**4. Therapeutic regimens designed to target BRCA2 defects**

Cells that are defective in BRCA1 and BRCA2 retain unresolved DSBs. This attribute, which is detrimental in terms of genomic instability and risk for cancer, is actually a potent target for inhibitors of Poly(ADP-ribose) polymerase, or PARP, in the eradication of transformed cells.

#### **4.1 Efficacy of PARP inhibitors in treating BRCA2-tumors**

PARPs are a family of 17 enzymes, with PARP-1 and -2 having been shown to be involved in DNA repair. PARP-1 is a nuclear protein with a zinc-finger DNA binding domain (Amir, Seruga et al.). It is responsible for binding to the sites of single-strand breaks, signaling damage at the site, and the initiating repair. The zinc finger domain binds to ssDNA breaks, cleaves NAD+, and attaches multiple ADP-ribose units to the protein. This results in an extremely negatively charged target which causes unwinding of the damaged DNA, followed by repair by the Base-Excision Repair (BER) pathway (Schreiber, Dantzer et al. 2006); (Ratnam and Low 2007)**.** However, PARP-1 has also been shown to serve as an antirecombinogenic factor at sites of damage where it has bound, thereby having implications on inhibiting HR-mediated repair (Amir, Seruga et al.), (Sandhu, Yap et al.). BRCA1 and -2 mutant cells are defective in repair of DSBs, and as a consequence, are sensitive to agents that induce DSBs. PARP-1 inhibitors have been shown to be effective in selectively targeting BRCA1 and -2 defective cells by converting SSBs, which have been induced by the use of chemotherapeutic agents, ionizing radiation, or occurring in normal cellular processes, such as stalled replication forks, to DSBs. The SSBs would have normally been identified and resolved by PARP-1 binding and the BER pathway; however, PARP-1 inhibitors prevent such resolution, and during DNA synthesis, the SSBs are converted to DSBs. The DSBs are normally resolved by HR-mediated repair involving BRCA1, and most important BRCA2, with the recombinase RAD51. However, this is deficient in BRCA-mutant cells and the addition of PARP inhibition enhances DNA-damage induced cell cycle arrest and apoptosis. This process eradicates the tumor cells.

#### **4.2 Development of PARP inhibitors**

The first PARP-1 inhibitor created was 3-aminobenzamide (3-AB). It causes inhibition of PARP-1 by competing with NAD+ as a substrate. However, 3-AB showed poor specificity

*BRCA2* Mutations and Consequences for DNA Repair 337

of concern. First and foremost, PARP inhibitors are still in the early stages of clinical testing. Therefore, the optimal dosage and duration of treatment have not been definitively determined. And, although PARP inhibitors are effective against BRCA-tumors, there is the potential for possible toxicity in normal tissues. In the Olaparib phase I study, DSB accumulations were observed in normal tissues (eyebrow hair follicles), (Drew and Plummer). In addition to toxicity, the inhibitors may disrupt DNA repair pathways in normal tissue from DNA damage acquired through sun exposure or other environmental agents (Ratnam and Low 2007). And, the potential for secondary cancers to occur through genomic instability from inhibition of PARP-1 is possible. In an in vivo study of PARP-1 deficiency, female mice developed mammary carcinoma (Tong, Yang et al. 2007); (Drew and Plummer). Furthermore, secondary mutations after PARP inhibitor treatment may lead to drug resistance. Previous reports have observed intragenic secondary mutations/deletions of BRCA2 occurring after treatment with PARP-1, and the anti-cancer agent cisplatin, which restored the open-reading frame and led to the expression of new BRCA2 isoforms. This resulted in reversal of the original BRCA2 mutation and resistance to PARP inhibitors

(Edwards, Brough et al. 2008), (Sakai, Swisher et al. 2008), (Drew and Plummer).

