1. PARP Inhibitors: Usurping DNA repair to target cancer
Lee Schwartzberg MD, FACP
Chief Medical Officer
The West Clinic

2. Question 1 DNA repair mechanisms are important in Cancer cells only
Both cancer and normal eukaryotic cells
Predominantly in rapidly growing cells like bone marrow precursors
Predominantly cancer cells with BRCA mutations

3. Question 2 PARP inhibitors have demonstrated activity in:
BRCA 1 mutation carrier breast cancer
BRCA 2 mutation carrier breast cancer
Triple negative breast cancer
1 and 3 only
1 and 2 only
All of the above

4. All cells are under constant risk of DNA damage
Ultraviolet light
Ionizing radiation
Man-made and natural chemicals
Reactive oxygen species
most are generated “endogenously”
10,000 Single Strand Breaks/ cell/day
~100,000,000,000,000,000 DNA lesions in a human body every day1-3
Mammalian cells are constantly at risk of damage to their DNA from multiple causes1, including ultraviolet light, ionizing radiation, reactive oxygen species, several endogenous and synthetic compounds, and during normal DNA replication.
Approximately 1x104 DNA lesions (single-strand breaks [SSBs]) are generated in a metabolically active mammalian cell each day.1,2 There are approximately 1x1013 cells in the human body, so this corresponds to a total of around 1x1017 DNA lesions per person, per day.1-3
References
1. Jackson SP. Biochem Soc Trans 2001; 29:
2. Lindahl T. Nature 1993; 362:
3. Jackson SP, Bishop CL. Drug Discovery World 2003; Fall:
1. Jackson SP. Biochem Soc Trans 2001;29:
2. Lindahl T. Nature 1993;362:
3. Jackson SP, Bishop CL. Drug Discovery World 2003;(Fall):41-45

5. Cellular Response To DNA Damage

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
P53, BRCA1, BRCA 2, ATM, Fanconi’s Anemia
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. DNA Excision Repair Mechanisms

8. Poly(ADP-Ribose) Polymerase (PARP)
A key role in the repair of DNA single-strand breaks
Through the base excision repair pathway (BER)
Binds directly to sites of DNA damage
Once activated, it uses NAD as a substrate, and generates large, branched chains of poly (ADP-ribose) polymers on multiple target proteins
Recruits other DNA repair enzymes
PAR
Lig3
XRCC1
PNK
Polß 8 8

9. Base Excision Repair

10. Inhibiting PARP-1 Increases Double-Strand DNA Damage
DNA single strand
break (SSB)
damage
XRCC1
LigIII
PNK 1
pol β
PARP
Inhibition of PARP-1 prevents
recruitment of DNA repair enzymes
leads to failure of SSB repair
-accumulation of SSBs
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:
During S-phase,
replication fork
is arrested at
site of SSB
Degeneration into
Double strand breaks

11. BRCA1 And 2 Are Required for Efficient Repair of Double Stranded DNA Breaks
DNA DSB
ATM/R
gH2AX
BRCA1
Rad50
MRE11
NBS1
Non-homologous end-joining
Homologous recombination
Ku 70/80
BRCA2
Rad 51
Cell survival
Cancer cell death
DNA-PKcs
RPA
Rad 52/4
ERCC1
XRCC3
XRCC4
Ligase IV
Predominant in G1
Error-prone
Gross Genomic instability
Major pathway for repair
Error-free
Cells with BRCA mutations are deficient in homologous recombination and lack the ability to efficiently repair DSBs.

