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Choline Kinase is a key component of the Kennedy pathway that converts choline into a number of structural and signalling lipids that are essential for cell growth and survival. One member of the family, Choline Kinase-α (ChoKα) is frequently up-regulated in human cancers, and expression of ChoKα is sufficient to transform cells. Consequently ChoKα has been studied as a potential target for therapeutic agents in cancer research. Despite great interest in the enzyme, mechanistic studies have not been reported. In this study, a combination of initial velocity and product inhibition studies, together with the kinetic and structural characterisation of a novel ChoKα inhibitor is used to support a mechanism of action for human ChoKα. Substrate and inhibition kinetics are consistent with an iso double displacement mechanism, in which the γ-phosphate from ATP is transferred to choline in two distinct steps via a phospho-enzyme intermediate. Co-crystal structures, and existing site-specific mutation studies, support an important role for Asp306, in stabilising the phospho-enzyme intermediate. The kinetics also indicate a distinct kinetic (isomerisation) step associated with product release, which may be attributed to a conformational change in the protein to disrupt an interaction between Asp306 and the phosphocholine product, facilitating product release. This study describes a mechanism for ChoKα that is unusual amongst kinases, and highlights the availability of different enzyme states that can be exploited for drug discovery.

DNA damaging agents such as radiotherapy and gemcitabine are frequently used for the treatment of pancreatic cancer. However, these treatments typically provide only modest benefit. Improving the low survival rate for pancreatic cancer patients therefore remains a major challenge in oncology. Inhibition of the key DNA damage response kinase ATR has been suggested as an attractive approach for sensitization of tumor cells to DNA damaging agents, but specific ATR inhibitors have remained elusive. Here we investigated the sensitization potential of the first highly selective and potent ATR inhibitor, VE-821, in vitro. VE-821 inhibited radiation- and gemcitabine-induced phosphorylation of Chk1, confirming inhibition of ATR signaling. Consistently, VE-821 significantly enhanced the sensitivity of PSN-1, MiaPaCa-2 and primary PancM pancreatic cancer cells to radiation and gemcitabine under both normoxic and hypoxic conditions. ATR inhibition by VE-821 led to inhibition of radiation-induced G 2/M arrest in cancer cells. Reduced cancer cell radiosurvival following treatment with VE-821 was also accompanied by increased DNA damage and inhibition of homologous recombination repair, as evidenced by persistence of γH2AX and 53BP1 foci and inhibition of Rad51 foci, respectively. These findings support ATR inhibition as a novel approach to improve the efficacy and therapeutic index of standard cancer treatments across a large proportion of pancreatic cancer patients.

Combined radiochemotherapy is the currently used therapy for locally advanced pancreatic ductal adenocarcinoma (PDAC), but normal tissue toxicity limits its application. Here we test the hypothesis that inhibition of ATR (ATM-Rad3-related) could increase the sensitivity of the cancer cells to radiation or chemotherapy without affecting normal cells. We tested VE-822, an ATR inhibitor, for in vitro and in vivo radiosensitization. Chk1 phosphorylation was used to indicate ATR activity, γH2AX and 53BP1 foci as evidence of DNA damage and Rad51 foci for homologous recombination activity. Sensitivity to radiation (XRT) and gemcitabine was measured with clonogenic assays in vitro and tumor growth delay in vivo. Murine intestinal damage was evaluated after abdominal XRT. VE-822 inhibited ATR in vitro and in vivo. VE-822 decreased maintenance of cell-cycle checkpoints, increased persistent DNA damage and decreased homologous recombination in irradiated cancer cells. VE-822 decreased survival of pancreatic cancer cells but not normal cells in response to XRT or gemcitabine. VE-822 markedly prolonged growth delay of pancreatic cancer xenografts after XRT and gemcitabine-based chemoradiation without augmenting normal cell or tissue toxicity. These findings support ATR inhibition as a promising new approach to improve the therapeutic ration of radiochemotherapy for patients with PDAC.

During his pontificate, Pius xi (1922–39) vigorously promoted the ‘export’ of the Italian model of Catholic Action to the rest of the Church as the organisational blueprint for Catholic lay activism, particularly in the battle against atheistic Communism. He was assisted by Mgr, later Cardinal, Giuseppe Pizzardo, the head of Italian Catholic Action. The successes and failures of this campaign are tracked through documents available since the opening of the files of the apostolic nunciatures and delegations and the Extraordinary Ecclesiastical Affairs section of the Secretariat of State for Pius xi's pontificate in the Vatican Archives.

