The biology of chronic myeloid leukemia2007mcb.med.uoc.gr/mysite/arxeia/grad-study_papadaki_leukaemia.pdf · Deininger, M. W. N. et al. Blood 2000;96:3343-3356 Abl protein (Abelson

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The biology of chronic The biology of chronic myeloid myeloid leukaemialeukaemia

Graduate ProgramGraduate ProgramCourse: Biology of cancerCourse: Biology of cancerApril 2007April 2007

MyeloproliferativeMyeloproliferative disordersdisorders

ClonalClonal disorders of disorders of haemopoiesishaemopoiesis that arise that arise in a in a haemopoietichaemopoietic stem cell or early stem cell or early progenitor cell. progenitor cell.

They are They are characterisedcharacterised by the by the dysregulateddysregulatedproduction of a particular lineage of mature production of a particular lineage of mature myeloid cells.myeloid cells.

They exhibit a variable tendency to progress They exhibit a variable tendency to progress to acute to acute leukaemialeukaemia..

Chronic Myeloid Chronic Myeloid LeukaemiaLeukaemia

PolycythemiaPolycythemia

Idiopathic Idiopathic ThrombocytosisThrombocytosis

MyelofibrosisMyelofibrosis

MyeloproliferativeMyeloproliferative disordersdisorders

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Chronic Myeloid Chronic Myeloid LeukaemiaLeukaemia(CML)(CML)

CML is classified as CML is classified as clonalclonal myeloproliferamyeloprolifera--tivetive expansion of transformed, primitive expansion of transformed, primitive haemopoietichaemopoietic progenitor cells.progenitor cells.

It involves myeloid, It involves myeloid, monocyticmonocytic, , erythroiderythroid, , megakaryocytic, Bmegakaryocytic, B--lymphoid and occasionallymphoid and occasional--lyly TT--lymphoid lineages. lymphoid lineages.

CML: Clinical manifestations CML: Clinical manifestations (1)(1)

Frequency:Frequency:15%15%--20% of cases of 20% of cases of leukaemialeukaemia in adults.in adults.

Annual Incidence:Annual Incidence:11--2 cases / 100.000, with a slight male predominance.2 cases / 100.000, with a slight male predominance.

Median age:Median age:4545--50 years50 years

Risk factors:Risk factors:Ionizing radiation.Ionizing radiation.

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Chronic phase

Median duration5–6 years

Accelerated phase

Median duration6–9 months

Blast crisis

Median survival3–6 months

Advanced phases

Faderl S, et al. Ann Intern Med. 1999;131:207-219.Pasternak G, et al. J Cancer Res Clin Oncol. 1998;124:643-660.



2020--50% of patients asymptomatic50% of patients asymptomatic

Increase of Increase of WBCsWBCs (shift to the left)(shift to the left)

SplenomegalySplenomegaly ±± hepatomegalyhepatomegaly or or adenopathyadenopathy

Fatigue, malaise, weight loss, sweating, Fatigue, malaise, weight loss, sweating, abdominal fullness, bleeding abdominal fullness, bleeding

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Sawyers, C. L. N Engl J Med 1999;340:1330-1340

Characteristics of Patients with Chronic Myeloid Leukemia at Presentation

CML: Bone marrowCML: Bone marrow

CML: Bone marrowCML: Bone marrow

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Normal Chronic phase CML

CML: Peripheral Blood SmearCML: Peripheral Blood Smear

NORMAL CML

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CML: Molecular BiologyCML: Molecular Biology

CML is the first CML is the first haematologicalhaematological malignancy malignancy to be associated with a specific chromosome to be associated with a specific chromosome abnormality, the Ph chromosome.abnormality, the Ph chromosome.

It is the hallmark of CML and is found in up It is the hallmark of CML and is found in up to 95% of patients. to 95% of patients.

It is also found in 5% of children and 15It is also found in 5% of children and 15--30% of adults with acute lymphoid 30% of adults with acute lymphoid leukaemialeukaemia and in 2% of patients with newly and in 2% of patients with newly diagnosed acute myeloid diagnosed acute myeloid leukaemialeukaemia..

