Distinct Differentiation Stages Research Articles

Differential effects of choline on TLR2/4 mediated signaling through possible regulation of toll-interacting protein in hepatocellular carcinoma cell lines

Abstract Objectives Toll-like receptor (TLR) mediated inflammatory status plays an important role in development and progression of hepatocellular carcinoma (HCC). Toll-interacting protein (TOLLIP) has an inhibitory effect on TLR-mediated inflammatory signalling and expression profile of TOLLIP varies between malignancies including HCC. Cholinergic anti-inflammatory pathway (CAP) is an endogenous mechanism that controls inflammatory status via α7nicotinic acetylcholine receptors (α7nAChR). This study aims to investigate the effect of CAP-acting agent choline on TOLLIP and its related TLR-mediated inflammatory response in HCC cells with distinct differentiation stages. Methods The expression patterns of α7nAChR, TLR2/4, TOLLIP, IL6,NFkB genes were evaluated by RT-PCR and ELISA in the presence of choline, along with the real-time cell proliferation and migration in HEP3B and SNU449 HCC cell lines. The interaction between choline and TOLLIP assessed by using in-silico analyses. Results Choline downregulated TOLLIP in Hep3B and SNU449 cells. However, the expressions of α7nAChR, NF-κB, IL-6, TLR2 and TLR4 showed a decreased pattern in well differentiated HEP3B cells, while an increased pattern in poorly differentiated SNU449 cells. Conclusions Choline might exert differential effects in TLR2/4-dependent signalling based on the differentiation stages of the HCC cells, suggesting its potential therapeutic effects in earlier stages of HCC which might be result of its partial modulation of TOLLIP.

Exploring the interactions between cardiomyocytes and endothelial cells to induce cardiac revascularization and regeneration

Abstract Funding Acknowledgements None. Background The adult heart is unable to regenerate after ischemic injury, primarily due to the incapacity of cardiomyocyte (CM) to re-enter in the cell cycle and endothelial cells (EC) to form new vessels. Interestingly, our recent data have unveiled that the decline of CM proliferation after birth is paralleled by a reduction in angiogenic potential. Nevertheless, the precise mechanisms governing angiogenesis inhibition in the adult heart remain elusive. We hypothesize that the terminal differentiation of CMs after birth leads to the paracrine suppression of EC proliferation. Purpose This study aims to identify membrane or secreted factors produced by adult CMs that inhibit EC proliferation and vessel formation in order to create new therapeutic strategies to induce cardiac revascularization and regeneration. Material and Methods To validate our hypothesis, ECs were co-cultured with CMs at distinct differentiation stages. Leveraging existing RNA sequencing datasets, we generated an interactome between ligands produced by CMs and receptors expressed by cardiac ECs. Through gain and loss of function studies, we identified pivotal anti-angiogenic factors and subsequently knocked-out the corresponding genes in Cas9 mice, with the aim of rescue the angiogenic potential of the adult heart. Concurrently, we assessed the ability of miR199a, a pro-proliferative miRNA known for inducing CM de-differentiation, to elicit EC proliferation and angiogenesis when delivered to the adult heart together with VEGF-A, utilizing adeno-associated viral vectors. Results Our findings revealed that adult CMs impaired EC proliferation. Comprehensive analysis identified 20 membrane and secreted factors, establishing 118 ligand-receptor interactions between CMs and ECs. Notably, KCNJ11 and CD74 emerged as key factors, as validated through gain and loss of function experiments. In vivo knockout of these genes in CMs led to significant increase in EC proliferation and the formation of neo-vessels. Furthermore, CM de-differentiation induced by miR199a demonstrated the capacity to promote EC proliferation and neo-vessel formation upon VEGF-A stimulation. Conclusions Collectively, this work demonstrated that fully mature CMs have a role in limiting angiogenic potential of the adult heart, likely explaining the failure of therapeutic angiogenesis approaches attempted so far. Our results suggest that innovative strategies targeting CM-EC cross-talk or inducing partial CM de-differentiation may open new therapeutic avenues in the field of cardiac regeneration.Adult CMs inhibit EC proliferationCMdedifferentiation rescues angiogenesis

Excess of body weight is associated with accelerated T-cell senescence in hospitalized COVID-19 patients

