Fanconi Anemia FA

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 ICL repair complexity. Fanconi anemia (FA): a rare autosomal recessive and X-linked genetic disorder characterized by congenital abnormalities, progressive bone marrow failure and increased risk of cancer, including leukemia. A novel DNA damage response pathway. See marked chromosomal instability and hypersensitivity to crosslinking agents. Repair of cis-platin, mitomycin C and other ICLs seems to use a combination of FA factors, NER factors and homologous recombination factors. DSBs can occur when a replication encounters a SS break, and this could require NHEJ factors to repair. Translesion synthesis may also be important. Details of repair process are unclear and may depend on the type of ICL.ICL repair complexity. Fanconi anemia (FA): a rare autosomal recessive and X-linked genetic disorder characterized by congenital abnormalities, progressive bone marrow failure and increased risk of cancer, including leukemia. A novel DNA damage response pathway. See marked chromosomal instability and hypersensitivity to crosslinking agents. Repair of cis-platin, mitomycin C and other ICLs seems to use a combination of FA factors, NER factors and homologous recombination factors. DSBs can occur when a replication encounters a SS break, and this could require NHEJ factors to repair. Translesion synthesis may also be important. Details of repair process are unclear and may depend on the type of ICL.

2. Classification and domain structures of Fanconi anaemia (FA) proteins. The FA proteins are classified into three groups on the basis of their roles in the monoubiquitylation of FA proteins FANCD2 and FANCI, as shown on the left. The brackets indicate that certain FA proteins can form complexes or subcomplexes (such as the FA core complex and the ID complex ) in which they work together as partners; inactivation of one such FA protein often affects the stability and nuclear localization of its partner proteins. ARM, armadillo repeat; BRC, the internal repeat domains of BRCA2; BRCA, breast cancer susceptibility; FAAP, FA-associated protein; P, phosphorylation; PALB2, partner and localizer of BRCA2; TPR, tetratricopeptide repeat motifs; Ub, ubiquitin; WD40, a repeat motif found in b-transducin and other proteins.Classification and domain structures of Fanconi anaemia (FA) proteins. The FA proteins are classified into three groups on the basis of their roles in the monoubiquitylation of FA proteins FANCD2 and FANCI, as shown on the left. The brackets indicate that certain FA proteins can form complexes or subcomplexes (such as the FA core complex and the ID complex ) in which they work together as partners; inactivation of one such FA protein often affects the stability and nuclear localization of its partner proteins. ARM, armadillo repeat; BRC, the internal repeat domains of BRCA2; BRCA, breast cancer susceptibility; FAAP, FA-associated protein; P, phosphorylation; PALB2, partner and localizer of BRCA2; TPR, tetratricopeptide repeat motifs; Ub, ubiquitin; WD40, a repeat motif found in b-transducin and other proteins.

3. FA proteins are conserved largely just among vertebrates A few FA proteins have been found in C. elegans and this may represent the core pathway components. Intactness of the FA pathway is often monitored based on the monoubiquitination of FANCD2. Most components are necessary for this to occur.A few FA proteins have been found in C. elegans and this may represent the core pathway components. Intactness of the FA pathway is often monitored based on the monoubiquitination of FANCD2. Most components are necessary for this to occur.

4. Biomarkers of FA pathway FANCD2 monoubiquitination blots are a standard test for the function of the FA pathway. Also help to partition genes into those required and not required for this step which roughly corresponds to those involved in sensing damage and those involved in reacting to it.FANCD2 monoubiquitination blots are a standard test for the function of the FA pathway. Also help to partition genes into those required and not required for this step which roughly corresponds to those involved in sensing damage and those involved in reacting to it.

