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Purpose: Enhanced tumor cell survival through expression of inhibitors of apoptosis (IAP) is a hallmark of cancer. Survivin, an IAP absent from most normal tissues, is overexpressed in many malignancies and associated with a poorer prognosis. We report the first-in-human dose study of LY2181308, a second-generation antisense oligonucleotide (ASO) directed against survivin mRNA.Patients and Methods: A dose-escalation study evaluating the safety, pharmacokinetics, and pharmacodynamics of LY2181308 administered intravenously for 3 hours as a loading dose on 3 consecutive days and followed by weekly maintenance doses. Patients were eligible after signing informed consent, had exhausted approved anticancer therapies and agreed to undergo pre- and posttreatment tumor biopsies to evaluate reduction of survivin protein and gene expression.Results: A total of 40 patients were treated with LY2181308 at doses of 100 to 1,000 mg. Twenty-six patients were evaluated at the recommended phase 2 dose of 750 mg, at which level serial tumor sampling and [11C]LY2183108 PET (positron emission tomography) imaging demonstrated that ASO accumulated within tumor tissue, reduced survivin gene and protein expression by 20% and restored apoptotic signaling in tumor cells in vivo. Pharmacokinetics were consistent with preclinical modeling, exhibiting rapid tissue distribution, and terminal half-life of 31 days.Conclusions: The tumor-specific, molecularly targeted effects demonstrated by this ASO in man underpin confirmatory studies evaluating its therapeutic efficacy in cancer.

Supplementary Figures S1-S2.

The Werner Syndrome helicase, WRN, is a promising therapeutic target in cancers with microsatellite instability (MSI). Long-term MSI leads to the expansion of TA nucleotide repeats proposed to form cruciform DNA structures, which in turn cause DNA breaks and cell lethality upon WRN downregulation. Here we employed biochemical assays to show that WRN helicase can efficiently and directly unfold cruciform structures, thereby preventing their cleavage by the SLX1-SLX4 structure-specific endonuclease. TA repeats are particularly prone to form cruciform structures, explaining why these DNA sequences are preferentially broken in MSI cells upon WRN downregulation. We further demonstrate that the activity of the DNA mismatch repair (MMR) complexes MutSα (MSH2-MSH6), MutSβ (MSH2-MSH3), and MutLα (MLH1-PMS2) similarly decreases the level of DNA cruciforms, although the mechanism is different from that employed by WRN. When combined, WRN and MutLα exhibited higher than additive effects in in vitro cruciform processing, suggesting that WRN and the MMR proteins may cooperate. Our data explain how WRN and MMR defects cause genome instability in MSI cells with expanded TA repeats, and provide a mechanistic basis for their recently discovered synthetic-lethal interaction with promising applications in precision cancer therapy.

Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-β-lactamase (MBL) and β-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its β-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors.

The SNM1 nucleases which help maintain genome integrity are members of the metallo-β-lactamase (MBL) structural superfamily. Their conserved MBL-β-CASP-fold SNM1 core provides a molecular scaffold forming an active site which coordinates the metal ions required for catalysis. The features that determine SNM1 endo- versus exonuclease activity, and which control substrate selectivity and binding are poorly understood. We describe a structure of SNM1B/Apollo with two nucleotides bound to its active site, resembling the product state of its exonuclease reaction. The structure enables definition of key SNM1B residues that form contacts with DNA and identifies a 5′ phosphate binding pocket, which we demonstrate is important in catalysis and which has a key role in determining endo- versus exonucleolytic activity across the SNM1 family. We probed the capacity of SNM1B to digest past sites of common endogenous DNA lesions and find that base modifications planar to the nucleobase can be accommodated due to the open architecture of the active site, but lesions axial to the plane of the nucleobase are not well tolerated due to constriction around the altered base. We propose that SNM1B/Apollo might employ its activity to help remove common oxidative lesions from telomeres.

DNA breaks, in particular double-strand breaks (DSBs), produced by radiation and radiomimetic agents are amongst the most toxic forms of DNA damage, in part because they are associated with extensive oxidative chemical modification at the break termini. Prior to the completion of DSB repair, the chemically modified termini associated with radiation-induced damage must be removed. A number of DNA processing enzymes have been implicated in the processing of these ‘dirty ends’, but our knowledge of this process remains limited. Here, we demonstrate a role for the 5’-3’ exonuclease SNM1A in this process. Cells disrupted for SNM1A are sensitive to radiation and radiomimetic agents and show defects in the resolution of the lethal DSB damage they induce. SNM1A is recruited and retained to the sites of complex DNA damage via the concerted action of three highly conserved interaction domains the PBZ, UBZ and PIP box, mediating interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. Consistent with its unprecedented capacity to digest DNA containing a wide range of chemically modified nucleotides, we found that SNM1A can resect DNA containing oxidative lesions representative of those induced by radiation damage at break termini. Taken together, our work reveals a crucial role for SNM1A in the repair of toxic DNA breaks.

Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons(1,2). The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.

The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14–nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14–nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14–nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3′-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14–nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12–nsp7–nsp8 (nsp12–7–8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14–nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14–nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.