BackgroundSeveral risk factors have been involved in the poor clinical progression of coronavirus disease-19 (COVID-19), including ageing, and obesity. SARS-CoV-2 may compromise lung function through cell damage and paracrine inflammation; and obesity has been associated with premature immunosenescence, microbial translocation, and dysfunctional innate immune responses leading to poor immune response against a range of viruses and bacterial infections. Here, we have comprehensively characterized the immunosenescence, microbial translocation, and immune dysregulation established in hospitalized COVID-19 patients with different degrees of body weight.ResultsHospitalised COVID-19 patients with overweight and obesity had similarly higher plasma LPS and sCD14 levels than controls (all p < 0.01). Patients with obesity had higher leptin levels than controls. Obesity and overweight patients had similarly higher expansions of classical monocytes and immature natural killer (NK) cells (CD56+CD16−) than controls. In contrast, reduced proportions of intermediate monocytes, mature NK cells (CD56+CD16+), and NKT were found in both groups of patients than controls. As expected, COVID-19 patients had a robust expansion of plasmablasts, contrasting to lower proportions of major T-cell subsets (CD4 + and CD8+) than controls. Concerning T-cell activation, overweight and obese patients had lower proportions of CD4+CD38+ cells than controls. Contrasting changes were reported in CD25+CD127low/neg regulatory T cells, with increased and decreased proportions found in CD4+ and CD8+ T cells, respectively. There were similar proportions of T cells expressing checkpoint inhibitors across all groups. We also investigated distinct stages of T-cell differentiation (early, intermediate, and late-differentiated – TEMRA). The intermediate-differentiated CD4 + T cells and TEMRA cells (CD4+ and CD8+) were expanded in patients compared to controls. Senescent T cells can also express NK receptors (NKG2A/D), and patients had a robust expansion of CD8+CD57+NKG2A+ cells than controls. Unbiased immune profiling further confirmed the expansions of senescent T cells in COVID-19.ConclusionsThese findings suggest that dysregulated immune cells, microbial translocation, and T-cell senescence may partially explain the increased vulnerability to COVID-19 in subjects with excess of body weight.

Comparative transcriptomics coupled to developmental grading via transgenic zebrafish reporter strains identifies conserved features in neutrophil maturation

Neutrophils are evolutionarily conserved innate immune cells playing pivotal roles in host defense. Zebrafish models have contributed substantially to our understanding of neutrophil functions but similarities to human neutrophil maturation have not been systematically characterized, which limits their applicability to studying human disease. Here we show, by generating and analysing transgenic zebrafish strains representing distinct neutrophil differentiation stages, a high-resolution transcriptional profile of neutrophil maturation. We link gene expression at each stage to characteristic transcription factors, including C/ebp-β, which is important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human samples confirms high molecular similarity of immature stages and discriminates zebrafish-specific from pan-species gene signatures. Applying the pan-species neutrophil maturation signature to RNA-sequencing data from human neuroblastoma patients reveals association between metastatic tumor cell infiltration in the bone marrow and an overall increase in mature neutrophils. Our detailed neutrophil maturation atlas thus provides a valuable resource for studying neutrophil function at different stages across species in health and disease.

Cell atlas of the Atlantic salmon spleen reveals immune cell heterogeneity and cell-specific responses to bacterial infection

The spleen is a conserved secondary lymphoid organ that emerged in parallel to adaptive immunity in early jawed vertebrates. Recent studies have applied single cell transcriptomics to reveal the cellular composition of spleen in several species, cataloguing diverse immune cell types and subpopulations. In this study, 51,119 spleen nuclei transcriptomes were comprehensively investigated in the commercially important teleost Atlantic salmon (Salmo salar L.), contrasting control animals with those challenged with the bacterial pathogen Aeromonas salmonicida. We identified clusters of nuclei representing the expected major cell types, namely T cells, B cells, natural killer-like cells, granulocytes, mononuclear phagocytes, endothelial cells, mesenchymal cells, erythrocytes and thrombocytes. We discovered heterogeneity within several immune lineages, providing evidence for resident macrophages and melanomacrophages, infiltrating monocytes, several candidate dendritic cell subpopulations, and B cells at distinct stages of differentiation, including plasma cells and an igt + subset. We provide evidence for twelve candidate T cell subsets, including cd4+ T helper and regulatory T cells, one cd8+ subset, three γδT subsets, and populations double negative for cd4 and cd8. The number of genes showing differential expression during the early stages of Aeromonas infection was highly variable across immune cell types, with the largest changes observed in macrophages and infiltrating monocytes, followed by resting mature B cells. Our analysis provides evidence for a local inflammatory response to infection alongside B cell maturation in the spleen, and upregulation of ccr9 genes in igt + B cells, T helper and cd8+ cells, and monocytes, consistent with the recruitment of immune cell populations to the gut to deal with Aeromonas infection. Overall, this study provides a new cell-resolved perspective of the immune actions of Atlantic salmon spleen, highlighting extensive heterogeneity hidden to bulk transcriptomics. We further provide a large catalogue of cell-specific marker genes that can be leveraged to further explore the function and structural organization of the salmonid immune system.