5. Simplified FA pathway. Complex 1 also called the core complex is assembled after replication fork stalling by a mechanism that is dependent upon ATR and includes phosphorylation of FANCM. Fork stalling triggers activation of ATR and phosphorylation of FA pathway components. Collapse of the fork and creation of the DSB will subsequently trigger ATM which can also phosphorylate many of the same components. Complex 1 functions as an E3 ligase to monoubiquitinate FANCD2 and FANCI. Mono-ubiquitination of these proteins is interdependent. Need FANCD2 to get FANCI modified and vice-versa. FANCD2 And FANCI nucleate the formation of Complex 2 which is a repair focus. Upon completion of repair, FANCD2 is de-ubiquitinated by USP1. Interestingly, USP1 also de-ubiquitinates PCNA which, as we previously discussed, may provide a signal to de-activate TLS polymerases. TLS polymerases have been demonstrated to co-localize with FA Complex 2 to DNA damage foci.Simplified FA pathway. Complex 1 also called the core complex is assembled after replication fork stalling by a mechanism that is dependent upon ATR and includes phosphorylation of FANCM. Fork stalling triggers activation of ATR and phosphorylation of FA pathway components. Collapse of the fork and creation of the DSB will subsequently trigger ATM which can also phosphorylate many of the same components. Complex 1 functions as an E3 ligase to monoubiquitinate FANCD2 and FANCI. Mono-ubiquitination of these proteins is interdependent. Need FANCD2 to get FANCI modified and vice-versa. FANCD2 And FANCI nucleate the formation of Complex 2 which is a repair focus. Upon completion of repair, FANCD2 is de-ubiquitinated by USP1. Interestingly, USP1 also de-ubiquitinates PCNA which, as we previously discussed, may provide a signal to de-activate TLS polymerases. TLS polymerases have been demonstrated to co-localize with FA Complex 2 to DNA damage foci.

6. A model for the participation of Fanconi anaemia (FA) proteins in crosslinked DNA-damage repair. FA proteins work together with other DNA-repair molecules to remove an interstrand crosslink (ICL) at a stalled replication fork. See text for details. One modification of the older model is that the unhooked crosslink is shown on the lagging-strand template of the replication fork, in contrast with the older model, which places the unhooked crosslink on the leading-strand template29. Studies of XPF�ERCC1 endonuclease cleavage patterns of crosslinked DNA substrates in vitro suggested that the unhooked crosslink should be on the lagging strand. Nevertheless, in both models, the same pathways (nucleotide excision repair (NER), FA, translesion synthesis and homologous recombination (HR)) are used to repair the crosslink and restart the replication fork. BLM, Bloom syndrome protein; BRCA, breast cancer susceptibility; ?H2AX, a histone H2A variant; PALB2, partner and localizer of BRCA2; Modifiedwith permission from Ref. 29 ? (2005) Cold Spring Harbor Laboratory Press.A model for the participation of Fanconi anaemia (FA) proteins in crosslinked DNA-damage repair. FA proteins work together with other DNA-repair molecules to remove an interstrand crosslink (ICL) at a stalled replication fork. See text for details. One modification of the older model is that the unhooked crosslink is shown on the lagging-strand template of the replication fork, in contrast with the older model, which places the unhooked crosslink on the leading-strand template29. Studies of XPF�ERCC1 endonuclease cleavage patterns of crosslinked DNA substrates in vitro suggested that the unhooked crosslink should be on the lagging strand. Nevertheless, in both models, the same pathways (nucleotide excision repair (NER), FA, translesion synthesis and homologous recombination (HR)) are used to repair the crosslink and restart the replication fork. BLM, Bloom syndrome protein; BRCA, breast cancer susceptibility; ?H2AX, a histone H2A variant; PALB2, partner and localizer of BRCA2; Modifiedwith permission from Ref. 29 ? (2005) Cold Spring Harbor Laboratory Press.

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

8. DNA damage response networks DNA repair is only a subset of the responses that cells utilize to respond to DNA damaging agents

9. Many of the DNA damage response networks have been uncovered based on human genetic disorders Note that for many of these disorders, the genes were originally cloned in humans and then subsequently studied in other species. As a result, the field is unique in that many of the antibodies and other reagents are specific to humans. Also, many of the disorders have multiple complementation groups that presage the subsequent unraveling of the underlying biochemical pathway.Note that for many of these disorders, the genes were originally cloned in humans and then subsequently studied in other species. As a result, the field is unique in that many of the antibodies and other reagents are specific to humans. Also, many of the disorders have multiple complementation groups that presage the subsequent unraveling of the underlying biochemical pathway.

