Translating DNA repair pathways into therapeutic targets: beyond the BRCA1/2 and PARP inhibitor saga. Jorge S Reis-Filho, MD PhD FRCPath

Transcription

1 Translating DNA repair pathways into therapeutic targets: beyond the BRCA1/2 and PARP inhibitor saga Jorge S Reis-Filho, MD PhD FRCPath

2 Summary How do PARP inhibitors work? Synthetic lethality Potential clinical use of PARP inhibitors Markers of response to PARP inhibitors Mechanisms of resistance

3 How do PARP inhibitors work?

4 Poly (ADP-Ribose) Polymerase (PARP)

5 When single strand breaks are not repaired they are converted into double strand breaks

6 BRCA1/2 inactivation and homologous recombination DNA repair defects SSB Inter-strand Crosslink repair Replication fork collapse Normal HR function Deficient HR HR-based repair Error prone repair or No repair (SSA or NHEJ) Survival Cell death due to chromosomal instability

7 Model of PARP inhibitor sensitivity

8 Model of PARP inhibitor sensitivity Wild type cells. Functional BRCA/RAD51 dependent gene conversion Replication fork restart RAD51 BRCA1 BRCA2 Rad51 foci Sister Chromatid Exchange Sister Chromatid Exchange

9 Model of PARP inhibitor sensitivity Wild type cells. Functional BRCA/RAD51 dependent gene conversion Replication fork restart RAD51 BRCA1 BRCA1-/- or BRCA2-/- cells. Failure of gene conversion. Replication fork failure BRCA2 Rad51 foci or, following error-prone DSB repair by NHEJ and/or SSA Sister Chromatid Exchange Sister Chromatid Exchange Complex rearrangements Arrest at G2/M checkpoint Cell Death

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

11 BRCA1 -/- and BRCA2 -/- cells are extremely sensitive to PARP inhibition BRCA1 +/+ BRCA1 +/- BRCA2 +/+ BRCA2 +/- BRCA1 -/- BRCA2 -/- No difference in sensitivity between heterozygous and wild-type BRCA cells Farmer et al. Nature 2005; Bryant et al. Nature 2005

12 Potential clinical use

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14 Metastatic familial breast and ovarian cancer PARP inhibitor phase I Strong family history Ovarian BRCA1-/23 mm 11 mm Fong et al. NEJM July 2009

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16 Basal-like and TN BRCA1 High grade Pushing borders Brisk lymphocytic infiltrate High proliferation rates ER - PR - HER2 - TP53 mutations

17 Addition to Iniparib to chemotherapy

18 Limited activity of PARP inhibitors in sporadic TNBC Confi rmed objective response BRCA1 or BRCA2 (n=8) Sporadic TNBC (n=15) 0 0 Complete response 0 0 Partial response 0 0 Stable disease 63% 13% Progressive disease 37% 80% N/A 0 7% Gelmon et al. Lancet Oncol 2011

19 INIPARIB is NOT a PARP inhibitor

20 Mendes-Pereira et al. EMBO Mol Med 2009; Dedes et al. Sci Transl Med 2010; Loveday et al. Nat Genet 2011 Loss of function of other genes also sensitises cells to PARP inhibitors PALB2 RAD51D Homologous recombination DNA repair MRE11A PTEN (?)

21 20 weeks 10 weeks Baseline

22 Markers of response to PARP inhibitors

23 Surrogate marker for HR competence PTEN? Epigenetic mechanisms Annunziata and O'Shaughnessy, Clin Canc Res 2010

24 RAD51 foci formation Potential marker of HR competence? H2Ax DNA damage marker RAD51 competent HR marker BRCA1/2 wild-type BRCA1/2 mut BRCA2 mut Gottipati et al, Cancer Res 2010; Graeser et al, Clin Canc Res 2010; Wilkerson et al, J Pathol 2011

25 RAD51 foci formation may be a predictor of response to chemotherapy Graeser et al, Clin Cancer Res 2010

26 PAR polymers identify HR-defective cells Depletion of HR proteins PARP hyperactivity increase in PAR polymers PARP inhibitor sensitivity Gottipati et al, Cancer Res 2010 HR competent cells PARP activity PAR polymer formation PARP inhibitor resistance

27 Mechanisms of resistance

28 Mechanisms of resistance to PARPi Over-expression of P-glycoprotein Rottenberg et al, PNAS 2008 Rescue of BRCA1 deficiency through TP53BP1 loss Bouwman et al, Nat Struct Mol Biol 2010 Chiarugi, Trends Pharmacol Sci 2012 Functional restoration of BRCA1/2 Edwards et al, Nature 2008; Sakai et al, Nature 2008

29 Take Home Messages PARP inhibitors Inhibit base excision repair In vitro and in vivo efficacy in HR deficient tumours Evidence of clinical activity in BRCA1/ BRCA2 patients Markers of response HR DNA repair defects Potential surrogates: RAD51 and PAR polymers Resistance is multifactorial

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31 Response in a Heavily Pretreated Breast Cancer Patient 46 year old patient., BRCA1 mutation carrier (c4154dela) 1. BC right (29y): pt1, pn0, M0, Histopathol.: Invasive ductal, HR neg. 2. BCT +radiation right 3. BC left (31 y): pt1, pn0, M0, Histopathol.:Invasive ductal, HR neg., Her2neg. 4. BCT + radiation left. 5. Local recurrence left. 1994: HR pos. 6. Hormone-therapy 7. Lung metastasis 1995 (33 y) 8. Lung surgery (R0) 9. Chemotherapy PBSO 2006 (44 y) 11. Inflammatory recurrence le Before surgery. FEC>Doc, Ablatio le. + TRAM 13. cutan metastasis chest both sides chemotherapy Progressive disease

32 Clinical Response to PARP Inhibitor Olaparib (AZD2281) 9 WEEKS single agent PARPi Tutt et al. Lancet 2010

33 Genetic instability in cancer is caused by defects in DNA repair pathways!

34 If primary DNA repair mechanisms are dysfunctional Single strand break Inter- strand Crosslink repair Several DNA lesions are converted into DNA double strand breaks during cell cycle

35 Functional restoration of BRCA2 by secondary or reversion mutations BRCA2 c.6174delt mutation carriers Edwards et al, Nature 2008; Sakai et al, Nature 2008

36 Functional restoration of BRCA1/2 by secondary or reversion mutations Ashworth, Cancer Res 2008

37 Up-regulation of P-glycoprotein efflux pumps (Abcb1a/b) Rottenberg et al, PNAS 2008

38 Loss of TP53BP1 Restoration of HR defects in Brca1-deleted cells Reversion of hypersensitivity of Brca1- deleted cells to DNA-damaging agents Bouwman et al, Nat Struct Mol Biol 2010

39 Principles of genotoxic therapies Chemotherapy kills proliferating cells Induces lesions in the DNA Proliferating cells accumulate damage DNA damage is sensed by cell cycle checkpoints Apoptosis or mitotic catastrophe

40 What do chemotherapy agents do?

41 Anthracyclines Chemotherapy agents Alkylators Replica>on inhibitors An>metabolites Bifunc>onal Monofunc>onal Radiotherapy and radiomime>cs Replica>on lesions Base damage DNA crosslinks Bulky adducts Double strand breaks Single strand breaks

42 Different DNA lesions are repaired by different mechanisms Single strand break Bulky adduct Double strand break A- G mismatch T- C mismatch Inser>on Dele>on Base excision repair Nucleo>de excision repair Homologous recombina>on Mismatch repair