EdiVoteStart EdiVoteStop Standard

Presentazione sul tema: "EdiVoteStart EdiVoteStop Standard"— Transcript della presentazione:

1 EdiVoteStart EdiVoteStop Standard Indicare quale tra queste affermazioni riguardante gli inibitori di PARP è SBAGLIATA: Olaparib ha mostrato attività antitumorale in monoterapia nei tumori con mutazioni di BRCA1 o BRCA2 Potenziano l’attività antitumorale della temozolomide inibendo il sistema di riparo per escissione delle basi Potenziano l’attività antitumorale dei derivati delle camptotecine, che inibiscono la Topoisomerasi 1, rendendo più lento il processo di riparo del DNA La maggior parte degli inibitori di PARP compete con il NAD per il legame al sito catalitico dell’enzima Potenziano l’attività antitumorale di trastuzumab (anticorpo monoclonale anti-HER2) nei tumori della mammella triple negative 20% 20% 000 20% 20% 010 20%

2 EdiVoteStart EdiVoteStop Standard Indicare quale tra queste affermazioni riguardante gli inibitori di PARP è SBAGLIATA: L’Iniparib è un inibitore di PARP irreversibile, non competitivo La mancata formazione di foci di RAD51 nel tumore in seguito a trattamento con inibitore di PARP potrebbe rappresentare un fattore predittivo di risposta al farmaco I tumori ovarici associati a mutazioni di BRCA1 o BRCA2 e sensibili ai composti del platino sono in genere più sensibili agli inibitori di PARP rispetto ai tumori refrattari al platino (cioè che vanno in progressione durante la terapia con composti del platino) Olaparib, un inibitore di PARP che supera la barriera emato-encefalica, in studi di fase II è stato associato a carboplatino e gemcitabina per il trattamento del glioblastoma multiforme La resistenza agli inibitori di PARP può essere dovuta a nuove mutazioni di BRCA1 o BRCA2, che ripristinano l’espressione della normale proteina corrispondente, o ad elevata espressione di pompe di efflusso della famiglia della Multi Drug Resistance 20% 20% 20% 000 20% 20% 010

3 PARP INHIBITORS FOR CANCER THERAPY Grazia Graziani University of Rome “Tor Vergata”
-Involvement of PARPs in DNA repair -Rationale for using PARP inhibitors as - chemosensitizers and radiosensitizers - in monotherapy for BRCA defective tumors -Clinical Trials with PARP inhibitors -Challenging tasks 3

4 POLY (ADP-ribose) polymerase (PARP)

5 1. Nicotinamide 2. Adenine NAD and ADP-ribose Nicotinamide

6 Glu and Lys of acceptor proteins
NAD+ Linkage at branch point PARP Binding of NAD+ to PARP Nicotinamide leaves Linkage in linear pADPr Capture by oxigen from Glu and Lys of acceptor proteins Acceptor protein

7 BIOLOGICAL EFFECTS DERIVING FROM PARP ACTIVATION
Protein Covalent poly(ADP-ribosyl)ation influences the function of target proteins Interaction of ADP-ribose polymers bound to activated PARP with polymer-binding consensus motif of partner proteins PAR oligomers, when cleaved from poly(ADPribosyl)ated proteins, confer distinct cellular effects Lowering of the cellular level of its substrate, NAD+ Protein PARP NAD+

8 DNA damage and repair Inflammatory responses Cell death pathways Mitotic apparatus function Poly (ADP-ribose) polymerase (PARP) Transcription Genome maintenance Aging

9 PARP-1 and PARP-2 are the only PARP activated by DNA breaks
PARP FAMILY MEMBERS NAD binding PARP-2 is 18 times less active than PARP-1 PARP-1 and PARP-2 are the only PARP activated by DNA breaks Hakmé et al., EMBO reports 9, 1094; 2008

