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Chronic Myelogenous Leukemia (CML) - History
• 1845- The first documented clinical description.
• 1960- The discovery of Philadelphia chromosome in CML cells
• 1970- The isolation of Abelson murine leukemia virus (A-MuLV).
• 1973- The discovery that Philadelphia chromosome
results from a reciprocal translocation of chromosomes 9 and 22.
• 1980-1983 The cloning of v-Abl oncogene in A-MuLV, cloning the c-Abl
proto-oncogene, locating c-Abl to chromosome 9, and demonstrating that
Abl encodes a protein tyrosine kinase.
• 1986- The cloning of BCR-ABL cDNA from CML cells.
• 1987-present: Understanding how BCR-ABL causes CML.
• 1996- The demonstration that imatinib (Gleevec) inhibits BCR-ABL
tyrosine kinase and CML growth in vitro.
• 2001- FDA approval of Gleevec for CML therapy.
• 2002-present : Gleevec resistance, CML stem cells
9p
Normal
Chromosomes
Reciprocal
Translocation
22p
Bcr
Bcr-Abl
22q
Ph1
Chromosome
9q
c-Abl
Myristoyl group
Gag
GAG
SH3
V-Abl
SH2
Y-kinase
SH2
Y-kinase
C-Abl
• V-Abl, but not c-Abl, Transforms Cells.
• V-Abl, but not c-Abl, is Tyr-Phosphorylated.
• ∆SH3-c-Abl becomes Tyr-Phosphorylated, and can
Transform Cells
Circa ~1990
v-Abl
BCR-ABL
Gag
NLSs
BCR
Abl(1a)
Abl(1b)
SH3
SH2
245
412
Y
Y
Kinase
NES
1 2 3
Domain
DNA Binding
CAP
Proline-rich motifs
Myristoyl group
Actin Binding
Mol Cell Biol. 1993 Dec;13(12):7587-95. (see also McWhirter and Wang, 1991)
A coiled-coil oligomerization domain of Bcr is essential for the
transforming function of Bcr-Abl oncoproteins.
McWhirter JR, Galasso DL, Wang JY.
Department of Biology, University of California, San Diego, La Jolla 92093-0116.
In Philadelphia chromosome-positive human leukemias, the c-abl proto-oncogene on chromosome 9
becomes fused to the bcr gene on chromosome 22, and chimeric Bcr-Abl proteins are produced.
The fused Bcr sequences activate the tyrosine kinase, actin-binding, and transforming functions of Abl.
Activation of the Abl transforming function has been shown to require two distinct domains of Bcr:
domain 1 (Bcr amino acids 1 to 63) and domain 2 (Bcr amino acids 176 to 242).
The amino acid sequence of domain 1 indicates that it may be a coiled-coil oligomerization domain.
We show here that domain 1 of Bcr forms a homotetramer.
Tetramerization of Bcr-Abl through Bcr domain 1 correlates with activation of the tyrosine kinase
and F-actin-binding functions of Abl.
Disruption of the coiled coil by insertional mutagenesis inactivates the oligomerization function
as well as the ability of Bcr-Abl to transform Rat-1 fibroblasts
or to abrogate interleukin-3 dependence in lymphoid cells.
These results strongly suggest that Bcr-Abl oligomers are the active entities in transformation.
N
BCR
Kinase
Function
Abl tyrosine kinase
SH3
PXXP
ATP
SH2 Peptide
YpXXP YXXP
ABL
Location
Cues
C
Three Nuclear Localization
Signals (NLS).
One Nuclear Export Signal (NES).
DNA-binding (bubble DNA).
Actin-binding (G and F).
ABL N-terminal Structure
Nagar, B., et al. (2003) Cell 112: 859
Cryrstal Structure of the N-terminal Region of Abl
v-Abl
BCR-ABL
Gag
BCR
Abl(1a)
Abl(1b)
SH3
245
412
Y
Y
SH2
Kinase
NLSs
NES
1 2 3
Domain
DNA Binding
Actin Binding
CAP
Proline-rich motifs
Myristoyl group
PD166326
SH3
N-lobe
Y
Y
CAP
SH2
C-lobe
Myristate
[Nagar et al, Cell, 2003]
ABL Inhibitors
• Welch, P.J. & Wang, J.Y.J. (1993)
RB binds the ATP-binding lobe of Abl kinase domain and
inhibits its kinase activity. Cell 75, 779-90.