Cytogenetic Abnormality of CML: Cytogenetic Abnormality of CML: the Ph Chromosomethe Ph Chromosome

1 2 3 4 5

6 7 8 10 119 12

13 14 15 16 17 18

19 20 21 22X Y

t(9;22)t(9;22)--(q34;q11)(q34;q11)

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Deininger, M. W. N. et al. Blood 2000;96:3343-3356

AblAbl protein protein ((AbelsonAbelson murinemurine Leukemia virus)Leukemia virus)

Regulation of cell cycleRegulation of cell cycle

Cellular response to Cellular response to genotoxicgenotoxic stressstress

Transmission of information about the cellular Transmission of information about the cellular environment through environment through integrinintegrin signalingsignaling

Generally, it appears that the Generally, it appears that the AblAbl protein serves a protein serves a complex role as a cellular module that integrates complex role as a cellular module that integrates signals from various signals from various extracellularextracellular and intracellular and intracellular sources and that influences decisions in regard to sources and that influences decisions in regard to cell cycle and apoptosis.cell cycle and apoptosis.

AblAbl protein protein ((AbelsonAbelson murinemurine Leukemia virus)Leukemia virus)

Regulation of cell cycleRegulation of cell cycle

Cellular response to Cellular response to genotoxicgenotoxic stressstress

Transmission of information about the cellular Transmission of information about the cellular environment through environment through integrinintegrin signalingsignaling

Generally, it appears that the Generally, it appears that the AblAbl protein serves a protein serves a complex role as a cellular module that integrates complex role as a cellular module that integrates signals from various signals from various extracellularextracellular and intracellular and intracellular sources and that influences decisions in regard to sources and that influences decisions in regard to cell cycle and apoptosiscell cycle and apoptosis..

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Faderl, S. et al. N Engl J Med 1999;341:164-172

Substrates of BCRSubstrates of BCR--ABLABL

AdapterAdapterAdapterAdapterAdapterAdapterAdapterAdapterCytoskeleton/cell membraneCytoskeleton/cell membraneCytoskeleton/cell membraneCytoskeleton/cell membraneCytoskeleton/cell membraneCytoskeleton/cell membraneMyeloid differentiationMyeloid differentiationRasRas--GTPaseGTPaseRasRas activation?activation?PhospholipasePhospholipaseSerine Serine kinasekinaseCytoplasmicCytoplasmic phosphatasephosphatase1414--33--3 protein3 proteinUnknownUnknownHaemopoieticHaemopoietic differentiationdifferentiation

P62P62DOKDOK

CrklCrklCrkCrkShcShcTalinTalinPaxillinPaxillinFakFakFesFesRasRas--GAPGAPGAPGAP--associated proteinsassociated proteinsPLCPLCγγPI3 PI3 KinaseKinaseSypSypBapBap--11CblCblVavVav

FunctionFunctionProteinProtein

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Altered adhesion Altered adhesion properiesproperiesof BCRof BCR--ABLABL--positive cellspositive cells

CML progenitor cells exhibit decreased adhesion to CML progenitor cells exhibit decreased adhesion to BM BM stromastroma cells and ECMcells and ECM

CML progenitor cells express an adhesionCML progenitor cells express an adhesion--inhibitory inhibitory variant of variant of ββ1 1 integrinintegrin that is not found in normal that is not found in normal progenitorsprogenitors

CrklCrkl, one of the most prominent tyrosine, one of the most prominent tyrosine--phosphorylatedphosphorylated proteins in proteins in BcrBcr--AblAbl transformed cells, transformed cells, is involved in the regulation of cell motility, and in is involved in the regulation of cell motility, and in integrinintegrin--mediated cell adhesion by association with mediated cell adhesion by association with other focal adhesion proteins such as other focal adhesion proteins such as paxillinpaxillin, , FakFak, , p130Cas and Hef1p130Cas and Hef1

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Inhibition of apoptosis in Inhibition of apoptosis in BCRBCR--ABLABL--positive cellspositive cells

Blockade of the release of Blockade of the release of cytochromecytochrome C from C from the mitochondria and thus the activation of the mitochondria and thus the activation of caspasescaspases..