10 BACKGROUND ON PARP’S AND DNA REPAIR
INVOLVEMENT OF PARP IN DNA REPAIR DNA damage signaling Recruitment and regulation of DNA repair components at DNA breaks Signaling to downstream effectors PARP-1 PARP-2 Histone H1 Histone H2B Cell cycle arrest Chromatin relaxation at DNA breaks DNA repair Genomic stability Cell survival

11 ELEVATED DNA DAMAGE INDUCES PARP-1 OVERACTIVATION
The Yin-yang of PARP activation ELEVATED DNA DAMAGE INDUCES PARP-1 OVERACTIVATION AND CELL DEATH PARP-1 overactivation Extensive DNA damage Glycolysis failure ATP depletion Necrosis 11

12 PARP INHIBITORS AS CHEMO- AND RADIOSENSITIZERS
INHIBITION OF DNA REPAIR PARP INHIBITORS AS CHEMO- AND RADIOSENSITIZERS Methylating agents: TEMOZOLOMIDE Topoisomerase I inhibitors Ionizing radiation

13 DACARBAZINE TEMOZOLOMIDE Spontaneous decomposition
Blood Brain Barrier DACARBAZINE TEMOZOLOMIDE P-450 Metabolic activation Spontaneous decomposition MethylTriazene Imidazol Carboxamide (MTIC)

14 TEMOZOLOMIDE and DNA METHYLATION
DNA damage 70% N7-Methyl-Guanine 9% N3-Methyl-Adenine 5% O6-Methyl-Guanine O6-Methyl-guanine- DNA methylransferase (MGMT) Base Excision Repair (BER) S-CH3 SH

15 THE CYTOTOXICITY OF TEMOZOLOMIDE IS DUE TO O6-METHYLGUANINE
O6-METHYLGUANINE TOXICITY REQUIRES INTACT MISMATCH REPAIR (MMR) Futile Cycle Theory MMR cannot find a correct base complementary to O6-Methyl-GUANINE: Block of replication Single and Double strand breaks Cytotoxicity requires: Low levels of O6-methylguanine DNA methyltrasferase (MGMT) A functional Mismatch Repair (MMR)

16 O6-MeG PARP INHIBITOR ENHANCES THE EFFICACY OF THE
METHYLATING AGENT TEMOZOLOMIDE Removal of O6-methylguanine S-Me High O6-Methylguanine- DNA methyltransferase SH O6-MeG RESISTANCE TO TEMOZOLOMIDE or TEMOZOLOMIDE O6-MeG Defective Mismatch Repair Lack of O6-MeG toxicity N3-MeA N7-MeG PARP-1 and 2 Base Excision Repair (BER) N3-MeA N7-MeG DNA repair PARP INHIBITOR Interruption of BER pathway Tumor cell survival Tumor cell death

17 PARP AND REPAIR OF N-METHYLATED PURINES AND
SINGLE STRAND BREAKS BY BASE EXCISION REPAIR (BER) METHYLATING AGENTS (N3-MethylAdenine, N7-MethylGuanine) IONIZING RADIATION INDIRECT SINGLE STRAND BREAK DIRECT SINGLE STRAND BREAK Removal of damaged base by glycosylase PARP INHIBITION PARP binds to strand break Strand interruption by AP endonuclease p24 PARP INHIBITION PARP binds to strand break PARP End processing XRCC1 PARP p24 Gap filling PARP CELL GROWTH ARREST Ligation PARP APOPTOSIS Short patch (1 nucleotide) Long patch (2-15 nucleotides) PERSISTANCE OF STRAND BREAKS

18 PARP inhibitors and topoisomerase I inhibitors
binding cleavage ligation release PARP-1 Poly (ADP-ribose) accelerates ligation Topoisomerase I inhibitor stabilizes cleavage complex and inhibits ligation Topoisomerase I PARP inhibitors and topoisomerase I inhibitors (Irinotecan and Topotecan) PARP inhibition enhances DNA damage induced by topoisomerase I inhibitors Fast repair + PARP inhibitors Active PARP Slow repair DNA damage Cancer Cell death Cell survival PARP Topo I Inhibitor