A NOVEL NON-CODING RNA-BASED APPROACH TO MODULATE OSTEOCLAST BEHAVIOUR

Osteoclasts (OCs) are multinucleated cells that play a pivotal role in skeletal development and bone remodeling. Abnormal activation of OCs contributes to the development of bone-related diseases, such as osteoporosis, bone metastasis and osteoarthritis. Restoring the normal function of OCs is crucial for bone homeostasis. Recently, RNA therapeutics emerged as a new field of research for osteoarticular diseases.The aim of this study is to use non-coding RNAs (ncRNAs) to molecularly engineer OCs and modulate their function. Specifically, we investigated the role of the microRNAs (namely miR-16) and long ncRNAs (namely DLEU1) in OCs differentiation and fusion.DLEU1/DLEU2 region, located at chromosome 13q14, also encodes miR-15 and miR-16. Our results show that levels of these ncRNA transcripts are differently expressed at distinct stages of the OCs differentiation. Specifically, silencing of DLEU1 by small interfering RNAs (siDLEU1) and overexpression of miR-16 by synthetic miRNA mimics (miR-16-mimics) led to a significant reduction in the number of OCs formed per field (OC/field), both at day 5 and 9 of the differentiation stage. Importantly, time-lapse analysis, used to track OCs behavior, revealed a significant decrease in fusion events after transfection with siDLEU1 or miR-16-mimics and an alteration in the fusion mode and partners. Next, we investigated the migration profile of these OCs, and the results show that only miR-16-mimics-OCs, but not siDLEU-OCs, have a lower percentage of immobile cells and an increase in cells with mobile regime, compared with controls. No differences in cell shape were found. Moreover, mass-spectrometry quantitative proteomic analysis revealed independent effects of siDLEU1 and miR-16-mimics at the protein levels. Importantly, DLEU1 and miR-16 act by distinct processes and pathways.Collectively, our findings support the ncRNAs DLEU1 and miR-16 as therapeutic targets to modulate early stages of OCs differentiation and, consequently, to impair OC fusion, advancing ncRNA-therapeutics for bone-related diseases.Acknowledgements: Authors would like to thank to AO CMF / AO Foundation (AOCMFS-21-23A). SRM and MIA are supported by FCT (SFRH/BD/147229/2019 and BiotechHealth Program; CEECINST/00091/2018/CP1500/CT0011, respectively).

IL-21R-STAT3 signalling initiates a differentiation program in uterine tissue-resident NK cells to support pregnancy

Tissue-resident Natural Killer (trNK) cells are crucial components of local immunity that activate rapidly upon infection. However, under steady state conditions, their responses are tightly controlled to prevent unwanted tissue damage. The mechanisms governing their differentiation and activation are not fully understood. Here, we characterise uterine trNK cells longitudinally during pregnancy by single cell RNA sequencing and find that the combined expression pattern of 4-1BB and CD55 defines their three distinct stages of differentiation in mice. Mechanistically, an IL-21R-STAT3 axis is essential for initiating the trNK cell differentiation. The fully differentiated trNK cells demonstrate enhanced functionality, which is necessary for remodelling spiral arteries in the decidua. We identify an apoptotic program that is specific to the terminal differentiation stage, which may preclude tissue damage by these highly activated trNK cells. In summary, uterine trNK cells become intensely active and effective during pregnancy, but tightly controlled via a differentiation program that also limits potential harm, suggesting an intricate mechanism for harnessing trNK cells in maintaining pregnancy.

Identification of Acute Myeloid Leukemia Cell Surface Therapeutic Targets Using Single Cell RNA Sequencing Supported By Surface Proteomics

Background: Acute myeloid leukemia (AML) comprises diverse genomic subgroups and remains hard to treat in most patients. Despite breakthroughs in the therapeutic arsenal in recent years, clinical usage of therapeutic antibodies or chimeric antigen receptor T (CAR-T) cells has been lagging in contrast to other hematological malignancies. In fact, CD33 represents the only antibody-based strategy approved for this disease to date, highlighting the need to identify new promising targets. AML cells span a wide range of aberrant myeloid differentiation programs, complexifying the identification, by bulk genomics, of targets expressed in the most immature leukemic cells. Aims and Methods: To identify the expression landscape of surface proteins in immature leukemic cells, we performed single-cell RNA sequencing (scRNA-seq, 10x 3' Reagent Kits) of primary human AML cells from 20 specimens of the Leucegene cohort enriched in intermediate and adverse genetic backgrounds ( KMT2A-rearranged n=5, chromosome 5 and/or 7 deletions (abn5/7, n=5) complex karyotype (n=4), NPM1/DNMT3A/FLT3-ITD triple-mutant (n=3) and others (n=3)). A Random Forest classifier was developed to unbiasedly classify AML cells into distinct differentiation stages using normal bone marrow-derived scRNA-seq data from the Human Cell Atlas (HCA) consortium. Genes were scored based on their probability of coding for proteins expressed at the cell surface using the SPAT algorithm developed by our group (https://doi.org/10.1101/2023.07.07.547075), retaining high score ones. To validate surface expression, we concomitantly analyzed the surface proteome (hereafter named surfaceome) of 100 primary human AML samples from the Leucegene cohort, including all 20 samples profiled by scRNA-seq. Results: After quality control, we profiled and characterized 103 690 high quality cells (mean of 5185 cells/sample). We trained a Random Forest classifier to annotate cells in a two step process, first identifying plasma cells based on a restricted list of genes abundantly expressed in these cells and subsequently assigning the remaining cells to one of 33 cell types. We performed a five-fold cross validation of the model and subsequently determined the accuracy of our classifier to be 92% on the test subset of the HCA data. Applied to our AML cell collection, a total of 35 053 cells (34%) were unbiasedly classified as Hematopoietic Stem Cell (HSC)-like, corresponding to the most phenotypically immature leukemic cells in each patient sample (ranging from 4 to 74 %). Accordingly, HSC-like AML cells preferentially express genes associated with normal HSCs, such as CD34, FAM30A, and SPINK2, and globally lack expression of mature lineages defining genes, further validating our classifier. The proportion of HSC-like cells varied among AML subgroups, and was lowest in KMT2A-r AML (median 19%) and highest in abn5/7 samples (46%). Integration of our AML atlas using Harmony algorithm preserved differentiation hierarchies across samples, with most cell types, including HSC-like cells, occupying a defined area in the low dimensional embedding. To identify new surface antigens specifically expressed in immature leukemic cells, we compared the high (≥8) SPAT score gene expression profile of AML HSC-like cells with that of normal HSC cells (HCA), and identified 60 genes significantly overexpressed in AML immature cells. Of those, 39 genes were also detected at the protein level by the surfaceome analysis, supporting their predicted expression at the cell surface in AML samples. 59% of these 39 genes (n=23) were detected in over 80% of the specimens analyzed by the surfaceome, and thus are nearly universally expressed in our AML cohort. To identify targets of therapies that could be repurposed, we next evaluated the relevance of our findings by querying the Thera-SAbDab database. Most interestingly, 8 of the 39 AML specific HSC markers are targeted by therapeutic antibodies FDA-approved or in clinical trials for the treatment of AML (n=4, IL3RA, FLT3, CD37 and TNFRSF10B) or other indications (n = 4). Conclusion Our genetically diverse AML single-cell atlas, supported by mass spectrometry, enables the identification of both subset-specific and pan-AML surface protein genes. These represent potential targets for antibody based strategy development or therapy repurposing in AML.

