Novel non-HAP class A HBV capsid assembly modulators have distinct in vitro and in vivo profiles.

IMPORTANCE: Chronic hepatitis B is the most important cause of liver cancer worldwide and affects more than 290 million people. Current treatments are mostly suppressive and rarely lead to a cure. Therefore, there is a need for novel and curative drugs that target the host or the causative agent, hepatitis B virus itself. Capsid assembly modulators are an interesting class of antiviral molecules that may one day become part of curative treatment regimens for chronic hepatitis B. Here we explore the characteristics of a particularly interesting subclass of capsid assembly modulators. These so-called non-HAP CAM-As have intriguing properties in cell culture but also clear virus-infected cells from the mouse liver in a gradual and sustained way. We believe they represent a considerable improvement over previously reported molecules and may one day be part of curative treatment combinations for chronic hepatitis B.

Class A capsid assembly modulator RG7907 clears HBV-infected hepatocytes through core-dependent hepatocyte death and proliferation.

BACKGROUND AND AIMS: Effective therapies leading to a functional cure for chronic hepatitis B are still lacking. Class A capsid assembly modulators (CAM-As) are an attractive modality to address this unmet medical need. CAM-As induce aggregation of the HBV core protein (HBc) and lead to sustained HBsAg reductions in a chronic hepatitis B mouse model. Here, we investigate the underlying mechanism of action for CAM-A compound RG7907. APPROACH AND RESULTS: RG7907 induced extensive HBc aggregation in vitro , in hepatoma cells, and in primary hepatocytes. In the adeno-associated virus (AAV)-HBV mouse model, the RG7907 treatment led to a pronounced reduction in serum HBsAg and HBeAg, concomitant with clearance of HBsAg, HBc, and AAV-HBV episome from the liver. Transient increases in alanine transaminase, hepatocyte apoptosis, and proliferation markers were observed. These processes were confirmed by RNA sequencing, which also uncovered a role for interferon alpha and gamma signaling, including the interferon-stimulated gene 15 (ISG15) pathway. Finally, the in vitro observation of CAM-A-induced HBc-dependent cell death through apoptosis established the link of HBc aggregation to in vivo loss of infected hepatocytes. CONCLUSIONS: Our study unravels a previously unknown mechanism of action for CAM-As such as RG7907 in which HBc aggregation induces cell death, resulting in hepatocyte proliferation and loss of covalently closed circular DNA or its equivalent, possibly assisted by an induced innate immune response. This represents a promising approach to attain a functional cure for chronic hepatitis B.

Machine learning-assisted fluoroscopy of bladder function in awake mice.

Understanding the lower urinary tract (LUT) and development of highly needed novel therapies to treat LUT disorders depends on accurate techniques to monitor LUT (dys)function in preclinical models. We recently developed videocystometry in rodents, which combines intravesical pressure measurements with X-ray-based fluoroscopy of the LUT, allowing the in vivo analysis of the process of urine storage and voiding with unprecedented detail. Videocystometry relies on the precise contrast-based determination of the bladder volume at high temporal resolution, which can readily be achieved in anesthetized or otherwise motion-restricted mice but not in awake and freely moving animals. To overcome this limitation, we developed a machine-learning method, in which we trained a neural network to automatically detect the bladder in fluoroscopic images, allowing the automatic analysis of bladder filling and voiding cycles based on large sets of time-lapse fluoroscopic images (>3 hr at 30 images/s) from behaving mice and in a noninvasive manner. With this approach, we found that urethane, an injectable anesthetic that is commonly used in preclinical urological research, has a profound, dose-dependent effect on urethral relaxation and voiding duration. Moreover, both in awake and in anesthetized mice, the bladder capacity was decreased ~fourfold when cystometry was performed acutely after surgical implantation of a suprapubic catheter. Our findings provide a paradigm for the noninvasive, in vivo monitoring of a hollow organ in behaving animals and pinpoint important limitations of the current gold standard techniques to study the LUT in mice.