Genome instability is a characteristic enabling factor for carcinogenesis. HelQ helicase is a component of human DNA maintenance systems that prevent or reverse genome instability arising during DNA replication. Here, we provide details of the molecular mechanisms that underpin HelQ function—its recruitment onto ssDNA through interaction with replication protein A (RPA), and subsequent translocation of HelQ along ssDNA. We describe for the first time a functional role for the non-catalytic N-terminal region of HelQ, by identifying and characterizing its PWI-like domain. We present evidence that this domain of HelQ mediates interaction with RPA that orchestrates loading of the helicase domains onto ssDNA. Once HelQ is loaded onto the ssDNA, ATP-Mg2+ binding in the catalytic site activates the helicase core and triggers translocation along ssDNA as a dimer. Furthermore, we identify HelQ-ssDNA interactions that are critical for the translocation mechanism. Our data are novel and detailed insights into the mechanisms of HelQ function relevant for understanding how human cells avoid genome instability provoking cancers, and also how cells can gain resistance to treatments that rely on DNA crosslinking agents.

Genome stability is essential for all cell types. However, defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons. The neuronal genome is subjected to a constant barrage of endogenous DNA damage due to high levels of oxidative metabolism in the central nervous system. Surprisingly, we know little about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here, we show that neurons, but not other post-mitotic cells, accumulate unexpectedly high numbers of DNA single-strand breaks (SSBs) at specific sites within the genome. These recurrent SSBs are found within enhancers, and trigger DNA repair through recruitment and activation of poly(ADP-ribose) polymerase-1 (PARP1) and XRCC1, the central SSB repair scaffold protein. Notably, deficiencies in PARP1, XRCC1, or DNA polymerase β elevate the localized incorporation of nucleotides, suggesting that the ongoing DNA synthesis at neuronal enhancers involves both short-patch and long-patch SSB repair processes. These data reveal unexpected levels of localized and continuous DNA single-strand breakage in neurons, suggesting an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.

The SARS-CoV-2 coronavirus (CoV) causes COVID-19, a current global pandemic. SARS-CoV-2 belongs to an order of Nidovirales with very large RNA genomes. It is proposed that the fidelity of CoV genome replication is aided by an RNA nuclease complex, formed of non-structural proteins 14 and 10 (nsp14-nsp10), an attractive target for antiviral inhibition. Here, we confirm that the SARS-CoV-2 nsp14-nsp10 complex is an RNase. Detailed functional characterisation reveals nsp14-nsp10 is a highly versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3’-terminus. We propose that the role of nsp14-nsp10 in maintaining replication fidelity goes beyond classical proofreading and purges the nascent replicating RNA strand of a range of potentially replication terminating aberrations. Using the developed assays, we identify a series of drug and drug-like molecules that potently inhibit nsp14-nsp10, including the known Sars-Cov-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for bifunctional inhibitors in the treatment of COVID-19.

DNA interstrand cross-links (ICLs) are an extremely toxic form of DNA damage that cells experience upon exposure to natural metabolites. Moreover, ICLs are cytotoxic lesions produced by a range of clinically important anticancer agents. Therefore, improving our understanding of ICL induction and processing has important implications in biology and medicine. The sensitive detection of ICLs in mammalian cells is challenging but has been aided by the development of a modified form of the single-cell gel electrophoresis (SCGE) assay, also known as the "comet assay." Here we describe this method and how it can be used to sensitively monitor the induction and removal of ICLs in single mammalian cells.

Unrepaired, or misrepaired, DNA damage can contribute to the pathogenesis of a number of conditions, or disease states; thus, DNA damage repair pathways, and the proteins within them, are required for the safeguarding of the genome. Human SNM1A is a 5′-to-3′ exonuclease that plays a role in multiple DNA damage repair processes. To date, most data suggest a role of SNM1A in primarily ICL repair: SNM1A deficient cells exhibit hypersensitivity to ICL-inducing agents (e.g. mitomycin C and cisplatin); and both in vivo and in vitro experiments demonstrate SNM1A and XPF-ERCC1 can function together in the ‘unhooking’ step of ICL repair. SNM1A further interacts with a number of other proteins that contribute to genome integrity outside canonical ICL repair (e.g. PCNA and CSB), and these may play a role in regulating SNM1As function, subcellular localisation, and post-translational modification state. These data also provide further insight into other DNA repair pathways to which SNM1A may contribute. This review aims to discuss all aspects of the exonuclease, SNM1A, and its contribution to DNA damage tolerance.

