Chronic Myelogenous Leukemia

Addis Ababa University College of Health Science,Department of Biochemistry

By: Yohannes Gemechu( B.Sc., MSc. Fellow)

December, 2014

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Outline

Introduction about Chronic Myeloid Leukemia (CML)Epidemiology of CML

Clinical Phases of CML

Pathogenesis of CML

Laboratory Features

Molecular target(treatment) for CML

Mechanism of Resistance of CML to Imatinib

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Chronic Myeloid Leukemia (CML)Chronic Myeloid Leukemia (CML)

Cancer of leukocytes(Leukemia).

In 1960, Nowell and Hungerford detected the

Philadelphia chromosome (22q-).

In 1973, Rowley identified the reciprocal translocation

involving chromosome 9 : t(9;22)(q34;q11).

In 1980s, the unique fusion gene termed BCR-ABL was

discovered. 01/12/15 3

Median age range at presentation: 45 to 55 years Incidence increases with age

Up to 30% of patients are >60 years old Slightly higher incidence in males

Male-to-female ratio—1.3:1 At presentation

50% diagnosed by routine laboratory tests 85% diagnosed during chronic phase

Accounts for 15-20% of adult leukemias Higher incidence noted in patients with heavy radiation

exposure

Epidemiology of CMLEpidemiology of CML

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(John K. University of Pennsylvania)

Normal Chronic phase CML

CML: Peripheral Blood Smear

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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 et al. 1999)

Clinical Course: Phases of Untreated CML

p53, Rb, p16, t(3;21), t(8;21), t(7;11)

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Pathogenesis of CMLPathogenesis of CML A single, pleuripotential, hematopoietic stem cell acquires a

Ph chromosome carrying the BCL-ABL fusion gene proliferative advantage

Constitutive expression by leukemic stem cell of growth factors ( Il-3, G-CSF)

CML cells survive longer due to defective apoptosis Close proximity of the BCR and ABL genes in

hematopoietic cells in interphase may favor translocations. Transformation from the chronic phase to blast phase is

associated with additional molecular changes ( activation of oncogenes or deletion of tumor-suppressor genes)

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Pathogenesis of CMLPathogenesis of CML

The classic BCR-ABL gene result from the fusion of parts of two normal genes ABL on Ch9 and BCR on Ch22.

Both genes are ubiquitously expressed in normal tissue, but their precise functions are not well defined.

Break occurs in ABL upstream of exon a2 and the major breakpoint cluster region of the BCR gene a 5’ portion of BCR and a 3’ portion of ABL are juxtaposed on a shortened Ch22.

The mRNA molecules transcribed from this hybrid gene contain one of two BCR-ABL junctions: e13a2 and e14a2 translated into p210BCR-ABL

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

The Translocation of t(9;22)(q34;q11) in CML

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Pathogenesis of CMLPathogenesis of CML

What causes the leukemogenic potential of p210bcr-abl? The constitutive activation of the ABL tyrosine kinase

activity by BCR

deregulated cellular proliferation

decreased adherence of leukemic cell to the stroma

reduced apoptotic response to mutagenetic stimuli Most crucial domain : the tyrosine kinase encoded by the

SRC-homology 1 (SH1) domain on ABL Various substrates have been found to bind to BCR-ABL

and to be tyrosine –phosphorylated by it.01/12/15 10

Pathophysiologic Result of the Expression of Pathophysiologic Result of the Expression of Bcr-AblBcr-Abl

01/12/15 11Bcr-Abl expression alone is necessary and sufficient for the development of CML

(Stephen et al., 2005)

BCR–ABL activation of STATBCR–ABL activation of STAT

The STAT participates in diverse processes, including cell growth, differentiation, apoptosis, fetal development, inflammation, and immune response. Ligand binding to cytokine or growth factor receptors initiates signaling events that result in STAT phosphorylation and subsequent translocation to the nucleus. STAT target genes include Bcl-xL and Mcl-1, substantiating an anti-apoptotic role for the activity of STAT transcription factors.