Alternative splicing of its 5′-UTR limits CD20 mRNA translation and enables resistance to CD20-directed immunotherapies

AbstractAberrant skipping of coding exons in CD19 and CD22 compromises the response to immunotherapy in B-cell malignancies. Here, we showed that the MS4A1 gene encoding human CD20 also produces several messenger RNA (mRNA) isoforms with distinct 5′ untranslated regions. Four variants (V1-4) were detected using RNA sequencing (RNA-seq) at distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant. During B-cell activation and Epstein-Barr virus infection, redirection of splicing from V1 to V3 coincided with increased CD20 positivity. Similarly, in diffuse large B-cell lymphoma, only V3, but not V1, correlated with CD20 protein levels, suggesting that V1 might be translation-deficient. Indeed, the longer V1 isoform contained upstream open reading frames and a stem-loop structure, which cooperatively inhibited polysome recruitment. By modulating CD20 isoforms with splice-switching morpholino oligomers, we enhanced CD20 expression and anti-CD20 antibody rituximab-mediated cytotoxicity in a panel of B-cell lines. Furthermore, reconstitution of CD20-knockout cells with V3 mRNA led to the recovery of CD20 positivity, whereas V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, in vitro CD20-directed chimeric antigen receptor T cells were able to kill both V3- and V1-expressing cells, but the bispecific T-cell engager mosunetuzumab was only effective against V3-expressing cells. To determine whether CD20 splicing is involved in immunotherapy resistance, we performed RNA-seq on 4 postmosunetuzumab follicular lymphoma relapses and discovered that in 2 of them, the downregulation of CD20 was accompanied by a V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies.

Histological Investigation of the Female Gonads of Chiropsalmus quadrumanus (Cubozoa: Cnidaria) Suggests Iteroparous Reproduction

The box jellyfish Chiropsalmus quadrumanus (Chirodropida: Cubozoa: Cnidaria) is common in warm waters. Although it is assumed that external fertilization is a characteristic of Chirodropida, the life history of C. quadrumanus is not yet known since its reproductive behavior has never been described, nor has the polyp has been found in nature. As a result, in the absence of documentation of reproductive behavior, we sought to test the hypothesis of external fertilization through a histological analysis of the female gonads. Herein, we analyze ten females collected in São Paulo and Rio de Janeiro (Brazil) and describe the gonadal organization and pattern of oocyte development. The discovery of four distinct stages of oocyte differentiation augments the scant existing reports of the structural and functional maturation of sex cells in Cubozoa species. Furthermore, the gonads of mature females comprise both mature (average diameter of 122 µm) and immature oocytes, suggesting that C. quadrumanus is iteroparous and exhibits multiple reproductive cycles during its life. Medusa bell size was not found to correlate with maturity state as even small females possessed a high percentage of oocytes in late vitellogenesis, suggesting that sexual maturation occurs rapidly in C. quadrumanus females.

MRLC controls apoptotic cell death and functions to regulate epidermal development during planarian regeneration and homeostasis.

