Junko Murai

Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors

Small-molecule inhibitors of PARP are thought to mediate their antitumor effects as catalytic inhibitors that block repair of DNA single-strand breaks (SSB). However, the mechanism of action of PARP inhibitors with regard to their effects in cancer cells is not fully understood. In this study, we show that PARP inhibitors trap the PARP1 and PARP2 enzymes at damaged DNA. Trapped PARP-DNA complexes were more cytotoxic than unrepaired SSBs caused by PARP inactivation, arguing that PARP inhibitors act in part as poisons that trap PARP enzyme on DNA. Moreover, the potency in trapping PARP differed markedly among inhibitors with niraparib (MK-4827) > olaparib (AZD-2281) >> veliparib (ABT-888), a pattern not correlated with the catalytic inhibitory properties for each drug. We also analyzed repair pathways for PARP-DNA complexes using 30 genetically altered avian DT40 cell lines with preestablished deletions in specific DNA repair genes. This analysis revealed that, in addition to homologous recombination, postreplication repair, the Fanconi anemia pathway, polymerase β, and FEN1 are critical for repairing trapped PARP-DNA complexes. In summary, our study provides a new mechanistic foundation for the rational application of PARP inhibitors in cancer therapy.

Stereospecific PARP trapping by BMN 673 and comparison with olaparib and rucaparib.

Anti-PARP drugs were initially developed as catalytic inhibitors to block the repair of DNA single-strand breaks. We recently reported that several PARP inhibitors have an additional cytotoxic mechanism by trapping PARP–DNA complexes, and that both olaparib and niraparib act as PARP poisons at pharmacologic concentrations. Therefore, we have proposed that PARP inhibitors should be evaluated based both on catalytic PARP inhibition and PARP–DNA trapping. Here, we evaluated the novel PARP inhibitor, BMN 673, and compared its effects on PARP1 and PARP2 with two other clinical PARP inhibitors, olaparib and rucaparib, using biochemical and cellular assays in genetically modified chicken DT40 and human cancer cell lines. Although BMN 673, olaparib, and rucaparib are comparable at inhibiting PARP catalytic activity, BMN 673 is ∼100-fold more potent at trapping PARP–DNA complexes and more cytotoxic as single agent than olaparib, whereas olaparib and rucaparib show similar potencies in trapping PARP–DNA complexes. The high level of resistance of PARP1/2 knockout cells to BMN 673 demonstrates the selectivity of BMN 673 for PARP1/2. Moreover, we show that BMN 673 acts by stereospecific binding to PARP1 as its enantiomer, LT674, is several orders of magnitude less efficient. BMN 673 is also approximately 100-fold more cytotoxic than olaparib and rucaparib in combination with the DNA alkylating agents methyl methane sulfonate (MMS) and temozolomide. Our study demonstrates that BMN 673 is the most potent clinical PARP inhibitor tested to date with the highest efficiency at trapping PARP–DNA complexes. Mol Cancer Ther; 13(2); 433–43. ©2013 AACR.

Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Repairs DNA Damage Induced by Topoisomerases I and II and Base Alkylation in Vertebrate Cells

Background: Tdp1 is a DNA repair enzyme conserved across eukaryotes. Results: Tdp1 repairs not only 3′-tyrosyl-DNA bonds and 3′-phosphoglycolates but also 5′-tyrosyl-DNA bonds and 3′-deoxyribose phosphates. Conclusion: The end processing functions of Tdp1 extend to the repair of Top2-DNA adducts and DNA breaks from base alkylation. Significance: Tdp1 has a broad range of DNA repair activities and is a potential drug target in anticancer therapy. Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3′-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3′-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1−/−) and Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1−/− cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H2O2, and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5′-phosphotyrosyl DNA ends when they mimic the 5′-overhangs of Top2cc. Tdp1 also processes 3′-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1−/− cells to MMS and H2O2. Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage.

