Transcript Slide 1

Targeting Survival and DNA
Repair Pathways in Chronic
Lymphocytic Leukemia
Dr. Lawrence Panasci
Potential Conflict of Interest
• Research Grant
– Luitpold Pharmaceuticals / 2007-2009
– Novartis Pharmaceuticals / 2009-
Targeting Survival And DNA Repair Pathways In
Chronic Lymphocytic Leukemia
Raquel Aloyz PhD
Dr Lawrence Panasci MD
Department of Oncology
& Program in Cancer Genetics
McGill university
Chronic Lymphocytic Leukemia (CLL)
• CLL is characterized by the accumulation in the blood of affected
patients quiescent B-lymphocytes in the G0/G1 phase of the cell
cycle.
• At an early stage of the disease, B-lymphocyte accumulation occurs
likely as a consequence of an undefined defect in the apoptotic
machinery rather than an increased proliferation of leukemic cells.
• While the patients often initially respond to conventional treatment
with chlorambucil or fludarabine, they eventually become resistant to
the drugs.
B-Cell Chronic Lymphocytic Leukemia (CLL)
1)
Is a disease characterized by the proliferation of abnormal, developmentally
regulated immature B lymphocytes that accumulate in the blood of affected
patients
2)
The nitrogen mustard (NM), chlorambucil (CLB), was commonly used as fist
line therapy for CLL with an initial response rate of 60-80%, often to low
dose CLB therapy, but eventually (usually after years of therapy), all CLL
patients become resistant to CLB
3)
Thus CLL is an excellent clinical model of NM drug resistance since:

an homogenous population of malignant B lymphocytes is easily obtainable

these malignant B lymphocytes are representative of the clinical status, i.e.
in-vitro/in-vivo NM resistance and

the chronic nature of the drug treatment allows for the development of drug
resistance
DNA Interstrand Crosslink Repair
Cyclophosphamide
Chlorambucil
Interstrand Crosslink
Removal
DNA INTERSTRAND
CROSSLINK (ICL)
NER
Non Limiting Step
Double
Strand
Break
DSB
Homologous
Recombinational
Repair
Non Homologous
End joining
Repair
Fig. 1
Christodoulopoulos, G. et al. Clin Cancer Res 1999;5:2178-2184
Copyright ©1999 American Association for Cancer Research
LINEAR REGRESSION Rad51 and Xrcc-3 Protein Level vs.
LD50Chlorambucil
In Chronic Lymphocytic Leukemia
summary
•
Resistance to DNA crosslinking agents is associated with:
a)
Accelerated repair of interstrand crosslinks induced by these
agents.
b)
Increased drug-induced-Rad51 foci density.
c)
3)
In CLL primary lymphocytes increased protein levels of
xrcc3/Rad51 and in epithelial cell lines increased levels of XPD and
xrcc3.
Overexpression of xrcc3 results in DNA cross linking
agent drug resistance.