Adult stem cells (ASCs) are pluripotent cells with the capacity to self-renew and constantly replace lost cells due to physiological turnover or injury. Understanding the molecular mechanisms of the precise coordination of stem cell proliferation and proper cell fate decision is important to regeneration and organismal homeostasis. The planarian epidermis provides a highly tractable model to study ASC complex dynamic due to the distinct spatiotemporal differentiation stages during lineage development. Here, we identified the myosin regulatory light chain (MRLC) homologue in the Dugesia japonica transcriptome. We found high expression levels of MRLC in wound region during regeneration and also expressed in late epidermal progenitors as an essential regulator of the lineage from neoblasts to mature epidermal cells. We investigated the function of MRLC using in situ hybridization, real-time polymerase chain reactionand double fluorescent and uncovered the potential mechanism. Knockdown of MRLC leads to a remarkable increase in cell death, causes severe abnormalities during regeneration and homeostasis and eventually leads to animal death. The global decrease in epidermal cell in MRLC RNAi animals induces accelerated epidermal proliferation and differentiation. Additionally, we find that MRLC is co-expressed with cdc42 and acts cooperatively to control the epidermal lineage development by affecting cell death. Our results uncover an important role of MRLC, as an inhibitor of apoptosis, involves in epidermal development.

GTAC enables parallel genotyping of multiple genomic loci with chromatin accessibility profiling in single cells

Understanding clonal evolution and cancer development requires experimental approaches for characterizing the consequences of somatic mutations on gene regulation. However, no methods currently exist that efficiently link high-content chromatin accessibility with high-confidence genotyping in single cells. To address this, we developed Genotyping with the Assay for Transposase-Accessible Chromatin (GTAC), enabling accurate mutation detection at multiple amplified loci, coupled with robust chromatin accessibility readout. We applied GTAC to primary acute myeloid leukemia, obtaining high-quality chromatin accessibility profiles and clonal identities for multiple mutations in 88% of cells. We traced chromatin variation throughout clonal evolution, showing the restriction of different clones to distinct differentiation stages. Furthermore, we identified switches in transcription factor motif accessibility associated with a specific combination of driver mutations, which biased transformed progenitors toward a leukemia stem cell-like chromatin state. GTAC is a powerful tool to study clonal heterogeneity across a wide spectrum of pre-malignant and neoplastic conditions.

211 Single cell transcriptomics of human epidermis reveals downregulation of structural genes associated with skin barrier development in aged skin

Epidermal keratinocytes are continuously dividing cells which, upon differentiation establish the outer skin barrier, the stratum corneum. In older age the epidermis becomes thinner, papillary protrusions flatten and the capacity for skin barrier repair decreases. Here we performed single-cell RNA sequencing of epidermal keratinocytes obtained by suction blisters from the volar forearm of younger (20-30) and older (65-70) healthy female individuals. Based on the expression of keratin (KRT) 5 and 10 and other structural proteins including filaggrin and corneodesmosin, we detected nine keratinocyte clusters at distinct differentiation stages amongst others.

Myeloid Progenitors Define Extent of Disease in Mouse Models of Histiocytic Disorders: BRAFV600E Expression in Monocyte Lineage Recapitulates Erdheim-Chester Phenotype

Langerhans cell histiocytosis (LCH) is a myeloid neoplastic disorder characterized by granulomatous lesions with CD207+ dendritic cells associated with a wide spectrum of clinical presentations. Activating MAPK pathway genes (most frequently BRAFV600E) are identified in lesion DCs as well as in precursor cells in bone marrow and peripheral blood in some cases. LCH typically arises in children, but can also occur de novo in adults. Erdheim-Chester disease (ECD) is a related histiocytic disorder characterized by CD163+ histiocytic lesions with activating MAPK mutations (most frequently BRAFV600E) arising primarily in adults. Unlike LCH, ECD lesion distribution frequently includes kidneys and heart. In order to test the impact of cell of origin on disease phenotype, we developed Cre-inducible mouse models in which BRAFV600E expression is enforced at distinct stages of differentiation: BRAFV600ECD11c and BRAFV600EMSaA3. ITGAX/CD11c is expressed across a range of myeloid dendritic cell and monocyte lineages, where MS4a3 is expressed exclusively in GMPs and GMP-derived cells (e.g. monocytes). BRAFV600E expression was enforced through Cre-mediated recombination to create BRAFV600ECD11c and BRAFV600EMSaA3 mice. Mice were analyzed at weeks 5, 10, 25, and 52 for body and organ weights and histology. Tissues were characterized by immunohistochemistry, gene expression, Luminex, CYTOF and RNASeq. BRAFV600ECD11c mice exhibited a more aggressive phenotype than BRAFV600EMS4A3 counterparts, though both developed systemic histiocytic infiltration. Kinetics of organomegaly, histiocytic infiltration, and systemic inflammation was much slower in the BRAFV600EMS4a3 mice. Notably, BRAFV600EMS4a3 demonstrated increased enrichment of CD207-negative macrophages in spleen, liver and bone marrow. BRAFV600EMS4a3 mice readily survived past 52 weeks, while BRAFV600ECD11c had 100% mortality by 16 weeks. Bulk RNASeq and Ingenuity Pathway Analysis identified relative decrease in macrophage functionality in BRAFV600EMSaA3 mice. Additionally, macrophage infiltrate was observed in heart, major blood vessels and kidneys of BRAFV600EMS4a3 where these organs were not affected in BRAFV600ECD11c. The same mutation that creates an aggressive LCH-like phenotype in CD11c-derived cells creates an indolent ECD-like phenotype in monocyte-derived cells. This study demonstrates a critical role for ontogeny in pathogenesis of histiocytic disorders. Further, the BRAFV600EMS4a3 mouse provides an ECD model for pre-clinical testing of therapeutic strategies.