Overall, the use of PARP inhibitors appears to be very promising in the treatment of BRCAtumors as a single agent, and as a chemotherapeutic/radiation sensitizer when used in combination with anticancer therapeutics or γ-radiation. The on-going clinical trials will provide more information about the aspects of PARP inhibitor usage that are presently vague, such as proper dosage and duration of treatment, possible effects on DNA repair mechanisms in normal cells, possible induction of secondary mutations, and acquired

Abeliovich, D., L. Kaduri, et al. (1997). "The founder mutations 185delAG and 5382insC in

Benson, F. E., A. Stasiak, et al. (1994). "Purification and characterization of the human Rad51

Bignell, G., G. Micklem, et al. (1997). "The BRC repeats are conserved in mammalian BRCA2

Buisson, R., A. M. Dion-Cote, et al. "Cooperation of breast cancer proteins PALB2 and

Carreira, A., J. Hilario, et al. (2009). "The BRC repeats of BRCA2 modulate the DNA-binding

Chalmers, A., P. Johnston, et al. (2004). "PARP-1, PARP-2, and the cellular response to low doses of ionizing radiation." *Int J Radiat Oncol Biol Phys* 58(2): 410-9. Chen, C. F., P. L. Chen, et al. (1999). "Expression of BRC repeats in breast cancer cells

loss of G(2)/M checkpoint control." *J Biol Chem* 274(46): 32931-5.

piccolo BRCA2 in stimulating homologous recombination." *Nat Struct Mol Biol*

disrupts the BRCA2-Rad51 complex and leads to radiation hypersensitivity and

protein, an analogue of E. coli RecA." *Embo J* 13(23): 5764-71.

BRCA1 and 6174delT in BRCA2 appear in 60% of ovarian cancer and 30% of earlyonset breast cancer patients among Ashkenazi women." *Am J Hum Genet* 60(3): 505-14. Amir, E., B. Seruga, et al. "Targeting DNA repair in breast cancer: a clinical and translational

**5. Conclusion** 

**6. References** 

resistance of tumors over the course of treatment.

update." *Cancer Treat Rev* 36(7): 557-65.

proteins." *Hum Mol Genet* 6(1): 53-8.

selectivity of RAD51." *Cell* 136(6): 1032-43.

17(10): 1247-54.

and inhibited de-novo purine synthesis (Purnell, Stone et al. 1980); (Drew and Plummer). Approximately, twenty years have passed since the synthesis of 3-AB, and the focus has been to create PARP-1 inhibitors with greater specificity for PARP-1 inhibition, only. In 2003, the PARP-1 inhibitor AG014699 was the first to enter clinical trials (Plummer and Calvert 2007), (Drew and Plummer). Xenograft studies showed significant delay of tumor growth when AG014699 was combined with irinotecan and irradiation and tumor regression when combined with temozolomide (Ratnam and Low 2007). There are presently at least eight PARP inhibitors in clinical trials (Drew and Plummer), (Amir, Seruga et al.), (Table 1). PARP inhibitors are effective at sensitizing tumor cells to other chemotherapeutic agents, and can be used as a combination therapy with platinums, temozolomide, topopisomerase I inhibitors, and γ-/X-radiation (Ratnam and Low 2007), (Curtin, Wang et al. 2004), (Miknyoczki, Jones-Bolin et al. 2003), (Nguewa, Fuertes et al. 2006), (Chalmers, Johnston et al. 2004), (Fernet, Ponette et al. 2000), (Veuger, Curtin et al. 2003). Due to PARP inhibitors effectively promoting cell cycle arrest and subsequent apoptosis, clinical trials are testing their efficacy as single-agents in the treatment of BRCA1- and BRCA2-tumors (Ratnam and Low 2007).


Table 1. PARP inhibitors presently in clinical trials

#### **4.3 Clinical implications of PARP inhibitor use**

In general, there is very high enthusiasm for the use of PARP inhibitors in the treatment of *BRCA2*-cancers. The requirement for specificity is met because the *BRCA1/2*-mutated cells are most sensitive to the inhibitors, due to their DNA repair defects, and the premise of "synthetic lethality", which is when two pathway defects alone are innocuous, but combined become lethal (Ratnam and Low 2007). The combination of impaired HRmediated repair due to the *BRCA1/2*-mutation and the inhibition of PARP-1 to signal the DNA breaks provides the "synthetic lethality" that is necessary for the efficacy of PARP inhibitors in the treatment of *BRCA*-tumors. Furthermore, the therapeutic benefit of PARP inhibitors appears to greatly outweigh the undesirable side effects; however, there are areas of concern. First and foremost, PARP inhibitors are still in the early stages of clinical testing. Therefore, the optimal dosage and duration of treatment have not been definitively determined. And, although PARP inhibitors are effective against BRCA-tumors, there is the potential for possible toxicity in normal tissues. In the Olaparib phase I study, DSB accumulations were observed in normal tissues (eyebrow hair follicles), (Drew and Plummer). In addition to toxicity, the inhibitors may disrupt DNA repair pathways in normal tissue from DNA damage acquired through sun exposure or other environmental agents (Ratnam and Low 2007). And, the potential for secondary cancers to occur through genomic instability from inhibition of PARP-1 is possible. In an in vivo study of PARP-1 deficiency, female mice developed mammary carcinoma (Tong, Yang et al. 2007); (Drew and Plummer). Furthermore, secondary mutations after PARP inhibitor treatment may lead to drug resistance. Previous reports have observed intragenic secondary mutations/deletions of BRCA2 occurring after treatment with PARP-1, and the anti-cancer agent cisplatin, which restored the open-reading frame and led to the expression of new BRCA2 isoforms. This resulted in reversal of the original BRCA2 mutation and resistance to PARP inhibitors (Edwards, Brough et al. 2008), (Sakai, Swisher et al. 2008), (Drew and Plummer).

#### **5. Conclusion**

336 DNA Repair

and inhibited de-novo purine synthesis (Purnell, Stone et al. 1980); (Drew and Plummer). Approximately, twenty years have passed since the synthesis of 3-AB, and the focus has been to create PARP-1 inhibitors with greater specificity for PARP-1 inhibition, only. In 2003, the PARP-1 inhibitor AG014699 was the first to enter clinical trials (Plummer and Calvert 2007), (Drew and Plummer). Xenograft studies showed significant delay of tumor growth when AG014699 was combined with irinotecan and irradiation and tumor regression when combined with temozolomide (Ratnam and Low 2007). There are presently at least eight PARP inhibitors in clinical trials (Drew and Plummer), (Amir, Seruga et al.), (Table 1). PARP inhibitors are effective at sensitizing tumor cells to other chemotherapeutic agents, and can be used as a combination therapy with platinums, temozolomide, topopisomerase I inhibitors, and γ-/X-radiation (Ratnam and Low 2007), (Curtin, Wang et al. 2004), (Miknyoczki, Jones-Bolin et al. 2003), (Nguewa, Fuertes et al. 2006), (Chalmers, Johnston et al. 2004), (Fernet, Ponette et al. 2000), (Veuger, Curtin et al. 2003). Due to PARP inhibitors effectively promoting cell cycle arrest and subsequent apoptosis, clinical trials are testing their efficacy as single-agents in the treatment of BRCA1- and BRCA2-tumors

**Agent Single/combination therapy Disease** 

BRCA-related tumors Solid tumors

Triple negative breast cancer Advanced solid tumors

> Solid tumors Melanoma

lymphoid malignancies

Melanoma Glioblastoma mutiforme

BRCA ovarian

Combination trials

Single agent Combination trials (gemcitabine/carboplatin)

Single agent Combination trials (temozolomide [TMZ]

Combination with TMZ

MK4827 Single agent Solid tumors

GPI21016 Combination with TMZ Solid tumors CEP-9722 Combination with TMZ Solid tumors

In general, there is very high enthusiasm for the use of PARP inhibitors in the treatment of *BRCA2*-cancers. The requirement for specificity is met because the *BRCA1/2*-mutated cells are most sensitive to the inhibitors, due to their DNA repair defects, and the premise of "synthetic lethality", which is when two pathway defects alone are innocuous, but combined become lethal (Ratnam and Low 2007). The combination of impaired HRmediated repair due to the *BRCA1/2*-mutation and the inhibition of PARP-1 to signal the DNA breaks provides the "synthetic lethality" that is necessary for the efficacy of PARP inhibitors in the treatment of *BRCA*-tumors. Furthermore, the therapeutic benefit of PARP inhibitors appears to greatly outweigh the undesirable side effects; however, there are areas

ABT-888 Single agent Solid tumors and

(Ratnam and Low 2007).

BSI-201

AG014699

Olaparib (AZD2281) Single agent

INO-1001 Single agent

Table 1. PARP inhibitors presently in clinical trials

**4.3 Clinical implications of PARP inhibitor use** 

Overall, the use of PARP inhibitors appears to be very promising in the treatment of BRCAtumors as a single agent, and as a chemotherapeutic/radiation sensitizer when used in combination with anticancer therapeutics or γ-radiation. The on-going clinical trials will provide more information about the aspects of PARP inhibitor usage that are presently vague, such as proper dosage and duration of treatment, possible effects on DNA repair mechanisms in normal cells, possible induction of secondary mutations, and acquired resistance of tumors over the course of treatment.

#### **6. References**


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**18** 

*U.S.A.* 

Xinrong Chen and Tao Chen

**Roles of MicroRNA in DNA Damage and Repair** 

DNA damage mainly results from either endogenous metabolic activity, such as oxidative stress, or environmental exposure, such as ionizing irradiation. In human cells, endogenous and exogenous genotoxic agents produce as many as 1 million molecular lesions per cell per day. If the unrepaired lesions occur in certain critical genes, they can cause mutations that

There are several different types of DNA damage, including DNA hydrolysis, DNA adduction, DNA crosslinking, and DNA strand breakage. DNA hydrolysis is the breaking of DNA through the addition of water. Hydrolysis of DNA bases consists of deamination, depurination, and depyrimidination. A DNA adduct is a piece of DNA covalently bonded to a chemical. DNA crosslinks are links formed within a single (intrastrand) or between strands of DNA (interstrand). There are two types of DNA strand breaks, single strand breaks and double strand breaks. DNA double strand breaks are particularly hazardous to

Cells respond to DNA damage through a variety of different mechanisms, such as apoptosis, senescence, and DNA repair. Excessive DNA damage induces apoptosis, or programmed cell death, that eliminates cells with heavily damaged DNA, thus protecting the organism from the mutations potentially induced by the damage. Unrepaired DNA damage is a driving force for senescence. Senescence serves as a functional alternative to apoptosis in cases where the physical presence of cells is required for spatial reasons. If DNA replication occurs before DNA damage is repaired, mutations can be formed in the cells. To prevent mutation formation, cells have developed DNA repair mechanisms to

There are several different types of DNA repair. They are direct reversal, base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end-joining (NHEJ), and homologous recombination repair (HRR). Direct reversal can remove DNA damage by chemically reversing it. Since the correction only occurs in one of the four bases and not the phosphodiester backbone, this type of repair does not need any DNA template. For example, methylation of guanine bases can be directly reversed by methyl guanine methyl transferase (MGMT) that removes the methyl group. BER amends damage to single nucleotides produced by oxidation, alkylation, or hydrolysis. NER corrects ethylation products, bulky DNA adducts, helix-distorting changes, such as thymine dimers, and single-strand breaks. MMR repairs mismatched bases in double-stranded DNA (e.g., A:C or G:T). HRR is a mechanism for DNA double-strand repair that reconstitutes the

the cells because they can lead to genome rearrangements. (Rich et al., 2000).

**1. Introduction** 

correct DNA.

can lead to tumors (Lodish H, 2004).

*National Center for Toxicological Research/US Food and Drug Administration* 

