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Developing tests for Bcr-Abl activity and Gleevec resistance in CML patients
Stephen J. Kron M.D.-Ph.D.The University of Chicago
A progress report on IMAT R33 CA103235, "Bcr-Abl kinase assays for STI571 sensitivity or response"
Janet Rowley M.D.U. Chicago
Lasker Award 1998
1973: A chromosome translocation in CML
Ph1
Cytogenetic testing for molecular diagnosis, monitoring
1982: Ph1 chromosome encodes BCR-ABL
BCR ABL
Molecular diagnosis and monitoring via unique transcriptTyrosine kinase enzyme: Active site = druggable target
Owen Witte M.D.-Ph.D.UCLA
SH3 SH2 SH1
ABL
BCRABLBCR
9 22
ABL
Ph1
BCR
Clusteredbreakpoints
Brian Druker M.D.Oregon Health Sci.Nobel Prize 200?
1997: Bcr-Abl kinase blocker kills CML cells and"cures" chronic phase patients
Imatinib mesylateGleevec (Novartis)
$2.2B in 2005
QuickTime™ and aCinepak decompressor
are needed to see this picture.
STI571
STI571+AblJ. Kuriyan
P-loop
C. Sawyers and others
2002: Imatinib resistant kinase mutations
1, F317L; 2, T315I; 3, F359; 4, M244; 5, G250; 6, Q252; 7, Y253; 8, E255; 9, M351; 10, E355; 11, V379; 12, L387; 13, H396
Imatinib
Newly approved & on the way
New clinical challenges
Cheaper, generic imatinibAMN107 Novartis NilotinibBMS354825 Bristol-Myers DasatinibCGP76030 Pfizer AP23464 AriadAZD0530 Astra Zeneca Phase ISKI-606 Wyeth-AyerstON012380 Onconova Phase IVX-680 Merck Phase I
and many more in development…
2006: A proliferation of new drugs, but no assays
Rapid testing for Imatinib resistanceSelection of second-line therapyIdentifying effective dosageDetermining failure of STI therapy
Methylcellulose assay for imatinib sensitivitySemi-solid matrix, supplemented with growth factors,allows individual progenitors to form discrete colonies
cell suspension in MethoCult 5 x 104 cells/35 mm dish
37° C, 14 to 16 d+
image colonies
1 10 100 µM imatinib0
BaF3/T315I
BaF3/Y253F
K562
D. Sher
Technology challenge: Measure Bcr-Abl activityCriteria for a useful kinase assay
Detect Bcr-Abl activity in whole cell lysateDynamic range to determine Ki for inhibitorsRapid, robust and simple assay, amenable to clinical labAdaptable to high throughput for screening, drug discovery
EIYAAPFAAKKK + ATP EIpYAAPFAAKKK + ADPAbl, BCR-ABL
Detect: ADPPhosphotyrosinePhosphopeptide
Abltide
Ignore: Cell lysateOther kinasesPhosphatases
Solid-phase assaysBeads Chips
versus
Glutathione Agarose Bead
Glutathione
GST CrkL domains
Cell Lysate
Anti-phosphotyrosineWestern blot
ATP
Proof-of-principleBead-based assay of Bcr-Abl in cell lysates
+/- Imatinib
Y
Y Y
P
GST-Abltide Y
GST-Abl SH3L-Abltide
Y
Y
GST-Crkl SH3n-Abltide
GST
– + – + – +
-p-Tyr
Memcode
High affinity substrates via Abl binding domain
Reaction on beads with c-Abl
SH3n
SH3L
D. Wu et al.
100 µM Imatinib
M IM pretreatmentpCrkleIF4E
pCrkl/eIF4E 1.0 1.0 1.0 1.0 .93 .95 .15 .15 (%)
00 1 1 10 10010 100
Bcr-Abl inhibition assay in K562 and CML cells
MC
M IM pretreatment
a-p-Tyr
MC
100 mM IM added Ğ
0
+
0
Ğ
1
Ğ
10
Ğ
100
Western blot -- K562 cell steady-state phosphorylation
Bead assay -- K562 cell Bcr-Abl activity
-P-Tyr
0 0.1 1 5 10 20 50 µM ImatinibAssay of Gleevec resistance in CML patient cells
IC50 ~ 10 µM
D. Wu, D. Sher
IC50 >> 50 µM
K562 cells
Imatinib resistant CML patient peripheral blood ficoll-paque extracted
EAIYAAPFAKKKGTGT
GT
GST SH3n
1) + cell extract, +ATP, +/- imatinib
2) anti-phosphotyrosine
3) anti-IgG-phycoerythrin
Well C2 Bead A mean fluorescenceBead B mean fluorescenceBead C mean fluorescence
A
B C
Rapid translation to clinic…Adapt kinase assay to Luminex technology
[imatinib mesylate]
0
200
400
600
800
1000
1200
1400
1600
0 µm 10 µm 50 µm 100 µm 1 mM w/oAP10
w/o cellextract
w/oAP10,extract
w/o4G10
Med
ian
Fluo
rese
nt In
tens
ityLuminex bead assay for imatinib sensitivity
~10 µg lysate/well~1 h reaction
~1 min to read
~50:1 S/N
K562 extract
IC50 ~ 20 µM
S. Petersen
SHSH
Activation by TCEP
Chip based on ez-rays commercial hydrogel slide
ez-rays slides, multiwell plates (Matrix Technologies)
HS
EAIYAAPFAKKKH2N
NH
O
NH
O
SSH NH
O
NH
O
SSH NH
O
NH
O
S
EAIYAAPFAKKKH2N
++
Bisacrylamide
Cys-Abltide
D. Wu
96 well ez-rayTM plates
0
10-2
10-1
1
10
102
103
µMdrug
- cell extract
*
High throughput Abl/Bcr-Abl activity assayc-Abl, Abltide, 10 µM ATP, 1 h @ 30° C
+ peptide - kinase1 2 3 4 5 6 7 8 9 10 1211
A
B
C
D
E
F
G
H
- peptide- kinase
- peptide+ kinase
+ PD180970, µM10-2 10-1 1 10
+ Imatinib, µM10-2 10-1 1 10
B6 StaurosporineB7 AG-494C11 PiceatannolC12 PP1D9 Ro 21-8220F1 5-iodotubercidinF4 PP2
G1 Erbstatin analogG2 Quercetin dihydrateG8 SP 600125G9 IndirubinG10 Indirubin 3' monoxime G12 KenpaulloneH1 Terreic acid
H2 TriciribineH3 BML-257H4 SC-514H5 BML-259H6 ApigeninH7 Erlotinib analogH8 Rapamycin
+ 1 µM Imatinib + 200 µM BioMol Kinase Inhibitor set
K562 cell extract, Abltide10 µM ATP, 1 h @ 30° C
IC50 ~ 10 µM
Acrylic chemistry--Super glue for proteins
Si O
O
OMe
OMeMeO
N
O
O
N
OO
O
NH2
NN
O
O
OH
OH
OH
Si O
O
O
O
O
Si
Si
O
O
O
O
O
O
glass slide
(3-acryloxypropyl)-trimethoxysilane
+6-((acrylo)amino) hexanoic acid,
succinimidyl ester
+
GFP, etc.
