1. Fanconi Anemia (FA) Rare, inherited chromosome instability disorder –Originally described by Guido Fanconi in 1927 Patients have diverse congenital abnormalities and cancer predisposition –Radial aplasia, short stature, hyperpigmentation, bone marrow failure Defines a novel DNA damage response pathway –Hypersensitivity to cross-linking agents –Mitomycin C and diepoxybutane Multiple complementation groups defined by sensitivity to cross-linking agents in cell fusion experiments –FANCA through FANCN –Two groups for FANCD, FANCD1 and FANCD2

2. Wang W. Nat Rev Genet 8:735-748, 2007

3. FA proteins are conserved largely just among vertebrates

4. Biomarkers of FA pathway Kennedy, R. D. et al. J Clin Oncol; 24:3799-3808 2006

5. Basic FA Pathway Response to stalled replication forks Monoubiquitination of FAND2/FANCI Activation of HR and TLS Reversal by de- ubiquitination via USP1 Wang W. Nat Rev Genet 8:735-748, 2007

7. The FA pathway displays a unique genetic relationship with breast cancer FANCD1/BRCA2-> BRCA2 mutations are associated with increased risk of breast cancer but breast cancer patients with biallelic mutations were not found. Turns out they get FA FANCJ/BACH1/BRIP1-> Originally identified as a DNA helicase that interacts with BRCA1 –Sporadic reports that variants in BACH1 affect breast cancer risk FANCN/PALB2-> (Partner and Localizer of BRCA2) originally identified as a regulator of BRCA2 –An insertion/deletion variant in PALB2 is associated with breast cancer risk Many of the FA genes are also mutated in or associated with risk of breast cancer

8. DNA damage response networks DNA repair is only a subset of the responses that cells utilize to respond to DNA damaging agents DamageDetectionSignaling cell cycle arrest DNA repair apoptosis Effectors

9. Many of the DNA damage response networks have been uncovered based on human genetic disorders Xeroderma pigmentosum (XP) Cockayne syndrome (CS) Trichothiodystrophy (TTD) Severe combined immunodeficiency (SCID) Ligase IV Syndrome (LIG4S) Ataxia Oculomoter Apraxia 1 (AOA1) Hereditary non-polyposis colon cancer (HNPCC) Fanconi anemia (FA) Ataxia-telangiectasia (A-T) Nijmegen breakage syndrome (NBS) Ataxia-telangiectasia-like disorder (ATLD) Seckel Syndrome Bloom syndrome (BS) Rothmund-Thomson syndrome (RTS) Werner syndrome (WS) Damage sensing and signaling DNA repair DNA topology

10. Complex relationship between genes that direct DNA damage responses and cancer risk Many inherited recessive disorders with mutations in specific response genes are cancer-prone Some recessive disorders may be characterized by increased cancer risk in heterozygotes Some genes predispose to cancer primarily through somatic mutation –E.g. BRCA1, BRCA2, p53 Common variants in some genes can also modulate risk of cancer or susceptibility to DNA damaging agents –Relevance to cancer risk and to cancer therapy

11. Effects of ionizing radiation Acute effects (first 72 hours) –Skin, hematopoetic system, gut Late effects (weeks to years) –Vascular damage, fibrosis, inflammation Significant population variation in responses to IR

12. Radiation Therapy One of the most effective therapies for cancer –Given a sufficiently high dose, any tumor can be sterilized Efficacy is limited by: –Complications –Tumor characteristics e.g., hypoxia Fractionation –Low dose radiation issues

13. Cellular effects of ionizing radiation Direct effects –Causes direct damage to DNA and proteins in cell –More likely when the beam of charged particles consists of alpha particles, protons and electrons Indirect effects –Causes damage by interacting with the cellular medium producing free radicals which, in turn, can damage DNA –Typical effect of X-rays or gamma rays Damage to DNA can include base loss or modification, single strand gaps or double-strand breaks

14. Finding genes that mediate responses to radiation damage Abundant evidence of genetic control of radiation responses –Interstrain differences in mice –Breed specific differences in dogs –Cell survival assays in humans Response to radiation is a complex trait –candidate genes from model systems? –Linkage/association mapping? –Extreme phenotypes?

15. Clinical features of Ataxia-Telangiectasia (AT) Progressive cerebellar ataxia Telangiectases High cancer incidence Hypersensitivity to ionizing radiation Chromosome instability Immunodeficiency Underdeveloped thymus Elevated serum alpha-fetoprotein Insulin resistance Progeria

16. Cerebellar degeneration in A-T

17. Telangectasias in A-T

18. Malignancies occurring in AT patients Stomach Liver Ovary Other Overall, ~40-50% of A-T patients will develop some kind of malignancy

19. Consequences of standard radiation therapy in an undiagnosed A-T patient

20. Phenotypes of AT cells in culture Radioresistant DNA synthesis (RDS) –Replication origin firing is not inhibited by IR Impaired survival after exposure to IR or radiomimetic chemicals Loss of cell cycle checkpoint control Increased frequency of IR induced chromosome aberrations Hyper-recombination

