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Inhibition of Poly(ADP)-Ribose Polymerase as a Therapeutic Strategy for Breast Cancer

Inhibition of Poly(ADP)-Ribose Polymerase as a Therapeutic Strategy for Breast Cancer

ABSTRACT: As knowledge increases about the processes underlying cancer, it is becoming feasible to design “targeted therapies” directed toward specific pathways that are critical to the genesis or maintenance of the malignant phenotype. Poly(ADP-ribose) polymerase (PARP) inhibitors are an example of this new framework. DNA damage repair is a complex and multifaceted process that is critical to cell survival. Members of the PARP family are central to specific DNA damage repair pathways, particularly the base excision repair (BER) pathway. PARP inhibition, with subsequent impairment of the BER mechanism, may enhance the cytotoxicity of agents that generate single-strand breaks in DNA, such as radiation and certain chemotherapy drugs. In addition, PARP inhibitors may induce death through “synthetic lethality” if the DNA repair mechanisms that rescue BER-deficient cells are themselves impaired. This mechanism is thought to underlie the impressive results of PARP inhibition in BRCA-associated breast and ovarian cancer, and may also account for the reported benefit of this approach in “triple-negative” breast cancer. This review will examine the current understanding of PARP inhibition as a treatment for breast cancer, ongoing clinical trials, and future directions for this new approach.

Poly-(ADP-ribose) polymerases (PARPs) are a family of enzymes that catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to acceptor proteins.[1,2] This transfer results in the creation of long, negatively charged, branched polymers on the acceptor proteins. The first PARP (PARP1) was described in 1963.[3] The family has since been expanded to include at least 17 members.[2] PARP1 and PARP2 are the most abundant members of the family, and appear to be the only ones localized to the nucleus. PARP1, the major nuclear PARP, is a 116-kDA protein with two N-terminal zinc-finger DNA-binding domains, a nuclear localization signal domain, and a BRCT-repeat automodification domain.[4] At the C-terminus, a catalytic domain containing the “PARP signature” identifies it as a member of the PARP superfamily.

Role of PARPs

PARPs play an important role in a number of cellular damage response pathways.[5] PARPs are involved in inflammation and, through NAD+-depletion, in triggering cell death in response to stresses such as ischemia. However, the function of PARPs that is most relevant to oncology is their role in DNA damage repair. PARP1 and PARP2 are the family members that participate in this process. Most is known about the action of PARP1. PARP2 appears to participate in the same processes, but it likely also has its own unique functions.[6]

Maintenance of genomic integrity is a critical cellular function. A number of exogenous and endogenous insults can result in DNA damage. To address these various forms of potentially lethal injury, cells have evolved a number of different repair pathways.[7] PARP1 is a major component of the base excision repair (BER) pathway, which is directed toward the repair of certain types of damage to the component bases of DNA and single-strand breaks in the DNA structure.[8,9] PARP1 senses and binds to “nicked” DNA through its N-terminal zinc-finger DNA-binding domains, after which it forms a homodimer. After binding, the catalytic activity is engaged and PARP1 automodifies by adding ADP-ribose moieties to the BRCT repeat domain. The extended negatively charged chain appears both to protect the break from further degradation (“anti-recombinogenic”) and to serve as a “beacon” for recruitment of BER effector proteins, such as XRCC1.

PARP1 also appears to modify local histone proteins (especially H1 and H2B) by poly(ADP)-ribosylation. The consequent negative charge may contribute to modification of local chromatin structure, resulting in improved access for repair proteins.[10] Chromatin modification may also be mediated through the action of other proteins, such as ALC1.[11]

PARP1 is not essential for survival. PARP1 knockout mice are viable and fertile.[12] However, these animals are sensitive to DNA-damaging agents such as N-methyl-N-nitrosourea (MNU) and ionizing radiation. Residual PARP2 activity may be compensating for the loss of PARP1, as PARP1/PARP2 double knockout mice are not viable.[13] Other DNA damage repair pathways are also likely to be involved in the rescue of cells with impaired base excision repair.