Methods to study adult hippocampal neurogenesis in humans and across the phylogeny.

The hippocampus hosts the continuous addition of new neurons throughout life-a phenomenon named adult hippocampal neurogenesis (AHN). Here we revisit the occurrence of AHN in more than 110 mammalian species, including humans, and discuss the further validation of these data by single-cell RNAseq and other alternative techniques. In this regard, our recent studies have addressed the long-standing controversy in the field, namely whether cells positive for AHN markers are present in the adult human dentate gyrus (DG). Here we review how we developed a tightly controlled methodology, based on the use of high-quality brain samples (characterized by short postmortem delays and ≤24 h of fixation in freshly prepared 4% paraformaldehyde), to address human AHN. We review that the detection of AHN markers in samples fixed for 24 h required mild antigen retrieval and chemical elimination of autofluorescence. However, these steps were not necessary for samples subjected to shorter fixation periods. Moreover, the detection of labile epitopes (such as Nestin) in the human hippocampus required the use of mild detergents. The application of this strictly controlled methodology allowed reconstruction of the entire AHN process, thus revealing the presence of neural stem cells, proliferative progenitors, neuroblasts, and immature neurons at distinct stages of differentiation in the human DG. The data reviewed here demonstrate that methodology is of utmost importance when studying AHN by means of distinct techniques across the phylogenetic scale. In this regard, we summarize the major findings made by our group that emphasize that overlooking fundamental technical principles might have consequences for any given research field.

Distinct metabolic states guide maturation of inflammatory and tolerogenic dendritic cells

Cellular metabolism underpins immune cell functionality, yet our understanding of metabolic influences in human dendritic cell biology and their ability to orchestrate immune responses is poorly developed. Here, we map single-cell metabolic states and immune profiles of inflammatory and tolerogenic monocytic dendritic cells using recently developed multiparametric approaches. Single-cell metabolic pathway activation scores reveal simultaneous engagement of multiple metabolic pathways in distinct monocytic dendritic cell differentiation stages. GM-CSF/IL4-induce rapid reprogramming of glycolytic monocytes and transient co-activation of mitochondrial pathways followed by TLR4-dependent maturation of dendritic cells. Skewing of the mTOR:AMPK phosphorylation balance and upregulation of OXPHOS, glycolytic and fatty acid oxidation metabolism underpin metabolic hyperactivity and an immunosuppressive phenotype of tolerogenic dendritic cells, which exhibit maturation-resistance and a de-differentiated immune phenotype marked by unique immunoregulatory receptor signatures. This single-cell dataset provides important insights into metabolic pathways impacting the immune profiles of human dendritic cells.

M 6 Aexpress-BHM: predicting m6A regulation of gene expression in multiple-groups context by a Bayesian hierarchical mixture model.

As the most abundant RNA modification, N6-methyladenosine (m6A) plays an important role in various RNA activities including gene expression and translation. With the rapid application of MeRIP-seq technology, samples of multiple groups, such as the involved multiple viral/ bacterial infection or distinct cell differentiation stages, are extracted from same experimental unit. However, our current knowledge about how the dynamic m6A regulating gene expression and the role in certain biological processes (e.g. immune response in this complex context) is largely elusive due to lack of effective tools. To address this issue, we proposed a Bayesian hierarchical mixture model (called m6Aexpress-BHM) to predict m6A regulation of gene expression (m6A-reg-exp) in multiple groups of MeRIP-seq experiment with limited samples. Comprehensive evaluations of m6Aexpress-BHM on the simulated data demonstrate its high predicting precision and robustness. Applying m6Aexpress-BHM on three real-world datasets (i.e. Flaviviridae infection, infected time-points of bacteria and differentiation stages of dendritic cells), we predicted more m6A-reg-exp genes with positive regulatory mode that significantly participate in innate immune or adaptive immune pathways, revealing the underlying mechanism of the regulatory function of m6A during immune response. In addition, we also found that m6A may influence the expression of PD-1/PD-L1 via regulating its interacted genes. These results demonstrate the power of m6Aexpress-BHM, helping us understand the m6A regulatory function in immune system.

Abstract 486: A systems biology approach to defining tumor heterogeneity, prognostic and targetable master regulator protein signatures from bulk and single cell RNA-seq in diffuse midline glioma (DMG)

Abstract Despite major advances in molecular profiling and numerous clinical trials, diffuse midline glioma (DMG) remains a fatal disease with median survival of only ~9 months and no identified effective drugs. To address this challenge, we leveraged network-based methodologies to dissect the heterogeneity of DMG tumors and to discover Master Regulator (MR) proteins representing pharmacologically accessible, mechanistic determinants of molecularly distinct DMG cell states. The study has produced the first DMG-specific regulatory network, reverse-engineered from 122 publicly available pediatric DMG RNA-seq profiles with ARACNe (Basso et al. Nat Genet 2005). Using this network, we measured protein activity for each sample via VIPER (Alvarez et al., Nat Genet 2016). Activity-based clustering identified 2 clusters, characterized by significant overall survival difference (&amp;gt;1 year, p-val=0.02 by χ2 analysis). Protein activity signatures were not significantly associated with tumor location and Histone3 mutation status. The most aberrantly activated MR proteins across all DMG patients (i.e., TOP2A, CENPF, BUB1B, FOXM1, GTSE1, MKI67, and E2F8), relative to normal caudate tissue from GTEx, were highly enriched in cell cycle regulation members, with samples in the worst outcome cluster showing significantly higher activity. Pharmacologically accessible MRs found to be significantly activated in subsets of patients (p-val&amp;lt;10E-5) include TOP2A, CHEK1, CDK2, and EZH2. To dissect DMG intra-tumor heterogeneity, we measured protein activity from published single-cell RNA-seq profiles of 6 DMG patients, using single-cell based regulatory networks. Activity-based analysis of tumor cells identified 8 clusters representing distinct differentiation and proliferation stages—i.e. astrocyte-like, oligodendrocyte-like, and multiple oligodendrocyte precursor cell (OPC)-like subpopulations. Consistent with bulk-based findings, these included an OPC-like-cycling population presenting highly aberrant activity of proliferative MRs (i.e. TOP2A, CENPF, FOXM1, E2F8, ZWINT, and CCNA2), suggesting this as a key DMG regulatory module (Tumor Checkpoint). We are working to define targetable MRs in these subpopulations, and generating RNA-seq profiles of SU-DIPG-VI and SF8628, two DMG cell lines showing protein activity similarity to &amp;gt;95% of patient samples by enrichment analysis (p-val &amp;lt; 10E-5), following perturbation with ~400 oncology drugs. This will allow us to identify drugs capable of inducing tumor demise by inverting the activity of the MRs of each tumor subpopulation, using the NY Dept. of Health approved OncoTreat algorithm (Alvarez et al., Nat Genet 2018). We are currently finalizing and validating both MR and drug predictions to nominate novel, much-needed therapeutic strategies. Citation Format: Ester Calvo Fernández, Junqiang Wang, Aaron T. Griffin, Luca Szalontay, Stergios Zacharoulis, Jovana Pavisic, Andrea Califano. A systems biology approach to defining tumor heterogeneity, prognostic and targetable master regulator protein signatures from bulk and single cell RNA-seq in diffuse midline glioma (DMG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 486.

Alternative Splicing Mediated by RNA-Binding Protein RBM24 Facilitates Cardiac Myofibrillogenesis in a Differentiation Stage-Specific Manner.

Mutations in genes encoding sarcomeric proteins lead to failures in sarcomere assembly, the building blocks of contracting muscles, resulting in cardiomyopathies that are a leading cause of morbidity and mortality worldwide. Splicing variants of sarcomeric proteins are crucial at different stages of myofibrillogenesis, accounting for sarcomeric structural integrity. RBM24 (RNA-binding motif protein 24) is known as a tissue-specific splicing regulator that plays an essential role in cardiogenesis. However, it had been unclear if the developmental stage-specific alternative splicing facilitated by RBM24 contributes to sarcomere assembly and cardiogenesis. Our aim is to study the molecular mechanism by which RBM24 regulates cardiogenesis and sarcomere assembly in a temporal-dependent manner. We ablated RBM24 from human embryonic stem cells (hESCs) using CRISPR/Cas9 techniques. Although RBM24-/- hESCs still differentiated into sarcomere-hosting cardiomyocytes, they exhibited disrupted sarcomeric structures with punctate Z-lines due to impaired myosin replacement during early myofibrillogenesis. Transcriptomics revealed >4000 genes regulated by RBM24. Among them, core myofibrillogenesis proteins (eg, ACTN2 [α-actinin 2], TTN [titin], and MYH10 [non-muscle myosin IIB]) were misspliced. Consequently, MYH6 (muscle myosin II) cannot replace nonmuscle myosin MYH10, leading to myofibrillogenesis arrest at the early premyofibril stage and causing disrupted sarcomeres. Intriguingly, we found that the ABD (actin-binding domain; encoded by exon 6) of the Z-line anchor protein ACTN2 is predominantly excluded from early cardiac differentiation, whereas it is consistently included in human adult heart. CRISPR/Cas9-mediated deletion of exon 6 from ACTN2 in hESCs, as well as forced expression of full-length ACTN2 in RBM24-/- hESCs, further corroborated that inclusion of exon 6 is critical for sarcomere assembly. Overall, we have demonstrated that RBM24-facilitated inclusion of exon 6 in ACTN2 at distinct stages of cardiac differentiation is evolutionarily conserved and crucial to sarcomere assembly and integrity. RBM24 acts as a master regulator to modulate the temporal dynamics of core myofibrillogenesis genes and thereby orchestrates sarcomere organization.

