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Clonal haematopoiesis, which is highly prevalent in older individuals, arises from somatic mutations that endow a proliferative advantage to haematopoietic cells. Clonal haematopoiesis increases the risk of myocardial infarction and stroke independently of traditional risk factors1. Among the common genetic variants that give rise to clonal haematopoiesis, the JAK2V617F (JAK2VF) mutation, which increases JAK–STAT signalling, occurs at a younger age and imparts the strongest risk of premature coronary heart disease1,2. Here we show increased proliferation of macrophages and prominent formation of necrotic cores in atherosclerotic lesions in mice that express Jak2VF selectively in macrophages, and in chimeric mice that model clonal haematopoiesis. Deletion of the essential inflammasome components caspase 1 and 11, or of the pyroptosis executioner gasdermin D, reversed these adverse changes. Jak2VF lesions showed increased expression of AIM2, oxidative DNA damage and DNA replication stress, and Aim2 deficiency reduced atherosclerosis. Single-cell RNA sequencing analysis of Jak2VF lesions revealed a landscape that was enriched for inflammatory myeloid cells, which were suppressed by deletion of Gsdmd. Inhibition of the inflammasome product interleukin-1β reduced macrophage proliferation and necrotic formation while increasing the thickness of fibrous caps, indicating that it stabilized plaques. Our findings suggest that increased proliferation and glycolytic metabolism in Jak2VF macrophages lead to DNA replication stress and activation of the AIM2 inflammasome, thereby aggravating atherosclerosis. Precise application of therapies that target interleukin-1β or specific inflammasomes according to clonal haematopoiesis status could substantially reduce cardiovascular risk. Accelerated atherosclerosis in a mouse model of clonal haematopoiesis is prevented by genetic interruption of AIM2 inflammasome activation or by inhibition of interleukin-1β.

Branching morphogenesis is a fascinating process whereby a simple network of biological tubes increases its complexity by adding new branches to existing ones, generating an enlarged structure of interconnected tubes. Branching morphogenesis has been studied extensively in animals and much has been learned about the regulation of branching at the cellular and molecular level. Here, we discuss studies of the Drosophila trachea and of the vertebrate vasculature, which have revealed how new branches are formed and connect (anastomose), leading to the establishment of complex tubular networks. We briefly describe the cell behaviour underlying tracheal and vascular branching. Although similar at many levels, the branching and anastomosis processes characterized thus far show a number of differences in cell behaviour, resulting in somewhat different tube architectures in these two organs. We describe the similarities and the differences and discuss them in the context of their possible developmental significance. We finish by highlighting some old and new data, which suggest that live imaging of the development of capillary beds in adult animals might reveal yet unexplored endothelial behaviour of endothelial cells.

Mutations in splicing factors (SF) are the predominant class of mutations in myelodysplastic syndrome (MDS), but convergent downstream disease drivers remain elusive. To identify common direct targets of missplicing by mutant U2AF1 and SRSF2, we performed RNA sequencing and enhanced version of the cross-linking and immunoprecipitation assay in human hematopoietic stem/progenitor cells derived from isogenic induced pluripotent stem cell (iPSC) models. Integrative analyses of alternative splicing and differential binding converged on a long isoform of GNAS (GNAS-L), promoted by both mutant factors. MDS population genetics, functional and biochemical analyses support that GNAS-L is a driver of MDS and encodes a hyperactive long form of the stimulatory G protein alpha subunit, Gαs-L, that activates ERK/MAPK signaling. SF-mutant MDS cells have activated ERK signaling and consequently are sensitive to MEK inhibitors. Our findings highlight an unexpected and unifying mechanism by which SRSF2 and U2AF1 mutations drive oncogenesis with potential therapeutic implications for MDS and other SF-mutant neoplasms. Significance: SF mutations are disease-defining in MDS, but their critical effectors remain unknown. We discover the first direct target of convergent missplicing by mutant U2AF1 and SRSF2, a long GNAS isoform, which activates G protein and ERK/MAPK signaling, thereby driving MDS and rendering mutant cells sensitive to MEK inhibition. This article is highlighted in the In This Issue feature, p. 587

Blood vessel morphogenesis is driven by coordinated endothelial cell behaviors, which depend on dynamic cell-cell interactions. Remodeling of endothelial cell-cell junctions promote morphogenetic cellular events while preserving vascular integrity. Here, we have analyzed the dynamics of endothelial cell-cell junctions during lumen formation in angiogenic sprouts. By live-imaging of the formation of intersegmental blood vessels in zebrafish, we demonstrate that lumen expansion is accompanied by the formation of transient finger-shaped junctions. Formation and maintenance of these junctional fingers are positively regulated by blood pressure whereas inhibition of blood flow prevents their formation. Using fluorescent reporters, we show that the tension-sensor Vinculin localizes to junctional fingers. Furthermore, loss of vinculin function, in vinculin a and -b double knockouts, prevents junctional finger formation in angiogenic sprouts, whereas endothelial expression of a vinculin transgene is sufficient to restore junctional fingers. Taken together, our findings suggest a mechanism in which lumen expansion during angiogenesis leads to an increase in junctional tension, which triggers recruitment of vinculin and formation of junctional fingers. We propose that endothelial cells may employ force-dependent junctional remodeling to react to changes in external forces to protect cell-cell contacts and to maintain vascular integrity during sprouting angiogenesis.

Acute myeloid leukemia (AML) is a clonal hematopoietic stem and progenitor cell malignancy characterized by poor clinical outcomes. Major histocompatibility complex class I polypeptide-related sequence A and B (MICA/B) are stress proteins expressed by cancer cells, and antibody-mediated inhibition of MICA/B shedding represents a novel approach to stimulate immunity against cancers. We found that the MICA/B antibody 7C6 potently inhibits the outgrowth of AML in 2 models in immunocompetent mice. Macrophages were essential for therapeutic efficacy, and 7C6 triggered antibody-dependent phagocytosis of AML cells. Furthermore, we found that romidepsin, a selective histone deacetylase inhibitor, increased MICB messenger RNA in AML cells and enabled subsequent stabilization of the translated protein by 7C6. This drug combination substantially increased surface MICA/B expression in a human AML line, pluripotent stem cell-derived AML blasts and leukemia stem cells, as well as primary cells from 3 untreated patients with AML. Human macrophages phagocytosed AML cells following treatment with 7C6 and romidepsin, and the combination therapy lowered leukemia burden in a humanized model of AML. Therefore, inhibition of MICA/B shedding promotes macrophage-driven immunity against AML via Fc receptor signaling and synergizes with an epigenetic regulator. These results provide the rationale for the clinical testing of this innovative immunotherapeutic approach for the treatment of AML.

Patient-derived induced pluripotent stem cells (iPSCs) have recently provided a new way to model acute myeloid leukemia (AML) and other myeloid malignancies. Here, we describe methods for the generation of patient-derived iPSCs from leukemia cells and for their subsequent directed in vitro differentiation into hematopoietic cells that recapitulate features of leukemia stem cells (LSCs) and leukemic blasts.

Summary: Leukemia stem cells (LSCs) are believed to have more distinct vulnerabilities than the bulk acute myeloid leukemia (AML) cells, but their rarity and the lack of universal markers for their prospective isolation hamper their study. We report that genetically clonal induced pluripotent stem cells (iPSCs) derived from an AML patient and characterized by exceptionally high engraftment potential give rise, upon hematopoietic differentiation, to a phenotypic hierarchy. Through fate-tracking experiments, xenotransplantation, and single-cell transcriptomics, we identify a cell fraction (iLSC) that can be isolated prospectively by means of adherent in vitro growth that resides on the apex of this hierarchy and fulfills the hallmark features of LSCs. Through integrative genomic studies of the iLSC transcriptome and chromatin landscape, we derive an LSC gene signature that predicts patient survival and uncovers a dependency of LSCs, across AML genotypes, on the RUNX1 transcription factor. These findings can empower efforts to therapeutically target AML LSCs.

Human cancers arise through an evolutionary process whereby cells acquire somatic mutations that drive them to outgrow normal cells and create successive clonal populations. “Bottom-up” human cancer evolution models could help illuminate this process, but their creation has faced significant challenges. Here we combined human induced pluripotent stem cell (iPSC) and CRISPR/Cas9 technologies to develop a model of the clonal evolution of acute myeloid leukemia (AML). Through the sequential introduction of 3 disease-causing mutations (ASXL1 C-terminus truncation, SRSF2P95L and NRASG12D), we obtained single, double and triple mutant iPSC lines that, upon hematopoietic differentiation, exhibit progressive dysplasia with increasing number of mutations, capturing distinct premalignant stages, including clonal hematopoiesis, myelodysplastic syndrome, and culminating in a transplantable leukemia. iPSC-derived clonal hematopoietic stem/progenitor cells recapitulate transcriptional and chromatin accessibility signatures of normal and malignant hematopoiesis found in primary human cells. By mapping dynamic changes in transcriptomes and chromatin landscapes, we characterize transcriptional programs driving specific stage transitions and identify vulnerabilities for early therapeutic targeting. Such synthetic “de novo oncogenesis” models can empower the investigation of multiple facets of the malignant transformation of human cells.

