Transcript Mechanisms

Mechanisms of
resistance in CML
Vernon Louw
Clinical Haematology
June 2010
1
Molecular basis CML
1882 – Sir Arthur Conan
Doyle published a case
in the Lancet
Thought that the
leukemia was caused
by malaria
Nowell PC et al. Science 1960; 132:1497
www.siteman.wustl.edu
6
Quintas-Cardama et al. Blood 2009
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•
p210BCR-ABL - CML
•
p190BCR-ABL - ALL
•
p230BCR-ABL - CNL
UpToDate v14.1
Substrate
Effector
Bcr-Abl
ADP
P
PPP
ATP
PPP
SIGNALING
Savage et al. NEJM 2002; Scheijen et al. Oncogene 2002
9
Quintas-Cardama et al. Blood 2009
10
Net effect
•Enhanced cellular proliferation
•Resistance to apoptosis
•Oncogenesis
11
Quintas-Cardama et al. Blood 2009
12
Imatinib
13
Bcr-Abl
P
ATP
PPP
Imatinib
mesylate
SIGNALING
Savage et al. NEJM 2002
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Resistance
15
16
Primary resistance
Failure to achieve responses at
defined endpoints
15 – 25%
Secondary (acquired) resistance
Loss of previously achieved
milestones
20-25%
17
1/3 patients may need alternative Rx to
imatinib due to:
resistance
intolerance
18
Mechanisms resistance
BCR-ABL dependent
BCR-ABL independent
19
BCR-ABL dependent mechanisms
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BCR-ABL dependent
BCR-ABL overexpression
BCR-ABL mutation
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BCR-ABL dependent
*BCR-ABL overexpression
In vitro demonstration
Cell lines from CML blast
crisis pts
developed resistant clones
serial passages in
imatinib-containing
cultures
elevated ABL kinase activity
due to genetic duplication of
Bcr-Abl sequence
Bcr-Abl
Le Coutre et al. Blood 2000; Gorre et al. Science 2001 22
BCR-ABL dependent
*BCR-ABL overexpression
More commonly seen in
advanced phase disease
Uncommon cause of
resistance
Bcr-Abl
Le Coutre et al. Blood 2000; Gorre et al. Science 2001 23
BCR-ABL dependent
*BCR-ABL mutations
Imatinib binds to the
Abl enzyme in the
inactive, closed
formation
Relatively rigid
structural
requirement for
entry and binding to
the ATP-binding site
24
BCR-ABL dependent
*BCR-ABL mutations
Point mutations
seen in 40 - 90% of
resistant patients
> 100 identified
Mutated Bcr-Abl
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BCR-ABL dependent
*BCR-ABL mutations
P-loop (residues 244 to
255)
docking site for
phosphate moieties
of ATP
most frequent (48% of
mutations in resistant
cases)
Destabilize
conformation
required for imatinib
binding
Branford S et al. Blood 2003; Jabbour E et al. Leukemia 2006
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BCR-ABL dependent
*BCR-ABL mutations
P-loop controversy
poor vs good
outcome
Branford S et al. Blood 2003; Jabbour E et al. Leukemia 2006
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BCR-ABL dependent
*BCR-ABL mutations
Catalytic domain
mutations (residues
350 to 363)
Can affect imatinib
binding
Branford S et al. Blood 2003; Jabbour E et al. Leukemia 2006
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BCR-ABL dependent
*BCR-ABL mutations
A-loop (residues 381 to
402)
key regulatory element of
the ABL1 kinase domain
Can adopt an
open/active or
closed/inactive
conformation
mutations prevent the
kinase from adopting the
inactive conformation
needed for imatinib
binding
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BCR-ABL dependent
*Bcr-Abl mutations
Imatinib binding site
T315I
seen in 4-19% of pts
with imatinib failure
impairs imatinib
binding to kinase
domain
complete resistance to
imatinib and 2nd
generation TKIs
Med survival 12/12
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BCR-ABL dependent
*BCR-ABL mutations
Mutations do not always
explain resistance
May activate different
downstream signalling
pathways
Very sensitive techniques
may detect mutations that
are not clinically relevant
•
Mutations have different
transforming capabilities,
e.g.
