Transient exposure to miR-203 enhances the differentiation capacity of established pluripotent stem cells.

Full differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) induces a transient expression of 2C markers that later results in expanded differentiation potency to multiple lineages, as well as improved efficiency in tetraploid complementation and human-mouse interspecies chimerism assays. Mechanistically, these effects are at least partially mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasure of DNA methylation. These data support the use of transient exposure to miR-203 as a versatile method to reset the epigenetic memory in PSCs, and improve their effectiveness in regenerative medicine.

SOCS3 deregulation contributes to aberrant activation of the JAK/STAT pathway in precursor T-cell neoplasms.

Despite the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway being frequently altered in T-ALL/LBL, no specific therapy has been approved for T-ALL/LBL patients with constitutive signalling by JAK/STAT, so there is an urgent need to identify pathway members that may be potential therapeutic targets. In the present study, we searched for JAK/STAT pathway members potentially modulated through aberrant methylation and identified SOCS3 hypermethylation as a recurrent event in T-ALL/LBL. Additionally, we explored the implications of SOCS3 deregulation in T-ALL/LBL and demonstrated that SOCS3 counteracts the constitutive activation of the JAK/STAT pathway through different molecular mechanisms. Therefore, SOCS3 emerges as a potential therapeutic target in T-ALL/LBL.

A Dual Role for FADD in Human Precursor T-Cell Neoplasms.

A reduction in FADD levels has been reported in precursor T-cell neoplasms and other tumor types. Such reduction would impact on the ability of tumor cells to undergo apoptosis and has been associated with poor clinical outcomes. However, FADD is also known to participate in non-apoptotic functions, but these mechanisms are not well-understood. Linking FADD expression to the severity of precursor T-cell neoplasms could indicate its use as a prognostic marker and may open new avenues for targeted therapeutic strategies. Using transcriptomic and clinical data from patients with precursor T-cell neoplasms, complemented by in vitro analysis of cellular functions and by high-throughput interactomics, our results allow us to propose a dual role for FADD in precursor T-cell neoplasms, whereby resisting cell death and chemotherapy would be a canonical consequence of FADD deficiency in these tumors, whereas deregulation of the cellular metabolism would be a relevant non-canonical function in patients expressing FADD. These results reveal that evaluation of FADD expression in precursor T-cell neoplasms may aid in the understanding of the biological processes that are affected in the tumor cells. The altered biological processes can be of different natures depending on the availability of FADD influencing its ability to exert its canonical or non-canonical functions. Accordingly, specific therapeutic interventions would be needed in each case.

More Insights on the Use of γ-Secretase Inhibitors in Cancer Treatment.

The NOTCH1 gene encodes a transmembrane receptor protein with activating mutations observed in many T-cell acute lymphoblastic leukemias (T-ALLs) and lymphomas, as well as in other tumor types, which has led to interest in inhibiting NOTCH1 signaling as a therapeutic target in cancer. Several classes of Notch inhibitors have been developed, including monoclonal antibodies against NOTCH receptors or ligands, decoys, blocking peptides, and γ-secretase inhibitors (GSIs). GSIs block a critical proteolytic step in NOTCH activation and are the most widely studied. Current treatments with GSIs have not successfully passed clinical trials because of side effects that limit the maximum tolerable dose. Multiple γ-secretase-cleavage substrates may be involved in carcinogenesis, indicating that there may be other targets for GSIs. Resistance mechanisms may include PTEN inactivation, mutations involving FBXW7, or constitutive MYC expression conferring independence from NOTCH1 inactivation. Recent studies have suggested that selective targeting γ-secretase may offer an improved efficacy and toxicity profile over the effects caused by broad-spectrum GSIs. Understanding the mechanism of GSI-induced cell death and the ability to accurately identify patients based on the activity of the pathway will improve the response to GSI and support further investigation of such compounds for the rational design of anti-NOTCH1 therapies for the treatment of T-ALL. IMPLICATIONS FOR PRACTICE: γ-secretase has been proposed as a therapeutic target in numerous human conditions, including cancer. A better understanding of the structure and function of the γ-secretase inhibitor (GSI) would help to develop safe and effective γ-secretase-based therapies. The ability to accurately identify patients based on the activity of the pathway could improve the response to GSI therapy for the treatment of cancer. Toward these ends, this study focused on γ-secretase inhibitors as a potential therapeutic target for the design of anti-NOTCH1 therapies for the treatment of T-cell acute lymphoblastic leukemias and lymphomas.

