Epigenetic Regulation_ Stem Cells and Cancer

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					   Epigenetic Regulation,
   Stem Cells and Cancer


              Rudolf Jaenisch
Whitehead Institute and Dept. of Biology, MIT,
                 Cambridge, MA
Development and Differentiation




                                   Genetic

                                      or

                              epigenetic changes?
      Levinson and Sweatt, 2005
Genetics vs. Epigenetics
Maintenance of DNA methylation and
        histone modification




                              Felsenfeld, 2007
Histone Modifications




               Allis, Jenuwein, Reinberg, 2007
One Genome, many Epigenomes




                   Allis, Jenuwein, Reinberg, 2007
            Three Definitions of
                Epigenetics

1. Transmission of information through meiosis or mitosis
   that is not based on DNA sequence
2. A mechanism for stable maintenance of gene
   expression states that involves physically “marking”
   the DNA or its associated proteins
3. Mitotically or meiotically heritable changes in gene
   expression that are not coded in the DNA itself


  Relevant for development and cancer
Epigenetic Regulation,
  a mechanism that
allows the genome to
 integrate
    – intrinsic with
    – environmental signals
    Epigenetics and
   Disease Relevance
Diet / environment alters epigenetic
  state of genome and may affect
  incidence of long latency / late stage
  diseases such as:

  – Cancer
  – Neuro-degenerative diseases

  Gene expression affected by diet?
     Pseudoagouti Allele: Mice with
          Variable Coat Color
Agouti gene (A): brownish coat of wild mice
    AI A P
       allele: insertion of IAP retroelement
           •
      coat variegates between yellow and wild type
           •
        color depends on methylation of IAP
                                          Expression
                                              (phenotype)
   A allele (wt):                               Skin

    AI A Pallele,   ****                               } normal
   methylated       IAP                         Skin


    AI A Pallele,                               Uiquitous:
                                                b
   un-methylated     IAP                       yellow, obese,
                                                    tumors
Environmental and epigenetic changes:
 short-term and long-term outcomes




                                Turner, 2007
Epigenetics, Environmental Stimuli
  and Disease: A few questions


How does diet affect long latency
   diseases?
    •   Diet strongly affects cancer incidence
          -- Mechanisms?
    •   How about neurodegenerative diseases
        such as AD, Parkinson’s?
          -- Not a “clonal” disease: how to study?
Nuclear Cloning, Stem Cells and
  Epigenetic Reprogramming


        Relevance for
    transplantation therapy
Medical Challenges and the
  Potential of Stem Cells

• Increasing population age:
  – Alzheimer, Parkinson, heart failure….

Potential solution:
 Regeneration, tissue repair

Problem: Suitable donor cells
      Tissue Repair by Cell
         Transplantation:
      Historical Perspective
• Bone marrow transplantation
  – Established medical treatment since the 70s to treat
    leukemia
  – Problem: finding suitable donors, immune rejection

• These problems are more serious for
  transplantation repair of other tissues

What is the goal stem cell research?
     To provide matched cells for
      “customized” tissue repair
                                                                    Adult
             Stem Cells:
                                                                                B
    A developmental hierarchy                               Blood               T

                                                                                WBC
             Embryonic
                                                                 ?
                                                                                 RBC

                                                                                 Plts
                                                             Mesenchymal-
                                                              Connective
     +
             Zygote                                                            Bone
           (TOTIPOTENTIAL)   Blastocyst
                                                                    ?           Muscle

                                                              Neural
                                                                                    Fat
                                                                            Neurons
                                                                            Oligoglia
                               Embryonic                                    Astroglia
                               Stem Cells
                                                             Liver/Pancreas
                                 In vitro                    Skin, Testes, Gut
G. Daley    Pluripotent                        All cell types
                             differentiation
     Sources of Pluripotent Cells




Surani and Reik, 2006
  ES Cells: A Model System for Development
                               Pluripotent
                           Embryonic Stem Cells




     Ectoderm                Endoderm               Mesoderm    Gametes/
                                                                Germ cells
                                                                             Multipotent
    Neural   Skin  Hepatic Intestinal Pancreatic Hematopoietic               progenitors
     stem    stem progenitor stem progenitor
     cells                                         stem cells                   Tissue
             cells   cells    cells     cells
                                                        Mesenchymal          precursors/
                                                          stem cells         tissue stem
                                                                                 cells

                   Hepatocytes
    Neurons Skin                        Pancreas   Blood     Bone     Egg
Oligodentrocytes                                           Cartilage Sperm    Somatic
                            Intestine
   Astrocytes                                               Muscle             Cells
                                                              Fat
         Potential of Embryonic Stem Cells




                  Tissue specific
                  precursor cells   Differentiated
  Embryonic                         somatic cells
  stem cells

self-renewal
pluripotency
               Fetal/adult
     ?         stem cells
                                                     Transplantation
                                       Tissue
                                     engineering
The interest in stem cells has grown exponentially

                                             GROWTH IN PUBLICATIONS
                                             Publications on stem cells
                                               ABOUT STEM 20 years
                                               over the last CELLS
                        1400

                        1200
Publications per year




                                                                                                  EMBRYONIC
                        1000                                                                      STEM CELLS
      PubMed




                        800                                                          Human
                                                                                    ES Cells
                        600  Mouse
                                                                                    Isolated
                            ES Cells
                                                                                                           HUMAN
                        400 Isolated
                                                                                                          ES CELLS

                        200
                                                                                                               ADULT
                                                                                                                STEM
                           0                                                                                    CELLS




                                                                                                                 6*
                          80

                                 82

                                        84

                                              86

                                                     8

                                                            0

                                                                   92

                                                                        94

                                                                               6

                                                                                     98

                                                                                            00

                                                                                                   02

                                                                                                          04
                                                      8

                                                             9




                                                                                9




                                                                                                                  0
                         19

                               19

                                      19

                                             19

                                                   19

                                                          19

                                                                 19

                                                                        19

                                                                             19

                                                                                    19

                                                                                          20

                                                                                                 20

                                                                                                        20

                                                                                                               20
                                                                    Year
                                                                     Year                                  *Projected
                                                                                                           G. Daley
  Therapeutic Limitations
  of Embryonic Stem Cells
• ES cells are derived from donated
  embryos:

 This causes immune rejection

One potential solution:
  – Nuclear cloning to create
    “customized” ES cells
 NT to create patient-specific ES cells
        Reproductive cloning
           Nuclear transfer

Donor                                          oocyte
 cell




        Nuclear
        Transfer
                                        Customized patient-specific
                                                 ES cell




                             Inner
                            Cell Mass
                                                        G. Daley
                   Blastocyst
   Nuclear Cloning is very
            Inefficient

• Most clones die soon after implantation

Question:
  Survival of NT clones dependent
   on differentiation state of donor
               cell type?
Loss of Nuclear Potency with
  Increasing Age of Donor
An old question:
   Is the genome of
terminally differentiated
cells reprogrammable by
   nuclear cloning?
 Nuclear Cloning of Terminally
     Differentiated Cells
     Genetic vs. epigenetic changes
A.   Monoclonal mice:
     •   B / T cells: visualization of Ig and TCR genomic
         rearrangements
           (Hochedlinger and Jaenisch, 2002)

B.   CNS: Cloning of postmitotic mature
     neurons
     •   Does brain development / neuronal functions involve
         epigenetic as well as genetic alterations?
           (Eggan et al, 2004; Li et al, 2004)

C.   Cancer:
     •   Can the malignant state be reversed?
           Distinction between epigenetic (= reversible) and
           genetic (= irreversible) changes in tumor
           (Hochedlinger et al, 2004; Blelloch et al, 2004)
   State of Donor Cell Differentiation
         and Efficiency of NT:
      Higher Survival of ES Cell Clones

Donor Cells                           Survival to adults
                                     (from cloned blastocysts)

Somatic cells                                      1-3 %
     Fibroblasts, Sertoli, cumulus cells

Terminally differentiated cells                 < .001 %
     B, T cells, neurons, cancer


ES cells                   15-25 %
  ES nuclei are easier to reprogram
    Lesson from Nuclear
         Cloning:

Differentiation state of donor cells
affects reprogramming efficiency


     Likely due to differences in
          epigenetic state
   Gene Expression and
Phenotype of Cloned Animals
1.   Widespread faulty gene expression
       •   4-5% of all genes
       •   30-50% of imprinted genes

2.   "Normal" appearing clones often develop serious
     abnormalities with age


      Faulty reprogramming may preclude the
        generation of normal cloned individuals

 However, offspring of clones are always normal
(The problem are not mutations but an abnormal epigenetic state)
Degree of Abnormalities in Clones:
 A continuum with few defined stages
                Implantation            Birth


                           Dead             Survivors
 Survival




                                                Long term Survivors:
                                                 Are they really
                                                   "normal"?




                       Age of clones


       Higher                                    Lower
                    Degree of abnormality
 Cloning of Humans
   Lessons from animal cloning:
• Even clones that survive to birth have often
     serious abnormalities and die later
• Widespread epigenetic dysregulation



  “Normal” clones may be the
          exception
Charlatans, Clowns and Publicity:
     Consequences for Legislation ?




Testifying in the
 United States
   Congress:
 Therapeutic Applications of Embryonic Stem Cells




                       Sexually      Embryonic
                       produced      stem cells
                        embryo


ES cells from IFV embryos: different from patient, immune rejections




                        Asexually   “Customized”
                        produced     embryonic
                         embryo      stem cells




“Customized” ES cells from cloned blastocysts: patient’s own cells
                                      Cell 2002, 109: 17-27

                                                              βThalassemia
             Egg      Tail Tip Cell                           Sickle cell anemia
                                                   Rag2-/-    Fanconi’s anemia
                                                              Leukemia




                                                                II. Derivation
                                                               of bone marrow
                                                                   cells and
I. Nuclear transfer                                            transplantation
    and ES cell                                                 into “patient”
     derivation



                               Gene Correction

                         Cloned                 Corrected
                         ES Cells                ES Cells            G. Daley
     Conclusion
In principle,
 therapeutic SCNT will work in humans
   to generate “customized” cell for
   treatment of
      – Parkinsons
      – Diabetes
      – Blood diseases…..


            BUT…..
    Problems with
  Therapeutic SCNT

1. Procedure too inefficient, costly
   for routine treatment

2. Ethical objections to using
   human eggs for therapy
“Customized” Cells for Tissue Repair:
          The key issue
Achieving reprogramming without
  the need for human eggs
  •   We need to understand the
      reprogramming rules



 The egg does not accomplish a
    miracle but a biochemical
             reaction
    Dedifferentiation and differentiation in the test tube:
       A strategy for cell based therapy

                                      Somatic cells
   Cells from                          Fibroblasts,
    patient                               Skin…


                                    Reprogramming
                                     in petri dish




                         “Reprogrammed” ES cell
                                     Differentiation
                                      in petri dish



 “Customized”            Differentiated cells for transplantation
                                Neurons, Muscle, β cells...
cells for therapy
Reversibility of the Epigenetic State




               Change in epigenetic state




granulocytes
   Different strategies to
    generate pluripotent
     from somatic cells

• Nuclear transplantation of
  somatic cell nuclei
• Fusion of ES and somatic cells
• Direct conversion of a somatic
  into an ”ES” like cell
 Somatic  embryonic epigenetic state
       Key question of
       Reprogramming:
•     Why is NT with somatic donor
      nuclei so inefficient?
•   What is the molecular circuitry
    that distinguishes pluripotent
    from somatic cells?
If we understand the key epigenetic switches of
    differentiation, we may eventually be able to
         convert one cell type into the other
          (“transdifferentiation, plasticity”)
Death of Clones after Implantation:
                       Questions
                Implantation                Birth




                        }
                            Dead
                              • Why              Survivors
                                      do most clones
 Survival




                                 die after implantation?
                               • Which genes not
                                 correctly reprogrammed?




                       Age of clones


       Higher                                       Lower
                    Degree of abnormality
 “Pluripotency” Genes
         Oct4, Nanog, Sox2
• Expressed in early embryo, ES
  cells, not in somatic cells
• Regulators of pluripotency and
  self-renewal
• Following NT their activation
  appears crucial for
  – Cloned embryos to survive after
    implantation 2002; Bortvin, 2003; Chambers, 2003; Mitsui, 2003)
     (Nichols, 1999; Boiani,
    Oct4, Nanog, Sox2:
Key regulators of stem cells

Issues:
 – Molecular control circuits of
   • Self-renewal
   • Pluripotency
 – Embryonic vs. adult stem cells
   • Similar / different regulation?
          Embryonic Stem (ES) Cells



                                    Key Properties:
                                    • Self-renewal
                                    • Pluripotent


                                  What controls
                                  ES cell
                                  identity?

Important implications for understanding nuclear reprogramming
            and directed differentiation of ES cells.
The molecular circuitry of
    pluripotency and
      self renewal
We have to understand the
 key factors that

 – Set up the pluripotent state
 – Maintain the pluripotent state
 – Induce differentiation of stem
   cells
        Genome-wide Location Analysis
Protein:DNA   interactions
                                       Oligonucleotide Promoter Array




                                    Oligos chosen to represent this region



                                                        Your Favorite Gene Here


                             Targets 8 kbp upstream and 2 kbp downstream of
                             transcription start sites for all annotated genes
R. Young
                             (~18K) in the human genome (RefSeq, Ensembl,
                             MGC, H-inv)
Oct4, Sox2, Nanog Co-Occupy Many Target Genes
               in Human ES Cells
Transcriptional Regulatory Hierarchy in Human ES Cells




                                    Activation
                                 Transcription Factors
                                 Chromatin Regulators
                                 Cell Cycle Regulators
                                  Signaling Molecules




                                   Repression
                                    Developmental
                                 Transcription Factors
 Insights from Core Regulatory Circuitry
      in Human Embryonic Stem Cells
Oct4, Sox2, and N a n o g:

• Co-occupy many target genes

• Associate with active genes encoding proliferation factors

• Associate with inactive genes encoding developmental
  transcription factors

• Contribute to specialized regulatory circuitry that provide
  important clues into ES cell pluripotency


                          Nanog Sox2
             self-renewal     Oct4


                      ES               Specialized cell
                      cell
       Polycomb Group (PcG) Proteins
•   Identified in Drosophila as regulators of homeotic (HOX)
    genes
•   Conserved from fly to human
•   Essential roles in early embryonic development
•   Maintain repressive gene expression patterns and cellular
    identity through epigenetic modification of chromatin
   Regulation of Pluripotency in
      Embryonic Stem Cells

Self-renewal,               Nanog
pluripotency                     Oct4

                     ES               differentiated
                     cell             cell

                            PcG proteins


  Oct4, Nanog and PcGs co-occupy a set of repressed genes
      that encodes developmental transcription factors.

  This set of target genes must be repressed
            to maintain pluripotency
             ES cell regulatory circuitry



                           Pol II



  self-
renewal




                           Suz12
          differentiated
               cells
Ethical / Political Problems
with Embryonic Stem Cells
  and Therapeutic SCNT:

   Scientific solutions?
      Any solutions?
The New Yorker
Alternatives to embryonic stem
             cells?

 Such as Adult Stem cells?

              or

 Do we need to use SCNT to
   generate “customized”
    ES cells for therapy?
           Different Approaches to obtain “Customized”
            Pluripotential Cells from Postnatal Animals
                                                 Promise Challenges

                                                 Reproductive               Clones are
                                                   cloning                  abnormal



                                                  Customized                  Ethics,
                                                   ES cells                 human eggs


                                                Reprogramming               Fused cells
                                                  in test tube                are 4n



                                                                        No evidence of
                                                    Simplicity
                                                                        reprogramming


                                                Spermatogonial
                                                                            Disturbed
                                                  Stem Cells
                                             (derived from spermatigonia)   Imprinting
                                              No ethical problems
                                                                              What are
                  reprogramming”
Transduction of “reprogramming”
                                                Reprogramming                  the key
  factors into somatic cells and
    selection for activation of                   in test tube               epigenetic
       pluripotency genes
                                                                             effectors?
    Dedifferentiation and differentiation in the test tube:
       A strategy for cell based therapy

