Transcript PowerPoint - Q-CROC

DNA damage in cancer
therapy
Lawrence Panasci MD
Department of Oncology
& Experimental Medicine
Segal Cancer Center-Lady Davis Institute
Jewish General Hospital-McGill university
Potential Conflict of Interest
• Dr. Lawrence C. Panasci
– None
Advances in cancer survival
1.Two recent cytotoxic agents,
taxanes and oxaliplatin
2. Herceptin, Rituximab, Gleevec
and perhaps Sutent
Repair of Specific DNA
Lesions
DNA DAMAGE
Repair of DNA Strand Breaks
DNA Repair in cancer and normal cells
Cytotoxic anticancer drugs
• DNA cross-linking agents, i.e. the platinum
compounds(cis-platinum and oxaliplatin)
and nitrogen mustards(chlorambucil) result
in cytotoxic interstrand cross-links(ICLs)
• Anthracyclines (adriamycin), etoposide
and irinotecan result in potentially lethal
double strand breaks (DSBs)
Repair of interstrand cross-links(ICLs)
and double strand breaks(DSBs)
• Repair of DSBs and ICLs may occur via
similar DNA repair pathways
• An intermediate step in repair of ICLs may
involve induction of DSBs
• ICLs are produced by DNA cross-linking
agents such as platinums and nitrogen
mustards
• DSBs are produced by ionizing
radiation(IR) or agents such as etoposide
and irinotecan
DNA DSB -induced activation of checkpoint and repair pathways.
Bolderson E et al. Clin Cancer Res 2009;15:6314
-6320
©2009 by American Association for Cancer Research
Rad-51 Directed DSB repair
Non Homologous End Joining Repair
Nitrogen Mustard (NM) Drug Resistance
in chronic lymphocytic leukemia (CLL)
(1)Lymphocytes from treated-resistant CLL
patients have an enhanced capacity to remove
DNA interstrand crosslinks formed by NMs such
as chlorambucil as compared with those from
untreated patients
(2)The development of chlorambucil (CLB)
resistance in CLL appears to be specifically
associated with cross-resistance to other
bifunctional alkylating agents, which produces
interstrand cross-links (ICL) in DNA.
(3)Thus, enhanced ICL-specific repair appears to
be one of the primary mechanisms of NM
resistance in CLL
Nitrogen Mustard (NM) Drug
Resistance in CLL (continued)
(4)DNA ICL and certain double-strand breaks
require information supplied by another
chromosome or chromatid for error-free repair.
(5)It is thought that ICL are removed from the DNA
of mammalian cells by the combined actions of
excision repair (NER) and recombination
systems.
(6)Our results suggest that the incision/excision
step of ICL repair is not rate-limiting in CLBresistant lymphocytes but instead is associated
with enhanced recombinational repair of ICL.
Nitrogen Mustard (NM) Drug Resistance
in CLL (continued)
(7) It is possible that the repair of ICLs involves an
intermediate step of DSB formation
(8) Both homologous recombinational repair (HRR)
and nonhomologous endjoining (NHEJ) are
implicated in the repair of DSBs
(9) Since CLL lymphocytes are largely
nonproliferative and since NHEJ can repair
DSBs in all phases of the cell cycle but HRR is
more involved in S/G2 phases of the cell cycle,
NHEJ is apt to be more prominent in CLL
lymphocytes
(10) NHEJ components include the Ku70/Ku80
heterodimer that is recruited to DSBs, followed
by DNA-PKcs, XRCC4, and ligase IV to
complete NHEJ.
Nitrogen Mustard (NM) Drug Resistance
in CLL(continued)
(11)DNA-dependent protein kinase (DNA-PK) is a
nuclear protein serine/threonine kinase
comprised of 3 subunits; a large catalytic subunit
of 460 KD, DNA-PKcs and a DNA binding
subunit, the Ku autoantigen (a dimer of Ku70
and Ku86 proteins).
(12)Mutations in the subunits result in doublestrand break repair defects, X-ray sensitivity,
impaired V(D) J recombination and nitrogen
mustard sensitivity.
