1. Princess Margaret Hospital, Co-Chair Gynecology, NCIC CTG
NEW AGENTS ON THE HORIZON: IMPLICATIONS FOR PHASE I, II & III TRIALS DNA Repair and PARP inhibitors Carol Aghajanian, Nicoletta Colombo & Amit Oza Acknowledgements: Stan Kaye and AZ for slides
Amit M. Oza
Professor of Medicine,
Princess Margaret Hospital,
University of Toronto
Co-Chair Gynecology, NCIC CTG

2. Types of DNA damage and repair
Type of
damage:
Single-
strand
breaks (SSBs)
Double-
strand
breaks (DSBs)
Bulky
adducts
O6-
alkylguanine
Insertions
& deletions
Mismatch
repair
Base
excision
repair
Recombinational
repair
Repair
pathway:
Nucleotide-
excision
repair
Direct reversal
DNA damage can occur in several different forms, including SSBs or double-strand breaks (DSBs).1
In higher eukaryotes, genomic stability is essential for healthy functioning and survival. DNA damage may induce mutations and can lead to cell death via apoptosis.2 Therefore, several repair mechanisms have evolved to maintain the integrity of the genome.1
Base excision repair (BER) is a key pathway in the repair of SSBs and is reliant on the enzyme poly(ADP-ribose) polymerase (PARP).1
For DSB repair, there are two predominant pathways:
Homologous recombination (HR) that involves a protein kinase, ataxia-telangiectasia mutated (ATM)
Non-homologous end-joining (NHEJ) that requires DNA-dependent protein kinase (DNA-PK).2
HR is the most accurate mechanism for repairing DSBs, whereas NHEJ is rarely error-free.2
Abbreviations on slide: AGT, O(6)-alkylguanine-DNA alkyltransferase; ATM, ataxia telangiectasia mutated; MLH1, MutL homolog; MSH2, MutS homolog; XP, xeroderma pigmentosum
References
1. Jackson SP, Bishop CL. Drug Discovery World 2003; Fall:
2. Jackson SP. Biochem Soc Trans 2001; 29: HR
NHEJ
Poly ADP Ribose Polymerase
Repair
enzymes:
ATM
DNA-PK
XP,
poly- merases
MSH2, MLH1
AGT
BRCA

3. PARP-1 is a key enzyme involved in the repair of single-strand DNA breaks
DNA damage
PARP
PAR chains are degraded via PARG
Binds directly to SSBs
Repaired DNA
PARP-1 is found in the cell’s nucleus and is a key component of BER, the DNA SSB repair process.1 PARP-1 activation is an immediate cellular response to metabolic, chemical, or radiation-induced DNA SSB damage.2
PARP-1 mediates the repair of SSBs via the activation and recruitment of repair enzymes.
Firstly, PARP-1 detects and signals the presence of an SSB by binding to the DNA adjacent to the damage. Once bound, PARP-1 catalyzes the cleavage of the coenzyme nicotinamide adenine dinucleotide (NAD+) into nicotinamide and ADP-ribose to produce highly charged branched chains of poly(ADP-ribose) (PAR).
These activities of PARP-1 serve to recruit other repair enzymes, such as DNA ligase III (LigIII) and DNA polymerase beta (polβ), and scaffolding proteins such as x-ray repair complementing gene 1 (XRCC1), to the site of damage, where together the enzymes repair the damaged DNA.2 DNA polymerase uses the complementary strand of DNA as a template to fix the SSB, and DNA ligase creates the final phosphodiester bond to fully repair the DNA.
After repair, the PAR chains are degraded via PAR glycohydrolase (PARG).
References
1. Hoeijmakers JH. Nature 2001; 411:
2. D’Amours D et al. J Biochem 1999; 342:
NAD+
Nicotinamide +pADPr
Repair enzymes
PAR
Once bound to damaged DNA, PARP modifies itself producing large branched chains of PAR

