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Background Preoperative L-lactatemia and heart rate have been suggested as prognostic indicators of outcome for cows with right dilatation of the abomasum or volvulus ( RDA/ AV). However, postoperative L-lactatemia has not been assessed as a potential prognostic tool. Objectives To determine the prognostic value of postoperative L-lactatemia ( LAC2), duration of treatment (Dt), relative L-lactatemia difference (compared with preoperative L-lactatemia [LAC1]) ([LAC2 − LAC1]/LAC2) and change in L-lactate over time ([LAC2 − LAC1]/Dt) as compared to preoperative findings ( LAC1 and heart rate [ HR]) as prognostic factors in dairy cows with RDA/ AV. Animals A total of 41 dairy cows were included: 19 with AV and 22 with RDA; 11 cows had a negative outcome ( NO) and 30 cows had a positive outcome ( PO) based on telephone follow-up with owners 30 days after surgery. Methods Prospective cohort study. Analysis was performed using logistic regression and comparison of area under the receiver operating characteristics curve ( AUC) using nonparametric tests. Results LAC1 > 1.4 mmol/L or LAC2 > 2.2 mmol/L had the same accuracy with sensitivity of 100% (95% CI, 75.1-100%) and specificity of 80% (95% CI, 61.4-92.3%) for predicting NO. The relative L-lactatemia difference ([LAC2 − LAC1]/LAC1) or lactate kinetics ([LAC2 − LAC1]/Dt) were not associated with prognosis. The AUC of the preoperative model (which included HR and lnLAC1) was 0.92 (95% CI, 0.83-1.0) and that of the postoperative model (including only lnLAC2) was 0.95 (95% CI, 0.88-1.0); these were not significantly different. Conclusions and Clinical Importance Postoperative L-lactatemia is helpful to predict outcome in cows with RDA/ AV. The short-term change in blood L-lactate is not a useful prognostic indicator , at least during the period of time spent on the farm for surgery and treatment. [ABSTRACT FROM AUTHOR]

EWS fusion oncoproteins underlie several human malignancies including Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive cancer driven by EWS-WT1 fusion proteins. Here we combine chromatin occupancy and 3D profiles to identify EWS-WT1-dependent gene regulation networks and target genes. We show that EWS-WT1 is a powerful chromatin activator controlling an oncogenic gene expression program that characterizes primary tumors. Similar to wild type WT1, EWS-WT1 has two isoforms that differ in their DNA binding domain and we find that they have distinct DNA binding profiles and are both required to generate viable tumors that resemble primary DSRCT. Finally, we identify candidate EWS-WT1 target genes with potential therapeutic implications, including CCND1, whose inhibition by the clinically-approved drug Palbociclib leads to marked tumor burden decrease in DSRCT PDXs in vivo. Taken together, our studies identify gene regulation programs and therapeutic vulnerabilities in DSRCT and provide a mechanistic understanding of the complex oncogenic activity of EWS-WT1., (© 2024. The Author(s).)

Huntington's disease (HD) is a rare genetic neurodegenerative disorder caused by an expansion of CAG repeats in the Huntingtin (HTT) gene. One hypothesis suggests that the mutant HTT gene contributes to HD neuropathology through transcriptional dysregulation involving microRNAs (miRNAs). In particular, the miR-132/212 cluster is strongly diminished in the HD brain. This study explores the effects of miR-132/212 deficiency specifically in adult HD zQ175 mice. The absence of miR-132/212 did not impact body weight, body temperature, or survival rates. Surprisingly, miR-132/212 loss seemed to alleviate, in part, the effects on endogenous Htt expression, HTT inclusions, and neuronal integrity in HD zQ175 mice. Additionally, miR-132/212 depletion led to age-dependent improvements in certain motor functions. Transcriptomic analysis revealed alterations in HD-related networks in WT- and HD zQ175-miR-132/212-deficient mice, including significant overlap in BDNF and Creb1 signaling pathways. Interestingly, however, a higher number of miR-132/212 gene targets was observed in HD zQ175 mice lacking the miR-132/212 cluster, especially in the striatum. These findings suggest a nuanced interplay between miR-132/212 expression and HD pathogenesis, providing potential insights into therapeutic interventions. Further investigation is needed to fully understand the underlying mechanisms and therapeutic potential of modulating miR-132/212 expression during HD progression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Nateghi, Keraudren, Boulay, Bazin, Goupil, Canet, Loiselle, St-Amour, Planel, Soulet and Hébert.)

The development of targeted therapies offers new hope for patients affected by incurable cancer. However, multiple challenges persist, notably in controlling tumor cell plasticity in patients with refractory and metastatic illness. Neuroblastoma (NB) is an aggressive pediatric malignancy originating from defective differentiation of neural crest-derived progenitors with oncogenic activity due to genetic and epigenetic alterations and remains a clinical challenge for high-risk patients. To identify critical genes driving NB aggressiveness, we performed combined chromatin and transcriptome analyses on matched patient-derived xenografts (PDXs), spheroids, and differentiated adherent cultures derived from metastatic MYCN nonamplified tumors. Bone marrow kinase on chromosome X (BMX) was identified among the most differentially regulated genes in PDXs and spheroids versus adherent models. BMX expression correlated with high tumor stage and poor patient survival and was crucial to the maintenance of the self-renewal and tumorigenic potential of NB spheroids. Moreover, BMX expression positively correlated with the mesenchymal NB cell phenotype, previously associated with increased chemoresistance. Finally, BMX inhibitors readily reversed this cellular state, increased the sensitivity of NB spheroids toward chemotherapy, and partially reduced tumor growth in a preclinical NB model. Altogether, our study identifies BMX as a promising innovative therapeutic target for patients with high-risk MYCN nonamplified NB.

Despite the recommendation of improving assessment objectivity and frequency, the use of immersive virtual reality to measure and quantify movement quality remains underexplored. In this study, we aimed to evaluate the reliability, validity and usability of an immersive virtual reality application, KinematicsVR, to assess upper limb kinematics among older adults with and without major neurocognitive disorder. The KinematicsVR involves the drawing of three-dimensional straight lines, circles and squares using a controller in a virtual environment. Twenty-eight older adults with or without major neurocognitive disorder were recruited. Reliability was evaluated through correlations on test-retest and validity through correlations between KinematicsVR variables and other functional tests (TEMPA, BBT-VR and Finger-Nose Test). The usability of the KinematicsVR was assessed with the System Usability Scale questionnaire. Kinematic indexes were compared between eight adults with major neurocognitive disorder and eight matched controls. Results indicated that most variables provided by the KinematicsVR had excellent reliability for tasks involving the drawing of straight lines and circles, but moderate reliability for tasks involving the drawing of squares. Secondary analyses showed that the usability of the application was excellent but few significant and strong correlations were observed between variables of the KinematicsVR and the scores of the TEMPA scale, Finger-Nose Test and BBT-VR. Adults with major neurocognitive disorder, when compared to other older adults, made larger and less linear hand movements. These findings provide perspectives for the use of immersive virtual reality to improve assessment frequency and objectivity through the autonomous measure of upper limb kinematics in older adults., (Copyright © 2023 IBRO. Published by Elsevier Inc. All rights reserved.)

Intrinsically disordered regions (IDRs) in DNA-associated proteins are known to influence gene regulation, but their distribution and cooperative functions in genome-wide regulatory programs remain poorly understood. Here we describe DisP-seq (disordered protein precipitation followed by DNA sequencing), an antibody-independent chemical precipitation assay that can simultaneously map endogenous DNA-associated disordered proteins genome-wide through a combination of biotinylated isoxazole precipitation and next-generation sequencing. DisP-seq profiles are composed of thousands of peaks that are associated with diverse chromatin states, are enriched for disordered transcription factors (TFs) and are often arranged in large lineage-specific clusters with high local concentrations of disordered proteins and different combinations of histone modifications linked to regulatory potential. We use DisP-seq to analyze cancer cells and reveal how disordered protein-associated islands enable IDR-dependent mechanisms that control the binding and function of disordered TFs, including oncogene-dependent sequestration of TFs through long-range interactions and the reactivation of differentiation pathways upon loss of oncogenic stimuli in Ewing sarcoma., (© 2023. The Author(s).)