Healthy adults empty their bladder many times a day with little thought. This seemingly simple process requires communication between the lower urinary tract and the central nervous system. About one in five adults experience conditions like urinary incontinence, urgency, or bladder pain caused by impairments in their lower urinary tract. Despite the harmful effects these conditions have on people's health and well-being, few good treatments are available. Mice are often used to study lower urinary tract conditions and treatments. One common technique is to fill a mouse's bladder using a catheter and measure changes in pressure as the bladder empties and refills. But these procedures and the anesthesia used during them may affect bladder function and skew results. Here, De Bruyn et al. have developed a new technique that allows scientists to measure bladder function in awake, freely moving mice. The mice's bladders were photographed using a specialized X-ray based fluoroscope that captured 30 images per second over the course of three hours. A machine learning algorithm was then applied which can automatically detect the circumference of the bladder in each captured image (over 30,000 in total) and quantify its volume. This makes it is possible to measure the bladder as it empties and fills even if the mice move between time frames. The new approach showed that 'gold standard' commonly used methods have a profound effect on the bladder. Surgical implantation of a catheter reduced the bladder to a quarter of its capacity. In addition, one of the most widely used anesthetic drugs in urinary tract research was found to affect the bladder's ability to drain. The technique created by De Bruyn et al. provides a new way to study lower urinary tract function and disease in awake, moving animals. This tool would be easy for other academic and pharmaceutical laboratories to implement, and may help scientists discover new therapies for lower urinary tract conditions.

Fetal hematopoietic stem cell homing is controlled by VEGF regulating the integrity and oxidative status of the stromal-vascular bone marrow niches.

Hematopoietic stem and progenitor cell (HSPC) engraftment after transplantation during anticancer treatment depends on support from the recipient bone marrow (BM) microenvironment. Here, by studying physiological homing of fetal HSPCs, we show the critical requirement of balanced local crosstalk within the skeletal niche for successful HSPC settlement in BM. Transgene-induced overproduction of vascular endothelial growth factor (VEGF) by osteoprogenitor cells elicits stromal and endothelial hyperactivation, profoundly impacting the stromal-vessel interface and vascular architecture. Concomitantly, HSPC homing and survival are drastically impaired. Transcriptome profiling, flow cytometry, and high-resolution imaging indicate alterations in perivascular and endothelial cell characteristics, vascular function and cellular metabolism, associated with increased oxidative stress within the VEGF-enriched BM environment. Thus, developmental HSPC homing to bone is controlled by local stromal-vascular integrity and the oxidative-metabolic status of the recipient milieu. Interestingly, irradiation of adult mice also induces stromal VEGF expression and similar osteo-angiogenic niche changes, underscoring that our findings may contribute targets for improving stem cell therapies.

Rectal organoid morphology analysis (ROMA) as a promising diagnostic tool in cystic fibrosis.

Diagnosing cystic fibrosis (CF) when sweat chloride is not in the CF range and less than 2 disease-causing CFTR mutations are found requires physiological CFTR assays, which are not always feasible or available. We developed a new physiological CFTR assay based on the morphological differences between rectal organoids from subjects with and without CF. In organoids from 167 subjects with and 22 without CF, two parameters derived from a semi-automated image analysis protocol (rectal organoid morphology analysis, ROMA) fully discriminated CF subjects with two disease-causing mutations from non-CF subjects (p<0.001). ROMA, feasible at all ages, can be centralised to improve standardisation.

AAV9-mediated gene delivery of MCT1 to oligodendrocytes does not provide a therapeutic benefit in a mouse model of ALS.

Oligodendrocyte dysfunction has been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by progressive motor neuron loss. The failure of trophic support provided by oligodendrocytes is associated with a concomitant reduction in oligodendroglial monocarboxylate transporter 1 (MCT1) expression and is detrimental for the long-term survival of motor neuron axons. Therefore, we established an adeno-associated virus 9 (AAV9)-based platform by which MCT1 was targeted mostly to white matter oligodendrocytes to investigate whether this approach could provide a therapeutic benefit in the SOD1G93A mouse model of ALS. Despite good oligodendrocyte transduction and AAV-mediated MCT1 transgene expression, the disease outcome of SOD1G93A mice was not altered. Our study further increases our current understanding about the complex nature of oligodendrocyte pathology in ALS and provides valuable insights into the future development of therapeutic strategies to efficiently modulate these cells.

