DNA DAMAGE RESPONSE&

PARP INHIBITION

FEBRUARY 2018

3

STEERING COMMITTEE

These slides summarise two BluePrint documents:

DDR (DNA Damage Response): • DDR BluePrint

PARPi (PARP inhibition): • PARPi BluePrint

These BluePrints have been developed under the guidance of a Steering Committee:

Dr. Judith • Balman a – Vall d’Hebron Institute of Oncology, Barcelona, Spain

Prof. Simon • Boulton – Francis Crick Institute, London, UK

Prof. Charlie • Gourley – University of Edinburgh Cancer Research Centre, Edinburgh, UK

Prof. Jonathan Ledermann • – UCL Cancer Institute, London, UK

Prof. • Sibylle Loibl – German Breast Group, Neu-Isenburg, Germany

Dr. Mark J. O’Connor • – AstraZeneca, Cambridge, UK

Prof. Eric • Pujade-Lauraine –Universite Paris Descartes - Hopitaux Universitaires Paris Centre site Hotel-Dieu, Paris, France

Dr. Violeta Serra • –Vall d’Hebron Institute of Oncology, Barcelona, Spain

4

FUNDING

AstraZeneca has provided a sponsorship grant towards this independent Programme. The content is not influenced by AstraZeneca and under the sole responsibility of the Steering Committee

DNA DAMAGE RESPONSE(DDR)

6DDR, DNA Damage Response

DDR – SENSING, SIGNALLING AND REPAIRING DNA DAMAGE

DNA damage can be sustained due to:

Endogenous factors•Spontaneous or enzymatic reactions•

Chemical modifications•

Replication errors•

Replication stress•

Exogenous factors•UV radiation•

Ionizing radiation•

Genotoxic chemicals•

Repair pathways to overcome this are collectively referred to as:DNA Damage Response

7

THE MAIN DNA REPAIR PATHWAYS, THE TYPE OF DNA LESIONS THEY REPAIR AND THEIR FUNCTION

DNA REPAIR MECHANISMS

BER, Base Excision Repair, NER, Nucleotide Excision Repair; MMR, Mismatch Repair; HRR, Homologous Recombination Repair; DSB, Double-strand breaks; NHEJ, Non-Homologous End Joining

DNA repair mechanism and type of lesion Repair function

Base Excision Repair (BER)

→ Single strand breaks

• Spontaneous hydrolytic decay products• Oxidative and alkylating modifications to bases or the

sugar phosphate backbone

Nucleotide Excision Repair (NER)

→ Bulky adducts

• Removes bulky DNA lesions formed by UV light, environmental mutagens and some cancer chemotherapeutic adducts (e.g. platinum compounds)

Mismatch Repair (MMR)

→ Mispaired bases

• Base-base mismatches and insertion/deletion mispairs generated during DNA replication and recombination or caused by oxidative DNA damage

Homologous Recombination Repair (HRR)

→ Double-strand breaks (DSB)

→ Inter/intra-strand cross links

• Repairs DSBs using as a template the undamaged homologous sequence provided by the “sister chromatid” (hence mostly error-free)

• Involved in the recovery of stalled or broken replication forks during DNA replication

Non-Homologous End Joining (NHEJ)

→ Double-strand breaks

• Direct re-ligation of broken DNA molecules (without the requirement for a homologoustemplate)

DNA Repair Mechanisms: the main DNA repair pathways, the type of DNA lesions they repair and their function

8

GENERATION OF ABERRANT REPLICATION FORK STRUCTURES CONTAINING SINGLE-STRANDED DNA, MANIFESTED IN A NUMBER OF FORMS

REPLICATION STRESS: TYPE OF DNA DAMAGE OCCURING DURING DNA REPLICATION

• If unrepaired, replication stress can lead to increased mutagenesis, ultimately resulting in genome instability – a hallmark of (pre-)cancerous cells

• Replication Stress Response: activation of DDR pathways preventing the collapse of the replication fork and subsequent generation of cytotoxic DNA Double Strand Breaks

