The poly (ADP-ribose) polymerase (PARP) inhibitor olaparib is FDA approved for the treatment of BRCA-mutated breast, ovarian and pancreatic cancers. Olaparib inhibits PARP1/2 enzymatic activity and ...traps PARP1 on DNA at single-strand breaks, leading to replication-induced DNA damage that requires BRCA1/2-dependent homologous recombination repair. Moreover, DNA damage response pathways mediated by the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia mutated and Rad3-related (ATR) kinases are hypothesised to be important survival pathways in response to PARP-inhibitor treatment. Here, we show that olaparib combines synergistically with the ATR-inhibitor AZD6738 (ceralasertib), in vitro, leading to selective cell death in ATM-deficient cells. We observe that 24 h olaparib treatment causes cells to accumulate in G2-M of the cell cycle, however, co-administration with AZD6738 releases the olaparib-treated cells from G2 arrest. Selectively in ATM-knockout cells, we show that combined olaparib/AZD6738 treatment induces more chromosomal aberrations and achieves this at lower concentrations and earlier treatment time-points than either monotherapy. Furthermore, single-agent olaparib efficacy in vitro requires PARP inhibition throughout multiple rounds of replication. Here, we demonstrate in several ATM-deficient cell lines that the olaparib and AZD6738 combination induces cell death within 1-2 cell divisions, suggesting that combined treatment could circumvent the need for prolonged drug exposure. Finally, we demonstrate in vivo combination activity of olaparib and AZD6738 in xenograft and PDX mouse models with complete ATM loss. Collectively, these data provide a mechanistic understanding of combined PARP and ATR inhibition in ATM-deficient models, and support the clinical development of AZD6738 in combination with olaparib.
Abstract
The protein kinase ATR plays pivotal roles in DNA repair, cell cycle checkpoint engagement and DNA replication. Consequently, ATR inhibitors (ATRi) are in clinical development for the ...treatment of cancers, including tumours harbouring mutations in the related kinase ATM. However, it still remains unclear which functions and pathways dominate long-term ATRi efficacy, and how these vary between clinically relevant genetic backgrounds. Elucidating common and genetic-background specific mechanisms of ATRi efficacy could therefore assist in patient stratification and pre-empting drug resistance. Here, we use CRISPR–Cas9 genome-wide screening in ATM-deficient and proficient mouse embryonic stem cells to interrogate cell fitness following treatment with the ATRi, ceralasertib. We identify factors that enhance or suppress ATRi efficacy, with a subset of these requiring intact ATM signalling. Strikingly, two of the strongest resistance-gene hits in both ATM-proficient and ATM-deficient cells encode Cyclin C and CDK8: members of the CDK8 kinase module for the RNA polymerase II mediator complex. We show that Cyclin C/CDK8 loss reduces S-phase DNA:RNA hybrid formation, transcription-replication stress, and ultimately micronuclei formation induced by ATRi. Overall, our work identifies novel biomarkers of ATRi efficacy in ATM-proficient and ATM-deficient cells, and highlights transcription-associated replication stress as a predominant driver of ATRi-induced cell death.
T cell cytokines contribute to immunity against Staphylococcus aureus, but the predominant T cell subsets involved are unclear. In an S. aureus skin infection mouse model, we found that the IL- 17 ...response was mediated by γδ T cells, which trafficked from lymph nodes to the infected skin to induce neutrophil recruitment, proinflammatory cytokines IL-1α, IL-1β, and TNF, and host defense peptides. RNA-seq for TRG and TRD sequences in lymph nodes and skin revealed a single clonotypic expansion of the encoded complementarity-determining region 3 amino acid sequence, which could be generated by canonical nucleotide sequences of TRGV5 or TRGV6 and TRDV4. However, only TRGV6 and TRDV4 but not TRGV5 sequences expanded. Finally, Vγ6⁺ T cells were a predominant γδ T cell subset that produced IL-17A as well as IL-22, TNF, and IFNγ, indicating a broad and substantial role for clonal Vγ6⁺Vδ4⁺ T cells in immunity against S. aureus skin infections.
There is heterogeneity in the names and anatomical descriptions of regional anesthetic techniques. This may have adverse consequences on education, research, and implementation into clinical ...practice. We aimed to produce standardized nomenclature for abdominal wall, paraspinal, and chest wall regional anesthetic techniques.
We conducted an international consensus study involving experts using a three-round Delphi method to produce a list of names and corresponding descriptions of anatomical targets. After long-list formulation by a Steering Committee, the first and second rounds involved anonymous electronic voting and commenting, with the third round involving a virtual round table discussion aiming to achieve consensus on items that had yet to achieve it. Novel names were presented where required for anatomical clarity and harmonization. Strong consensus was defined as ≥75% agreement and weak consensus as 50% to 74% agreement.