• Wen, S.T. & Van Etten, R.A. (1996)
PAG/PrdxI binds Abl SH3 domain and inhibits its kinase
activity. Genes Dev 11, 2456-67.
• Woodring, P.J., Hunterm T. & Wang, J. Y. J. (2001)
F-actin binds the C-terminus of Abl and inhibits its kinase
activity. J. Biol. Cell. 276: 27104-27110.
A Model for the Regulation of Abl Kinase:
Auto-inhibition and Co-inhibition
Catalytically inactive
Equilibrium of
Low to medium activities
Coinhibitor 1
Abl
Abl
Autoinhibited
High Activity
(Transforming)
P
Abl
P
Co-inhibitors
Abl
Activated
Abl
Coinhibitor 2
Uninhibited
Coinhibited
Wang, J.Y.J.
NCB, 2004
Partitioning of Latent Abl Kinase into
Implications:
Distinct Signaling Complexes
by its Co-Inhibitors
RB-Abl complex:
Abl Transduces Signals that Disrupt
RB-Abl Interaction:
e.g., RB phosphorylation, RB degradation.
F-actin-Abl complex:
Abl Transduces Signals that Disrupt
F-actin-Abl Interaction: e.g., Cell adhesion.
Prdx-Abl complex:
Abl Transduces Signals that Disrupt
Prdx-Abl interaction: oxidative stress?
Master Protein Kinase
Slave Protein Kinase
Signal
(ligand, second messenger)
Signals
(various)
Kinase
Kinase
Protein Substrates
Substrates
BCR-ABL phosphorylates proteins that are substrates
or non-substrates of Abl tyrosine kinase.
• BCR-ABL contains BCR-sequences.
• BCR-ABL does not enter the nucleus.
Biological Activity Reported for BCR-ABL
• Many!
• Abrogates the cytokine requirement for cell survival..
•Abrogates the adhesion requirement for cell proliferation.
• Does not abrogate the serum requirement for growth.
• Stimulates random motility.
• Phosphorylates different proteins in different cell lines.
N
BCR
Kinase
Function
Abl tyrosine kinase
SH3
PXXP
ATP
SH2 Peptide
YpXXP YXXP
ABL
Location
Cues
C
Three Nuclear Localization
Signals (NLS).
One Nuclear Export Signal (NES).
DNA-binding (bubble DNA).
Actin-binding (G and F).
Imatinib mesylate is the active component of Gleevec
C29H31N7O.CH4SO3, relative molecular mass is 589.7.
Cohen, M. H. et al. Clin Cancer Res 2002;8:935-942
Copyright ©2002 American Association for Cancer Research
Imatinib-resistant BCR-ABL
kinase domain mutations
N Shah, C Sawyers et al.
CANCER CELL 2, 2002
Imatinib-resistant mutations at ABL kinase domain
❶ F317L
❷ T315I
❸ F359V
❹
❺
❻
❼
❽
9
10
11
12
13
N Shah, C Sawyers, et al.
Cancer Cell 2, 2002
M244V
G250E
Q252H/R
Y253F/H
E255K
M351T
E355G
V379I
L387M
H396R
Mathematical models suggest
CML stem cells to be refractory to Imatinib
F Michor, et al. Nature 435, 2005
I Roeder, et al. Nature Med 12, 2006
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Stem cells, cancer, and cancer stem cells
Tannishtha Reya, Sean J. Morrison, Michael F. Clarke and
Irving L. Weissman. Nature 414, 105-111 (1 November 2001)
The current model of hematopoiesis
CML Stem Cells
* CML is a disease of the hematopoietic stem cells (HSC)
• During Chronic Phase (CP), Ph1-chromosome can be found in
mature blood cells of various myeloid and lymphoid lineage.
• Gleevec is effective during CP, but ineffective if disease has
progressed to accelerated phase (AP) or Blast Crisis (BC).
• CML stem cell identified in myeloid blast crisis patient samples.