DNA INTERSTRAND
CROSSLINK (ICL)
NER
Double
Strand
Break
DSB
BRCA1
RAD51
NUCLEAR
FOCI
c-Abl
Sensitization to
ICL-inducing agents
Decreased Rad51 Foci
Untreated
B-lymphocyte
Homologous
Recombinational
Repair
CLB treated
B-Lymphocytes
DNA DAMAGE
BRCA1
ATM
c-Abl
Activation
RAD51
c-Abl
Activation
BRCA1
Tyr 315
RAD51
RAD52
1-c-Abl positively regulates Rad51-related Homologous Recombinational Repair
2-Homologous recombinational Repair is implicated in CLB drug sensitivity in CLL
We investigate the effect of the c-abl inhibitor Imatinib/STI571 in CLB
cytotoxicity in CLL lymphocytes
STI571
Tyr 412
REGULATED
Tyr 245
ACTIVE
Determination of Drug Synergy Using the MTT Assay
CLB
y = 106.63e-0.026x
R2 = 0.9648
IC50=29.12mM
y = 109.53e-0.0469x
R2 = 0.9797
IC50=13.72mM
120
100
80
80
% of Control
60
40
120
100
80
20
60
40
50
0
100
60
20
0
0
IC50= 2.8 mM
40
20
0
20
40
0
60
0
STI571 (mM)
CLB (mM)
5
10
CLB (mM)
IC50
CLB
5mM STI571
120
IC50= 2.8 mM
100
% of Control
% of Control
100
CLB
5mM STI571
% of Control
120
STI571
80
60
40
I=
20
0
0
10
I< 1 Synergy
20
30
CLB (mM)
40
IC50 CLB
IC50 CLB
+
[ STI571]
IC50 STI571
50
I=1 Additive
I> 1 Antagonism
= 0.46
15
STI571(Imatinib) Sensitizes CLL cells to CLB (Chlorambucil)
Independently of the clinical status
CLB IC50 mM Imatinib IC50 mM
CLB IC50 mM+
I Value
5mM Imatinib
CLB IC50 mM+
10 mM Imatinib
I Value
Patient U1
14.6
55.93
3.35; (4.0)a
0.32
3.35; (4.9) a
0.32
Patient U2
28.49
29.79
2.90; (9.6) a
0.27
2.96; (9.6) a
0.27
a
0.37
4.70; (4.2)
a
0.37
0.58
21.20; (2.5) a
0.59
0.75
a
0.96
3
Patient U
20.02
36.8
4.70; (4.2)
Patient U4
54.2
101.8
21.20; (2.6) a
5
a
Patient U
18.08
47.9
11.70; (1.5)
Patient U6
7.84
46.7
0.49
Patient U
12.27
44.9
3.04; (2.5) a
79.9
13.80; (1.3)
ND
6.5
70.1
Patient T1
9.29
33.29
1.20; (7.7) a
0.28
1.09; (8.5) a
7
Patient T
2
Patient T
3
Patient T
4
79.7
32.2
Patient T
5
49.2
34
23.1
5.56
16.5
53.06
CLB IC50 mM Imatinib IC50 µM
3.00; (7.7)
a
3.54; (1.5)
a
5.93
0.42
1.10; (21.0)
0.72
a
2.67; (2.0)
59.00; (1.3)
a
0.88
35.20; (2.2)
17.20; (2.8)
a
0.49
ND
0.41
a
a
0.65
0.66
0.75
CLB IC50 CLB+Imatinib µM
I Value
WSU
34.00±2.80
13.72±2.75
5.95±0.17**; (5.7) a ; 5µM STI571b
3.15±1.82**; (10.7) a ; 10µM STI571b
0.530.82
I83
40.66±2.80
33.73±4.19
17.30±1.00*; (2.3) a 1.5µM STI571b
25.40±1.53*; (1.6) a ; 3µM STI571b
0.450.74
Summary of the mechanisms of action of Gleevec alone or
in combination with Chlorambucil in malignant CLLlymphocytes
Chlorambucil
Constitutive
c-Abl
activation of Lyn
Fludarabine
•Inhibition of transcription
DNA
damage
Anti apoptotic signalling
•Inhibition of DNA synthesis
(cycling cells)
Rad51-dependant
DNA repair
Death
Survival
Survival
APOPTOSIS
c-abl kinase
inhibition
c-abl kinase
Src kinase
inhibition
inhibition
Dasatinib
Gleevec
Survival
Survival
A phase I -II Clinical Trial is in process to assess the
effect of Gleevec in combination with CLB
A phase I-II trial of Gleevec (imatinib mesylate)
in combination with chlorambucil in previously
treated chronic lymphocytic leukemia (CLL)
patients
Study Protocol
Sponsor:
Novartis Pharmaceuticals
Group/Participating Institutions:
Jewish General Hospital
Hopital Notre-Dame, CHUM
Hopital Charles Lemoyne
Investigators:
Jonathan Hebb, MD, MSc
Sarit Assouline, MD
Lawrence Panasci, MD
Pierre DesJardins, MD
Stephen Caplan, MD
Raquel Aloyz, PhD
Rationale
•
•
•
•
•
•
There is a synergistic effect of imatinib on CLB mediated cytotoxicity in CLL
cells in vitro.