Recurrent chromosomal deletions spanning several megabases are often found in hematological malignancies. The ability to engineer deletions in model systems to functionally study their effects on the phenotype would enable, first, determination of whether a given deletion is pathogenic or neutral and, second, identification of the critical genes. Incomplete synteny makes modeling of deletions of megabase scale challenging or impossible in the mouse or other model organisms. Furthermore, despite the breakthroughs in targeted nuclease technologies in recent years, engineering of megabase-scale deletions remains challenging and has not been achieved in normal diploid human cells. Large deletions of the long arm of chromosome 7 (chr7q) occur frequently in myelodysplastic syndrome (MDS) and are associated with poor prognosis. We previously found that we can model chr7q deletions in human induced pluripotent stem cells (iPSCs) using a modified Cre-loxP strategy. However, this strategy did not afford control over the length and boundaries of the engineered deletions, which were initiated through random chromosome breaks. Here we developed strategies enabling the generation of defined and precise chromosomal deletions of up to 22 Mb, using two different strategies: "classic" Cre-loxP recombination and CRISPR/Cas9-mediated DNA cleavage. As proof of principle, we illustrate that phenotypic characterization of the hematopoiesis derived from these iPSCs upon in vitro differentiation allows further definition of the critical region of chr7q whose hemizygosity impairs hematopoietic differentiation potential. The strategies we present here can be broadly applicable to engineering of diverse chromosomal deletions in human cells.

Summary Connecting specific cancer genotypes with phenotypes and drug responses constitutes the central premise of precision oncology but is hindered by the genetic complexity and heterogeneity of primary cancer cells. Here, we use patient-derived induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 genome editing to dissect the individual contributions of two recurrent genetic lesions, the splicing factor SRSF2 P95L mutation and the chromosome 7q deletion, to the development of myeloid malignancy. Using a comprehensive panel of isogenic iPSCs—with none, one, or both genetic lesions—we characterize their relative phenotypic contributions and identify drug sensitivities specific to each one through a candidate drug approach and an unbiased large-scale small-molecule screen. To facilitate drug testing and discovery, we also derive SRSF2-mutant and isogenic normal expandable hematopoietic progenitor cells. We thus describe here an approach to dissect the individual effects of two cooperating mutations to clinically relevant features of malignant diseases., Graphical Abstract, Highlights • A panel of isogenic iPSCs with SRSF2 P95L mutation and del(7q) was generated • The contributions of each genetic lesion to phenotype were separated • Each genetic lesion could be linked to specific therapeutic vulnerabilities • Expandable hematopoietic progenitor cell lines facilitate drug testing, Papapetrou and colleagues develop a comprehensive panel of isogenic iPSC lines with SRSF2 P95L mutation and chr7q deletion. They use these cells to identify cellular phenotypes contributed by each genetic lesion and therapeutic vulnerabilities specific to each one and develop expandable hematopoietic progenitor cell lines to facilitate drug discovery.

The somatic hotspot mutation SF3B1K700E is characteristically found in myelodysplastic syndrome with ring sideroblasts (MDS-RS) and frequently occurs as an isolated mutation. However, our understanding of how this mutation drives MDS pathogenesis remains limited. To explore the downstream consequences of the SF3B1K700E mutation and its role in disease pathogenesis, we generated a panel of isogenic SF3B1K700E and SF3B1WT induced pluripotent stem cell (iPSC) lines from 3 MDS-RS patients with isolated SF3B1K700E mutation (3 SF3B1K700E and 3 SF3B1WT lines per patient, total 18). Upon hematopoietic differentiation, SF3B1K700E cells exhibited lower growth and colony-forming ability, compared to SF3B1WTcells, recapitulating hallmark phenotypes of MDS cells. To investigate the effects of the SF3B1K700E mutation on the transcriptome and chromatin landscape, we performed RNA- and ATAC- sequencing in purified CD34+/CD45+ hematopoietic stem/progenitor cells (HSPCs) derived from the panel of the 18 isogenic SF3B1K700E and SF3B1WT iPSC lines. Principal component analysis (PCA) and hierarchical clustering based on gene expression grouped the iPSC lines primarily by genotype (SF3B1K700E vs SF3B1WT) and secondarily by genetic background. To assess the impact of the SF3B1K700E mutation at the exon, transcript and gene level, we developed an analytical framework integrating differential splicing with differential transcript usage and differential gene expression analyses. We thus discovered 59 splicing events linked to 34 genes (most statistically significant events that also mapped to differentially used transcripts and differentially expressed genes). This SF3B1K700E splicing signature includes genes previously reported as mis-spliced in SF3B1K700E cells (e.g BRD9, ABCB7), as well as novel genes. We tested this signature against a published dataset of primary MDS patient samples (Pellagatti et al.). PCA based on the inclusion level of the splicing events of our signature separated SF3B1-mutated MDS patients from patients without splicing factor mutations (SF-WT) or healthy individuals. Furthermore, it identified one patient erroneously annotated as SF-WT that clustered together with the SF3B1-mutated patients, who had a, previously overlooked, 6bp in-frame deletion spanning the K700E hotspot. By comparing the chromatin accessibility profiles of SF3B1K700E and SF3B1WT iPSC-HSPCs to those defined in primary human cell types along the hematopoietic hierarchy (Corces et al.), we found that the chromatin landscape of SF3B1K700E HSPCs resembled more this of megakaryocyte-erythroid progenitor cells (MEPs) and erythroid cells, whereas that of SF3B1WT HSPCs resembled more granulocyte-monocyte progenitors (GMPs) and monocytes. This finding may underlie the more prominent involvement of the erythroid lineage in the pathology and clinical presentation of MDS-RS. To interrogate transcriptional programs in SF3B1K700E mutant cells, we performed transcription factor (TF) motif enrichment analysis. Motifs enriched in ATAC-Seq peaks more accessible in SF3B1K700E cells that were linked to genes upregulated in SF3B1K700E cells, included motifs of several TFs with known roles in hematopoiesis (GATA, ETS, STAT, AP-1). Unexpectedly, motifs of the TEAD TFs were also enriched. The TEAD family of TFs are best known as effectors of the Hippo signaling pathway, with important roles in various biological processes and malignancies, albeit no clear links to adult hematopoiesis or hematologic disease. TEAD2 and TEAD4 were upregulated in SF3B1-mutant, compared to the WT, iPSC-HSPCs and TEAD transcriptional activity, measured with a luciferase reporter construct, was higher in SF3B1K700E, compared to SF3B1WTiPSC-HSPCs. We did not find expression or activation of YAP or TAZ, which bind to DNA as a complex with TEAD upon Hippo pathway activation. These results support a Hippo-independent increase of TEAD expression and activity in SF3B1K700E cells. In summary, we generated a panel of isogenic patient-derived iPSCs that allowed us to comprehensively characterize the transcriptome and chromatin landscape of SF3B1K700E HSPCs in an isogenic system, derive a splicing signature of SF3B1K700E and identify the TEAD TF as a new transcriptional regulator of SF3B1K700Emutant HSPCs. G. Asimomitis, A.G. Deslauriers: shared 1 st authorship E. Papaemmanuil, E.P. Papapetrou: shared senior authorship Disclosures Deslauriers: Novo Nordisk A/S: Current Employment. Hellström-Lindberg: Celgene: Research Funding. Papaemmanuil: Isabl Technologies: Divested equity in a private or publicly-traded company in the past 24 months; Kyowa Hakko Kirin Pharma: Consultancy.

Human cancers arise through the sequential acquisition of somatic mutations that create successive clonal populations. Human cancer evolution models could help illuminate this process and inform therapeutic intervention at an early disease stage, but their creation has faced significant challenges. Here, we combined induced pluripotent stem cell (iPSC) and CRISPR-Cas9 technologies to develop a model of the clonal evolution of acute myeloid leukemia (AML). Through the stepwise introduction of three driver mutations, we generated iPSC lines that, upon hematopoietic differentiation, capture distinct premalignant stages, including clonal hematopoiesis (CH) and myelodysplastic syndrome (MDS), culminating in a transplantable leukemia, and recapitulate transcriptional and chromatin accessibility signatures of primary human MDS and AML. By mapping dynamic changes in transcriptomes and chromatin landscapes, we characterize transcriptional programs driving specific transitions between disease stages. We identify cell-autonomous dysregulation of inflammatory signaling as an early and persistent event in leukemogenesis and a promising early therapeutic target.

Monosomy 7 and deletion of 7q, known as del(7q), are common clonal cytogenetic abnormalities associated with high-grade myelodysplastic syndrome (MDS) arising in inherited and acquired bone marrow failure. Current nontransplant approaches to treat marrow failure may be complicated by stimulation of clonal outgrowth. To study the biological consequences of del(7q) within the context of a failing marrow, we generated induced pluripotent stem cells (iPSCs) derived from patients with Shwachman-Diamond syndrome (SDS), a bone marrow failure disorder with MDS predisposition, and genomically engineered a 7q deletion. The TGF-β pathway was the top differentially regulated pathway in transcriptomic analysis of SDS versus SDSdel(7q) iPSCs. SMAD2 phosphorylation was increased in SDS relative to wild-type cells, consistent with hyperactivation of the TGF-β pathway in SDS. Phospho-SMAD2 levels were reduced following 7q deletion in SDS cells and increased upon restoration of 7q diploidy. Inhibition of the TGF-β pathway rescued hematopoiesis in SDS iPSCs and in bone marrow hematopoietic cells from SDS patients while it had no impact on the SDSdel(7q) cells. These results identified a potential targetable vulnerability to improve hematopoiesis in an MDS predisposition syndrome and highlighted the importance of the germline context of somatic alterations to inform precision medicine approaches to therapy.

Angiogenesis is a fundamental developmental process and a hallmark of cancer progression. Receptor tyrosine kinases (RTK) are targets for cancer therapy which may include their action as anti-angiogenic agents. Derazantinib (DZB) is an inhibitor of the fibroblast growth factor receptors (FGFRs) 1&ndash, 3 as well as other kinase targets including vascular endothelial growth factor receptor 2 (VEGFR2), colony stimulating factor-1 receptor (CSF1R) and platelet-derived growth factor beta receptor (PDGFRbeta). This study aimed to investigate the effect of DZB on blood vessel morphogenesis and to compare its activity to known specific FGFR and VEGFR inhibitors. For this purpose, we used the developing vasculature in the zebrafish embryo as a model system for angiogenesis in vivo. We show that DZB interferes with multiple angiogenic processes that are linked to FGF and VEGF signalling, revealing a potential dual role for DZB as a potent anti-angiogenic treatment.

The myelodysplastic syndromes (MDS) are common myeloid malignancies. Mutations in genes involved in pre-mRNA splicing (SF3B1, SRSF2, U2AF1 and ZRSR2) are the most common mutations found in MDS. There is evidence that some spliceosomal components play a role in the maintenance of genomic stability. Splicing is a transcription coupled process; splicing factor mutations affect transcription and may lead to the accumulation of R-loops (RNA-DNA hybrids with a displaced single stranded DNA). Mutations in the splicing factors SRSF2 and U2AF1 have been recently shown to increase R-loops formation in leukemia cell lines, resulting in increased DNA damage, replication stress and activation of the ATR-Chk1 pathway. SF3B1 is the most frequently mutated splicing factor gene in MDS, but a role for mutated SF3B1 in R-loop accumulation and DNA damage has not yet been reported in hematopoietic cells. We have investigated the effects of the common SF3B1 K700E mutation on R-loop formation and DNA damage response in MDS and leukemia cells. R-loop signals and the DNA damage response were measured by immunofluorescence staining using S9.6 and anti-γ-H2AX antibodies respectively. Firstly, we studied K562 (myeloid leukemia) cells with the SF3B1 K700E mutation and isogenic SF3B1 K700K wildtype (WT) K562 cells. K562 cells with SF3B1 mutation showed a significant increase in the number of S9.6 foci [Fold change (FC) 2.01, p To investigate whether the observed DNA damage and ATR activation in SF3B1 mutant K562 cells result from induced R-loops, we overexpressed RNASEH1 (encoding an enzyme that degrades the RNA in RNA:DNA hybrids) to resolve R-loops in these cells. RNASEH1 overexpression significantly reduced the number of S9.6 (FC 0.51, p In summary, our results show that the SF3B1 mutation leads to accumulation of R-loops and associated DNA damage resulting in activation of the ATR pathway in MDS and leukemia cells. Thus different mutated splicing factors have convergent effects on R-loop elevation leading to DNA damage. Moreover, our data suggest that Chk1 inhibition, alone or in combination with splicing modulators, may represent a novel therapeutic strategy to target SF3B1 mutant cells. Disclosures Schuh: Janssen: Speakers Bureau; Verastem: Speakers Bureau; Kite: Speakers Bureau; Gilead: Speakers Bureau; Seattle Genetics: Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau; Bristol-Myers Squibb: Research Funding; AbbVie: Consultancy, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau. Wiseman:Novartis, Celgene: Consultancy, Honoraria.

Selenocysteine (Sec) is inserted into proteins by recoding a UGA stop codon followed by a selenocysteine insertion sequence (SECIS). UGA recoding by the Sec machinery is believed to be very inefficient owing to RF2-mediated termination at UGA. Here we show that recoding efficiency in vivo is 30-40% independently of the cell growth rate. Efficient recoding requires sufficient selenium concentrations in the medium. RF2 is an unexpectedly poor competitor of Sec. We recapitulate the major characteristics of SECIS-dependent UGA recoding in vitro using a fragment of fdhF-mRNA encoding a natural bacterial selenoprotein. Only 40% of actively translating ribosomes that reach the UGA codon insert Sec, even in the absence of RF2, suggesting that the capacity to insert Sec into proteins is inherently limited. RF2 does not compete with the Sec incorporation machinery; rather, it terminates translation on those ribosomes that failed to incorporate Sec. The data suggest a model in which early recruitment of Sec-tRNA(Sec)-SelB-GTP to the SECIS blocks the access of RF2 to the stop codon, thereby prioritizing recoding over termination at Sec-dedicated stop codons.

Acid ecto-phosphatase activity has been implicated in Leishmania donovani promastigote virulence. In the present study, we report data contributing to the molecular/structural and functional characterization of the L. donovani LdMAcP (L. donovani membrane acid phosphatase), member of the histidine acid phosphatase (HAcP) family. LdMAcP is membrane-anchored and shares high sequence identity with the major secreted L. donovani acid phosphatases (LdSAcPs). Sequence comparison of the LdMAcP orthologues in Leishmania sp. revealed strain polymorphism and species specificity for the L. donovani complex, responsible for visceral leishmaniasis (Khala azar), proposing thus a potential value of LdMAcP as an epidemiological or diagnostic tool. The extracellular orientation of the LdMAcP catalytic domain was confirmed in L. donovani promastigotes, wild-type (wt) and transgenic overexpressing a recombinant LdMAcP–mRFP1 (monomeric RFP1) chimera, as well as in transiently transfected mammalian cells expressing rLdMAcP–His. For the first time it is demonstrated in the present study that LdMAcP confers tartrate resistant acid ecto-phosphatase activity in live L. donovani promastigotes. The latter confirmed the long sought molecular identity of at least one enzyme contributing to this activity. Interestingly, the L. donovani rLdMAcP–mRFP1 promastigotes generated in this study, showed significantly higher infectivity and virulence indexes than control parasites in the infection of J774 mouse macrophages highlighting thereby a role for LdMAcP in the parasite's virulence., Acid ecto-phosphatase activity has been linked to Leishmania donovani virulence. In the present study, we confirm the molecular identity and characterize molecular and functional properties of an enzyme contributing to this activity, the LdMAcP, an L. donovani specific membrane histidine acid phosphatase (HAcP).

Chromosomal deletions associated with human diseases, such as cancer, are common, but synteny issues complicate modeling of these deletions in mice. We use cellular reprogramming and genome engineering to functionally dissect the loss of chromosome 7q (del(7q)), a somatic cytogenetic abnormality present in myelodysplastic syndromes (MDS). We derive del(7q)- and isogenic karyotypically normal induced pluripotent stem cells (iPSCs) from hematopoietic cells of MDS patients and show that the del(7q) iPSCs recapitulate disease-associated phenotypes, including impaired hematopoietic differentiation. These disease phenotypes are rescued by spontaneous dosage correction and can be reproduced in karyotypically normal cells by engineering hemizygosity of defined chr7q segments in a 20-Mb region. We use a phenotype-rescue screen to identify candidate haploinsufficient genes that might mediate the del(7q)- hematopoietic defect. Our approach highlights the utility of human iPSCs both for functional mapping of disease-associated large-scale chromosomal deletions and for discovery of haploinsufficient genes.

Connexins are the primary components of gap junctions, providing direct links between cells in many physiological processes, including cell migration and cancer metastasis. Exactly how cell migration is controlled by gap junctions remains a mystery. To shed light on this, we investigated the role of Connexin43 in collective cell migration during embryo development using the neural crest, an embryonic cell population whose migratory behavior has been likened to cancer invasion. We discovered that Connexin43 is required for contact inhibition of locomotion by directly regulating the transcription of N-cadherin. For this function, the Connexin43 carboxy tail interacts with Basic Transcription Factor 3, which mediates its translocation to the nucleus. Together, they bind to the n-cad promotor regulating n-cad transcription. Thus, we uncover an unexpected, gap junction-independent role for Connexin43 in collective migration that illustrates the possibility that connexins, in general, may be important for a wide variety of cellular processes that we are only beginning to understand.HighlightsCx43 regulates collective directional migration of neural crest cellsCx43 carboxy tail controls cell polarity via n-cad regulationCx43 carboxy tail localises at the nucleus and that depends on BTF3BTF3 and Cx43 carboxy tail directly interact to bind and regulate n-cad promoter activity

Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo., Cell migration is essential for development, but its deregulation causes metastasis. The Scar/WAVE complex is absolutely required for lamellipodia and is a key effector in cell migration, but its regulation in vivo is enigmatic. Lamellipodin (Lpd) controls lamellipodium formation through an unknown mechanism. Here, we report that Lpd directly binds active Rac, which regulates a direct interaction between Lpd and the Scar/WAVE complex via Abi. Consequently, Lpd controls lamellipodium size, cell migration speed, and persistence via Scar/WAVE in vitro. Moreover, Lpd knockout mice display defective pigmentation because fewer migrating neural crest-derived melanoblasts reach their target during development. Consistently, Lpd regulates mesenchymal neural crest cell migration cell autonomously in Xenopus laevis via the Scar/WAVE complex. Further, Lpd’s Drosophila melanogaster orthologue Pico binds Scar, and both regulate collective epithelial border cell migration. Pico also controls directed cell protrusions of border cell clusters in a Scar-dependent manner. Taken together, Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo.

