Microbiota-Induced Type I Interferons Instruct a Poised Basal State of Dendritic Cells.

Environmental signals shape host physiology and fitness. Microbiota-derived cues are required to program conventional dendritic cells (cDCs) during the steady state so that they can promptly respond and initiate adaptive immune responses when encountering pathogens. However, the molecular underpinnings of microbiota-guided instructive programs are not well understood. Here, we report that the indigenous microbiota controls constitutive production of type I interferons (IFN-I) by plasmacytoid DCs. Using genome-wide analysis of transcriptional and epigenetic regulomes of cDCs from germ-free and IFN-I receptor (IFNAR)-deficient mice, we found that tonic IFNAR signaling instructs a specific epigenomic and metabolic basal state that poises cDCs for future pathogen combat. However, such beneficial biological function comes with a trade-off. Instructed cDCs can prime T cell responses against harmless peripheral antigens when removing roadblocks of peripheral tolerance. Our data provide fresh insights into the evolutionary trade-offs that come with successful adaptation of vertebrates to their microbial environment.

Arginase-1 Deletion in Erythrocytes Promotes Vascular Calcification via Enhanced GSNOR (S-Nitrosoglutathione Reductase) Expression and NO Signaling in Smooth Muscle Cells.

BACKGROUND: Erythrocytes (red blood cells) participate in the control of vascular NO bioavailability. The purpose of this study was to determine whether and how genetic deletion of ARG1 (arginase-1) affects vascular smooth muscle cell NO signaling, osteoblastic differentiation, and atherosclerotic lesion calcification. METHODS: Atherosclerosis-prone mice with conditional, erythrocyte-restricted deletion of ARG1 (apoE-/- red blood cell.ARG1 knockout) were generated and vascular calcification studied using molecular imaging of the osteogenic activity agent OsteoSense, Alizarin staining or immunohistochemistry, qPCR of osteogenic markers and ex vivo assays. RESULTS: Atherosclerotic lesion size at the aortic root did not differ, but calcification was significantly more pronounced in apoE-/- mice lacking erythrocyte ARG1. Incubation of murine and human VSMCs with lysed erythrocyte membranes from apoE-/- red blood cell. ARG1-knockout mice accelerated their osteogenic differentiation, and mRNA transcripts of osteogenic markers decreased following NO scavenging. In addition to NO signaling via sGC (soluble guanylyl cyclase), overexpression of GSNOR (S-nitrosoglutathione reductase) enhanced degradation of S-nitrosoglutathione to glutathione and reduced protein S-nitrosation of HSP (heat shock protein)-70 were identified as potential mechanisms of vascular smooth muscle cell calcification in mice lacking ARG1 in erythrocytes, and calcium phosphate deposition was enhanced by heat shock and prevented by GSNOR inhibition. Messenger RNA levels of enzymes metabolizing the arginase products L-ornithine and L-proline also were elevated in VSMCs, paralleled by increased proliferation, myofibroblast marker and collagen type 1 expression. CONCLUSIONS: Our findings support an important role of erythrocyte ARG1 for NO bioavailability and L-arginine metabolism in VSMCs, which controls atherosclerotic lesion composition and calcification.

TREND-DB-a transcriptome-wide atlas of the dynamic landscape of alternative polyadenylation.

Alternative polyadenylation (APA) profoundly expands the transcriptome complexity. Perturbations of APA can disrupt biological processes, ultimately resulting in devastating disorders. A major challenge in identifying mechanisms and consequences of APA (and its perturbations) lies in the complexity of RNA 3' end processing, involving poorly conserved RNA motifs and multi-component complexes consisting of far more than 50 proteins. This is further complicated in that RNA 3' end maturation is closely linked to transcription, RNA processing and even epigenetic (histone/DNA/RNA) modifications. Here, we present TREND-DB (http://shiny.imbei.uni-mainz.de:3838/trend-db), a resource cataloging the dynamic landscape of APA after depletion of >170 proteins involved in various facets of transcriptional, co- and post-transcriptional gene regulation, epigenetic modifications and further processes. TREND-DB visualizes the dynamics of transcriptome 3' end diversification (TREND) in a highly interactive manner; it provides a global APA network map and allows interrogating genes affected by specific APA-regulators and vice versa. It also permits condition-specific functional enrichment analyses of APA-affected genes, which suggest wide biological and clinical relevance across all RNAi conditions. The implementation of the UCSC Genome Browser provides additional customizable layers of gene regulation accounting for individual transcript isoforms (e.g. epigenetics, miRNA-binding sites and RNA-binding proteins). TREND-DB thereby fosters disentangling the role of APA for various biological programs, including potential disease mechanisms, and helps identify their diagnostic and therapeutic potential.

