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Cells have evolved a complex network of biochemical pathways, collectively known as the DNA damage response (DDR), to prevent detrimental mutations from being passed on to their progeny. The DDR coordinates DNA repair with cell-cycle checkpoint activation and other global cellular responses. Genes encoding DDR factors are frequently mutated in cancer, causing genomic instability, an intrinsic feature of many tumours that underlies their ability to grow, metastasize and respond to treatments that inflict DNA damage (such as radiotherapy). One instance where we have greater insight into how genetic DDR abrogation impacts on therapy responses is in tumours with mutated BRCA1 or BRCA2. Due to compromised homologous recombination DNA repair, these tumours rely on alternative repair mechanisms and are susceptible to chemical inhibitors of poly(ADP-ribose) polymerase (PARP), which specifically kill homologous recombination-deficient cancer cells, and have become a paradigm for targeted cancer therapy. It is now clear that many other synthetic-lethal relationships exist between DDR genes. Crucially, some of these interactions could be exploited in the clinic to target tumours that become resistant to PARP inhibition. In this Review, we discuss state-of-the-art strategies for DDR inactivation using small-molecule inhibitors and highlight those compounds currently being evaluated in the clinic.Genes encoding DNA damage response factors are frequently mutated in cancer, causing genomic instability and presenting opportunities for therapeutic intervention. This Review discusses state-of-the-art strategies for DNA damage response inactivation using small-molecule inhibitors.

Background Polyadenylation plays a key role in producing mature mRNAs in eukaryotes. It is widely believed that the poly(A)-binding proteins (PABs) uniformly bind to poly(A)-tailed mRNAs, regulating their stability and translational efficiency. Results We observe that the homozygous triple mutant of broadly expressed Arabidopsis thaliana PABs, AtPAB2, AtPAB4, and AtPAB8, is embryonic lethal. To understand the molecular basis, we characterize the RNA-binding landscape of these PABs. The AtPAB-binding efficiency varies over one order of magnitude among genes. To identify the sequences accounting for the variation, we perform poly(A)-seq that directly sequences the full-length poly(A) tails. More than 10% of poly(A) tails contain at least one guanosine (G); among them, the G-content varies from 0.8 to 28%. These guanosines frequently divide poly(A) tails into interspersed A-tracts and therefore cause the variation in the AtPAB-binding efficiency among genes. Ribo-seq and genome-wide RNA stability assays show that AtPAB-binding efficiency of a gene is positively correlated with translational efficiency rather than mRNA stability. Consistently, genes with stronger AtPAB binding exhibit a greater reduction in translational efficiency when AtPAB is depleted. Conclusions Our study provides a new mechanism that translational efficiency of a gene can be regulated through the G-content-dependent PAB binding, paving the way for a better understanding of poly(A) tail-associated regulation of gene expression.

Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a severely debilitating disease of unknown pathogenesis consisting of a variety of symptoms including severe fatigue. The objective of the study was to examine the efficacy and safety of a TLR-3 agonist, rintatolimod (Poly I: C(12)U), in patients with debilitating CFS/ME. A Phase III prospective, double-blind, randomized, placebo-controlled trial comparing twice weekly IV rintatolimod versus placebo was conducted in 234 subjects with long-standing, debilitating CFS/ME at 12 sites. The primary endpoint was the intra-patient change from baseline at Week 40 in exercise tolerance (ET). Secondary endpoints included concomitant drug usage, the Karnofsky Performance Score (KPS), Activities of Daily Living (ADL), and Vitality Score (SF 36). Subjects receiving rintatolimod for 40 weeks improved intra-patient placebo-adjusted ET 21.3% (p = 0.047) from baseline in an intention-to-treat analysis. Correction for subjects with reduced dosing compliance increased placebo-adjusted ET improvement to 28% (p = 0.022). The improvement observed represents approximately twice the minimum considered medically significant by regulatory agencies. The rintatolimod cohort vs. placebo also reduced dependence on drugs commonly used by patients in an attempt to alleviate the symptoms of CFS/ME (p = 0.048). Placebo subjects crossed-over to receive rintatolimod demonstrated an intra-patient improvement in ET performance at 24 weeks of 39% (p = 0.04). Rintatolimod at 400 mg twice weekly was generally well-tolerated. Rintatolimod produced objective improvement in ET and a reduction in CFS/ME related concomitant medication usage as well as other secondary outcomes. ClinicalTrials.gov NCT00215800.

Poly-ADP-ribosylation (PARylation) is a fully reversible post-translational modification with key roles in cellular physiology. Due to the multi-domain structure of poly(ADP-ribose) polymerase-1 (PARP1) and the highly dynamic nature of the PARylation reaction, studies on the biochemical mechanism and structural dynamics remain challenging. Here, we report label-free, time-resolved monitoring of PARP1-dependent PARylation using ATR-FTIR spectroscopy. This includes PARP1 activation by binding to DNA strand break models, NAD + substrate binding, PAR formation, and dissociation of automodified PARP1 from DNA. Analyses of PARP1 activation at different DNA models demonstrate a strong positive correlation of PARylation and PARP1 dissociation, with the strongest effects observed for DNA nicks and 3’ phosphorylated ends. Moreover, by examining dynamic structural changes of PARP1, we reveal changes in the secondary structure of PARP1 induced by NAD + and PARP inhibitor binding. In summary, this approach enables holistic and dynamic insights into PARP1-dependent PARylation with molecular and temporal resolution. The mechanism of PARP1-dependent poly-ADP-ribosylation in response to DNA damage is still under debate. Here, the authors use ATR-FTIR spectroscopy to provide time-resolved insights into the molecular details of this process under near physiological conditions.

Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification involved in multiple biological processes, including DNA damage repair. This modification is catalyzed by poly(ADP-ribose) polymerase (PARP) family of enzymes. PARylation is composed of both linear and branched polymers of poly(ADP-ribose) (PAR). However, the biochemical mechanism of polymerization and biological functions of branched PAR chains are elusive. Here we show that PARP2 is preferentially activated by PAR and subsequently catalyzes branched PAR chain synthesis. Notably, the direct binding to PAR by the N-terminus of PARP2 promotes the enzymatic activity of PARP2 toward the branched PAR chain synthesis. Moreover, the PBZ domain of APLF recognizes the branched PAR chain and regulates chromatin remodeling to DNA damage response. This unique feature of PAR-dependent PARP2 activation and subsequent PARylation mediates the participation of PARP2 in DNA damage repair. Thus, our results reveal an important molecular mechanism of branched PAR synthesis and a key biological function of branched PARylation. PARP1 and PARP2 of the PARP family enzymes are involved in DNA damage response. Here the authors report PARP2 activation mechanisms and its role in the formation of branched poly(ADP-ribose) chains in response to DNA damage.

Selective inhibitors of PARP1 and PARP2 (PARP1/2) are used to treat cancer patients with deficiencies in the repair of DNA via homologous recombination. Here we provide a perspective on the reported potencies of the most studied of these inhibitors (olaparib, talazoparib, niraparib, rucaparib, and veliparib) in vitro and in vivo and how these numbers relate to the known structures of these inhibitors bound to the active sites of PARP1 and PARP2. We suggest that the phenomenon of PARP trapping is primarily due to the inhibition of the catalytic activity of PARP1 and that the basis for the higher potency of talazoparib compared to the other inhibitors lies in its more extensive network of interactions with conserved residues in the active site. We also consider the potential role of the recently characterized protein “Histone PARylation Factor 1” (HPF1), which interacts with PARP1/2 to form a shared active site, for the design of the next generation of inhibitors of PARP1/2.

The anti-cancer drug target poly(ADP-ribose) polymerase 1 (PARP1) and its close homologue, PARP2, are early responders to DNA damage in human cells 1 , 2 . After binding to genomic lesions, these enzymes use NAD + to modify numerous proteins with mono- and poly(ADP-ribose) signals that are important for the subsequent decompaction of chromatin and the recruitment of repair factors 3 , 4 . These post-translational modifications are predominantly serine-linked and require the accessory factor HPF1, which is specific for the DNA damage response and switches the amino acid specificity of PARP1 and PARP2 from aspartate or glutamate to serine residues 5 – 10 . Here we report a co-structure of HPF1 bound to the catalytic domain of PARP2 that, in combination with NMR and biochemical data, reveals a composite active site formed by residues from HPF1 and PARP1 or PARP2 . The assembly of this catalytic centre is essential for the addition of ADP-ribose moieties after DNA damage in human cells. In response to DNA damage and occupancy of the NAD + -binding site, the interaction of HPF1 with PARP1 or PARP2 is enhanced by allosteric networks that operate within the PARP proteins, providing an additional level of regulation in the induction of the DNA damage response. As HPF1 forms a joint active site with PARP1 or PARP2, our data implicate HPF1 as an important determinant of the response to clinical PARP inhibitors. Assembly of a catalytic centre formed by HPF1 bound to PARP1 or PARP2 is essential for protein ADP-ribosylation after DNA damage in human cells.

Up to 30% of patients with metastatic castration-resistant prostate cancer have deleterious mutations in genes involved in homologous recombination repair of DNA damage. The use of the PARP inhibitor olaparib in such patients was associated with longer progression-free survival and a longer time to pain progression than control therapy.

Patients with newly diagnosed advanced ovarian cancer were randomly assigned to receive daily niraparib, a PARP inhibitor, or placebo as maintenance therapy after having had a response to platinum-based chemotherapy. Progression-free survival was significantly longer in the niraparib group than in the placebo group, with some increase in the frequency of myelosuppression and nausea.

Using an adaptive trial design to minimize the exposure of patients to inactive agents and to detect more active regimens sooner, investigators found that adding veliparib and carboplatin to standard therapy improved outcome in triple-negative breast cancer. Breast cancer is genetically and clinically heterogeneous, which makes it challenging to identify effective patient-specific therapies. Although mortality due to breast cancer in the United States has decreased, more than 40,000 women in the United States still die from this disease each year. 1 Further decreases in mortality will require therapeutic options that target biologic properties of tumors and can be delivered early enough in the disease course to make a clinical difference. The neoadjuvant approach facilitates the evaluation of an individual patient's response to treatment and holds promise for the development of experimental therapies for disease while it is still . . .