Exploiting the passenger ACO1-deficiency arising from 9p21 deletions to kill T-cell lymphoblastic neoplasia cells.

Precursor T-cell lymphoblastic neoplasms are aggressive malignancies in need for more effective and specific therapeutic treatments. A significant fraction of these neoplasms harbor deletions on the locus 9p21, targeting the tumor suppressor CDKN2A but also deleting the aconitase 1 (ACO1) gene, a neighboring housekeeping gene involved in cytoplasm and mitochondrial metabolism. Here we show that reducing the aconitase activity with fluorocitrate decreases the viability of T-cell lymphoblastic neoplasia cells in correlation to the differential aconitase expression. The consequences of the treatment were evidenced in vitro using T-cell lymphoblastic neoplasia cell lines exhibiting 9p21 deletions and variable levels of ACO1 expression or activity. Similar results were observed in melanoma cell lines, suggesting a true potential for fluorocitrate in different cancer types. Notably, ectopic expression of ACO1 alleviated the susceptibility of cell lines to fluorocitrate and, conversely, knockdown experiments increased susceptibility of resistant cell lines. These findings were confirmed in vivo on athymic nude mice by using tumor xenografts derived from two T-cell lines with different levels of ACO1. Taken together, our results indicate that the non-targeted ACO1 deficiency induced by common deletions exerts a collateral cellular lethality that can be used as a novel therapeutic strategy in the treatment of several types of cancer.

S194-P-FADD as a marker of aggressiveness and poor prognosis in human T-cell lymphoblastic lymphoma.

T-cell lymphoblastic lymphoma is a haematological disease with an urgent need for reliable prognostic biomarkers that allow therapeutic stratification and dose adjustment. The scarcity of human samples is responsible for the delayed progress in the study and the clinical management of this disease, especially compared with T-cell acute lymphoblastic leukaemia, its leukemic counterpart. In the present work, we have determined by immunohistochemistry that S194-P-FADD protein is significantly reduced in a cohort of 22 samples from human T-cell lymphoblastic lymphoma. Notably, the extent of such reduction varies significantly among samples and has revealed determinant for the outcome of the tumour. We demonstrate that Fas-associated protein with death domain (FADD) phosphorylation status affects protein stability, subcellular localization and non-apoptotic functions, specifically cell proliferation. Phosphorylated FADD would be more stable and preferentially localized to the cell nucleus; there, it would favour cell proliferation. We show that patients with higher levels of S194-P-FADD exhibit more proliferative tumours and that they present worse clinical characteristics and a significant enrichment to an oncogenic signature. This supports that FADD phosphorylation may serve as a predictor for T-cell lymphoblastic lymphoma aggressiveness and clinical status. In summary, we propose FADD phosphorylation as a new biomarker with prognostic value in T-cell lymphoblastic lymphoma.

The use of PanDrugs to prioritize anticancer drug treatments in a case of T-ALL based on individual genomic data.