BackgroundCongenital dyserythropoietic anaemia type I (CDA-I) is a hereditary anaemia caused by biallelic mutations in the widely expressed genes CDAN1 and C15orf41. Little is understood about either protein and it is unclear in which cellular pathways they participate.MethodsGenetic analysis of a cohort of patients with CDA-I identifies novel pathogenic variants in both known causative genes. We analyse the mutation distribution and the predicted structural positioning of amino acids affected in Codanin-1, the protein encoded by CDAN1. Using western blotting, immunoprecipitation and immunofluorescence, we determine the effect of particular mutations on both proteins and interrogate protein interaction, stability and subcellular localisation.ResultsWe identify six novel CDAN1 mutations and one novel mutation in C15orf41 and uncover evidence of further genetic heterogeneity in CDA-I. Additionally, population genetics suggests that CDA-I is more common than currently predicted. Mutations are enriched in six clusters in Codanin-1 and tend to affect buried residues. Many missense and in-frame mutations do not destabilise the entire protein. Rather C15orf41 relies on Codanin-1 for stability and both proteins, which are enriched in the nucleolus, interact to form an obligate complex in cells.ConclusionStability and interaction data suggest that C15orf41 may be the key determinant of CDA-I and offer insight into the mechanism underlying this disease. Both proteins share a common pathway likely to be present in a wide variety of cell types; however, nucleolar enrichment may provide a clue as to the erythroid specific nature of CDA-I. The surprisingly high predicted incidence of CDA-I suggests that better ascertainment would lead to improved patient care.

DNA interstrand cross-links (ICLs) are an extremely toxic form of DNA damage that cells experience upon exposure to natural metabolites. Moreover, ICLs are cytotoxic lesions produced by a range of clinically important anticancer agents. Therefore, improving our understanding of ICL induction and processing has important implications in biology and medicine. The sensitive detection of ICLs in mammalian cells is challenging but has been aided by the development of a modified form of the single-cell gel electrophoresis (SCGE) assay, also known as the “comet assay.” Here we describe this method and how it can be used to sensitively monitor the induction and removal of ICLs in single mammalian cells.

Zinc ion dependent β-lactamases (MBLs) catalyze the hydrolysis of almost all β-lactam antibiotics and resist the action of clinically available β-lactamase inhibitors. We report how application of in silico fragment-based molecular design employing thiol-mediated metal anchorage leads to potent MBL inhibitors. The new inhibitors manifest potent inhibition of clinically important B1 subfamily MBLs, including the widespread NDM-1, IMP-1 and VIM-2 enzymes; with lower potency, some of them also inhibit clinically relevant Class A and D serine-β-lactamases. The inhibitors show selectivity for bacterial MBL enzymes compared to human MBL fold nucleases. Co-crystallization of one inhibitor, which shows potentiation of meropenem activity against MBL-expressing Enterobacteriaceae, with VIM-2 reveals an unexpected binding mode, involving interactions with residues from conserved active site bordering loops.

SUMMARY DNA interstrand crosslinks (ICLs) are extremely cytotoxic, but the mechanism of their repair remains incompletely understood. Using Xenopus egg extracts, we previously showed that repair of a cisplatin ICL is triggered when two replication forks converge on the lesion. After CDC45/MCM2-7/GINS (CMG) ubiquitylation and unloading by the p97 segregase, FANCI-FANCD2 promotes DNA incisions by XPF-ERCC1, leading to ICL unhooking. Here, we report that, during this cell-free ICL repair reaction, one of the two converged forks undergoes reversal. Fork reversal fails when CMG unloading is inhibited, but it does not require FANCI-FANCD2. After one fork has undergone reversal, the opposing fork that still abuts the ICL undergoes incisions. Our data show that replication fork reversal at an ICL requires replisome disassembly. We present a revised model of ICL repair that involves a reversed fork intermediate., In Brief DNA interstrand crosslinks (ICLs) are extremely cytotoxic lesions that are mainly repaired in a replication-coupled manner. Using a cell-free system, Amunugama et al. report that, during ICL repair, replication forks undergo reversal. Fork reversal requires replicative CMG helicase unloading.

We report the design and optimisation of novel oligonucleotide substrates for a sensitive fluorescence assay for high-throughput screening and functional studies of the DNA repair enzyme, XPF-ERCC1, with a view to accelerating inhibitor and drug discovery.

The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.

Understanding the control of viral infections is of broad importance. Chronic hepatitis C virus (HCV) infection causes decreased expression of the iron hormone hepcidin, which is regulated by hepatic bone morphogenetic protein (BMP)/SMAD signalling. We found that HCV infection and the BMP/SMAD pathway are mutually antagonistic. HCV blunted induction of hepcidin expression by BMP6, probably via tumour necrosis factor (TNF)-mediated downregulation of the BMP co-receptor haemojuvelin. In HCV-infected patients, disruption of the BMP6/hepcidin axis and genetic variation associated with the BMP/SMAD pathway predicted the outcome of infection, suggesting that BMP/SMAD activity influences antiviral immunity. Correspondingly, BMP6 regulated a gene repertoire reminiscent of type I interferon (IFN) signalling, including upregulating interferon regulatory factors (IRFs) and downregulating an inhibitor of IFN signalling, USP18. Moreover, in BMP-stimulated cells, SMAD1 occupied loci across the genome, similar to those bound by IRF1 in IFN-stimulated cells. Functionally, BMP6 enhanced the transcriptional and antiviral response to IFN, but BMP6 and related activin proteins also potently blocked HCV replication independently of IFN. Furthermore, BMP6 and activin A suppressed growth of HBV in cell culture, and activin A inhibited Zika virus replication alone and in combination with IFN. The data establish an unappreciated important role for BMPs and activins in cellular antiviral immunity, which acts independently of, and modulates, IFN.