19 PARP inhibitors as anti-angiogenic agents
Chronic inflammation PARP INHIBITOR ACTIVATION OF PARP HIF-1 AP-1 PROLIFERATION OF INFLAMMATORY CELLS INDUCTION OF ANGIOGENESIS INCREASED TUMOR GROWTH NF-kB PARP inhibitors might interfere with tumor growth at different levels Modulating the transcriptional activation of different tumor related transcription factors tumorigenesis and angiogenesis Tentor et al., Eur J Cancer. 2007, 43:2124

20 PARP INHIBITORS AS MONOTHERAPY
Anti-tumor activity in tumor cells with defective repair of DNA double strand breaks by Homologous Recombination due to BRCA mutations: THE CONCEPT OF “SYNTHETIC LETHALITY” Bryant et al. Nature 2005, 434:913.; Farmer et al., Nature 2005, 434:917

21 Synthetic lethality Two genes are synthetically lethal if loss of function of either gene alone is compatible with cell survival, but inactivation of both cause cell death. A cancer-related mutated gene can sensitise tumour cells to a drug that specifically inhibits its “synthetic lethal partner”. The same drug should not affect normal cells, thus allowing higher therapeutic selectivity Modified by Fong et al. NEJM : 123 Ferrari E. et al., Eur J Cancer 46, , 2010

22 BRCA The mutation increases the risk of ovarian cancer up to 60% (BRCA1 60%, BRCA %) and breast cancer up to 84% (BRCA1 84%, BRCA %). BRCA2, however, contributes less to the risk of ovarian cancer, but more to the risk of male breast cancer and pancreatic cancer. Only 5% of breast cancers and 10% of ovarian cancers are associated with a known genetic mutation. Loss of the second allele will lead to a cell not capable of the repair of DNA double strand breaks.

23 Homologous Recombination relies on the presence of an intact sister chromatid to act as template for correct repair of the break without loss of sequence information DSB is recognized by the MRN complex (Mre11–Rad50–Nbs1), that keeps the DNA ends in close proximity to each other and is required for end resection to generate the long 3’-ssDNA overhangs required for HR. BRCA1, BRCA2 and BARD1 allow recruitment of the Rad51 protein which coats the ssDNA segment displacing RPA and forming a RAD51 nucleoprotein filament. RAD51 nucleoprotein filament catalyzes strand invasion and initiates the homology search on the intact sister chromatid. The eukaryotic genome is under constant stress, one result of which is the constant generation of DNA damage. DNA damage can result from both endogenous (e.g. reactive oxygen species and cytosine deamination) and exogenous (e.g. ultraviolet radiation, ionizing radiation and chemicals) sources. HR is responsible for the repair of DNA double-strand breaks and DNA interstrand cross-links and for accurate chromosome segration. The process of HR is initiated at the site of DNA breaks and gaps and involves a search for homologous sequences promoted by Rad51 and auxiliary proteins followed by the subsequent invasion of broken DNA ends into the homologous duplex DNA that then serves as a template for repair. 23

24 WHY DO PARP INHIBITORS SPARE NORMAL CELLS?
Normal Cells: BRCA+/+ or BRCA +/- (heterozygous mutation) Spontaneous Oxidative DNA damage: Single-Strand Breaks (104/cell/day) Base excision + PARP INHIBITOR Base excision Repair (PARP-1): repair of Single Strand Breaks (SSB) Generation of Double Strand Breaks (DSB) Collapsed replication fork Homologous Recombination error-free repair Repair of Double Strand Breaks (DSB) SSB DSB CELL SURVIVAL