4. Inhibiting PARP-1 increases double-strand DNA damage
DNA SSB
XRCC1
LigIII
PNK 1
pol β
PARP
Inhibition of PARP-1 prevents
recruitment of repair factors
to repair SSB
Inhibition of PARP-1 activity prevents the recruitment of DNA repair enzymes and leads to failure of SSB repair and accumulation of SSBs.
During the S-phase of the cell cycle, the replication fork is arrested at the site of an SSB, which then degenerates into a DSB. In normal cells, this triggers activation of the HR pathway to repair the DSB.1
Abbreviation on slide: PNK 1, polynucleotide kinase 1
Reference
1. Helleday T et al. Cell Cycle 2005; 4:
Replication (S-phase)
DNA DSB

5. Selective effect of PARP-1 inhibition on cancer cells with BRCA1 or BRCA2 mutation
DSB in DNA
Normal cell
BRCA-deficient cancer cell
DSB repaired effectively via HR pathway
Deficient HR pathway – DSB not repaired
When PARP-1 is inhibited, SSBs collapse replication forks leading to an increase in DSBs.1,2
In normal replicating cells, DSBs are repaired by the HR pathway. BRCA1 or BRCA2 are a vital part of the HR mechanism as without these proteins, the replication fork cannot be restarted.3
In cells that are deficient in HR repair (eg breast or ovarian cancer cells with a BRCA1 or BRCA2 deficiency), the inhibition of PARP activity leads to an increase in DSBs that cannot be repaired by this mechanism.
DSBs can induce genomic instability and cell-death mechanisms such as apoptosis.2 In addition, many of these DSBs would be repaired by other, error-prone mechanisms, leading to large numbers of aberrations and this in turn would also ultimately lead to cell death.
The effect of PARP inhibition would be highly specific to tumor cells that have a HR deficiency (eg BRCA1 or BRCA2 deficiency). This results in selective cell killing and an increased therapeutic ratio; normal cells have a working HR repair pathway and so would not be affected in this way.
References
1. Bryant HE et al. Nature 2005; 434:
2. Helleday T et al. Cell Cycle 2005; 4:
3. Lomonosov M et al. Genes Dev 2003; 17:
Cell survival
Cancer cell death

6. x x Tumour Selective Synthetic Lethality Lethality Normal or
heterozygote
for HR defect
HR deficient
e.g. BRCA1/2-/-
DNA DAMAGE
DNA DAMAGE x
HR NHEJ SSA BER NER etc
HR NHEJ SSA BER NER etc x
PARPi
PARPi
Error prone repair
Genomic instability
Cell death
Lethality

7. RESPONSE TO AZD 2281, Parp inhibitor - OLAPARIB BY PLATINUM-FREE INTERVAL
Total
Platinum sensitive
Platinum resistant
Platinum refractory
No. of evaluable patients 46 10 25 11
Responders by RECIST
13 (28%)
5 (50%)
8 (32%)
0 (0%)
Responders by GCIG CA125
18 (39%)
8 (80%)
2 (18%)
Responders by either RECIST or GCIG criteria
21 (46%)
11 (44%)
SD (> 4 cycles)
9 (15%)
1 (10%)
4 (16%)
1 (9%)
Median duration of response in weeks (range)
31 (10-96)
31 (16-96)
29 (10-84)
39 (27-51)

8. 23 mm
Strong family history
Ovarian BRCA1-/-

9. Breast BRCA? Ovarian BRCA1-/-
12 mm
6.8 mm
Breast BRCA?
Ovarian BRCA1-/-
6.5 mm
3 mm

10. CORRELATION OF PLATINUM SENSITIVITY WITH RESPONSE TO OLAPARIB
Sensitive
Resistant
Refractory
Platinum-free interval (months)
CR/PR SD >4 months PD

11. SINGLE AGENT TREATMENT WITH OLAPARIB
Well tolerated oral therapy not associated with the typical toxicities of chemotherapy
Clear evidence of beneficial tumour response in BRCA mutated ovarian cancer patients
46% (21/46 pts) response rate (RECIST or GCIG CA125)
15% meaningful disease stabilisation
Total clinical benefit rate of 61%
Median response duration: 8 m
Randomized trials now underway

12. POTENTIAL OF PARP INHIBITOR (SINGLE AGENT) IN SPORADIC OVARIAN CANCER
Question: What proportion of ovarian cancer patients will have BRCA1/2 dysfunction, either due to mutation of either gene or for other reasons, e.g. methylation of this or related genes?
Answer: • approx 15% of sporadic ovarian cancers have mutation of either gene; in serous histological subtypes, proportion is 18%
• approx 15-20% more cases have BRCA dysfunction, through methylation, etc.
• approx 10% have FANCF methylation
Therefore: potentially half the cases of serous ovarian ca could benefit from targeted single agent treatment
- how can these be identified?