Phosphate groups are often crucial to biological activity and interactions of oligonucleotides, but confer poor membrane permeability. In addition, the group's lability to enzymatic hydrolysis is an obstacle to its use in therapeutics and in biological tools. We present the synthesis of N‐oxyamide and squaramide modifications at the 5’‐end of oligonucleotides as phosphate replacements and their biological evaluation using the 5’‐exonuclease SNM1A. The squaryl diamide modification showed minimal recognition as a 5’‐phosphate mimic; however, modest inhibition of SNM1A, postulated to occur through metal coordination at the active site, was observed. Their facile incorporation after solid‐phase synthesis and recognition by the exonuclease makes squaryl diamides attractive neutral 5’‐phosphate replacements for oligonucleotides. This work is the first example of squaryl diamide modifications at the 5’‐terminal position of oligonucleotides and of the potential use of modified oligonucleotides to bind to the metal center of SNM1A.

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is critical for survival and genome stability of individual cells and organisms, but also contributes to the genetic diversity of species. A vital step in HR is MRN-CtIP-dependent end resection, which generates the 3' single-stranded DNA overhangs required for the subsequent strand exchange reaction. Here, we identify EXD2 (also known as EXDL2) as an exonuclease essential for DSB resection and efficient HR. EXD2 is recruited to chromatin in a damage-dependent manner and confers resistance to DSB-inducing agents. EXD2 functionally interacts with the MRN complex to accelerate resection through its 3'-5' exonuclease activity, which efficiently processes double-stranded DNA substrates containing nicks. Finally, we establish that EXD2 stimulates both short- and long-range DSB resection, and thus, together with MRE11, is required for efficient HR. This establishes a key role for EXD2 in controlling the initial steps of chromosomal break repair.

PARP1 regulates the repair of DNA single-strand breaks generated directly, or during base excision repair (BER). However, the role of PARP2 in these and other repair mechanisms is unknown. Here, we report a requirement for PARP2 in stabilising replication forks that encounter BER intermediates through Fbh1-dependent regulation of Rad51. Whereas PARP2 is dispensable for tolerance of cells to SSBs or homologous recombination dysfunction, it is redundant with PARP1 in BER. Therefore, combined disruption of PARP1 and PARP2 leads to defective BER, resulting in elevated levels of replication-associated DNA damage owing to an inability to stabilise Rad51 at damaged replication forks and prevent uncontrolled DNA resection. Together, our results demonstrate how PARP1 and PARP2 regulate two independent, but intrinsically linked aspects of DNA base damage tolerance by promoting BER directly, and by stabilising replication forks that encounter BER intermediates., PARP1 has a well characterised role in DNA break repair and base excision repair, whereas the role of PARP2 is less well understood. Here, the authors show a requirement for PARP2 in stabilising replication forks that encounter base excision repair intermediates.

Mgm101 has well-characterized activity for the repair and replication of the mitochondrial genome. Recent work has demonstrated a further role for Mgm101 in nuclear DNA metabolism, contributing to an S-phase specific DNA interstrand cross-link repair pathway that acts redundantly with a pathway controlled by Pso2 exonuclease. Due to involvement of FANCM, FANCJ and FANCP homologues (Mph1, Chl1 and Slx4), this pathway has been described as a Fanconi anemia-like pathway. In this pathway, Mgm101 physically interacts with the DNA helicase Mph1 and the MutSα (Msh2/Msh6) heterodimer, but its precise role is yet to be elucidated. Data presented here suggests that Mgm101 functionally overlaps with Rad52, supporting previous suggestions that, based on protein structure and biochemical properties, Mgm101 and Rad52 belong to a family of proteins with similar function. In addition, our data shows that this overlap extends to the function of both proteins at telomeres, where Mgm101 is required for telomere elongation during chromosome replication in rad52 defective cells. We hypothesize that Mgm101 could, in Rad52-like manner, preferentially bind single-stranded DNAs (such as at stalled replication forks, broken chromosomes and natural chromosome ends), stabilize them and mediate single-strand annealing-like homologous recombination event to prevent them from converting into toxic structures.

Radiotherapy and chemotherapy are effective treatment methods for many types of cancer, but resistance is common. Recent findings indicate that antiviral type I interferon (IFN) signaling is induced by these treatments. However, the underlying mechanisms still need to be elucidated. Expression of a set of IFN-stimulated genes comprises an IFN-related DNA damage resistance signature (IRDS), which correlates strongly with resistance to radiotherapy and chemotherapy across different tumors. Classically, during viral infection, the presence of foreign DNA in the cytoplasm of host cells can initiate type I IFN signaling. Here, we demonstrate that DNA-damaging modalities used during cancer therapy lead to the release of ssDNA fragments from the cell nucleus into the cytosol, engaging this innate immune response. We found that the factors that control DNA end resection during double-strand break repair, including the Bloom syndrome (BLM) helicase and exonuclease 1 (EXO1), play a major role in generating these DNA fragments and that the cytoplasmic 3'-5' exonuclease Trex1 is required for their degradation. Analysis of mRNA expression profiles in breast tumors demonstrates that those with lower Trex1 and higher BLM and EXO1 expression levels are associated with poor prognosis. Targeting BLM and EXO1 could therefore represent a novel approach for circumventing the IRDS produced in response to cancer therapeutics.

