1. PARP Inhibition: A New Approach To Cancer Therapy? Dr. Geert Kolvenbag
Date

2. Potential Conflict of Interest
Employee and Shareholder /
AstraZeneca
Date

3. PARP Inhibition: A New Therapeutic Approach?
Geert J.C.M. Kolvenbag MD PhD
Global Product Vice President
AstraZeneca

4. Targeting DNA Repair in Oncology
DNA damage occurs all the time in all cells
DNA repair defects lead to increased
cancer susceptibility and increased
sensitivity to DNA damaging agents
Why is DNA
repair a good
target?
Normal cells have multiple DNA repair
pathways but some are lost in cancer cells
Inhibiting DNA repair in cancer cells that have impaired repair pathways leads to selective
cell killing and an increased therapeutic ratio

5. Inducing Synthetic Lethality in Cancer Cells
Normal Cell
Full complement
of repair pathways
Pathway B inhibitor
Survival AB
Alternative DNA
repair pathways
available
Pre-cancerous Cell
DNA damage leads to continuous activation of pathway A AB
Death B
Cancer Cell
Selective pressure loss of pathway A,
genetic instability,
reliance on pathway B
A number of DNA repair pathways have evolved to deal with the different types of DNA damage encountered by a human cell. These (DNA repair) pathways are normally induced when required and there is a level of redundancy that means more than one pathway may be capable of dealing with a particular type of DNA damage.
An early step in tumourigenesis is oncogene activation of one or more DNA repair pathways resulting in cell cycle checkpoint activation. This represents a barrier to uncontrolled cell growth
Loss of activated DNA repair pathways/checkpoint response results in the removal of a barrier to uncontrolled growth and is likely to be selected for. A consequence of this is increased genomic instability and an increased likelihood of progression to cancer formation. The loss of one DNA repair pathway can increase the dependence on a remaining pathway
By targeting a remaining DNA repair pathway dependency in the cancer cells, tumour specific cell killing by synthetic lethality can be achieved

6. Cancer Cells are Highly Susceptible to DNA Repair Inhibition
Undergo deregulated proliferation
less time for DNA repair than in normal cells
Grow under stress, which causes ongoing DNA damage
Have DNA repair defects
mutator phenotype
allow growth despite ongoing genome instability
Are reliant on the DNA repair pathways they still retain
Eukaryotic cells have evolved mechanisms to monitor the integrity of their genome and repair damaged DNA before the cell cycle progresses. The mechanisms that monitor for damaged DNA are intimately linked with cell-cycle events and the process known as “checkpoint” control.
Loss of checkpoint function predisposes a cell to acquire selective oncogenic mutations and, therefore, it is an important prognostic indicator. People with genetic instability disorders that cause defects in checkpoint function have an increased incidence of many cancers.1
Cancer cells undergo deregulated proliferation, losing cell-cycle control and resting in the G0 phase of the cell cycle for little or no time. Thus, there is less opportunity for DNA repair to occur than in normal cells.
Growth under stress increases the risk of DNA damage in cancer cells. In addition, the genomic instability associated with cancer cell formation (“mutator phenotype”) results in an increased DNA mutation rate.2
Many DNA repair pathways are lost or deficient in cancer cells. In particular, breast or ovarian tumor cells with hereditary breast cancer associated gene (BRCA) 1 or BRCA2 mutations are HR deficient and so they lack the ability to efficiently repair DSBs.3
Cancer cells continue to grow despite ongoing genomic instability and so the repair pathways they still retain become integral to their survival. Interest in targeting these remaining DNA repair pathways in cancer cells as a selective anti-cancer therapy is developing.
References
1. Khanna K. J Natl Cancer Inst 2000; 92:
2. Bielas JH, Loeb LA. Environ Mol Mutagen 2005; 45:
3. Lomonosov M et al. Genes Dev 2003; 17:

7. Focus on DDR Pathways for SSBs/DSBs
Type of
damage:
Repair
pathway:
enzymes:
Bulky
adducts
Insertions
& deletions
O6-
alkylguanine
Nucleotide-
excision
repair
Mismatch
Direct reversal
XP,
poly- merases
MSH2, MLH1
AGT
Base
excision
repair
Single-
strand
breaks (SSBs)
PARP
Double-
strand
breaks (DSBs)
Recombinational
repair
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
ATM
BRCA
DNA-PK

8. Mechanisms of Action of Olaparib
PARP
Replicating cells
olaparib
SSBs increased by dacarbazine, temozolomide and topotecan
DSBs increased by platinums
Mechanism 2:
Potentiation
Survival
Normal cell
Repair by Homologous Recombination
No effective repair (No HR pathway)
Cell death
Cancer cell with HRD
DNA SSBs occur all the time (in the order of 10,000 per cell per day) and PARP plays a pivotal role in repairing them
Olaparib, by inhibiting PARP activity, results in unrepaired SSBs persisting into S phase of the cell cycle where they are converted into more genotoxic DNA DSBs during the replication process
In normal cells with a functional Homologous Recombination (HR) pathway, these DSBs are effectively repaired with a high degree of fidelity. In cancer cells that are HR repair pathway deficient (HRD), these DSBs persist or are dealt with by error-prone pathways resulting in an unsupportable level of genomic instability and cancer cell death
Consequently, single agent Olaparib can in an HRD tumour result in tumour-specific cell killing
Olaparib can also potentiate the effects of Ionising radiation and chemotherapies that induce DNA damage, representing a second mechanism of action.
Mechanism 1:
Tumor specific killing by olaparib