En 1997, le manque de données disponibles sur les Orchidaceae Juss. d'Afrique centrale atlantique et l'importance des menaces pesant sur la forêt africaine, comme sur les épiphytes qu'elles abritent, nous ont amenés à entreprendre une vaste campagne d'échantillonnage sur l'ensemble de cette région. L'examen du matériel récolté et des collections historiques a permis des avancées taxonomiques sur un grand nombre d'espèces endémiques de la région, ou jusqu'à présent considérées comme telles, et a abouti à la mise à jour de leurs aires de distribution. Des mises en synonymie sont proposées : Ancistrorhynchus constrictus Szlach. & Olszewski est inclus dans Ancistrorhynchus capitatus (Lindl.) Summerh. ; Bulbophyllum mayombeense Garay est mis en synonymie de Bulbophyllum schinzianum Kraenzl. var. schinzianum ; et Bulbophyllum kupense P.J.Cribb & B.J.Pollard devient synonyme de Bulbophyllum teretifolium Schltr. Une néotypification est faite pour cette dernière espèce, de même que pour Liparis goodyeroides Schltr. La distribution géographique de plusieurs taxons est modifiée : Aerangis megaphylla Summerh. est bien endémique de l'île d'Annobon, Polystachya superposita Rchb.f. est de nouveau considéré comme endémique du Cameroun et Liparis suborbicularis Summerh. n'est plus signalé qu_au Nigeria et au Cameroun. Trois nouvelles signalisations sont mentionnées : Aerangis bouarensis Chiron pour le Cameroun, Bulbophyllum schinzianum Kraenzl. var. schinzianum pour la République du Congo et Liparis goodyeroides Schltr. pour le Liberia. Les statuts de conservation selon les catégories et critères de l'IUCN sont donnés pour chaque taxon.

This article analyses the parallels between the role played by the Church, first during the Crisis of the Liberal State in the early twentieth century and then during the transition from the Christian Democratic regime to the ‘bi-polar’ Second Republic more than 70 years later. It explores both the particular, contingent forces at work in each, and the underlying explanations as to why the Church was able to successfully exploit these two processes of transition in the political history of Italy to its advantage. It concludes by arguing that the experience of these two crises demonstrates that the Church is not only a powerful force in Italian civil society but also effectively ‘a state within a state’ in relation to the functioning of Italy's political structures.

DNA-damaging agents are among the most frequently used anticancer drugs. However, they provide only modest benefit in most cancers. This may be attributed to a genome maintenance network, the DNA damage response (DDR), that recognizes and repairs damaged DNA. ATR is a major regulator of the DDR and an attractive anticancer target. Herein, we describe the discovery of a series of aminopyrazines with potent and selective ATR inhibition. Compound 45 inhibits ATR with a K(i) of 6 nM, shows >600-fold selectivity over related kinases ATM or DNA-PK, and blocks ATR signaling in cells with an IC(50) of 0.42 μM. Using this compound, we show that ATR inhibition markedly enhances death induced by DNA-damaging agents in certain cancers but not normal cells. This differential response between cancer and normal cells highlights the great potential for ATR inhibition as a novel mechanism to dramatically increase the efficacy of many established drugs and ionizing radiation.

Background & Aims Non-alcoholic fatty liver disease is a progressive condition comprising steatosis, steatohepatitis, and cirrhosis. Caspase activation mediates apoptosis and the inflammatory response. Studies demonstrate increased apoptotic activity in NASH although its pathophysiological importance is uncertain. We sought to determine the effects of irreversible pan-caspase inhibition in murine models of established steatosis (high fat diet, HFD) and steatohepatitis (methionine-choline deficient diet, MCD). Methods In one study arm, male C3H/HeN mice were fed HFD; in the other, Db/Db mice were fed MCD. Once disease was established, animals were randomised to receive caspase inhibitor (VX-166), TPGS/PEG vehicle or no additional therapy until the end of the study. Biochemical and histological indices were examined to determine NASH activity and tissue oxidative stress. Apoptotic activity and cell turnover were assessed immunohistochemically by staining for caspase-cleaved CK-18 and PCNA. Results MCD and HFD significantly increased apoptosis, which was reduced by VX-166 treatment. VX-166 did not reduce steatosis but reduced histological inflammation, serum ALT levels, and oxidative stress, particularly in the MCD model. TPGS/PEG vehicle also exhibited some anti-inflammatory activity. Conclusions In both models, VX-166 inhibited apoptosis and reduced histological inflammatory infiltrate although there was a more modest impact on other indices of liver injury. In addition, TPGS/PEG vehicle also exhibited some anti-inflammatory activity, likely through the antioxidant effects of vitamin E and changes in gut flora/mucosal interactions. These data suggest that caspase inhibition may represent a valid therapeutic approach; however, further studies to assess the long-term value of more selective caspase inhibition are merited.