For a majority of patients with non-small cell lung cancer with EGFR mutations, treatment with EGFR inhibitors (EGFRi) induces a clinical response. Despite this initial reduction in tumor size, residual disease persists that leads to disease relapse. Elucidating the preexisting biological differences between sensitive cells and surviving drug-tolerant persister cells and deciphering how drug-tolerant cells evolve in response to treatment could help identify strategies to improve the efficacy of EGFRi. In this study, we tracked the origins and clonal evolution of drug-tolerant cells at a high resolution by using an expressed barcoding system coupled with single-cell RNA sequencing. This platform enabled longitudinal profiling of gene expression and drug sensitivity in response to EGFRi across a large number of clones. Drug-tolerant cells had higher expression of key survival pathways such as YAP and EMT at baseline and could also differentially adapt their gene expression following EGFRi treatment compared with sensitive cells. In addition, drug combinations targeting common downstream components (MAPK) or orthogonal factors (chemotherapy) showed greater efficacy than EGFRi alone, which is attributable to broader targeting of the heterogeneous EGFRi-tolerance mechanisms present in tumors. Overall, this approach facilitates thorough examination of clonal evolution in response to therapy that could inform the development of improved diagnostic approaches and treatment strategies for targeting drug-tolerant cells., Significance: The evolution and heterogeneity of EGFR inhibitor tolerance are identified in a large number of clones at enhanced cellular and temporal resolution using an expressed barcode technology coupled with single-cell RNA sequencing., (©2023 American Association for Cancer Research.)

The interaction of RB with chromatin is key to understanding its molecular functions. Here, for first time, we identify the full spectrum of chromatin-bound RB. Rather than exclusively binding promoters, as is often described, RB targets three fundamentally different types of loci (promoters, enhancers, and insulators), which are largely distinguishable by the mutually exclusive presence of E2F1, c-Jun, and CTCF. While E2F/DP facilitates RB association with promoters, AP-1 recruits RB to enhancers. Although phosphorylation in CDK sites is often portrayed as releasing RB from chromatin, we show that the cell cycle redistributes RB so that it enriches at promoters in G1 and at non-promoter sites in cycling cells. RB-bound promoters include the classic E2F-targets and are similar between lineages, but RB-bound enhancers associate with different categories of genes and vary between cell types. Thus, RB has a well-preserved role controlling E2F in G1, and it targets cell-type-specific enhancers and CTCF sites when cells enter S-phase., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Oncogenic fusion proteins generated by chromosomal translocations play major roles in cancer. Among them, fusions between EWSR1 and transcription factors generate oncogenes with powerful chromatin regulatory activities, capable of establishing complex gene expression programs in permissive precursor cells. Here we define the epigenetic and 3D connectivity landscape of Clear Cell Sarcoma, an aggressive cancer driven by the EWSR1-ATF1 fusion gene. We find that EWSR1-ATF1 displays a distinct DNA binding pattern that requires the EWSR1 domain and promotes ATF1 retargeting to new distal sites, leading to chromatin activation and the establishment of a 3D network that controls oncogenic and differentiation signatures observed in primary CCS tumors. Conversely, EWSR1-ATF1 depletion results in a marked reconfiguration of 3D connectivity, including the emergence of regulatory circuits that promote neural crest-related developmental programs. Taken together, our study elucidates the epigenetic mechanisms utilized by EWSR1-ATF1 to establish regulatory networks in CCS, and points to precursor cells in the neural crest lineage as candidate cells of origin for these tumors., (© 2022. The Author(s).)

Repeat elements can be dysregulated at a genome-wide scale in human diseases. For example, in Ewing sarcoma, hundreds of inert GGAA repeats can be converted into active enhancers when bound by EWS-FLI1. Here we show that fusions between EWS and GGAA-repeat-targeted engineered zinc finger arrays (ZFAs) can function at least as efficiently as EWS-FLI1 for converting hundreds of GGAA repeats into active enhancers in a Ewing sarcoma precursor cell model. Furthermore, a fusion of a KRAB domain to a ZFA can silence GGAA microsatellite enhancers genome wide in Ewing sarcoma cells, thereby reducing expression of EWS-FLI1-activated genes. Remarkably, this KRAB-ZFA fusion showed selective toxicity against Ewing sarcoma cells compared with non-Ewing cancer cells, consistent with its Ewing sarcoma-specific impact on the transcriptome. These findings demonstrate the value of ZFAs for functional annotation of repeats and illustrate how aberrant microsatellite activities might be regulated for potential therapeutic applications., Competing Interests: DECLARATION OF INTERESTS J.K.J. has, or had during the course of this research, financial interests in several companies developing gene-editing or epigenetic-editing technology: Beam Therapeutics, Blink Therapeutics, Chroma Medicine, Editas Medicine, EpiLogic Therapeutics, ETx Inc., Excelsior Genomics, Hera Biolabs, Monitor Biotechnologies, Pairwise Plants, Poseida Therapeutics, SeQure Dx Inc., and Verve Therapeutics. J.K.J.’s interests were reviewed and are managed by Massachusetts General Hospital and Mass General Brigham in accordance with their conflict of interest policies. J.K.J. is a consultant for Beam Therapeutics, Chroma Medicine, ETx Inc., Pairwise Plants, SeQure Dx Inc., and Verve Therapeutics and is a scientific co-founder of these companies and of Blink Therapeutics, Editas Medicine, EpiLogic Therapeutics, Excelsior Genomics, and Monitor Biotechnologies. G.B., J.K.J., M.N.R., and Y.E.T. are inventors on a patent application that encompasses work described in this paper. J.K.J. and Y.E.T. are inventors on patents or patent applications describing various gene- and epigenetic-editing technologies. M.N.R. receives research support from ACD and Merck Serono for work unrelated to this study. S.I. and S.P.G. are employees of Verve Therapeutics. J.K.J. is a member of the Advisory Board of Cell Genomics.

Background: Herpes virus remains dormant in human cells and could reactivate under immunosuppressed conditions, such as prolonged critical illnesses. The phenomenon of viral replication during intensive care is well known, even in patients without a history of immunosuppression, but it usually does not have a clinical impact. Systemic reactivation leads to viral DNA in blood. It remains unclear whether this replication is a marker of morbimortality or a true pathogenic process. Therefore, it is unclear what medical treatment is most appropriate for simple replication. In organ damage suspected to be induced by herpes virus, there is no consensus on the most appropriate treatment duration. Here, we report a rarely described case of multiorgan failure implicating herpes simplex virus and discuss its treatment., Case Report: A 53-year-old Caucasian immunosuppressed woman was admitted to the intensive care unit for septic shock. She presented pneumonia due to Klebsiella pneumoniae. Two weeks after admission, she showed multiorgan failure with acute respiratory distress syndrome and circulation failure. She had digestive and cutaneous lesions typical of herpes simplex virus 1. Blood and respiratory polymerase chain reaction was strongly herpes simplex virus-1 positive. No other bacteria, fungi, or viruses were found. The evolution was rapidly favorable after the initiation of antiviral treatment. Treatment was stopped after 3 weeks of well-conducted antiviral therapy. Curative-dose treatment was interrupted despite continuous strongly positive blood polymerase chain reaction results. In this context, prophylactic treatment was continued., Conclusion: We report an exceptional presentation of multiorgan failure in the intensive care unit due to herpes simplex virus-1. The diagnosis was made based on typical herpes simplex virus-1 visceral lesions and the absence of other responsible microorganisms. Intense viral replication is a key diagnostic element. There is no consensus regarding the most appropriate treatment duration, but such decisions should not be based on blood polymerase chain reaction., (© 2021. The Author(s).)

Synovial sarcoma (SyS) is an aggressive neoplasm driven by the SS18-SSX fusion, and is characterized by low T cell infiltration. Here, we studied the cancer-immune interplay in SyS using an integrative approach that combines single-cell RNA sequencing (scRNA-seq), spatial profiling and genetic and pharmacological perturbations. scRNA-seq of 16,872 cells from 12 human SyS tumors uncovered a malignant subpopulation that marks immune-deprived niches in situ and is predictive of poor clinical outcomes in two independent cohorts. Functional analyses revealed that this malignant cell state is controlled by the SS18-SSX fusion, is repressed by cytokines secreted by macrophages and T cells, and can be synergistically targeted with a combination of HDAC and CDK4/CDK6 inhibitors. This drug combination enhanced malignant-cell immunogenicity in SyS models, leading to induced T cell reactivity and T cell-mediated killing. Our study provides a blueprint for investigating heterogeneity in fusion-driven malignancies and demonstrates an interplay between immune evasion and oncogenic processes that can be co-targeted in SyS and potentially in other malignancies.

Synovial sarcoma (SyS) is an aggressive mesenchymal malignancy invariably associated with the chromosomal translocation t(X:18; p11:q11), which results in the in-frame fusion of the BAF complex gene SS18 to one of three SSX genes. Fusion of SS18 to SSX generates an aberrant transcriptional regulator, which, in permissive cells, drives tumor development by initiating major chromatin remodeling events that disrupt the balance between BAF-mediated gene activation and polycomb-dependent repression. Here, we developed SyS organoids and performed genome-wide epigenomic profiling of these models and mesenchymal precursors to define SyS-specific chromatin remodeling mechanisms and dependencies. We show that SS18-SSX induces broad BAF domains at its binding sites, which oppose polycomb repressor complex (PRC) 2 activity, while facilitating recruitment of a non-canonical (nc)PRC1 variant. Along with the uncoupling of polycomb complexes, we observed H3K27me3 eviction, H2AK119ub deposition and the establishment of de novo active regulatory elements that drive SyS identity. These alterations are completely reversible upon SS18-SSX depletion and are associated with vulnerability to USP7 loss, a core member of ncPRC1.1. Using the power of primary tumor organoids, our work helps define the mechanisms of epigenetic dysregulation on which SyS cells are dependent., (© 2020 Boulay et al.)

HIC1 ( Hypermethylated In Cancer 1 ) a tumor suppressor gene located at 17p13.3, is frequently deleted or epigenetically silenced in many human tumors. HIC1 encodes a transcriptional repressor involved in various aspects of the DNA damage response and in complex regulatory loops with P53 and SIRT1. HIC1 expression in normal prostate tissues has not yet been investigated in detail. Here, we demonstrated by immunohistochemistry that detectable HIC1 expression is restricted to the stroma of both normal and tumor prostate tissues. By RT-qPCR, we showed that HIC1 is poorly expressed in all tested prostate epithelial lineage cell types: primary (PrEC), immortalized (RWPE1) or transformed androgen-dependent (LnCAP) or androgen-independent (PC3 and DU145) prostate epithelial cells. By contrast, HIC1 is strongly expressed in primary PrSMC and immortalized (WMPY-1) prostate myofibroblastic cells. HIC1 depletion in WPMY-1 cells induced decreases in α-SMA expression and contractile capability. In addition to SLUG , we identified stromal cell-derived factor 1/C-X-C motif chemokine 12 ( SDF1/ CXCL12) as a new HIC1 direct target-gene. Thus, our results identify HIC1 as a tumor suppressor gene which is poorly expressed in the epithelial cells targeted by the tumorigenic process. HIC1 is expressed in stromal myofibroblasts and regulates CXCL12/SDF1 expression, thereby highlighting a complex interplay mediating the tumor promoting activity of the tumor microenvironment. Our studies provide new insights into the role of HIC1 in normal prostatic epithelial-stromal interactions through direct repression of CXCL12 and new mechanistic clues on how its loss of function through promoter hypermethylation during aging could contribute to prostatic tumors., Competing Interests: CONFLICTS OF INTEREST Authors have no conflicts of interest to declare., (Copyright: © 2020 Dubuissez et al.)