25 Spontaneous Oxidative DNA damage:
WHY DO PARP INHIBITORS SELECTIVELY KILL TUMOR CELLS WITH MUTATED BRCA AND DEFECT IN THE REPAIR OF DNA DOUBLE STRAND BREAKS? TUMOR cells: BRCA-/- (homozygous mutation) Spontaneous Oxidative DNA damage: Single-Strand Breaks (104/cell/day) Base excision + PARP INHIBITOR Base excision Repair (PARP-1): repair of Single Strand Breaks (SSB Generation of Double Strand Breaks (DSB) Collapsed replication fork Homologous Recombination error-free repair PERSISTANCE of Double Strand Breaks (DSB) SSB DSB CELL DEATH

26 Lack of PARP-mediated BER is a synthetic lethal partner of complete BRCA1 or BRCA2 functional loss
RATIONALE FOR USING PARP INHIBITORS IN MONOTHERAPY IN BRCA ASSOCIATED OVARIAN OR BREAST CANCER Iglehart JD and Silver DP The New England Journal of Medicine 3612:189, 2009

27 SPORADIC TUMOURS WITH BRCA-LIKE CLINICAL PROPERTIES: TRIPLE NEGATIVE BREAST CANCERS
15% of breast cancers The overlap of BRCA1-associated breast cancers with the TNBC phenotype is significant. There is a clinical and pathological likeness between TRIPLE NEGATIVE, BASAL-LIKE breast cancers and hereditary BRCA1 breast cancers

28 BASAL-LIKE AND TRIPLE NEGATIVE BREAST CANCER
Her++ Normal-like Luminal 75% triple negative breast cancers are also basal-like 80-90% of hereditary BRCA1 breast carcinoma have features similar to basal-like tumors HER2 OVEREXPRESSING and BASAL-LIKE have the worst prognosis Basal-like tumors are mitotically active, high-grade, invasive, and associated with younger age The basal-like phenotype is ER, PR and HER2-negative, and either EGFR or CK 5/6 positive Perou CM , Nature 406: 747, 2000

29 Possible mechanisms of developing BRCAness in sporadic cancers
Increased expression of proteins that inactivate BRCA1 (ID4) BRCA1 promoter methylation (10-15% sporadic breast cancers and 5-30% sporadic ovarian cancers) FANCF promoter methylation Defects in other Fanconi’s anemia complex proteins Decreased expression of other proteins involved in HR, including Rad51, ATM PTEN deficiency Curr Probl Cancer 35:7, 2011

30 PARP INHIBITION BY NICOTINAMIDE ANALOGUES
PARP inhibitors and clinical trials PARP INHIBITION BY NICOTINAMIDE ANALOGUES CATALYTIC DOMAIN NAD+ binding H O O NH2 N N N X X Nicotinamide PARP inhibitor

31 PARP INHIBITORS IN CLINICAL TRIALS Route of administration
Agent Company Route of administration Phase Olaparib (PARP-1/2 IC50<2 nM) Astra Zeneca oral I-II-III Veliparib PARP-1/2 IC50 5 nM Abbott Oral (BBB) I-II Iniparib (PARP-1 mM range) BiPar/Sanofi Aventis IV II-III AG014699 (PARP-1/2 IC50 1,4 nM) Pfizer IV (oral) E7016 (PARP-1/2 IC50 50 nM) Eisai I CEP-9722 (PARP-1 IC50 20 nM PARP-2 IC50 6 nM) Cephalon INO-1001 (PARP-1/2 1 nM) MK-4827 (PARP-1/2 IC50 3,8 nM) Merck Iniparib AG014699 Nicotinamide newer PARP inhibitors are much more potent and specific for the reason that most of the compounds in this category are composed of carboxamide attached to an aromatic ring or the carbamoyl group built in a polyaromatic heterocyclic skeleton to form a fused aromatic lactam or imide which enhances the potency and specificity Olaparib Veliparib

32 First clinical trials with AG14699 and INO-1001
One of the most exciting classes of compounds in oncology—inhibitors of the DNA repair enzyme poly-(ADP ribose) polymerase (PARP)—suffered a double blow, with Sanofi-aventis and AstraZeneca announcing disappointing results for their respective drugs. 2003-5 First clinical trials with AG14699 and INO-1001 Graziani et al., Pharm. Res. 2005 Discovery of the activity in BRCA-defective tumors

