Chronic Myelogenous Leukemia (CML) - History
• 1845- The first documented clinical description.
• 1960- The discovery of Philadelphia chromosome in CML cells
• 1973- The discovery that Philadelphia chromosome results from a reciprocal translocation of chromosomes 9 and 22.
• 1986- The cloning of BCR-ABL cDNA from CML cells.
• 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.
• 1970- The isolation of Abelson murine leukemia virus (A-MuLV).
• 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
• 1987-present: Understanding how BCR-ABL causes CML.
Normal Chromosomes9p
9q
22p
22q
c-Abl
Bcr
ReciprocalTranslocation
Ph1Chromosome
Bcr-Abl
SH3 SH2 Y-kinase
Gag
Myristoyl groupMyristoyl group
GAGGAG SH2 Y-kinaseV-AblV-Abl
C-AblC-Abl
Circa ~1990Circa ~1990
• 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
• 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
YY
NLSsNLSs
11 22 33
Proline-rich motifsProline-rich motifs
NESNES245245 412412
CAPCAP
Abl(1b)Abl(1b)
Abl(1a)Abl(1a)
BCR-ABLBCR-ABL
v-Ablv-Abl Gag
BCR
SH3 SH2 Kinase Domain DNA Binding Actin Binding
Myristoyl groupMyristoyl group
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.
Mol Cell Biol. 1993 Dec;13(12):7587-95. (see also McWhirter and Wang, 1991)
ABL
KinaseFunctionKinase
Function
LocationCues
LocationCues
NN
CC
SH3PXXPSH3
PXXP
SH2YpXXP
SH2YpXXP
ATPATP
PeptideYXXP
PeptideYXXP
Abl tyrosine kinaseAbl tyrosine kinase
Three Nuclear Localization
Signals (NLS). One Nuclear Export Signal (NES).
DNA-binding (bubble DNA). Actin-binding (G and F).
Three Nuclear Localization
Signals (NLS). One Nuclear Export Signal (NES).
DNA-binding (bubble DNA). Actin-binding (G and F).
BCR
ABL N-terminal StructureABL N-terminal StructureABL N-terminal StructureABL N-terminal Structure
Nagar, B., et al. (2003) Nagar, B., et al. (2003) CellCell 112: 859 112: 859
YY
PD166326PD166326N-lobeN-lobe
C-lobeC-lobe
MyristateMyristateSH2SH2
SH3SH3
YY
NLSsNLSs
11 22 33
Proline-rich motifsProline-rich motifs
NESNES245245 412412
CAPCAPAbl(1b)Abl(1b)
Abl(1a)Abl(1a)
BCR-ABLBCR-ABL
v-Ablv-Abl Gag
BCR
SH3 SH2 Kinase Domain DNA Binding Actin Binding
Myristoyl groupMyristoyl group
CAPCAP
[Nagar et al, Cell, 2003][Nagar et al, Cell, 2003]
Cryrstal Structure of the N-terminal Region of Abl
• • Welch, P.J. & Wang, J.Y.J. (1993) Welch, P.J. & Wang, J.Y.J. (1993) RB binds the ATP-binding lobe of Abl kinase domain and RB binds the ATP-binding lobe of Abl kinase domain and
inhibits its kinase activityinhibits its kinase activity. . CellCell 75, 779-90. 75, 779-90.
• • Wen, S.T. & Van Etten, R.A. (1996) Wen, S.T. & Van Etten, R.A. (1996) PAG/PrdxI binds Abl SH3 domain and inhibits its kinase PAG/PrdxI binds Abl SH3 domain and inhibits its kinase
activity.activity. Genes DevGenes Dev 11, 2456-67. 11, 2456-67.
• • Woodring, P.J., Hunterm T. & Wang, J. Y. J. (2001) Woodring, P.J., Hunterm T. & Wang, J. Y. J. (2001) F-actin binds the C-terminus of Abl and inhibits its kinase F-actin binds the C-terminus of Abl and inhibits its kinase
activityactivity. . J. Biol. Cell. 276: 27104-27110J. Biol. Cell. 276: 27104-27110. .