Cluster of differentiation 44 promotes osteosarcoma progression in mice lacking the tumor suppressor Merlin.

Merlin is a versatile tumor suppressor protein encoded by the NF2 gene. Several lines of evidence suggest that Merlin exerts its tumor suppressor activity, at least in part, by forming an inhibitory complex with cluster of differentiation 44 (CD44). Consistently, numerous NF2 mutations in cancer patients are predicted to perturb the interaction of Merlin with CD44. We hypothesized that disruption of the Merlin-CD44 complex through loss of Merlin, unleashes putative tumor- or metastasis-promoting functions of CD44. To evaluate the relevance of the Merlin-CD44 interaction in vivo, we compared tumor growth and progression in Cd44-positive and Cd44-negative Nf2-mutant mice. Heterozygous Nf2-mutant mice were prone to developing highly metastatic osteosarcomas. Importantly, while the absence of the Cd44 gene had no effect on the frequency of primary osteosarcoma development, it strongly diminished osteosarcoma metastasis formation in the Nf2-mutant mice. In vitro assays identified transendothelial migration as the most prominent cellular phenotype dependent on CD44. Adhesion to endothelial cells was blocked by interfering with integrin α4ß1 (very late antigen-4, VLA-4) on osteosarcoma cells and CD44 upregulated levels of integrin VLA-4 ß1 subunit. Among other putative functions of CD44, which may contribute to the metastatic behavior, the passage through the endothelial cells also appears to be critical in vivo, as CD44 significantly promoted formation of lung metastasis upon intravenous injection of osteosarcoma cells into immunocompromised mice. Altogether, our results strongly suggest that CD44 plays a metastasis-promoting role in the absence of Merlin.

A non-invasive diagnostic assay for rapid detection and characterization of aberrant mRNA-splicing by nonsense mediated decay inhibition.

BACKGROUND: Interpretation of genetic variants detected by sequencing of genomic DNA, which may cause splicing defects, regularly requires mRNA analysis. Usually, only bioinformatic testing is provided, because simple and non-invasive assay protocols are lacking. Furthermore, the detection of mis-splicing is often hampered by nonsense mediated mRNA decay (NMD). METHODS: Starting from a case of Pompe disease with two potential splicing variants an assay for the analysis of splice defects in general was developed. We analyzed the transcripts from the gene of interest by standard methods after short-term culture of the patient's lymphocytes in the presence and absence of a NMD inhibitor. Variant and wild type transcript expression were quantified by allele specific PCR in the patient and both parents and the expression ratio with/without NMD inhibition was calculated for each transcript. RESULTS: NMD detection in lymphocytes was optimized and evaluated by analyzing a naturally occurring NMD transcript. Several compounds inhibited NMD successfully, including potential therapeutic agents. Sample storage for up to 4 days at room temperature prior to lymphocyte isolation did not affect results. In a proof of concept we identified two candidate variants as severe splicing variants in a patient with Pompe disease, but the strategy can also be used to screen for any mis-spliced transcripts prone to NMD. CONCLUSIONS: We developed a simple, non-invasive assay for the detection and characterization of potential splicing variants. This is essential, because early and near-term diagnosis and disease classification is required to facilitate therapy in many genetic diseases.

p38 MAPK controls prothrombin expression by regulated RNA 3' end processing.

Thrombin is a key protease involved in blood coagulation, complement activation, inflammation, angiogenesis, and tumor invasion. Although induced in many (patho-)physiological conditions, the underlying mechanisms controlling prothrombin expression remained enigmatic. We have now discovered that prothrombin expression is regulated by a posttranscriptional regulatory mechanism responding to stress and inflammation. This mechanism is triggered by external stimuli that activate p38 MAPK. In turn, p38 MAPK upmodulates canonical 3' end processing components and phosphorylates the RNA-binding proteins FBP2 and FBP3, which inhibit 3' end processing of mRNAs, such as prothrombin mRNA, that bear a defined upstream sequence element (USE) in their 3'UTRs. Upon phosphorylation, FBP2 and FBP3 dissociate from the USE, making it accessible to proteins that stimulate 3' end processing. We provide in vivo evidence suggesting the importance of this mechanism in inflammatory hypercoagulation and tumor invasion. Regulated 3' end processing thus emerges as a key mechanism of gene regulation with broad biological and medical implications.

sCLIP-an integrated platform to study RNA-protein interactomes in biomedical research: identification of CSTF2tau in alternative processing of small nuclear RNAs.