Despite multiple calls for reform, the curriculum for first-year college chemistry at many universities across the world is still mostly fact-based and encyclopedic, built upon a collection of isolated topics, oriented too much towards the perceived needs of chemistry majors, focused too much on abstract concepts and algorithmic problem solving, and detached from the practices, ways of thinking, and applications of both chemistry research and chemistry education research in the 21st century. This paper describes an alternative way of conceptualizing the introductory chemistry curriculum for science and engineering majors by shifting the focus from learning chemistry as a body of knowledge to understanding chemistry as a way of thinking. Starting in 2007, we have worked on the development and implementation of a new curriculum intended to: promote deeper conceptual understanding of a minimum core of fundamental ideas instead of superficial coverage of multiple topics; connect core ideas between the course units by following well-defined learning progressions; introduce students to modern ways of thinking and problem-solving in chemistry; and involve students in realistic decision-making and problem-solving activities.

The identification of a novel series of Aurora kinase inhibitors and exploitation of their SAR is described. Replacement of the initial quinazoline core with a pyrimidine scaffold and modification of substituents led to a series of very potent inhibitors of cellular proliferation. MK-0457 (VX-680) has been assessed in Phase II clinical trials in patients with treatment-refractory chronic myelogenous leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) containing the T315I mutation.

(2007). A Court in Exile: The Vatican, 1870–1929. The Court Historian: Vol. 12, No. 1, pp. 35-47.

It was the Catholic priest and leader of the Italian People's Party, Luigi Sturzo, who first coined the term ‘clerico‐fascism’, rather than ‘clerical fascism’, back in 1922. He applied it to former...

(S)-1-((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide (VX-765) is an orally absorbed prodrug of (S)-3-({1-[(S)-1-((S)-2-{[1-(4-amino-3-chlorophenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidin-2yl]-methanoyl}-amino)-4-oxo-butyric acid (VRT-043198), a potent and selective inhibitor of interleukin-converting enzyme/caspase-1 subfamily caspases. VRT-043198 exhibits 100- to 10,000-fold selectivity against other caspase-3 and -6 to -9. The therapeutic potential of VX-765 was assessed by determining the effects of VRT-043198 on cytokine release by monocytes in vitro and of orally administered VX-765 in several animal models in vivo. In cultures of peripheral blood mononuclear cells and whole blood from healthy subjects stimulated with bacterial products, VRT-043198 inhibited the release of interleukin (IL)-1beta and IL-18, but it had little effect on the release of several other cytokines, including IL-1alpha, tumor necrosis factor-alpha, IL-6 and IL-8. In contrast, VRT-043198 had little or no demonstrable activity in cellular models of apoptosis, and it did not affect the proliferation of activated primary T cells or T-cell lines. VX-765 was efficiently converted to VRT-043198 when administered orally to mice, and it inhibited lipopolysaccharide-induced cytokine secretion. In addition, VX-765 reduced disease severity and the expression of inflammatory mediators in models of rheumatoid arthritis and skin inflammation. These data suggest that VX-765 is a novel cytokine inhibitor useful for treatment of inflammatory diseases.

In this case, we report difficulty in the conduct of carotid surgery during an earthquake. With the epicenter >200 miles away, intraoperative movement was subtle and initially attributed to recovery from neuromuscular blockade. More significant seismic motion in a hospital is accompanied by additional challenges that are discussed.