UpregulationUpregulation of Bclof Bcl--2 (via 2 (via RasRas-- or PI3K).or PI3K).

Transcriptional activation of Transcriptional activation of BclxLBclxL by Stat5. by Stat5.

PhosphorylationPhosphorylation and therefore inactivation of and therefore inactivation of the prothe pro--apoptotic protein Bad.apoptotic protein Bad.

CML: CELLULAR BIOLOGYCML: CELLULAR BIOLOGY

Myeloid progenitor cells expand in various stages of Myeloid progenitor cells expand in various stages of maturation, are released prematurely into the peripheral maturation, are released prematurely into the peripheral blood and home to blood and home to extramedullaryextramedullary locations.locations.

Alterations in the Alterations in the proliferativeproliferative capacity of immature capacity of immature haemopoietichaemopoietic progenitors and a shift in the balance progenitors and a shift in the balance between self renewal and differentiation.between self renewal and differentiation.

Defective adherence of immature Defective adherence of immature haemopoietichaemopoieticprogenitors to marrow progenitors to marrow stromalstromal elements.elements.

Activation of Activation of antiapoptoticantiapoptotic pathways.pathways.

CML BMCML BM

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CML PBCML PB

Chronic phase

Median duration5–6 years

Accelerated phase

Median duration6–9 months

Blast crisis

Median survival3–6 months

Advanced phases

Faderl S, et al. Ann Intern Med. 1999;131:207-219.Pasternak G, et al. J Cancer Res Clin Oncol. 1998;124:643-660.

CML: Clinical manifestationsCML: Clinical manifestations

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Faderl, S. et al. N Engl J Med 1999;341:164-172

MOLECULAR AND CELLULAR EVENTS MOLECULAR AND CELLULAR EVENTS IN DISEASE TRANSFORMATION (1)IN DISEASE TRANSFORMATION (1)

Minor Minor cytogeneticcytogenetic changeschangesMonosomiesMonosomies of chromosomes 7, 17, Yof chromosomes 7, 17, YTrisomiesTrisomies of chromosomes 17, 21of chromosomes 17, 21Translocation t(3;21)(q26;q22)Translocation t(3;21)(q26;q22)

Major Major cytogeneticcytogenetic changeschangesTrisomyTrisomy 8, 8, isochromosomeisochromosome i(17q)i(17q)

MOLECULAR AND CELLULAR EVENTS MOLECULAR AND CELLULAR EVENTS IN DISEASE TRANSFORMATION (2)IN DISEASE TRANSFORMATION (2)

Abnormalities in p53 (17p13)Abnormalities in p53 (17p13)RB1 (13q14)RB1 (13q14)cc--MYC (8q24)MYC (8q24)p16 (9p21)p16 (9p21)RASRASAMLAML--EVIEVI--1 t(3;21)(q26;q22)1 t(3;21)(q26;q22)

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ACUTE ACUTE TRANSFORMATIONTRANSFORMATIONThe rate of response to standardinduction chemotherapy forpatients in the myeloid blasticphase is approximately 20%, andthe rate of complete remission isless than 10%.

In patients in the lymphoidblastic phase, the rate ofresponse is approximately 50%, but remissions are transient andthe median survival is 9 to 12 months.

DIAGNOSIS OF CML (1)DIAGNOSIS OF CML (1)

CytogeneticCytogenetic analysisanalysis

Reveals the Ph chromosome in 95% of patients. Reveals the Ph chromosome in 95% of patients.

Valuable for demonstrating additional abnormalities.Valuable for demonstrating additional abnormalities.

BM cells are required.BM cells are required.