12. The Concept of Synthetic Lethality
(BRCA)
(PARP)
Ashworth, A. J Clin Oncol; 26:

13. BRCA1 and BRCA2 -/- cells are very sensitive to PARP inhibition
Increased levels of chromosomal aberrations in PARP inhibitor treated BRCA2 -/- cells
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
Wild type
Control
+ PARP inhibitor
In vitro and in vivo studies have investigated the potential therapeutic effect of PARP inhibition on cells that have deficient DNA repair systems.1,2
One of these studies by Farmer et al used mouse embryonic stem (ES) cells lacking BRCA1 or BRCA2.1 BRCA1 and BRCA2 are essential for efficient DNA DSB repair by HR3 and mutations in these genes predispose to breast, ovarian, and other cancers.4 In HR-deficient cells, the activity of PARP-1 reduces the DNA damage burden.
Farmer et al demonstrated that BRCA2 (and BRCA1)-deficient mouse ES cells were extremely sensitive to PARP inhibition.1 As a result of HR deficiency in these cells, the addition of PARP inhibitors led to chromosomal instability, cell-cycle arrest, and subsequent cell death.1 The effects were rapid and irreversible.
Furthermore, to investigate the possibility that PARP inhibition may be used to treat BRCA2-deficient tumors, Bryant et al2 conducted a xenograph study in nude mice. The results indicated that BRCA2-deficient tumors were susceptible to PARP inhibition alone.2
References
1. Farmer H et al. Nature 2005; 434:
2. Bryant HE et al. Nature 2005; 434:
3. Tutt A, Ashworth A. Trends Mol Med 2002; 8:
4. Wooster R, Weber BL. N Engl J Med 2003; 348:
BRCA2 -/-
Control
+ PARP inhibitor
Farmer H et al. Nature 2005;434:
Personal communication, Alan Ashworth

14. PARP Inhibitors in Clinical Development
Differing chemical structures
Differing toxicity
Differing schedules and routes of administration

15. Chemotherapeutic Agents: Double Strand DNA Breaks
Alkylators
DNA interstrand cross-links  double strand (DS) DNA breaks
Cyclophosphamide
Platinums
Forms adducts with DNA
Cisplatin
Carboplatin
Oxaliplatin
Topoisomerase I poisons
Arrest of DNA replication forks
Etoposide
Irinotecan
Topotecan
Mitoxantrone
Topoisomerase II poisons
DNA interstrand cross-linking, generation of O2 free radicals
Doxorubicin
Epirubicin
Bleomycin
Directly damages DNA  DS DNA breaks
Kennedy R et al. JNCI 2004; 96:

16. PARP Inhibitors in BRCA 1/2 Mutated Tumors

17. Phase I Trial of Olaparib in Patients with Solid Tumors
Escalation and expansion phase, n = 60
Recommended phase II dose: 400 mg PO BID
Toxicities
Nausea (32%), fatigue (30%), vomiting (20%), taste alteration (13%), anorexia (12%), anemia (5%)
Clinical activity = 12/19 patients with BRCA mutations
Tumor
BRCA
No. of pts
Response
Breast 2
1 CR, 1 SD
Ovarian
1 or 2 8
8 PRs
Fallopian tube 1 PR
Prostate
Fong PC et al. N Engl J Med 2009; 361:

18. Phase II Trial of Olaparib in BRCA-deficient Metastatic Breast Cancer
Eligibility
Confirmed BRCA1 or 2 mutation
Stage IIIB/C or IV BC after progression ≥ 1 prior chemotherapy for advanced disease
(Non-randomized sequential cohorts)
Cohort 1
Olaparib 400 mg po bid (MTD)
28-day cycles
Cohort 2*
Olaparib 100 mg po bid (maximal PARP inhibition)
28-day cycles
Primary Endpoint: Response rate
* Following an interim review, patients in the 100 mg bid cohort were permitted to crossover to receive 400 mg bid
Tutt A et al. J Clin Oncol 2009;27(18S):803s (abstr CRA501)

19. Olaparib in BRCA-deficient Metastatic Breast Cancer: Select Toxicities
Olaparib 400 mg BID
(n = 27)
Olaparib 100 mg BID
Grade 1/2
Grade 3
Fatigue
15 (56)
4 (15)
2 (7)
Nausea
11 (41)
5 (19)
Vomiting
7 (26)
3 (11)
6 (22)
Headache
10 (37)
1 (4)
Constipation
8 (30)
Tutt A et al. J Clin Oncol 2009;27(18S):803s (abstr CRA501)