Background: Return to Driving and Employment are goals for many stroke survivors. These person-centered goals are often the motivators for participation in outpatient rehabilitation. However, standardized measure sets and patient assessments are not in place or required for outpatient rehabilitation programs. As such, few outpatient rehabilitation programs measure and report patient centered outcomes like return to work or employment in addition to functional status and disease management measures. Methods: Prospective observational study of recovery of stroke patients treated between 12/2011-1/2015 in an interdisciplinary outpatient rehabilitation program that addresses health literacy, risk factors, physical, psychosocial, cognitive, communicative and vocational issues. Results: Among 96 consecutive patients (69 ischemic, 27 hemorrhages), with admit NIHSS scores 0-16, there were 59 men, 37 women, with an age range of 18-90 years. At baseline(admission to the outpatient program), 36.5% were aphasic(communication impairment), 88% had impairments and needed assistance with activities of daily living (55% and 33% with modified Rankin Scale [mRS] Scores of 3 and 4, respectively), compared with mild impairments (12%mRS 1-2). Of 46 patients working pre-stroke, 48% had returned to work and an additional 13% were work capable at discharge. Of 86 patients driving pre-stroke, 38% returned to driving at completion of the program and 27% began using public transportation. Stroke Impact Scale (SIS) stroke recovery Domain scores increased an average of 38%, SIS ADL scores 33%, SIS mood scores 17%. Cardiovascular and medication education sessions with the Nurse Practitioner(NP) were based on AHA guidelines that included Life’s Simple 7’s curriculum. Stroke Knowledge scores improved an average of 14% and stroke risk factor knowledge improved an average of 19% across program participants. Program treatment also emphasized blood pressure control and exercise, with all but 1 patient receiving an average of 23 physical therapy visits over the duration of program involvement. Compared with baseline, 6 minute walk and Berg Balance scores improved an average of 79% and 33%, respectively. Blood pressures(BPs) measured during the 1st 75% of PT visits were compared to BPs during the last 25%. 56% of patients had fewer moderate(140-159/91-99) to severe(>160/100) BPs and more normal( Conclusions: Participants in our interdisciplinary community-based rehabilitation program demonstrated improvements in stroke knowledge, physical function, blood pressure control, and mood, while also returning to work, driving, and participation in the community. Comparative research and multi-site studies are needed.

ATR is an attractive target in cancer therapy because it signals replication stress and DNA lesions for repair and to S/G2 checkpoints. Cancer-specific defects in the DNA damage response (DDR) may render cancer cells vulnerable to ATR inhibition alone. We determined the cytotoxicity of the ATR inhibitor VE-821 in isogenically matched cells with DDR imbalance. Cell cycle arrest, DNA damage accumulation and repair were determined following VE-821 exposure. Defects in homologous recombination repair (HRR: ATM, BRCA2 and XRCC3) and base excision repair (BER: XRCC1) conferred sensitivity to VE-821. Surprisingly, the loss of different components of the trimeric non-homologous end-joining (NHEJ) protein DNA-PK had opposing effects. Loss of the DNA-binding component, Ku80, caused hypersensitivity to VE-821, but loss of its partner catalytic subunit, DNA-PKcs, did not. Unexpectedly, VE-821 was particularly cytotoxic to human and hamster cells expressing high levels of DNA-PKcs. High DNA-PKcs was associated with replicative stress and activation of the DDR. VE-821 suppressed HRR, determined by RAD51 focus formation, to a greater extent in cells with high DNA-PKcs. Defects in HRR and BER and high DNA-PKcs expression, that are common in cancer, confer sensitivity to ATR inhibitor monotherapy and may be developed as predictive biomarkers for personalised medicine.