Sixty expert Collaborators participated in this study. After three rounds and clarification, harmonization, and introduction of novel nomenclature, strong consensus was achieved for the names of 16 block names and weak consensus for four names. For anatomical descriptions, strong consensus was achieved for 19 blocks and weak consensus was achieved for one approach. Several areas requiring further research were identified.
Harmonization and standardization of nomenclature may improve education, research, and ultimately patient care. We present the first international consensus on nomenclature and anatomical descriptions of blocks of the abdominal wall, chest wall, and paraspinal blocks. We recommend using the consensus results in academic and clinical practice.
Daytime atmospheric oxidation chemistry is conventionally considered to be driven primarily by the OH radical, formed via photolytic sources. In this paper we examine how, during winter when ...photolytic processes are slow, chlorine chemistry can have a significant impact on oxidative processes in the urban boundary layer. Photolysis of nitryl chloride (ClNO2) provides a significant source of chlorine atoms, which enhances the oxidation of volatile organic compounds (VOCs) and the production of atmospheric pollutants.
We present a set of observations of ClNO2 and HONO made at urban locations in central England in December 2014 and February 2016. While direct emissions and in-situ chemical formation of HONO continue throughout the day, ClNO2 is only formed at night and is usually completely photolyzed by midday. Our data show that, during winter, ClNO2 often persists through the daylight hours at mixing ratios above 10–20 ppt (on average). In addition, relatively high mixing ratios of daytime HONO (>65 ppt) provide a strong source of OH radicals throughout the day.
The combined effects of ClNO2 and HONO result in sustained sources of Cl and OH radicals from sunrise to sunset, which form additional ozone, PAN, oxygenated VOCs, and secondary organic aerosol. We show that radical sources such as ClNO2 and HONO can lead to a surprisingly photoactive urban atmosphere during winter and should therefore be included in atmospheric chemical models.
Display omitted
•High ClNO2 concentrations at sunrise cause it to persist until sunset above 10–20 ppt.•Daytime HONO accounts for over 90% of OH formation in the winter urban atmosphere.•Sustained sources of Cl and OH throughout the day enhance the oxidation of VOCs.•Production of O3 and secondary pollutants increases and continues into the afternoon.
Radiotherapy is an effective anticancer treatment, but combinations with targeted agents that maximize efficacy while sparing normal tissue are needed. Here, we assess the radiopotentiation profiles ...of DNA damage response inhibitors (DDRi) olaparib (PARP1/2), ceralasertib (ATR), adavosertib (WEE1), AZD0156 (ATM), and KU-60648 (DNA-PK). We performed a radiotherapy combination screen and assessed how drug concentration and cellular DDR deficiencies influence the radiopotentiation ability of DDRi. We pre-selected six lung cancer cell lines with different genetic/signaling aberrations (including mutations in
and
) and assessed multiple concentrations of DDRi in combination with a fixed radiotherapy dose by clonogenic assay. The effective concentration of DDRi in radiotherapy combinations is lower than that required for single-agent efficacy. This has the potential to be exploited further in the context of DDR deficiencies to increase therapeutic index and we demonstrate that low concentrations of AZD0156 preferentially sensitized p53-deficient cells. Moreover, testing multiple concentrations of DDRi in radiotherapy combinations indicated that olaparib, ceralasertib, and adavosertib have a desirable safety profile showing moderate increases in radiotherapy dose enhancement with increasing inhibitor concentration. Small increases in concentration of AZD0156 and particularly KU-60648, however, result in steep increases in dose enhancement. Radiopotentiation profiling can inform on effective drug doses required for radiosensitization in relation to biomarkers, providing an opportunity to increase therapeutic index. Moreover, multiple concentration testing demonstrates a relationship between drug concentration and radiotherapy effect that provides valuable insights that, with future
validation, can guide dose-escalation strategies in clinical trials.
Coordination cage catalysis has commonly relied on the endogenous binding of substrates, exploiting the cavity microenvironment and spatial constraints to engender increased reactivity or interesting ...selectivity. Nonetheless, there are issues with this approach, such as the frequent occurrence of product inhibition or the limited applicability to a wide range of substrates and reactions. Here we describe a strategy in which the cage acts as an exogenous catalyst, wherein reactants, intermediates and products remain unbound throughout the course of the catalytic cycle. Instead, the cage is used to alter the properties of a cofactor guest, which then transfers reactivity to the bulk-phase. We have exemplified this approach using photocatalysis, showing that a photoactivated host-guest complex can mediate 4 + 2 cycloadditions and the aza-Henry reaction. Detailed
photolysis experiments show that the cage can both act as a photo-initiator and as an on-cycle catalyst where the quantum yield is less than unity.