DDR, DNA Damage Response; ssDNA, single-stranded DNA

9DDR, DNA Damage Response

DDR PLAYS AN IMPORTANT ROLE IN HEALTH AND DISEASEApproximately 450 human DDR genes code for proteins with roles in physiological processes including:

• Immune receptor diversity

• Production of gametes for sexual reproduction

• Telomere homeostasis

• Aging

Deregulation of DDR is involved in the etiology of diseases such as:

• Genetic disorders

• Neurodegenerative disorders

• Immune pathologies

• Infertility

• Cardiovascular disease

• Metabolic syndrome

• Cancer

10

EXAMPLES OF DDR GENES INVOLVED IN CANCER

DDR IS OFTEN DYSREGULATED IN CANCER

DDR, DNA Damage Response; BER, Base Excision Repair, NER, Nucleotide Excision Repair; MMR, Mismatch Repair; HRR, Homologous Recombination Repair; NHEJ, Non-Homologous End Joining

DNA repair mechanism

Gene examples Cancer predisposing syndrome

BEROGG1 Renal, breast and lung cancer

XRCC1 Non-small cell lung cancer

NER

ERCC1 Lung and skin cancer, and glioma

XPXeroderma pigmentosum predisposing to skin cancer. Also increased risk of bladder and lung cancer

MMR MSH2,MLH1Lynch syndrome predisposing to colorectal cancer as well as endometrial, ovarian, stomach, small intestine, hepatobiliary tract, upper urinary tract, brain and skincancer

HRR

BRCA1,BRCA2Increased risk of breast, ovarian, prostate, pancreatic, as well as gastrointestinal and haematological cancer, and melanoma

FANCGroup of proteins associated with Fanconi anaemia predisposing to squamous cell carcinomas and acute myeloid leukaemia (e.g. FANCA, FANCB)

NHEJKU70 Breast, colorectal and lung cancer

KU80 Lung cancer

Cell cycle checkpoints

ATM Ataxia-telangiectasia predisposing to leukaemia, breast and pancreatic cancer

ATR Leukaemia, lymphoma, gastric and endometrial cancer

11DDR, DNA Damage Response; BRCA, Breast Cancer Susceptibility Protein; c-MYC, Myc proto-oncogene; K-Ras, Ras proto-oncogene; ROS, Reactive Oxygen Species

DDR DEREGULATION MAY LEAD TO CANCER THROUGH VARIOUS MECHANISMS

• Dysregulation of one or more DDR pathways driving genetic instability; e.g. by BRCA1/2 mutation

• Increased levels of replication stress leading to increased mutagenesis; e.g. by overexpression of oncogenes such as cyclin E, c-Myc or K-Ras

• Increased levels of endogenous damage generated by high ROS

• Any combination of the above, and/or sustained by the application of anticancer therapies

12

DDR CAN BE EXPLOITED IN CANCER THERAPY

DDR-targeting agents: maximize DNA damage in G1 and S-phase & prevent repair in G2

→ Ensuring the maximum amount of DNA damage is taken into mitosis

DDR, DNA Damage Response

CELL CYCLE AND DDR TARGETING: THREE KEY CELL CYCLE CHECKPOINTS AND ASSOCIATED PROTEINS ARE TARGETED BY SMALL MOLECULE INHIBITORS

13BRCA, Breast Cancer Susceptibility Protein; PARP, Poly(ADP-ribose) Polymerase

SYNTHETIC LETHALITY

• When one DNA repair pathways is defective, cells can repair DNA damage by switching to an alternative repair mechanism

• Cells are unable to survive when one mechanism is defective (e.g. BRCA mutation) and another is blocked by pharmacological inhibition (PARP inhibition)

14DDR, DNA Damage Response

DDR: RAPIDLY EMERGING AND PROMISING AREA FOR FUTURE ANTICANCER THERAPY

• Efficacy of DDR inhibitors may increase when combined with other DNA damaging agents

• The hope is that DDR inhibitors will find further use in clinical practice, in addition to or replacing chemotherapy