Polycomb repressive complex 2 (PRC2) allows the deposition of H3K27me3. PRC2 facultative subunits modulate its activity and recruitment such as hPCL3/PHF19, a human ortholog of Drosophila Polycomb-like protein (PCL). These proteins contain a TUDOR domain binding H3K36me3, two PHD domains and a "Winged-helix" domain involved in GC-rich DNA binding. The human PCL3 locus encodes the full-length hPCL3L protein and a shorter isoform, hPCL3S containing the TUDOR and PHD1 domains only. In this study, we demonstrated by RT-qPCR analyses of 25 prostate tumors that hPCL3S is frequently up-regulated. In addition, hPCL3S is overexpressed in the androgen-independent DU145 and PC3 cells, but not in the androgen-dependent LNCaP cells. hPCL3S knockdown decreased the proliferation and migration of DU145 and PC3 whereas its forced expression into LNCaP increased these properties. A mutant hPCL3S unable to bind H3K36me3 (TUDOR-W50A) increased proliferation and migration of LNCaP similarly to wt hPCL3S whereas inactivation of its PHD1 domain decreased proliferation. These effects partially relied on the up-regulation of genes known to be important for the proliferation and/or migration of prostate cancer cells such as S100A16, PlexinA2 , and Spondin1 . Collectively, our results suggest hPCL3S as a new potential therapeutic target in castration resistant prostate cancers., Competing Interests: CONFLICTS OF INTEREST The authors declare no disclosure of potential conflicts of interest.

Intact store-operated calcium entry (SOCE) mechanisms ensure the maintenance of Ca 2+ homeostasis in cardiomyocytes while their dysregulation promotes the development of cardiomyopathies. To better understand this calcium handling process in cardiomyocytes, we sought to identify unknown protein partners of stromal interaction molecule 1 (STIM1), a main regulatory protein of SOCE. We identified the muscle-related coiled-coil protein (MURC), also known as Cavin-4, as a candidate and showed that MURC interacts with STIM1 in cardiomyocytes. This interaction occurs via the HR1 and ERM domains of MURC and STIM1, respectively. Our results also demonstrated that the overexpression of MURC in neonatal rat ventricular myocytes (NRVM) is sufficient to potentiate SOCE and that its HR1 domain is required to mediate this effect. Interestingly, the R140W-MURC mutant, a missense variant of the HR1 domain associated with human dilated cardiomyopathy, exacerbates the SOCE increase in NRVM. Although the endogenous expression of STIM1 and Ca 2+ channel Orai1 is not modulated under these conditions, we showed that MURC increases the interaction between these proteins under resting conditions. Our study provides novel evidence that MURC regulates SOCE by interacting with STIM1 in cardiomyocytes. In addition, we identified a first potential mechanism by which the R140W mutation of MURC may contribute to calcium mishandling and the development of cardiomyopathies., (Copyright © 2019. Published by Elsevier B.V.)

Tumor-stromal communication within the microenvironment contributes to initiation of metastasis and may present a therapeutic opportunity. Using serial single-cell RNA sequencing in an orthotopic mouse prostate cancer model, we find up-regulation of prolactin receptor as cancer cells that have disseminated to the lungs expand into micrometastases. Secretion of the ligand prolactin by adjacent lung stromal cells is induced by tumor cell production of the COX-2 synthetic product prostaglandin E2 (PGE2). PGE2 treatment of fibroblasts activates the orphan nuclear receptor NR4A (Nur77), with prolactin as a major transcriptional target for the NR4A-retinoid X receptor (RXR) heterodimer. Ectopic expression of prolactin receptor in mouse cancer cells enhances micrometastasis, while treatment with the COX-2 inhibitor celecoxib abrogates prolactin secretion by fibroblasts and reduces tumor initiation. Across multiple human cancers, COX-2, prolactin, and prolactin receptor show consistent differential expression in tumor and stromal compartments. Such paracrine cross-talk may thus contribute to the documented efficacy of COX-2 inhibitors in cancer suppression., Competing Interests: Conflict of interest statement: Massachusetts General Hospital has filed for patent protection for the circulating tumor cell inertial focusing (iChip) technology., (Copyright © 2019 the Author(s). Published by PNAS.)

Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Various types of repetitive sequences are dysregulated in cancer. In Ewing sarcoma, the oncogenic fusion protein EWS-FLI1 induces chromatin features typical of active enhancers at GGAA microsatellite repeats, but the function of these sites has not been directly demonstrated. Here, by combining nascent transcription profiling with epigenome editing, we found that a subset of GGAA microsatellite repeats is transcriptionally active in Ewing sarcoma and that silencing individual repeats abolishes local nascent transcription and leads to markedly reduced expression of putative target genes. Epigenome silencing of these repeat sites does not affect gene expression in unrelated cells, can prevent the induction of gene expression by EWS-FLI1, and, in the case of a GGAA repeat that controls SOX2 expression from a distance of 470 kb, is sufficient to impair the growth of Ewing sarcoma xenografts. Using an experimental approach that is broadly applicable to testing different types of repetitive genomic elements, our study directly demonstrates that specific repeat microsatellites can have critical gene regulation functions in cancer and thus represent tumor-specific vulnerabilities that may be exploited to develop new therapies., (© 2018 Boulay et al.; Published by Cold Spring Harbor Laboratory Press.)

Alterations in transcriptional regulators can orchestrate oncogenic gene expression programs in cancer. Here, we show that the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, which is mutated in over 20% of human tumors, interacts with EWSR1, a member of a family of proteins with prion-like domains (PrLD) that are frequent partners in oncogenic fusions with transcription factors. In Ewing sarcoma, we find that the BAF complex is recruited by the EWS-FLI1 fusion protein to tumor-specific enhancers and contributes to target gene activation. This process is a neomorphic property of EWS-FLI1 compared to wild-type FLI1 and depends on tyrosine residues that are necessary for phase transitions of the EWSR1 prion-like domain. Furthermore, fusion of short fragments of EWSR1 to FLI1 is sufficient to recapitulate BAF complex retargeting and EWS-FLI1 activities. Our studies thus demonstrate that the physical properties of prion-like domains can retarget critical chromatin regulatory complexes to establish and maintain oncogenic gene expression programs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Medulloblastoma is the most frequent malignant pediatric brain tumor and is divided into at least four subgroups known as WNT, SHH, Group 3, and Group 4. Here, we characterized gene regulation mechanisms in the most aggressive subtype, Group 3 tumors, through genome-wide chromatin and expression profiling. Our results show that most active distal sites in these tumors are occupied by the transcription factor OTX2. Highly active OTX2-bound enhancers are often arranged as clusters of adjacent peaks and are also bound by the transcription factor NEUROD1. These sites are responsive to OTX2 and NEUROD1 knockdown and could also be generated de novo upon ectopic OTX2 expression in primary cells, showing that OTX2 cooperates with NEUROD1 and plays a major role in maintaining and possibly establishing regulatory elements as a pioneer factor. Among OTX2 target genes, we identified the kinase NEK2, whose knockdown and pharmacologic inhibition decreased cell viability. Our studies thus show that OTX2 controls the regulatory landscape of Group 3 medulloblastoma through cooperative activity at enhancer elements and contributes to the expression of critical target genes. Significance: The gene regulation mechanisms that drive medulloblastoma are not well understood. Using chromatin profiling, we find that the transcription factor OTX2 acts as a pioneer factor and, in cooperation with NEUROD1, controls the Group 3 medulloblastoma active enhancer landscape. OTX2 itself or its target genes, including the mitotic kinase NEK2, represent attractive targets for future therapies. Cancer Discov; 7(3); 288-301. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 235 ., (©2017 American Association for Cancer Research.)

T lymphocytes from Gimap5 lyp/lyp rats carrying a recessive mutation in the GTPase of immune-associated protein 5 ( Gimap5 ) gene undergo spontaneous apoptosis. Molecular mechanisms underlying this survival defect are not yet clear. We have shown that Gimap5 lyp/lyp T lymphocytes display reduced calcium influx following T cell antigen receptor (TCR) stimulation that was associated with impaired buffering of calcium by mitochondria. Here, we investigated the subcellular localization of GIMAP5 and its influence on Ca 2+ response in HEK293T cells and T lymphocytes. The more abundantly expressed GIMAP5v2 localizes to the lysosome and certain endosomal vesicles. Gimap5 lyp/lyp T lymphocytes showed increased accumulation of calcium in the lysosomes as evidenced by Gly-Phe β-naphthylamide (GPN) triggered Ca 2+ release. As a corollary, GPN-induced Ca 2+ flux was decreased in HEK293T cells expressing GIMAP5v2. Strikingly, TCR stimulation of rat, mouse, and human T lymphocytes increased lysosomal calcium content. Overall, our findings show that lysosomes modulate cellular Ca 2+ response during T cell activation and that GIMAP5 regulates the lysosomal Ca 2+ compartment in T lymphocytes.