This effect occurred at concentrations of imatinib (<10mm) that are clinically
achievable.
Drug sensitivity in CLL lymphocytes is determined in part by the repair capacity
of nitrogen mustard-induced DNA interstrand cross links (ICLs).
The regulation of this repair mechanism has been associated with a c-abl
mediated phosphorylation of Rad51 which is involved in repair of CLB –
induced ICLs.
Imatinib inhibits c-abl activity; imatinib may sensitize CLL cells to CLB through
inhibition of c-abl mediated DNA-repair pathways.
Imatinib in vitro inhibits c-abl, with a resultant decrease in c-abl mediated
Rad51 phosphorylation in CLB-treated CLL lymphocytes. Encouraging results
from these in vitro studies provide a basis for initiating a phase I/II clinical
study.
STUDY OBJECTIVES
Primary Objectives
• To determine the maximum tolerated dose of Gleevec in combination with
chlorambucil(CLB).
• To determine the toxicities of Gleevec in combination with CLB.
Secondary Objectives
• To determine the efficacy of Gleevec at the MTD in combination with CLB.
• To determine the peak/steady state plasma concentration of Gleevec at
each level, but mainly at the MTD.
• To determine the amount of Gleevec sensitization of CLB in-vitro in
pretreatment lymphocytes and correlate these results with in-vivo anti-tumor
activity.
• To determine the duration of response in patients who respond to Gleevec
and CLB at the MTD.
SAMPLE SIZE
•
Up to 18 patients will be enrolled in the phase I portion of this study, with
three patients tested at each dose level of Gleevec.
•
Cohorts will be expanded to 6 patients if there is one dose limiting toxicity in
the first three patients enrolled in a given cohort.
•
Once the maximum tolerated dose has been determined, a total of 16
patients will be enrolled in the phase II component of the study.
PATIENT POPULATION
Patients with CLL in whom treatment is clinically indicated and who
have been previously treated with one or more of the following
regimens:
• CLB, with a progression free survival of at least 6 months.
• Fludarabine or any fludarabine containing regimen.
• Any other treatment regimen including monoclonal antibodies,
corticosteroids, immunotherapies, or radiation.
Patient Eligibility
•
Patients with B-cell chronic lymphocytic leukemia (a) Rai Stage 0-II with
indication for treatment by NCI Working Group Criteria; or (b) Rai Stage III
or IV.
•
The diagnosis of CLL must be pathologically verified according to the WHO
classification of hematological malignancies.
•
Received a minimum of one prior chemotherapy regimen. Additionally, prior
treatment with corticosteroids, immunotherapies, monoclonal antibodies or
radiation therapy is permitted.
•
WBC count of > 25 x 109/L.
DOSAGE REGIMEN
• Gleevec at three dose levels: 300mg, 400mg, or 600mg
daily for 10 days (from day 1 to day 10), and a total fixed
dose of CLB 8 mg/m2 daily x 5 days (day 3 to day 7) will
be administered. The treatment will be administered
every 28 days. Patients may receive up to 6 cycles of
therapy.