• Combinations of multiple DDR inhibitors or combining DDR inhibitors with antiangiogenic agents or immune checkpoint inhibitors pose interesting novel strategies

15

REFERENCES

• Brown JS, Kaye SB, Yap TA. PARP inhibitors: the race is on. Br J Cancer. 2016;114(7):713-5

• Brown JS, O’Carrigan B, Jackson SP, Yap TA. Targeting DNA Repair in Cancer: Beyond PARP Inhibitors. Cancer Discov. 2017;7(1):20-37

• Curtin NJ. DNA repair dysregulation from cancer driver to therapeutic target. Nat Rev Cancer. 2012;12(12):801-17

• Dobbelstein M, Sørensen CS. Exploiting replicative stress to treat cancer. Nat Rev Drug Discov. 2015;14(6):405-23

• Friedberg EC. A brief history of the DNA repair field. Cell Res. 2008;18(1):3-7

• Jackson SP, Bartek J. The DNA-damage response in human biology and disease. Nature. 2009;461(7267):1071-8

• Jeggo PA, Pearl LH, Carr AM. DNA repair, genome stability and cancer: a historical perspective. Nat Rev Cancer.2016;16(1):35-42

• Kaelin WG Jr. The concept of synthetic lethality in the context of anticancer therapy. Nat Rev Cancer. 2005;5(9):689-98

• Nijman SM. Synthetic lethality: general principles, utility and detection using genetic screens in human cells. FEBS Lett. 2011;585(1):1-6

• O’Connor MJ. Targeting the DNA Damage Response in Cancer. Mol Cell. 2015;60(4):547-60

• Pearl LH, Schierz AC, Ward SE, Al-Lazikani B, Pearl FM. Therapeutic opportunities within the DNA damage response. Nat Rev Cancer. 2015;15(3):166-80

• Roos WP, Thomas AD, Kaina B. DNA damage and the balance between survival and death in cancer biology. Nat Rev Cancer. 2016;16(1):20-33

• Zeman MK, Cimprich KA. Causes and consequences of replication stress. Nat Cell Biol. 2014;16(1):2-9

16

ABBREVIATIONS

ATM Ataxia telangiectasia mutated kinase LOH Loss of heterozygosity

ATR Ataxia-and Rad-related kinase LST Large-scale state transitions

ATRi Ataxia-and Rad-related kinase inhibitor MLH1 MutL homologue 1

BER Base excision repair MMR Mismatch repair

BIR Break-induced replication MSH2 MutS protein homolog 2

BRCA1/2 Breast cancer 1/2 susceptibility protein NER Nucleotide excision repair

Chk1/2 Checkpoint kinase 1/2 NHEJ Non-homologous end joining

c-MYC Myc proto-oncogene NSCLC Non-small cell lung cancer

Chk2 Checkpoint kinase 2 OGG1 8-Oxoguanine glycosylase

DDR DNA damage response PARP Poly(ADP-ribose) polymerase

DNA Deoxyribonucleic acid PARPi Poly(ADP-ribose) polymerase inhibitor

DNA-PK DNA-dependent protein kinase RAD51 DNA repair protein

DSBs Double strand breaks ROS Reactive Oxygen Species

ERCC1 ERCC excision repair 1 RPA Replication protein A

FANC Fanconi anemia complementation groups SCLC Small cell lung cancer

G1 (phase) Gap/ growth phase 1 ssDNA Single-stranded DNA

G2 (phase) Gap/ growth phase 2 TAI Telomeric allelic imbalance

H2AX Variant of histone H2 TLS Translesion synthesis

HRR Homologous recombination repair UV Ultraviolet

IHC Immunohistochemistry WEE1 WEE1 G2 checkpoint kinase

K-Ras Ras proto-oncogene XP Xeroderma pigmentosum

XRCC1 X-ray repair cross-complementing protein 1

PARP INHIBITION(PARPi)

18

PARP: KEY MEDIATOR OF DNA REPAIR

DNA damage is detected by PARP1, triggering its activation and cleavage of NAD+ generating nicotinamide and ADP-ribose