Summary ATR and CHK1 maintain cancer cell survival under replication stress and inhibitors of both kinases are currently undergoing clinical trials. As ATR activity is increased after CHK1 inhibition, we hypothesized that this may indicate an increased reliance on ATR for survival. Indeed, we observe that replication stress induced by the CHK1 inhibitor AZD7762 results in replication catastrophe and apoptosis, when combined with the ATR inhibitor VE-821 specifically in cancer cells. Combined treatment with ATR and CHK1 inhibitors leads to replication fork arrest, ssDNA accumulation, replication collapse, and synergistic cell death in cancer cells in vitro and in vivo. Inhibition of CDK reversed replication stress and synthetic lethality, demonstrating that regulation of origin firing by ATR and CHK1 explains the synthetic lethality. In conclusion, this study exemplifies cancer-specific synthetic lethality between two proteins in the same pathway and raises the prospect of combining ATR and CHK1 inhibitors as promising cancer therapy., Graphical Abstract, Highlights • Synthetic lethality within the same pathway is via the induction of replication catastrophe • New origin firing and replication stress induced by CHK1 inhibitor reliance on ATR • ATR and CHK1 inhibitor combination results in cancer-specific cytotoxicity • ATR and CHK1 inhibitor combination could be used broadly across cancer indications, Sanjiv et al. report synthetic lethality in cancer cells induced by the inhibition of two kinases, ATR and CHK1, operating in the same signaling pathway. This combination therapy could potentially be used for the treatment of various cancer types due to cancer-specific cytotoxicity.

Cyperus microcristatus, a new species from western Cameroon, is described and illustrated. Its taxonomic affinities are discussed. Cyperus microcristatus is most similar to C. densicaespitosus (syn. Kyllinga pumila) in general appearance, but differs in its more perennial habit, its smaller spikelets and glumes, which are more prominently winged. Notes on habitat, ecology, distribution and conservation are provided. It is assessed as Critically Endangered, following IUCN (2001).

This paper examines coastal management policies in southern Spain in the context of the state's role in the promotion of sustainable development practices. It recognises the critical importance of the coast as a landscape resource for both visitors and residents and, in addition, the latter's rights to enjoy access to it as public property. Attention is directed at both physical protection of the coast through erosion control programmes and urban planning policies, with special emphasis on the 1988 Shores Act that attempts to treat coastal planning in a more integrated manner than has historically been the case. The underlying rationale to introduce a more environmentally sensitive treatment of the coast is discussed together with the attempts to confront illegal occupancy through re-establishment of public ownership and control of land-use in the coastal zone. Positive achievements in beach management, and changing attitudes to the law, are seen as evidence that the message of sustainability is beginning...

A new rare species, Scleria afroreflexa. is descriped from an upland area (1500 m) of South west Cameroon. It is most similar to S. sheilae Rayn., another rare species only known from one collection from a rocky habitat at 800 m near Yaounde in southeastern Cameroon. S. afroreflexa differs, however, in its more dense inflorescence with reflexed branches, its more hairy leaves, smaller achenes and in its montane habitat. Notes on habitat, ecology, distribution and conservation are given for the two species.

DanShen is a Chinese medicine that is used to treat cardiovascular disorders. DanShen is moderately to strongly protein bound, mainly to albumin. Because impaired protein binding of albumin‐bound drugs in uremia has been reported, we studied protein binding of DanShen by measuring the digoxin‐like immunoreactive component of this Chinese medicine. We observed a significantly higher percentage of free fraction of DanShen in uremic sera in vitro. Impaired protein binding of DanShen was also observed in sera from patients with liver disease, who had elevated concentrations of bilirubin. Treating uremic sera with activated charcoal significantly improved the protein binding of DanShen, indicating that uremic compounds are responsible for the impaired protein binding of DanShen. On the other hand, when various amounts of bilirubin were added to aliquots of the normal pool supplemented with DanShen, we observed only a modest displacement of DanShen from the protein‐binding sites by bilirubin, indicating that hypoalbuminemia may play a major role in impaired protein binding of DanShen in sera with elevated bilirubin concentrations. We conclude that protein binding of DanShen is lower in uremic sera and in sera with elevated bilirubin concentrations. J. Clin. Lab. Anal. 17:179–183, 2003. © 2003 Wiley‐Liss, Inc.

This paper examines some of the Spanish coastal management policies that have relevance for the small business enterprises located on the sea-front. Physical protection of the coast through erosion control programmes and urban planning policies are considered, with special emphasis on the 1988 Shores Act that attempts to treat coastal planning more holistically than has historically been the case. Business awareness and response to the coastal actions are investigated through a survey of 150 small businesses in four localities on the Costa del Sol. Results indicate a lack of universal understanding and approval of management policies and objectives, although significant geographical variations exist in views expressed. Forced relocation of businesses has influenced attitudes, as have perceptions that money has not been effectively spent. However, there is clearly much misunderstanding of work that has been effected, and much might be gained through improved dissemination of information by both lo...