Super-resolution microscopy reveals majorly mono- and dimeric presenilin1/γ-secretase at the cell surface.

γ-Secretase is a multi-subunit enzyme whose aberrant activity is associated with Alzheimer's disease and cancer. While its structure is atomically resolved, γ-secretase localization in the membrane in situ relies mostly on biochemical data. Here, we combined fluorescent tagging of γ-secretase subunits with super-resolution microscopy in fibroblasts. Structured illumination microscopy revealed single γ-secretase complexes with a monodisperse distribution and in a 1:1 stoichiometry of PSEN1 and nicastrin subunits. In living cells, sptPALM revealed PSEN1/γ-secretase mainly with directed motility and frequenting 'hotspots' or high track-density areas that are sensitive to γ-secretase inhibitors. We visualized γ-secretase association with substrates like amyloid precursor protein and N-cadherin, but not with its sheddases ADAM10 or BACE1 at the cell surface, arguing against pre-formed megadalton complexes. Nonetheless, in living cells PSEN1/γ-secretase transiently visits ADAM10 hotspots. Our results highlight the power of super-resolution microscopy for the study of γ-secretase distribution and dynamics in the membrane.

Defective Sec61α1 underlies a novel cause of autosomal dominant severe congenital neutropenia.

BACKGROUND: The molecular cause of severe congenital neutropenia (SCN) is unknown in 30% to 50% of patients. SEC61A1 encodes the α-subunit of the Sec61 complex, which governs endoplasmic reticulum protein transport and passive calcium leakage. Recently, mutations in SEC61A1 were reported to be pathogenic in common variable immunodeficiency and glomerulocystic kidney disease. OBJECTIVE: Our aim was to expand the spectrum of SEC61A1-mediated disease to include autosomal dominant SCN. METHODS: Whole exome sequencing findings were validated, and reported mutations were compared by Western blotting, Ca2+ flux assays, differentiation of transduced HL-60 cells, in vitro differentiation of primary CD34 cells, quantitative PCR for unfolded protein response (UPR) genes, and single-cell RNA sequencing on whole bone marrow. RESULTS: We identified a novel de novo missense mutation in SEC61A1 (c.A275G;p.Q92R) in a patient with SCN who was born to nonconsanguineous Belgian parents. The mutation results in diminished protein expression, disturbed protein translocation, and an increase in calcium leakage from the endoplasmic reticulum. In vitro differentiation of CD34+ cells recapitulated the patient's clinical arrest in granulopoiesis. The impact of Q92R-Sec61α1 on neutrophil maturation was validated by using HL-60 cells, in which transduction reduced differentiation into CD11b+CD16+ cells. A potential mechanism for this defect is the uncontrolled initiation of the unfolded protein stress response, with single-cell analysis of primary bone marrow revealing perturbed UPR in myeloid precursors and in vitro differentiation of primary CD34+ cells revealing upregulation of CCAAT/enhancer-binding protein homologous protein and immunoglobulin heavy chain binding protein UPR-response genes. CONCLUSION: Specific mutations in SEC61A1 cause SCN through dysregulation of the UPR.

Evidence for an alternative fatty acid desaturation pathway increasing cancer plasticity.

Most tumours have an aberrantly activated lipid metabolism1,2 that enables them to synthesize, elongate and desaturate fatty acids to support proliferation. However, only particular subsets of cancer cells are sensitive to approaches that target fatty acid metabolism and, in particular, fatty acid desaturation3. This suggests that many cancer cells contain an unexplored plasticity in their fatty acid metabolism. Here we show that some cancer cells can exploit an alternative fatty acid desaturation pathway. We identify various cancer cell lines, mouse hepatocellular carcinomas, and primary human liver and lung carcinomas that desaturate palmitate to the unusual fatty acid sapienate to support membrane biosynthesis during proliferation. Accordingly, we found that sapienate biosynthesis enables cancer cells to bypass the known fatty acid desaturation pathway that is dependent on stearoyl-CoA desaturase. Thus, only by targeting both desaturation pathways is the in vitro and in vivo proliferation of cancer cells that synthesize sapienate impaired. Our discovery explains metabolic plasticity in fatty acid desaturation and constitutes an unexplored metabolic rewiring in cancers.

Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aß Pool.

γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aß that contains longer Aß; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aß further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aß42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.

Identification of drugs that restore primary cilium expression in cancer cells.

The development of cancer is often accompanied by a loss of the primary cilium, a microtubule-based cellular protrusion that functions as a cellular antenna and that puts a break on cell proliferation. Hence, restoration of the primary cilium in cancer cells may represent a novel promising approach to attenuate tumor growth. Using a high content analysis-based approach we screened a library of clinically evaluated compounds and marketed drugs for their ability to restore primary cilium expression in pancreatic ductal cancer cells. A diverse set of 118 compounds stimulating cilium expression was identified. These included glucocorticoids, fibrates and other nuclear receptor modulators, neurotransmitter regulators, ion channel modulators, tyrosine kinase inhibitors, DNA gyrase/topoisomerase inhibitors, antibacterial compounds, protein inhibitors, microtubule modulators, and COX inhibitors. Certain compounds also dramatically affected the length of the cilium. For a selection of compounds (Clofibrate, Gefitinib, Sirolimus, Imexon and Dexamethasone) their ability to restore ciliogenesis was confirmed in a panel of human cancer cell line models representing different cancer types (pancreas, lung, kidney, breast). Most compounds attenuated cell proliferation, at least in part through induction of the primary cilium, as demonstrated by cilium removal using chloral hydrate. These findings reveal that several commonly used drugs restore ciliogenesis in cancer cells, and warrant further investigation of their antineoplastic properties.

High-content analysis of α-synuclein aggregation and cell death in a cellular model of Parkinson's disease.

BACKGROUND: Alpha-synuclein (α-SYN) aggregates represent a key feature of Parkinson's disease, but the exact relationship between α-SYN aggregation and neurodegeneration remains incompletely understood. Therefore, the availability of a cellular assay that allows medium-throughput analysis of α-SYN-linked pathology will be of great value for studying the aggregation process and for advancing α-SYN-based therapies. NEW METHOD: Here we describe a high-content neuronal cell assay that simultaneously measures oxidative stress-induced α-SYN aggregation and apoptosis. RESULTS: We optimized an automated and reproducible assay to quantify both α-SYN aggregation and cell death in human SH-SY5Y neuroblastoma cells. COMPARISON WITH EXISTING METHODS: Quantification of α-SYN aggregates in cells has typically relied on manual imaging and counting or cell-free assays, which are time consuming and do not allow a concurrent analysis of cell viability. Our high-content analysis method for quantification of α-SYN aggregation allows simultaneous measurements of multiple cell parameters at a single-cell level in a fast, objective and automated manner. CONCLUSIONS: The presented analysis approach offers a rapid, objective and multiparametric approach for the screening of compounds and genes that might alter α-SYN aggregation and/or toxicity.

High content analysis at single cell level identifies different cellular responses dependent on nanomaterial concentrations.

A mechanistic understanding of nanomaterial (NM) interaction with biological environments is pivotal for the safe transition from basic science to applied nanomedicine. NM exposure results in varying levels of internalized NM in different neighboring cells, due to variances in cell size, cell cycle phase and NM agglomeration. Using high-content analysis, we investigated the cytotoxic effects of fluorescent quantum dots on cultured cells, where all effects were correlated with the concentration of NMs at the single cell level. Upon binning the single cell data into different categories related to NM concentration, this study demonstrates, for the first time, that quantum dots activate both cytoprotective and cytotoxic mechanisms, resulting in a zero net result on the overall cell population, yet with significant effects in cells with higher cellular NM levels. Our results suggest that future NM cytotoxicity studies should correlate NM toxicity with cellular NM numbers on the single cell level, as conflicting mechanisms in particular cell subpopulations are commonly overlooked using classical toxicological methods.