21. A-T fibroblasts exhibit radio-resistant DNA synthesis 051020 0 40 60 80 100 Radiation Dosage (Gy) % Control DNA Synthesis LM217 (Normal) GM637 (Normal) AT4BI (ATC) AT3BI (ATA)

22. Impaired colony formation after irradiation of A-T fibroblasts 0123 0.1 0.2 0.5 1 2 5 10 20 50 100 Dose (Gy) % Surviving GM637 AT Grp A

23. Radiation survival of A-T, A-T carriers and other disorders in a colony survival assay

24. ATM, the gene mutated in A-T, is a PIK related kinase

25. Atm transphosphorylates itself following DNA damage coincident with the release of active monomers From Bakkenist and Kastan. 2003. Nature 421:499-506.

26. PIK related kinases are recruited to damage sites by a conserved mechanism NBS1 Mre11 RAD50 ATM P ATR ATRIP P DNA-PK Ku80 Ku70 P Ku80 Ku70 DNA-PK P Conserved sequences in the C-terminal end of interacting partners, NBS1, ATRIP and Ku 80 are required for recruitment of ATM, ATR, and DNA-PK, respectively.

27. ATM regulates the mammalian cellular response to DNA DSBs ATM P P53 Apoptosis P G1/S Checkpoint S phase checkpoint Nibrin P Homologous recombination repair Non- homologous end-joining repair Rad50 Mre11 DNA-PK Ku80 Ku70 Xrcc 4 Ligase IV BRCA1 Rad51 Chk2 P FANCD2 P P P P P ARTEMIS DNA double-strand break P P P

29. More than 700 proteins are phosphorylated by ATM in response to radiation

30. Incidence of female breast cancer in blood relatives of A-T patients Swift et al. NEJM 325:1831-1836, 1991

31. ATM regulates the mammalian cellular response to DNA DSBs ATM P P53 Apoptosis P G1/S Checkpoint S phase checkpoint Nibrin P Homologous recombination repair Non- homologous end-joining repair Rad50 Mre11 DNA-PK Ku80 Ku70 Xrcc 4 Ligase IV BRCA1 Rad51 Chk2 P FANCD2 P P P P P ARTEMIS DNA double-strand break P P P

32. ATM and breast cancer All studies of A-T families reveal increased breast cancer incidence in carriers Sporadic individuals who carry A-T causing mutations appear to be at increased risk of breast cancer –These alleles appear to be highly penetrant but rare in the population The mechanism whereby ATM predisposes to breast cancer is unknown –Evidence for dominant negative effects, LOH, or epigenetic silencing effects Overall, ATM is a risk factor for breast cancer but not a significant one on a population basis.

33. Can knowledge of DNA damage response pathways be used to inform cancer therapy? Genetic prediction of sensitivity to radio- or chemotherapeutic agents Treatment-related risks of second cancers Radiosensitizers –Targeting molecules in response pathways –Radiation or hypoxic activation Tumors frequently inactivate pathways that trigger DNA repair, cell cycle checkpoints or apoptosis –Can this be exploited as a therapeutic strategy? Synthetic lethality –Analogy to yeast where many synthetic lethal partners of DNA repair genes exist –Hartwell-> MMR and TLS

34. Some small molecule inhibitors of DNA damage response pathways PARP inhibitors –Multiple companies and indications MGMT inhibitor –Melanoma, colorectal cancers DNA-PKcs inhibitors ATM inhibitor

35. BRCA1 and BRCA2 The primary genetic risk factors for breast cancer BRCA1 or BRCA2 mutation are dominant with subsequent epigenetic silencing or loss of the normal allele in the tumor

36. Estimated mutation frequencies in general breast cancer populations Caution: frequencies and risks remain a contentious area

37. Targeting BRCA deficiency As we have seen, HR plays a key role in the repair of stalled replication forks –BRCA1 and BRCA2 deficient cells are particularly sensitive to agents that trigger fork stalling ICL inducing agents such as mitomycin C, cisplatin or carboplatin Single strand breaks can also lead to fork stalling –Would inhibition of single strand break response be synthetically lethal with HR inhibition? “BRCAness”

38. PARP inhibition and BRCA deficiency Caveat: As therapy becomes more finely targeted, the potential for resistance to therapy potentially becomes greater.

39. Resistance to PARP inhibitors Reversion of BRCA2 mutations Edwards et al. Nature 451: 1111-1115, 2008

40. Cisplatin resistance in tumors Sakai et al. Nature 451:1116-1120,2008 Ovarian tumors with BRCA2 mutations highly sensitive to cisplatin –However, resistance develops over time Analysis of resistant tumor line revealed reversion of BRCA2 mutation Further revertants selected in culture Recurrent tumor in a patient had LOH and reverting mutation of BRCA2

41. Synthetic lethal screens for new drug targets Drug selection for synthetic lethality with PARP inhibitor High sensitivity of recipient cell to PARP inhibitor improves sensitivity of the assay Screened kinases only as druggable targets Novel target CDK5