RNA-binding proteins (RBPs) are central for gene expression by controlling the RNA fate from birth to decay. Various disorders arising from perturbations of RNA-protein interactions document their critical function. However, deciphering their function is complex, limiting the general functional elucidation of this growing class of proteins and their contribution to (patho)physiology. Here, we present sCLIP, a simplified and robust platform for genome-wide interrogation of RNA-protein interactomes based on crosslinking-immunoprecipitation and high-throughput sequencing. sCLIP exploits linear amplification of the immunoprecipitated RNA improving the complexity of the sequencing-library despite significantly reducing the amount of input material and omitting several purification steps. Additionally, it permits a radiolabel-free visualization of immunoprecipitated RNA. In a proof of concept, we identify that CSTF2tau binds many previously not recognized RNAs including histone, snoRNA and snRNAs. CSTF2tau-binding is associated with internal oligoadenylation resulting in shortened snRNA isoforms subjected to rapid degradation. We provide evidence for a new mechanism whereby CSTF2tau controls the abundance of snRNAs resulting in alternative splicing of several RNAs including ANK2 with critical roles in tumorigenesis and cardiac function. Combined with a bioinformatic pipeline sCLIP thus uncovers new functions for established RBPs and fosters the illumination of RBP-protein interaction landscapes in health and disease.

Processing and transcriptome expansion at the mRNA 3' end in health and disease: finding the right end.

The human transcriptome is highly dynamic, with each cell type, tissue, and organ system expressing an ensemble of transcript isoforms that give rise to considerable diversity. Apart from alternative splicing affecting the "body" of the transcripts, extensive transcriptome diversification occurs at the 3' end. Transcripts differing at the 3' end can have profound physiological effects by encoding proteins with distinct functions or regulatory properties or by affecting the mRNA fate via the inclusion or exclusion of regulatory elements (such as miRNA or protein binding sites). Importantly, the dynamic regulation at the 3' end is associated with various (patho)physiological processes, including the immune regulation but also tumorigenesis. Here, we recapitulate the mechanisms of constitutive mRNA 3' end processing and review the current understanding of the dynamically regulated diversity at the transcriptome 3' end. We illustrate the medical importance by presenting examples that are associated with perturbations of this process and indicate resulting implications for molecular diagnostics as well as potentially arising novel therapeutic strategies.

The Importance of Thrombin in Cerebral Injury and Disease.

There is increasing evidence that prothrombin and its active derivative thrombin are expressed locally in the central nervous system. So far, little is known about the physiological and pathophysiological functions exerted by thrombin in the human brain. Extra-hepatic prothrombin expression has been identified in neuronal cells and astrocytes via mRNA measurement. The actual amount of brain derived prothrombin is expected to be 1% or less compared to that in the liver. The role in brain injury depends upon its concentration, as higher amounts cause neuroinflammation and apoptosis, while lower concentrations might even be cytoprotective. Its involvement in numerous diseases like Alzheimer's, multiple sclerosis, cerebral ischemia and haemorrhage is becoming increasingly clear. This review focuses on elucidation of the cerebral thrombin expression, local generation and its role in injury and disease of the central nervous system.

Plasma levels of complement components C5 and C9 are associated with thrombin generation.