33 First PARP inhibitor showing activity in BRCA-related
ovarian and breast cancers Olaparib (Astra-Zeneca): clinical development in MONOTHERAPY Phase 1 Phase II Phase III Suspended phase III in breast cancer: focus on ovarian Advanced Breast Cancer BRCA mutations (ICEBERG1) Advanced Ovarian Cancer BRCA mutations (ICEBERG2) Platinum resistant ovarian cancer (ICEBERG 3) vs Lip Dox BRCA mutated or Recurrent High Grade Ovarian or Known BRCA mutated or Triple Negative Breast Cancer Advanced solid tumours with BRCA mutations Disseminated colorectal cancer Advanced Solid tumors

34 OLAPARIB: Phase II in BRCA1 OR BRCA2 MUTATED ADVANCED BREAST CANCER (ICEBERG 1)
Tutt A et al., The Lancet 376, 235, 2010 54 recurrent or advanced cancer patients with a median of 3 previous chemotherapy regimens Low grade toxicity: 41% fatigue (grade 1 and 2) and nausea (grade 1 and 2) 41% objective response and tolerability of olaparib at 400 mg twice daily compares favorably with expected levels of activity (20–30% or less; 22% 100 mg) and toxicity of most chemotherapy agents used in patients previously treated with anthracycline and taxanes. Mean PFS 6 mo (3.8 mo 100 mg).

35 Before Olaparib After Olaparib
Recurrent BRCA Triple neg. 3m before and 2m after olaparib CT scan of a BRCA Triple neg: arrows point to lung metastases before and after 180 d from olaparib PET-CT scan of a BRCA Triple neg: arows point to hylar lympnodes and pleural disease before Olaparib Tutt A et al., The lancet 376, 235, 2010

36 OLAPARIB: Phase II in BRCA1 OR BRCA2 MUTATIONS ADVANCED OVARIAN CANCER (ICEBERG 2)
90% sporadic and 10% have a predisposing genetic defect 90% of patients with familial predisposition carry the BRCA defect. Although women with BRCA-associated ovarian cancer might have higher response rates to chemotherapy and improved survival rates than do those with sporadic ovarian cancer, most with stages III and IV will ultimately relapse and die despite available therapies. Up until now, knowledge of a BRCA mutation has not affected the selection of treatment for ovarian cancer. Phase II: in 57 patients with 2 doses with a median of 3 previous chemotherapy regimens Phase II: in 57 patients with 2 doses with a median of 3 previous chemotherapy regimens 400 mg more effective than 100 mg: the efficacy comparison has to be cautious because patients allocation was not randomized (overall response rate 33% versus 13%). Mean PFS 5.8 mo (1.8 mo 100 mg). Mild to moderate adverse effects Audeh MW et al., The Lancet 376, 245, 2010

37 OLAPARIB: Phase II in BRCA1 or BRCA2 MUTATIONS ADVANCED OVARIAN CANCER PLATINUM RESISTANT (ICEBERG 3) The PFS were 6.5 vs 8.8 vs 7.1 months for 200 mg, 400 mg, and liposomal doxorubicin arm, respectively. Olaparib did not reach the primary objective of improving PFS partly because of a better PFS seen in the liposomal doxorubicin arm than expected. Twice as many grade 3 toxicities were seen in the liposomal doxorubicin arm. Although reported as a negative study, this trial still shows consistent response and decreased toxicity with the use of single-agent olaparib in BRCA mutation ovarian cancer patients. KAYE S et al., Ann Oncol 21:viii176, 537P (suppl 2), 245, 2010