20. Olaparib in BRCA-deficient Metastatic Breast Cancer: Results
Best percent change from baseline in target lesions by genotype
Median 3 prior lines of therapy
ITT cohort
400 mg BID
N = 27
100 mg BID
ORR
11 (41%)
6 (22%) CR
1 (4%) PR
10 (37%)
Median PFS
5.7 mo
( )
3.8 mo
(1.9 – 5.6)
Tutt A et al. J Clin Oncol 2009;27(18S):803s (abstr CRA501)

21. PARPi Monotherapy in BRCA Mutated tumors
Drug
Phase
Dose
Tumor N
CBR (%)
RR (%)
MDR (MOS)
PFS (MOS)
Olapirib 1
Varies
Ovarian 50 46 40
6.5 NR 2
400 mg BID 33 35
9.6
100 mg
BID 24 13
9.0
400 mg
Breast 27 41
5.7 22
3.8
MK-4827 19 45 4

22. Prior response to platinum may predict response to olaparib in BRCA mutated Ovarian Cancer
Gelmon K, et al J Clin Onc 2010

23. PARP Inhibitors beyond BRCA mutation carriers

24. Triple Negative Breast Cancer (TNBC)
‘Triple negative’: ER-negative, PR-negative, HER2-negative
Depending on thresholds used to define ER and PR positivity and methods for HER2 testing
TNBC accounts for 10–17% of all breast carcinomas
Significantly more aggressive than other molecular subtype tumors
Higher relapse rate than other subtypes
No specific targeted therapy
Reis-Filho JS, et al. Histopathology 2008;52:

25. Triple Negative/Basal-Like1,2,3
TNBC Shares Clinical and Pathologic Features with BRCA-1-Related Breast Cancers (“BRCAness”)
Characteristics
Hereditary BRCA1
Triple Negative/Basal-Like1,2,3
ER/PR/HER2 status
Negative
TP53 status
Mutant
BRCA1 status
Mutational inactivation*
Diminished expression*
Gene-expression pattern
Basal-like
Tumor histology
Poorly differentiated
(high grade)
Chemosensitivity to DNA-damaging agents
Highly sensitive
*BRCA1 dysfunction due to germline mutations, promoter methylation, or overexpression of HMG or ID44
1Perou et al. Nature. 2000; 406:
2Cleator et al.Lancet Oncol 2007;8:235-44
3Sorlie et al. Proc Natl Acad Sci U S A 2001;98:
4 Miyoshi et al. Int J Clin Oncol 2008;13:

26. Targeting DNA Repair Pathway in TNBC
Clustering analyses of microarray RNA expression have shown that familial BRCA-1 tumors strongly segregate with basal-like/ triple-negative tumors
Suggests that sporadic TNBC may have acquired defects in BRCA1-related functions in DNA repair
Basal-like
= BRCA1+
= BRCA2+
Sorlie T et al. PNAS 2003;100:

27. Predictors of Response to Cisplatin in TNBC
Silver, D. P. et al. J Clin Oncol; 28:

28. Phase II Study of the PARP inhibitor Iniparib in Combination with Gemcitabine/Carboplatin in Triple Negative Metastatic Breast Cancer
Background and Rationale
PARP1
Upregulated in majority of triple negative human breast cancers1
Iniparib (BSI-201)
Small molecule IV PARP inhibitor
Potentiates effects of chemotherapy-induced DNA damage
No dose-limiting toxicities in Phase I studies of BSI-201 alone or in combination with chemotherapy
Marked and prolonged PARP inhibition in PBMCs
O’Shaughnessy J, et al. NEJM 2011