•
Y253F > E255K (P-loop) >
unmutated BCR-ABL1 >
T315I (binding site)>
H396P (activation loop) >
M351T (catalytic domain)
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Mutations at seven residues
account for 85% of all
resistance-associated
mutations
P-loop
M244V, G250E,
Y253F/H, E255K/V
Imatinib binding site
T315I
Catalytic domain
M351T, F359V
32
Mutations have been documented prior to TKI Rx
Suggests that pre-existing mutations do not acquire a
survival advantage until subjected to a TKI
No difference in pre-existing mutational status in pts who
have relapsed
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BCR-ABL independent mechanisms
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BCR-ABL independent
Leukemia cell related
Patient-related (Pseudoresistance)
Heterogeneity of CML
cells
Reduced levels of hOCT1
Poor compliance
Pharmacological
Poor absorption GIT
Drug interactions
Binding with plasma
components
Increased levels of
exporter (PgP)
Alternative signalling
pathway activation
Epigenetic modification
Clonal evolution
Quiescent stem cells
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BCR-ABL Independent
*Drug concentration
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BCR-ABL Independent
*Drug concentration
•
Possible association
between trough plasma
levels and CCyR and
MMR rates
•
No prospective data
•
Requires further
validation
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Compliance
38
THE
GOAL
≥ 95%
COMPLIANCE
FOR PATIENTS
WITH LIFETHREATENING
DISEASES
39
Compliance
mg taken /
mg
prescribed
40
Suboptimal compliance
Doses < 300-400mg
Plasma concentration too low
Malignant cells not eliminated
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In developed
countries compliance
with prescribed
medication
WHO reports
compliance rates:
51% with
antihypertensives
averages only 50% in
pts with chronic
illness
40%-70% with
antidepressants
43% with asthma
medications
37%-83% with
HIV/AIDS medications
Estimated that
3-10% of pts never fill
their prescriptions,
and
>30% of all
prescription refills are
never obtained
Haynes, 2002; WHO 2003
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125,000 AMERICANS DIE EACH
YEAR BECAUSE OF MEDICATION
NON-COMPLIANCE
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PRESCRIPTION COMPLIANCE
3502
patients
(CML and
GIST)
14 months
of
pharmacy
data
analyzed
Average
compliance
over 14
months of
observation
was 75%
Only 41%
of patients
more than
90%
compliant
On average
patients
consume
about 20%
less than
their
prescribed
dose
CML
patients
more
compliant
than GIST
Tsang et al. ASH 2006
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•
Men and women aged 55-70 most compliant
•
Men < 25 and women 35-45 least compliant
Tsang et al. ASH 2006
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•
Pts initially treated with 400 or 300mg/d had the
highest compliance (89%)
Tsang et al. ASH 2006
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3500
Patients
3000
COMPLIANCE DECAY
2921
2500
2000
1500
1000
685
500
0
0-1 1-2
2-3
3-4
4-5
5-6
6-7
7-8
8-9 9-10 10-11 11-12 12-13 13+
Months
• Adherence* is near 100% through month 4
• Declines from 94% at month 5 to 23% at month 14
• Only 57% of patients were still on imatinib @ 12 months
*Time on therapy without significant gaps in refills.
Tsang J et al. J Clin Oncol. 2006;24:330s. Abstract 6119.
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Compliance decay – why?
Unpleasant side-effects
Pts do not fully
understand importance of
taking medication
prescribed
Forget to take pills
Cancer patients
overestimate compliance
to oral Rx by a factor of 2
in discussion with
physicians
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Intake
Similar
time
each
day.
With
food
With
fulll
glass of
water.
Can put
tablets
in 1/2
cup of
water or
apple
juice
Drink
plenty
of
noncaff
einecontaini
ng
liquid
Take a
missed
dose
ASAP.
49
Absorption
Substrate for
intestinal ATPbinding cassette
transporters
ABCB1 and
ABCG2
Extent absorption
may be affected by
Primarily
absorbed from
small intestine
• GI abnormalities or
disease
• Drugs interfering with
transporters
• NOT affected by food
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Distribution
Circulating imatinib 95% bound to
plasma proteins
Mainly albumin and
alpha-1-acid
glycoprotein (AAG)
AAG levels may
influence imatinib
pharmacokinetics
Individual differences
in AAG and plasma
protein binding may
explain some of the
inter-patient
variability in the
observed total
plasma exposure to
imatinib.
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BCR-ABL Independent
*Drug binding
•
α1-AGP binds basic
drugs
•
Sequesters imatinib
in plasma
•
Reduces ability of
imatinib to inhibit the
ABL kinase
52
BCR-ABL Independent
*Drug binding
•
•
•
Role in resistance
implicated in mouse
models
Elevated AGP in tumourbearing mice associated
with PD despite imatinib
Rx
Competitive binder
erythromycin reversed the
effect
•
AGP levels shown to be
elevated in pts with CML
•
Further increased with
disease progression
•
Lack of clinical data
demonstrating variable
outcomes in patients
•
Clinical role of AGP
remains undefined
53
Distribution
Rapidly and
extensively
distributed into
tissues
Minimal penetration
to the central
nervous system
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Metabolism
Metabolized
by cytochrome
P450 (CYP)
3A4 and 3A5
Imatinib
exposure
influenced by
• drug-drug
interactions
• CYP3A4
inhibitors or
inducers
Major
metabolite, Ndesmethyl
metabolite
(CGR74588)
• Similar activity to
parent drug
55
Metabolism
CYP3A4 inhibitors:
drugs that may INCREASE
imatinib plasma
concentrations
CYP3A4 inducers:
drugs that may DECREASE
imatinib plasma
concentrations
Clarithromycin
Carbamazepine
Erythromycin
Phenytoin
Ketoconazole
Dexamethasone
Itraconazole
Phenobarbitone
Grapefruit
Rifampicin
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Elimination
Therefore
once daily
dose
•
Faeces
(predominant)
•
Urine
Elimination
half-life
imatinib +/18 hours
Can be
dosed
twice daily
to
Steady state reached
within one week
57
BCR-ABL Independent
*Drug import
hOCT1 responsible for imatinib uptake in cell
hOCT1 expression and inhibition correlated
with intracellular imatinib concentration
Differential in vitro susceptibility to imatinib
based upon expression and function of hOCT1
Thomas et al. Blood 2004; White et al. Blood 2006
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BCR-ABL Independent
*Drug import
Reduced levels of hOCT1 found in BM mononuclear cells in pts
failing to achieve at least a minor CyR after 10/12 on imatinib
Study of 70 pts demonstrated a significant difference in CCyR @
6/12, PFS and OS based upon pre-Rx hOCT1 level
TOPS trial showed reduced MMR rate @ 12/12 in pts with lower
hOCT1 levels receiving standard-dose imatinib compared with highdose imatinib
SNPs of hOCT1 associated with poor response to imatinib
Crossman et al. Blood 2005; Wang et al. Clin Pharmacol Ther 2008; White et al. Blood 2008 [abstract]
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BCR-ABL Independent
*Drug efflux
•
•
Increased expression PGP efflux
pump (MDR gene) associated with
exposure to increasing doses of
imatinib
•
Associated with 6-fold amplification
of the BCR-ABL gene product
•
Not differentiated in study between
effect of amplification and PGP
expression on resistance
SNPs in MDR gene with different
MMR rates in pts on imatinib
Mahon et al. Blood 2000; Dulucq et al. Blood 2008
60
BCR-ABL Independent
*Drug efflux
•
PGP inhibitors could modulate the activity of the export
protein and restore susceptibility to previous resistant cell
lines
•
siRNA also successfully used in vitro to
•
Restore imatinib susceptibility
•
