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

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

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

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

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 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.

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

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

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

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

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)

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

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

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

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)

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, 2000 (M. Barbacid lab) Wild-type +/+ mice and retrovirus Wolff lab p15INK4b +/- and retrovirus 18% same p15INK4b -/- and retrovirus 15%

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

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

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

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

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

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 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.