Rationale for Poly(ADP-ribose) Polymerase (PARP) Inhibitors in Combination Therapy with Camptothecins or Temozolomide Based on PARP Trapping versus Catalytic Inhibition

We recently showed that poly(ADP-ribose) polymerase (PARP) inhibitors exert their cytotoxicity primarily by trapping PARP-DNA complexes in addition to their NAD+-competitive catalytic inhibitory mechanism. PARP trapping is drug-specific, with olaparib exhibiting a greater ability than veliparib, whereas both compounds are potent catalytic PARP inhibitors. Here, we evaluated the combination of olaparib or veliparib with therapeutically relevant DNA-targeted drugs, including the topoisomerase I inhibitor camptothecin, the alkylating agent temozolomide, the cross-linking agent cisplatin, and the topoisomerase II inhibitor etoposide at the cellular and molecular levels. We determined PARP-DNA trapping and catalytic PARP inhibition in genetically modified chicken lymphoma DT40, human prostate DU145, and glioblastoma SF295 cancer cells. For camptothecin, both PARP inhibitors showed highly synergistic effects due to catalytic PARP inhibition, indicating the value of combining either veliparib or olaparib with topoisomerase I inhibitors. On the other hand, for temozolomide, PARP trapping was critical in addition to catalytic inhibition, consistent with the fact that olaparib was more effective than veliparib in combination with temozolomide. For cisplatin and etoposide, olaparib only showed no or a weak combination effect, which is consistent with the lack of involvement of PARP in the repair of cisplatin- and etoposide-induced lesions. Hence, we conclude that catalytic PARP inhibitors are highly effective in combination with camptothecins, whereas PARP inhibitors capable of PARP trapping are more effective with temozolomide. Our study provides insights in combination treatment rationales for different PARP inhibitors.

PARP1–TDP1 coupling for the repair of topoisomerase I–induced DNA damage

Poly(ADP-ribose) polymerases (PARP) attach poly(ADP-ribose) (PAR) chains to various proteins including themselves and chromatin. Topoisomerase I (Top1) regulates DNA supercoiling and is the target of camptothecin and indenoisoquinoline anticancer drugs, as it forms Top1 cleavage complexes (Top1cc) that are trapped by the drugs. Endogenous and carcinogenic DNA lesions can also trap Top1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1), a key repair enzyme for trapped Top1cc, hydrolyzes the phosphodiester bond between the DNA 3′-end and the Top1 tyrosyl moiety. Alternative repair pathways for Top1cc involve endonuclease cleavage. However, it is unknown what determines the choice between TDP1 and the endonuclease repair pathways. Here we show that PARP1 plays a critical role in this process. By generating TDP1 and PARP1 double-knockout lymphoma chicken DT40 cells, we demonstrate that TDP1 and PARP1 are epistatic for the repair of Top1cc. The N-terminal domain of TDP1 directly binds the C-terminal domain of PARP1, and TDP1 is PARylated by PARP1. PARylation stabilizes TDP1 together with SUMOylation of TDP1. TDP1 PARylation enhances its recruitment to DNA damage sites without interfering with TDP1 catalytic activity. TDP1–PARP1 complexes, in turn recruit X-ray repair cross-complementing protein 1 (XRCC1). This work identifies PARP1 as a key component driving the repair of trapped Top1cc by TDP1.

Regulation of the Fanconi anemia pathway by a SUMO-like delivery network

The USP1/UAF1 complex deubiquitinates the Fanconi anemia protein FANCD2, thereby promoting homologous recombination and DNA cross-link repair. How USP1/UAF1 is targeted to the FANCD2/FANCI heterodimer has remained unknown. Here we show that UAF1 contains a tandem repeat of SUMO-like domains in its C terminus (SLD1 and SLD2). SLD2 binds directly to a SUMO-like domain-interacting motif (SIM) on FANCI. Deletion of the SLD2 sequence of UAF1 or mutation of the SIM on FANCI disrupts UAF1/FANCI binding and inhibits FANCD2 deubiquitination and DNA repair. The USP1/UAF1 complex also deubiquitinates PCNA-Ub, and deubiquitination requires the PCNA-binding protein hELG1. The SLD2 sequence of UAF1 binds to a SIM on hELG1, thus targeting the USP1/UAF1 complex to its PCNA-Ub substrate. We propose that the regulated targeting of USP1/UAF1 to its DNA repair substrates, FANCD2-Ub and PCNA-Ub, by SLD-SIM interactions coordinates homologous recombination and translesion DNA synthesis.

Differential and Common DNA Repair Pathways for Topoisomerase I- and II-Targeted Drugs in a Genetic DT40 Repair Cell Screen Panel

Clinical topoisomerase I (Top1) and II (Top2) inhibitors trap topoisomerases on DNA, thereby inducing protein-linked DNA breaks. Cancer cells resist the drugs by removing topoisomerase-DNA complexes, and repairing the drug-induced DNA double-strand breaks (DSB) by homologous recombination and nonhomologous end joining (NHEJ). Because numerous enzymes and cofactors are involved in the removal of the topoisomerase-DNA complexes and DSB repair, it has been challenging to comprehensively analyze the relative contribution of multiple genetic pathways in vertebrate cells. Comprehending the relative contribution of individual repair factors would give insights into the lesions induced by the inhibitors and genetic determinants of response. Ultimately, this information would be useful to target specific pathways to augment the therapeutic activity of topoisomerase inhibitors. To this end, we put together 48 isogenic DT40 mutant cells deficient in DNA repair and generated one cell line deficient in autophagy (ATG5). Sensitivity profiles were established for three clinically relevant Top1 inhibitors (camptothecin and the indenoisoquinolines LMP400 and LMP776) and three Top2 inhibitors (etoposide, doxorubicin, and ICRF-193). Highly significant correlations were found among Top1 inhibitors as well as Top2 inhibitors, whereas the profiles of Top1 inhibitors were different from those of Top2 inhibitors. Most distinct repair pathways between Top1 and Top2 inhibitors include NHEJ, TDP1, TDP2, PARP1, and Fanconi Anemia genes, whereas homologous recombination seems relevant especially for Top1 and, to a lesser extent, for Top2 inhibitors. We also found and discuss differential pathways among Top1 inhibitors and Top2 inhibitors. Mol Cancer Ther; 13(1); 214–20. ©2013 AACR.

TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs

Chain-terminating nucleoside analogs (CTNAs) that cause stalling or premature termination of DNA replication forks are widely used as anticancer and antiviral drugs. However, it is not well understood how cells repair the DNA damage induced by these drugs. Here, we reveal the importance of tyrosyl–DNA phosphodiesterase 1 (TDP1) in the repair of nuclear and mitochondrial DNA damage induced by CTNAs. On investigating the effects of four CTNAs—acyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT) and zalcitabine (ddC)—we show that TDP1 is capable of removing the covalently linked corresponding CTNAs from DNA 3′-ends. We also show that Tdp1−/− cells are hypersensitive and accumulate more DNA damage when treated with ACV and Ara-C, implicating TDP1 in repairing CTNA-induced DNA damage. As AZT and ddC are known to cause mitochondrial dysfunction, we examined whether TDP1 repairs the mitochondrial DNA damage they induced. We find that AZT and ddC treatment leads to greater depletion of mitochondrial DNA in Tdp1−/− cells. Thus, TDP1 seems to be critical for repairing nuclear and mitochondrial DNA damage caused by CTNAs.

TDP2 promotes repair of topoisomerase I-mediated DNA damage in the absence of TDP1

The abortive activity of topoisomerases can result in clastogenic and/or lethal DNA damage in which the topoisomerase is covalently linked to the 3′- or 5′-terminus of a DNA strand break. This type of DNA damage is implicated in chromosome translocations and neurological disease and underlies the clinical efficacy of an important class of anticancer topoisomerase ‘poisons’. Tyrosyl DNA phosphodiesterase-1 protects cells from abortive topoisomerase I (Top1) activity by hydrolyzing the 3′-phosphotyrosyl bond that links Top1 to a DNA strand break and is currently the only known human enzyme that displays this activity in cells. Recently, we identified a second tyrosyl DNA phosphodiesterase (TDP2; aka TTRAP/EAPII) that possesses weak 3′-tyrosyl DNA phosphodiesterase (3′-TDP) activity, in vitro. Herein, we have examined whether TDP2 contributes to the repair of Top1-mediated DNA breaks by deleting Tdp1 and Tdp2 separately and together in murine and avian cells. We show that while deletion of Tdp1 in wild-type DT40 cells and mouse embryonic fibroblasts decreases DNA strand break repair rates and cellular survival in response to Top1-induced DNA damage, deletion of Tdp2 does not. However, deletion of both Tdp1 and Tdp2 reduces rates of DNA strand break repair and cell survival below that observed in Tdp1−/− cells, suggesting that Tdp2 contributes to cellular 3′-TDP activity in the absence of Tdp1. Consistent with this idea, over-expression of human TDP2 in Tdp1−/−/Tdp2−/−/− DT40 cells increases DNA strand break repair rates and cell survival above that observed in Tdp1−/− DT40 cells, suggesting that Tdp2 over-expression can partially complement the defect imposed by loss of Tdp1. Finally, mice lacking both Tdp1 and Tdp2 exhibit greater sensitivity to Top1 poisons than do mice lacking Tdp1 alone, further suggesting that Tdp2 contributes to the repair of Top1-mediated DNA damage in the absence of Tdp1. In contrast, we failed to detect a contribution for Tdp1 to repair Top2-mediated damage. Together, our data suggest that Tdp1 and Tdp2 fulfil overlapping roles following Top1-induced DNA damage, but not following Top2-induced DNA damage, in vivo.

Smad7 Inhibits Chondrocyte Differentiation at Multiple Steps during Endochondral Bone Formation and Down-regulates p38 MAPK Pathways

Bone morphogenetic proteins (BMPs) play critical roles at various stages in endochondral bone formation. In vitro studies have demonstrated that Smad7 regulates transforming growth factor-β and BMP signals by inhibiting Smad pathways in chondrocytes. However, the in vivo roles of Smad7 during cartilage development are unknown. To investigate distinct effects of Smad7 at different stages during chondrocyte differentiation, we generated a series of conditional transgenic mice that overexpress Smad7 in chondrocytes at various steps of differentiation by using the Cre/loxP system. We generated Col11a2-lacZfloxed-Smad7 transgenic mice and mated them with three types of Cre transgenic mice to obtain Smad7Prx1, Smad711Enh, and Smad711Prom conditional transgenic mice. Smad7Prx1 mice overexpressing Smad7 in condensing mesenchymal cells showed disturbed mesenchymal condensation associated with decreased Sox9 expression, leading to poor cartilage formation. Smad711Enh mice overexpressing Smad7 in round chondrocytes showed decreased chondrocyte proliferation rates. Smad711Prom mice overexpressing Smad7 in flat chondrocytes showed inhibited maturation of chondrocytes toward hypertrophy. Micromass culture of mesenchymal cells showed that BMP-induced cartilaginous nodule formation was down-regulated by overexpression of Smad7, but not Smad6. Overexpression of Smad7, but not Smad6, down-regulated the phosphorylation of p38 MAPKs. Our data provide in vivo evidence for distinct effects of Smad7 at different stages during chondrocyte differentiation and suggest that Smad7 in prechondrogenic cells inhibits chondrocyte differentiation possibly by down-regulating BMP-activated p38 MAPK pathways.