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 We will look at two examples. First, we will consider pathways involved in the response to radiation damage focusing on the role of ATM. Second, we will consider how genetic changes in tumors that affect damage response genes might be used advantageously for therapy.We will look at two examples. First, we will consider pathways involved in the response to radiation damage focusing on the role of ATM. Second, we will consider how genetic changes in tumors that affect damage response genes might be used advantageously for 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 ofAtaxia-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

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. Radio resistant DNA synthesis assays DNA synthesis by a pulse-chase approach after a high dose of radiation. The curve for normal cells has two components, a steep initial drop at lower doses and then a gradual diminution from there. These are thought to correspond to inhibition of fork firing and elongation, respectively. Initially, it was thought that this phenotype was specific to A-T but it has now been found to generally reflect impairment of the S-phase progression checkpoint.Radio resistant DNA synthesis assays DNA synthesis by a pulse-chase approach after a high dose of radiation. The curve for normal cells has two components, a steep initial drop at lower doses and then a gradual diminution from there. These are thought to correspond to inhibition of fork firing and elongation, respectively. Initially, it was thought that this phenotype was specific to A-T but it has now been found to generally reflect impairment of the S-phase progression checkpoint.

22. Colony survival is a fundamental approach in toxicity testing. The basic problem is that cells do not die immediately from many insults. Therefore, simple counting does not accurately reflect the clonality of survival. In an irradiated culture you will have cells that survive with little pausing or impairment and these will continue to divide pretty much unabated. Other cells my pause and then continue. Some cells may pause and then ultimately die. Finally, some may die very quickly. Response depends on where cells are in the cell cycle, how much damage they have sustained, where the damage has occurred and how well it was repaired.Colony survival is a fundamental approach in toxicity testing. The basic problem is that cells do not die immediately from many insults. Therefore, simple counting does not accurately reflect the clonality of survival. In an irradiated culture you will have cells that survive with little pausing or impairment and these will continue to divide pretty much unabated. Other cells my pause and then continue. Some cells may pause and then ultimately die. Finally, some may die very quickly. Response depends on where cells are in the cell cycle, how much damage they have sustained, where the damage has occurred and how well it was repaired.

23. Radiation sensitivity varies substantially in the population. Disorder not directly related to DSB repair may be somewhat sensitive to radiation.Radiation sensitivity varies substantially in the population. Disorder not directly related to DSB repair may be somewhat sensitive to radiation.

24. ATM, the gene mutated in A-T, is a PIK related kinase PIK related kinases are large serine-threonine kinases involved in DNA damage sensing, signaling and repair. All are activated via phosphorylation on critical residues. This phosphorylation may be an autophosphorylation event or may be carried out by another PIK related kinase. ATM is activated by the presence of DSBs. This response is rapid, occurring within a minute or so, and saturates even at fairly low doses within 60 minutes. ATR is activated primarily by stalled replication forks. ATR activation occurs somewhat later after radiation damage and may reflect either fork stalling subsequent to radiation damage or cross-phosphorylation by ATM. DNA-PK we have seen before and noted that it can autophosphorylate or be phosphorylated by ATM. Finally, ATR can cross-phosphorylate ATM due to fork stalling but ATM can also be activated directly by DSBs occurring at collapsed forks.PIK related kinases are large serine-threonine kinases involved in DNA damage sensing, signaling and repair. All are activated via phosphorylation on critical residues. This phosphorylation may be an autophosphorylation event or may be carried out by another PIK related kinase. ATM is activated by the presence of DSBs. This response is rapid, occurring within a minute or so, and saturates even at fairly low doses within 60 minutes. ATR is activated primarily by stalled replication forks. ATR activation occurs somewhat later after radiation damage and may reflect either fork stalling subsequent to radiation damage or cross-phosphorylation by ATM. DNA-PK we have seen before and noted that it can autophosphorylate or be phosphorylated by ATM. Finally, ATR can cross-phosphorylate ATM due to fork stalling but ATM can also be activated directly by DSBs occurring at collapsed forks.