• • Welch, P.J. & Wang, J.Y.J. (1993) Welch, P.J. & Wang, J.Y.J. (1993) RB binds the ATP-binding lobe of Abl kinase domain and RB binds the ATP-binding lobe of Abl kinase domain and
inhibits its kinase activityinhibits its kinase activity. . CellCell 75, 779-90. 75, 779-90.
• • Wen, S.T. & Van Etten, R.A. (1996) Wen, S.T. & Van Etten, R.A. (1996) PAG/PrdxI binds Abl SH3 domain and inhibits its kinase PAG/PrdxI binds Abl SH3 domain and inhibits its kinase
activity.activity. Genes DevGenes Dev 11, 2456-67. 11, 2456-67.
• • Woodring, P.J., Hunterm T. & Wang, J. Y. J. (2001) Woodring, P.J., Hunterm T. & Wang, J. Y. J. (2001) F-actin binds the C-terminus of Abl and inhibits its kinase F-actin binds the C-terminus of Abl and inhibits its kinase
activityactivity. . J. Biol. Cell. 276: 27104-27110J. Biol. Cell. 276: 27104-27110. .
ABL InhibitorsABL InhibitorsABL InhibitorsABL Inhibitors
PP
PPAblAbl
High Activity(Transforming)High Activity
(Transforming)
ActivatedActivated
Equilibrium ofLow to medium activities
Equilibrium ofLow to medium activities
Catalytically inactiveCatalytically inactive
Co-inhibitorsCo-inhibitors
UninhibitedUninhibited
AblAbl
AutoinhibitedAutoinhibited
AblAbl
CoinhibitedCoinhibited
AblAbl
AblAbl
Coinhibitor 1Coinhibitor 1
Coinhibitor 2Coinhibitor 2
A Model for the Regulation of Abl Kinase: Auto-inhibition and Co-inhibition
A Model for the Regulation of Abl Kinase: Auto-inhibition and Co-inhibition
Wang, J.Y.J.NCB, 2004Wang, J.Y.J.NCB, 2004
Implications:Implications:Partitioning of Latent Abl Kinase into
Distinct Signaling Complexesby its Co-Inhibitors
Partitioning of Latent Abl Kinase into Distinct Signaling Complexes
by its Co-Inhibitors
RB-Abl complex: Abl Transduces Signals that Disrupt
RB-Abl Interaction: e.g., RB phosphorylation, RB degradation.
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.
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?
Prdx-Abl complex: Abl Transduces Signals that Disrupt
Prdx-Abl interaction: oxidative stress?
Master Protein Kinase
Signal (ligand, second messenger)
Kinase
Protein Substrates
Slave Protein Kinase
Signals(various)
Kinase
Substrates
BCR-ABL phosphorylates proteins that are substrates or non-substrates of Abl tyrosine kinase.
BCR-ABL phosphorylates proteins that are substrates or non-substrates of Abl tyrosine kinase.
• BCR-ABL contains BCR-sequences.• BCR-ABL contains BCR-sequences.
• BCR-ABL does not enter the nucleus.• 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.
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.
ABL
KinaseFunctionKinase
Function
LocationCues
LocationCues
NN
CC
SH3PXXPSH3
PXXP
SH2YpXXP
SH2YpXXP
ATPATP
PeptideYXXP
PeptideYXXP
Abl tyrosine kinaseAbl tyrosine kinase
Three Nuclear Localization
Signals (NLS). One Nuclear Export Signal (NES).
DNA-binding (bubble DNA). Actin-binding (G and F).
Three Nuclear Localization
Signals (NLS). One Nuclear Export Signal (NES).
DNA-binding (bubble DNA). Actin-binding (G and F).
BCR
Copyright ©2002 American Association for Cancer Research
Cohen, M. H. et al. Clin Cancer Res 2002;8:935-942
C29H31N7O.CH4SO3, relative molecular mass is 589.7.