Metabolic Changes in Venetoclax Resistance Are Determined By Differentiation State in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive hematologic malignancy arising from the transformation of immune T-cell lymphocytes. Early T-cell progenitor (ETP-ALL) is a subgroup particularly associated with chemoresistance and a high risk for relapse. Recently, it was shown that ETP-ALL is dependent on the expression of the anti-apoptotic protein BCL-2, and is sensitive to inhibition with ABT-199, a BCL-2 specific BH3 mimetic 1,2. However, one issue with a targeted agent, such as ABT-199, is the development of acquired resistance. Interestingly, there have been numerous high impact papers connecting ABT-199 resistance to altered oxidative phosphorylation (OXPHOS) 3,4. While there are relatively few studies into T-ALL metabolism, there is evidence that aerobic glycolysis, the conversion of glucose to lactate, is greater in proliferating T-cells than in T-ALL and that NOTCH signalling can drive mitochondrial OXPHOS 5. A recent study showed that the transcription factor RUNX2 altered T-ALL metabolism, increasing both glycolysis and OXPHOS and enhancing leukemic cell migration 6. However, there has been relatively little research into the metabolic profile of T-ALL at distinct stages of differentiation. The aim of this study was to determine the role of ABT-199 resistance in altering metabolism and determine if that was due to the differentiation state of the T-ALL.ABT-199 R LOUCY cells were generated by chronic exposure to increasing concentrations of ABT-199 administered every two days. The EZH2 KO Jurkat cell lines were previously generated through CRISPR-Cas9 engineering 7. In order to assess the metabolic profile, cells were attached to a 96 well plate using CellTak and the extracellular acidification rate (ECAR) and oxidative phosphorylation (OXPHOS) was measured on a Seahorse Bioscience XF96 Extracellular Flux Analyzer. Anti-apoptotic dependence was measured using BH3 profiling and cell death by Annexin V/propidium iodide staining. The mitochondrial structure was visualized using transmission electron microscopy.Previously, we generated ABT-199 resistant ETP-ALL LOUCY cells (ABT-199 R) following continuous exposure to ABT-199 over a prolonged period of several months 8. The ABT-199 R cells showed dependence on BCL-XL for survival and sensitivity to the BCL-XL inhibitor WEHI 539. The ABT-199 R cells showed evidence of differentiation to a more mature T-cell. The ABT-199 R cells had increased surface CD3 (sCD3) expression and CD1A expression, along with increased expression of TAL1 and LMO2 genes compared to parental LOUCY cells. Interestingly, the ABT-199 R cells showed enhanced basal respiration, ATP production and max respiration compared to the parental cells. Indeed, analysis of the expression of OXPHOS complexes showed increased expression of complexes I-IV in the ABT-199 R cells, compared to the parental controls. Indeed, the parental LOUCY cells appeared to have reduced cristae number and length compared to the ABT-199R cells.Next, we assessed if inhibiting OXPHOS with a series of inhibitors (oligomycin, rotenone, antimycin) could sensitize the ABT-199 R LOUCY cells to ABT-199. However, we did not detect any changes to sensitivity of ABT-199. This led us to hypothesize that perhaps the changes in OXPHOS were due differentiation state of the LOUCY cells. We confirmed that more typical T-ALL cell lines (JURKAT and CEM-CCRF) had higher OXPHOS than the ETP-ALL cell line LOUCY and had higher expression of the OXPHOS complexes I-IV by Western blotting.To assess if de-differentiation of a more typical T-ALL cell line would cause a reduction in OXPHOS we turned to the EZH2 knockout (K/O) Jurkat cells 7. We found that EZH2 KO2 showed a reduction in the differentiation markers CD1A and CD3 on the cell surface and TAL1 gene expression, compared to WT control Jurkats. Next, we assessed the OXPHOS and found that the de-differentiated EZH2 cells had reduced OXPHOS compared to the parental controls, with altered mitochondrial structure. Suggesting, that de-differentiation of typical T-ALL cell line reduces OXPHOS.In this study we show that metabolic phenotype is linked to the maturation stage of T-ALL. We believe that the altered metabolism identified in ABT-199 resistance is linked to the selection of a more mature cell type. Highlighting, that altered metabolism may not be a driver of resistance to ABT-199 but a consequence of the maturation stage of the resistant cell. DisclosuresDi Grande: Novartis: Current Employment. Leon: BenevolentAI: Current Employment. Mansour: Astellas: Consultancy, Honoraria; Janssen: Consultancy. Bond: Haematology Association of Ireland Award funded by Novartis: Research Funding. Ni Chonghaile: AbbVie: Research Funding.