The document discusses chronic myelogenous leukemia (CML), detailing its epidemiology, clinical phases, and molecular pathogenesis, including the crucial BCR-ABL fusion gene. It emphasizes the role of various signaling pathways activated by BCR-ABL, treatment options like Imatinib, and mechanisms of resistance observed in patients. Additionally, it outlines laboratory features and diagnostic criteria for CML.

1. 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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2. 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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3. 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

4.  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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5. (John K. University of Pennsylvania)
Normal Chronic phase CML
CML: Peripheral Blood Smear
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6. Chronic phase
Median duration
5–6 years
Accelerated
phase
Median duration
6–9 months
Blast crisis
Median survival
3–6 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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7. 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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8. 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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9. 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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10. 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.
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11. Pathophysiologic Result of the Expression ofPathophysiologic Result of the Expression of
Bcr-AblBcr-Abl
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Bcr-Abl expression alone is necessary and sufficient for the development of CML
(Stephen et al., 2005)

12. 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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13. 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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14. 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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15. 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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16. 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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17.  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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18.  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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19. Fig. : Signaling pathways impacted by BCR-ABL expression. (Patel et
al., 2010)
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20. 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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21. 01/12/15 21ref

22. 01/12/15 22

23. 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%
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24. 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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25. 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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26. 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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27. 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

28. Mechanisms of Imatinib ResistanceMechanisms of Imatinib Resistance
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Primary resistancePrimary resistance
failure to achieve preset hematologic and/or
cytogenetic milestones
rates higher in accelerated and blast phase disease
Secondary resistanceSecondary resistance
loss of a previously achieved hematologic or
cytogenetic milestone
rates may be 10-15% on Imatinib, but become
rarer as time on therapy progresses
rates higher in accelerated and blast phase disease

29. 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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30. 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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31. 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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32. 01/12/15 32
(Patel et al., 2010)

33. 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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34. 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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35. Thank
U 01/12/15 35