The procedure is timeThe procedure is time--consuming and only 20consuming and only 20--25 cells 25 cells in metaphase are examined per sample.in metaphase are examined per sample.

DIAGNOSIS OF CML (2)DIAGNOSIS OF CML (2)

FISH (Fluorescence In Situ FISH (Fluorescence In Situ HybdridizationHybdridization))

Uses doubleUses double--color probes for the detection of Ph color probes for the detection of Ph chromosome. chromosome.

PB cells can be analyzed. PB cells can be analyzed.

Fast techniqueFast technique

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Fluorescence in situ hybridization (FISH)

Interphase Metaphase

Courtesy of Charles L. Sawyers, MD, UCLA.

Labeled DNA probe(fluorescent)

Nuclear DNA

bcr-abl geneSeparate DNA strands and add labeled probe

Labeled bcr-abl gene

Molecular Methods for Detecting bcr-ablon the Ph Chromosome

Goals of treatment of CMLGoals of treatment of CML

Elimination of the Ph chromosomeElimination of the Ph chromosomeComplete cytogenetic responseComplete cytogenetic responseMolecular responseMolecular response

Other therapeutic goalsOther therapeutic goalsHematologic responseHematologic responseDisappearance of Disappearance of splenomegalysplenomegalyElimination of symptomsElimination of symptoms

TREATMENT OF CMLTREATMENT OF CML

1860: Arsenic Potassium1860: Arsenic Potassium1902: Irradiation1902: Irradiation1930: Spleen irradiation1930: Spleen irradiation1952: 1952: BusulphanBusulphan1966: 1966: HydroxyureaHydroxyurea1981: Transplantation1981: Transplantation1983: Interferon1983: Interferon2001:Tyrosine 2001:Tyrosine kinasekinase inhibitorsinhibitors

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Results of Allogeneic Bone Marrow Transplantation in Patients with Chronic Myeloid Leukemia in Chronic Phase

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IFNAR1IFNAR1IFNAR2IFNAR2

IFNIFN--αα

PPPPPP

JAKJAK--11Tyk2Tyk2

SHP1SHP1

ΥΠΟΔΟΧΕΑΣΥΠΟΔΟΧΕΑΣ ΙΝΤΕΡΦΕΡΟΝΗΣΙΝΤΕΡΦΕΡΟΝΗΣ--ααΡΡΟΛΟΣΟΛΟΣ ΤΗΣΤΗΣ ΦΩΣΦΑΤΑΣΗΣΦΩΣΦΑΤΑΣΗΣ SHPSHP--1 1

SHP1SHP1 ΦωσφαστάσηΦωσφαστάση

Tyk2Tyk2

JAKJAK--11ΚινάσεςΚινάσεςτυροσίνηςτυροσίνης

• Ενεργοποιείται αναστρέψιμα μετα απόενεργοποίηση του υποδοχέα της IFN-α

• Επιδρά ως αρνητικός ρυθμιστής τηςμεταβίβασης σήματος διαφόρων κυτταροκινών(IL4, IL13, EPO)

• Ενδεχομένως παίζει ρόλο στηνFAS εξαρτώμενη απόπτωση

Mechanisms of action of Mechanisms of action of IFNIFNαα

ImmunomodylatoryImmunomodylatory,, antiproliferativeantiproliferative, , antiogeantioge--neticnetic activitiesactivities

Indirectly influences the survival of CML cellsIndirectly influences the survival of CML cells

Restoration of defective Restoration of defective cytoadhesioncytoadhesion of CML of CML cells to BM microenvironmentcells to BM microenvironment

Augmentation of the Augmentation of the cytotoxicitycytotoxicity of NK cellsof NK cells

Inhibition of cytokines through Inhibition of cytokines through paracrineparacrine loopsloops

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Protein Protein kinaseskinases as therapeutic targetsas therapeutic targets

Protein Protein kinaseskinases are enzymes that transfer are enzymes that transfer phosphate from ATP to specific amino acids on phosphate from ATP to specific amino acids on substrate proteins.substrate proteins.