38 Olaparib (Astra-Zeneca): clinical development in combination therapy
Phase 1 Phase II Phase III Advanced ovarian cancer (+carboplatin and paclitaxel) Recurrent Ovarian or Triple-Negative Breast Cancer(+cediranib) I-II BRCA1/BRCA2-associated, hereditary, or triple negative metastatic or unresectable breast cancer or ovarian epithelial cancer.(+carboplatin) Triple Negative Breast Cancer and advanced ovarian cancer (+carboplatin and or paclitaxel ) Metastatic melanoma (+dacarbazine) Pancreatic cancer (+gemcitabine) Recurrent or metastatic gastric cancer (+paclitaxel) Breast, ovarian cervix, uterine unresponsive; men with BRCA metastatic breast (+carboplatin) Advanced solid tumours (+ topotecan or bevacizumb or cisplatin) Advanced colorectal cancer (+irinotecan) One of the concerns regarding the addition of PARP inhibitors to chemotherapy is the potential of enhancing toxicity

39 INIPARIB: A NON-COMPETITIVE PARP INHIBITOR
Iniparib (Sanofi-Aventis): clinical development Phase 1 Phase II Phase III Triple Negative metastatic breast cancer (+gemcitabine-carboplatin) Non Small Cell Lung Cancer (+gemcitabine-carboplatin) Triple Negative breast cancer neoadjuvant (+gemcitabine-carboplatin or paclitaxel) Platinum-Sensitive or -Resistant Recurrent Ovarian CancerNon Small Cell Lung Cancer (+gemcitabine-carboplatin) or monotherapy in BRCA Triple negative Brain metastases (+ irinotecan) Glioblastoma (+temozolomide) Advanced or recurrent uterine carcinosarcma (+paclitaxel and carboplatin) Advanced Solid tumors (+chemotherapy) Iniparib is an irreversible inhibitor It ejects the Zn from the first zinc finger of PARP-1 inactivating the enzyme and degrades PARP-1 activating a protease The first PARP inhibitor that has shown survival advantage in TNBC phase II

40 Phase II: INIPARIB PLUS CHEMOTHERAPY IN METASTATIC TRIPLE NEGATIVE BREAST CANCER
123 patients (>50% previously received taxanes and antracycline-based therapy and 10% bevacizumab) randomized to receive: Iniparib (5,6 mg/kg d1,4,8,11) + gemcitabine and carboplatin every 21 d Gemcitabine (1000 mg/m2) and carboplatin No differences in myelotoxicity between the two treatment arms About 15% of breast cancer; good response in the neo-adjuvant setting to taxanes and antracyclines. When metastatic disease develops the median survival is of approximately 1 year. No standard-of-care therapy exists for patients with metastatic triple-negative breast cancer. Current phase III comparing carboplatin versus docetaxel It shares clinical and pathological features with hereditary BRCA1-related breast cancers In sporadic triple-negative there is a dysregulation of BRCA1 or other components of HR Gemcitabine + carboplatin give 26-34% response rates G+C+Iniparib G+C P Response rate 52% 32% 0.02 Overall Survival (mo) 12,3(9,8-21,5) 7,7( ) 0,01 Adapted from O’Shaughnessy JO et al., The New England Journal of medicine 364: 205, 2011

41 INIPARIB AND PHASE III IN TNBC
Sanofi-Aventis and its subsidiary, BiPar Sciences, at the end of January 2011 announced that a randomized Phase III trial evaluating Iniparib in 519 patients with metastatic disease did not meet the pre-specified criteria for significance for co-primary endpoints of overall survival and progression-free survival. The overall safety analysis indicates that the addition of iniparib did not significantly add to the toxicity profile of gemcitabine and carboplatin.