Drugs that produce covalent interstrand cross-links (ICLs) in DNA remain central to the treatment of cancer, but the cell cycle checkpoints activated by ICLs have received little attention. We have used the fission yeast, Schizosaccharomyces pombe, to elucidate the checkpoint responses to the ICL-inducing anticancer drugs nitrogen mustard and mitomycin C. First we confirmed that the repair pathways acting on ICLs in this yeast are similar to those in the main organisms studied to date (Escherichia coli, budding yeast, and mammalian cells), principally nucleotide excision repair and homologous recombination. We also identified and disrupted the S. pombe homologue of the Saccharomyces cerevisiae SNM1/PSO2 ICL repair gene and found that this activity is required for normal resistance to cross-linking agents, but not other forms of DNA damage. Survival and biochemical analysis indicated a key role for the "checkpoint Rad" family acting through the chk1-dependent DNA damage checkpoint in the ICL response. Rhp9-dependent phosphorylation of Chk1 correlates with G(2) arrest following ICL induction. In cells able to bypass the G(2) block, a second-cycle (S-phase) arrest was observed. Only a transient activation of the Cds1 DNA replication checkpoint factor occurs following ICL formation in wild-type cells, but this is increased and persists in G(2) arrest-deficient mutants. This likely reflects the fraction of cells escaping the G(2) damage checkpoint and arresting in the subsequent S phase due to ICL replication blocks. Disruption of cds1 confers increased resistance to ICLs, suggesting that this second-cycle S-phase arrest might be a lethal event.

AS-I-145 is a novel achiral seco-amino-cyclopropylbenz[e]indolone (seco-amino-CBI) analogue of duocarmycin that has evolved from an alternative strategy of designing CC-1065/duocarmycin agents lacking the characteristic chiral center of the natural agents. The sequence specificity of this compound was assessed by a Taq polymerase stop assay, identifying the sites of covalent modification on plasmid DNA. The adenine-N3 adducts were confirmed at AT-rich sequences using a thermally induced strand cleavage assay. These studies reveal that this compound retains the inherent sequence selectivity of the related natural compounds. The AS-I-145 sensitivity of yeast mutants deficient in excision and post-replication repair (PRR) pathways was assessed. The sensitivity profile suggests that the sequence-specific adenine-N3 adducts are substrates for nucleotide excision repair (NER) but not base excision repair (BER). Single-strand ligation PCR was employed to follow the induction and repair of the lesions at nucleotide resolution in yeast cells. Sequence specificity was preserved in intact cells, and adduct elimination occurred in a transcription-coupled manner and was dependent on a functional NER pathway and Rad18. The involvement of NER as the predominant excision pathway was confirmed in mammalian DNA repair mutant cells. AS-I-145 showed good in vivo antitumor activity in the National Cancer Institute standard hollow fiber assay and was active against the human breast MDA-MD-435 xenograft when administered i.v. or p.o. Its novel structure and in vivo activity renders AS-I-145 a new paradigm in the design of novel achiral analogues of CC-1065 and the duocarmycins. [Mol Cancer Ther 2007;6(10):2708–18]

Cockayne syndrome (CS) is a premature aging disorder characterized by photosensitivity, impaired development and multisystem progressive degeneration, and consists of two strict complementation groups, A and B. Using a yeast two-hybrid approach, we identified the 5'-3' exonuclease SNM1A as one of four strong interacting partners of CSB. This direct interaction was confirmed using purified recombinant proteins-with CSB able to modulate the exonuclease activity of SNM1A on oligonucleotide substrates in vitro-and the two proteins were shown to exist in a common complex in human cell extracts. CSB and SNM1A were also found, using fluorescently tagged proteins in combination with confocal microscopy and laser microirradiation, to be recruited to localized trioxsalen-induced ICL damage in human cells, with accumulation being suppressed by transcription inhibition. Moreover, SNM1A recruitment was significantly reduced in CSB-deficient cells, suggesting coordination between the two proteins in vivo. CSB-deficient neural cells exhibited increased sensitivity to DNA crosslinking agents, particularly, in a non-cycling, differentiated state, as well as delayed ICL processing as revealed by a modified Comet assay and γ-H2AX foci persistence. The results indicate that CSB coordinates the resolution of ICLs, possibly in a transcription-associated repair mechanism involving SNM1A, and that defects in the process could contribute to the post-mitotic degenerative pathologies associated with CS.