Imatinib mesylate is the active component of Gleevec
N Shah, C Sawyers et al. CANCER CELL 2, 2002
Imatinib-resistant Imatinib-resistant BCR-ABLBCR-ABL kinase domain mutationskinase domain mutations
❶ F317L❷ T315I❸ F359V
❹ M244V❺ G250E❻ Q252H/R❼ Y253F/H❽ E255K
M351T E355G V379I L387M H396R
10
11
12
13
9
N Shah, C Sawyers, N Shah, C Sawyers, et alet al. . Cancer CellCancer Cell 2, 2002 2, 2002
Imatinib-resistant mutations at ABL kinase domainImatinib-resistant mutations at ABL kinase domain
Mathematical models suggest Mathematical models suggest CML stem cells to be refractory to Imatinib CML stem cells to be refractory to Imatinib
F Michor, F Michor, et al. et al. Nature Nature 435, 2005435, 2005I Roeder, I Roeder, et al. et al. Nature Med Nature Med 12, 200612, 2006
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
Stem cells, cancer, and cancer stem cells Tannishtha Reya, Sean J. Morrison, Michael F. Clarke andIrving 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 hasprogressed to accelerated phase (AP) or Blast Crisis (BC).
• CML stem cell identified in myeloid blast crisis patient samples. Jamieson et al, 2004
Increased Nuclear -catenin in Blast Crisis CML Granulocyte-Macrophage Progenitors
n = 6 n = 3 n = 4Jamieson et al, New Engl J Med 2004;351:657-67
CML-like phenotypes CML-like phenotypes in mHPC/p210-transplanted micein mHPC/p210-transplanted mice
mHPC/GFP mHPC/p210 87 (21) mg 782 (84) mg
n=6 n=12
Peripheral Blood smearof mHPC/p210 mice (7w)
(Wright-Giemsa staining)
Spleen (8w)
HistopathologyHistopathology(H&E staining)(H&E staining) at 8 Weeks at 8 Weeks Post-Post-transplantationtransplantation
BoneMarrow
Liver
mHPC/GFP (8w) mHPC/p210 (8w)
Spleen
Schedule of Imatinib-treatmentSchedule of Imatinib-treatmentIrradiated Irradiated 2200-recipients-recipients
Day 0 16 27 28 ~Day 0 16 27 28 ~
TransplantationTransplantation 1 x 101 x 106 6 BM cellsBM cells from leukemic mice (1from leukemic mice (100))
11 days11 days
Imatinib-treatmentImatinib-treatment AM: 50 mg/kg, p.o.AM: 50 mg/kg, p.o. PM: 100 mg/kg, p.o.PM: 100 mg/kg, p.o.
v.s. Vehicle controlv.s. Vehicle control
Effects of ImatinibEffects of Imatinib
0
10000
20000
30000
40000
WB
C (/
l)
GFP p210 Vehicle Imatinib
Vehicle Imatinib
Sp
lee
n s
ize
s (m
g)
GFP p210 Vehicle Imatinib
Vehicle Imatinib
0
200
400
600
P0.01 P0.01
GFP p210
0
20
40
60
80
100
GM
P (%
)
mHPC/GFP mHPC/p210
P0.01 P=0.11
BM
GF
P (
%)
0
2
4
6
Transplantation of Imatinib-treated Transplantation of Imatinib-treated mouse bone marrow induced leukemiamouse bone marrow induced leukemia
30-#1 (Day 38, 30)
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
587 (146) mg n=5
Spleen
BoneMarrow
GFP GFP
71.5 % 55.5 %
85.3 % 80.8 %
30-#1 (Day 38, 30) 30-#2 (Day 41, 30)
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
In the Cytoplasm: In the Cytoplasm:
BCR-ABL inhibits apoptosis.BCR-ABL inhibits apoptosis.
OncogenicOncogenic
In the Nucleus:In the Nucleus: (not where it is usually found) (not where it is usually found)
BCR-ABL induces apoptosis.BCR-ABL induces apoptosis.