A major recent discovery from large-scale sequencing studies was that over half of Myelodysplastic Syndromes (MDS) patients harbor mutations in splicing factor (SF) genes. SF mutations are the most common class of mutations in MDS and occur early in the course of the disease. These strongly suggest that SF mutations are key to the pathogenesis of MDS and can provide new therapeutic opportunities. However, identifying the downstream effects of SF mutations that are critical for the development of MDS presents a big challenge due to the cellular and genetic heterogeneity of primary patient samples, the unavailability of immortalized cell lines harboring SF mutations in the native genomic context and the limited conservation of alternative splicing isoforms between mice and humans. We previously showed that SF-mutant induced pluripotent stem cells (iPSCs) generated from MDS patients recapitulate key features of the disease upon differentiation into hematopoietic lineages, including cellular phenotypes (increased cell death, decreased clonogenicity and dysplastic morphology), sensitivity to splicing modulating drugs and the altered RNA binding specificity of mutant SFs (Chang et al. Stem Cell Reports, 2018). To further investigate the effects of SF mutations, we used CRISPR to introduce each of the 3 main canonical SF mutations (SRSF2 P95L, SF3B1 K700E, U2AF1 S34F) in the same normal iPSC line N-2.12 that we previously derived and extensively characterized in terms of pluripotency, genetic integrity and hematopoietic differentiation potential. The derivative iPSC lines contain the 3 SF mutations in isogenic conditions in the context of a diploid genome, in a heterozygous state, with both the normal and mutant alleles expressed at physiological and equal levels. To uncover potential new therapeutic targets and gain insights into the downstream effects of SF mutations, we set up CRISPR knockout (KO) lethality screens in hematopoietic progenitor cells (HPCs) derived from these SF-mutant iPSCs. We began with a gRNA library containing 224 gRNAs targeting 57 kinase genes (4 gRNAs per gene). The library was assembled and packaged in a lentiviral backbone also expressing GFP. Cas9 together with mCherry was expressed from a separate lentiviral vector. iPSCs were differentiated along the hematopoietic lineage, transduced on day 11, coinciding with the onset of the emergence of CD34+/CD45+ HPCs, and further cultured for up to day 27 to allow "dropout" of lethal genes, read out by next-generation sequencing (NGS). We titrated the lentiviral vectors to obtain transduction efficiency of nearly 100% for the Cas9 vector and up to 40-50% for the gRNA library (in order to obtain the highest percentage of cells harboring a single gRNA) in 500,000 HPCs, to ensure representation of the library of at least 500 cells per gRNA. To avoid population bottlenecks, we ensured that at least 500,000 cells were present in the culture at all times. All library gRNAs were present in the transduced cells and their distribution correlated tightly with that of the lentiviral supernatant. Technical repeats of independently prepared DNA samples, independent PCR reactions and independently generated NGS reads showed high reproducibility and absence of batch effects. The library was screened in 3 independent clones harboring each of the 3 canonical SF mutations, as well as in 3 clones of the parental normal line. This design allows the identification of potential convergent genes or pathways downstream of the 3 SF mutations and exclusion of non-synthetically lethal targets (which would also drop out in the isogenic normal cells). CRISPR scores were calculated as the average of the log of the final vs initial abundance of all gRNAs per gene, and showed a distribution consistent with the expectation that the majority of the gRNAs do not have a major impact on cell viability. Experiments with evidence of random genetic drifts from the CRISPR scores distribution were excluded from the analyses. Initial hits, defined as kinase genes with targeting gRNAs consistently depleted in SF-mutant lines of all 3 genotypes, but not in the normal isogenic cells are being validated with individual gene knockout and small molecule inhibition. In parallel, we are setting up CRISPR screens in expandable HPCs (eHPCs) derived from iPSCs. The latter can be expanded in culture for several weeks and could enable screening of larger or even genome-wide gRNA libraries. Disclosures No relevant conflicts of interest to declare.

Myeloproliferative neoplasms (MPN) are characterized by the excessive production of one or more myeloid lineages and a propensity to progress to acute leukemia. In 2013, mutations in the CALR gene, encoding calreticulin, were identified in patients with MPN, mutually exclusive to the previously identified JAK2 and MPL (TPO-R) mutations. CALR mutations are frameshift mutations - typically a 52-bp deletion (type 1) or a 5-bp insertion (type 2) - that result in a novel C-terminus. The discovery of mutations in a ubiquitously expressed multifunctional protein like calreticulin was unanticipated. Subsequent studies found that CALR mutations lead to activation of JAK/STAT, mediated through aberrant interactions between mutant CALR and MPL, thus presenting an excellent opportunity for targeted therapy. However, the mechanism of MPL activation remains largely unexplained with prior studies using cell lines with exogenous expression of CALR and MPL following transfection. To create a more physiological cellular model to study the effects of CALR mutations, we established multiple iPSC lines from two patients with CALR-mutant MPN - one type 1-like (del34) and one type 2 (ins5) -, as well as from one patient with JAK2V617F MPN. All iPSC lines were confirmed to harbour the CALR or JAK2V617F mutation found in the corresponding patient, to express mutant calreticulin, as detected by flow cytometry using an antibody which specifically recognizes the novel calreticulin C-terminus, and to be karyotypically normal. Genetically matched iPSC lines with WT JAK2 could also be generated from the JAK2V617F (but not the CALR-mutant) patient cells in the same reprogramming round. CRISPR gene editing was used to generate isogenic CALR-corrected lines from both CALR-mutant patients. Furthermore, in order to facilitate biochemical studies, we used CRISPR to introduce a V5 epitope tag in one allele of the endogenous mutant or WT CALR gene, in mutant and isogenic corrected iPSC lines, respectively. We optimized an in vitro differentiation protocol for efficient derivation of megakaryocyte (MK) progenitors from iPSCs and found disease-relevant phenotypes, mainly TPO-independent MK colony formation in semi-solid media, which is the phenotypic hallmark of ex vivo primary MPN cells. In the absence of TPO, JAK2 V617F, CALR-mutant type 1-like and CALR-mutant type 2 iPSCs generated 52.1%, 58.7±22.2% and 59.8±3.6%, respectively, of the number of MK colonies generated in the presence of TPO, as opposed to 10%, 8.8±1.8% and 0.5±0.9%, respectively, for the matched WT JAK2, the corrected CALR-mutant type 1-like and the corrected CALR-mutant type 2 iPSCs. Isolated CALR mutant iPSC-derived CD41a+ MK progenitors had increased phosphorylation of STAT5 following cytokine starvation as compared to isogenic corrected and non-isogenic normal cells. CALR-mutant cells expressed equal transcript levels of the WT and mutant CALR alleles. However, mutant CALR protein levels were severely reduced, at levels 1~12% of those of the WT protein. This is consistent with previous studies documenting instability of mutant calreticulin. Transcriptomics (RNA-seq) and proteomics analyses of CD41a+-sorted MK progenitors derived from CALR mutant and isogenic corrected iPSCs are ongoing. These iPSC models offer the opportunity to study the effects of CALR mutations in a cellular context with both MPL and CALR (WT or mutant) expressed from their endogenous loci. They thus provide a powerful platform to investigate the disease mechanisms underlying CALR-mutant MPNs and to perform small molecule and genetic (CRISPR) screens to identify new therapeutic targets. Disclosures Iancu-Rubin: Merck: Research Funding; Incyte: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding. Hoffman:Incyte: Research Funding; Merus: Research Funding; Formation Biologics: Research Funding; Janssen: Research Funding; Summer Road: Research Funding.

