Targeting Treatment Naïve CML Stem/Progenitor
Cells from Imatinib-Nonresponders by
Combination Treatment with JAK2 (BMS-911543)
and ABL Inhibitors
Leon (Hanyang Lin), Min Chen, Katharina
Rothe, Matthew V. Lorenzi, Adrian Woolfson
and Xiaoyan Jiang
Terry Fox Laboratory, BC Cancer Agency
Department of Medicine, UBC
Vancouver, Canada
Discovery Medicine Oncology, Bristol-Myers
Squibb, Princeton, United States
Experimental Medicine Program Student Research Day 2014-06-25
Chronic Myeloid Leukemia (CML)
Chronic Phase
(CP)
3-5 years
Accelerated Phase
(AP)
0.1-1 years
Blast Phase
(BP)
Adapted from Ren R, Nature Reviews, 2005
Chronic Myeloid Leukemia (CML)
p210BCR-ABL
BCR
SH2
Y1294
SH3
S/T kinase
Rho-GEF
Y177
ABL
Tyrosine NLS DNA Actin
BD
BD
Kinase
Ras
PI3K
JAK2
MAPK
AKT
STAT5
Increased proliferation
Decreased apoptosis
Sattler et al, Cytokine Growth Factor Rev 8:63, 1997
Massive accumulation of myeloid
cells in circulation
(Major characteristic of CML)
Challenges in CML Treatment:
CML Stem Cells Are Insensitive to TKIs
IM
IM
IM
Current 1st line: Imatinib
Major problems associated with TKI therapy:
Alternatives: Dasatinib, Nilotinib
- Early relapse, acquired drug resistance
- Ineffectiveness in eliminating CML stem cells
Druker BJ Blood , 2008
TKI Resistance of CML Stem Cells
AHI-1-BCR-ABL-JAK2 Complex
Regulates TKI Response/resistance of
CD34+ CML Cells
BCR-ABL-dependent
mechanisms:
Increased BCR-ABL expression
BCR-ABL mutations
Increased drug efflux and reduced
drug influx
Imatinib 5 µM
% inhibition relative
to untreated cells
Current Challenges:
Ctrl
AHI-1sh4
80
60
40
20
0
Genomic instability
BCR-ABL-independent
mechanisms:
Not addicted to BCR-ABL for survival
Protected by BM microenvironment
Activation of pro-survival pathways:
Autophagy, Wnt//β-catenin, etc
Jiang et al, J Natl Cancer Inst, 2007; Chomel et al, Oncotarget, 2011
Corbin et al, JCI, 2011; Hamilton et al, Blood 2012
Zhou et al, J Exp Med, 2008
Hypothesis and Objectives
Stem cells are
insensitive to TKIs
Relapse
TKIs
TKIs
BCR-ABL
JAK2
AHI-1
Combined suppression of BCR-ABL and JAK2 activities to
destabilize this protein complex may be more effective at
eliminating CML stem cells
Combination Treatment with ABL and JAK2 Inhibitors Is
More Effective at Reducing pSTAT5 Levels in CML Cells
K562
1.2
1
1
2
3
4
5
6
7
8
pSTAT5
GAPDH
2 hours after drug treatment
K562 DMSO Ctrl
K562 1.0 µM BMS
K562 0.05 µm IM
K562 IM + BMS
K562R DMSO Ctrl
K562R 1.0 µM BMS
K562R 0.05 µm IM
K562R IM + BMS
0.6
0.4
Relative pSTAT5 levels
STAT5
1
2
3
4
5
6
7
8
P < 0.05
0.8
0.2
0
1
2
3
4
K562R (IM-resistant cells)
1.2
P < 0.05
1
0.8
P < 0.05
0.6
0.4
0.2
0
5
6
7
8
n=3
Combination Treatment Results in a Significant
Reduction in Colony Formation of CML Cells
Total CFC
Methylcellulose
± inhibitors
80
60
40
20
0
No drug
12 days
37ºc
IM
BMSBMS911543 911543
+ IM
K562R
120
100
80
200 cells 200 cells
60
40
Small colonies: <50 cells
20
Medium colonies: 50-300 cells
