# Presentation: 930097

## 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"*

## 1973:  A chromosome translocation in CML

- Janet Rowley M.D.
- U. Chicago
- Lasker Award 1998

**Ph1**

- Cytogenetic testing for molecular diagnosis, monitoring

## 1982: Ph1 chromosome encodes BCR-ABL

- BCR

- ABL

- Molecular diagnosis and monitoring via unique transcript
- Tyrosine kinase enzyme: Active site = druggable target

- Owen Witte M.D.-Ph.D.
- UCLA

- SH3 SH2 SH1

- ABL

- BCR

- ABL

- BCR

- 9

- 22

- ABL

- Ph1

- BCR

- Clustered
- breakpoints

## 1997: Bcr-Abl kinase blocker kills CML cells and

- Brian Druker M.D.
- Oregon Health Sci.
- Nobel Prize 200?

- "cures" chronic phase patients

- Imatinib mesylate
- Gleevec (Novartis)
- $2.2B in 2005

- STI571

- STI571+Abl
- J. Kuriyan

## 2002:  Imatinib resistant kinase mutations

- P-loop

- C. Sawyers and others

- 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

## 2006: A proliferation of new drugs, but no assays

**New clinical challenges**

**Cheaper, generic imatinib**

- AMN107         Novartis	     Nilotinib
- BMS354825   Bristol-Myers    Dasatinib
- CGP76030     Pfizer
- AP23464        Ariad
- AZD0530        Astra Zeneca    Phase I
- SKI-606          Wyeth-Ayerst
- ON012380     Onconova          Phase I
- VX-680           Merck                Phase I
-    and many more in development...

- Rapid testing for Imatinib resistance
- Selection of second-line therapy
- Identifying effective dosage
- Determining failure of STI therapy

## Methylcellulose assay for imatinib sensitivity

- Semi-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 imatinib

- 0

- BaF3/T315I

- BaF3/Y253F

- K562

- D. Sher

## Technology challenge: Measure Bcr-Abl activity

**Criteria for a useful kinase assay**
- 	Detect Bcr-Abl activity in whole cell lysate
- 	Dynamic range to determine Ki for inhibitors
- 	Rapid, robust and simple assay, amenable to clinical lab
- 	Adaptable to high throughput for screening, drug discovery

- EIYAAPFAAKKK + ATP

- EIpYAAPFAAKKK + ADP

- Abl, BCR-ABL

- Detect:
-       ADP
-       Phosphotyrosine
-       Phosphopeptide

- Abltide

- Ignore:
-       Cell lysate
-       Other kinases
-       Phosphatases

- Solid-phase assays

- Beads

- Chips

- versus

## Proof-of-principle

- Glutathione
- Agarose Bead

- Glutathione

- GST  CrkL domains

- Cell Lysate

- Anti-phosphotyrosine
- Western blot

- ATP

- Bead-based assay of Bcr-Abl in cell lysates

- +/- Imatinib

- Y

- Y

- Y

- P

## High affinity substrates via Abl binding domain

- GST-Abltide

- GST-Abl SH3L-Abltide

- GST-Crkl SH3n-Abltide

- GST

- GST-Abltide

- GST-AblSH3L-Abltide

- GST-Crkl SH3n-Abltide

-  –     +

-  –     +

-  –      +

- -p-Tyr

- Memcode

- Reaction on beads
- with c-Abl

- SH3n

- SH3L

- D. Wu et al.

- 100 μM Imatinib

## Bcr-Abl inhibition assay in K562 and CML cells

**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 Imatinib

**Assay 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

## Rapid translation to clinic...

- EAIYAAPFAKKK

- GT

- GT

- GT

- GST

- SH3n

- 1) + cell extract, +ATP, +/- imatinib
- 2) anti-phosphotyrosine
- 3) anti-IgG-phycoerythrin

- Well C2
- Bead A  mean fluorescence
- Bead B  mean fluorescence
- Bead C  mean fluorescence

- A

- B

- C

- Adapt kinase assay to Luminex technology

## Luminex 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

## Chip based on ez-rays commercial hydrogel slide

- Activation
- by TCEP

- ez-rays slides, multiwell plates (Matrix Technologies)

