Retroviral Insertional Mutagenesis and Cancer in Animal Models

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					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
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
Nucleus               Integration is essentially random
       provirus         throughout the genome

      Integration     Cell division required for efficient
   into genomic DNA    integration
                    Cellular Genome
        Provirus                   Proto-oncogene

 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




                provirus




Virus integrated at the 3’ end of gene---enhancer activation

                                          provirus
      Insertional Mutagenesis
Type of genes                  Species- virus

  growth factors                  avian ALV
  growth factor receptors         rodent MuLV, MMTV, IAP
  cytoplasmic kinases             feline FeLV
  transcription factors


                 Disease

                     myeloid leukemia
                     lymphoid leukemia
                     erythroleukemia
                     mammary carcinomas
       How Insertional Mutagenesis Leads to Leukemia


Normal progenitor
                                    Insertional mutagenesis
   blood cells



Preleukemic phase
                                    Additional oncogenic event(s)
with progression




Leukemia- malignant                       Rapid Expansion
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                   Latency after     Latency after
   after virus      Incidence (%)   Virus (days)      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

 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)
                          BK



                               Proviruses in the
                                 Mml locus
                          BK   or unknown locus

                          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.
 virus                                       virus

                   Human
                   Oncogene                                    x
                                                              Tumor
                                                             suppressor



      Transgenic mouse expressing an          Knockout mouse with deleted
      activated oncogene                      tumor suppressor




 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        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
             100                                               WT with ENU or retrovirus
                                                               KI with re tro virus
                                                               KI untrea ted
              90

              80
% Survival




              70                                                Cbf-MYH11 alone

              60
                                Cbf-MYH11
                                 + Retrovirus 4070A
              50

              40

              30
                   0        5          10              15      20            25
                                    time afte r tr e atme nt
                                           (month)
                                                                     Collaboration between the
                                                                     Paul Liu and Linda Wolff labs
                                                                     (unpublished)
     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

                                      NONE                           Latres et al. EMBO J 19:3496,
p15Ink4b -/-                                                         2000 (M. Barbacid lab)
                                      (Extramedulary myelopoiesis,
                                      lymphoid hyperplasia)
Wild-type   +/+ mice and retrovirus   NONE                           Wolff lab



p15INK4b     +/-   and retrovirus     18%                            same



p15INK4b -/- and retrovirus           15%                            same
Can non-replicating virus such as a vector cause leukemia
    through the process of 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.



2. Later evidence that malignant transformation due to
     Retroviral insertional mutagenesis
        Erythroleukemia Induced by Friend Virus in Mice

     Fr-SFFV           env: Recombination    Fr-MuLV (replication competent
                       deletion, insertion
                                                        helper-virus)
 LTR     gag     pol     env        LTR      LTR     gag    pol    env         LTR


                        gp52




                                             2nd stage transformation
1st Stage                                    Demonstrated by:

   Expansion of erythroblasts                1.transplantation to other mice
     in spleen due to gp52                   2.growth outside of the spleen
                                               in the omentum-autonomy

2nd Stage
Malignant transformation of erythroblasts
-block in differentiation (due to helper-virus?)
          Production of helper-free virus
   Transfect SFFV                                          Test for lack of
         DNA                                                 helper virus

                            SFFV          SFFV




          pMov-               SFFV                         NIH3T3 cells
                                      SFFV                                 5 days
                                                 -2
                                                                 NIH3T3 with supes
                                                                         -2 -2
        -2                                                             SFFV SFFV


  Packaging cell line
Mann, Mulligan, Baltimore
   Cell 33:153, 1983           gp85env

                                   gp52
                                                                                         gp52



                                           Wolff and Ruscetti, Science 228: 1549, 1985
          Injection of help-free SFFV
                    into mice
   SFFV                        Tests for lack of replicating virus


                                         Spleen
                                     Cell free extracts

Enlarged spleen
Erythroblast hyperplasia                          NIH3T3 cells
And malignant transformation



                                No disease         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.

				
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