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BCR–ABL activation of STAT Cont’dBCR–ABL activation of STAT Cont’d

BCR–ABL-positive CML cell lines display constitutive phosphorylation and activation of STAT-1 and STAT-5.

STAT-5 activation induces upregulation of the serine/threonine kinase Pim-1 and the anti-apoptotic genes of the Bcl-2 family, A1 and Bcl-xL.

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BCR–ABL activation of NF-ΚbBCR–ABL activation of NF-Κb

The Nuclear Factor-κB (NF-κB) families of pleiotropic transcription factors function as dimers.The IκB proteins negatively regulate NF-κB by sequestering it to the cytoplasm. Phosphorylation and subsequent degradation of IκB relieves NF-κB to translocate to the nucleus. The constitutive activation of NF-κB is frequently observed in various cancers, and correlates with resistance of tumor cells to apoptosis. The NF-κB anti-apoptotic target genes include those from the Bcl-2 family (Bcl-xL, BFL1) and the inhibitors of apoptosis proteins, IAP1, IAP2, and XIAP.

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BCR–ABL activation of the Ras pathwayBCR–ABL activation of the Ras pathway

The Ras pathway regulates various aspects of cellular growth both in the context of normal and cancer cells.Activating mutations in Ras, or changes in molecular components that comprise Ras signaling, are found in most human cancers including leukemias, and result in increased cellular proliferation and survival.

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BCR–ABL activation of the PI3-K/Akt pathwayBCR–ABL activation of the PI3-K/Akt pathway

BCR–ABL activation of the PI3-K/Akt pathway Signal transduction pathways play a central role in survival, proliferation, differentiation, adhesion, metabolism, and motility.Upon its activation by growth factor tyrosine kinase receptors, PI3-K phosphorylates PIP2 to form PIP3. The formation of PIP3 can be reversed by the phosphatase and tensin homolog deleted on chromosome 10 (PTEN).

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PIP3 provides a platform for the recruitment of kinases, such as the serine/threonine kinases Akt, 3-phosphoinositide- dependent protein kinase-1 (PDK1), and others via their pleckstrin homology (PH) domains.

Akt is phosphorylated at distinct residues, namely at threonine 308 by PDK1, and at serine 473 by PDK2 (mTORC2).

Activated Akt regulates numerous cellular substrates, resulting in cell growth, survival, and suppression of apoptosis.

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Pharmacological inhibitors of PI3-K (LY294002 and Wortmannin) synergize with imatinib in inducing apoptosis of both chronic and blast crisis C L cells.

Combination of a PDK-1 inhibitor (OSU-03012), which inhibits Akt activation, with imatinib resulted in apoptosis even in cells expressing the BCR–ABL T315I imatinib-resistant mutant.

Besides substantiating a role for PI3-K/Akt signaling in BCR–ABL-mediated transformation and leukemogenesis, some of these observations also indicate that PI3-K/Akt activation is potentially a crucial event in BCR–ABL-mediated resistance to imatinib.

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Fig. : Signaling pathways impacted by BCR-ABL expression. (Patel et al., 2010)

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Laboratory featuresLaboratory features The hemoglobin concentration is decreased Nucleated red cells in blood film The leukocyte count above 25000/μl (often

above 100000/μl), granulocytes at all stages of development

Hypersegmentated neutrophils The basophiles count is increased The platelet count is normal or increased Neutrophils alkaline phosphatase activity is low

or absent (90%)

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Lab features

Bone marrow Hypercellular (reduced fat

spaces) Myeloid:erythroid ratio –

10:1 to 30:1 (N : 2:1) Myelocyte predominant

cell, blasts less 10% Megakaryocytes increased &

dysplastic Increase reticulin fibrosis in

30-40%01/12/15 23

Molecular TargetsMolecular Targets

Target for inhibition: Tyrosine kinase By blocking the ATP site, no phosphate groups would

be transferred to tyrosine residues on the BCR-ABL substrate

unphosphorylated substrate protein would not be able to undergo a conformational change to allow it to associate with downstream effectors

the downstream reactions would then be impeded interrupting transmission of the oncogenic signal to

the nucleus.