(13)The role of DNA-PK was investigated vis-à-vis
nitrogen mustard drug resistance in CLL
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 is a specific DNA-PKi
• Wortmannin, a nonspecific 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 90fold more active against PI 3-K than DNA-PK or ATM, NU7026
is more selective for DNA-PK with a 60-fold 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.
NU7026 in CLL
• (1-10uM)NU7026 synergistically(<1) sensitizes
CLL lymphocytes to CLB in almost all(>15/19
samples). Similar sensitization occurs in the I83
CLL cell line.
• This is associated with NU7026 inhibition of
CLB-induced DNA-PK activity both in I83 cells
and CLL lymphocytes( using FACS analysis)
and NU7026 inhibition of repair of DSBs (using
gH2AX )
NU7026 pharmacokinetics
Following intravenous administration to mice at 5 mg kg,
NU7026 undergoes rapid plasma clearance and this is
largely attributed to extensive metabolism.
Bioavailability following interperitoneal (i.p.) and p.o.
administration at 20 mg kg(-1) was 20 and 15%,
respectively.
Investigation of NU7026 metabolism profiles in plasma and
urine indicated that the compound undergoes multiple
hydroxylations.
NU7026 will have to be administered four times per day at
100 mg kg(-1) i.p. in order to obtain drug exposure
required for radiosensitisation.
Icos compounds
• Icos corp made a series of IC compounds which
are specific inhibitors of DNA-PK
• These compounds also have problems with their
pharmacokinetic parameters
• These compounds were acquired by Luitpold
corporation and they have modified these
compounds to improve the pharmacokinetic
parameters.
• We have investigated the original IC compounds
in CLL lymphocytes in colloboration with the
Luitpold corporation.
IC compounds compared to
NU7026
• The IC compounds compare favorably
with NU7026.
• IC486241 appears to have the most
favorable profile but all the IC compounds
are active.
• IC486241 sensitizes CLL lymphocytes to
chlorambucil similar the results with
Nu7026
• The Luitpold modification of the IC
compounds should render them more
useful clinically
DNA-PK in colon cancer therapy
• Irinotecan and oxaliplatin are major
anticancer agents utilized in the treatment of
metastatic colon cancer
• We utilized two divergent colon cancer cell
lines, the mismatch-repair deficient, p53
normal HCT-116 and the mismatch repair
competent, dysfunctional p53 mutant HT-29
• Nu7026 and IC486241(ICC) specific DNA-PK
inhibitors were utilized to test for synergy
DNA-PK inhibitors in colon
cancer
• Modest reductions in IC50 values and a lack
of synergy were observed with oxaliplatin in
the presence of nontoxic concentrations of
Nu7026 or IC486241
• In contrast, significant synergy was observed
with SN38(activated irinotecan) in the
presence of nontoxic concentrations of the
inhibitors
• This was associated with decreased
activation of DNA-PK and greater DNA
damage (comet assay) with presumably less
DNA repair
Homologous Recombinational Repair
(HRR) in chlorambucil resistance in CLL
• Since chlorambucil-induced ICLs are more
rapidly repaired in resistant CLL
lymphocytes, we also investigated the role
of HRR
• HRR can remove the ICLs in an error free
fashion by utilizing non-damaged sister
chromosones which are most readily
available during DNA replication and cell
division
Mechanisms of drug resistance
Panasci L et al. Clin Cancer Res 2001;7:454-461
©2001 by American Association for Cancer Research
CLB induces Rad51 Foci in primary CLL lymphocytes
Christodoulopoulos G et al. Clin Cancer Res 1999;5:21782184
©1999 by American Association for Cancer Research
CLB-induced Foci correlates with CLB resistance
Christodoulopoulos G et al. Clin Cancer Res 1999;5:21782184
©1999 by American Association for Cancer Research
c-abl phosphorylates Rad51
DNA DAMAGE
BRCA1
c-Abl
ATM
Activation
RAD51
c-Abl
Activation
BRCA1
Tyr 315
RAD51
RAD52
c-abl modulates Rad51-directed DNA
Repair
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/Gleevec in CLB cytotoxicity in
CLL lymphocytes
Determination of Drug Synergy Using the MTT Assay
How we assess synergy
CLB
y = 109.53e
2
R = 0.9797
IC50=13.72M
120
100
80
80
% of Control
60
40
120
100
80
20
60
40
50
0
100
60
20
0
0
IC50= 2.8  M
40
20
0
20
40
0
60
0
STI571 (M)
CLB (M)
5
10
CLB (M)
IC50
CLB
5M STI571
120
IC50= 2.8 M
100
% of Control
% of Control
-0.0469x
y = 106.63e-0.026x
R2 = 0.9648
IC50=29.12M
100
CLB
5M STI571
% of Control
120
STI571
80
60
40
I=
20
0
0
10
I< 1 Synergy
20
30
CLB (M)
40
IC50 CLB
IC50 CLB
+
[ STI571]
IC50 STI571
50
I=1 Additive
I> 1 Antagonism
= 0.46
15
CLB induces phosphorylation of
Ra51
Imatinib reduces CLB-induced
Rad51 phosphorylation
Mechanisms of imatinib
cytotoxicity
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
Survival
APOPTOSIS
c-abl kinase
inhibition
c-abl kinase
Src kinase
inhibition
inhibition
Dasatinib
Gleevec
Survival
Mechanisms of imatinib cytotoxicity
c-Abl inhibition sensitizes CLL lymphocytes to
chlorambucil independently of the clinical status
(untreated vs. treated ). The mechanisms involves
increased apoptosis and decreased homologous
recombinational repair.
STI571
Chlorambucil
C-abl activation
C-Abl inhibition by STI571 (imatinib) decreases
chlorambucil induced-RAD51 phosphorylation
and Rad51 Foci
Rad51phosphorylation
Apoptosis
c-Abl inhibition by STI571(gleevec) in the
absence of DNA damage results in CLL
lymphocytes apoptosis
Rad51 Foci
STI571 and chlorambucil cytotoxicity appear to
be mediated by different mechanisms
DNA repair
STI571 is a promising agent to utilize with
chlorambucil in the treatment of CLL patients
Drug sensitization
Phase I trial of imatinib and
chlorambucil in previously treated CLL
• : The three dose levels studied included imatinib at 300, 400, or 600
mg/day. Imatinib was given on days 1-10 and chlorambucil (8 mg/m2 daily)
was given on days 3-7 of a 28-day cycle (up to 6 cycles).
• Eleven patients participated in this study. Low grade gastrointestinal
toxicities were observed in a dose-dependent manner. Forty-five percent
of patients responded (2 unconfirmed CRs and 3 PRs). Two responding
patients were fludarabine-refractory
• in vitro IC50 of chlorambucil in the presence of 5 uM imatinib in CLL
lymphocytes correlated with the decrease in lymphocyte counts on day
15.
• Imatinib plasma concentrations achieved in patients were in the range of
those effective in in vitro sensitization
• The combination of chlorambucil and imatinib in patients with previously
treated CLL was well tolerated and showed evidence of clinical efficacy.
Future studies
• DNA-PKcs detailed crystal structure
determined ( Nature 2010 463:118-21)–
better inhibitors?
• We are designing combi-molecules with Dr
Jean-Claude combining c-abl inhibitors and
bifunctional alkylating agents
• The first molecule incorporating imatinib and
a chlorambucil-like molecule(ZRF4) has
increased activity in-vitro against CLL
lymphocytes than the individual components
• Newer more potent c-abl inhibitors will be
utilized to exploit this concept
Acknowledgements
•
•
•
•
•
Lilian Amrein, PhD Research Associate
Veronica Marignac, PhD Research Associate
David Davidson, PhD postdoctoral fellow
Jeremy Grenier Mcgill student
Collaborators: Dr Raquel Aloyz & Dr Jean-Yves
Masson
Reference on NHEJ/HR/MRN
Lamarche B FEBS letters 584:3682, 2010