BACKGROUND: Acute T-cell lymphoblastic leukaemia (T-ALL) is an aggressive disorder derived from immature thymocytes. The variability observed in clinical responses on this type of tumours to treatments, the high toxicity of current protocols and the poor prognosis of patients with relapse or refractory make it urgent to find less toxic and more effective therapies in the context of a personalized medicine of precision. METHODS: Whole exome sequencing and RNAseq were performed on DNA and RNA respectively, extracted of a bone marrow sample from a patient diagnosed with tumour primary T-ALL and double negative thymocytes from thymus control samples. We used PanDrugs, a computational resource to propose pharmacological therapies based on our experimental results, including lists of variants and genes. We extend the possible therapeutic options for the patient by taking into account multiple genomic events potentially sensitive to a treatment, the context of the pathway and the pharmacological evidence already known by large-scale experiments. RESULTS: As a proof-of-principle we used next-generation-sequencing technologies (Whole Exome Sequencing and RNA-Sequencing) in a case of diagnosed Pro-T acute lymphoblastic leukaemia. We identified 689 disease-causing mutations involving 308 genes, as well as multiple fusion transcript variants, alternative splicing, and 6652 genes with at least one principal isoform significantly deregulated. Only 12 genes, with 27 pathogenic gene variants, were among the most frequently mutated ones in this type of lymphoproliferative disorder. Among them, 5 variants detected in CTCF, FBXW7, JAK1, NOTCH1 and WT1 genes have not yet been reported in T-ALL pathogenesis. CONCLUSIONS: Personalized genomic medicine is a therapeutic approach involving the use of an individual's information data to tailor drug therapy. Implementing bioinformatics platform PanDrugs enables us to propose a prioritized list of anticancer drugs as the best theoretical therapeutic candidates to treat this patient has been the goal of this article. Of note, most of the proposed drugs are not being yet considered in the clinical practice of this type of cancer opening up the approach of new treatment possibilities.

RNA-Seq reveals the existence of a CDKN1C-E2F1-TP53 axis that is altered in human T-cell lymphoblastic lymphomas.

BACKGROUND: Precursor T-cell lymphoblastic lymphomas (T-LBL) are rare aggressive hematological malignancies that mainly develop in children. As in other cancers, the loss of cell cycle control plays a prominent role in the pathogenesis in these malignancies that is primarily attributed to loss of CDKN2A (encoding protein p16INK4A). However, the impact of the deregulation of other genes such as CDKN1C, E2F1, and TP53 remains to be clarified. Interestingly, experiments in mouse models have proven that conditional T-cell specific deletion of Cdkn1c gene may induce a differentiation block at the DN3 to DN4 transition, and that the loss of this gene in the absence of Tp53 led to aggressive thymic lymphomas. RESULTS: In this manuscript, we demonstrated that the simultaneous deregulation of CDKN1C, E2F1, and TP53 genes by epigenetic mechanisms and/or the deregulation of specific microRNAs, together with additional impairing of TP53 function by the expression of dominant-negative isoforms are common features in primary human T-LBLs. CONCLUSIONS: Previous experimental work in mice revealed that T-cell specific deletion of Cdkn1c accelerates lymphomagenesis in the absence of Tp53. If, as expected, the consequences of the deregulation of the CDKN1C-E2F1-TP53 axis were the same as those experimentally demonstrated in mouse models, the disruption of this axis might be useful to predict tumor aggressiveness, and to provide the basis towards the development of potential therapeutic strategiesin human T-LBL.

Transcriptomic analysis reveals sex-specific differences in the expression of Dcl1 and Fis1 genes in the radio-adaptive response of thymocytes to TRP53-mediated apoptosis.

BACKGROUND: Radio-Adaptive Response (RAR) is a biological defense mechanism whereby exposure to low dose ionizing radiation (IR) mitigates the detrimental effects of high dose irradiation. RAR has been widely observed in vivo using as endpoint less induction of apoptosis. However, sex differences associated with RAR and variations between males and females on global gene expression influenced by RAR have not been still investigated. In addition, the response to radiation-induced apoptosis is associated with phosphorylation of TRP53 at both the serine 15 (ser-18 in the mouse) and serine 392 (ser-389 in mice) residues, but the role of these two phosphorylated forms in male and female RAR remains to be elucidated. RESULTS: We analyzed the effect of administering priming low dose radiation (0.075 Gy of X-rays) prior to high dose radiation (1.75 Gy of γ-rays) on the level of caspase-3-mediated apoptosis and on global transcriptional expression in thymocytes of male and female mice. Here, we provide the first evidence of a differential sex effect of RAR on the reduction of thymocyte apoptosis with males showing lesser levels of caspase-3-mediated apoptosis than females. Analysis of transcriptomic profiles of 1944 genes involved in apoptosis signaling in radio-adapted thymocytes identified 17 transcripts exhibiting differential expression between both sexes. Among them, Dlc1 and Fis1 are closely related to the apoptosis mediated by the TRP53 protein. Our data demonstrate that overexpression of Dlc1 and Fis1 occur concomitantly with a highest accumulation of phosphoserine-18-TRP53 and caspase-3 in radio-adapted thymocytes of female mice. In an opposite way, both down-modulation of Fis1 and phosphoserine-389-TRP53 accumulation appear to be associated with protection from thymocyte apoptosis mediated by caspase-3 in males. CONCLUSIONS: Transcriptomic analysis performed in this work reveals for the first time sex-specific differences in gene expression influenced by RAR. Our results also suggest a sex-dependent dual role for phosphoserine-18-TRP53 and phosphoserine-389-TRP53 in the regulation of the radio-adaptive response in mouse thymocytes.

Putative association between the -1415 T/C polymorphism of spermidine/spermine N1-acetyltransferase (SSAT1) gene and alcohol use disorders in women and men.

BACKGROUND: The activity of N-methyl-D-aspartate (NMDA) glutamate receptor, which responds to the levels of polyamines, modifies the neurotoxicity caused by ethanol. We aimed to investigate if the functionality of the spermidine/spermine N1-acetyltransferase (SSAT1) gene could be associated with a differential risk for alcoholism. METHODS: We studied a sample of 586 subjects: 104 alcohol-dependent patients, 273 patients with psychiatric disorders but without substance dependence, and 209 healthy controls. After gender stratification, the allele frequency distribution of the SSAT1 gene was compared between these three groups. RESULTS: In females, the TC genotype was significantly more frequent in alcohol-dependent patients than in non-alcohol-dependent psychiatric controls (χ(2 )= 7.509 df = 2, p = 0.023). A trend was found when alcohol-dependent females were compared with the healthy control group (χ(2 )= 4.897 df = 2, p = 0.086). No statistical differences were found among the males. DISCUSSION AND CONCLUSION: Gender differences in the regulation of SSAT1 gene expression may possibly be due to gender-specific effects of stress, ethanol toxicity, and/or polyamines levels. Further studies are needed to confirm our findings.

PIM1 is a potential therapeutic target for the leukemogenic effects mediated by JAK/STAT pathway mutations in T-ALL/LBL.

Precursor T-cell neoplasms (T-ALL/LBL) are aggressive hematological malignancies that arise from the malignant transformation of immature thymocytes. Despite the JAK/STAT pathway is recurrently altered in these neoplasms, there are not pharmacological inhibitors officially approved for the treatment of T-ALL/LBL patients that present oncogenic JAK/STAT pathway mutations. In the effort to identify potential therapeutic targets for those patients, we followed an alternative approach and focused on their transcriptional profile. We combined the analysis of molecular data from T-ALL/LBL patients with the generation of hematopoietic cellular models to reveal that JAK/STAT pathway mutations are associated with an aberrant transcriptional profile. Specifically, we demonstrate that JAK/STAT pathway mutations induce the overexpression of the PIM1 gene. Moreover, we show that the pan-PIM inhibitor, PIM447, significantly reduces the leukemogenesis, as well as the aberrant activation of c-MYC and mTOR pathways in cells expressing different JAK/STAT pathway mutations, becoming a potential therapeutic opportunity for a relevant subset of T-ALL/LBL patients.

Down-regulation of specific miRNAs enhances the expression of the gene Smoothened and contributes to T-cell lymphoblastic lymphoma development.

Inappropriate activation of the GLI/hedgehog (GLI/Hh) signalling occurs in several human cancers, including haematological neoplasms. However, little is known about its relevance in precursor T-cell lymphoblastic lymphomas (T-LBL) development. Moreover, the mechanisms whereby GLI/Hh signalling is activated in haematological malignancies are not fully clear. Here, we show that the gene Smoothened (SMO), the only non-redundant gene of this pathway, is up-regulated in mouse and human T-LBL. Interestingly, down-regulation of micro-RNAs mmu-miR-30a and mmu-miR-141 as well as hsa-miR-193b clearly contributes to enhance the expression of this gene in mouse and human lymphomas and, subsequently, to activate the GLI/Hh signalling. Activation of the GLI/Hh signalling in T-LBL promotes cell survival and proliferation, since inhibition of the pathway using short-hairpin-RNA-mediated SMO knockdown, or cyclopamine as a specific antagonist, significantly reduces these cellular processes. These findings suggest that sustained SMO up-regulation may contribute to T-LBL development and advocate the use of specific SMO inhibitors or microRNAs-based therapies as an attractive possibility to treat an important subset of T-LBL.

CYP2D6 poor metabolizer status might be associated with better response to risperidone treatment.

The variability in the antipsychotic response is, to some extent, genetically determined. Several studies have attempted to establish a role for genetic variation in genes coding pharmacokinetic and pharmacodynamic targets, but to date, no definite genetic predictive marker has been identified. We aimed to explore the putative role of 19 genetic variants and risperidone clinical improvement in 76 White schizophrenic inpatients, measured as change in Positive and Negative Syndrome Scale (PANSS). CYP2D6 poor metabolism was significantly associated with greater clinical improvement in total PANSS and a trend was also found for MDR1 3435C>T to higher total PANSS scores in 3435T carriers. This study suggests the importance that genetic variability on pharmacokinetic factors may have in risperidone response and gives evidence for the need for further investigation in order to establish the actual predictive value and clinical utility that CYP2D6 genotyping might have in risperidone therapy management.

Combinatorial effects of microRNAs to suppress the Myc oncogenic pathway.

Many mammalian transcripts contain target sites for multiple miRNAs, although it is not clear to what extent miRNAs may coordinately regulate single genes. We have mapped the interactions between down-regulated miRNAs and overexpressed target protein-coding genes in murine and human lymphomas. Myc, one of the hallmark oncogenes in these lymphomas, stands out as the up-regulated gene with the highest number of genetic interactions with down-regulated miRNAs in mouse lymphomas. The regulation of Myc by several of these miRNAs is confirmed by cellular and reporter assays. The same approach identifies MYC and multiple Myc targets as a preferential target of down-regulated miRNAs in human Burkitt lymphoma, a pathology characterized by translocated MYC oncogenes. These results indicate that several miRNAs must be coordinately down-regulated to enhance critical oncogenes, such as Myc. Some of these Myc-targeting miRNAs are repressed by Myc, suggesting that these tumors are a consequence of the unbalanced activity of Myc versus miRNAs.

EPHA7, a new target gene for 6q deletion in T-cell lymphoblastic lymphomas.

Cryptic deletions at chromosome 6q are common cytogenetic abnormalities in T-cell lymphoblastic leukemia/lymphoma (T-LBL), but the target genes have not been formally identified. Our results build on detection of specific chromosomal losses in a mouse model of γ-radiation-induced T-LBLs and provide interesting clues for new putative susceptibility genes in a region orthologous to human 6q15-6q16.3. Among these, Epha7 emerges as a bona fide candidate tumor suppressor gene because it is inactivated in practically all the T-LBLs analyzed (100% in mouse and 95.23% in human). We provide evidence showing that Epha7 downregulation may occur, at least in part, by loss of heterozygosity (19.35% in mouse and 12.5% in human) or promoter hypermethylation (51.61% in mouse and 43.75% in human) or a combination of both mechanisms (12.90% in mouse and 6.25% in human). These results indicate that EPHA7 might be considered a new tumor suppressor gene for 6q deletions in T-LBLs. Notably, this gene is located in 6q16.1 proximal to GRIK2 and CASP8AP2, other candidate genes identified in this region. Thus, del6q seems to be a complex region where inactivation of multiple genes may cooperatively contribute to the onset of T-cell lymphomas.

Quantifying telomeric lncRNAs using PNA-labelled RNA-Flow FISH (RNA-Flow).

Here we present a method to detect and quantify long non-coding RNAs, in particular those related to telomeres. By coupling the specificity of a peptide nucleic acid (PNA) probe with flow cytometry we have quantified cellular levels of TERRA and TERC lncRNAs in culture cell lines and PBMCs. This easy-to-use method appointed RNA-Flow allows reliable lncRNA quantification with broad applications in basic research and clinical diagnostics. In addition, the staining protocol presented here was proven useful for the detection and quantification of such lncRNAs on unfixed cells using confocal microscopy.

Patterns of Differentially Expressed circRNAs in Human Thymocytes.

Circular RNAs (circRNAs) are suggested to play a discriminative role between some stages of thymocyte differentiation. However, differential aspects of the stage of mature single-positive thymocytes remain to be explored. The purpose of this study is to investigate the differential expression pattern of circRNAs in three different development stages of human thymocytes, including mature single-positive cells, and perform predictions in silico regarding the ability of specific circRNAs when controlling the expression of genes involved in thymocyte differentiation. We isolate human thymocytes at three different stages of intrathymic differentiation and determine the expression of circRNAs and mRNA by RNASeq. We show that the differential expression pattern of 50 specific circRNAs serves to discriminate between the three human thymocyte populations. Interestingly, the downregulation of RAG2, a gene involved in T-cell differentiation in the thymus, could be simultaneously controlled by the downregulation of two circRNASs (hsa_circ_0031584 and hsa_circ_0019079) through the hypothetical liberation of hsa-miR-609. Our study provides, for the first time, significant insights into the usefulness of circRNAs in discriminating between different stages of thymocyte differentiation and provides new potential circRNA-miRNA-mRNA networks capable of controlling the expression of genes involved in T-cell differentiation in the thymus.

Differential molecular response in mice and human thymocytes exposed to a combined-dose radiation regime.

In the quest for more effective radiation treatment options that can improve both cell killing and healthy tissue recovery, combined radiation therapies are lately in the spotlight. The molecular response to a combined radiation regime where exposure to an initial low dose (priming dose) of ionizing radiation is administered prior to a subsequent higher radiation dose (challenging dose) after a given latency period have not been thoroughly explored. In this study we report on the differential response to either a combined radiation regime or a single challenging dose both in mouse in vivo and in human ex vivo thymocytes. A differential cell cycle response including an increase in the subG1 fraction on cells exposed to the combined regime was found. Together with this, a differential protein expression profiling in several pathways including cell cycle control (ATM, TP53, p21CDKN1A), damage response (γH2AX) and cell death pathways such as apoptosis (Cleaved Caspase-3, PARP1, PKCδ and H3T45ph) and ferroptosis (xCT/GPX4) was demonstrated. This study also shows the epigenetic regulation following a combined regime that alters the expression of chromatin modifiers such as DNMTs (DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3L) and glycosylases (MBD4 and TDG). Furthermore, a study of the underlying cellular status six hours after the priming dose alone showed evidence of retained modifications on the molecular and epigenetic pathways suggesting that the priming dose infers a "radiation awareness phenotype" to the thymocytes, a sensitization key to the differential response seen after the second hit with the challenging dose. These data suggest that combined-dose radiation regimes could be more efficient at making cells respond to radiation and it would be interesting to further investigate how can these schemes be of use to potential new radiation therapies.