Basic mechanisms of leukemogenesis Basic mechanisms of leukemogenesis QuickTime¢â€‍¢ and a TIFF (LZW)

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  • Basic mechanisms of leukemogenesis

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    J. Schwaller MD, University Hospital Basel [J.Schwaller@unibas.ch]

  • Basic molecular mechanisms of leukemogenesis

    Part I

    What is leukemia?

    Genetic aberrations leading to leukemia a) Alterations in tyrosine kinases (BCR/ABL) b) Alterations in transcriptional regulators (CBF, RARα)

    Cooperative model - therapeutic consequences

    Part II

    Leukemic stem cells

  • Blood formation: hematopoietic hierarchy

    Self renewal

    Self renewal

    Myeloid Lymphoid

    Stem cells

  • Normal (Np, Ly) Normal (Np, Eos)

    Normal (Ba) Normal (Ly)

    Normal cells as detected in smear of peripheral blood.

  • Leukemia: cancer of the hematopoietic system

    Too many hematopoietic cells with normal or blocked differentiation occupy the hematopoietic organs such as peripheral blood, bone marrow, and spleen.

    The main consequence is functional insufficiency of the blood cell forming system resulting in anemia, infection, and bleeding episodes.

  • LEUKEMIA = LEUKOS = “White blood”

  • Acute leukemiaChronic leukemia

    Block in normal cell differentiation

    Normal differentiation

    Clinic: rapidClinic: gradual

    Leukemia: cancer from blood-forming system often associated with an increase in white blood cells

  • “Leukemia” summarizes a large number of different diseases

    Chronic myeloproliferative disorders - Chronic myeloid leukemia (CML) - Polycythemia vera (PV) - Essential thrombocythemia (ET) - Hypereosinophilc syndrome (HES) - Chronic idiopathic myelofibrosis (OMF)

    Myelodysplastic/Myeloproliferative disorders (e.g. CMML) Myelodysplastic syndromes (MDS)

    Acute myeloid leukemia (AML)

    B-cell acute lymphoblastic leukemia (ALL)

    T-cell/NK-cell acute lymphoblastic leukemia

    Chronic lymphoproliferative disorders (CLL)

  • Leukemia: cancer of the hematopoietic system

    CLL

    CML

    CML: chronic myeloid leukemia; CLL: chronic lymphocytic leukemia;

    QuickTime™ and a TIFF (LZW) decompressor

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    Chronic myeloid leukemia: affects all myeloid lineages: differentiation maintained

  • Leukemia: cancer of the hematopoietic system

    AML

    ALL

    Acute leukemia: differentiation block at progenitor stage

    AML: acute myeloid leukemia ALL: acute lymphoblastic leukemia;

  • MPO +/- CD13/CD33+, ev. CD22/CD79α (B), CD3 (T)

    CD34+, CD13+, ev. CD19+, ev. CD56+

    HLA-DR-, CD33/13+ CD65/CD15 +/-, ev CD2+

    CD34+, CD13+, ev. CD2+ HLA-DR+/-, CD34+/-, CD14- GlycoA+, CD36+, CD41-

    CD41/CD61+, CD13/CD33+/- CD7-

    Immunophenotype

    5q-, 7q-, +13, rearr. 2p, 12p t(8;21)(q22;q22): AML1/ETO t(15;17)(q22;q11): PML/RARα

    In(16)(p13;q22) t(16;16)(p13q22) CBFβ/SMMHC (MYH11) -5, -7, -12p, +8 +21; t(1;22)(p13;q13): OTT/MAL1

    Karyotype & Molecular genetics

    AML-FAB-M0 AML-FAB-M2 AML-FAB-M3

    AML-FAB-M4 AML-FAB-M6 AML-FAB-M7

    Morphology

  • chromosomal translocationschromosomal translocations –– fusion proteinfusion protein –– abnormal gene expressionabnormal gene expression

    numerical chromosomal aberrationsnumerical chromosomal aberrations –– deletions, deletions, aneuploidyaneuploidy

    MutationsMutations (point(point--, ITD..) in known , ITD..) in known oncogenesoncogenes or tumor suppressor genes.or tumor suppressor genes.

    Somatic mutations in leukemia

  • FIG. 13

    Metaphases from Leukemia case 4 (male); 46 chromosomes. Note: minute chromosome (arrow), x 2700.

    Chromosome studies on normal and leukemic human leukocytes [Peter C. Nowell & David. A. Hungerford, J. Nat. Cancer Inst. (25):85-109, 1960]

  • OLIGO S/T KINASEA B DBL/CDC24 PH RACGAP CAP SH3 SH2 SH1 KINASE NLS DNA ACTIN

    SH3 SH2 SH1 KINASE NLS DNA ACTINOLIGO S/T KINASEA B

    SH3 SH2 SH1 KINASE NLS DNA ACTIN

    SH3 SH2 SH1 KINASE NLS DNA ACTIN

    OLIGO S/T KINASEA B DBL/CDC24 PH

    S/T KINASEA B DBL/CDC24 PH RACOLIGO

    N-

    N-

    N-

    N-

    -C -CN-

    -C

    -C

    -C

    e1a2 p185

    b2a2 b3a2 p210

    c3a2 p230

    m-bcr p185

    M-bcr p210

    μ-bcr p210 abl

    m-bcr M-bcr μ-bcr abl

    e1 e1’e2 b2b3 c3 1b 1a a2 a3

    BCR Abelson kinase (ABL)