9. Hypothesis
In situations where the DNA repair is compromised inhibition of PARP will lead to synthetic lethality of the cell
DNA repair factors deficient in functioning:
BRCA gene deficient in genotype or phenotype
Other Homologues Recombination Repair factors deficient in functioning (HRD) , eg ATM, MDC1, MRE11
In presence of DNA damaging agents
Chemotherapy
Radiotherapy

10. Olaparib: An oral inhibitor of Poly (ADP-ribose) Polymerase (PARP)
IC50 on PARP-1 = 4.9 nM
IC50 on PARP-2 ≈ 5nM
IC50 on PARP-3 ≈ 50nM
IC50 on Tankyrase >1M
olaparib (AZD2281; KU )
Favorable PK
Good bioavailability across species
Tumor PK -Significant levels at 24 hrs following single oral dose

11. Does the PARP inhibition result in therapeutic effects
In vitro
In vivo
Clinical response

12. Targeted inhibition  selective and less toxic therapy
Increased Sensitivity of BRCA1-/- and BRCA2-/- Cells to PARP Inhibition
BRCA1-/-
BRCA1+/+
BRCA1+/-
BRCA2-/-
BRCA2+/+
BRCA2+/-
No difference in sensitivity between heterozygous and wild-type BRCA cells
Targeted inhibition  selective and less toxic therapy
Farmer et al. Nature 2005; 434:917-21

13. Mean number of chromatid PARP inhibitor concentration (M)
BRCA 1 & 2 -/- ES Cells are Very
Sensitive to PARP Inhibition
Increased levels of chromosomal aberrations in PARP inhibitor treated BRCA2 -/- cells WT
BRCA-/- 1 2 3 4
BRCA2-/-
+ PARPi
Mean number of chromatid
aberrations per cell
Chromatid breaks
Complex aberrations
BRCA2 +/-
BRCA2 -/-
Wild type
Log surviving fraction - 4 3 2 1
PARP inhibitor concentration (M)
10-9
10-8
10-7
10-6
10-5
10-4
Farmer et al. Nature 2005; 434:917-21

14. KU95 Cell Line Panel: Olaparib SensitivityIC
data by tumor type 50
RAD51 DNA damage induced foci
To assess the correlation between HRD and response to Olaparib, a cross-tumour panel of 95 cancer cell lines has been analysed in vitro using the clonogenic formation assay
There is a continuum of response to Olaparib and all 95 cell lines are in the process of being characterized for genomic rearrangements, global gene expression and the analysis of the HR pathway by looking at specific HR factors at the RNA and protein level as well as the ability of the cell lines to form RAD51 foci in response to DNA damage
HRD and Sensitive
HR Proficient and Resistant

15. HRD is Strongly Linked with Cancer
Breast
Ovarian
H&N
NSCLC
GI, HCC
Pancreas
Paediatrics
BRCA1
ATM
Mre11
FANC
BRCA
MDC1
ATM
TN Breast
ATM /MRE11
Serous Ovarian
NSCLC
Head & Neck
CRC
MRE11
BRCA2
MDC1
CHK2
BRCA1
ATM
BRCA2
Mre11
ATM
Mre11
MDC1
A number of HR factors have been linked with cancer – in particular with breast and ovarian cancers
Moreover, a small number of HR factors have been implicated in a number of different cancers
The literature based estimates suggests a high prevalence of HRD in the cancer segments TN breast and serous ovarian, as well as suggesting a significant prevalence in Head & Neck, NSCLC and CRC tumours as well
Consequently, both TN breast cancer and serous ovarian cancer represent good target tumour segments to test the Olaparib: HRD hypothesis in the clinic
Mre11
BRCA1
BRCA2
FANC

16. CFA Analysis of Breast Cancer Lines using Olaparib
25 cell lines from the Slamon breast cancer panel
Alan Lau; Richard Finn & Dennis Slamon

17. Response to Olaparib by HR Status
Triple Negative cell lines (n=14)
HRD (n=12)
%Sensitive (< 1µM)
%Insensitive
%Sensitive (< 1µM)
%Insensitive
25.00
43.75
56.25
75.00
ER-, PR -, Her2+ cell lines (n=11)
HR proficient (n=13)
%Sensitive (< 1µM)
%Insensitive
%Sensitive (< 1µM)
%Insensitive
22.22
0.00
77.78
100.00