Increase intracellular imatinib concentration
Che et al. Cancer letters 2002; Kotaki et al. Cancer letters 2003;
Rumpold et al. Exp Hematol 2005; Widmer et al. Leukemia 2007
61
BCR-ABL Independent
*Drug efflux
•
•
Other studies failed to find:
•
Association clinical response and PGP expression levels
•
Difference PGP levels in pre-Rx and post-Rx BM mononuclear
cells in pts achieving CCyR vs pts with less than a minor CyR
Role of PGP as a relevant marker of resistance in
population studies questioned
Lange et al. Blood 2003; Crossman et al. Blood 2005
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BCR-ABL Independent
*Alternative signaling pathway activation
Quintas-Cardama et al. Blood 2009
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BCR-ABL Independent
*Alternative signaling pathway activation
•
SRC family kinases LYN
and HCK upregulation
associated with:
•
Imatinib resistance
•
Lymphoid blast phase
•
Stabilizes BCR-ABL1 in
the active conformation to
which imatinib can not bind
64
BCR-ABL Independent
*Epigenetic modification
•
Imatinib resistance shown in a cell line through the
concurrent upregulation of Class I and II deacetylases
(HDACs) and downregulation of histone
acetyltransferases
•
Rx of these cells with an HDAC inhibitor restored the
acetylation pattern of several proteins and altered the
apoptotic threshold
•
TKIs + HDAC inhibitors showed a synergistic effect on the
level of apoptosis in cells from CML pts
Lee et al. J Pharmacol Exp Ther 2007; Kircher et al. Eur J Haematol 2009; Fiskus et al. Blood 2006
65
BCR-ABL Independent
*Clonal evolution
•
Usually indicates
transformation to an
advanced phase
•
Most common:
•
Additional Ph+
•
Trisomy 8
•
Isochromosome 17q
Secondary genetic
alterations
Clonal evolution
von Bubnoff et al. Leukemia. 2003;17:829.
66
BCR-ABL Independent
*Clonal evolution
•
Decreased response rate to
imatinib
•
Increased haematological
relapse and reduced OS
•
Reflects genetic instability of
CML progenitors
•
Haematological resistance
more common with clonal
evolution(58%) than with
BCR-ABL1 mutations (45%)
•
Clonal evolution more
common in blast phase
(73%) than kinase domain
mutations (30%).
•
More kinase domain
mutations in pts with clonal
evolution (58%) than in pts
with Ph+ metaphases only
von Bubnoff et al. Leukemia. 2003;17:829.
67
BCR-ABL Independent
*Quiescent stem cells
•
Primitive CD34+CD38cells in G0 phase cell cycle
•
<1% of CD34+ cells at dx
•
Inherent insensitivity to
imatinib
•
Postulated to sustain the
disease with the constant
potential to escalate
Mughal et al. Frontiers in Bioscience 2006
68
BCR-ABL Independent
*Quiescent stem cells
•
Resistance QSCs
multifactorial:
•
Altered drug influx and
efflux mechanisms (low
OCT1 and high PgP)
•
•
Increased BCR-ABL1
transcript levels in the
absence of BCR-ABL1
gene amplification
Decreased BCR-ABL1
transcript degradation
•
Cell migration defects due
to down-regulation of
CXCR4 by BCR-ABL1
overexpression
•
Promotes the migration of
CML cells to BM stroma
•
Causes G0-G1 cell cycle
arrest
•
Preserve survival of
quiescent CML progenitor
cells
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Summary
Bixby et al. Hematology 2009
70
Further investigation needed into
the roles of:
• Genomic instability
• Faulty DNA repair
• Quiescent stem cells
• Tumor suppressors
• Cross-talk between BCR-ABL1 and
oncogenic signaling pathways
71
Good news
•
Many novel agents directed at every area of
the pathogenetic pathway in development
72
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Mechanisms resistance
•
Bcr-Abl-independent
•
Successful inhibition Bcr-Abl kinase, yet
pt resistant to imatinib
•
•
Most common in primary resistance
Bcr-Abl-dependent
•
Reactivation of Bcr-Abl kinase after
successful inhibition before
•
Most common in pts who relapse on
Rx with imatinib
74