Single-strand breaks may result in unattached double-strand ends if they are unrepaired when encountered by a replication fork. To avoid this result, the cell engages repair mechanisms that are involved in the repair of double-strand DNA breaks, such as homologous recombination repair and nonhomologous end-joining.[14] Homologous recombination repair is a high-fidelity mechanism that is activated during later S phase, when a sister chromatid is available to serve as a template for resolving the double-strand break. Nonhomologous end-joining is less accurate, and therefore more likely to result in genomic instability, but it is available throughout the cell cycle. Bone marrow cells from PARP1 knockout mice demonstrate an increased prevalence of sister chromatid exchanges under both baseline and stress conditions, indicating that homologous recombination repair mechanisms are engaged in response to endogenous and exogenous DNA damage when PARP1 is absent.[12] The availability of this repair mechanism is thought to be responsible for the continued viability of cells in which PARP1 function is lost.

Synthetic Lethality

FIGURE 1
Figure 1
Schematic Illustration of Synthetic Lethality

As above, double-strand break repair pathways, especially homologous recombination repair, appear to be critical to rescuing cells that are lacking PARP. Therefore, one would predict that cells with defects in homologous recombination repair would be sensitive to loss of single-strand repair capability through inhibition of PARP activity.[15] Conversely, cells that are competent in repairing double-strand breaks would, like PARP1 knockout mice, experience little effect from loss of PARP function, at least under normal conditions.

Synthetic lethality is the phenomenon whereby loss of products from two different genes results in cell death, which does not occur if either gene product is present and functional (Figure 1). The products of BRCA1 and BRCA2 are known to be critical to double-strand DNA break repair mediated by homologous recombination.[16] If PARP activity is lost in BRCA-deficient cells, such as BRCA-associated breast and ovarian cancers, one would therefore expect that exogenous or endogenous DNA damage that is normally addressed by the base excision repair pathway would result in genomic instability and cell death, since the rescue pathway is not available in such cells. Nonmalignant cells from BRCA mutation carriers should be relatively unaffected, however, because these cells still have functional BRCA gene product and are competent in double-strand break repair.

In 2005, two groups clearly demonstrated that inhibition of PARP causes synthetic lethality in BRCA-deficient cells.[17,18] Farmer et al demonstrated that the PARP inhibitors NU1025 and AG14361 were highly cytotoxic in BRCA2-deficient VC-8 cells. In addition, small interfering RNA (siRNA) “knockdown” of BRCA2 in MCF-7 and MDA-MB-231 cells resulted in sensitivity to PARP inhibition.[18] Similarly, Bryant et al showed that BRCA-deficient embryonic stem (ES) cells are sensitive to PARP inhibition, whereas heterozygous or wild-type cells are not affected.[17] Subsequent studies in mice demonstrated that PARP inhibition had a significant impact on orthotopic BRCA null breast cancers transplanted from conditional mutant mice.[19-21]

Taken together, these studies provided the preclinical rationale for studying PARP inhibitors as single agents in patients with BRCA-associated breast and ovarian cancer (see below). The concept of treating cancer by causing synthetic lethality is profoundly novel, and is distinct from the more traditional approaches of chemo- or radiosensitization (ie, accentuation or inhibition of repair of a specific type of damage) or combination therapy (ie, which employs agents that have different mechanisms to cause cellular damage through more than one pathway).

In theory, synthetic lethality through PARP inhibition may be applicable beyond the special circumstance of BRCA-associated cancer. Cells with deficiencies in double-strand DNA break repair resulting from other genetic alterations should also be sensitive to this approach. For example, Lord and colleagues performed a siRNA screen to identify other genes that, when inhibited, conferred sensitivity to PARP inhibition.[22] The same group has described sensitivity to PARP inhibition in cells with null PTEN mutations.[23] These early studies suggest that the synthetic lethality mechanism may be applicable to a wide range of malignancies beyond those arising in BRCA mutation carriers.