Jamieson et al, 2004
Increased Nuclear b-catenin in Blast Crisis
CML Granulocyte-Macrophage Progenitors
n=6
n=3
n=4
Jamieson et al, New Engl J Med 2004;351:657-67
CML-like phenotypes
in mHPC/p210-transplanted mice
Spleen (8w)
mHPC/GFP
mHPC/p210
87 (21) mg 782 (84) mg
n=6
n=12
Peripheral Blood smear
of mHPC/p210 mice
(7w)
(Wright-Giemsa staining)
mHPC/GFP (8w)
Bone
Marrow
Histopathology
(H&E staining)
at 8 Weeks
PostSpleen
transplantation
Liver
mHPC/p210 (8w)
Irradiated
20-recipients
Schedule of Imatinib-treatment
11 days
Day 0
16
27 28 ~
Transplantation
Imatinib-treatment
1 x 106 BM cells
from leukemic mice (10)
AM: 50 mg/kg, p.o.
PM: 100 mg/kg, p.o.
v.s. Vehicle control
Effects of Imatinib
WBC (/l)
40000
30000
20000
10000
0
GFP
P0.01
Spleen sizes (mg)
P0.01
600
400
200
0
Vehicle
Imatinib
p210
GFP
P0.01
Vehicle
Imatinib
p210
P=0.11
6
80
GMP (%)
BM GFP (%)
100
60
40
4
2
20
0
GFP
Vehicle
Imatinib
p210
0
mHPC/GFP
Vehicle
Imatinib
mHPC/p210
Transplantation of Imatinib-treated
mouse bone marrow induced leukemia
30-#1 (Day 38, 30)
30-#1 (Day 38, 30)
71.5 %
30-#2 (Day 41, 30)
55.5 %
Spleen
587 ( 146) mg
n=5
85.3 %
80.8 %
GFP
GFP
Bone
Marrow
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
In the Cytoplasm:
BCR-ABL inhibits apoptosis.
Oncogenic
In the Nucleus:
(not where it is usually found)
BCR-ABL induces apoptosis.
Anti-oncogenic
Vigneri & Wang, Nat. Med. 2001
Extra Cellular Matrix, PDGF
Outside
actin dynamics
F-actin
Abl
Abl
Cytoplasm
Nucleus
NES
NLS
DNA
Abl Damage
TNF
APOPTOSIS
Inactive
Abl
RB
Abl
Inactive
TNF-induced apoptosis of
mouse thymocytes requires Abl
and is blocked by Rb-MI
Chau et al, MCB, 2004
Abl contributes to TNF-induced apoptosis
Chau et al, MCB, 2004.
LMB
Covalent modification of CRM1 by Leptomycin B
CRM1=exportin-1
T Kau, P Silver et al.
Nature Reviews Cancer 4, 2004
Trapping BCR-ABL in the Nucleus:
Leptomycin B
NES
**
NLS
**
*STI571
cytoplasm
Nucleus
Nuclear BCR-ABL kinase kills:
Leptomycin B
NES
Death
cytoplasm
Nucleus
Imatinib
or
LMB
Extensive
Washing
Count viable cells
every 2 days
or
Imatinib + LMB
48 hours
16 days
Complete Media,
NO DRUGS
1x10
1x10
Un-Induced for
TonB (+IL3) BCR-ABL
Expression
8
non-treated
Imatinib
7
LMB
Imatinib+LMB
1x10
1x10
1x10
6
5
4
2
4
6
8 10 12 14 days
TonB/BCR-ABL (-IL3)
1x10
1x10
1x10
1x10
8
Induced for
BCR-ABL
Expression
non-treated
Imatinib
LMB
6
4
2
Imatinib+LMB
0
2
4 6 8 10 12 14 16 days
TonB/BCR-ABL (+IL3)
1x10
1x10
1x10
1x10
8
Induced for
BCR-ABL
Expression
Imatinib
non-treated
6
LMB
4
2
Imatinib+LMB
0
2 4
6 8 10 12 14 days
K562 (-IL3)
1x10
8
non-treated
Imatinib
1x10
1x10
1x10
6
LMB
4
2
Imatinib+LMB
0
3
5
7
9 11 13 15 days
Questions1. How can the mathematical prediction
depicted in Fig. 4A (sc panel) of the Michor et al
paper be tested in the clinic?
2. What may account for the innate resistance
of CML stem cells to imatinib?
3. What may account for the activation of
cell death by nuclear BCR-ABL?