The The phosphorylationphosphorylation process leads to activation process leads to activation of signal transduction pathways which have of signal transduction pathways which have critical role in a variety of bicritical role in a variety of biοοlogic processes logic processes including cell growth, differentiation, and death.including cell growth, differentiation, and death.

Protein Protein kinaseskinases are composed of two are composed of two subfamilies, the protein serinesubfamilies, the protein serine--threoninethreoninekinaseskinases and the protein tyrosine and the protein tyrosine kinaseskinases..

A selective tyrosine kinase A selective tyrosine kinase inhibitor ofinhibitor of–– KITKIT–– BcrBcr--AblAbl–– PDGFRA/BPDGFRA/B

Druker et al. Nat Med. 1996;2:561.

Class: PhenylaminopyrimidinesClass: Phenylaminopyrimidines

C29H31N7O•CH4SO3

MW 589.7

GlivecGlivec / / GleevecGleevecImatinibImatinib MesylateMesylate

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Savage, D. G. et al. N Engl J Med 2002;346:683-693

Mechanism of Action of BCR-ABL and of Its Inhibition by Imatinib

Mechanism of action of STIMechanism of action of STI

ImatinibImatinib is a specific and potent inhibitor of a small family is a specific and potent inhibitor of a small family of tyrosine of tyrosine kinaseskinases, including , including BcrBcr--AblAbl..

ImatinibImatinib acts specifically by blocking the binding site for ATP in acts specifically by blocking the binding site for ATP in the the AblAbl kinasekinase, thereby preventing the , thereby preventing the phosphorylationphosphorylation of of tyrosine residues on substrate proteins.tyrosine residues on substrate proteins.

By inhibiting By inhibiting phosphorylationphosphorylation, , imatinibimatinib prevents the activation prevents the activation of signal transduction pathways that induce the of signal transduction pathways that induce the leukaemicleukaemictransformation to CML.transformation to CML.

ImatinibImatinib is not entirely selective for the is not entirely selective for the BcrBcr--AblAbl tyrosine tyrosine kinasekinase; it also inhibits the receptor tyrosine ; it also inhibits the receptor tyrosine kinaseskinases for for plateletplatelet--derived growth factor (PDGF) and stem cell factor derived growth factor (PDGF) and stem cell factor (SCF), Kit, and inhibits PDGF(SCF), Kit, and inhibits PDGF-- and SCFand SCF--mediated cellular mediated cellular events.events.

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Goldman JM. Blood. 2005; 106 (11) Abs 163, ASH 2005 Oral presentation

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International Randomized trial of Interferon/Ara-C versus STI571 (IRIS)

Imatinib

IFN-α + Ara-C

Randomization Crossover

Progression defined as:• Increasing WBC count• Loss of MCR or CHR• Accelerated phase or blast crisis• Death during treatment

1106 CML-CP patients enrolled from June 2000 to January 2001

14 (3%)

358 (65%)

397 pts (72%) remain on first-line imatinib

18 pts (3%) still on first-line IFN

O'Brien, S. G. et al. N Engl J Med 2003;348:994-1004

Kaplan-Meier Estimate of the Time to a Major Cytogenetic Response

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Kantarjian, H. M. et. al. Ann Intern Med 2006;145:913-923

Outcome with Frontline Imatinib Therapy: 54-Month Follow-up of the International Randomized Interferon versus STI571 Trial*

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DIAGNOSIS AND DIAGNOSIS AND MONITORING OF CMLMONITORING OF CML

Hughes, T. P. et al. N Engl J Med 2003;349:1423-1432

Estimated Percentages of All Study Patients in Each Group with a Reduction from Base Line in BCR-ABL Transcript Levels of at Least 3 Log, 2 Log, or Less Than 2 Log after 12 Months

39

12

31

39

41

8

22

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Year 1 Year 4

>= 4 log reduction

3 - < 4 log reduction

2 - < 3 log reduction

< 2 log reduction

8

Degree (%) of Degree (%) of BCRBCR--ABLABL log log reduction in 124 reduction in 124 CCyRCCyR pts pts

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Approach to the Treatment of Patients with CML in Chronic Phase

Peggs, K. et al. N Engl J Med 2003;348:1048-1050

Algorithm for Treating Chronic Myeloid Leukemia in Patients under the Age of 60 Years

??

