Increased Replication Stress Determines ATR Inhibitor Sensitivity in Neuroblastoma Cells.

Despite intensive high-dose multimodal therapy, high-risk neuroblastoma (NB) confers a less than 50% survival rate. This study investigates the role of replication stress in sensitivity to inhibition of Ataxia telangiectasia and Rad3-related (ATR) in pre-clinical models of high-risk NB. Amplification of the oncogene MYCN always imparts high-risk disease and occurs in 25% of all NB. Here, we show that MYCN-induced replication stress directly increases sensitivity to the ATR inhibitors VE-821 and AZD6738. PARP inhibition with Olaparib also results in replication stress and ATR activation, and sensitises NB cells to ATR inhibition independently of MYCN status, with synergistic levels of cell death seen in MYCN expressing ATR- and PARP-inhibited cells. Mechanistically, we demonstrate that ATR inhibition increases the number of persistent stalled and collapsed replication forks, exacerbating replication stress. It also abrogates S and G2 cell cycle checkpoints leading to death during mitosis in cells treated with an ATR inhibitor combined with PARP inhibition. In summary, increased replication stress through high MYCN expression, PARP inhibition or chemotherapeutic agents results in sensitivity to ATR inhibition. Our findings provide a mechanistic rationale for the inclusion of ATR and PARP inhibitors as a potential treatment strategy for high-risk NB.

Poly(ADP-Ribose) Glycohydrolase (PARG) vs. Poly(ADP-Ribose) Polymerase (PARP) - Function in Genome Maintenance and Relevance of Inhibitors for Anti-cancer Therapy.

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that catalyze the addition of poly(ADP-ribose) (PAR) subunits onto themselves and other acceptor proteins. PARPs are known to function in a large range of cellular processes including DNA repair, DNA replication, transcription and modulation of chromatin structure. Inhibition of PARP holds great potential for therapy, especially in cancer. Several PARP1/2/3 inhibitors (PARPi) have had success in treating ovarian, breast and prostate tumors harboring defects in the homologous recombination (HR) DNA repair pathway, especially BRCA1/2 mutated tumors. However, treatment is limited to specific sub-groups of patients and resistance can occur, limiting the use of PARPi. Poly(ADP-ribose) glycohydrolase (PARG) reverses the action of PARP enzymes, hydrolysing the ribose-ribose bonds present in poly(ADP-ribose). Like PARPs, PARG is involved in DNA replication and repair and PARG depleted/inhibited cells show increased sensitivity to DNA damaging agents. They also display an accumulation of perturbed replication intermediates which can lead to synthetic lethality in certain contexts. In addition, PARG is thought to play an important role in preventing the accumulation of cytoplasmic PAR and therefore parthanatos, a caspase-independent PAR-mediated type of cell death. In contrast to PARP, the therapeutic potential of PARG has been largely ignored. However, several recent papers have demonstrated the exciting possibilities that inhibitors of this enzyme may have for cancer treatment, both as single agents and in combination with cytotoxic drugs and radiotherapy. This article discusses what is known about the functions of PARP and PARG and the potential future implications of pharmacological inhibition in anti-cancer therapy.

MYCN expression induces replication stress and sensitivity to PARP inhibition in neuroblastoma.

This study investigates the influence expression of the MYCN oncogene has on the DNA damage response, replication fork progression and sensitivity to PARP inhibition in neuroblastoma. In a panel of neuroblastoma cell lines, MYCN amplification or MYCN expression resulted in increased cell death in response to a range of PARP inhibitors (niraparib, veliparib, talazoparib and olaparib) compared to the response seen in non-expressing/amplified cells. MYCN expression slowed replication fork speed and increased replication fork stalling, an effect that was amplified by PARP inhibition or PARP1 depletion. Increased DNA damage seen was specifically induced in S-phase cells. Importantly, PARP inhibition caused a significant increase in the survival of mice bearing MYCN expressing tumours in a transgenic murine model of MYCN expressing neuroblastoma. Olaparib also sensitized MYCN expressing cells to camptothecin- and temozolomide-induced cell death to a greater degree than non-expressing cells. In summary, MYCN expression leads to increased replication stress in neuroblastoma cells. This effect is exaggerated by inhibition of PARP, resulting in S-phase specific DNA damage and ultimately increased tumour cell death. PARP inhibition alone or in combination with classical chemotherapeutics is therefore a potential therapeutic strategy for neuroblastoma and may be more effective in MYCN expressing tumours.