•
Dose Level
Gleevec
CLB
-1
300mg
6mg/m2
1
300mg
8mg/m2
2
400mg
8mg/m2
3
600mg
8mg/m2
No of patients
3*
3*
3*
3*
Lymphocyte counts for CLL patients on protocol GL-CLB-001
(treatment started at Week 0)
120
Dose Level 1 (300 mg Gleevec)
03-01
03-02
03-03
100
80
60
40
Lymphocyte counts (x10^9/L)
20
0
-1
1
2
3
4
5
6
7
8
12
16
20
24
36
300
Dose Level 2 (400 mg Gleevec)
250
03-04
01-02
200
150
100
50
0
-1
1
2
3
4
5
6
7
8
12
16
20
24
36
24
36
350
Dose Level 3 (600 mg Gleevec)
300
01-03
03-05
03-06
01-04
01-05
250
200
150
100
50
0
-1
1
2
3
4
5
6
Weeks
7
8
12
16
20
Dose
Level
Patient
#
Age
Current
Staging
Prior Tx
Response on
Gleevec/Chlora
mbucil at Cycle
3
Response on
Gleevec/Chlora
mbucil at Cycle
5
Best Response
at Follow-up
Long Term
Follow-Up
Plasma
Concentration*
2 - 4h
uM
1
300mg
03-01
L-G
71
Rai II
Chlorambucil (PR)
SD
PR
PR (6 mth F/U)
Pt developed
Hodgkins
Died of
pneumonitis rltd
to bleomycin
1
300mg
03-02
R-C
71
Rai II
Fludarabine (PR)
Cycloph/Flud (CR)
SD
off study
(AE
neutropenia)
SD (6 mth F/U)
PD (9 mth F/U)
6 - 9.7
1
300mg
03-03
M-B
57
Rai II
Fludarabine (PR)
PR
PR
CR (1 mth F/U)
CR (9 mth F/U)
3 - 4.4
2
400mg
03-04
T-P
86
Rai III
Fludarabine (CR)
SD
Increased
Lymphocytes
-
-
3.8 - 6.2
2
400mg
01-01
LHT
49
bulky
adenop
-
-
-
4.5 - 8.9
2
400mg
01-02
L-S
73
Rai III
Chlorambucil (PR)
Chlorambucil (PR)
Fludarabine (PR)
PR
PR
FU not done. Pt
decision
PD (6 mth F/U)
7.0 -08.1
3
600mg
01-03
F-Z
80
Rai III
Chlorambucil (PR)
CLB/Pred (PR)
PR
off study (SAE
- disseminated
herpes)
-
-
N/A
3
600mg
03-05
L-L
78
Rai I
Fludarabine (CR)
PR
PR
PR
3
600mg
03-06
M-H
59
Rai 1
Fludarabine (PR)
Cycloph/Flud (SD)
R-FCM (PR)
R-CHOP (PR)
3*
600mg
01-04
M-D
77
Rai IV
Chlorambucil (PR)
Fludarabine (CR)
Cycloph (PD)
3*
600mg
01-05
P-O
3*
600mg
10 - 14
PD at Cycle 4
Rai I
76
Rai II
Fludarabine (CR)
Chlorambucil (PR)
Cycloph/Fludar (PR)
CLB (Unk, 1 course)
Flud (Unk, 1 course)
Flud/Retuximab (Tox
to chemo after C1)
Unk Offstudy at C2
(SAEpneumonia)
5.0 - 5.6
N/A
Off-study at
C1
-
-
-
N/A
-
-
-
Not available
(low platelets)
PD at
Cycle3
DNA Interstrand Crosslink Repair
Cyclophosphamide
Chlorambucil
Interstrand Crosslink
Removal
DNA INTERSTRAND
CROSSLINK (ICL)
NER
Non Limiting Step
Double
Strand
Break
DSB
Homologous
Recombinational
Repair
Non Homologous
End joining
Repair
Non Homologous End Joining (NHEJ) Pathway
DNA damage recognition and processing:
•H2X2
• Ku70/80
•DNA-PKcs
•Artemis
Ligation:
• Ku70/80
•DNA-PKcs
•Ligase IV/xrcc4
LINEAR REGRESSION Ku86 Protein Levels and DNA-PK activity
vs LD50Chlorambucil
In Chronic Lymphocytic Leukemia
6
y = 0.024x + 0.625
r=0.5225
2
Ku86 Levels
DNA-PK Activity (Arbitrary Units)
2.5
1.5
1
0.5
y=7.51x+4.0
r=0.875
5
4
3
2
1
0
0
10
20
Chlorambucil IC50 (mM)
30
0
10
20
30
40
50
60
70
Chlorambucil IC50 (mM)
Muller C et al. Blood. 1998 Oct 1;92(7):2213-9
NHEJ in CLL NM drug resistance
• KU80 protein levels and DNA-PK activity
correlate directly with CLB drug resistance
in-vitro in CLL lymphocytes
• These results suggest that NHEJ may play
a role in CLB drug resistance in CLL
• In order to investigate this, we utilized
relatively specific inhibitors of DNA-PK
NU7026
• Wortmannin, a nospecific DNA-PK inhibitor’ sensitizes
CLL lymphocytes to chlorambucil.