42 AG014699 (Pfizer): clinical development
Increased myelosuppression that required temozolomide dose reduction (in 32% of patients) AG (Pfizer): clinical development Phase 1 Phase II Phase III Ovarian breast cancer BRCA associated Monotherapy BRCA 1 or BRCA 2 Mutation With Locally Advanced or Metastatic Breast or Advanced Ovarian Cancer (+cisplatin) Metastatic Melanoma (+temozolomide) Adv. Solid tumours (+carboplatin + carboplatin and paclitaxel +cisplatin and pemetrexed +epirubicin and cyclophosphamide) (chemotherapy-naïve)

43 VeIiparib (Abbott): clinical development (32 clinical studies) Phase 1
Phase II Phase III Heavily treated metastatic colorectal cancer (+temozolomide) Metastatic Breast Cancer (+temozolomide) Hepatocellular carcinoma (+temozolomide) Relapsed or Refractory Ovarian Cancer or Primary Peritoneal Cancer (+topotecan) Metastatic prostate cancer (+temozolomide) Pediatric recurrent refractory CNS Tumors (+temozolomide) Advanced solid tumors and/or lymphoma (+folfiri or temozolomide or gemcitabine and carboplatin, cyclophosphamide, topotecan) Acute Leukemia (+temozolomide) Newly Diagnosed Stage II, Stage III, or Stage IV Ovarian Epithelial Cancer, Fallopian Tube Cancer, or Primary Peritoneal Cancer (+carboplain, paclitaxel and bevacizumab) Refractory BRCA-Positive Ovarian, Primary Peritoneal or Ovarian High-Grade Serous Carcinoma, Fallopian Tube Cancer, Triple-Negative Breast Cancer, and Low-Grade Non-Hodgkin's Lymphoma (+cyclophosphamide) Recurrent ovarian cancer, fallopian tube cancer, or primary peritoneal cancer or metastatic breast cancer (+liposomal doxorubicin) Primary and metastatic brain tumours (+whole brain irradiation) Monotherapy: Leukemia/lymphoma/solid tumors, Recurrent and/or Metastatic Breast Cancer

44 VELIPARIB AND PHASE 0 TRIALS
PHASE 0 CLINICAL TRIAL AND VELAPARIB Poly(ADP) RIBOSE IN : PBMC AND TUMOR BIOPSIES Kummar et al., J Clin Oncol ;27: VELIPARIB AND PHASE 0 TRIALS The first Phase 0 study under FDA new Exploratory Investigational New Drug was performed (a first-in-human clinical trial conducted under an exploratory IND that has no therapeutic or diagnostic intent and involves very limited human exposure). Veliparib was chosen because it has a wide therapeutic index and a validated PD assay. The primary study end-point was target modulation by the PARP inhibitor measuring POLY(ADP-RIBOSE) levels in perypheral blood mononuclear cells (PBMC) and tumour biopsies.

45 RESISTANCE TO PARP INHIBITORS
Secondary mutations of BRCA genes altering the reading frame to wild-type Selection of pre-existing resistance clones Mutations acquired during treatment in cells genetically unstable due to the lack of HR

46 SENSITIVITY OF OVARIAN CANCER TO PLATINUM AGENTS AND USE OF PARP INHIBITORS
Resistance to platinum is associated with decreased sensitivity to PARP inhibitors since platinum sensitivity may be in part related to BRCAness. Platinum-resistant tumors without secondary BRCA2 mutations may remain sensitive to PARP inhibitors Platinum resistance: disease progression within 6 months of prior platinum therapy Platinum refractory disease: disease progression on platinum therapy Fong PC et al., Journal of Clnical Oncology 28, 2512, 2010: Olaparib induced objective response 46.2% in platinum sensitive patients (6/13), 33.5% in platinum-resistant patients (8/24), 0% in platinum-refractory patients(0/13)

47 RESISTANCE TO PARP INHIBITORS
Up-regulation of p-glycoprotein (Pgp) efflux pump

48 CHALLENGING TASKS (1) To determine the optimal chemotherapy backbone to combine with PARP inhibitors (2) To balance the benefit and toxicity risk when PARP inhibitors are used in combination therapies (3) To define the most effective schedule of administration (i.e., continuous versus intermittent dosing with chemotherapy: higher myelotoxicity; concomitant or sequence treatment)