18. Olaparib Inhibits Growth of HRD Tumors in vivo
Aaron Cranston (KuDOS) & Richard Finn (UCLA)

19. Olaparib in Spontaneous BRCA2-Deficient Tumors
Vehicle
PARPi qdx28 i.p. 50mg/kg
Mean RTV
day 28 = 15.3
Mean RTV
day 28 = 1.20
BRCA2-deficient KO Mice

20. From Targeted Therapy to the Olaparib Phase I Study
Oral, small molecule PARP inhibitor
IC50 for PARP1 enzyme in the low nM range
Phase I trial began at RMH then NKI; later expanded to other centres
Escalation phase: All tumor types
Primary objectives of safety and tolerability
Expansion phase: BRCA mutation carriers (HR deficient) especially ovarian cancer
Further assessment of efficacy

21. Overall Recruitment Escalation Phase (n=46)1,2
Various tumor types; BRCA carrier status not mandatory
10 dose level cohorts:
10mg daily given for 2 out of 3 weeks
600mg bid continuous dosing
11 BRCA carrier ovarian cancer
Expansion phase (n=52) at 200mg bid continuous2
Confirmed BRCA mutation carriers
39 ovarian cancer
1Fong et al. Proceedings of ASCO 2006
2Yap et al. Proceedings of ASCO 2007

22. Demographics BRCA-Mutated Ovarian Cancer Subpopulation
Characteristics
Number
BRCA1 / BRCA2 / Family history
41 / 8 / 1
Median age (range)
52 (37-80) yrs
ECOG PS 0-1 47
Median duration from diagnosis to treatment (range)
4.7 (0.5–16) yrs
Platinum status
Sensitive (PD > 6 months after platinum)
Resistant (PD ≤ 6 months after platinum)
Refractory (PD on platinum or on completion of platinum) 10 27 13
Median no. of prior systemic therapies (range)
3 (1-8)

23. Toxicities first 60 patients, all tumor types)
Most toxicities were Grade 1-2 (≥95%)
Most common toxicities were:
nausea 28%, vomiting 18%, dysgeusia 13%, anorexia 12%
fatigue 28%
Grade 3-4 toxicities were rare:
myelosuppression (≤5%)
nausea and vomiting (2-3%)
CNS: dizziness or mood changes (2-3%)
Pattern of toxicity similar in BRCA mutation carriers

24. Dose Limiting Toxicities (DLT)
Dose (mg)/ Schedule
Tumour type
DLT
Outcome
400 bid continuous
Ovarian Ca
G3 low mood and
G3 fatigue
Resolved within 24 hours of drug discontinuation
Recurred with re-challenge
600 bid continuous
Mesothelioma
G4 thrombocytopenia
Resolved 2 weeks after drug discontinuation
Breast Ca
G3 somnolence
G1 on lower dose
Maximum Tolerated Dose (MTD) = 400mg bid

25. Response to 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)
6 (13%)
1 (10%)
4 (16%)
1 (9%)
Median duration of response in weeks (range)
24 (10-77)
23 (16-77)
24 (10-65)
26 (20-32)

26. Platinum Sensitivity Correlated with Response to Olaparib
Sensitive
Resistant
Refractory
Platinum-free interval (months)
CR/PR SD >4 months PD

27. 23 mm

28. 21mm
16mm

29. Ovarian BRCA1-/- Breast BRCA? 12 mm 6.8 mm 3 mm 6.5 mm 21.05.07
Breast BRCA?
Ovarian BRCA1-/-
6.5 mm
3 mm

30. Olaparib Resistance Pre-clinical Clinical
Over expression of pgp (olaparib is pgp substrate)
Reactivating BCRA mutation
Clinical
Todate no evidence of PARP inhibitor resistence
Note: Platinum resistence has been shown due to reactivating BRCA mutation

31. Olaparib Overcoming Drug Resistance
Pre-clinical
Overcome TMZ resistence
Potentiation of chemotherapy, e.g. TMZ
Clinical
No data yet

32. Delivery of the right drug, at the right dose to the right patient
Summary
AZD2281 is a potent inhibitor of PARP and has impressive clinical activity in BRCA patients with breast and ovarian cancer
The drug has additional potential to benefit a larger group of patients with HRD tumors
Patient selection is key to the success of this project and is a paradigm for personalized health care
The development of biomarkers and a diagnostic are complex but pivotal to:
Delivery of the right drug, at the right dose to the right patient

33. Acknowledgements The patients and their families Cancer Research UK
Royal Marsden Hospital
Janet Hanwell
Dimitrios Magkos
Netherlands Cancer Institute
Jana van der Sar
Marja Voogel
Edinburgh Cancer Centre
UZ Brussel Oncologisch Centrum
International Hereditary Cancer Centre, Poland
Jan Lubinski
Cancer Research UK
Institute of Cancer Research/ Breakthrough Breast Cancer Research UK
Andrew Tutt
Pei-Jun Wu
Alan Ashworth
AstraZeneca
John Stone
Mark O’Connor
Helen Swaisland
Peter Mortimer
Jim Carmichael
Clinical teams
Theradex UK
FECS/AACR/ASCO Methods in Clinical Cancer Research Workshop, Flims, 2005