Successive addition of ADP-ribose units results in PAR polymers adjacent to the DNA breaks

These highly negatively charged polymers form a scaffold that recruits critical proteins for DNA repair

PARP, Poly(ADP-ribose) Polymerase; NAD+, nicotinamide adenine dinucleotide; PAR, poly (ADP-ribose)

19

PARPi IN TREATMENT OF CANCER

PARP, Poly(ADP-ribose) Polymerase; DSB, Double Strand Break; HRR, Homologous Recombination Repair

PARP inhibitors act by inhibitingthe enzymatic activity, and by trapping PARP on DNA

In HRR deficient cells, trapped PARP results in replication fork collapse and finally cell death

20

THREE PARP INHIBITORS APPROVED FOR CLINICAL USE IN OVARIAN CANCER

PARPi Approval Indication

Olaparib* (Lynparza™, AstraZeneca)

• EMA (Dec2014): As monotherapy for maintenance treatment of platinum-sensitive relapsed BRCA-mutated (germline and/or somatic) high-grade serous ovarian cancer patients who are in response (complete response or partial response) to platinum-based chemotherapy.

• FDA (Dec2014): As monotherapy for the treatment of germline BRCA1/2 mutated (as detected by an FDA-approved test) advanced ovarian cancer patients that have been treated with three or more prior lines of chemotherapy (capsule formulation).

• FDA (Aug2017): As maintenance treatment of adult patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer, who are in a complete or partial response to platinum-basedchemotherapy (tablet formulation).

• FDA (Jan2018): For use in patients with deleterious or suspected deleterious gBRCAm, HER2-negative metastatic breast cancer who have been previously treated with chemotherapy in the neoadjuvant, adjuvant or metastatic setting. Patients with HR+ breast cancer should have been treated with a prior endocrine therapy or be considered inappropriate for endocrine therapy. Patients are selected for therapy based on an FDA-approved CDx.

Rucaparib (Rubraca™,

ClovisOncology)

• FDA (Dec2016): As monotherapy for the treatment of patients with deleterious BRCA mutation (germline and/or somatic) associated with advanced ovarian cancer who have been treated with two or more chemotherapies (patient selection using an FDA-approved CDx for Rubraca™).

Niraparib (Zejula™, Tesaro)

• FDA (Mar2017): As monotherapy for maintenance treatment of patients with recurrent epithelial ovarian, fallopian tube or primary peritoneal cancer, whose tumours have a complete or partial response toplatinum-based chemotherapy.

• EMA (Sept2017): As monotherapy for the maintenance treatment of adult patients with platinum-sensitive relapsed high grade serous epithelial ovarian, fallopian tube, or primary peritoneal cancer who are in response (complete or partial) to platinum-based chemotherapy.

gBRCAm, germline BRCA-mutated; HER2, human epidermal growth factor receptor 2; HR+, hormone receptor positive; CDx, companion diagnostic

21

EVOLVING CONCEPT IN EVALUATING EFFICACY IN MAINTENANCE SETTING, EXTENSIVELY EMPLOYED IN CLINICAL TRIALS WITH PARP INHIBITORS

INTERMEDIATE CLINICAL ENDPOINTS

Clinical trials with PARPi have implemented intermediate clinical endpoints, complementing PFS in determining efficacy of therapies in settings with prolonged PFSor with post-progression survival benefit in multiple subsequent lines of therapies

PFS, Progression Free Survival; TFST, Time to First Subsequent Therapy; PFS2, Progression Free Survival 2; TSST, Time to Second Subsequent Therapy; OS, Overall Survival; PARPi, Poly(ADP-ribose) Polymerase inhibitor; Tx, treatment

Diagram adapted from Matulonis et al., Cancer 2015

22

EFFICACY OF PARP INHIBITORS IN OVARIAN CANCER

In advanced ovarian cancer olaparib, niraparib and rucaparib display similar efficacy, the magnitude of this benefit may depend on:

• In the maintenance setting: existence of a germline/somatic BRCA1/2 mutation versus absence of any homologous recombination deficiency

• In the monotherapy setting: on number of previous lines of treatment received

PARP, Poly(ADP-ribose) Polymerase; BRCA, Breast Cancer Susceptibility Protein

23

OlympiAD: OLAPARIB IN METASTATIC BREAST CANCER PATIENTSWITH gBRCA MUTATION

EFFICACY OF PARP INHIBITORS IN BREAST CANCER

AEs, Adverse Events; HR, Hazard Ratio; ORR, Objective Response Rate; PFS, Progression Free Survival; TPC, Treatment of Physician’s Choice; gBRCA, germline Breast Cancer Susceptibility Protein; FDA, US Food and Drug Administration

Robson et al., N Engl J Med 2017

In January 2018, the FDA approved olaparib as monotherapy for the treatment of patients with germline BRCA1/2 metastatic breast cancer after chemotherapy or endocrine treatment using an FDA-approved companion diagnostic

ORR(%)

Median PFS(months)

PFS2(months)

Grade ≥3 AEs(%)

Olaparib 59.9 7.0 13.2 36.6

Treatment of Physician’sChoice

28.8 4.2 9.3 50.5

HR – 0.58; P=0.0009 0.57; P=0.0033 –

24

ALL APPROVED PARPi CARRY A SIMILAR SAFETY PROFILE

SAFETY OF PARP INHIBITORS

AE, Adverse Event. 1Gourley et al, ASCO 2017; 2Pujade-Lauraine et al., Lancet Oncol 2017; 3Mirza et al., New Engl J Med 2016; 375(22):2154-2164; 4Coleman et al., Lancet 2017; 390:1949–1961.*5similar incidence in control arm [24(2)]; *6incidence in control arm 1%; *7incidence higher in control arm (4%); *8similar incidence in control arm (1%)

% AE Incidence – any grade (% AE Incidence – Grade 3/Grade 4)

Olaparib Niraparib Rucaparib

AE Preferred TermStudy 19(1)

(400 mg bid capsule)SOLO2(2)

(300 mg bid tablet)NOVA(3)

(300 mg od)ARIEL2(4)

(600 mg bid)

Anaemia 21 (6) 44 (20) 50 (25) 37 (19)

Neutropenia 7 (4) 20 (5) 30 (20) 18 (7)

Thrombocytopenia 4 (1) 8 (0) 61 (34) 28 (5)

Nausea 71 (2) 76 (3) 74 (3) 75 (4)

Fatigue 63 (9) 66 (4) 60 (8) 69 (7)

Vomiting 35 (2) 37 (3) 34 (2) 37 (4)

Diarrhoea 27 (2)*5 33 (1) 32 (1)

Dysgeusia – 27 (0) 10 (0) 39 (0)

Headache – 25 (1) 26 (0) 18 (<1)

Decreased appetite – 22 (11) 25 (0) 23 (1)

Constipation – 21 (0) 40 (1) 37 (2)

Transaminase elevation – 5 – 34 (10)

Hypertension – – 19 (8) –

Hypotension – – – –

MDS/AML 2*6 2*7 1*8 –

25

PARPi HAVE SHOWN ACTIVITY IN CANCER TYPES OTHER THAN OVARIAN CANCER

PARP, Poly(ADP-ribose) Polymerase

26

PARPi ARE FIRMLY ESTABLISHED AS EFFECTIVE THERAPIES FOR SELECTED PATIENTS WITH OVARIAN CANCER

• The first PARP inhibitor is now also approved for the treatment of breast cancer

• The therapeutic reach of PARPi is likely to expand to include other cancer types in the near future (prostate cancer)

• Evolving biological insight within the overall context of DDR, replication stress, and immunological responses will allow us to use these agents in the most appropriate clinical setting to improve patient care