49 CHALLENGING TASKS BRCA SIGNATURE Konstantinopoulos e al., JCO 2010, 28:355 (4) To define more precisely the patient population most likely to respond: potential signature of BRCAness (e.g. 60-gene assay to identify BRCAness profile) (5) Potential prophylactic use for BRCA1 and BRCA2 mutation carriers: potential long-term toxicity (6) PARP-1 expression/activity levels???? (HR-deficient cancers have higher PARP-1 levels) (7) To discover and validate candidate biomarkers to predict responders [i.e., γH2AX, RAD51 (as a marker of intact HR), germline DNA studies,etc.]

50 gH2AX and RAD51 and HR function
PRESENCE OF gH2AX AND DECREASE OF RAD51 INDICATE REDUCED HR FUNCTION AND SENSITIVITY TO PARP INHIBITORS gH2AX and RAD51 and HR function A key component in DNA repair is the histone protein H2AX, which becomes rapidly phosphorylated to form large numbers of γH2AX at nascent DSB, creating a focus where proteins involved in DNA repair and chromatin remodeling accumulate (pink spots). Rad51 is a crucial downstream protein involved in HR repair, which is relocalized within the nucleus in response to DNA damage to form distinct foci that are thought to represent assemblies of proteins at these sites of HR repair (green spots).

51 CHALLENGING TASKS: HOW TO OVERCOME RESISTANCE TO PARP INHIBITORS
6-THYOGUANINE: it is not substrate of Pgp and reactivation of HR is not sufficient to repair lesions induced by 6-Thyoguanine Inhibitors of Pgp Proteasome inhibitors: down-regulation of Pgp

52 WHAT IS GOINGO TO BE THE FUTURE OF PARP INHIBITORS?
Trials directed at subsets of patients who are most likely to respond to PARP inhibitors Trials in combination with chemotherapeutic agents for which preclinical studies have indeed shown synergy with PARP inhibitors

53 EdiVoteStart EdiVoteStop Standard Indicare quale tra queste affermazioni riguardante gli inibitori di PARP è SBAGLIATA: Olaparib ha mostrato attività antitumorale in monoterapia nei tumori con mutazioni di BRCA1 o BRCA2 Potenziano l’attività antitumorale della temozolomide inibendo il sistema di riparo per escissione delle basi Potenziano l’attività antitumorale dei derivati delle camptotecine, che inibiscono la Topoisomerasi 1, rendendo più lento il processo di riparo del DNA La maggior parte degli inibitori di PARP compete con il NAD per il legame al sito catalitico dell’enzima Potenziano l’attività antitumorale di trastuzumab (anticorpo monoclonale anti-HER2) nei tumori della mammella triple negative 20% 20% 000 20% 20% 010 20%

54 EdiVoteStart EdiVoteStop Standard Indicare quale tra queste affermazioni riguardante gli inibitori di PARP è SBAGLIATA: L’Iniparib è un inibitore di PARP irreversibile, non competitivo La mancata formazione di foci di RAD51 nel tumore in seguito a trattamento con inibitore di PARP potrebbe rappresentare un fattore predittivo di risposta al farmaco I tumori ovarici associati a mutazioni di BRCA1 o BRCA2 e sensibili ai composti del platino sono in genere più sensibili agli inibitori di PARP rispetto ai tumori refrattari al platino (cioè che vanno in progressione durante la terapia con composti del platino) Olaparib, un inibitore di PARP che supera la barriera emato-encefalica, in studi di fase II è stato associato a carboplatino e gemcitabina per il trattamento del glioblastoma multiforme La resistenza agli inibitori di PARP può essere dovuta a nuove mutazioni di BRCA1 o BRCA2, che ripristinano l’espressione della normale proteina corrispondente, o ad elevata espressione di pompe di efflusso della famiglia della Multi Drug Resistance 20% 20% 20% 000 20% 20% 010