DNA Fiber Assay for the Analysis of DNA Replication Progression in Human Pluripotent Stem Cells.

Human pluripotent stem cells (PSC) acquire recurrent chromosomal instabilities during prolonged in vitro culture that threaten to preclude their use in cell-based regenerative medicine. The rapid proliferation of pluripotent cells leads to constitutive replication stress, hindering the progression of DNA replication forks and in some cases leading to replication-fork collapse. Failure to overcome replication stress can result in incomplete genome duplication, which, if left to persist into the subsequent mitosis, can result in structural and numerical chromosomal instability. We have recently applied the DNA fiber assay to the study of replication stress in human PSC and found that, in comparison to somatic cells states, these cells display features of DNA replication stress that include slower replication fork speeds, evidence of stalled forks, and replication initiation from dormant replication origins. These findings have expanded on previous work demonstrating that extensive DNA damage in human PSC is replication associated. In this capacity, the DNA fiber assay has enabled the development of an advanced nucleoside-enriched culture medium that increases replication fork progression and decreases DNA damage and mitotic errors in human PSC cultures. The DNA fiber assay allows for the study of replication fork dynamics at single-molecule resolution. The assay relies on cells incorporating nucleotide analogs into nascent DNA during replication, which are then measured to monitor several replication parameters. Here we provide an optimized protocol for the fiber assay intended for use with human PSC, and describe the methods employed to analyze replication fork parameters. © 2020 Wiley Periodicals LLC. Basic Protocol 1: DNA fiber labeling Basic Protocol 2: DNA fiber spreading Basic Protocol 3: Immunostaining Support Protocol 1: Microscopy/data acquisition Support Protocol 2: Data analysis.

Radiosensitization with an inhibitor of poly(ADP-ribose) glycohydrolase: A comparison with the PARP1/2/3 inhibitor olaparib.

Upon DNA binding the poly(ADP-ribose) polymerase family of enzymes (PARPs) add multiple ADP-ribose subunits to themselves and other acceptor proteins. Inhibitors of PARPs have become an exciting and real prospect for monotherapy and as sensitizers to ionising radiation (IR). The action of PARPs are reversed by poly(ADP-ribose) glycohydrolase (PARG). Until recently studies of PARG have been limited by the lack of an inhibitor. Here, a first in class, specific, and cell permeable PARG inhibitor, PDD00017273, is shown to radiosensitize. Further, PDD00017273 is compared with the PARP1/2/3 inhibitor olaparib. Both olaparib and PDD00017273 altered the repair of IR-induced DNA damage, resulting in delayed resolution of RAD51 foci compared with control cells. However, only PARG inhibition induced a rapid increase in IR-induced activation of PRKDC (DNA-PK) and perturbed mitotic progression. This suggests that PARG has additional functions in the cell compared with inhibition of PARP1/2/3, likely via reversal of tankyrase activity and/or that inhibiting the removal of poly(ADP-ribose) (PAR) has a different consequence to inhibiting PAR addition. Overall, our data are consistent with previous genetic findings, reveal new insights into the function of PAR metabolism following IR and demonstrate for the first time the therapeutic potential of PARG inhibitors as radiosensitizing agents.

Specific killing of DNA damage-response deficient cells with inhibitors of poly(ADP-ribose) glycohydrolase.

Poly(ADP-ribosylation) of proteins following DNA damage is well studied and the use of poly(ADP-ribose) polymerase (PARP) inhibitors as therapeutic agents is an exciting prospect for the treatment of many cancers. Poly(ADP-ribose) glycohydrolase (PARG) has endo- and exoglycosidase activities which can cleave glycosidic bonds, rapidly reversing the action of PARP enzymes. Like addition of poly(ADP-ribose) (PAR) by PARP, removal of PAR by PARG is also thought to be required for repair of DNA strand breaks and for continued replication at perturbed forks. Here we use siRNA to show a synthetic lethal relationship between PARG and BRCA1, BRCA2, PALB2, FAM175A (ABRAXAS) and BARD1. In addition, we demonstrate that MCF7 cells depleted of these proteins are sensitive to Gallotannin and a novel and specific PARG inhibitor PDD00017273. We confirm that PARG inhibition increases endogenous DNA damage, stalls replication forks and increases homologous recombination, and propose that it is the lack of homologous recombination (HR) proteins at PARG inhibitor-induced stalled replication forks that induces cell death. Interestingly not all genes that are synthetically lethal with PARP result in sensitivity to PARG inhibitors, suggesting that although there is overlap, the functions of PARP and PARG may not be completely identical. These data together add further evidence to the possibility that single treatment therapy with PARG inhibitors could be used for treatment of certain HR deficient tumours and provide insight into the relationship between PARP, PARG and the processes of DNA repair.