‘BRCA-ness’ and Triple-Negative Breast Cancer

The preclinical studies described above suggested that PARP inhibitors would be effective as single agents in the treatment of BRCA-deficient cancers. Classically, such cancers are associated with germline BRCA mutations. Somatic mutations in BRCA1 or BRCA2 appear to be rare in nonhereditary breast or ovarian cancer, although loss of heterozygosity of the genomic regions encompassing these genes is not uncommon. A number of studies have demonstrated reduced expression of BRCA1 protein, however, in certain nonhereditary breast cancers, especially poorly differentiated disease.[24,25] Reduced expression appears to result from either somatic BRCA1 promoter hypermethylation or downregulation consequent to overexpression of regulatory proteins such as Id4.[26-29]

Sporadic breast cancers with reduced BRCA1 expression are usually poorly differentiated tumors that do not express estrogen or progesterone receptors and do not overexpress HER2 (“triple-negative” breast cancer, or TNBC). In addition, many cases of TNBC (but not all) demonstrate a “basal-like” gene-expression pattern, as do most breast cancers arising in BRCA1-mutation carriers. These commonalities have led some to suggest that a subset of TNBC may have a BRCA1 pathway defect that is severe enough to compromise homologous recombination repair.[30] If this is the case, then a subset of TNBC may be sensitive to synthetic lethality induced by PARP inhibition.

A number of caveats should be mentioned with regard to this hypothesis. There is incomplete phenotypic overlap between TNBC, “basal-like” breast cancer defined by gene-expression pattern, and the cancers that arise in BRCA1 mutation carriers.[31] Not all TNBC or “basal-like” cancers are necessarily associated with reduced BRCA1 expression. Even in those that are, it is unclear to what degree homologous recombination repair is compromised. It is therefore premature to assume that PARP inhibition alone will be broadly effective in TNBC by the same synthetic lethality mechanism as in BRCA-associated disease. However, this does not preclude the possibility that the agents may enhance the toxicity of existing chemotherapy agents by increasing the number of double-strand breaks that must be repaired.

PARP Inhibition in Combination Therapy

As a therapeutic strategy, PARP inhibition may have merit even if it does not lead to synthetic lethality by leveraging a defect in homologous recombination repair. Numerous chemotherapy agents (as well as ionizing radiation) induce DNA damage that is usually repaired by the base excision repair pathway, and inhibition of that pathway would be expected to result in increased cytotoxicity. As described above, PARP1 knockout mice were sensitive to treatment with MNU or ionizing radiation, even though they were phenotypically normal and had a normal lifespan in the absence of that stress.[12] This suggests that the ability of the double-strand break repair system to compensate for loss of PARP function is not infinite, and that combinations that increase the stress on that system may be more effective. PARP1 and PARP2 may also have as yet undefined roles in DNA damage repair that, if inhibited, could increase the cytotoxicity of various agents. All of these considerations justify studying the newer generation of potent PARP inhibitors in combinations with conventional cytotoxic agents.

Reference Guide

Therapeutic Agents
Mentioned in This Article
ABT-888
AGO14699
BSI-201
Carboplatin
Gemcitabine (Gemzar)
INO-1001
MK4827
Nicotinamide
Olaparib (AZD2281, KU0059436)
Paclitaxel
Temozolomide (Temodar)
Topotecan
Brand names are listed in parentheses only if a drug is not available generically and is marketed as no more than two trademarked or registered products. More familiar alternative generic designations may also be included parenthetically.

At the present time, the most actively investigated partners in combination with PARP inhibitors are platinum-based agents. The major form of DNA damage induced by these drugs is bulky adducts that are repaired by the nucleotide excision repair pathway.[32] However, they also cause double-strand cross-links that are repaired by double-strand break repair pathways. Cells that are defective in double-strand break repair, such as those lacking BRCA1 or BRCA2, are extremely sensitive to platinum-based agents.[33] Preclinical studies of combination therapy with PARP inhibitors and platinum agents in orthotopic transplants of breast cancers from conditional BRCA mutant mice have shown enhanced efficacy (and toxicity).[19,34] In this situation, the lack of homologous recombination repair capability leads to cell death after exposure to the aggregate insult of double-strand breaks resulting from the inability to repair platinum-induced cross-links and similar breaks resulting from replication fork collapse at the site of single-strand breaks that are not repaired due to PARP inhibition.

If there is a homologous repair defect in TNBC, one would expect specific platinum sensitivity in that setting as well. Clinical studies examining this hypothesis are not conclusive. As noted above, the effectiveness of combination platinum/PARP inhibitor therapy may not require a homologous recombination repair defect. Combining a platinum agent with a PARP inhibitor might increase effectiveness by increasing double-strand breaks to the point where even a competent repair system is overwhelmed. By extension, PARP inhibitors may enhance the effectiveness of nonplatinum agents, and combinations may be effective in settings other than those in which a homologous recombination repair defect is predicted to exist.

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