    Chromosome 22q11 Chromosome 9q34

    OLIGO S/T KINASEA B DBL/CDC24 PH RACGAP CAP SH3 SH2 SH1 KINASE NLS DNA ACTIN

    SH3 SH2 SH1 KINASE NLS DNA ACTINOLIGO S/T KINASEA B

    SH3 SH2 SH1 KINASE NLS DNA ACTIN

    SH3 SH2 SH1 KINASE NLS DNA ACTIN

    OLIGO S/T KINASEA B DBL/CDC24 PH

    S/T KINASEA B DBL/CDC24 PH RACOLIGO

    N-

    N-

    N-

    N-

    -C -CN-

    -C

    -C

    -C

    e1a2 p185

    b2a2 b3a2 p210

    c3a2 p230

    m-bcr p185

    M-bcr p210

    μ-bcr p210 abl

    m-bcr M-bcr μ-bcr abl

    e1 e1’e2 b2b3 c3 1b 1a a2 a3

    BCR Abelson kinase (ABL)

    Chromosome 22q11 Chromosome 9q34

    1983-86: Identification of the BCR/ABL fusion product that is present in close to 100% of CML cases.

  • [Mittelman et al., Nat. Rev. Cancer 2007]

    Clonal chromosomal abnormalities in human cancer

  • + +

    Chromosomal translocations: principle

    Promoter exchange: Deregulated expression

    P

    Oncogenic fusion genes

  • Genetic aberrations in leukemogenesis

    Functional classification

    Affecting cellular growth & survival

    Protein tyrosine kinases and related signaling mediators

    X-ABL, X-PDGFR FLT3 JAK2

  • Most frequent PTK alterations in leukemia

    ABL BCR-ABL CML (>90%) (9q22) (p210/p190/p230) B-ALL (>15%)

    PDGFβR TEL-PDGFβR CMML, EoCMPD (5q31-33) HIP-PDGFβR EoL, MDS+Eo

    H4-PDGFβR Rap5-PDGFβR CEV14-PDGFβR

    FLT3 FLT3-ITD AML (>20%) (13q12) FLT3-TKD AML/ALL

    X-FLT3 MPD/AML (rare)

  • Normal blood smear

    CML - chronic phase CML - blast crisis

    Chronic myeloid leukemia (CML)

    - >90% Ph+ = t(9;22) = BCR/ABL

    - expansion of the myeloid lineage with “normal” maturation

    - inevitable transformation from a chronic phase into a blast crisis resembling AML/ALL.

  • Class I mutationClass I mutation e.g. BCR/ABL

    ProliferationProliferation SurvivalSurvival

    CHRONIC PHASE CML

    HSC

    NORMAL

    Chronic myeloid leukemia (CML) is induced by t(9;22) [“Philadelphia chromosome”] in hematopoietic stem cells

  • Experimental approaches to assess the transforming properties of a PTK fusion

    N - - CTyrosine kinase domainDi/Oligomerization

    N - - CTyrosine kinase domainDi/Oligomerization x

    N - - CTyrosine kinase domainDi/Oligomerization x

    - cytokines (days)

    N um

    be r o

    f v ia

    bl e

    ce lls

    A) in vitro

    TEL JAK2LTR LTR

    Retroviral infection

    BMT

    Transgenic animals

    B) in vivo

    “Leukemia”

    [Growth-factor dependent cell lines]

  • 5’- -3’

    N- -C N- -C

    P PP

    P PP Di-/ oligomerization Auto-/ trans tyrosine-phosphorylation

    RAS/MAPKRAS/MAPK PI3K/PKB(AKT)PI3K/PKB(AKT) JAK/STATJAK/STAT NFNF--κκBB

    M a l i g n a n t p h e n o t y p e

    Malignant transformation by protein tyrosine kinase fusions through multiple signal transduction pathways

    PTKs: ABL, ABL, PDGFPDGFββRR, JAK2, JAK2...

    5’ partners: BCRBCR,, TELTEL, ….

  • Imatinib mesylate - STI-571 - GleevecTM

    Imatinib

    P TYR

    TYR

    P P

    P

    P

    BCR-ABL Substrat

    ATP Auto-phoshorylation

    Trans-phosphorylation

    P P

    P

    BCR-ABL

    [Schindler et al., Science, 289:1938, 2000]

  • Druker B, et al., 2006

    CML Therapy: the impact of imatinib

  • M

    Imatinib Mesylate STI-571

    mdr

    Gene Amplification

    Targeted Mutations (E255V/K,

    T315I)

    MDR expression

    AGP

    Alpha1 acid- glycoprotein binding (?)

    Apoptosis

    BCR-ABL

    Imatinib mesylate [STI-571/GleevecTM]: Resistance

    Multifunctional resistance mechanisms (variable latency)

    STI

  • XX--ABLABL XX--PDGFPDGFββRR

    Small molecule PTK inhibitors

    ??

    Primary and secondary drug resistance

    Signaling pathwaysSignaling pathways

    “where to interfere? “...

  • [Van Etten RA, 2007]

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    [Rosnet & Birnbaum, 2007]

    Protein tyrosine kinase fusion genes in human myeloproliferative disorders

  • Functional classification of genetic alterations in leukemia

    Conferring proliferative and/or survival advantage but do not affect differentiation

    “Class I mutations”

    Tyrosine kinase fusions

    - ABL - JAK2

    Gain of function point mutations

    - FLT3 - RAS - KIT - PTPN11

    Myeloproliferative disease (MPD, “CML-like”)

    “S