3507 Background: Survivin is an inhibitor of apoptosis and its overexpression in cancer has been associated with poor survival. LY2181308 (LY), a novel modified antisense oligonucleotide is a specific inhibitor of survivin. The safety and PK profile of LY from this first-in-human study was presented at ASCO 2008. We now present the PD data from this trial. Methods: Patients with advanced or metastatic malignancies who had failed previous anti-tumor treatments were enrolled. Three consecutive IV loading doses given over 3 hours were followed by weekly maintenance doses. Pre- and post-dosing biopsies were mandated to test for evidence of modification of the target at doses where a PD effect was expected. Biopsies were taken 48 hours after the last loading dose by CT-guided fine needle biopsy. Tumor tissue was paraffin-embedded for immunohistochemistry (IHC) and survivin gene expression. Given the finite amount of biopsy material, the quantification of survivin protein was prioritized. In addition, at one site, pre- and post-dosing endobronchial sampling was conducted in NSCLC patients, with the aim of quantifying levels of native survivin protein, and assessing changes in cell cycle profile in freshly isolated tumor cells using FACS analysis. Results: Out of the 34 patients enrolled, 22 patients had a pre- and posttreatment biopsy. Results from IHC indicated that survivin expression was reduced in the nucleus and cytoplasm in 11/17 and 5/14 evaluable pairs, respectively. LY was detected in neoplastic and non-neoplastic tumor cells in 5/16 and 15/16 evaluable pairs respectively. Gene expression analysis indicated a reduction in survivin expression by 20% to 50% in 11/15 evaluable pairs. Analysis of the fresh tumor material from endobronchial sampling revealed that 2 out of 3 patients with NSCLC had a near-complete elimination of survivin-positive cells accompanied by an increase in the fraction of cells with a sub-G1 DNA content, consistent with cell death. Conclusions: In this study, we were able to demonstrate the presence of the ASO in tumor tissue and confirm a reduction in survivin protein and gene expression. These findings demonstrate the proof of principle of antitumor activity of LY and provide the rationale for phase II studies. [Table: see text]

RNA-directed antisense and interference therapeutics are a promising treatment option for cancer. The demonstration of depletion of target proteins within human tumors in vivo using validated methodology will be a key to the application of this technology. Here, we present a flow cytometric-based approach to quantitatively determine protein levels in solid tumor material derived by fiber optic brushing (FOB) of non-small cell lung cancer (NSCLC) patients. Focusing upon the survivin protein, and its depletion by an antisense oligonucleotide (ASO) (LY2181308), we show that we can robustly identify a subpopulation of survivin positive tumor cells in FOB samples, and, moreover, detect survivin depletion in tumor samples from a patient treated with LY2181308. Survivin depletion appears to be a result of treatment with this ASO, because a tumor treated with conventional cytotoxic chemotherapy did not exhibit a decreased percentage of survivin positive cells. Our approach is likely to be broadly applicable to, and useful for, the quantification of protein levels in tumor samples obtained as part of clinical trials and studies, facilitating the proof-of-principle testing of novel targeted therapies.

Efficient interstrand crosslink (ICL) repair in yeast depends on the Pso2/Snm1 protein. Pso2 is a member of the highly conserved metallo-β-lactamase structural family of nucleases. Mammalian cells possess three SNM1/Pso2 related proteins, SNM1A, SNM1B/Apollo, and SNM1C/Artemis. Evidence that SNM1A and SNM1B contribute to ICL repair is mounting, whereas Artemis appears to primarily contribute to non-ICL repair pathways, particularly some double-strand break repair events. Yeast Pso2 and all three mammalian SNM1-family proteins have been shown to possess nuclease activity. Here, we review the biochemical, genetic, and cellular evidence for the SNM1 family as DNA repair factors, focusing on ICL repair. Environ. Mol. Mutagen. 2010. © 2010 Wiley-Liss, Inc.

Bacterial metallo-β-lactamases (MBLs) are involved in resistance to β-lactam antibiotics including cephalosporins. Human SNM1A and SNM1B are MBL superfamily exonucleases that play a key role in the repair of DNA interstrand cross-links, which are induced by antitumour chemotherapeutics, and are therefore targets for cancer chemosensitization. We report that cephalosporins are competitive inhibitors of SNM1A and SNM1B exonuclease activity; both the intact β-lactam and their hydrolysed products are active. This discovery provides a lead for the development of potent and selective SNM1A and SNM1B inhibitors.

DNA interstrand cross-links (ICLs) pose a significant threat to genomic and cellular integrity by blocking essential cellular processes, including replication and transcription. In mammalian cells, much ICL repair occurs in association with DNA replication during S phase, following the stalling of a replication fork at the block caused by an ICL lesion. Here, we review recent work showing that the XPF-ERCC1 endonuclease and the hSNM1A exonuclease act in the same pathway, together with SLX4, to initiate ICL repair, with the MUS81-EME1 fork incision activity becoming important in the absence of the XPF-SNM1A-SLX4-dependent pathway. Another nuclease, the Fanconi anemia-associated nuclease (FAN1), has recently been implicated in the repair of ICLs, and we discuss the possible ways in which the activities of different nucleases at the ICL-stalled replication fork may be coordinated. In relation to this, we briefly speculate on the possible role of SLX4, which contains XPF and MUS81- interacting domains, in the coordination of ICL repair nucleases.