Ever since Leo XIII promulgated his encyclical Rerum novarum, ‘On the Conditions of the Working Classes’, in 1891, successive popes have added to the corpus of Catholic teaching on social/ labour questions. Pius X, for example, published an encyclical specifically addressing the vexed question of ‘interconfessional’ Christian trade unions in Germany, and Pius XI published no fewer than three encyclicals on social questions in the space of twelve months – Quadragesimo anno of May 1931, Nova impendet of October 1931, and Cantate Christi compulsi of May 1932. Recent popes, John XXIII, Paul VI, and John Paul II, have been equally prolific in their commentaries on the labour question.

DNA damaging agents, such as cisplatin, gemcitabine or ionizing radiation (IR), represent the cornerstone for the treatment of cancer. However, for many patients they provide only modest benefit. One reason for this is the presence of highly effective cellular processes that detect and repair damaged DNA. The kinase ataxia telangiectasia mutated (ATM) and rad3 related (ATR) is a key mediator for one such cellular repair process that responds to replication stress (RS). RS arises when the DNA replication machinery stalls, leading to production of ssDNA, which directly recruits ATR. This occurs at difficult to replicate sites of the genome, when nucleotides are limiting, or when the replication machinery encounters DNA lesions. RS levels are often elevated in cancer cells, for example due to expression of oncogenes that drive unregulated proliferation, an hypoxic environment, or from treatment with DNA damaging drugs and ionizing radiation (IR). Once recruited to sites of RS ATR mediates activation of cell cycle checkpoints, stabilization of the stalled replication fork and repair of damaged DNA by homologous recombination (HR). Unresolved RS often leads to lethal double strand breaks. Over the past few years a number of potent and selective inhibitors of ATR have been reported and are widely used as pre-clinical tools to assess ATR inhibition as an anti-cancer approach. In vitro, inhibition of ATR potentiates the cytotoxic activity of many DNA damaging drugs and IR in many cancer cell lines. In stark contrast non-cancer cells tolerate inhibition of ATR with just transient and rapidly reversible growth arrest. This ability to tolerate ATR inhibition has been attributed to activation of a compensatory damage response mediated by the ATR homolog ATM. Accordingly, defects in ATM pathway function, for example from loss of expression in ATM or defects in a principle ATM substrate, p53, can confer cell sensitivity to ATR inhibition. In human cancer cell line, and patient-derived tumor, mouse xenografts, ATR inhibition markedly enhances the efficacy of a range of DNA damaging chemotherapies and IR with minimal impact on body weight loss. ATR inhibition has also shown single agent activity in certain cancer cell lines that have high background RS levels from expression of oncogenes such as Ras, defects elsewhere in the DNA repair network (e.g., ERRC1), or reliance on the HR-dependent alternative lengthening of telomeres (ALT) mechanism of telomere maintenance. Finally, ATR inhibition has recently been shown to have benefit when combined with targeted agents that impair other components of the DNA damage response. The best characterized of these are inhibitors of poly ADP-ribose polymerase (PARP), which is involved in the repair of single strand breaks and replication fork dynamics. Three ATR inhibitors are in clinical development, progressing as monotherapy and in combination with DNA damaging drugs and IR. Preliminary evidence of clinical activity has recently been reported for the first-in-class agent VX-970. Citation Format: John R. Pollard. Discovery and characterization of potent and selective inhibitors of ATR kinase as anti-cancer agents. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA11.

Camptothecin and its derivatives, topotecan and irinotecan, are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs killing cancer cells by producing replication-associated DNA double-strand breaks, and the indenoisoquinoline LMP-400 (indotecan) is a novel Top1 inhibitor in clinical trial. To develop novel drug combinations, we conducted a synthetic lethal siRNA screen using a library that targets nearly 7,000 human genes. Depletion of ATR, the main transducer of replication stress, came as a top candidate gene for camptothecin synthetic lethality. Validation studies using ATR siRNA and the ATR inhibitor VE-821 confirmed marked antiproliferative synergy with camptothecin and even greater synergy with LMP-400. Single-cell analyses and DNA fiber combing assays showed that VE-821 abrogates the S-phase replication elongation checkpoint and the replication origin–firing checkpoint induced by camptothecin and LMP-400. As expected, the combination of Top1 inhibitors with VE-821 inhibited the phosphorylation of ATR and Chk1; however, it strongly induced γH2AX. In cells treated with the combination, the γH2AX pattern changed over time from the well-defined Top1-induced damage foci to an intense peripheral and diffuse nuclear staining, which could be used as response biomarker. Finally, the clinical derivative of VE-821, VX-970, enhanced the in vivo tumor response to irinotecan without additional toxicity. A key implication of our work is the mechanistic rationale and proof of principle it provides to evaluate the combination of Top1 inhibitors with ATR inhibitors in clinical trials. Cancer Res; 74(23); 6968–79. ©2014 AACR.