25. Atm transphosphorylates itself following DNA damage coincident with the release of active monomers Mechanism of ATM activation. Continued controversy as to whether autophosphorylation represents the recognition of damage or whether damage recognition results in phosphorylation. Mechanism of ATM activation. Continued controversy as to whether autophosphorylation represents the recognition of damage or whether damage recognition results in phosphorylation.

26. PIK related kinases are recruited to damage sites by a conserved mechanism In the case of each of the three PIK related kinases there appears to be a common mechanism of recruitment. An accessory molecule binds to a conserved structure on the kinase and recruits it to the site of damage. However, these mechanisms and the order of the steps involved are controversial and remain unresolved. In the case of each of the three PIK related kinases there appears to be a common mechanism of recruitment. An accessory molecule binds to a conserved structure on the kinase and recruits it to the site of damage. However, these mechanisms and the order of the steps involved are controversial and remain unresolved.

27. ATM regulates the mammalian cellular response to DNA DSBs Just a small number of the proteins phosphorylated by ATM. Note that many are proteins we have seen before and/or are involved in cancer or cancer predisposition.Just a small number of the proteins phosphorylated by ATM. Note that many are proteins we have seen before and/or are involved in cancer or cancer predisposition.

28. Even more protein substrates of ATM.Even more protein substrates of ATM.

29. More than 700 proteins are phosphorylated by ATM in response to radiation Probably the definitive treatment of ATM/ATR signaling.Probably the definitive treatment of ATM/ATR signaling.

30. Incidence of female breast cancer in blood relatives of A-T patients We briefly mentioned heterozygous risk in recessivel repair disorders previously. A-T is one of those disorders where simply the reduction in protein, or some dominant negative effect of the mutated allele is associated with an increased risk of cancer.We briefly mentioned heterozygous risk in recessivel repair disorders previously. A-T is one of those disorders where simply the reduction in protein, or some dominant negative effect of the mutated allele is associated with an increased risk of cancer.

31. ATM regulates the mammalian cellular response to DNA DSBs The mechanism of ATM�s effect on breast cancer risk is unresolved. It could be due to its regulation of known risk factors like BRCA1, BRCA2 and CHEK2.The mechanism of ATM�s effect on breast cancer risk is unresolved. It could be due to its regulation of known risk factors like BRCA1, BRCA2 and CHEK2.

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 Up until just a few years ago, there was insufficient information to try to generate new classes of chemotherapeutic agents by rational design.Up until just a few years ago, there was insufficient information to try to generate new classes of chemotherapeutic agents by rational design.

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 Now there are a bunch of inhibitors in various phase 1 and phase 2 trials. Recall PARP�s vague role in recognizing and responding to single strand breaks. MGMT triggers the direct reversal of alkylation damage. Thus, might synergize in patients with MMR defects.Now there are a bunch of inhibitors in various phase 1 and phase 2 trials. Recall PARP�s vague role in recognizing and responding to single strand breaks. MGMT triggers the direct reversal of alkylation damage. Thus, might synergize in patients with MMR defects.

35. BRCA1 and BRCA2The primary genetic risk factors for breast cancer BRCA1 and BRCA2 are the primary genes responsible for inherited breast cancer. As we have seen, both are involved DNA repair particularly by HR. Raises the interesting question of why repair mutants result in a limited spectrum of cancers. BRCA2 and, to a lesser extent, BRCA1, also increase risk for ovarian cancer. MMR defects have a similarly narrow spectrum of cancers.BRCA1 and BRCA2 are the primary genes responsible for inherited breast cancer. As we have seen, both are involved DNA repair particularly by HR. Raises the interesting question of why repair mutants result in a limited spectrum of cancers. BRCA2 and, to a lesser extent, BRCA1, also increase risk for ovarian cancer. MMR defects have a similarly narrow spectrum of cancers.

36. Estimated mutation frequencies in general breast cancer populations Some perspective is warranted. BRCA1 and BRCA2, while the major breast cancer genes, only account for a small percentage of total breast cancer, approximately 2-5%.Some perspective is warranted. BRCA1 and BRCA2, while the major breast cancer genes, only account for a small percentage of total breast cancer, approximately 2-5%.