                                      Somatic cells
   Cells from                          Fibroblasts,
    patient                               Skin…


                                    Reprogramming
                                     in petri dish




                         “Reprogrammed” ES cell
                                     Differentiation
                                      in petri dish



 “Customized”            Differentiated cells for transplantation
                                Neurons, Muscle, β cells...
cells for therapy
Epigenetics, Cancer and
 the Reversibility of the
    Malignant State
Progressive promoter methylation and
   selection for silencing in tumors




                               Jones and
                              Baylin, 2006
Genes that are frequently hypermethylated
        and /or mutated in cancer




        Only mutated
       Only methylated       Jones and Baylin, 2006
    Methylated and mutated
Epigenetic Alterations
     and Cancer:

• How stable is the
  malignant state?
• Reversible by nuclear
  transfer into egg?
                 The Concept

     Normal                          Tumorigenesis
     development

        embryonic cell                     normal cell
                               genetic
                               changes
epigenetic                        +
                 Nuclear                           ? Nuclear
 changes                      epigenetic
                 cloning                           cloning ?
                               changes

        differentiated cell                malignant cell
      Nuclear Cloning and Cancer
                                  Eggs
   Tu m o r donor c e l ls      injected
                                           Blastocysts ES cell lines


       Lymphomas,                 1450           30               0
        leukemias

             p53-/- spontaneous
  Solid      breast cancer          189          23               0
             (cell line CK5)
 tumors
             Ras-inducible melanoma 600           8              2



 Somatic
               Total              2515          6 1( 2 . 5 % )   2 (3 %)
 tumors


Embryonal      METT1,
                                   435          32 ( 7 % )       17 ( 5 3 %
carcinomas     P19, F9
                                                                       )
Melanoma donor



                 NT ES cell derived
                  from melanoma
Melanoma Derived ES Cells form Chimeric Pup




                                         GFP




                                         light



         kidney    heart     intestine
Chimeric mouse from Melanoma Donor
          Derived ES cell
Coat color and tumors in chimeras




   Chimera # 1   Chimera # 3   Chimera # 6




   Chimera # 2   Chimera # 4   Chimera # 8
  Reprogramming of Cancer Cell
           Genome
Malignant tumor cell genome                  can be
      reprogrammed by nuclear transfer into pluripotent
      ES cells with the potential to differentiate into
      most if not all somatic tissues



Key question:
  •     NT ES cell derived from tumor cell or non-tumor
        support cell?

Approach:
  •     CGH analysis of donor cells, NT ES cells and
        chimeras (tumors and fibroblasts)
                   CGH Analysis of Melanoma Donor and
                     their Cloned ES Cell Derivatives

                  R545
 Donor




                 parental
                   R545
                   SCID
                  tumor
                  R545-1
                 NT ES cells

                Melanoma
R545-1 NT ES
 cell derived




                 Rhabdo-
                myosarcoma

                  MPNST

                 Fibroblasts




                   Chr 8
                               R545 parental   R545-1 NT ES
Reprogramming of Cancer
      Cell Genome

Malignant tumor cell genome
 –   Can be reprogrammed by nuclear
     transfer into pluripotent ES cells with
     the potential to differentiate into most if
     not all somatic tissues

 –   Tumor phenotype largely determined by
     epigenetic changes
Somatic Stem cells / Cancer Stem Cells




                       Pardal, Clarke, Morrison, 2003
Epigenetic silencing, stem cells and cancer




                           Baylin and Jones, 2007
Epigenetic modifications in cancer are
   reversible: relevance for therapy




                         Allis, Jenuwein, Reinberg, 2007
                  M. Guenther
L. Boyer          T. Lee
K. Plath          M. Cole
A. Meissner       S. Johnston
Y. Yamada         R. Jenner
C. Beard          B. Chevalier
T. Brambrink      J. Zucker
R. Blelloch       S. Levine
Z. Wang           T. Volkert
                  R. Young
M. Wernig
                  D. Gifford
L. Medeiros
                  M. Kyba
M. Ray
                  G. Daley
A. Tajonar
K. Hochedlinger
K. Eggan          C. Brennan
                  M. Kim
                  L. Chin (Harvard)

				
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