Imatinib Bcr-Abl Mutated Bcr-Abl Clonal evolution

bcr-abl gene amplification/overexpression

Mutations in thekinase domain

Secondary geneticalterations

Cell growth independentof Bcr-Abl activityCell growth dependent on Bcr-Abl activity

Von Bubnoff N, et al. Leukemia. 2003;17:829-838.

Potential Mechanisms of Resistance to Imatinib

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Mechanisms for resistance Mechanisms for resistance to to ImatinibImatinib

Overexpression and amplification of the BCR-ABL gene locus.

BCR-ABL gene mutations.

Activation of BCR-ABL–independent pathways, suchas members of the Src kinase family.

Binding of imatinib to serum -1 acid glycoprotein.

Increased drug efflux through the multidrugresistance gene.


BCR-ABL characterized mutants associated with clinical resistance to imatinib

Goldman, J. M. et al. NEJM 2003;349:1451

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Spectrum of Tyrosine Kinase Inhibition for Imatinib and Novel Compounds*


Initial Phase II Data for Second-line Therapy with Dasatinib after Imatinib Failure*

BCR-ABL

ALTERNATIVE STRATEGIES TO ALTERNATIVE STRATEGIES TO TARGET ABLTARGET ABL--BCRBCR

Rapamycin

RASRAS PI3KPI3K

RAFRAF

MEKMEK

AKTAKT

mTORmTOR

ERKERK

BAY 43-9006

Cl-1040

HspHsp 9090 17-AAG

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Summary (1)Summary (1)

The characteristic genetic abnormality of CML, the Ph1 chr is present in the BM cells of more than 90% of patients with CMLand in 15% to 30% of adult patients with ALL.

It results from a reciprocal chromosomal translocation betweenthe long arms of chromosomes 9 and 22.

This process fuses the ABL gene on chr 9 with the BCR gene onchr 22, generating an oncogene that encodes the BCR-ABLprotein, a constitutively active, cytoplasmic form of ABL kinase.

The activity of this fusion protein is no longer under theregulatory control mechanisms for ABL and induces malignantdisease by activating multiple cytoplasmic and nuclear signaltransduction pathways that influence the growth and survival ofhematopoietic cells.

The targets for BCR-ABL include members of the Ras, phosphatidylinositol-3 kinase (PI3K)/Akt, and Jak/Stat signalingpathways, which regulate cell proliferation and apoptosis.

BCR-ABL abrogates cell dependence on external growth factors andalters the cell adhesion properties by modulating expression andactivation of focal adhesion kinase and associated proteins.

The kinase also has diverse effects on the DNA repair which maypromote additional chromosomal alterations and mutations involvedin the progression of the disease and may play a role in theaggressive nature of late-stage CML.

The The SrcSrc family family kinaseskinases may play an important role in latemay play an important role in late--stage stage disease, functioning downstream and upstream of BCRABL and in disease, functioning downstream and upstream of BCRABL and in pathways that are independent of BCRpathways that are independent of BCR--ABL.ABL.

Summary (2)Summary (2)

Imatinib selectively inhibits BCR-ABL by occupying the ABL domain adenosine triphosphate–binding site; it maintains the protein in an inactive conformation, thereby inhibiting its tyrosine kinase activity.

Imatinib Targets the Cause of CML

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A simplified illustration of BCR-ABL and Src family kinase involvement in oncogenic signaling pathways

Documents

The biology of chronic myeloid leukemia2007mcb.med.uoc.gr/mysite/arxeia/grad-study_papadaki_leukaemia.pdf · Deininger, M. W. N. et al. Blood 2000;96:3343-3356 Abl protein (Abelson