13. Randomized Phase II/III
Phase I-II studies
Phase I:
Combination with - platinum, topotecan
Phase II:
Single agent BRCA +/-
Randomized Phase II/III
Post chemo consolidation/maintenance
Combination/maintenance
Carbo/taxol +/- Parp inhibitor

14. Parp Inhibition
Compelling efficacy data in hereditary ovarian cancer patients
Studies in hereditary ovarian cancer.
High grade serous histology – “BRCAness”
Without BRCA mutations
Combination studies
Chemotherapy
Targeted agents

15. PARP Inhibitors in Clinical Trials
Agent
Company
Strategy
Administration
AG014699
Pfizer
Combination* IV
KU59436
AstraZeneca-Kudos
Single
Oral
ABT-888
Abbott
BSI-201
BiPar
Combinations
INO-1001
Inotek-Genentech MK
Merck
Single agent and combination
GPI 21016
MGI Pharma
Adapted Ratnam K, Low JA Clin Cancer Res 2007;13:

16. FURTHER DEVELOPMENT OF OLAPARIB - 1S E
olaparib 400 mg bd cont
Patients with advanced ovarian cancer with BRCA-1 or 2 mutations, relapsed within 12 months of platinum-based chemotherapy
olaparib 200 mg bd cont
caelyx/doxil 50 mg/m2 q4 weekly
n = 90, recruitment complete
primary end point = PFS
statistical analysis: combined olaparib arms vs caelyx/doxil, aimed at detecting incr. in PFS from 4 m to 7.3 m (HR 0.55, 80% power)

17. FURTHER DEVELOPMENT OF OLAPARIB - 2S E
Patients with serous ovarian cancer, responding to 2nd or 3rd line platinum-based chemo, with CR/PR (penultimate treatment-free interval >6 m)
olaparib 400 mg bd
until PD
placebo until PD
n = 250
- BRCA mutation not necessary
Primary end point: progression-free survival
Recruitment now underway

18. PATIENT SELECTION FOR SINGLE AGENT
Predictive biomarker:
immunohistochemistry, with BRCA 1/2 antibodies
functional (ex vivo) test for loss of HR (RAD 51 foci-formation)
molecular signature (gene array)
and/or: background of • repeated response to platinum-based chemo
• prolonged survival (>5 yrs)
• serous histology

19. OLAPARIB AND ITS ROLE IN SPORADIC CANCER – AN ANECDOTE
57-yr-old pt with metastatic endometrial cancer, first diagnosed in 1999, no family history
had multiple lines of prior chemotherapy, including platinum-based treatment on 7 occasions (responding each time with progressively shorter duration)
BRCA testing: negative for mutations
began olaparib 400 mg b.d. (in bioavailability study) with progressive disease (liver, spleen, pleura) 6 weeks after last platinum-based treatment
2 days later: developed symptoms of brain mets
continued olaparib and no other treatment

20. After 6 weeks treatment, complete resolution of symptoms, radiological response in brain, pleural met
2/12/08
21/1/09
2/12/08
17/3/09

21. KEY ISSUES FOR FUTURE DEVELOPMENTS OF PARP INHIBITORS IN OVARIAN CANCER
• What determines resistance to (single agent) PARP inhibitor treatment?
• recent data indicate return of BRCA function through intragenic deletion of mutation (in BRCA2) (Edwards et al, Nature 2008)
• clinical data now beginning to emerge; suggest that platinum sensitivity is retained; for example (our longest responder) ….

22. BRCA1-/- OVARIAN CANCER PATIENT
Carboplatin +
Caelyx
Olaparib
98% CA125 decline
86% CA125 decline
initial prolonged response to olaparib, then radiological and CA125 progression
subsequent response to carboplatin/caelyx