• Wortmannin is a noncompetitive, irreversible inhibitor of
DNA-PK , whereas NU7026 (2-(morpholin-4-yl)benzo[h]chomen-4-one) is competitive inhibitor of the
ATP site of DNA-PK
• Although Wortmannin is primarily a PI 3-K inhibitor,
being 90-fold more active against PI 3-K than DNA-PK or
ATM, NU7026 is more selective for DNA-PK with a 60fold greater potency against this enzyme than PI 3-K and
inactive against both ATM and ATR. Thus, in contrast
to Wortmannin, NU7026 demonstrates excellent
specificity for DNA-PK.
Effects of NU7026 and CLB
on survival and DNA-PK phosphorylation
a
b
DNA-PK expression in I83 cell line after
48h drugs treatment. NT: Untreated
Bar: 10mM
Amrein L et al J Pharmacol Exp Ther. 2007 Jun;321(3):848-55.
c
NU7026 (a DNA-PK inhibitor)
Sensitizes Primary B-CLL Lymphocytes to CLB in Vitro
•We determine the IC50 of CLB or NU7026 alone and CLB in combination with 1, 5
or 10 mM NU7026 in vitro in a cohort of 19 B-CLL patients (14 untreated and 5
treated patients)
•The IC50 (mM) range for the drugs was:
CLB
7.14 to 61.17 (I value: 0,4-2,0)
NU7026
17.35 to 67.48 (non toxic (>100 mM) in 50 % of patients)
•NU7026 sensitizes the B-CLL lymphocytes to chlorambucil in all the patients but
one. The effect of 1, 5 or 10 mM NU7026 on chlorambucil sensitivity was
synergistic (I value<1) in 14 patients additive in one patient (I=1) and antagonistic
(I>1).
I=
I< 1 Synergy
IC50 CLB
IC50 CLB
+
[ NU7026]
IC50 Nu7026
I=1 Additive
I> 1 Antagonism
Amrein L et al J Pharmacol Exp Ther. 2007 Jun;321(3):848-55.
Effect of NU7026 on CLB cytotoxicity in lymphocytes from CLL patients
Patients
IC50
(mM)CLB
alone
IC50 (mM)
NU7026
alone
IC50 (mM)
CLB+1mM
NU7026
Synergy
Value I
IC50 (mM)
CLB+5mM
NU7026
Synergy
Value I
IC50 (mM)
CLB+10mM
NU7026
Synergy
Value I
U1
16±4.3
17±3.2
11± 3.5 (1.4)a
0.76±0.17
6±2.9 (2.6)a
0.68±0.10
3±0.4 (4.6)a
0.79±0.14
U2
27±3.7
> 100
23± 1.3 (1.2)a
0.86±0.14
12±2.8 (2.2)a
0.46±0.21
4±0.3 (7.6)a
0.13±0.03
U3
17±3.1
> 100
17± 6.4 (1.0)a
0.99±0.19
17±1.1 (1.0)a
1.00±0.12
13±3.6 (1.4)a
0.72±0.16
U4
47±5.4
41±0.2
42±4.9 (1.1)a
0.92±0.21
34±6.4 (1.4)a
0.84±0.20
7±1.3 (6.3)a
0.40±0.09
U5
25±2.0
65±4.7
29±2.3 (0.9)a
1.17±0.16
26±1.1 (1.0)a
1.11±0.18
11±2.4 (2.2)a
0.60±0.06