acrylic-labeled surface
acrylic-labeled protein
Acrylated glass Acrylated protein
S. Brueggemeier
Si O
O
O
O
O
Si
Si
O
O
O
O
O
O
NN
O
O
N
N
NO
O
NO
O
NH2O
n
NH2O
n
CO
NH2
Si O
O
O
O NH2O
n
NH2O
n
O
Si
Si
O
O
O
O
O
O
NH2O
n
CO
NH2
CO
NH
CH2
NH
CO
NH2
O
acrylamide
N N
O O
bisacrylamide
UV or APSpolymerization
Copolymerization in situ Acrychip
Quantitative detection of Bcr-Ablinhibition by Imatinib
0
20
40
60
80
100
0 100 200 300 400
[Inhibitor] (M)
ECL
Ave
rage
Gra
y Va
lue
from
ph
osph
oryl
ated
tyro
sine GST-Crkl (both SH3's)
GST-Crkl (full length)
IC50 ~ 20 M
Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry
+ -
ABI 4700
25 kV
+
++
++
Desorption/Ionization
Detector
+ +
+
Time-of-flight tube
Label-free detection of peptide phosphorylation
80 au
m/z
Inte
nsity
Peptide Phospho-peptide
1357 1437
1 µl spot with 10 µM Abltide, c-Abl, 1 h at 30 °C
Photocleavable peptide array with MALDI read-off
NH
NH
OO2N
NH2
O NH
OO2N+
UV
-NPA photolinker Photocleavable peptide arrays
L. Parker et al.
In development: Multiplexed “lawn format” assay
Polyacrylamide copolymerAbltide EAIYAAPFAKKK-NPA-Cys-acrylamideSrctide GEEPLYWSFPAKKK-NPA-Cys-acrylamideetc..
1200 1220 1240 1260 1280 1300Mass (m/z)
1819.6
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
4700 Linear S pec #1 MC[BP = 1248.7, 1820]
1248.6241
mass (m/z)
Abltide
1220 12801200 13001240 1260
20 µM Abltide-NPA-CysCHCA matrixLinear positive mode
no Abltide-Cys+ cAbl
100 µM Abltide-Cys+ cAbl
500 µM Abltide-Cys+ cAbl
anti-pTyr "blot"~1 cm
Immunodetection
MALDI detectionfrom copolymerized pad
Hydrogel pads in lawn or well geometry
0
100
inte
nsity
X. Shi
Toward an integrated assay system:Cotter lab (JHU) mini-MALDI-TOF mass spec
Bench-top MALDI-TOF kinase activity microarray scanner?
?
R.J. Cotter et al. 1999, 2003
4.0 inches
New assays, new geometries, new technologies
Beads
Chips
MethoCult methylcellulose colony forming cell assayFunctional assay of growth inhibition by drugsSlow, low-throughput
Glutathione agarose/GST fusion phosphorylation assaySimple, sensitive, robust (Stratagene SignalScout)Low throughput
Luminex glutathione bead/GST fusion phosphoryation assaySimple, semi-quantitative, high throughput, easy multiplexingDedicated reader, low sensitivity
Acrylamide copolymerization GST fusion phosphoryation assayRobust, high signal to noiseLow sensitivity, difficult multiplexing
ez-rays 96 well hydrogel peptide phosphorylation assaySimple, semi-quantitative, medium throughputLow sensitivity, difficult multiplexing
Photocleavable peptide array with MALDI read-offRobust, semi-quantitative, high throughput, easy multiplexingDedicated reader, low sensitivity
Plates
AssaysSteve Kron
Ding WuXiangfu ShiDavid Rhee
Jennifer CampbellShariska Petersen
Cells/PatientsWendy StockDorie Sher
Matthew Myers
Peptides/MALDISteve Kent
Laurie ParkerVivian TienSurface chemistry
Sean PalecekShawn Brueggemeier
FundingNIH NCI IMAT R33 CA103235NIH Roadmap R01 HG3864
NSF Chicago MRSEC
ReagentsA. ImamotoJ. KuriyanB. DrukerJ. Groffen
C. Sawyers
The Team & Acknowledgements