e13587 Background: Oxaliplatin is first-line chemotherapy for colorectal, gastric, and esophageal cancers. Aim was to identify key determinants of oxaliplatin sensitivity and optimise these biomarkers to select patients for chemotherapy. Methods: High-throughput screening of oxaliplatin sensitivity was performed in a Schizosaccharomyces pombe deletion library of 229 DNA repair strains and Chinese Hamster Ovary (CHO) cell lines with mutations in specific proteins. Biopsies were taken from 50 patients with esophageal cancer, who then received two cycles of oxaliplatin and fluorouracil chemotherapy prior to surgery. Levels of DNA repair proteins were quantified by immunohistochemistry and by qRT-PCR. Results: Twelve lead biomarkers were identified from the preclinical models. In CHO cells, XPF and ERCC1 mutants were approximately 30 times more sensitive than the WT cells (p

Background: The nucleases hSNM1A and hSNM1B are implicated in DNA interstrand cross-link repair. Results: hSNM1A and hSNM1B were biochemically characterized using undamaged and cross-linked DNA. A real-time assay for the nucleases suitable for inhibitor identification was developed. Conclusion: Preferential hSNM1A activation by high molecular weight and cross-linked DNA was observed. Significance: This work provides a basis for hSNM1A inhibitor development for improved cancer therapy., Human SNM1A and SNM1B/Apollo have both been implicated in the repair of DNA interstrand cross-links (ICLs) by cellular studies, and SNM1B is also required for telomere protection. Here, we describe studies on the biochemical characterization of the SNM1A and SNM1B proteins. The results reveal some fundamental differences in the mechanisms of the two proteins. Both SNM1A and SNM1B digest double-stranded and single-stranded DNA with a 5′-to-3′ directionality in a reaction that is stimulated by divalent cations, and both nucleases are inhibited by the zinc chelator o-phenanthroline. We find that SNM1A has greater affinity for single-stranded DNA over double-stranded DNA that is not observed with SNM1B. Although both proteins demonstrate a low level of processivity on low molecular weight DNA oligonucleotide substrates, when presented with high molecular weight DNA, SNM1A alone is rendered much more active, being capable of digesting kilobase-long stretches of DNA. Both proteins can digest past ICLs induced by the non-distorting minor groove cross-linking agent SJG-136, albeit with SNM1A showing a greater capacity to achieve this. This is consistent with the proposal that SNM1A and SNM1B might exhibit some redundancy in ICL repair. Together, our work establishes differences in the substrate selectivities of SNM1A and SNM1B that are likely to be relevant to their in vivo roles and which might be exploited in the development of selective inhibitors.

One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. Here, we show that purified human SNM1A (hSNM1A), which exhibits a 59-39 exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replicationassociated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A- and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells. © 2011 by Cold Spring Harbor Laboratory Press.

HEL308 is a superfamily II DNA helicase, conserved from archaea through to humans. HEL308 family members were originally isolated by their similarity to the Drosophila melanogaster Mus308 protein, which contributes to the repair of replication-blocking lesions such as DNA interstrand cross-links. Biochemical studies have established that human HEL308 is an ATP-dependent enzyme that unwinds DNA with a 3' to 5' polarity, but little else is know about its mechanism. Here, we show that GFP-tagged HEL308 localizes to replication forks following camptothecin treatment. Moreover, HEL308 colocalizes with two factors involved in the repair of damaged forks by homologous recombination, Rad51 and FANCD2. Purified HEL308 requires a 3' single-stranded DNA region to load and unwind duplex DNA structures. When incubated with substrates that model stalled replication forks, HEL308 preferentially unwinds the parental strands of a structure that models a fork with a nascent lagging strand, and the unwinding action of HEL308 is specifically stimulated by human replication protein A. Finally, we show that HEL308 appears to target and unwind from the junction between single-stranded to double-stranded DNA on model fork structures. Together, our results suggest that one role for HEL308 at sites of blocked replication might be to open up the parental strands to facilitate the loading of subsequent factors required for replication restart.

Ino80 facilitates restoration of nucleosome structure during NER-mediated repair of UV-induced lesions., Chromatin structure is modulated during deoxyribonucleic acid excision repair, but how this is achieved is unclear. Loss of the yeast Ino80 chromatin-remodeling complex (Ino80-C) moderately sensitizes cells to ultraviolet (UV) light. In this paper, we show that INO80 acts in the same genetic pathway as nucleotide excision repair (NER) and that the Ino80-C contributes to efficient UV photoproduct removal in a region of high nucleosome occupancy. Moreover, Ino80 interacts with the early NER damage recognition complex Rad4–Rad23 and is recruited to chromatin by Rad4 in a UV damage–dependent manner. Using a modified chromatin immunoprecipitation assay, we find that chromatin disruption during UV lesion repair is normal, whereas the restoration of nucleosome structure is defective in ino80 mutant cells. Collectively, our work suggests that Ino80 is recruited to sites of UV lesion repair through interactions with the NER apparatus and is required for the restoration of chromatin structure after repair.