An important component of wildlife management and conservation is monitoring the health and population size of wildlife species. Monitoring the population size of an animal group can inform ...researchers of habitat use, potential changes in habitat and resulting behavioral adaptations, individual health, and the effectiveness of conservation efforts. Arboreal monkeys are difficult to monitor as their habitat is often poorly accessible and most monkey species have some degree of camouflage, making them hard to observe in and below the tree canopy. Surveys conducted using uninhabited aerial vehicles (UAVs) equipped with thermal infrared (TIR) cameras can help overcome these limitations by flying above the canopy and using the contrast between the warm body temperature of the monkeys and the cooler background vegetation, reducing issues with impassable terrain and animal camouflage. We evaluated the technical and procedural elements associated with conducting UAV-TIR surveys for arboreal and terrestrial macaque species. Primary imaging missions and analyses were conducted over a monkey park housing approximately 160 semi-free-ranging Japanese macaques (
Macaca fuscata
). We demonstrate Repeat Station Imaging (RSI) procedures using co-registered TIR image pairs facilitate the use of image differencing to detect targets that were moving during rapid sequence imaging passes. We also show that 3D point clouds may be generated from highly overlapping UAV-TIR image sets in a forested setting using structure from motion (SfM) image processing techniques. A point cloud showing area-wide elevation values was generated from TIR imagery, but it lacked sufficient point density to reliably determine the 3D locations of monkeys.
Ca
2+
entry
via
Orai1 store-operated Ca
2+
channels in the plasma membrane is critical to cell function, and Orai1 loss causes severe immunodeficiency and developmental defects. The tetraspanins are ...a superfamily of transmembrane proteins that interact with specific ‘partner proteins’ and regulate their trafficking and clustering. The aim of this study was to functionally characterize tetraspanin Tspan18. We show that Tspan18 is expressed by endothelial cells at several-fold higher levels than most other cell types analyzed. Tspan18-knockdown primary human umbilical vein endothelial cells have 55-70% decreased Ca
2+
mobilization upon stimulation with the inflammatory mediators thrombin or histamine, similar to Orai1-knockdown. Tspan18 interacts with Orai1, and Orai1 cell surface localization is reduced by 70% in Tspan18-knockdown endothelial cells. Tspan18 overexpression in lymphocyte model cell lines induces 20-fold activation of Ca
2+
-responsive nuclear factor of activated T cell (NFAT) signaling, in an Orai1-dependent manner. Tspan18-knockout mice are viable. They lose on average 6-fold more blood in a tail-bleed assay. This is due to Tspan18 deficiency in non-hematopoietic cells, as assessed using chimeric mice. Tspan18-knockout mice have 60% reduced thrombus size in a deep vein thrombosis model, and 50% reduced platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury. Histamine- or thrombin-induced von Willebrand factor release from endothelial cells is reduced by 90% following Tspan18-knockdown, and histamine-induced increase of plasma von Willebrand factor is reduced by 45% in Tspan18-knockout mice. These findings identify Tspan18 as a novel regulator of endothelial cell Orai1/Ca
2+
signaling and von Willebrand factor release in response to inflammatory stimuli.
Understanding how cell identity transitions occur and whether there are multiple paths between the same beginning and end states are questions of wide interest. Here we show that acquisition of naive ...pluripotency can follow transcriptionally and mechanistically distinct routes. Starting from post-implantation epiblast stem cells (EpiSCs), one route advances through a mesodermal state prior to naive pluripotency induction, whereas another transiently resembles the early inner cell mass and correspondingly gains greater developmental potency. These routes utilize distinct signaling networks and transcription factors but subsequently converge on the same naive endpoint, showing surprising flexibility in mechanisms underlying identity transitions and suggesting that naive pluripotency is a multidimensional attractor state. These route differences are reconciled by precise expression of Oct4 as a unifying, essential, and sufficient feature. We propose that fine-tuned regulation of this “transition factor” underpins multidimensional access to naive pluripotency, offering a conceptual framework for understanding cell identity transitions.
Display omitted
•Reprogramming routes differ transcriptionally and mechanistically•Reprogramming intermediates resemble different developmental stages•Distinct routes converge on precise Oct4 regulation to permit identity transition•Precise Oct4 expression is sufficient for reprogramming of EpiSCs and fibroblasts
Stuart et al. report distinct routes of reprogramming to naive pluripotency. These differ in their transcriptional trajectories, mechanistic requirements, and developmental parallels, thus demonstrating considerable flexibility for a given cell identity transition to occur. Distinct routes converge on precise Oct4 expression, which is necessary and sufficient for naive pluripotency induction.
PDF