Acute Myeloid Leukemia (AML) develops through the stepwise acquisition of mutations by hematopoietic stem or progenitor cells in a process of clonal evolution, which drives disease initiation, progression and relapse. In recent years, large scale sequencing of patients with AML yielded nearly complete catalogues of the driver mutations, uncovered their temporal occurrences and classified AML subgroups based on mutational profiles. The next daunting challenge is to understand how these mutations contribute to the development of Myelodysplastic Syndrome (MDS) and AML and how the order of their acquisition impacts disease progression. To study the individual and cooperative effects of driver mutations in MDS and AML and model clonal evolution, we developed iPSC models by introducing mutations in a stepwise manner through serial CRISPR/Cas9-mediated gene editing. We selected two mutational paths, representing de novo AML and secondary AML from preexisting MDS, respectively: (a) the combination DNMT3AR882H, NPM1c and FLT3ITD, the most common 3-gene co-mutation in AML, associated with poor prognosis; and (b) the combination SRSF2P95L, ASXL1 C-terminus truncation and NRASG12D, which represents the "chromatin-spliceosome" AML genomic classification group. The normal iPSC line N-2.12, previously extensively characterized in terms of pluripotency, genetic integrity and hematopoietic differentiation potential, was used as the parental line. Each gene editing step entailed screening of single cell clones by RFLP analysis, confirmation of correct targeting and presence of an intact untargeted allele by DNA sequencing, a second round of single-cell cloning to ensure clonality and karyotyping to exclude chromosomal abnormalities. At least two clones from each step were subjected to hematopoietic differentiation to test for potential phenotypic discrepancies and one representative clone was selected for the next gene editing step. For the DNMT3AR882H, FLT3ITD, SRSF2P95L and NRASG12D mutations, CRISPR-mediated homology directed repair (HDR) was used. ASXL1mutant lines were generated by CRISPR/Cas9 targeting of the beginning of exon 12 of ASXL1 and selection of clones with frame-shifting indels. NPM1c was introduced with a dox-inducible lentiviral vector. We thus derived a panel of single, double and triple mutant clones representing each step of the clonal evolution of AML.Single mutant clones (DNMT3AR882H and ASXL1-truncated) had completely normal hematopoietic differentiation potential (similar to that of the parental normal line and other non-isogenic normal lines), which is not surprising given that both these mutations are also found in individuals with clonal hematopoiesis without any hematopoietic defects. The double mutant SRSF2P95L-ASXL1 cells showed moderately impaired early hematopoietic differentiation potential with colony-forming ability reduced to approximately 50% of normal. In contrast, both triple mutant lines (DNMT3AR882H-FLT3ITD-NPM1c and SRSF2P95L-ASXL1-NRASG12D) showed a more severe differentiation block with markedly reduced colony-forming ability and prolonged growth in culture for over 11 weeks, in striking contrast to normal iPSC-derived hematopoietic progenitor cells (HPCs) which completely arrest their proliferation after 4 weeks. Transplantation of SRSF2P95L-ASXL1-NRASG12DHPCs into NSG mice resulted in detectable engraftment after 9 weeks in all transplanted animals. In conclusion, we successfully employed CRISPR/Cas to introduce driver mutations into iPSCs in a stepwise manner to "de novo" reconstruct the development of AML. These panels of iPSCs capture the distinct mutational steps along the evolution of AML in a clonal state and isogenic conditions. They should enable mechanistic studies into the processes of leukemogenesis and the effects of specific mutations acting at distinct stages of this progression, their cooperation and the order by which they are acquired. They should also prove a powerful tool to investigate the minimal genetic requirements for leukemia development, the potential need for cooperating epigenetic insults and the effects of the cellular context in myeloid transformation. Disclosures No relevant conflicts of interest to declare.

Connexins are the primary components of gap junctions, providing direct links between cells under many physiological processes. Here, we demonstrate that in addition to this canonical role, Connexins act as transcriptional regulators. We show that Connexin 43 (Cx43) controls neural crest cell migration in vivo by directly regulating N-cadherin transcription. This activity requires interaction between Cx43 carboxy tail and the basic transcription factor-3, which drives the translocation of Cx43 tail to the nucleus. Once in the nucleus they form a complex with PolII which directly binds to the N-cadherin promoter. We found that this mechanism is conserved between amphibian and mammalian cells. Given the strong evolutionary conservation of connexins across vertebrates, this may reflect a common mechanism of gene regulation by a protein whose function was previously ascribed only to gap junctional communication.

Transgenesis of human pluripotent stem cells (hPSCs) can enable and empower a variety of studies in stem cell research, including lineage tracing and functional genetics studies. While in recent years much progress has been made in the development of tools for gene targeting, little attention has been given to the identification of sites in the human genome where transgenes can be inserted and reliably expressed. In order to find human genomic sites capable of supporting long-term and high-level transgene expression in hPSCs, we performed a lentiviral screen in human induced pluripotent stem cells (iPSCs). We isolated 40 iPSC clones each harboring a single vector copy and characterized the level of transgene expression afforded by each unique integration site. We selected one clone, LiPS-A3 with an integration site in chromosome 15 maintaining robust expression without silencing and demonstrate that different transgenes can be inserted therein rapidly and efficiently through recombinase-mediated cassette exchange (RMCE). The LiPS-A3 line can greatly facilitate the insertion of reporter and other genes in hPSCs. Targeting transgenes in the LiPS-A3S genomic locus can find broad applications in stem cell research and possibly cell and gene therapy.

Loss of the RNA-binding fragile X protein [fragile X mental retardation protein (FMRP)] results in a spectrum of cognitive deficits, the fragile X syndrome (FXS), while aging individuals with decreased protein levels present with a subset of these symptoms and tremor. The broad range of behavioral deficits likely reflects the ubiquitous distribution and multiple functions of the protein. FMRP loss is expected to affect multiple neuronal proteins and intracellular signaling pathways, whose identity and interactions are essential in understanding and ameliorating FXS symptoms. We used heterozygous mutants and targeted RNA interference-mediated abrogation inDrosophilato uncover molecular pathways affected by FMRP reduction. We present evidence that FMRP loss results in excess metabotropic glutamate receptor (mGluR) activity, attributable at least in part to elevation of the protein in affected neurons. Using high-resolution behavioral, genetic, and biochemical analyses, we present evidence that excess mGluR upon FMRP attenuation is linked to the cAMP decrement reported in patients and models, and underlies olfactory associative learning and memory deficits. Furthermore, our data indicate positive transcriptional regulation of the flyfmr1gene by cAMP, via protein kinase A, likely through the transcription factor CREB. Because the human Fmr1 gene also contains CREB binding sites, the interaction of mGluR excess and cAMP signaling defects we present suggests novel combinatorial pharmaceutical approaches to symptom amelioration upon FMRP attenuation.

Monosomy 7 or deletion of 7q (del(7q)) are common cytogenetic abnormalities in pediatric MDS. Monosomy 7/del(7q) frequently arises in the context of inherited bone marrow failure (BMF) syndromes such as Shwachman Diamond Syndrome (SDS), an autosomal recessive disorder caused by biallelic mutations in the SBDS gene. Monosomy 7/del(7q) is associated with high grade MDS and a high risk of malignant transformation to acute myelogenous leukemia, a major cause of morbidity and mortality for patients with inherited BMF. The basis for this propensity to develop monosomy 7/del(7q) remains unclear. Whether monosomy 7/del(7q) functions as a driver of MDS, or is merely an associated marker of clonal progression in BMF, remains a critical question. The aim of this study is to investigate the molecular consequences of del(7q) in the context of BMF with the goal of developing more effective treatments. The lack of synteny between murine and human chromosome 7 has posed a major barrier to modeling monosomy 7/del(7q). Therefore, we utilized SDS patient-derived induced pluripotent stem cells (SDS-iPSC), which recapitulate the hematopoietic defects of this BMF syndrome. Independent SDS-iPSC lines were derived from two patients with SDS. Proliferationof SDS-iPSCs was reduced relative to wild type controls without a concomitant increase in cell death. Compared to normal iPSC controls, SDS iPSC-derived CD34+ cells exhibited reduced hematopoietic differentiation to CD45+ cells, decreased erythroid and myeloid hematopoietic progenitor colony formation in methylcellulose, and impaired terminal myeloid differentiation. To study the molecular and biologic effects of del(7q), a deletion of the MDS-associated region of the long arm of chromosome 7 was genomically engineered using a previously published modified Cre-Lox approach. The deletion of 7q in SDS iPSCs spanned band q11.23 to q36.3. The SDS-del(7q) iPSCs expressed stem cell markers and formed teratomas as efficiently as their isogenic SDS iPSC controls.Deletion of 7q failed to confer a relative growth advantage even within the context of BMF. Indeed, proliferation of the SDS-del(7q) iPSCs was reduced below that of both the isogenic SDS iPSCs and normal controls without an increase in cell death. To investigate the effect of del(7q) on hematopoiesis, iPSC-derived CD34+ cells were assayed for their ability to undergo hematopoietic differentiation. SDS-del(7q) CD34+ cells demonstrated reduced differentiation to CD45+ cells, reduced methylcellulose hematopoietic progenitor colony formation and impaired terminal myeloid differentiation compared with isogenic SDS CD34+ cells. These data demonstrate that deletion of 7q fails to confer a relative growth advantage relative to isogenic SDS iPSCs and results in a further impairment of hematopoiesis. To gain insight into the mechanisms of del7q-associated clonal evolution in SDS, we performed RNA sequencing (RNAseq) of SDS and SDS-del(7q) iPSC. Expression of TGFβ pathways and their downstream targets were reduced in SDS del(7q) iPSCs compared to isogenic SDS iPSC controls. This observation was intriguing because single cell RNAseq analysis of primary SDS bone marrow cells revealed that TGFβ pathway is hyperactivated in SDS. Additionally, a quantitative DNA aptamer-based proteomic analysis demonstrated elevated levels of TGFβ ligands in plasma from six SDS patients compared to 6 age-matched controls. Western blot analysis showed increased P-SMAD2 levels in SDS iPSCs compared to SDS-del(7q) and normal controls, while total levels of SMAD2 were unchanged. Pharmacological targeting of TFGβ with small molecule inhibitors resulted in selective improvement of SDS hematopoietic colony formation without stimulating outgrowth of the isogenic SDS-del(7q) cells. Although del(7q) does not seem to confer an advantage in growth or hematopoiesis in vitro, we are testing whether the deletion of 7q might confer relative resistance to elevated TGFβ levels in vivo, resulting in an overall survival advantage of the del7q clone.These results demonstrate that the TGFβ pathway is activated in SDS, and that deletion of 7q reverses the TGFβ pathway hyperactivation in SDS. Inhibition of TGFβ selectively rescues hematopoiesis in SDS but not in isogenic del7q cells, suggesting a potential strategy to treat bone marrow failure without stimulating del7q clonal outgrowth. Disclosures Stegmaier: Novartis: Consultancy, Research Funding. Shimamura: TransCellular Therapeutics: Other: Spouse is majority shareholder.

