome
- “helper-virus”

- Rapid disease

- Disease caused by
- Insertional mutagenesis

## During replication:integrate in DNA

**Integration **
**into genomic DNA**

**Nucleus**

**Integration is essentially random**
**   ****throughout the genome **
**Cell division required for efficient**
**   ****integration**

**provirus**

## Cellular Genome

**LTR**

**LTR**

***gag*****       *****pol       env***

**Proto-oncogene**

**mRNA**

**protein**

**Provirus**

**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**

## Onocogenes Activated

## 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

## Clonal progression

**Normal progenitor**
**blood cells**

**Preleukemic**** phase**
**with progression**

**Leukemia- malignant **
**transformation**

***Insertional mutagenesis***

***How Insertional Mutagenesis Leads to Leukemia***

## 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)**
**	**

## 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 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***

## 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 in***
***The Myb locus***

***EcoRI / Viral LTR probe***

***Mml1***

***Mml1***

***Mml1***

## 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 deleted**
**tumor suppressor**

**Human**
**Oncogene**

**   ****Tumor**
** ****suppressor**

- x

**virus**

**virus**

**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**

## 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 sequence**
**knockin at the mouse Cbf******** ****locus**

## Use of retroviruses in acceleration of AML    in mice expressing Cbf-MYH11

**Cbf********-MYH11 alone**

**Cbf********-MYH11**
**   ****+  Retrovirus 4070A**

**Retrovirus 4070A**

**Collaboration between the**
**Paul Liu and Linda Wolff labs**
**(unpublished)**

- Use of retroviruses in acceleration of AML    in mice expressing Cbf-MYH11

## Retrovirus Provides Second Hit in Validation of a Proposed Human Tumor Suppressor (p15INK4b) in Leukemia

| 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 |

**p15INK4b is Hypermethylation in 80% Human AML **

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

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

## Scan paper titles

**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.**

## Friend SFFV disease

**LTR**

**LTR**

***gag*****       *****pol       env***

**LTR**

**LTR**

***gag*****       *****pol       env***

***env****:* Recombination
- deletion, insertion

**Fr-SFFV**

**Fr-MuLV (replication competent **
**                    ****helper-virus)**

**Erythroleukemia Induced by Friend Virus in Mice**

**gp52**

## Production of helper-free virus

**pMov-******

******-2**
**Packaging cell line**
**Mann, Mulligan, Baltimore**
**Cell 33:153, 1983**

**Transfect SFFV**
**           ****DNA**

**SFFV**

**SFFV**

**SFFV**

**SFFV**

**NIH3T3 cells**

**Test**** ****for lack of **
**helper virus**

**5 days**

**gp85****env**

**gp52**

**gp52**

******-2**

**SFFV**

**SFFV**

**NIH3T3 with supes **

******-2**

******-2**

**SFFV**

**SFFV**

**Wolff and Ruscetti,  Science 228: 1549, 1985**

## Injection of help-free SFFV into mice

**SFFV**

**Enlarged spleen**
**Erythroblast hyperplasia **
**And malignant transformation**

## 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.**

-  induced murine erythroleukemias. Nature 331: 277,1988

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

## 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.**