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� BRCA1 and BRCA2 predispose to breast cancer by a classic tumor suppressor model. One allele carries a germline mutation. The other allele is lost somatically in tumor tissue. This may be by deletion, mutation or epigenetic silencing. Although BRCA1 or BRCA2 carriers are relatively rare, there is some evidence for �BRCAness� in tumors, i.e, there may be additional tumors that are defective in HR for other, unestablished, reasons.BRCA1 and BRCA2 predispose to breast cancer by a classic tumor suppressor model. One allele carries a germline mutation. The other allele is lost somatically in tumor tissue. This may be by deletion, mutation or epigenetic silencing. Although BRCA1 or BRCA2 carriers are relatively rare, there is some evidence for �BRCAness� in tumors, i.e, there may be additional tumors that are defective in HR for other, unestablished, reasons.

38. PARP inhibition and BRCA deficiency PARP inhibition and BRCA deficiency. A model for the selective effects of PARP inhibition on cells lacking wild-type BRCA1 or BRCA2. (a) PARP is important for the repair of DNA lesions, including DNA SSBs, by BER. When PARP activity is impaired, DNA SSBs persist. These SSBs encounter a DNA replication fork, arrest occurs resulting in fork collapse and/or a DSB. BRCA1 and BRCA2 in association with RAD51 are involved in the repair of such lesions by HR enabling restart of a collapsed replication fork. The excess number of replication fork arrests associated with loss of PARP function leads to an increase in sister chromatid recombination events and sister chromatid exchanges. (b) In the absence of functional BRCA1 or BRCA2, sister chromatid recombination and the formation of RAD51 foci are severely impaired. This leads to the utilization of error-prone RAD51-independent mechanisms such as non-homologous end joining (NHEJ) or single-strand annealing (SSA), and complex chromatid rearrangement results. Cells harboring these rearrangements may permanently arrest or undergo apoptosis. Confocal images of BRCA-deficient cells or wild-type BRCA cells are shown. RAD51 foci revealed by immunofluoresence are indicated in red. Nuclear staining with DAPI is indicated in blue.PARP inhibition and BRCA deficiency. A model for the selective effects of PARP inhibition on cells lacking wild-type BRCA1 or BRCA2. (a) PARP is important for the repair of DNA lesions, including DNA SSBs, by BER. When PARP activity is impaired, DNA SSBs persist. These SSBs encounter a DNA replication fork, arrest occurs resulting in fork collapse and/or a DSB. BRCA1 and BRCA2 in association with RAD51 are involved in the repair of such lesions by HR enabling restart of a collapsed replication fork. The excess number of replication fork arrests associated with loss of PARP function leads to an increase in sister chromatid recombination events and sister chromatid exchanges. (b) In the absence of functional BRCA1 or BRCA2, sister chromatid recombination and the formation of RAD51 foci are severely impaired. This leads to the utilization of error-prone RAD51-independent mechanisms such as non-homologous end joining (NHEJ) or single-strand annealing (SSA), and complex chromatid rearrangement results. Cells harboring these rearrangements may permanently arrest or undergo apoptosis. Confocal images of BRCA-deficient cells or wild-type BRCA cells are shown. RAD51 foci revealed by immunofluoresence are indicated in red. Nuclear staining with DAPI is indicated in blue.

39. Resistance to PARP inhibitorsReversion of BRCA2 mutations 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.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.

40. 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 Complementary study to the last one showing that chemotherapeutic drugs like cisplatin, whose use against breast cancer was largely for empiric reasons, act through a similar mechanism.Complementary study to the last one showing that chemotherapeutic drugs like cisplatin, whose use against breast cancer was largely for empiric reasons, act through a similar mechanism.

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 Although there is some concern that defective DNA repair may only be transiently necessary for tumor growth, the effectiveness of drugs like cisplatin or PARP inhibitors in BRCA deficient cells encourages the use of synthetic lethality to select new drug targets. The ability to choose from multiple drugs that may have limited side effects might allow reversion to be overcome.Although there is some concern that defective DNA repair may only be transiently necessary for tumor growth, the effectiveness of drugs like cisplatin or PARP inhibitors in BRCA deficient cells encourages the use of synthetic lethality to select new drug targets. The ability to choose from multiple drugs that may have limited side effects might allow reversion to be overcome.