Constitutive hippocampal cholesterol loss underlies poor cognition in old rodents.

Cognitive decline is one of the many characteristics of aging. Reduced long-term potentiation (LTP) and long-term depression (LTD) are thought to be responsible for this decline, although the precise mechanisms underlying LTP and LTD dampening in the old remain unclear. We previously showed that aging is accompanied by the loss of cholesterol from the hippocampus, which leads to PI3K/Akt phosphorylation. Given that Akt de-phosphorylation is required for glutamate receptor internalization and LTD, we hypothesized that the decrease in cholesterol in neuronal membranes may contribute to the deficits in LTD typical of aging. Here, we show that cholesterol loss triggers p-Akt accumulation, which in turn perturbs the normal cellular and molecular responses induced by LTD, such as impaired AMPA receptor internalization and its reduced lateral diffusion. Electrophysiology recordings in brain slices of old mice and in anesthetized elderly rats demonstrate that the reduced hippocampal LTD associated with age can be rescued by cholesterol perfusion. Accordingly, cholesterol replenishment in aging animals improves hippocampal-dependent learning and memory in the water maze test.

Regulation of branching dynamics by axon-intrinsic asymmetries in Tyrosine Kinase Receptor signaling.

Axonal branching allows a neuron to connect to several targets, increasing neuronal circuit complexity. While axonal branching is well described, the mechanisms that control it remain largely unknown. We find that in the Drosophila CNS branches develop through a process of excessive growth followed by pruning. In vivo high-resolution live imaging of developing brains as well as loss and gain of function experiments show that activation of Epidermal Growth Factor Receptor (EGFR) is necessary for branch dynamics and the final branching pattern. Live imaging also reveals that intrinsic asymmetry in EGFR localization regulates the balance between dynamic and static filopodia. Elimination of signaling asymmetry by either loss or gain of EGFR function results in reduced dynamics leading to excessive branch formation. In summary, we propose that the dynamic process of axon branch development is mediated by differential local distribution of signaling receptors. DOI: http://dx.doi.org/10.7554/eLife.01699.001.

The fragile X protein binds mRNAs involved in cancer progression and modulates metastasis formation.

The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression.

Role of PFKFB3-driven glycolysis in vessel sprouting.

Vessel sprouting by migrating tip and proliferating stalk endothelial cells (ECs) is controlled by genetic signals (such as Notch), but it is unknown whether metabolism also regulates this process. Here, we show that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the glycolytic activator PFKFB3 in ECs impaired vessel formation. Mechanistically, PFKFB3 not only regulated EC proliferation but also controlled the formation of filopodia/lamellipodia and directional migration, in part by compartmentalizing with F-actin in motile protrusions. Mosaic in vitro and in vivo sprouting assays further revealed that PFKFB3 overexpression overruled the pro-stalk activity of Notch, whereas PFKFB3 deficiency impaired tip cell formation upon Notch blockade, implying that glycolysis regulates vessel branching.

Integration of Microfractionation, qNMR and zebrafish screening for the in vivo bioassay-guided isolation and quantitative bioactivity analysis of natural products.

