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BackgroundThe stimulation and expansion of antigen-specific T cells ex vivo enables the targeting of a multitude of cancer antigens. However, clinical scale T-cell expansion from rare precursors requires repeated stimulations ex vivo leading to T-cell terminal effector differentiation and exhaustion that adversely impact therapeutic potential. We leveraged immune checkpoint blockade relevant to antigen-specific CD8+ human T cells to improve the expansion and function of T cells targeting clinically relevant antigens.MethodsA clinically-compliant protocol relying on peptide-pulsed monocyte-derived dendritic cells and cytokines was used to expand antigen-specific CD8+ targeting the oncogenic Epstein-Barr virus (EBV) and the tumor associated antigen (TAA) Wilms Tumor 1 (WT1) protein. The effects of antibody-mediated blockade of immune checkpoints applied to the cultures (T-cell expansion, phenotypes and function) were assessed at various time points. Genomic studies including single cell RNA (scRNA) sequencing and T-cell receptor sequencing were performed on EBV-specific T cells to inform about the impact of immune checkpoint blockade on the clonal distribution and gene expression of the expanded T cells.ResultsSeveral immune checkpoints were expressed early by ex vivo expanded antigen-specific CD8+ T cells, including PD-1 and TIM-3 with co-expression matching evidence of T-cell dysfunction as the cultures progressed. The introduction of anti-PD-L1 (expressed by the dendritic cells) and anti-TIM-3 antibodies in combination (but not individually) to the culture led to markedly improved antigen-specific T-cell expansion based on cell counts, fluorescent multimer staining and functional tests. This was not associated with evidence of T-cell dysfunction when compared to T cells expanded without immune checkpoint blockade. Genomics studies largely confirmed these results, showing that double blockade does not impart specific transcriptional programs or patterns on TCR repertoires. However, our data indicate that combined blockade may nonetheless alter gene expression in a minority of clonotypes and have donor-specific impacts.ConclusionsThe manufacturing of antigen-specific CD8+ T cells can be improved in terms of yield and functionality using blockade of TIM-3 and the PD-L1/PD-1 axis in combination. Overcoming the deleterious effects of multiple antigenic stimulations through PD-L1/TIM-3 blockade is a readily applicable approach for several adoptive-immunotherapy strategies.

Glycine 34-to-tryptophan (G34W) substitutions in H3.3 arise in approximately 90% of giant cell tumor of bone (GCT). Here, we show H3.3 G34W is necessary for tumor formation. By profiling the epigenome, transcriptome, and secreted proteome of patient samples and tumor-derived cells CRISPR–Cas9-edited for H3.3 G34W, we show that H3.3K36me3 loss on mutant H3.3 alters the deposition of the repressive H3K27me3 mark from intergenic to genic regions, beyond areas of H3.3 deposition. This promotes redistribution of other chromatin marks and aberrant transcription, altering cell fate in mesenchymal progenitors and hindering differentiation. Single-cell transcriptomics reveals that H3.3 G34W stromal cells recapitulate a neoplastic trajectory from a SPP1+ osteoblast-like progenitor population toward an ACTA2+ myofibroblast-like population, which secretes extracellular matrix ligands predicted to recruit and activate osteoclasts. Our findings suggest that H3.3 G34W leads to GCT by sustaining a transformed state in osteoblast-like progenitors, which promotes neoplastic growth, pathologic recruitment of giant osteoclasts, and bone destruction. Significance: This study shows that H3.3 G34W drives GCT tumorigenesis through aberrant epigenetic remodeling, altering differentiation trajectories in mesenchymal progenitors. H3.3 G34W promotes in neoplastic stromal cells an osteoblast-like progenitor state that enables undue interactions with the tumor microenvironment, driving GCT pathogenesis. These epigenetic changes may be amenable to therapeutic targeting in GCT. See related commentary by Licht, p. 1794. This article is highlighted in the In This Issue feature, p. 1775

Neurodegenerative disorders are complex multifactorial diseases that have poorly understood selective vulnerabilities among discrete cell populations. We performed single cell RNA sequencing of whole hippocampi from the rTg4510 mouse tauopathy model, which expresses a P301L MAPT mutation at two time points—before and after the onset of pathology. One population of neurons showed a robust size reduction in both the young and the old transgenic animals. Differential expression of genes expressed in this group of neurons suggested an enrichment in granule cell neurons. We identified genes that characterize this population of neurons using Pareto optimization of the specificity and precision of gene pairs for the population of interest. The resulting optimal marker genes were overwhelmingly associated with neuronal projections and their expression was enriched in the dentate gyrus suggesting that the rTg4510 mouse is a good model for Pick’s disease. This observation suggested that the tau mutation affects the population of neurons associated with neuronal projections even before overt tau inclusions can be observed. Out of the optimal pairs of genes identified as markers of the population of neurons of interest, we selected Purkinje cell protein 4 (Pcp4+) and Syntaxin binding protein 6 (Stxbp6+) for experimental validation. Single-molecule RNA fluorescence in situ hybridization confirmed preferential expression of these markers and localized them to the dentate gyrus.

Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here, we show that 50% of G34R/V-tumours (n=95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. While considered gliomas, G34R/V-tumours actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V-mutations impair neuronal differentiation. The lineage-of-origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V-tumours harbour dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell-fate specification. G34R/V may become dispensable for tumour maintenance, while mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumours. G34R/V-gliomas are neuronal malignancies, where interneuron progenitors are stalled in differentiation by G34R/V-mutations, and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signalling.

Oncogenic transcription factors are commonly activated in acute leukemias and subvert normal gene expression networks to reprogram hematopoietic progenitors into preleukemic stem cells, as exemplified by LIM-only 2 (LMO2) in T-cell acute lymphoblastic leukemia (T-ALL). Whether or not these oncoproteins interfere with other DNA-dependent processes is largely unexplored. Here, we show that LMO2 is recruited to DNA replication origins by interaction with three essential replication enzymes: DNA polymerase delta (POLD1), DNA primase (PRIM1), and minichromosome 6 (MCM6). Furthermore, tethering LMO2 to synthetic DNA sequences is sufficient to transform these sequences into origins of replication. We next addressed the importance of LMO2 in erythroid and thymocyte development, two lineages in which cell cycle and differentiation are tightly coordinated. Lowering LMO2 levels in erythroid progenitors delays G1-S progression and arrests erythropoietin-dependent cell growth while favoring terminal differentiation. Conversely, ectopic expression in thymocytes induces DNA replication and drives these cells into cell cycle, causing differentiation blockade. Our results define a novel role for LMO2 in directly promoting DNA synthesis and G1-S progression.

In eucaryotes, gene expression is regulated by microRNAs (miRNAs) which bind to messenger RNAs (mRNAs) and interfere with their translation into proteins, either by promoting their degradation or inducing their repression. We study the effect of miRNA interference on each gene using experimental methods, such as microarrays and RNA-seq at the mRNA level, or luciferase reporter assays and variations of SILAC at the protein level. Alternatively, computational predictions would provide clear benefits. However, no algorithm toward this task has ever been proposed. Here, we introduce a new algorithm to predict genome-wide expression data from initial transcriptome abundance. The algorithm simulates the miRNA and mRNA hybridization competition that occurs in given cellular conditions, and derives the whole set of miRNA::mRNA interactions at equilibrium (microtargetome). Interestingly, solving the competition improves the accuracy of miRNA target predictions. Furthermore, this model implements a previously reported and fundamental property of the microtargetome: the binding between a miRNA and a mRNA depends on their sequence complementarity, but also on the abundance of all RNAs expressed in the cell, i.e. the stoichiometry of all the miRNA sites and all the miRNAs given their respective abundance. This model generalizes the miRNA-induced synchronistic silencing previously observed, and described as sponges and competitive endogenous RNAs.

© 2015 Elsevier Inc. Axon regeneration in the CNS requires reactivating injured neurons' intrinsic growth state and enabling growth in an inhibitory environment. Using an inbred mouse neuronal phenotypic screen, we find that CAST/Ei mouse adult dorsal root ganglion neurons extend axons more on CNS myelin than the other eight strains tested, especially when pre-injured. Injury-primed CAST/Ei neurons also regenerate markedly in the spinal cord and optic nerve more than those from C57BL/6 mice and show greater sprouting following ischemic stroke. Heritability estimates indicate that extended growth in CAST/Ei neurons on myelin is genetically determined, and two whole-genome expression screens yield the Activin transcript Inhba as most correlated with this ability. Inhibition of Activin signaling in CAST/Ei mice diminishes their CNS regenerative capacity, whereas its activation in C57BL/6 animals boosts regeneration. This screen demonstrates that mammalian CNS regeneration can occur and reveals a molecular pathway that contributes to this ability. Omura et al. screened for neuronal growth on myelin and find CAST/Ei inbred mice are uniquely able to regenerate axons in the injured CNS through Activin signaling. Enhancing Activin signaling confers a CNS-regenerative capacity in normally non-regenerative mouse strains.

Summary: Peripheral nerve regeneration after injury requires a broad program of transcriptional changes. We investigated the basis for the enhanced nerve regenerative capacity of the CAST/Ei mouse strain relative to C57BL/6 mice. RNA sequencing of dorsal root ganglia (DRG) showed a CAST/Ei-specific upregulation of Ascl1 after injury. Ascl1 overexpression in DRG neurons of C57BL/6 mice enhanced their neurite outgrowth. Ascl1 is regulated by miR-7048-3p, which is downregulated in CAST/Ei mice. Inhibition of miR-7048-3p enhances neurite outgrowth. Following injury, CAST/Ei neurons largely retained their mature neuronal profile as determined by single-cell RNA- seq, whereas the C57BL/6 neurons acquired an immature profile. These findings suggest that one facet of the enhanced regenerative phenotype is preservation of neuronal identity in response to injury. : Lisi et al. find that increased Ascl1 expression is associated with the superior regenerative capacity of CAST/Ei mice. Single-cell RNA-seq demonstrated that CAST/Ei mice retained more mature neurons after injury than did C57BL/6 mice, thereby suggesting that genetic drivers of the enhanced regenerative phenotype include mechanisms to preserve neuronal identity after injury. Keywords: single-cell analyses, RNA sequencing, regeneration, dorsal root ganglia, CAST/Ei, Ascl1, miR-7048

Anti-infection drugs target vital functions of infectious agents, including their ribosome and other essential non-coding RNAs. One of the reasons infectious agents become resistant to drugs is due to mutations that eliminate drug-binding affinity while maintaining vital elements. Identifying these elements is based on the determination of viable and lethal mutants and associated structures. However, determining the structure of enough mutants at high resolution is not always possible. Here, we introduce a new computational method, MC-3DQSAR, to determine the vital elements of target RNA structure from mutagenesis and available high-resolution data. We applied the method to further characterize the structural determinants of the bacterial 23S ribosomal RNA sarcin–ricin loop (SRL), as well as those of the lead-activated and hammerhead ribozymes. The method was accurate in confirming experimentally determined essential structural elements and predicting the viability of new SRL variants, which were either observed in bacteria or validated in bacterial growth assays. Our results indicate that MC-3DQSAR could be used systematically to evaluate the drug-target potentials of any RNA sites using current high-resolution structural data.

© 2016 Elsevier Inc. Long non-coding RNAs (lncRNAs) are a diverse and poorly conserved category of transcripts that have expanded greatly in primates, particularly in the brain. We identified an lncRNA, which has acquired 16 microRNA response elements for miR-143-3p in the Catarrhini branch of primates. This lncRNA, termed LncND (neurodevelopment), is expressed in neural progenitor cells and then declines in neurons. Binding and release of miR-143-3p by LncND control the expression of Notch receptors. LncND expression is enriched in radial glia cells (RGCs) in the ventricular and subventricular zones of developing human brain. Downregulation in neuroblastoma cells reduced cell proliferation and induced neuronal differentiation, an effect phenocopied by miR-143-3p overexpression. Gain of function of LncND in developing mouse cortex led to an expansion of PAX6+ RGCs. These findings support a role for LncND in miRNA-mediated regulation of Notch signaling within the neural progenitor pool in primates that may have contributed to the expansion of cerebral cortex.

We report the results of a first, collective, blind experiment in RNA three-dimensional (3D) structure prediction, encompassing three prediction puzzles. The goals are to assess the leading edge of RNA structure prediction techniques; compare existing methods and tools; and evaluate their relative strengths, weaknesses, and limitations in terms of sequence length and structural complexity. The results should give potential users insight into the suitability of available methods for different applications and facilitate efforts in the RNA structure prediction community in ongoing efforts to improve prediction tools. We also report the creation of an automated evaluation pipeline to facilitate the analysis of future RNA structure prediction exercises.

Despite an increasing number of experimentally determined RNA structures, the gap between the number of structures and that of RNA families is still growing. To overcome this limitation, efficient and reliable RNA modeling methodologies must be developed. In order to reach this goal, here, we show how triloop sequence–structure relationships have been inferred through a systematic analysis of all triloops found in available high-resolution structures. The structural annotation of all triloops allowed us to define discrete states of the triloop's conformational space, and therefore an explicit sequence-to-structure relation. The sequence–structure relationships inferred from this explicit relation are presented in a convenient modeling table that provides a limited set of possible three-dimensional structures given any triloop sequence. The table is indexed by the two nucleotides that form the triloop's flanking base pair, since they are shown to provide the most information about the triloop three-dimensional structures. We also report the observations in the X-ray crystallographic structures of important conformational variations, which we believe might be the result of RNA dynamic.

Substrate recognition by the VS ribozyme involves a magnesium-dependent loop/loop interaction between the SLI substrate and the SLV hairpin from the catalytic domain. Recent NMR studies of SLV demonstrated that magnesium ions stabilize a U-turn loop structure and trigger a conformational change for the extruded loop residue U700, suggesting a role for U700 in SLI recognition. Here, we kinetically characterized VS ribozyme mutants to evaluate the contribution of U700 and other SLV loop residues to SLI recognition. To help interpret the kinetic data, we structurally characterized the SLV mutants by NMR spectroscopy and generated a three-dimensional model of the SLI/SLV complex by homology modeling with MC-Sym. We demonstrated that the mutation of U700 by A, C, or G does not significantly affect ribozyme activity, whereas deletion of U700 dramatically impairs this activity. The U700 backbone is likely important for SLI recognition, but does not appear to be required for either the structural integrity of the SLV loop or for direct interactions with SLI. Thus, deletion of U700 may affect other aspects of SLI recognition, such as magnesium ion binding and SLV loop dynamics. As part of our NMR studies, we developed a convenient assay based on detection of unusual 31P and 15N N7 chemical shifts to probe the formation of U-turn structures in RNAs. Our model of the SLI/SLV complex, which is compatible with biochemical data, leads us to propose novel interactions at the loop I/loop V interface.

Oncogenic transcription factors are major drivers in acute leukemias. These oncogenes are believed to subvert normal cell identity via the establishment of gene expression programs that dictate cell differentiation and growth. The LMO2 oncogene, which is commonly activated in T-cell acute lymphoblastic leukemia (T-ALL), has a well-established function in transcription regulation. We and others previously demonstrated that LMO1 or LMO2 collaborate with the SCL transcription factor to activate a self-renewal program that converts non self-renewing progenitors into pre-leukemic stem cells. Here we demonstrate a non-transcriptional role of LMO2 in controlling cell fate by directly promoting DNA replication, a hitherto unrecognized mechanism that might also account for its oncogenic properties. To address the question whether LMO2 controls other functions via protein-protein interactions, we performed a proteome-wide screen for LMO2 interaction partners in Kit+ Lin- cells. In addition to known LMO2-interacting proteins such as LDB1 and to proteins associated with transcription, we unexpectedly identified new interactions with three essential DNA replication enzymes, namely minichromosome 6 (MCM6), DNA polymerase delta (POLD1) and DNA primase (PRIM1). First, we show that in Kit+ hematopoietic cells (TF-1), all components of the pre-replication complex co-immunoprecipitate with LMO2 but not with SCL, suggesting a novel SCL-independent function. Second, LMO2 is recruited to DNA replication origins in these cells together with MCM5. Third, tethering LMO2 to synthetic DNA sequences is sufficient to transform these into origins of replication. Indeed, we show by DNA capture that LMO2 fused to the DNA binding domain of GAL4 is sufficient to recruit DNA replication proteins to GAL4 binding sites on DNA. In vivo, this recruitment is sufficient to drive DNA replication in a manner which is dependent on the integrity of the GAL4 binding sites. These results provide unambiguous evidence for a role of LMO2 in directly controlling DNA replication. Cell cycle and cell differentiation are tightly coordinated during normal hematopoiesis, both during erythroid differentiation and during thymocyte development. We next addressed the functional importance of LMO2 in these two lineages. Erythroid cell differentiation proceeds through different stages from the CD71+Ter119- to the CD71-Ter119+. These stages are also distinguishable by morphological criteria. We observe that LMO2 protein levels directly correlate with the proportion of cells in S phase, i.e. both LMO2 levels and the proportions of cycling cells decrease with terminal erythroid differentiation. Strikingly, lowering LMO2 levels in fetal liver erythroid progenitors via shRNAs decreases the proportion of cells in S phase and arrests Epo-dependent cell growth. Despite a drastic decrease in the numbers of erythroid precursors, these cells differentiate readily to the CD71-Ter119+ stage. Therefore, LMO2 levels dictate cell fate in the erythroid lineage, by favoring DNA replication at the expense of terminal maturation. Conversely, ectopic expression in thymocytes induces DNA replication and drives cells into cell cycle, causing differentiation blockade. Our results define a novel role for the oncogenic transcription factor LMO2 in directly promoting DNA synthesis. To our knowledge, this is the first evidence for a non-transcriptional function of the LMO2 oncogene that drives cell cycle at the expense of differentiation, favouring progenitor cell expansion in the thymus, and causing T-ALL when ectopically expressed in the T lineage. We propose that the non-transcriptional control of DNA replication uncovered here for LMO2 may be a more common function of oncogenic transcription factors than previously appreciated. Disclosures No relevant conflicts of interest to declare.

The SCL and LMO oncogenes are frequently activated in childhood T cell acute leukemia (T-ALL). SCL is a bHLH transcription factor that forms heterodimers with other members, specifically HEB and E2A. SCL can either activate or repress transcription but its mechanism of action as an oncogene remains to be clarified. Ectopic expression of SCL and LMO in transgenic mice causes thymocyte differentiation arrest during the preleukemic phase with aberrant differentiation at the DN3–DN4 stage, prior to the acquisition of CD4 and CD8. We therefore took several approaches to define the mechanism underlying this differentiation arrest. Using a CD3e-deficient background to eliminate preTCR signaling effects, an event that is not essential for leukemogenesis, we first analyzed global gene expression of pre-leukemic SCLtg/LMOtg DN3 thymocytes against their non-transgenic littermates via the Affymetrix platform. In this context, SCL/LMO globally affects the expression of both positive and negative targets of E2A/HEB. These results were confirmed by real-time quantitative PCR, showing that SCL/LMO repress E2A/HEB activity. Second, bioinformatic analysis of genes significantly affected by SCL/LMO allowed us to identify phylogenetically conserved HEB/E2A binding sites within the promoter regions of half of these genes. E2A/HEB binding was directly assessed through chromatin immunoprecipitation of primary thymocytes, confirming known target genes and revealing novel ones. This assay also showed that SCL associates with HEB and/or E2A on DNA. Moreover, structure function analysis indicate that transcription inhibition by SCL depends on its HLH domain but not on LMO interaction. We therefore conclude that SCL inhibits thymocyte differentiation by inhibiting E2A/HEB targets that include genes required for thymocyte differentiation, cell signalling, DNA repair and replication. Finally, using a genetic approach we show that SCL/LMO collaborates with HEB haploinsufficiency in inducing leukemia. Our observations therefore reveal that the repression of HEB/E2A by SCL/LMO is a crucial step in T cell transformation. Support from CIHR and FRSQ.