BACKGROUND: The thrombin generation assay (TGA) evaluates the potential of plasma to generate thrombin over time, providing a global picture of an individual's hemostatic balance. OBJECTIVES: This study aimed to identify novel biological determinants of thrombin generation using a multiomics approach. METHODS: Associations between TGA parameters and plasma levels of 377 antibodies targeting 236 candidate proteins for cardiovascular risk were tested using multiple linear regression analysis in 770 individuals with venous thrombosis from the Marseille Thrombosis Association (MARTHA) study. Proteins associated with at least 3 TGA parameters were selected for validation in an independent population of 536 healthy individuals (Etablissement Français du Sang Alpes-Méditerranée [EFS-AM]). Proteins with strongest associations in both groups underwent additional genetic analyses and in vitro experiments. RESULTS: Eighteen proteins were associated (P < 1.33 × 10⁻4) with at least 3 TGA parameters in MARTHA, among which 13 demonstrated a similar pattern of associations in EFS-AM. Complement proteins C5 and C9 had the strongest associations in both groups. Ex vivo supplementation of platelet-poor plasma with purified C9 protein had a significant dose-dependent effect on TGA parameters. No effect was observed with purified C5. Several single nucleotide polymorphisms associated with C5 and C9 plasma levels were identified, with the strongest association for the C5 missense variant rs17611, which was associated with a decrease in C5 levels, endogenous thrombin potential, and peak in MARTHA. No association of this variant with TGA parameters was observed in EFS-AM. CONCLUSION: This study identified complement proteins C5 and C9 as potential determinants of thrombin generation. Further studies are warranted to establish causality and elucidate the underlying mechanisms.

Post-transcriptional control of haemostatic genes: mechanisms and emerging therapeutic concepts in thrombo-inflammatory disorders.

The haemostatic system is pivotal to maintaining vascular integrity. Multiple components involved in blood coagulation have central functions in inflammation and immunity. A derailed haemostasis is common in prevalent pathologies such as sepsis, cardiovascular disorders, and lately, COVID-19. Physiological mechanisms limit the deleterious consequences of a hyperactivated haemostatic system through adaptive changes in gene expression. While this is mainly regulated at the level of transcription, co- and posttranscriptional mechanisms are increasingly perceived as central hubs governing multiple facets of the haemostatic system. This layer of regulation modulates the biogenesis of haemostatic components, for example in situations of increased turnover and demand. However, they can also be 'hijacked' in disease processes, thereby perpetuating and even causally entertaining associated pathologies. This review summarizes examples and emerging concepts that illustrate the importance of posttranscriptional mechanisms in haemostatic control and crosstalk with the immune system. It also discusses how such regulatory principles can be used to usher in new therapeutic concepts to combat global medical threats such as sepsis or cardiovascular disorders.

3' end mRNA processing: molecular mechanisms and implications for health and disease.

Recent advances in the understanding of the molecular mechanism of mRNA 3' end processing have uncovered a previously unanticipated integrated network of transcriptional and RNA-processing mechanisms. A variety of human diseases impressively reflect the importance of the precision of the complex 3' end-processing machinery and gene specific deregulation of 3' end processing can result from mutations of RNA sequence elements that bind key specific processing factors. Interestingly, more general deregulation of 3' end processing can be caused either by mutations of these processing factors or by the disturbance of the well-coordinated equilibrium between these factors. From a medical perspective, both loss of function and gain of function can be functionally relevant, and an increasing number of different disease entities exemplifies that inappropriate 3' end formation of human mRNAs can have a tremendous impact on health and disease. Here, we review the mechanistic hallmarks of mRNA 3' end processing, highlight the medical relevance of deregulation of this important step of mRNA maturation and illustrate the implications for diagnostic and therapeutic strategies.

Pathophysiological Role and Diagnostic Potential of R-Loops in Cancer and Beyond.

R-loops are DNA-RNA hybrids that play multifunctional roles in gene regulation, including replication, transcription, transcription-replication collision, epigenetics, and preserving the integrity of the genome. The aberrant formation and accumulation of unscheduled R-loops can disrupt gene expression and damage DNA, thereby causing genome instability. Recent links between unscheduled R-loop accumulation and the abundance of proteins that modulate R-loop biogenesis have been associated with numerous human diseases, including various cancers. Although R-loops are not necessarily causative for all disease entities described to date, they can perpetuate and even exacerbate the initially disease-eliciting pathophysiology, making them structures of interest for molecular diagnostics. In this review, we discuss the (patho) physiological role of R-loops in health and disease, their surprising diagnostic potential, and state-of-the-art techniques for their detection.

The Bidirectional Link Between RNA Cleavage and Polyadenylation and Genome Stability: Recent Insights From a Systematic Screen.