Use of the HPRT gene to study nuclease-induced DNA double-strand break repair.

Understanding the mechanisms of chromosomal double-strand break repair (DSBR) provides insight into genome instability, oncogenesis and genome engineering, including disease gene correction. Research into DSBR exploits rare-cutting endonucleases to cleave exogenous reporter constructs integrated into the genome. Multiple reporter constructs have been developed to detect various DSBR pathways. Here, using a single endogenous reporter gene, the X-chromosomal disease gene encoding hypoxanthine phosphoribosyltransferase (HPRT), we monitor the relative utilization of three DSBR pathways following cleavage by I-SceI or CRISPR/Cas9 nucleases. For I-SceI, our estimated frequencies of accurate or mutagenic non-homologous end-joining and gene correction by homologous recombination are 4.1, 1.5 and 0.16%, respectively. Unexpectedly, I-SceI and Cas9 induced markedly different DSBR profiles. Also, using an I-SceI-sensitive HPRT minigene, we show that gene correction is more efficient when using long double-stranded DNA than single- or double-stranded oligonucleotides. Finally, using both endogenous HPRT and exogenous reporters, we validate novel cell cycle phase-specific I-SceI derivatives for investigating cell cycle variations in DSBR. The results obtained using these novel approaches provide new insights into template design for gene correction and the relationships between multiple DSBR pathways at a single endogenous disease gene.

Chemical genetic analyses of quantitative changes in Cdk1 activity during the human cell cycle.

Cyclin-dependent kinase 1 (Cdk1) controls cell proliferation and is inhibited by promising anticancer agents, but its mode of action and the consequences of its inhibition are incompletely understood. Cdk1 promotes S- and M-phases during the cell-cycle but also suppresses endoreduplication, which is associated with polyploidy and genome instability. The complexity of Cdk1 regulation has made it difficult to determine whether these different roles require different thresholds of kinase activity and whether the surge of activity as inhibitory phosphates are removed at mitotic onset is essential for cell proliferation. Here, we have used chemical genetics in a human cell line to address these issues. We rescued cells lethally depleted of endogenous Cdk1 with an exogenous Cdk1 conferring sensitivity to one ATP analogue inhibitor (1NMPP1) and resistance to another (RO3306). At no 1NMPP1 concentration was mitosis in rescued clones prevented without also inducing endoreduplication, suggesting that these two key roles for Cdk1 are not simply controlled by different Cdk1 activity thresholds. We also rescued RO3306-resistant clones using exogenous Cdk1 without inhibitory phosphorylation sites, indicating that the mitotic surge of Cdk1 activity is dispensable for cell proliferation. These results suggest that the basic mammalian cycle requires at least some qualitative changes in Cdk1 activity and that quantitative increases in activity need not be rapid. Furthermore, the viability of cells that are unable to undergo rapid Cdk1 activation, and the strong association between endoreduplication and impaired proliferation, may place restrictions on the therapeutic use of a Cdk1 inhibitors.

Atypically low spontaneous sister chromatid exchange formation in uveal melanoma.

Uveal melanoma (UM) is the most common primary intraocular cancer of adults and is characterized by several well-established chromosomal changes. More recently, a specific mutation of guanine nucleotide binding protein Gq alpha subunit (GNAQ) has also been identified in a proportion of UM. Although some of these alterations have been suggested to be early changes, the genetic alterations responsible for the development of UM have yet to be clearly determined. Cancers are characterized by increased genetic instability, and analysis of established cancer cell lines and blood from cancer patients has universally been associated with an increased level of sister chromatid exchange (SCE). We have observed that the spontaneous frequency of SCE in primary cultures of UM and UM-derived cell lines is decreased below normal baseline levels, a phenomenon unique to UM when compared with multiple other cancers. This finding was specific to the tumor and not found in lymphocytes from the patients. Although we cannot exclude the possibility that low SCE (LSCE) is peculiar to the uveal melanocytes lineage, as it was consistently observed in all UM studied, regardless of other genetic defects, we propose that this phenomenon contributes to the molecular pathogenesis of UM.