U6
24±6.3
67±2.7
11±3.2 (2.2)a
0.47±0.05
2.6±0.3 (9.2)a
0.18±0.04
2.3±0.8 (10.1)a
0.25±0.05
U7
52±2.8
48±5.6
29±7.8 (1.8)a
0.58±1.13
19±3.8 (2.7)a
0.47±0.07
17±3 (3.0)a
0.55±0.03
U8
9±0.8
20±2.0
24±2.3 (0.4)a
2.73±0.22
34±1.5 (0.3)a
4.05±0.20
16±4.1 (0.6)a
2.27±0.57
U9
44±3.5
> 100
29±1.4 (1.5)a
0.65±0.05
31±2.8 (1.4)a
0.71±0.14
28±1.3 (1.6)a
0.63±0.05
U10
61±3.7
39±4.8
25±1.3 (2.5)a
0.43±0.04
9.6±4.4 (6.4)a
0.28±0.02
6.8±1.1 (9.0)a
0.37±0.01
U11
7.1±2.3
20±1.7
5.1±1.0 (1.4)a
0.77±0.19
4.4±0.9 (1.6)a
0.87±0.04
2.9±0.07 (2.4)a
0.92±0.15
U12
32±3.3
33±3.9
22±1.7 (1.5)a
0.72±0.02
13±1.7 (2.5)a
0.55±0.01
12±1.6 (2.7)a
0.67±0.01
U13
31±3.1
> 100
26±2.5 (1.2)a
0.83±0.14
27±0.2 (1.1)a
0.89±0.07
21±1.1 (1.4)a
0.70±0.11
U14
12±2.8
> 100
5.9±0.2 (2.1)a
0.48±0.12
8.8±1.8 (1.4)a
0.72±0.18
6.3±1.6 (1.9)a
0.52±0.02
T1
24±5.5
24±6.2
12±3.4 (2.0)a
0.54±0.15
6.9±1.4 (3.5)a
0.49±0.09
5.9±1.9 (4.0)a
0.66±0.18
T2
59±2.8
> 100
49±5.2 (1.2)a
0.84±0.05
40±2.7 (1.5)a
0.68±0.03
17±4.5 (3.4)a
0.30±0.05
T3
32±2.2
> 100
30±3.1 (1.1)a
0.95±0.13
22±3.7 (1.4)a
0.70±0.07
14±1.3 (2.2)a
0.45±0.01
T4
43±6.0
> 100
66±5.8 (0.6)a
1.54±0.12
48±12 (0.9)a
1.11±0.37
29±2.3 (1.5)a
0.67±0.20
T5
16±0.7
> 100
10±0.8 (1.4)a
0.71±0.07
6.9±0.4 (2.1)a
0.48±0.05
5.3±1.0 (2.7)a
0.37±0.07
Using the MTT assay, we evaluated the effect of NU7026 on CLB cytotoxicity in malignant B lymphocytes
from CLL patients. The I-value, I<1 or I>1, indicates that the CLB and NU7026 act synergistically or
antagonistically, respectively. a Ratio between CLB IC50 alone/CLB IC50 in the presence of NU7026.
The results are expressed as the mean value ± s.d. Amrein L et al J Pharmacol Exp Ther. 2007 Jun;321(3):848-55.
Fig. 1
Panasci, L. et al. Clin Cancer Res 2001;7:454-461
Copyright ©2001 American Association for Cancer Research
Dr Aloyz and myself would like to
Acknowledge the following scientists
James Johnston
Spencer Gibson
Manitoba CLL tissue bank
Lilian Amrein
Annette Hollmann
Tiffany A Hernandez
David Davidson
CIHR- “ Dasatinib Cytotoxicity and sensitization to standard therapy in
CLL” Operating Grant to R Aloyz
&
Leukemia & Lymphoma Society “Inhibition of DNA-PK to improve the
efficacy of CLB in CLL”. Translational Research Grant to L Panasci