The integrity of the genome is governed by multiple processes to ensure optimal survival and to prevent the inheritance of deleterious traits. While significant progress has been made to characterize components involved in the DNA Damage Response (DDR), little is known about the interplay between RNA processing and the maintenance of genome stability. Here, we describe the emerging picture of an intricate bidirectional coupling between RNA processing and genome integrity in an integrative manner. By employing insights from a recent large-scale RNAi screening involving the depletion of more than 170 components that direct (alternative) polyadenylation, we provide evidence of bidirectional crosstalk between co-transcriptional RNA 3'end processing and the DDR in a manner that optimizes genomic integrity. We provide instructive examples illustrating the wiring between the two processes and show how perturbations at one end are either compensated by buffering mechanisms at the other end, or even propel the initial insult and thereby become disease-eliciting as evidenced by various disorders.

A novel rationale for targeting FXI: Insights from the hemostatic microRNA targetome for emerging anticoagulant strategies.

Therapeutic targeting of blood coagulation is a challenging task as it interferes with the delicate balance of pro- and anticoagulant activities. Anticoagulants are employed in millions of thrombophilic patients worldwide each year. The treatment and prevention of venous thromboembolism has changed drastically. Traditional vitamin K antagonists are being replaced by direct oral anticoagulants (DOACs), which selectively target coagulation factors Xa or IIa. However for a growing population with comorbidities satisfying therapeutic options are still lacking and the quest for novel therapeutics continues. Recently, targeting factors XI or XII have emerged as new therapeutic strategies. As these factors play important roles in thrombosis, yet are essentially dispensable for hemostasis, these strategies may overcome the obstacle of treating or preventing thrombosis without affecting hemostasis. Based on the recent elucidation of the hemostatic microRNA targetome, we introduce and discuss a hitherto unrecognized rationale for the therapeutic targeting of factor XI. This is based on mimicking endogenous factor XI expression control by therapeutic delivery of microRNA mimics. We discuss the functional difference between various gene-targeting approaches, and propose the hemostatic system to represent an ideal model for assessment of the efficacy and safety of such therapeutic components, ushering in a novel therapeutic era with broad applicability.

TRENDseq-A highly multiplexed high throughput RNA 3' end sequencing for mapping alternative polyadenylation.

Alternative polyadenylation (APA) is a widespread and highly dynamic mechanism of gene regulation. It affects more than 70% of all genes, resulting in transcript isoforms with distinct 3' end termini. APA thereby considerably expands the diversity of the transcriptome 3' end (TREND). This leads to mRNA isoforms with profoundly different physiological effects, by affecting protein output, production of distinct protein isoforms, or modulating protein localization. APA is globally regulated in various conditions, including developmental and adaptive programs. Since perturbations of APA can disrupt biological processes, ultimately resulting in most devastating disorders, querying the APA landscape is crucial to decipher underlying mechanisms, resulting consequences and potential diagnostic and therapeutic implications. Here we provide a detailed step-by-step protocol for TRENDseq, a method for transcriptome-wide high-throughput sequencing of polyadenylated RNA 3' ends in a highly multiplexed fashion. TRENDseq exploits linear amplification of the starting material to improve sensitivity while significantly reducing the amount of input material. It thereby represents a powerful tool to study APA in numerous experimental set-ups and/or limited human samples in a highly multiplexed and reproducible manner.

No Evidence for Classic Thrombotic Microangiopathy in COVID-19.

BACKGROUND: Coronavirus disease-2019 (COVID-19) triggers systemic infection with involvement of the respiratory tract. There are some patients developing haemostatic abnormalities during their infection with a considerably increased risk of death. MATERIALS AND METHODS: Patients (n = 85) with SARS-CoV-2 infection attending the University Medical Center, Mainz, from 3 March to 15 May 2020 were retrospectively included in this study. Data regarding demography, clinical features, treatment and laboratory parameters were analyzed. Twenty patients were excluded for assessment of disseminated intravascular coagulation (DIC) and thrombotic microangiopathy (TMA) due to lack of laboratory data. RESULTS: COVID-19 patients (n = 65) were investigated, 19 with uncomplicated, 29 with complicated, and 17 with critical course; nine (13.8%) died. Seven patients showed overt DIC according to the ISTH criteria. The fibrinogen levels dropped significantly in these patients, although not below 100 mg/dl. Hallmarks of TMA, such as thrombocytopenia and microangiopathic haemolytic anaemia, were not detected in any of our COVID-19 patients. ADAMTS13 activity was mildly to moderately reduced in 4/22 patients, all having strongly elevated procalcitonin levels. CONCLUSION: DIC occurred in 7/65 COVID-19 patients but fibrinogen and platelet consumption were compensated in almost all. ADAMTS13 assays excluded TTP and hallmarks of classic TMA were absent in all investigated patients. We hypothesize that the lacking erythrocyte fragmentation and only mild platelet consumption in severe COVID-19 are due to a microangiopathy predominantly localized to the alveolar microcirculation with a low blood pressure gradient.