Anti-oncogenicAnti-oncogenic
Vigneri & Wang, Nat. Med. 2001Vigneri & Wang, Nat. Med. 2001
AblAbl
AblAblAblF-actinF-actin
Extra Cellular Matrix, PDGFExtra Cellular Matrix, PDGF
AblAbl
Inactive
RBRB
Inactive
NLSNLSNESNES
NucleusNucleus
CytoplasmCytoplasm
OutsideOutside
actin dynamicsactin dynamics
APOPTOSISAPOPTOSIS
DNA Damage
DNA Damage
AblAblTNFTNF
TNF-induced apoptosis of mouse thymocytes requires Abl
and is blocked by Rb-MI
TNF-induced apoptosis of mouse thymocytes requires Abl
and is blocked by Rb-MI
Chau et al, MCB, 2004Chau et al, MCB, 2004
Abl contributes to TNF-induced apoptosisAbl contributes to TNF-induced apoptosis
Chau et al, MCB, 2004.Chau et al, MCB, 2004.
LMB
Covalent modification of CRM1 by Leptomycin BCovalent modification of CRM1 by Leptomycin B
T Kau, P Silver et al. Nature Reviews Cancer 4, 2004
CRM1=exportin-1
Trapping BCR-ABL in the Nucleus:Trapping BCR-ABL in the Nucleus:
cytoplasmcytoplasm NucleusNucleus
** ** ** **NLSNLS
NESNES
Leptomycin BLeptomycin B
*STI571*STI571
Nuclear BCR-ABL kinase kills:Nuclear BCR-ABL kinase kills:
cytoplasmcytoplasm NucleusNucleus
NESNES
Leptomycin BLeptomycin B
DeathDeath
ImatinibImatinib
LMBLMB
Imatinib + LMBImatinib + LMB
Count viable cells every 2 days
Count viable cells every 2 days
48 hours48 hours
16 days16 days
oror
oror
Extensive WashingExtensive Washing
Complete Media, NO DRUGS
Complete Media, NO DRUGS
TonB (+IL3)TonB (+IL3)
22 44 66 88 1010 1212 14 days14 days
non-treatednon-treated
LMBLMBImatinibImatinib
Imatinib+LMBImatinib+LMB
1x101x1088
1x101x1077
1x101x1066
1x101x1055
1x101x1044
Un-Induced forBCR-ABL Expression
Un-Induced forBCR-ABL Expression
TonB/BCR-ABL (-IL3)TonB/BCR-ABL (-IL3)
22 44 66 88 1010 1212 1414 16 days16 days
non-treatednon-treated
LMBLMBImatinibImatinib
Imatinib+LMBImatinib+LMB
1x101x1088
1x101x1066
1x101x1044
1x101x1022
00
Induced forBCR-ABLExpression
Induced forBCR-ABLExpression
TonB/BCR-ABL (+IL3)TonB/BCR-ABL (+IL3)
22 44 66 88 1010 1212 14 days14 days
non-treatednon-treated
LMBLMB
ImatinibImatinib
Imatinib+LMBImatinib+LMB
1x101x1088
1x101x1066
1x101x1044
1x101x1022
00
Induced forBCR-ABLExpression
Induced forBCR-ABLExpression
K562 (-IL3)K562 (-IL3)
33 55 77 99 1111 1313 15 days15 days
non-treatednon-treated
LMBLMB
ImatinibImatinib
Imatinib+LMBImatinib+LMB
1x101x1088
1x101x1066
1x101x1044
1x101x1022
00
Questions-
1. How can the mathematical predictiondepicted in Fig. 4A (sc panel) of the Michor et al
paper be tested in the clinic?
2. What may account for the innate resistanceof CML stem cells to imatinib?
3. What may account for the activation ofcell death by nuclear BCR-ABL?
Questions-
1. How can the mathematical predictiondepicted in Fig. 4A (sc panel) of the Michor et al
paper be tested in the clinic?
2. What may account for the innate resistanceof CML stem cells to imatinib?
3. What may account for the activation ofcell death by nuclear BCR-ABL?