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Durvalumab is a programmed death-ligand 1 (PD-L1) inhibitor with clinical activity in advanced urothelial cancer (AUC) 1 . AUC is characterized by several recurrent targetable genomic alterations 2 – 5 . This study ( NCT02546661 , BISCAY) combined durvalumab with relevant targeted therapies in biomarker-selected chemotherapy-refractory AUC populations including: (1) fibroblast growth factor receptor (FGFR) inhibitors in tumors with FGFR DNA alterations (FGFRm); (2) pharmacological inhibitor of the enzyme poly-ADP ribose polymerase (PARP) in tumors with and without DNA homologous recombination repair deficiency (HRRm); and (3) TORC1/2 inhibitors in tumors with DNA alteration to the mTOR/PI3K pathway 3 – 5 .This trial adopted a new, biomarker-driven, multiarm adaptive design. Safety, efficacy and relevant biomarkers were evaluated. Overall, 391 patients were screened of whom 135 were allocated to one of six study arms. Response rates (RRs) ranged 9–36% across the study arms, which did not meet efficacy criteria for further development. Overall survival (OS) and progression-free survival (PFS) were similar in the combination arms and durvalumab monotherapy arm. Biomarker analysis showed a correlation between circulating plasma-based DNA (ctDNA) and tissue for FGFRm. Sequential circulating tumor DNA analysis showed that changes to FGFRm correlated with clinical outcome. Our data support the clinical activity of FGFR inhibition and durvalumab monotherapy but do not show increased activity for any of the combinations. These findings question the targeted/immune therapy approach in AUC. The adaptive, biomarker-driven BISCAY trial evaluating durvalumab with targeted agents in patients with metastatic urothelial carcinoma based on tumor genomic alterations finds no added clinical benefit over durvalumab monotherapy.

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.

PARP1 detection of DNA strand breaks allosterically leads to PARP1 synthesis of poly(ADP-ribose) modifications that signal DNA damage. HPF1 engages activated PARP1 to control modification site selection. Understanding of the mechanism of DNA break detection and catalytic activation is incomplete, due largely to limited structural information for full-length PARP1. Here, single-particle cryo-EM provides views of the full complement of PARP1 domains engaging a DNA single-strand break in the presence of HPF1 and a fragment of binding partner Timeless. Cryo-EM, single-molecule DNA dynamics, and small-angle X-ray scattering analysis indicate that PARP1 remains dynamic even when the multi-domain structure is organized on a DNA break, with the minimal catalytic region displaying high mobility relative to domains engaging damage. We propose that the organization of PARP1 domains on a DNA break releases a tethered, constitutively active catalytic region to modify molecules in a radius surrounding the DNA break site. PARP1 is a first responder to DNA damage. Here, multiple cryo-EM strategies and biophysical tools analyze PARP1 and partner HPF1 detecting a DNA break, revealing a striking mobility of the PARP1 catalytic domain relative to the DNA binding domains.

PARP1 is recognized for its role as a DNA damage sensor and its involvement in inflammatory diseases, but its impact on prostatitis remains unclear. We aimed to elucidate how PARP1 affects prostatitis progression. Our results showed that in 1% carrageenan-induced prostatitis mouse model, Parp1 −/− prostatitic mice showed less pathological damage, decreased prostate weight, and lower inflammatory indices, decreased macrophage and neutrophil infiltration, down-regulated the expression of pro-inflammatory cytokines (IL-6, IL-12p70, CCL2, TNF) and up-regulated anti-inflammatory cytokine IL-10 in prostate tissue. The expression of NF-κB, TNF, and IL-6 mRNA in the prostate tissue of Parp1 −/− prostatitic mice decreased. In vitro experiments revealed that M1(CD206 − CD86 +) macrophage in LPS-induced macrophage of Parp1 −/− mice decreased, as did iNOS, TNF, IL-6 and NF-κB mRNA expression. Mechanically, treatment with the PARP1 inhibitor (AG14361) led to a significant reduction in NF-κB mRNA and Phospho-NF-κB P65 protein expression in macrophages. Following intervention with NF-κB inhibitors (Bay 11–7082), both IL-6 protein and mRNA levels were markedly diminished, meanwhile the secretion of IL-6, IL-10, IL-12p70, CCL2, IFN-γ, and TNF exhibited a pronounced dose-dependent decrease. Collectively, these findings indicated that PARP1 exacerbates carrageenan-induced prostatitis by promoting M1 macrophages polarization via the NF-κB pathway, suggesting PARP1 could be a potential therapeutic target for macrophage-based treatments in prostatitis.