0
Large colonies: >300 cells
No drug
IM
BMSBMS911543 911543
+ IM
No. of CFC per 200 input cells
CML cell lines 120
(600 cells) 100
K562
60
50
40
30
20
10
0
No. of CFC per 200 input cells
K562
80
70
60
50
40
30
20
10
0
P < 0.05
Small
P < 0.05
Medium
Large
No drug
IM
BMSBMS911543 911543
+ IM
K562R
P < 0.05
P < 0.05
No drug
IM
BMSBMS911543 911543
+ IM
P-STAT5 relative
to untreated cells (%)
Combination Treatment Is More Effective at Reducing
pSTAT5 and pCRKL Levels in
CD34+ CML Cells
P<0.01
100
-H, SSC-H subset
P<0.05
100
80
60
20
60
100
100
100
100
100
40
20
0
0
101
102
FL4-H: APC
103
104
C-H, SSC-H subset
No drug
72 ctrl
pstat5
DA 72 DA pstat5
BMS72 BMS pstat5
DA +72
BMSDA+BMS pstat5
IgG Ctrl
72 2 only
P-CRKL relative
to untreated cells (%)
BMS
100
100
100
100
100
100
P<0.05
80
72 ctrl pstat5
72 DA pstat5
72 BMS pstat5
72 DA+BMS pstat5
72 2 only
40
P<0.05
IM
DA
IM+BMS DA+BMS
100
BMS
IM
P<0.01
60
40
40
20
20
0
0
BMS
IM
DA
P<0.05
80
60
IM+BMS DA+BMS
48 hrs treatment
IM+BMS DA+BMS
P<0.05
100
80
DA
BMS
IM
DA
IM+BMS DA+BMS
72 hrs treatment
n=4
Combination Treatment Results in a Significant
Increase in Apoptosis of CD34+ CML Cells
10
4
0.34
15.8
10
4
10
3
10
2
10
1
0.33
PI
10
3
10
2
10
1
10
FL3-H: FL3 PI
FL3-H: FL3 PI
Ctrl
0
79.1
410
0
10
4.77
10
1
0.52
Ungated
Ctrl
Event Count: 10000
3
10
2
10
10
FL4-H: Annexin APC
3
10
2
10
1
10
0
10
Ungated
DA
Event Count: 10000
71
0
4
5.02
0
1
2
3
10
46
4
BMS+DA
66.7
0
BMS
10 4 10
10
10
10
10
FL4-H: Annexin APC
0.39
26.6
Ungated
BMS
Event Count: 10000
3
10
FL3-H: FL3 PI
FL3-H: FL3 PI
DA
10
23.7
6.19
10
1
2
10
10
FL4-H: Annexin APC
3
10
2
10
1
10
0
10
4
46.5
10
0
Ungated
BMS+DA
Event Count: 10000
Annexin V
10
2
10
10
FL4-H: Annexin APC
3
10
P<0.05
30
20
48 hrs
10
0
BMS
IM
DA
NL BMS BMS BMS
+ IM + DA + NL
P<0.05
P<0.05
P<0.05
40
30
20
7.05
1
Apoptotic cells above untreated (%)
40
72 hrs
4
10
0
BMS
IM
DA
NL BMS BMS BMS
+ IM + DA + NL
n=3
Combination Treatment Is More Effective at Reducing
Colony Formation in Treatment-naïve CD34+ CML Cells
from Subsequent IM-nonresponders
CML blood
samples
Methylcellulose
± inhibitors
Select
CD34+ cells
CD34+ cells
BFU-E
P<0.001
60
P<0.05
P<0.01
40
20
P<0.001
80
P<0.001
P<0.001
60
40
20
BMS IM
100
80
Total CFC
0
12 days
% CFCs relative to
untreated cells
% CFCs relative to
untreated cells
100
% CFCs relative to
untreated cells
100
DA
CFU-GM
80
NL BMS BMS BMS
+ IM + DA + NL
P<0.001
P<0.001
P<0.01
60
40
20
0
0
BMS IM
DA
NL BMS BMS BMS
+ IM + DA + NL
BMS IM
DA
NL BMS BMS BMS
+ IM + DA + NL
n=10
Combination Treatment Is More Effective at
Eliminating More Primitive CML Cells from Subsequent
IM-nonresponders
CML blood
samples
Select
CD34+ cells
CD34+ cells
On feeders for
6 wks  inhibitors
Methylcellulose
Total LTC-IC derived CFCs relative to
untreated cells (%)
140
120
100
80
P<0.05
60
P<0.05
40
20
0
Ctrl
BMS
IM
DA
AMN
BMS +
IM
BMS +
DA
BMS +
AMN
12 days
37ºc
n=3
% of CFCs relative to untreated cells
