Members of the poly (ADP-ribose) polymerase (PARP) superfamily of enzymes catalyze the post-translational modification of proteins using β‑NAD⁺ as a substrate to successively add ADP-ribose moieties onto target proteins: a process termed PARsylation. In the 1960s, this posttranslational modification was first characterized with the identification of PARP1 and its role in DNA repair. Subsequently, an additional 16 members of the PARP superfamily were identified, each of which possesses a structurally similar PARP catalytic domain (Figure 1). Furthermore, in addition to its well-studied role in DNA repair, PARsylation has now been shown to modulate processes as diverse as cellular proliferation, apoptosis, DNA methylation, transcriptional regulation and WNT signaling.

Figure 1. The members of PARP family. ANK, ankyrin; mART, mono (ADP-ribose) transferase domain; SAM, sterile alpha module domain; REG, regulatory domain; VIT, vault protein inter-alpha-trypsin domain; VWA, Von Willebrand factor type A domain.

The overview of PARP family

Based on new proposed nomenclature by Hottiger et al., the human PARP (hPARP) family is classified into three groups depending on their motifs and functions: (1) PARP 1-5: have a conserved glutamate residue (Glu988); (2) PARP 6-8, 10-12 and 14-16: are putative mono-(ADP-ribose) polymerases and (3) PARP 9 and 13 which do not have PARP signature motif that binds NAD⁺ nor do they have Glu988 implying that they are inactive.

Out of 17 members, PARP-1 (113 kDa) was the first characterized and extensively studied enzyme recognized to play an essential role in DNA repair. PARP-1 and PARP-2 share ~69% homology in the catalytic domain and they are documented as important proteins in DNA repair system, while PARP-3 is reported to be a mono-ADP- ribosylating enzyme by Loseva and group. PARP-4, also known as Vault PARP, is a ribonucleoprotein complex having PARylation activity and it is thought to be involved in multidrug resistance of tumor and intracellular transport. Tankyrase-1 (TRF-1- interacting ankyrin-related ADP-ribose polymerase-1), also known as PARP5a, is identified to enhance telomere elongation by telomerase. Other PARP homologues show structural and functional differences. Tankyrase-2 lacks N-terminal HPS (His-Pro-Ser) domain, but it may share some overlapping functions with tankyrase-1. Other PARP family members like tiPARP, PARP-12 and PARP-13 share PARP catalytic, WWE and CX8CX5CX3-like zinc finger domains. PARP-13 has been reported to be an important regulator of cellular mRNA via regulation of miRNA activity. The next subgroup which includes PARP-9/BAL1, PARP-14/BAL2/CoaSt6 and PARP-15/BAL3 are macro-PARPs, characterized by macro domains positioned before the PARP domain. This domain is found to be involved in transcriptional repression and X-chromosome inactivation, suggesting it as a transcription factor. The RNA recognition motif (RRM) and the Gly-rich domain of PARP-10 are known to help in binding of RNA with proto-oncoprotein c-Myc. Other PARP family members such as PARP-6, PARP-8, PARP-11 and PARP-16 have been identified but their functions are still elusive, though PARP-8 and 16 have been recently shown to be involved in assembly or maintenance of membranous organelles.

PARP and cancer

DNA is continually damaged by environmental exposures and endogenous activities (such as DNA replication errors), which cause diverse lesions, including single-strand breaks (SSBs) and double-strand breaks (DSBs). PARP1, a widely and abundantly expressed member of the PARP family, facilitates DNA repair by binding to SSBs and recruiting DNA repair proteins to the site of damage. Nevertheless, genetic ablation of PARP1 expression in mouse models did not increase the development or early onset of tumors. Studies revealed that, in the absence of PARP1, spontaneous SSBs collapse replication forks and result in DSBs, thus triggering DNA repair by homologous recombination pathways; this compensatory repair mechanism probably explains the normal susceptibility of Parp1-deficient mice to cancer.

The use of an inhibitor of a DNA-repair enzyme to selectively kill tumor cells with deficient homologous recombination in the absence of an exogenous DNA-damaging agent represented a new concept in cancer therapy. This concept is an example of synthetic lethality, a phenomenon that arises when combined mutation or blockade of two or more genes leads to cell death, whereas a mutation (or blockade) of only one of these genes does not affect viability. The synthetic lethal treatment approach is built around a mutation that does not induce cell death (for example, in breast cancer type 1 susceptibility protein (BRCA) genes), although the mutation might confer a phenotype (such as defective homologous recombination); however, the underlying mutation provides the opportunity to therapeutically target additional pathways (PARP proteins) to achieve lethality (Figure 2).

Figure 2. The role of PARP inhibitors in synthetic lethality.

PARP-1 inhibition does not cause cell lethality by itself, as the cell has an intact HR pathway for DNA repair. Cells that have a mutated BRCA1 or BRCA2 genes as in the case of breast cancer or those that are deficient in BRCA1 or BRCA2 proteins like sporadic cancers are found to be defective in their ability to repair DNA through HR and henceforth depend on error-prone NHEJ. This results in amplification of DNA instability and chromosomal aberrations eventually causing cell death (Figure 3a). This concept of synthetic lethality has been implemented upon in cancer therapeutics. In cases of ovarian and breast cancer, treatment with PARP-1 inhibitors Olaparib and Veliparib (Approach A) has found positive clinical results. Epigenetic modulation or artificial inactivation of BRCA pathway (Approach 2a) in cases of sporadic cancer along with the use of PARPi plays a key to therapeutics. This synergistic inhibition of DNA repair poses as a double-hit mechanism for cancer cell death. PARPi can also be used in combination with chemotherapy and radiation (Approach 2b) to render the cells prone to cell death under enhanced damaged conditions as in cases of non-Hodgkin lymphoma cell line, use of PARPi in combination with both external beam radiation and I-tositumomab; radio sensitization with veliparib in head and neck carcinoma cell lines and lung cancer xenograft models; or with niraparib in neuroblastoma cell lines, and whole brain radiation in cases of brain metastases (Figure 3b).

Figure 3. PARP-1 and cancer therapy.

Concluding remarks

Inhibiting PARP activity uncovers the potential of PARP inhibitors as promising candidates for cancer therapy, particularly in BRCA1/2-mutated cancers, alone or in combination with cytotoxic drugs. P53-deficient breast cancer cells treated with a PARP inhibitor happen to lose resistance to an apoptosis-promoting, clinically active anti-tumor agent called doxorubicin. However, these PARP inhibitors have several side effects that are toxic to the cell as the reports clearly show PARP-1’s role in physiological conditions. Hence, to harness the therapeutic potential of PARP-1, studies are required to find out new inhibitors with least side effects. Therefore, PARP-1 has now opened new avenues for researchers to understand PARP-1’s multifunctional role in the cell which would eventually aid to further expand the utility of PARP family and its inhibition in therapeutics.