// Amy B. Hall 1,* , Dave Newsome 1,* , Yuxin Wang 1,* , Diane M. Boucher 1 , Brenda Eustace 1 , Yong Gu 1 , Brian Hare 1 , Mac A. Johnson 1 , Howard Li 1 , Sean Milton 1 , Cheryl E. Murphy 1 , Darin Takemoto 1 , Crystal Tolman 1 , Mark Wood 1 , Peter Charlton 2 , Jean-Damien Charrier 2 , Brinley Furey 1 , Julian Golec 2 , Philip M. Reaper 2 and John R. Pollard 2 1 Vertex Pharmaceuticals Inc, Boston, MA, USA 2 Vertex Pharmaceuticals (Europe) Ltd, Milton Park, Abingdon, Oxfordshire, UK * These authors contributed equally to this work Correspondence: John R Pollard , email: // Keywords : ATR, DNA damage response, VX-970 Received : June 9, 2014 Accepted : July 2, 2014 Published : July 3, 2014 Abstract Platinum-based DNA-damaging chemotherapy is standard-of-care for most patients with lung cancer but outcomes remain poor. This has been attributed, in part, to the highly effective repair network known as the DNA-damage response (DDR). ATR kinase is a critical regulator of this pathway, and its inhibition has been shown to sensitize some cancer, but not normal, cells in vitro to DNA damaging agents. However, there are limited in vivo proof-of-concept data for ATR inhibition. To address this we profiled VX-970, the first clinical ATR inhibitor, in a series of in vitro and in vivo lung cancer models and compared it with an inhibitor of the downstream kinase Chk1. VX-970 markedly sensitized a large proportion of a lung cancer cell line and primary tumor panel in vitro to multiple DNA damaging drugs with clear differences to Chk1 inhibition observed. In vivo VX-970 blocked ATR activity in tumors and dramatically enhanced the efficacy of cisplatin across a panel of patient derived primary lung xenografts. The combination led to complete tumor growth inhibition in three cisplatin-insensitive models and durable tumor regression in a cisplatin-sensitive model. These data provide a strong rationale for the clinical evaluation of VX-970 in lung cancer patients.

Background ATR, which signals DNA damage to S/G2 cell cycle checkpoints and for repair, is an attractive target in cancer therapy. ATR inhibitors are being developed and a pharmacodynamic assay is needed to support clinical studies. Methods Phosphorylation of ATR targets, Chk1 and H2AX, was evaluated in MCF7 and K562 cells, human volunteer PBMCs and whole blood by Western blot, immunofluorescence microscopy and flow cytometry after DNA damage. The effect of cell cycle phase, ATR knockdown and inhibition on these phosphorylation events was determined. Results Hydroxyurea, UV and 4NQO induced Chk1 and H2AX phosphorylation in MCF7 and K562 cells. UV/4NQO activation of ATR was detectable in non-cycling cells. Chk1 phosphorylation was reduced by ATR knockdown and reflects ATR activity for 3 h, H2AX phosphorylation after UV/4NQO is ATR-dependent for 1 h but increasingly ATM and DNA-PK-dependent at later time points. In isolated PBMCs both phospho-targets were detectable after UV/4NQO but in PBMCs from whole blood treated with 4NQO only H2AX was detectable. Conclusion PhosphoChk1 and H2AX are useful biomarkers for ATR inhibition using a variety of immuno-detection methods, but timing may be critical. Importantly, ATR activity is detectable in non-cycling PBMCs allowing them to be used as a surrogate tissue for biomarker measurement. In PBMCs from whole blood treated with 4NQO phosphoH2AX was the most useful biomarker of ATR activity and a clinically viable pharmacodynamic assay for ATR inhibitors has been developed.