Aim: To determine the immunoreactivity of cholecystokinin (CCK) during the development of the human fetal pancreas and pancreatic adenocarcinoma, given that, CCK positive cells were demonstrated either in its embryonic anlage or in pancreatic cancer. In order to obtain possible parallels in the expression pattern of neoplastic cells in adults (well – moderately – poorly differentiated), we investigated the pattern of CCK expression in the pancreatic tissue during the various stages of development and compared these with the proliferation of tissue assessed by proliferating cell nuclear antigen (PCNA) immunohistochemistry. Experimental design: Tissue sections from 15 pancreatic fetal specimens, and equal number of ductal adenocarcinoma specimens, were assessed using immunohistochemical methods for CCK. Results: The density of positive cells in the primitive exocrine ductal walls and outgrowing buds was significantly higher than the relevant density in the neoplastic pancreatic tissue of mixed (ductal – endocrine) and pure ductal type (p1=0.004, p2

The antigens encoded by the major histocompability complex (HLA-DR) are cell glycoproteins that play a fundamental role in the regulation of the immune response. The prognosis of ovarian cancer is dependent on the histological type and on the clinical stage at diagnosis. Our study reports the value of HLA-DR antigen as a prognostic marker of ovarian serous adenocarcinoma. We studied 31 cases of serous ovarian cystadenoma, 12 cases of serous ovarian borderline cystadenoma, and 39 cases of well-differentiated cystadenocarcinoma for HLA-DR monoclonal antigen. We also studied the T helper marker (CD4) in the tumor stroma of the relevant cases, given that it is now known that the dependence of immune responsiveness on the class II antigens reflects the central role of these molecules in presenting antigen to T helper cells. HLA-DR was expressed in 20 of 31 cystadenomas (64.5%), 4 of 12 borderline tumors (33.3%), and in 10 of 39 invasive carcinomas (25.6%). CD4 was expressed in 9 of 31 cystadenomas (29%), 5 of 12 borderline tumors (42%), and in 26 of 39 invasive carcinomas (67%). There was a statistically significant difference for the two examined antigens in cystadenomas ( p

To evaluate the biomagnetic activity of a low-grade mucosa-associated lymphoid tissue (MALT-) type gastric lymphoma, and to determine if this procedure could be an adjunct to endoscopic ultrasonography (EUS) in the assessment of the disease pre- and postsurgically.A 47-year-old female with a 2-year clinical history of a low-grade MALT-type gastric lymphoma associated with Helicobacter pylori was examined. The disease showed no histologic remission after eradication of the H. pylori infection and subsequent treatment with chemotherapy and the patient was classified as stage II EA. A decision for surgical resection was made. Biomagnetic waveform recordings were made in the target area before and after surgery and the Fourier analysis of these recordings was performed.The gastric lymphoma biomagnetic waveforms showed high amplitudes (1.8 pT) before and low amplitudes (0.6 pT) after resection. The corresponding Fourier analysis demonstrated that the maximum spectral power of the presurgical measurement was elevated (mean 235 +/- 847 pT2/Hz) compared with that obtained postsurgically (mean 725 +/- 89 pT2/Hz). The difference was of statistical significance ( p0.0001, t-test).Biomagnetic monitoring of gastric lymphoma, which is an entirely new diagnostic modality, could be a supplement to EUS for assessing remission or persistence of disease with medical treatment during follow up.

Aim: The maturation of most T- lymphocyte precursors takes place within the meshwork of thymic epithelial cells. Different steps of this process can be defined by immunologic phenotyping. The prothymocytes are positive for the terminal deoxynucleotidyl transferase (TdT) and give rise to cortical thymocytes, which express CD1, CD2, CD3, CD5, and both CD4 and CD8. These CD4 and CD8 double-positive cortical thymocytes differentiate into two lineages: CD4+ or CD8+ lymphocytes of the thymic medulla, by the tenth week of gestation. Our study points towards the determination of the CD8 cytotoxic/suppressor capacity of the fetal thymus in Down’s syndrome. Experimental design: A quantitative comparison of T-lymphocytes (CD3, CD4, and CD8) in the thymic parenchyme in embryos after voluntary abortion during 2nd trimester of gestation and embryos with Down’s syndrome, respectively, was performed. Results: Our results showed: 1) A statistically significant depletion in the total number of T-cells (CD3 positive) in the cases of embryos with Down’s syndrome over those after voluntary abortion, during the second trimester of gestation (p

The presence of myoid cells (MCs) – stromal cells with myogenic differentiation – in human bone marrow (BM) has been observed during hematopoiesis in embryonic life, whereas during adult life, it is strictly related to various pathologic conditions. The aim of this study was to examine the presence, distribution and quantitation of cells in the stroma of the BM expressing α-smooth muscle actin (α-SMA) in reactive BM plasmacytosis and plasma cell myeloma. For this reason, a series of 10 trephine BM biopsies from patients with plasma cell myeloma and 10 specimens from healthy controls with reactive plasmacytosis were examined for the presence of stromal cells with myoid differentiation, using a monoclonal antibody recognizing α-SMA. Our results suggest that the incidence of MCs and subsequent fibrosis rises in plasma cell myeloma and in a lesser degree in the BM stroma of individuals with reactive plasmacytosis.

Myelodysplastic syndrome (MDS) caused by monosomy 7 or del(7q) is a frequent clonal abnormality that arises in the context of inherited bone marrow failure syndromes, such as Shwachman Diamond Syndrome (SDS). Monosomy 7/del(7q) also develops in a subset of patients with acquired aplastic anemia or de novo MDS in the general population. Monosomy 7/del(7q) is associated with high grade MDS and a high risk of malignant transformation, most frequently to acute myelogenous leukemia (AML). Bone marrow failure and clonal evolution to MDS and AML remain major causes of morbidity and mortality for individuals with SDS. Currently, the only curative therapy for these hematological complications is a hematopoietic stem cell transplant. Prognosis is extremely poor once SDS patients develop leukemia. The basis for this propensity to develop monosomy 7 clones remains unclear. The longterm aim of this study is to understand the molecular mechanisms underlying leukemia predisposition and develop more effective treatments. Whether monosomy 7/del(7q) functions as a driver of MDS, or is merely an associated marker of clonal progression in bone marrow failure remains a critical question. The lack of synteny between murine versus human chromosome 7 has posed a major barrier to the development of mouse models of monosomy 7/del(7q). To study the biological and molecular consequences of monosomy 7/del(7q) in SDS, induced pluripotent stem cells (iPSCs) were generated from bone marrow mononuclear cells of two patients with SDS. Each patient harbored homozygous c.258+2 T>C mutations in the canonical splice donor site of intron 2 in the SBDS gene. The SDS-iPSCs retained the pathogenic homozygous IVS2+2 T>C SBDS mutations, expressed stem cell markers, formed teratomas, and expressed reduced levels of SBDS protein similar to levels noted in the primary patient samples. Proliferation of 4 distinct SDS-iPSC clones derived from two different patients was reduced relative to wild type controls without an increase in cell death. SDS-iPSC formed smaller embryoid bodies with reduced production of CD34+ hematopoietic stem/progenitor cells. Hematopoietic differentiation from CD34+ to CD45+ cells was also impaired. Preliminary data suggest that SDS-iPSCs retain the capacity to give rise to hematopoietic stem/progenitor cells and early myeloid progenitor cells in vitro. These populations were also observed in primary SDS patient-derived bone marrow samples. Because the number of CD34+ cells derived from SDS-iPSCs are limiting, a previously reported 5 transcrition factor re-specification system was used to expand multilineage hematopietic progenitors for further characterization. SDS iPSCs were able to differentiate into an expandable CD34+ population in vitro. Further studies to characterize the hematopoietic impairment in SDS iPSC and primary marrow samples are ongoing. To model del(7q) in SDS iPSCs, a deletion of the MDS-associated long arm of chromosome 7 was genomically engineered using a previously published modified Cre-Lox approach. The deletion of 7q at locus (11.2) was confirmed by karyotyping and by qPCR across chromosome 7. The SDS (del7q) iPSCs retained the SBDS pathogenic mutations, expressed stem cell markers, and formed teratomas. Proliferation of the SDS del(7q) iPSC was markedly impaired compared to isogenic SDS iPSCs. No increase in cell death was observed in the SDS del7q iPSCs. Studies are in progress to determine the effects of del7q on hematopoiesis. Investigation is ongoing to determine the molecular consequences of deleting 7q. These isogenic SDS+/- del(7q) iPS models provide a platform to study the role of 7q loss in clonal evolution from bone marrow failure and to screen for novel therapeutic compounds or pathways to treat bone marrow failure and MDS. Disclosures No relevant conflicts of interest to declare.