BMS-911543 Has Less Toxicity on CD34+ Normal Bone
Marrow Cells than CML Cells
Total CFC
100.0
NBM
CML
80.0
60.0
P<0.001
P<0.001
P<0.01
40.0
20.0
0.0
BMS
IM
DA
NL
BMS + IM BMS + BMS + NL
DA
n=7
In Vivo Combination Effects
NSG mice
age 7-10
weeks
% Survival
Day 55
Sub-lethal
cesium
irradiation
Oral gavage drug
treatments
Days after transplantation
Weight (grams)
I.V. injection
of BV173 cells
P<0.001
Days after transplantation
In Vivo Combination Effects
BMS
0.03
0.21
Ctrl
DA DA+BMS
Vehicle
BMS
DA
DA+BMS
Spleen
0.19
0.03
CD19
1.0 mm
0.02
Liver
1.9
3.15
4.53
1.62
1.47
1.0mm
Vehicle
28.9
2
101
10
2
101
100
200
400
600
800
FSC-H: FSC-Height
1000
10
2
101
100
0
100
0
200
400
600
800
FSC-H: FSC-Height
DA
4
1000
0
200
DA+BMS
0.53
104
1.34
103
FL2-H: CD19 PE
10
10
33
103
FL2-H: CD19 PE
CD19
FL2-H: CD19 PE
103
BMS
104
400
600
800
FSC-H: FSC-Height
10
3
10
2
10
1
10
0
1000
103
FL2-H: CD19 PE
104
0.07
FL2-H: CD19 PE
Ctrl
104
BM
10
2
101
100
0
200
400
600
800
FSC-H: FSC-Height
1000
0
200
400
600
800
FSC-H: FSC-Height
1000
FSC
Fold difference relative
to vehicle
Day 55
Ctrl Vehicle
BM
1.2
1
0.8
0.6
0.4
0.2
0
BCR-ABL transcript levels
Spleen
1.5
1
0.5
ND
ND
0
ND
1.2
1
0.8
0.6
0.4
ND ND 0.2
0
Liver
P<0.01
P<0.05
ND
In Vivo Combination Effects
NSG mice
age 7-10
weeks
% Survival
Day 70
Sub-lethal
cesium
irradiation
Oral gavage drug
treatments
Days after transplantation
Weight (grams)
I.V. injection
of BV173 cells
P<0.001
Days after transplantation
In Vivo Combination Effects
Vehicle
Day 70
DA
Vehicle
DA+BMS
DA
DA+BMS
Spleen
0.14
0.11
0.03
CD19
1.0 mm
Liver
5.55
4.17
10
3
10
2
10
1
10
0
DA
104
103
102
101
400
600
800
FSC-H: FSC-Height
1000
102
101
100
200
DA+BMS
1.17
104
26.3
103
0
Fold difference relative
to vehicle
1.0mm
FL2-H: CD19 PE
FL2-H: CD19 PE
CD19
BM
Vehicle
34.7
4
FL2-H: CD19 PE
10
1.78
100
0
200
400
600
800
FSC-H: FSC-Height
1000
0
FSC
200
400
600
800
FSC-H: FSC-Height
1000
BCR-ABL transcript levels
1.2
1
0.8
0.6
0.4
0.2
0
BM
P<0.001
Spleen
1.5
1
0.5
0
P<0.01
1.2
1
0.8
0.6
0.4
0.2
0
Liver
P<0.001
Conclusions
Y177 P
Y177 P
JAK2
BCR-ABL
AHI-1
STAT5
Stem cell proliferation
& survival
TKIs
BCR-ABL
JAK2
AHI-1
STAT5
TKI
response
Stem cell proliferation
& survival
TKI
response
Combination treatment:
• More effectively reduces pSTAT5 and pCRKL levels
• Increases apoptotic cells and inhibits colony growth in CD34+ CML cells
• Prolongs survival of leukemic mice
Targeting both BCR-ABL and JAK2 activities may be a more effective
therapeutic option for CML patients
Acknowledgments
Jiang Lab Members:
Dr. Xiaoyan Jiang
Dr. Min Chen
Katharina Rothe
Dr. Kevin Lin
Sharmin Esmailzadeh
Will Liu
Rachel Huang
Damian Lai
CML Working Group:
Stem Cell Assay Lab: Dr. Connie Eaves
Karen Lambie
Helen Nakamoto
Kyi Min Saw
Animal Resource
Centre
FACS Facility
Dr. Donna Forrest
BMS:
Dr. Matthew Lorenzi
Dr. Adrian Woolfson
Collaborators:
Dr. T. Holyoake, Scotland
Dr. A.Turhan, France
Dr. R. Arlinghaus, Houston