PURPOSE: Enhanced tumor cell survival through expression of inhibitors of apoptosis (IAP) is a hallmark of cancer. Survivin, an IAP absent from most normal tissues, is overexpressed in many malignancies and associated with a poorer prognosis. We report the first-in-human dose study of LY2181308, a second-generation antisense oligonucleotide (ASO) directed against survivin mRNA. PATIENTS AND METHODS: A dose-escalation study evaluating the safety, pharmacokinetics, and pharmacodynamics of LY2181308 administered intravenously for 3 hours as a loading dose on 3 consecutive days and followed by weekly maintenance doses. Patients were eligible after signing informed consent, had exhausted approved anticancer therapies and agreed to undergo pre- and posttreatment tumor biopsies to evaluate reduction of survivin protein and gene expression. RESULTS: A total of 40 patients were treated with LY2181308 at doses of 100 to 1,000 mg. Twenty-six patients were evaluated at the recommended phase 2 dose of 750 mg, at which level serial tumor sampling and [(11)C]LY2183108 PET (positron emission tomography) imaging demonstrated that ASO accumulated within tumor tissue, reduced survivin gene and protein expression by 20% and restored apoptotic signaling in tumor cells in vivo. Pharmacokinetics were consistent with preclinical modeling, exhibiting rapid tissue distribution, and terminal half-life of 31 days. CONCLUSIONS: The tumor-specific, molecularly targeted effects demonstrated by this ASO in man underpin confirmatory studies evaluating its therapeutic efficacy in cancer.

Interstrand cross-links (ICLs) prevent DNA strand separation and, therefore, transcription and replication, making them extremely cytotoxic. The precise mechanism by which ICLs are removed from mammalian genomes largely remains elusive. Genetic evidence implicates ATR, the Fanconi anemia proteins, proteins required for homologous recombination, translesion synthesis, and at least two endonucleases, MUS81-EME1 and XPF-ERCC1. ICLs cause replication-dependent DNA double-strand breaks (DSBs), and MUS81-EME1 facilitates DSB formation. The subsequent repair of these DSBs occurs via homologous recombination after the ICL is unhooked by XPF-ERCC1. Here, we examined the effect of the loss of either nuclease on FANCD2 monoubiquitination to determine if the nucleolytic processing of ICLs is required for the activation of the Fanconi anemia pathway. FANCD2 was monoubiquitinated in Mus81(-/-), Ercc1(-/-), and XPF-deficient human, mouse, and hamster cells exposed to cross-linking agents. However, the monoubiquitinated form of FANCD2 persisted longer in XPF-ERCC1-deficient cells than in wild-type cells. Moreover, the levels of chromatin-bound FANCD2 were dramatically reduced and the number of ICL-induced FANCD2 foci significantly lower in XPF-ERCC1-deficient cells. These data demonstrate that the unhooking of an ICL by XPF-ERCC1 is necessary for the stable localization of FANCD2 to the chromatin and subsequent homologous recombination-mediated DSB repair.

Pso2/Snm1 plays a key role in the repair of DNA interstrand cross-links in yeast. Human cells possess three orthologues of Pso2; SNM1A, SNM1B/Apollo and SNM1C/Artemis. Studies using mammalian cells disrupted or depleted for these genes have yielded equivocal evidence that any of these is a true functional homologues of the yeast gene. Here we show that ectopic expression of only one of the three human orthologues, hSNM1A, effectively suppresses the sensitivity of yeast pso2 (snm1) disruptants to cross-linking agents. Two other phenotypes of the pso2 mutants are also partially rescued by ectopic expression of hSNM1A, namely the double-strand repair break defect observed during cross-link processing in pso2 cells, as well as the spontaneous intrachromatid recombination defect of pso2 msh2 double mutants. Finally, we show that recombinant hSNM1A is a 5′-exonuclease, as also recently reported for the yeast Pso2 protein. Together our data suggest that hSnm1A is a functional homologue of yeast Pso2/Snm1.

The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-β-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-β-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions.

β-Lactams are the most successful antibacterials, but their effectiveness is threatened by resistance, most importantly by production of serine- and metallo-β-lactamases (MBLs). MBLs are of increasing concern because they catalyze the hydrolysis of almost all β-lactam antibiotics, including recent-generation carbapenems. Clinically useful serine-β-lactamase inhibitors have been developed, but such inhibitors are not available for MBLs. l-Captopril, which is used to treat hypertension via angiotensin-converting enzyme inhibition, has been reported to inhibit MBLs by chelating the active site zinc ions via its thiol(ate). We report systematic studies on B1 MBL inhibition by all four captopril stereoisomers. High-resolution crystal structures of three MBLs (IMP-1, BcII, and VIM-2) in complex with either the l- or d-captopril stereoisomer reveal correlations between the binding mode and inhibition potency. The results will be useful in the design of MBL inhibitors with the breadth of selectivity required for clinical application against carbapenem-resistant Enterobacteriaceae and other organisms causing MBL-mediated resistant infections.

DNA interstrand cross-links (ICLs) present a formidable challenge to the cellular repair apparatus, but to date ICL repair pathways have proved difficult to dissect genetically. It now appears that this is partly the result of a high degree of cell cycle phase selectivity in the choice of ICL pathway employed. Here we review recent results showing that Polymerase ζ, aspecialised translesion polymerase, plays an important role during ICL repair in G1 phase yeast cells, and that PCNA modification by ubiquitin is a key regulator of its activity. Given that this reaction can occur outside the context of S-phase, these results imply a more general role for PCNA modification in the control of DNA repair pathways through the cell cycle, which is dependent on the type of damage or repair intermediate encountered.