A major recent advance for the biology of Myelodysplastic Syndrome (MDS) was the discovery of recurrent mutations in genes encoding splicing factors (SFs). SRSF2 is a SF that binds to RNA sequences called exonic splicing enhancers (ESEs) to promote inclusion of exons containing these motifs. Mutations of SRSF2 are found in 20-30% of MDS patients, are associated with adverse prognosis and are almost always heterozygous missense mutations (P95 L/R/H). This strongly suggests a gain or alteration of function mechanism, corroborated by recent findings by us and others in a knockin mouse model and in K562 cells expressing mutant SRSF2, showing that the SRSF2 P95H mutation changes the normal RNA binding specificity of SRSF2 (Kim et al. Cancer Cell, 2015; Zhang et al. PNAS, 2015). While mis-splicing of hematopoietic regulators, such as EZH2, was proposed as a potential downstream mechanism of SF mutations, systematically identifying the key downstream mediators remains a challenge. No human hematopoietic cell lines harboring SF mutations in the context of a normal diploid genome exist, primary MDS cells afford limited experimental opportunities and mouse modeling is likely to miss disease relevant targets, as only a quarter of regulated alternative splicing events are conserved between human and mouse. Our lab has pioneered the modeling of MDS with human induced pluripotent stem cells (iPSCs). We previously derived iPSCs with the SRSF2 P95L mutation and a chr7q deletion (del7q), as well as normal iPSCs from residual normal hematopoietic cells from the same MDS patient (Kotini et al. Nat. Biotech, 2015). To develop a model of mutant SRSF2, we used CRISPR/Cas9 technology to generate a panel of isogenic iPSCs with or without the SRSF2 P95 mutation, isolated or with the cooperating del(7q), all in the same genetic background, by both introducing the SRSF2 P95L mutation in normal iPSCs and correcting it in MDS-iPSCs from the same patient. Both patient-derived and genetically engineered SRSF2 P95L-iPSCs showed decreased growth and increased cell death of hematopoietic progenitor cells (HPCs), decreased clonogenic capacity and features of morphologic dysplasia. This phenotype is consistent with the SRSF2 mutation being an early, potentially initiating, event, supported by its frequent presence in the dominant clone and in individuals with clonal hematopoiesis of indeterminate potential (CHIP) without overt MDS. Using a competitive cell growth assay, we found that the splicing inhibitor E7107, as well as small molecule inhibitors of kinases modulating splicing, preferentially inhibit the growth of SRSF2 mutant, but not of isogenic normal, iPSC-derived HPCs. To investigate the effects of mutant SRSF2 in mRNA splicing, we performed RNA sequencing of purified CD34+, CD34+/CD45+ HPCs and undifferentiated iPSCs from mutant and isogenic wild-type (WT) iPSCs. SRSF2 mutant cells exhibited genome-wide alterations in ESE preferences, recapitulating the altered RNA binding found in patient cells, with mutant SRSF2 preferentially recognizing a CCNG motif versus a GGNG motif, while WT SRSF2 binds to both with similar affinity. Genes found mis-spliced in the SRSF2 mutant cells included previously reported genes of potential disease relevance, like EZH2 and FYN. While the majority of differentially spliced genes overlapped among the 3 cell states, cell type-specific differences were also noted, highlighting the importance of performing these analyses in the appropriate cell type. Importantly, iPSC-derived HPCs recapitulated a higher percentage of the mis-spliced events observed in patient cells than either the knockin mouse or the K562 models, thus capturing disease-relevant splicing alterations more faithfully than other models. To identify critical direct targets of mutant SRSF2, we used a second round of CRISPR/Cas9 gene editing to introduce a 3xFLAG epitope tag at the carboxyl terminus of the endogenous SRSF2 locus in isogenic SRSF2mutant and WT iPSCs. CLIP-seq experiments in undifferentiated cells and HPCs derived from them are underway to identify genes differentially bound by the mutant vs the WT SRSF2. The model we describe here will be valuable for dissecting the pathogenesis of MDS with SF mutations, testing drugs and identifying new therapeutic targets for drug development. Disclosures No relevant conflicts of interest to declare.

Magnetoencephalography (MEG) recordings were evaluated for 25 healthy female volunteers, in five different gustatory states: normal, sweet, bitter, sour and salty. The study population was divided in two groups according to age: group A (10-19 years old) and group B (20-30 years old). There was a higher count of low frequencies (2 Hz) and a lower count of high frequencies (7 Hz) with increasing age, in all studied states. We compared each state for the frequencies of 2 Hz and 7 Hz between the two groups. Statistically significant differences were found in the normal and sweet states for the frequencies of 2 Hz and 7 Hz and in the salty taste in the frequency of 7 Hz. We also intra-compared the five states in group A and the five states in group B for the 2 Hz and 7 Hz frequencies. The results were not statistically significant. A differentiation in the distribution of the frequencies with increasing age may provide new insights into the age-dependence of taste quality brain centers.

Human pluripotent stem cells (hPSCs) hold great promise for cell therapy. However, a major concern is the risk of tumor formation by residual undifferentiated cells contaminating the hPSC-derived cell product. Suicide genes could safeguard against such adverse events by enabling elimination of cells gone astray, but the efficacy of this approach has not yet been thoroughly tested. Here, we engineered a lentivirally encoded herpes simplex virus thymidine kinase (HSVtk) with expression restricted to undifferentiated hPSCs through regulation by the let7 family of miRNAs. We show that induced pluripotent stem cells (iPSCs) expressing a let7-regulated HSVtk transgene are selectively killed by ganciclovir (GCV), whereas differentiated cells are fully protected. However, in contrast to previous studies, we find that in vivo GCV administration results in longer latency but does not prevent teratoma formation by iPSCs expressing either a constitutive or a let7-regulated HSVtk, without evidence of silencing of the HSVtk. Clonal analyses of iPSCs expressing HSVtk revealed frequent emergence of GCV resistance which, at least in some cases, could be attributed to preexisting inactivating mutations in the HSVtk coding sequence, selected for upon GCV treatment. Our findings have important consequences for the future use of suicide genes in hPSC-based cell therapies.

The 5-methyl cytosine (5meC) genomic methylation patterns play crucial roles in mammalian development and are altered in cancer. The enzymes that create, maintain and modify the DNA methylation patterns are the DNA methyltransferases (Dnmts) which are all encoded by essential genes. The de novo Dnmts -Dnmt3a and Dnmt3b- establish the DNA methylation patterns early in mammalian development by introducing DNA methylation marks where no previous methylation exists. These enzymes do not exhibit affinity for specific DNA sequences, thus their recruitment to specific DNA loci and their activities must be tightly regulated. In particular, Dnmt3a2 -one of the two protein isoforms produced by the Dnmt3a locus- is the most abundant DNA methyltransferase in mouse Embryonic Stem Cells. To identify Dnmt3a (and DNA methylation) regulators we have searched for Dnmt3a2 interacting proteins in mESCs by pull down and Mass Spectrometry. The DEAD box p68/Ddx5 RNA helicase was identified to directly interact with Dnmt3a1 and Dnmt3a2 in vitro and in vivo. We have created a mutant Ddx5 (Ddx5 MUT ) protein exhibiting an altered nuclear localization pattern compared to the wt protein. Both wt and mutant Ddx5 interact directly in vitro and co localize in vivo with Dnmt3a proteins. Our data suggest that the Dnmt3a/Ddx5 interaction might be significant for modulating the DNA methylation/demethylation dynamics in vivo.

Dnmt3a1 and Dnmt3a2 are two de novo DNA methyltransferases expressed in mouse embryonic stem cells (mESCs). They differ in that a 219-amino-acid (aa) amino (N)-terminal noncatalytic domain is present only in Dnmt3a1. Here, we examined the unique functions of Dnmt3a1 in mESCs by targeting the coding sequence of the Dnmt3a1 N-terminal domain tagged with enhanced green fluorescent protein (GFP) for insertion into the mouse Rosa26 locus. Using these targeted cells (GFP-3a1Nter), we showed that Dnmt3a1 was efficiently recruited to the silenced Oct3/4 and activated Vtn (vitronectin) gene promoters via its unique N-terminal domain. This recruitment affected the two genes in contrasting ways, compromising Oct3/4 gene promoter DNA methylation to prevent consolidation of the silent state while significantly reducing Vtn transcription. We used this negative effect of the Dnmt3a1 N-terminal domain to investigate the extent of transcriptional regulation by Dnmt3a1 in mESCs by using microarrays. A small group of all-trans retinoic acid (tRA)-inducible genes had lower transcript levels in GFP-3a1Nter cells than in wild-type mESCs. Intriguingly, this group included genes that are important for fetal nutrition, placenta development, and metabolic functions and is enriched for a distinct set of imprinted genes. We also identified a larger group of genes that showed higher transcript levels in the GFP-3a1Nter-expressing cells than in wild-type mESCs, including pluripotency factors and key regulators of primordial germ cell differentiation. Thus, Dnmt3a1 in mESCs functions primarily as a negative and to a lesser extent as a positive regulator of transcription. Our findings suggest that Dnmt3a1 positively affects transcription of specific genes at the promoter level and targets chromosomal domains to epigenetically silence gene clusters in mESCs.