Emerging Roles of RNA 3'-end Cleavage and Polyadenylation in Pathogenesis, Diagnosis and Therapy of Human Disorders.

A crucial feature of gene expression involves RNA processing to produce 3' ends through a process termed 3' end cleavage and polyadenylation (CPA). This ensures the nascent RNA molecule can exit the nucleus and be translated to ultimately give rise to a protein which can execute a function. Further, alternative polyadenylation (APA) can produce distinct transcript isoforms, profoundly expanding the complexity of the transcriptome. CPA is carried out by multi-component protein complexes interacting with multiple RNA motifs and is tightly coupled to transcription, other steps of RNA processing, and even epigenetic modifications. CPA and APA contribute to the maintenance of a multitude of diverse physiological processes. It is therefore not surprising that disruptions of CPA and APA can lead to devastating disorders. Here, we review potential CPA and APA mechanisms involving both loss and gain of function that can have tremendous impacts on health and disease. Ultimately we highlight the emerging diagnostic and therapeutic potential CPA and APA offer.

Macrophage protease-activated receptor 2 regulates fetal liver erythropoiesis in mice.

Deficiencies in many coagulation factors and protease-activated receptors (PARs) affect embryonic development. We describe a defect in definitive erythropoiesis in PAR2-deficient mice. Embryonic PAR2 deficiency increases embryonic death associated with variably severe anemia in comparison with PAR2-expressing embryos. PAR2-deficient fetal livers display reduced macrophage densities, erythroblastic island areas, and messenger RNA expression levels of markers for erythropoiesis and macrophages. Coagulation factor synthesis in the liver coincides with expanding fetal liver hematopoiesis during midgestation, and embryonic factor VII (FVII) deficiency impairs liver macrophage development. Cleavage-insensitive PAR2-mutant mice recapitulate the hematopoiesis defect of PAR2-deficient embryos, and macrophage-expressed PAR2 directly supports erythroblastic island function and the differentiation of red blood cells in the fetal liver. Conditional deletion of PAR2 in macrophages impairs erythropoiesis, as well as increases inflammatory stress, as evidenced by upregulation of interferon-regulated hepcidin antimicrobial peptide. In contrast, postnatal macrophage PAR2 deficiency does not have any effect on steady-state Kupffer cells, bone marrow macrophage numbers, or erythropoiesis, but erythropoiesis in macrophages from PAR2-deficient mice is impaired following hemolysis. These data identify a novel function for macrophage PAR2 signaling in adapting to rapid increases in blood demand during gestational development and postnatal erythropoiesis under stress conditions.

Downregulation of Notch signaling by gamma-secretase inhibition can abrogate chemotherapy-induced apoptosis in T-ALL cell lines.

Activation of Notch1 signaling plays an important role in the pathogenesis of precursor T-cell lymphoblastic leukemia (T-ALL). The Notch1 receptor is cleaved and activated via the gamma-secretase complex. Downregulation of Notch1 signaling by gamma-secretase inhibitors (GSIs) thus represents a potential novel therapeutic approach. In this study, we analyzed the response of four T-ALL cell lines to compound E, a potent gamma-secretase inhibitor, and to the combination of compound E with vincristine, daunorubicin, L-asparaginase (L-ASP), and dexamethasone (DEX). We identified two distinct types of responses: In type 1 cell lines, represented by TALL1 and HSB2, GSI-induced apoptosis followed cell cycle arrest and enhanced the induction of apoptosis caused by DEX and L-ASP. In type 2 cell lines, represented by CEM and Jurkat J6, GSI caused neither cell cycle block nor cell death. Notably, the combination of GSI with chemotherapy-induced resistance by decreasing apoptosis. In type 2 cells, GSI induced the upregulation of Bcl-xl mRNA and protein, which was thus identified as a candidate mechanism for the inhibition of apoptosis. In conclusion, the data presented here caution against clinical use of a combination treatment of GSI and chemotherapy in T-ALL.