How pathologic α-synuclein (α-syn) leads to neurodegeneration in Parkinson's disease (PD) remains poorly understood. Kam et al. studied the α-syn preformed fibril (α-syn PFF) model of sporadic PD (see the Perspective by Brundin and Wyse). They found that pathologic α-syn–activated poly(adenosine 5′-diphosphate–ribose) (PAR) polymerase–1 (PARP-1) and inhibition of PARP or knockout of PARP-1 protected mice from pathology. The generation of PAR by α-syn PFF–induced PARP-1 activation converted α-syn PFF to a strain that was 25-fold more toxic, termed PAR–α-syn PFF. An increase of PAR in the cerebrospinal fluid and evidence of PARP activation in the substantia nigra of PD patients indicates that PARP activation contributes to the pathogenesis of PD through parthanatos and conversion of α-syn to a more toxic strain. Science , this issue p. eaat8407 ; see also p. 521 Poly(ADP-ribose) polymerase-1 (PARP-1) accelerates the formation of pathological α-synuclein, resulting in cell death. The pathologic accumulation and aggregation of α-synuclein (α-syn) underlies Parkinson’s disease (PD). The molecular mechanisms by which pathologic α-syn causes neurodegeneration in PD are not known. Here, we found that pathologic α-syn activates poly(adenosine 5′-diphosphate–ribose) (PAR) polymerase-1 (PARP-1), and PAR generation accelerates the formation of pathologic α-syn, resulting in cell death via parthanatos. PARP inhibitors or genetic deletion of PARP-1 prevented pathologic α-syn toxicity. In a feed-forward loop, PAR converted pathologic α-syn to a more toxic strain. PAR levels were increased in the cerebrospinal fluid and brains of patients with PD, suggesting that PARP activation plays a role in PD pathogenesis. Thus, strategies aimed at inhibiting PARP-1 activation could hold promise as a disease-modifying therapy to prevent the loss of dopamine neurons in PD.

The observation that BRCA1 - and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP–ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination 1 . The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein–DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins 1 – 4 . To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor 1 . Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair 5 . Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukaemia could provide an opportunity to exploit these findings therapeutically. Mutations in all three genes encoding ribonuclease H2 sensitize cells to poly(ADP–ribose) polymerase inhibitors by compromising ribonucleotide excision repair.

The pro-apoptotic tumor suppressor BIN1 inhibits the activities of the neoplastic transcription factor MYC, poly (ADP-ribose) polymerase-1 (PARP1), and ATM Ser/Thr kinase (ATM) by separate mechanisms. Although BIN1 deficits increase cancer-cell resistance to DNA-damaging chemotherapeutics, such as cisplatin, it is not fully understood when BIN1 deficiency occurs and how it provokes cisplatin resistance. Here, we report that the coordinated actions of MYC, PARP1, and ATM assist cancer cells in acquiring cisplatin resistance by BIN1 deficits. Forced BIN1 depletion compromised cisplatin sensitivity irrespective of Ser15-phosphorylated, pro-apoptotic TP53 tumor suppressor. The BIN1 deficit facilitated ATM to phosphorylate the DNA-damage-response (DDR) effectors, including MDC1. Consequently, another DDR protein, RNF8, bound to ATM-phosphorylated MDC1 and protected MDC1 from caspase-3-dependent proteolytic cleavage to hinder cisplatin sensitivity. Of note, long-term and repeated exposure to cisplatin naturally recapitulated the BIN1 loss and accompanying RNF8-dependent cisplatin resistance. Simultaneously, endogenous MYC was remarkably activated by PARP1, thereby repressing the BIN1 promoter, whereas PARP inhibition abolished the hyperactivated MYC-dependent BIN1 suppression and restored cisplatin sensitivity. Since the BIN1 gene rarely mutates in human cancers, our results suggest that simultaneous inhibition of PARP1 and ATM provokes a new BRCAness-independent synthetic lethal effect and ultimately re-establishes cisplatin sensitivity even in platinum-refractory cancer cells.

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.

Resistance to androgen deprivation therapy, or castration-resistant prostate cancer (CRPC), is often accompanied by metastasis and is currently the ultimate cause of prostate cancer-associated deaths in men. Recently, secondary hormonal therapies have led to an increase of neuroendocrine prostate cancer (NEPC), a highly aggressive variant of CRPC. Here, we identify that high levels of cell surface receptor Trop2 are predictive of recurrence of localized prostate cancer. Moreover, Trop2 is significantly elevated in CRPC and NEPC, drives prostate cancer growth, and induces neuroendocrine phenotype. Overexpression of Trop2 induces tumor growth and metastasis while loss of Trop2 suppresses these abilities in vivo. Trop2-driven NEPC displays a significant up-regulation of PARP1, and PARP inhibitors significantly delay tumor growth and metastatic colonization and reverse neuroendocrine features in Trop2-driven NEPC. Our findings establish Trop2 as a driver and therapeutic target for metastatic prostate cancer with neuroendocrine phenotype and suggest that high Trop2 levels could identify cancers that are sensitive to Trop2-targeting therapies and PARP1 inhibition.

DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD⁺ (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD⁺ to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate–ribose) polymerase], a critical DNA repair protein. As mice age and NAD⁺ concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD⁺. Thus, NAD⁺ directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging.