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Chimeric antigen receptor (CAR) T-cell therapy represents a revolutionary approach to induce long-lasting remission in patients with B-cell malignancies not responding to conventional therapies. Nevertheless, possible severe side effects, including cytokine release syndrome (CRS), neurotoxicity and macrophage activation syndrome, whose management is still challenging, as well as lack of pathophysiological experimental models to investigate novel interventions, limit the widespread use of this therapy. In light of these considerations, we developed a comprehensive humanized mouse model to investigate the role of IFNγ neutralization, provided by the clinically approved monoclonal antibody, emapalumab, in controlling severe toxicity related to CAR T cells. We demonstrated that emapalumab reduces the pro-inflammatory environment in the animal model, allowing severe CRS control and preventing brain damage, characterized by multifocal hemorrhages. Furthermore, we proved that IFNγ inhibition does not affect the ability of CAR.CD19 T cells to eradicate CD19+ lymphoma cells, both in vitro and in vivo.

Purpose Patient-derived cancer cell lines can be very useful to investigate genetic as well as epigenetic mechanisms of transformation and to test new drugs. In this multi-centric study, we performed genomic and transcriptomic characterization of a large set of patient-derived glioblastoma (GBM) stem-like cells (GSCs). Methods 94 (80 I surgery/14 II surgery) and 53 (42 I surgery/11 II surgery) GSCs lines underwent whole exome and trascriptome analysis, respectively. Results Exome sequencing revealed TP53 as the main mutated gene (41/94 samples, 44%), followed by PTEN (33/94, 35%), RB1 (16/94, 17%) and NF1 (15/94, 16%), among other genes associated to brain tumors. One GSC sample bearing a BRAF p.V600E mutation showed sensitivity in vitro to a BRAF inhibitor. Gene Ontology and Reactome analysis uncovered several biological processes mostly associated to gliogenesis and glial cell differentiation, S − adenosylmethionine metabolic process, mismatch repair and methylation. Comparison of I and II surgery samples disclosed a similar distribution of mutated genes, with an overrepresentation of mutations in mismatch repair, cell cycle, p53 and methylation pathways in I surgery samples, and of mutations in receptor tyrosine kinase and MAPK signaling pathways in II surgery samples. Unsupervised hierarchical clustering of RNA-seq data produced 3 clusters characterized by distinctive sets of up-regulated genes and signaling pathways. Conclusion The availability of a large set of fully molecularly characterized GCSs represents a valuable public resource to support the advancement of precision oncology for the treatment of GBM.

Fcγ RIIA (CD32A) and their ligands, including the immunoglobulin Fc fragment and pentraxins, are key players in a variety of innate immune responses. Still unclear is whether additional ligands of CD32A do exist. The objective of this study is to demonstrate that CD32A-chimeric receptor (CR) can be utilized for the identification of CD32A cell surface ligand(s). Among fifteen cancer cell lines tested, CD32A-CR T cells recognized three of breast cancer (BC) including the MDA-MB-468 and one colorectal carcinoma (HT29) in the absence of targeting antibodies. Conjugation of sensitive BC cells with CD32A-CR T cells induced CD32A polarization and down-regulation, CD107 release, and mutual cell eliminationin vitro. Conversely, normal fibroblasts and myoblasts were not affected while normal HUVEC cells promoted CD32A down-regulation. CD32A-CR T cell activity was not inhibited by human IgGs or human serum, but; it was rather enhanced by cetuximab antibody. RNAseq analysis of sensitive vs resistant BC cells identified a fingerprint of 42 genes predicting the sensitivity of BC cells to CD32A-CR T cells and their association with favorable prognostic significance in advanced BC patients. Our data also identify ICAM 1 as a major regulator of CD32A-CR T cell-mediated cytotoxicity. Finally, CD32A-CR T cell administration protected immunodeficient mice from subcutaneous growth of MDA-MB-468 cells in the absence of tumor-specific antibodies. These data indicate that CD32A-CR can be utilized for the identification of (1) cell surface CD32A ligand(s); (2) rational therapeutic strategies to target BC; and (3) novel transcriptomic signatures prognostically relevant for advanced BC patients.

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.One sentence summary:DNA recombination requires RNA editing of DNA:RNA hybrids to ease their melting and facilitate DNA end resection

MicroRNAs (miRNAs) are a class of ~22 nt noncoding RNAs playing essential roles in the post-transcriptional regulation of gene expression, cell proliferation, and cell differentiation and are often found deregulated in several diseases including cancer.The A-to-I RNA editing, mediated by ADAR enzymes, is a diffuse post-transcriptional mechanism that converts the genetically coded adenosine (A) into inosine (I) at the RNA level. Among different RNA targets, the ADAR enzymes can also edit miRNA precursors. Specifically, a single nucleotide change (A/I) lying within the mature miRNA can alter the miRNA binding specificity and redirect the edited miRNA to a different mRNA target. In several cancer types a consistent deregulation of A-to-I RNA editing machinery also involves important miRNAs (either oncomiRs or tumor-suppressor miRNAs). Herein we describe a combined in silico and experimental approach for the detection of edited miRNAs and the identification and validation of their target genes potentially involved in cancer progression or invasion.

Glioblastoma (GBM) is the most aggressive, infiltrative, and lethal brain tumor in humans. Despite the extensive advancement in the knowledge about tumor progression and treatment over the last few years, the prognosis of GBM is still very poor due to the difficulty of targeting drugs or anticancer molecules to GBM cells. The major challenge in improving GBM treatment implicates the development of a targeted drug delivery system, capable of crossing the blood–brain barrier (BBB) and specifically targeting GBM cells. Aptamers possess many characteristics that make them ideal novel therapeutic agents for the treatment of GBM. They are short single-stranded nucleic acids (RNA or ssDNA) able to bind to a molecular target with high affinity and specificity. Several GBM-targeting aptamers have been developed for imaging, tumor cell isolation from biopsies, and drug/anticancer molecule delivery to the tumor cells. Due to their properties (low immunogenicity, long stability, and toxicity), a large number of aptamers have been selected against GBM biomarkers and tested in GBM cell lines, while only a few of them have also been tested in in vivo models of GBM. Herein, we specifically focus on aptamers tested in GBM in vivo models that can be considered as new diagnostic and/or therapeutic tools for GBM patients’ treatment.

MicroRNAs (miRNAs) are a class of ~22 nt noncoding RNAs playing essential roles in the post-transcriptional regulation of gene expression, cell proliferation, and cell differentiation and are often found deregulated in several diseases including cancer.The A-to-I RNA editing, mediated by ADAR enzymes, is a diffuse post-transcriptional mechanism that converts the genetically coded adenosine (A) into inosine (I) at the RNA level. Among different RNA targets, the ADAR enzymes can also edit miRNA precursors. Specifically, a single nucleotide change (A/I) lying within the mature miRNA can alter the miRNA binding specificity and redirect the edited miRNA to a different mRNA target. In several cancer types a consistent deregulation of A-to-I RNA editing machinery also involves important miRNAs (either oncomiRs or tumor-suppressor miRNAs). Herein we describe a combined in silico and experimental approach for the detection of edited miRNAs and the identification and validation of their target genes potentially involved in cancer progression or invasion.

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution., This work was funded by an R + D + I grant from the Agencia Estatal de Investigación PID2019-104195GB-I00/ AEI/10.13039/501100011033 (P.H.), R + D + I grants from the Spanish Ministry of Economy and Competitiveness (SAF2016-74855-P to P.H. and BFU2016-75058-P to A.A.), the European Research Council (ERC2014 AdG669898 TARLOOP to A.A.), the Associazione Italiana Ricerca sul Cancro-AIRC (AIRC IG 22080 to A.G.), The Swedish Research Council (grants 2015-04553 to N.V.), The Swedish Cancer Society (grant CAN 2016/460 to N.V.) and the European Union (FEDER). AR is, and FM-N and RP-C were, funded with FPU fellowships from the Spanish Ministry of Education and S.S. by a Juan de la Cierva contract from the Spanish Ministry of Economy and Competitiveness. J.D.P. and M.E-C. were supported by the Department of Molecular Biosciences, The Wenner-Gren Institute at Stockholm University. J.D-P. and R.P-C. were recipients of short-term EMBO fellowships (STF- 7513-2018 and STF-7764-2018). The mutational results shown here are in whole or part based upon the data generated by the TCGA Research Network: https://www.cancer.gov/tcga. All the bioinformatic analyses were performed using custom scripts that were run on the High performance computing cluster provided by the Centro Informático Cienti ́fico de Andalucía (CICA). CABIMER is supported by the regional government of Andalucia (Junta de Andalucía).

Summary: Memory B cells (MBCs) epitomize the adaptation of the immune system to the environment. We identify two MBC subsets in peripheral blood, CD27dull and CD27bright MBCs, whose frequency changes with age. Heavy chain variable region (VH) usage, somatic mutation frequency replacement-to-silent ratio, and CDR3 property changes, reflecting consecutive selection of highly antigen-specific, low cross-reactive antibody variants, all demonstrate that CD27dull and CD27bright MBCs represent sequential MBC developmental stages, and stringent antigen-driven pressure selects CD27dull into the CD27bright MBC pool. Dynamics of human MBCs are exploited in pregnancy, when 50% of maternal MBCs are lost and CD27dull MBCs transit to the more differentiated CD27bright stage. In the postpartum period, the maternal MBC pool is replenished by the expansion of persistent CD27dull clones. Thus, the stability and flexibility of human B cell memory is ensured by CD27dull MBCs that expand and differentiate in response to change. : Grimsholm et al. show that CD27dull and CD27bright represent sequential MBC developmental stages. T cell- and germinal center (GC)-independent CD27dull MBCs are the plastic source of strongly selected and GC-dependent CD27bright MBCs. CD27dull MBCs, able to expand and differentiate in response to change, ensure stability and flexibility of human B cell memory. Keywords: memory B cells, pregnancy, immunological memory, CD27, VH repertoire, immunodeficiency, aging, spleen, vaccine, germinal center

Memory B cells (MBCs) epitomize the adaptation of the immune system to the environment. We identify two MBC subsets in peripheral blood, CD27

Background Adenosine-to-inosine (A-to-I) RNA editing is an essential post-transcriptional mechanism mediated by ADAR enzymes that have been recently associated with cancer. Results Here, we characterize the inosinome signature in normal brain and de novo glioblastoma (GBM) using new metrics that re-stratify GBM patients according to their editing profiles and indicate this post-transcriptional event as a possible molecular mechanism for sexual dimorphism in GBM. We find that over 85% of de novo GBMs carry a deletion involving the genomic locus of ADAR3, which is specifically expressed in the brain. By analyzing RNA editing and patient outcomes, an intriguing gender-dependent link appears, with high editing of Alus shown to be beneficial only in male patients. We propose an inosinome-based molecular stratification of GBM patients that identifies two different GBM subgroups, INO-1 and INO-2, which can identify novel high-risk gender-specific patient groups for which more aggressive treatments may be necessary. Conclusions Our data provide a detailed picture of RNA editing landscape in normal brain and GBM, exploring A-to-I RNA editing regulation, disclosing unexpected editing implications for GBM patient stratification and identification of gender-dependent high-risk patients, and suggesting COG3 I/V as an eligible site for future personalized targeted gene therapy. Electronic supplementary material The online version of this article (10.1186/s13059-019-1647-x) contains supplementary material, which is available to authorized users.

Objective Adenosine-to-inosine (A-to-I) RNA editing of Alu retroelements is a primate-specific mechanism mediated by adenosine deaminases acting on RNA (ADARs) that diversifies transcriptome by changing selected nucleotides in RNA molecules. We tested the hypothesis that A-to-I RNA editing is altered in rheumatoid arthritis (RA). Methods Synovium expression analysis of ADAR1 was investigated in 152 RA patients and 50 controls. Peripheral blood mononuclear cells derived from 14 healthy subjects and 19 patients with active RA at baseline and after 12-week treatment were examined for ADAR1p150 and ADAR1p110 isoform expression by RT-qPCR. RNA editing activity was analysed by AluSx+ Sanger-sequencing of cathepsin S, an extracellular matrix degradation enzyme involved in antigen presentation. Results ADAR1 was significantly over-expressed in RA synovium regardless of disease duration. Similarly, ADAR1p150 isoform expression was significantly increased in the blood of active RA patients. Individual nucleotide analysis revealed that A-to-I RNA editing rate was also significantly increased in RA patients. Both baseline ADAR1p150 expression and individual adenosine RNA editing rate of cathepsin S AluSx+ decreased after treatment only in those patients with good clinical response. Upregulation of the expression and/or activity of the RNA editing machinery were associated with a higher expression of edited Alu-enriched genes including cathepsin S and TNF receptor-associated factors 1,2,3 and 5. Conclusion A previously unrecognized regulation and role of ADAR1p150-mediated A-to-I RNA editing in post-transcriptional control in RA underpins therapeutic response and fuels inflammatory gene expression, thus representing an interesting therapeutic target., Highlights • The RNA editing enzyme ADAR1 is increased in rheumatoid arthritis. • ADAR1-induced Alu A-to-I RNA editing is increased in active RA and decreases after treatment only in responding patients. • Association of increased A-to-I RNA editing with pro-inflammatory gene expression suggests a role in chronic inflammation. • Interrupting the pro-inflammatory ADAR1p150-induced Alu RNA editing may comprise an interesting therapeutic target in RA.

Recent studies have reported the emerging role of microRNAs (miRNAs) in human cancers. We systematically characterized miRNA expression and editing in the human brain, which displays the highest number of A-to-I RNA editing sites among human tissues, and in de novo glioblastoma brain cancer. We identified 299 miRNAs altered in their expression and 24 miRNAs differently edited in human brain compared to glioblastoma tissues. We focused on the editing site within the miR-589–3p seed. MiR-589–3p is a unique miRNA almost fully edited (∼100%) in normal brain and with a consistent editing decrease in glioblastoma. The edited version of miR-589–3p inhibits glioblastoma cell proliferation, migration and invasion, while the unedited version boosts cell proliferation and motility/invasion, thus being a potential cancer-promoting factor. We demonstrated that the editing of this miRNA is mediated by ADAR2, and retargets miR-589–3p from the tumor-suppressor PCDH9 to ADAM12, which codes for the metalloproteinase 12 promoting glioblastoma invasion. Overall, our study dissects the role of a unique brain-specific editing site within miR-589–3p, with important anticancer features, and highlights the importance of RNA editing as an essential player not only for diversifying the genomic message but also for correcting not-tolerable/critical genomic coding sites.