Chronic myeloid leukemia (CML), also known as chronic myelogenous leukemia, is a type of
cancer that starts in the blood-forming cells of the bone marrow and invades the blood.
Each human cell contains 23 pairs of chromosomes. Most cases of CML start when a "swapping"
of chromosomal material (DNA) occurs between chromosomes 9 and 22 during cell division due
to attack of DNA by radiation or other damage. Part of chromosome 9 goes to 22 and part of 22
goes to 9. This is known as a translocation and gives rise to a chromosome 22 that is shorter than
normal. This new abnormal chromosome is known as the Philadelphia chromosome.
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Chronic myelogenous leukemia
1. Chronic Myelogenous Leukemia
Addis Ababa University College of Health
Science,Department of Biochemistry
By: Yohannes Gemechu( B.Sc., MSc. Fellow)
December, 2014
01/12/15 1
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
01/12/15 2
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
01/12/15 4
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)
01/12/15 6
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)
01/12/15 7
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
01/12/15 8
9. Faderl, S. et al. N Engl J Med 1999;341:164-172
The Translocation of t(9;22)(q34;q11) in CML
01/12/15 9
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.
01/12/15 10
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.
01/12/15 12
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.
01/12/15 13
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.
01/12/15 14
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.
01/12/15 15
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).
01/12/15 16
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.
01/12/15 17
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.
01/12/15 18
19. Fig. : Signaling pathways impacted by BCR-ABL expression. (Patel et
al., 2010)
01/12/15 19
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%)
01/12/15 20
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%
01/12/15 23
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.
01/12/15 24
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
01/12/15 25
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
01/12/15 26
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
01/12/15 27
Imatinib Mesylate (Gleevec, STI571) Mechanism of action
28. Mechanisms of Imatinib ResistanceMechanisms of Imatinib Resistance
01/12/15 28
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
01/12/15 29
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
01/12/15 30
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.
01/12/15 31
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.
01/12/15 33
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.
01/12/15 34
CML: a Progressive and Fatal Disease
CML progresses through 3 phases of shortening duration characterized by worsening clinical features and laboratory findings and increasing refractoriness to therapy. These stages include chronic phase, accelerated phase, and blast crisis. The majority of patients present in chronic phase and then progress to accelerated phase; however, 25% to 40% of patients progress directly from chronic phase to terminal blast crisis without evidence of a transitional
accelerated phase.1,2
� Chronic phase. There are less than 10% to 15% blasts in peripheral blood and bone marrow, and the white blood cell (WBC) count at presentation is typically elevated to ≥20 x 109/L. The cutoff for blasts in all Novartis studies was 15%. Signs and symptoms may initially be mild and develop as the disease progresses. The chronic phase of CML may last 5 to 6 years before the disease accelerates.1,3,4
� Accelerated phase. There are more than 10% to 15% (but less than 30%) blasts in peripheral blood or bone marrow.
The less favorable cutoff of 15% was used in all Novartis studies. Symptoms may increase and include unexplained fever, bone pain, splenomegaly, and hepatomegaly. Basophilia, decreased platelet counts, and cytogenetic progression may also be observed. Cytogenetic abnormalities such as duplication of the Ph chromosome, iso17q, or trisomy 8 (+8) are the most common additional chromosomal abnormalities described during cytogenetic progression.
The accelerated phase may last 6 to 9 months.1,3
� Blast crisis. There are more than 30% blasts in peripheral blood or bone marrow and increased symptomatology, especially relating to anemia and infection, central nervous system (CNS) disease, lymphadenopathy, and bleeding.
Approximately 50% of patients have myeloid blast crisis, 25% have lymphoid blast crisis, and 25% are mixed.5 Patients with CML in blast crisis have a poor prognosis because of the lack of effective therapy. This phase is rapidly fatal, with a median survival of 3 to 6 months.2,5
References
1. Hill JM, Meehan KR. Chronic myelogenous leukemia. Curable with early diagnosis and treatment. Postgrad Med. 1999;106:149-152, 157-159.
2. Faderl S, Kantarjian HM, Talpaz M. Chronic myelogenous leukemia: update on biology and treatment. Oncology (Huntingt). 1999;13:169-180.
3. Faderl S, Talpaz M, Estrov Z, et al. Chronic myelogenous leukemia: biology and therapy.Ann Intern Med. 1999;131:207-219.
4. Pasternak G, Hochhaus A, Schultheis B, et al. Chronic myelogenous leukemia: molecular and cellular aspects. J Cancer Res Clin Oncol. 1998;124:643-660.
5. Cortes JE, Talpaz M, Kantarjian H. Chronic myelogenous leukemia: a review. Am J Med. 1996;100:555- 570.