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Molecular TargetsMolecular Targets Imatinib Mesylate (Gleevec, STI571): a small molecule

that inhibits the kinase activity of all proteins that contain ABL, ABL-related gene protein, PDGFR, as well as c-kit receptor.

It was first approved in 2001. It occupies the ATP binding site in the SH1 domain of

the BCR-ABL oncoprotein. It inhibits cellular growth and induces apoptosis. Other targeted therapies being investigated:

The more specific Tyrosine Kinase inhibitors such as the dual SRC-ABL inhibitor : Dasatanib

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

Translocation Leading to the Philadelphia (Ph) Chromosome and the Role of BCR-ABL in the Pathogenesis of CML (Panel A) and the Effect of Normal (Panel B) and Abnormal (Panel C) c-kit

Function on Platelet-Derived Growth Factor and Gastrointestinal Stromal Tumors

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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

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Imatinib Mesylate (Gleevec, STI571) Mechanism of action

Mechanisms of Imatinib ResistanceMechanisms of Imatinib Resistance

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Primary resistancePrimary resistancefailure to achieve preset hematologic and/or cytogenetic milestonesrates higher in accelerated and blast phase disease

Secondary resistanceSecondary resistance loss of a previously achieved hematologic or cytogenetic milestonerates may be 10-15% on Imatinib, but become rarer as time on therapy progressesrates higher in accelerated and blast phase disease

Resistance MechanismsResistance Mechanisms

1) Bcr-Abl Kinase mutations 50 known mutations within Abl sequence which inhibits

Imatinib from binding

mutations identified in 30-80% of individuals with resistant disease

E.g. T315I point mutation prevents imatinib mesylate from binding to the ATP-binding domain

2) Bcr-Abl duplication duplication of the Bcr-Abl sequence has been identified in

cell lines with Im resistance

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Resistance Mechanisms Cont’dResistance Mechanisms Cont’d

3) Pgp over-expression export pump of many chemotherapeutics leading

to lower intracellular Im concentration

4) hOct-1(Human Organic Cation Transporter-1) under-expression import pump for Im which may lead to lower

intracellular levels of IM

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Resistance Mechanisms Cont’dResistance Mechanisms Cont’d

5) Src-Family kinase (SFK) expression activation may circumnavigate the Bcr-Abl

‘addiction’ of the transformed cell

6) High plasma levels of α1 Acid Glycoprotein (AGP). AGP binds imatinib mesylate at physiological

concentrations in vitro and in vivo, and blocks the ability of imatinib mesylate to inhibit BCR/ABL kinase activity in a dose-dependent manner.

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(Patel et al., 2010)

References

Faderl S.,Talpaz M., Estrov Z. and Kantarjian HM . (1999)

Chronic myelogenous leukemia: biology and therapy. Ann Intern

Med;131:207-219.

Pasternak G., Hochhaus A., Schultheis B. and Hehlmann R.

(1998) Chronic myelogenous leukemia:molecular and cellular

aspects. J Cancer Res Clin Oncol;124:643-660. 

Patel D., Suthar M., Patel V. and Singh R. (2010) BCR ABL

Kinase Inhibitors for Cancer Therapy. Inter Jour of Pharma Sci

and Drug Res; 2(2): 80-90.

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References

Sawyers CL . (1999) Chronic myeloid leukemia. N Engl J

Med;340:1330–1340. 

Stephen B. Marley and Myrtle Y. Gordon. (2005) Chronic

myeloid leukaemia: stem cell derived but progenitor cell

driven. Clinical Science ;109:13-25.

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Thank

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