-   S

**+**

**+**

- Bisacrylamide

- Cys-Abltide

- D. Wu

## High throughput Abl/Bcr-Abl activity assay

- 0

- 10-2

- 10-1

- 1

- 10

- 102

- 103

- PD 180970

- PD 166326

- PD 173955

- Imatinib

- AG957

- Genestein

- μM
- drug

- -  cell extract

- *

- c-Abl, Abltide, 10 μM ATP, 1 h @ 30° C

- + peptide - kinase

- 1

- 2

- 3

- 4

- 5

- 6

- 7

- 8

- 9

- 10

- 12

- 11

- A

- B

- C

- D

- E

- F

- G

- H

- - peptide
- - kinase

- - peptide
- + kinase

- + PD180970, μM
- 10-2  10-1   1    10

-   + Imatinib, μM
- 10-2  10-1   1     10

- B6     Staurosporine
- B7     AG-494
- C11   Piceatannol
- C12   PP1
- D9     Ro 21-8220
- F1     5-iodotubercidin
- F4     PP2

- G1    Erbstatin analog
- G2    Quercetin dihydrate
- G8    SP 600125
- G9    Indirubin
- G10  Indirubin 3' monoxime
- G12  Kenpaullone
- H1    Terreic acid

- H2    Triciribine
- H3    BML-257
- H4    SC-514
- H5    BML-259
- H6    Apigenin
- H7    Erlotinib analog
- H8    Rapamycin

- + 1 μM Imatinib + 200 μM BioMol Kinase Inhibitor set

- K562 cell extract, Abltide
- 10 μM ATP, 1 h @ 30° C

- IC50 ~ 10 μM

## Acrylic chemistry--Super glue for proteins

- 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

## Copolymerization in situ       Acrychip

- UV or APS
- polymerization

- Copolymerization *in situ       *Acrychip

## Quantitative detection of Bcr-Abl

- inhibition by Imatinib

- IC50 ~ 20 M

## Label-free detection of peptide phosphorylation

- Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry

- +

- -

- 25 kV

- Detector

- Time-of-flight tube

- 80 au

- m/z

- Intensity

- Peptide

- Phospho-
- peptide

- 1357

- 1437

## Photocleavable peptide array with MALDI read-off

- 1 μl spot with 10 μM Abltide, c-Abl, 1 h at 30 °C

- UV

- -NPA photolinker

- Photocleavable peptide arrays

- L. Parker et al.

## In development: Multiplexed “lawn format” assay 

- Polyacrylamide copolymer
- Abltide  EAIYAAPFAKKK-NPA-Cys-acrylamide
- Srctide  GEEPLYWSFPAKKK-NPA-Cys-acrylamide
- etc..

- mass (m/z)

- Abltide

- 1220

- 1280

- 1200

- 1300

- 1240

- 1260

- 20 μM Abltide-NPA-Cys
- CHCA matrix
- Linear positive mode

- no Abltide-Cys
- + cAbl

- 100 μM Abltide-Cys
- + cAbl

- 500 μM Abltide-Cys
- + cAbl

- anti-pTyr "blot"

- ~1 cm

**Immunodetection**

**MALDI detection**
**from copolymerized pad**

**Hydrogel pads in lawn or well geometry**

- 0

- 100

- intensity

- 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

## New assays, new geometries, new technologies

**MethoCult methylcellulose colony forming cell assay**
- 	Functional assay of growth inhibition by drugs
- 	Slow, low-throughput
**Glutathione agarose/GST fusion phosphorylation assay**
- 	Simple, sensitive, robust (Stratagene SignalScout)
- 	Low throughput
**Luminex glutathione bead/GST fusion phosphoryation assay**
- 	Simple, semi-quantitative, high throughput, easy multiplexing
- 	Dedicated reader, low sensitivity
**Acrylamide copolymerization GST fusion phosphoryation assay**
- 	Robust, high signal to noise
- 	Low sensitivity, difficult multiplexing
**ez-rays 96 well hydrogel peptide phosphorylation assay**
- 	Simple, semi-quantitative, medium throughput
- 	Low sensitivity, difficult multiplexing
**Photocleavable peptide array with MALDI read-off**
- 	Robust, semi-quantitative, high throughput, easy multiplexing
- 	Dedicated reader, low sensitivity

## The Team & Acknowledgements

- Steve Kron
- Ding Wu
- Xiangfu Shi
- David Rhee
- Jennifer Campbell
- Shariska Petersen

**Cells/Patients**
- Wendy Stock
- Dorie Sher
- Matthew Myers

**Peptides/MALDI**
- Steve Kent
- Laurie Parker
- Vivian Tien

**Surface chemistry**
- Sean Palecek
- Shawn Brueggemeier

**Funding**
**NIH NCI IMAT R33 CA103235**
- NIH Roadmap R01 HG3864
- NSF Chicago MRSEC

**Reagents**
- A. Imamoto
- J. Kuriyan
- B. Druker
- J. Groffen
- C. Sawyers