Natural products (NPs) are an attractive source of chemical diversity for small-molecule drug discovery. Several challenges nevertheless persist with respect to NP discovery, including the time and effort required for bioassay-guided isolation of bioactive NPs, and the limited biomedical relevance to date of in vitro bioassays used in this context. With regard to bioassays, zebrafish have recently emerged as an effective model system for chemical biology, allowing in vivo high-content screens that are compatible with microgram amounts of compound. For the deconvolution of the complex extracts into their individual constituents, recent progress has been achieved on several fronts as analytical techniques now enable the rapid microfractionation of extracts, and microflow NMR methods have developed to the point of allowing the identification of microgram amounts of NPs. Here we combine advanced analytical methods with high-content screening in zebrafish to create an integrated platform for microgram-scale, in vivo NP discovery. We use this platform for the bioassay-guided fractionation of an East African medicinal plant, Rhynchosia viscosa, resulting in the identification of both known and novel isoflavone derivatives with anti-angiogenic and anti-inflammatory activity. Quantitative microflow NMR is used both to determine the structure of bioactive compounds and to quantify them for direct dose-response experiments at the microgram scale. The key advantages of this approach are (1) the microgram scale at which both biological and analytical experiments can be performed, (2) the speed and the rationality of the bioassay-guided fractionation - generic for NP extracts of diverse origin - that requires only limited sample-specific optimization and (3) the use of microflow NMR for quantification, enabling the identification and dose-response experiments with only tens of micrograms of each compound. This study demonstrates that a complete in vivo bioassay-guided fractionation can be performed with only 20 mg of NP extract within a few days.

KIF1Bß transports dendritically localized mRNPs in neurons and is recruited to synapses in an activity-dependent manner.

KIF1Bß is a kinesin-like, microtubule-based molecular motor protein involved in anterograde axonal vesicular transport in vertebrate and invertebrate neurons. Certain KIF1Bß isoforms have been implicated in different forms of human neurodegenerative disease, with characterization of their functional integration and regulation in the context of synaptic signaling still ongoing. Here, we characterize human KIF1Bß (isoform NM015074), whose expression we show to be developmentally regulated and elevated in cortical areas of the CNS (including the motor cortex), in the hippocampus, and in spinal motor neurons. KIF1Bß localizes to the cell body, axon, and dendrites, overlapping with synaptic-vesicle and postsynaptic-density structures. Correspondingly, in purified cortical synaptoneurosomes, KIF1Bß is enriched in both pre- and postsynaptic structures, forming detergent-resistant complexes. Interestingly, KIF1Bß forms RNA-protein complexes, containing the dendritically localized Arc and Calmodulin mRNAs, proteins previously shown to be part of RNA transport granules such as Purα, FMRP and FXR2P, and motor protein KIF3A, as well as Calmodulin. The interaction between KIF1Bß and Calmodulin is Ca(+2)-dependent and takes place through a domain mapped at the carboxy-terminal tail of the motor. Live imaging of cortical neurons reveals active movement by KIF1Bß at dendritic processes, suggesting that it mediates the transport of dendritically localized mRNAs. Finally, we show that synaptic recruitment of KIF1Bß is activity-dependent and increased by stimulation of metabotropic or ionotropic glutamate receptors. The activity-dependent synaptic recruitment of KIF1Bß, its interaction with Ca(2+) sensor Calmodulin, and its new role as a dendritic motor of ribonucleoprotein complexes provide a novel basis for understanding the concerted co-ordination of motor protein mobilization and synaptic signaling pathways.

Loss of PPP2R2A inhibits homologous recombination DNA repair and predicts tumor sensitivity to PARP inhibition.

Reversible phosphorylation plays a critical role in DNA repair. Here, we report the results of a loss-of-function screen that identifies the PP2A heterotrimeric serine/threonine phosphatases PPP2R2A, PPP2R2D, PPP2R5A, and PPP2R3C in double-strand break (DSB) repair. In particular, we found that PPP2R2A-containing complexes directly dephosphorylated ATM at S367, S1893, and S1981 to regulate its retention at DSB sites. Increased ATM phosphorylation triggered by PPP2R2A attenuation dramatically upregulated the activity of the downstream effector kinase CHK2, resulting in G(1) to S-phase cell-cycle arrest and downregulation of BRCA1 and RAD51. In tumor cells, blocking PPP2R2A thereby impaired the high-fidelity homologous recombination repair pathway and sensitized cells to small-molecule inhibitors of PARP. We found that PPP2R2A was commonly downregulated in non-small cell lung carcinomas, suggesting that PPP2R2A status may serve as a marker to predict therapeutic efficacy to PARP inhibition. In summary, our results deepen understanding of the role of PP2A family phosphatases in DNA repair and suggest PPP2R2A as a marker for PARP inhibitor responses in clinic.