PARPi, Poly(ADP-ribose) Polymerase inhibitor

Bao• Z, Cao C, Geng X, Tian B, Wu Y, Zhang C, Chen Z, Li W, Shen H, Ying S. Effectiveness and safety of poly (ADP-ribose) polymerase inhibitors in cancer therapy: A systematic review and meta-analysis. Oncotarget.2016;7(7):7629-39

Dr• ean A, Lord CJ, Ashworth A. PARP inhibitor combination therapy. Crit Rev Oncol Hematol. 2016;108:73-85

Hakm• e A, Wong HK, Dantzer F, Schreiber V. The expanding field of poly(ADP-ribosyl)ation reactions. ‘Protein Modifications: Beyond the Usual Suspects’ Review Series. EMBO Rep. 2008;9(11):1094-100

Konecny• GE, Kristeleit RS. PARP inhibitors for BRCA1/2-mutated and sporadic ovarian cancer: current practice and future directions. Br J Cancer. 2016;115(10):1157-73

Lord CJ, Ashworth A. Mechanisms of resistance to therapies targeting BRCA• -mutant cancers. Nat Med. 2013;19(11):1381-8

Meehan RS, Chen AP. New treatment option for ovarian cancer: PARP inhibitors. • Gynecol Oncol Res Pract. 2016;3:3

Michels• J, Vitale I, Saparbaev M, Castedo M, Kroemer G. Predictive biomarkers for cancer therapy with PARP inhibitors. Oncogene. 2014;33(30):3894-907

Schreiber V, • Dantzer F, Ame JC, de Murcia G. Poly(ADP-ribose): novel functions for an old molecule. Nat Rev Mol Cell Biol. 2006;7(7):517-28

Sonnenblick• A, de Azambuja E, Azim HA Jr, Piccart M. An update on PARP inhibitors-moving to the adjuvant setting. Nat Rev Clin Oncol. 2015;12(1):27-41

Vyas S, Chang P. New PARP targets for cancer therapy. Nat Rev Cancer. • 2014;14(7):502-9

27

REFERENCES

28

ABBREVIATIONS

AE Adverse Event MMEJ Microhomology-mediated end joining

ADP Adenosine diphosphate NAD+ Nicotinamide adenine dinucleotide

ALT Alanine aminotransferase NHEJ Non-homologous end joining

ASCO American Society of Clinical Oncology ORR Objective Response Rate

AST Aspartate aminotransferase OS Overall survival

ATM Ataxia telangiectasia mutated kinase PALB2 Partner and localizer of BRCA2

ATR Ataxia-and Rad-related kinase PAR Poly(ADP-ribose)

BER Base excision repair PARP Poly(ADP-ribose) polymerase

BRCA1/2 Breast cancer 1/2 susceptibility protein PARPi Poly(ADP-ribose) polymerase inhibitor

CDx Companion diagnostic pCR Pathological complete response

Chk2 Checkpoint kinase 2 PD-1 PDCD1, programmed cell death protein 1

CI Confidence intervals PD-L1 Programmed cell death protein-ligand 1

DDR DNA damage response PFS Progression free survival

DNA Deoxyribonucleic acid pgP P-glycoprotein

DSB Double strand break RAD51 RAD51 recombinase

EMA European Medicines Agency TFST Time to first subsequent therapy or death

FDA Food and Drug Administration (US) TKI Tyrosine kinase inhibitor

gBRCA Germline BRCA1 and/2 mutation TLS Translesional synthesis

g/sBRCA Germline/somatic BRCA1 and/2 mutation TSST Time to second subsequent therapy or death

HRD Homologous Recombination Deficiency Tx Therapies

HRR Homologous Recombination Repair VEGF Vascular Endothelial Growth Factor

HR Hazard ratio VEGFR Vascular Endothelial Growth Factor Receptor

LOH Loss of heterozygosity

Dr. Antoine Lacombe Pharm D, MBAPhone: +41 79 529 42 79antoine.lacombe@cor2ed.com

COR2EDBodenackerstrasse 174103 Bottmingen SWITZERLAND

Dr. Froukje SosefMDPhone: +31 6 2324 3636froukje.sosef@cor2ed.com