Treatment of EGFR-mutant lung cancer with erlotinib results in dramatic tumor regression but it is invariably followed by drug resistance. In characterizing early transcriptional changes following drug treatment of mutant EGFR-addicted cells, we identified the stem cell transcriptional regulator SOX2 as being rapidly and specifically induced, both in vitro and in vivo. Suppression of SOX2 sensitizes cells to erlotinib-mediated apoptosis, ultimately decreasing the emergence of acquired resistance, whereas its ectopic expression reduces drug-induced cell death. We show that erlotinib relieves EGFR-dependent suppression of FOXO6, leading to its induction of SOX2, which in turn represses the pro-apoptotic BH3-only genes BIM and BMF. Together, these observations point to a physiological feedback mechanism that attenuates oncogene addiction-mediated cell death associated with the withdrawal of growth factor signaling and may therefore contribute to the development of resistance.

Background: Blood L-lactate concentration (LAC) can be used for various diagnostic purposes in cattle. As multiple handheld analyzers for LAC exist, it is important to validate their use in cattle in comparison with reference laboratory blood analyzers., Objectives: The objectives of this study were to validate the handheld Lactate Pro meter (LacP) including reproducibility, and compare the measurements with the StatProfile (StatP) as a gold standard. In addition, diagnostic sensitivity and specificity, and the impact of HCT on LAC measured by both analyzers were assessed., Methods: A cohort of 64 cattle with acute medical and surgical conditions was studied. Whole blood samples in heparin lithium tubes were analyzed upon arrival with both StatP and LacP. Twenty-three samples were immediately retested to assess intra-assay coefficient of variation (CV). The HCT values were also recorded., Results: The LAC using LacP was highly correlated with the StatP (r = 0.9736 [95% confidence interval [CI]: 0.9562-0.9841]). The LacP underestimated LAC (mean difference:-0.9 mmol/L, 95% CI:-3.1 mmol/L to 1.3 mmol/L). The intra-assay CV was excellent (4.77%). No significant correlation was observed between LacP or StatP and HCT (P = .39 and .09, respectively). Sensitivity and specificity for LacP were 91.7% (95% CI: 76.4-97.8%) and 100% (83.4-100%, cutoff of 4 mmol/L), and 78.6% (58.5-90.9%) and 100% (87.0-100%, cutoff of 6 mmol/L)., Conclusions: The LacP handheld lactate meter can be used safely and reliably cow-side, although it underestimates LAC value when compared with a standard laboratory analyzer especially for LAC ≥ 10.0 mmol/L. The LAC value was not influenced by HCT in this study., (© 2014 American Society for Veterinary Clinical Pathology.)

The aberrant transcription factor EWS-FLI1 drives Ewing sarcoma, but its molecular function is not completely understood. We find that EWS-FLI1 reprograms gene regulatory circuits in Ewing sarcoma by directly inducing or repressing enhancers. At GGAA repeat elements, which lack evolutionary conservation and regulatory potential in other cell types, EWS-FLI1 multimers induce chromatin opening and create de novo enhancers that physically interact with target promoters. Conversely, EWS-FLI1 inactivates conserved enhancers containing canonical ETS motifs by displacing wild-type ETS transcription factors. These divergent chromatin-remodeling patterns repress tumor suppressors and mesenchymal lineage regulators while activating oncogenes and potential therapeutic targets, such as the kinase VRK1. Our findings demonstrate how EWS-FLI1 establishes an oncogenic regulatory program governing both tumor survival and differentiation., (Copyright © 2014 Elsevier Inc. All rights reserved.)