29. Phase II TNBC Study: Treatment Schema
Metastatic TNBC
N = 120
RANDOMIZE
1st -3rd line MBC
Eligible
BSI-201 (5.6 mg/kg, IV, d 1, 4, 8, 11)
Gemcitabine (1000 mg/m2, IV, d 1, 8)
Carboplatin (AUC 2, IV, d 1, 8)
Gemcitabine (1000 mg/m2, IV, d 1, 8)
Carboplatin (AUC 2, IV, d 1, 8)
21-Day
Cycle
RESTAGING
Every 2 Cycles
* Patients randomized to gem/carbo alone could crossover to receive gem/carbo + BSI-201 at disease progression

30. Safety – Hematologic Toxicity Phase II Gem Carbo +/- Iniparib
BSI Gem/Carbo
(n = 57)
Grade 2
Grade 3
Grade 4
Anemia, n (%)
12 (20.3%)
7 (11.9%)
(0.0%) 15
(26.3%) 7
(12.3%)
Thrombocytopenia, n (%) 6
(10.2%) 4
(7.0%)
(10.5%)
Neutropenia, n (%) 18
(30.5%)
13 (22.0%)
7 (12.3%)
(31.6%)
Febrile neutropenia, n (%) 3
(5.1%) 1
(1.7%)
RBC treatment*, n (%) 5
(8.5%) 2
(3.4%)
(5.3%)
(8.8%)
(3.5%)
G-CSF Use, n (%)
(1.8%)
*Transfusion and/or EPO use
O’Shaughnessy J, et al. NEJM 2011

31. Safety – Non-Hematologic Toxicity Phase II Gem Carbo +/- Iniparib
BSI Gem/Carbo
(n = 57)
Grade 2
Grade 3
Grade 4
Nausea, n (%)
10 (16.9%) 2
(3.4%)
(0.0%) 7
(12.3%)
Vomiting, n (%) 9
(15.3%) 4
(7.0%) 1
(1.8%)
Fatigue, n (%) 10
(16.9%) 6
(10.2%)
10 (17.5%)
Neuropathy, n (%)
Diarrhea, n (%)
(1.7%)
O’Shaughnessy J, et al. NEJM 2011

32. Final Results: Phase II: Gem Carbo +/- Iniparib in TNBC
O’Shaughnessy J et.al. NEJM 2011

33. Final Results: Phase II Gem Carbo +/- Iniparib in TNBC
O’Shaughnessy J, et.al. NEJM 2011

34. Phase I: Olaparib + Paclitaxel in 1st and 2nd line MBC
BKG: Olaparib single agent activity in BRCA 1/2 mutated MBC
Olaparib + paclitaxel, N=19, 70% 1st line, unselected for BRCA mutations
33-40% RR; no CRs
Median PFS: months
Hematologic toxicity high, requires G-CSF
Dose reductions common
Unclear whether combination be taken forward

37. Resistance to PARP Inhibitors: Reversion of BRCA2 mutations
Partial function of BRCA2 is restored and cells become competent for homologous recombination repair
Selection of resistant clones from a BRCA2 mutated cell line reveals that resistance to PARP inhibitors is associated with reversion of BRCA2 mutation. Investigators go on to show that partial function of BRCA2 is restored and that cells become competent for HR repair. A number of important points from this study: (1) Validates the mechanism of action of PARP inhibitors in BRCA2 deficient patients, (2) PARP inhibition unlikely to be a “magic bullet”, (3) Continued HR deficiency is not required for tumor survival and development.
Edwards SL et al. Nature 2008; 451:

38. The Future of PARP inhibitors: Many Unanswered Questions
Can we use these agents more broadly?
To treat other tumors with specific DNA repair defects, i.e. sporadic loss of BRCA 1/2, tumors with PTEN mutations
Challenge is to identify them
Timing of PARP inhibitor in relation to cytotoxic agent (before it, with it, how long to continue it?)

39. Conclusions
Targeting DNA repair mechanisms in tumor cells is a rational target
PARP is an integral enzyme in DNA repair
Multiple PARP inhibitors are available
Preliminary results show activity in BRCA mutated cancers (Breast and Ovarian)
Preliminary results show activity of iniparib with chemotherapy in TNBC
Phase III results forthcoming