We investigated the localization of current sources for spontaneous magnetoencephalographic (MEG) data in the frequency domain. MEGs were evaluated in three different states: (i) physiological condition; (ii) sweet taste, and (iii) salt taste. Low frequencies can be seen in the maps obtained with the sweet taste, whereas in the physiological and salt taste, the maps show higher frequencies in the majority of channels. A differentiation in the spatial distribution of the frequencies provides novel insights into the identification of taste quality with the MEG systems.

International audience; To ensure a high fidelity during translation, threonyl-tRNA synthetases (ThrRSs) harbor an editing domain that removes noncognate L-serine attached to tRNAThr. Most archaeal ThrRSs possess a unique editing domain structurally similar to D-aminoacyl-tRNA deacylases (DTDs) found in eubacteria and eukaryotes that specifically removes D-amino acids attached to tRNA. Here, we provide mechanistic insights into the removal of noncognate L-serine from tRNAThr by a DTD-like editing module from Pyrococcus abyssi ThrRS (Pab-NTD). High-resolution crystal structures of Pab-NTD with pre- and post-transfer substrate analogs and with L-serine show mutually nonoverlapping binding sites for the seryl moiety. Although the pre-transfer editing is excluded, the analysis reveals the importance of main chain atoms in proper positioning of the post-transfer substrate for its hydrolysis. A single residue has been shown to play a pivotal role in the inversion of enantioselectivity both in Pab-NTD and DTD. The study identifies an enantioselectivity checkpoint that filters opposite chiral molecules and thus provides a fascinating example of how nature has subtly engineered this domain for the selection of chiral molecules during translation.

Aim: To determine the expression of CMV-associated antigen in the human decidual endometrial stromal cells in spontaneous abortions with no evidence of maternal relapse during the first trimester of gestation. Experimental design: We examined 15 placentas resulting from intrauterine fetal death after spontaneous abortion during the 8th, 10th, and 12th week of gestation respectively, and in which CMV reactivation was ruled out from serological evaluation of the pregnant women at admission, versus equal controls after voluntary abortion following well-documented maternal viral recurrence. In addition, a panel of monoclonal antibodies for the identification of leukocytes (CD45/LCA), B-lymphocytes (CD20/L-26), and T-lymphocytes (CD45RO/UCHL1), was performed. All women received hormonal medication to support gestation, in the cases of spontaneous abortions. Results: Immunohistochemical examination using a specific antibody against cytomegalovirus showed large multinucleated infected cells with intranuclear inclusions, located primarily in the decidual stroma within a lymphoplasmacytic infiltrate in the cases of spontaneous abortions. No evidence of infection was observed in the chorionic villi. In the cases of voluntary abortions same findings were observed in the relevant areas, and a strong evidence of infection was observed in the chorionic villi. Conclusion: This study demonstrates 1) that the decidual endometrial stromal cells can express the CMV-associated antigen prior to serological manifestation of the viral replication, 2) the expression of the antigen is higher in cases of hormonal administration to support gestation. In these cases a mild mononuclear infiltrate of UCHL1 (T marker) positive cells, accompanies the CMV-associated antigen positive cells.

Objective: To determine the role of tenascin-C in fetal hepatic hematopoietic proliferation and differentiation in different stages of development. Study design: We examined and compared the immunohistochemical expression of tenascin-C in the hepatic stromal portal fields in the first, second, and third trimester of gestation respectively, in relation to the appearance of CD34 progenitor hematopoietic, stromal progenitor and vascular endothelial positive cells. Results: Our results demonstrated a quantitative difference in the second trimester of gestation concerning the expression of tenascin-C in the connective tissue stroma of the hepatic portal fields over the equivalent expression of the protein in the first (P

Loss of the entire or part of one copy of chromosome 7 [del(7/7q)] is a recurrent cytogenetic abnormality in MDS. Its strong association with previous exposure to alkylating agents, consistently poor response to therapy and discrete gene expression profile strongly suggest that del(7/7q)-MDS is a distinct disease in the MDS spectrum whose pathogenesis is intimately linked to loss of chr7q genetic material. Understanding the role of chr7q loss in cell biology can provide key insights into the pathogenesis of MDS and leukemogenesis. Narrowing down the responsible region on chr7q presents a challenge that has proved intractable with existing approaches. Chr7q deletions physically mapped in large cohorts of patients are typically very large and dispersed along most of the length of chr7q. Modeling in the mouse is problematic for reasons of synteny. Deletion of the syntenic region of one commonly deleted region (7q22 CDR) failed to demonstrate a phenotype. Several lines of evidence point to haploinsufficiency of chr7q genetic material – rather than a 2-hit model – as the underlying mechanism in del(7q)-MDS. No inactivating mutations of candidate 7q genes have been detected by resequencing the remaining allele and haploinsufficiency of coding and miRNA genes has been strongly linked to the pathogenesis of del(5q)-MDS. Haploinsufficiency can only be assessed through functional studies (and not genomic technologies), but these are currently hindered by the lack of a clearly described del(7q)-associated phenotype. With recent advances in human pluripotent stem cell (hPSC) research and genetic engineering technologies, reverse human genetics in an isogenic setting by disruption of genomic elements into their cognate genomic and cellular context - hitherto unthinkable for the human genome – are now a realistic prospect. To determine the impact of hemizygous chr7q loss on the cellular phenotype, we have generated isogenic del(7q)- and normal hPSCs by engineering deletions spanning variable overlapping regions encompassing the entire length of chr7q using adeno-associated virus (AAV)-mediated gene targeting combined with Cre-lox technology. Specifically, we targeted two inverted loxP sites together with a positive (puro) and a negative (HSVtk) selection marker in a near-telomeric region of chromosome 7q (7q36.3) into the H1 hESC line, as well as a karyotypically normal iPSC line (line 2-12) derived from BMMCs of a patient with del(7q)-MDS. Following transient expression of Cre recombinase and ganciclovir selection, clones were screened by qPCR probing different regions along the length of chr7. Four H1-derived and four 2-12-derived clones were selected following screening of 24 and 34 clones, respectively, and after excluding clones with additional chromosomal abnormalities by karyotyping and the exact extent of their chr7q deletions was mapped by aCGH. We focused our phenotypic characterization on two cellular phenotypes that we recently reported in del(7q)-iPSCs derived from MDS BMMCs: cell proliferation and in vitro hematopoietic differentiation potential. 7 of the 8 clones harboring deletions spanning variable lengths along the entire chr7q had a lower (by ½ log) proliferation rate than their corresponding isogenic parental lines. All these clones also exhibited a markedly reduced differentiation potential along all hematopoietic lineages and almost absent clonogenic capacity in methylcellulose. These cellular phenotypes are highly similar to those we find in our del(7q)-MDS-iPSCs. Notably, one of the eight clones (2-12.Cre-44), harboring a smaller deletion spanning 7q11.21-7q.31.1 retained comparable proliferation and differentiation capacity to that of normal isogenic and non-isogenic hPSCs. In conclusion, our results demonstrate that hemizygous loss of chr7q material recapitulates the cellular phenotypes of impaired proliferation and hematopoietic differentiation that we find in del(7q)-MDS-iPSCs, supporting a haploinsufficiency pathogenesis of del(7q)-MDS. Correlation of the phenotypes with the boundaries of chr7q deletions in our collection of hESC and iPSC clones points to a region spanning 7q31.1-7q36.1 as the critical region in del(7q)-MDS. Further studies in additional clones harboring smaller chr7q deletions will further narrow down the responsible region and guide prioritization of candidate genes. Disclosures: No relevant conflicts of interest to declare.

Despite past and ongoing research efforts, the pathogenetic mechanisms of MDS remain far from understood sufficiently to point to single genes or molecular pathways as putative targets for generation of better mouse models or drug development. In lack of clear targets, the manipulation of disease-associated phenotypes may at present be the best opportunity for disease study and therapeutic intervention. Furthermore, because cellular phenotypes are more proximal to molecular disease mechanisms than phenotypes seen at the level of the tissue or organism (i.e. the murine or human hematopoietic system), they may provide more sensitive and relevant readouts of the disease process. With the advent of patient-specific induced pluripotent stem cells (iPSCs), disease models based on cellular phenotypes (“disease-in-a-dish”) can now be developed and their unique properties promise to revolutionize disease study and drug development. Unlimited cell numbers of biologically relevant cells can be obtained relatively easily and cost-effectively. Here we present a new MDS model based on patient-specific iPSCs and its use in two phenotype-driven screens: (a) a focused genetic screen and (b) a high-throughput chemical screen. We have derived multiple MDS-iPSC lines with deletions of chromosomes 7q or 20q (characteristic chromosomal deletions that we are using as markers of iPSCs derived from the MDS clone) from bone marrow (BM) of 3 patients with MDS and 1 patient with sAML. We also derived isogenic karyotypically normal iPSC lines in parallel from the same BM samples. We identified two cellular phenotypes specific to the MDS-iPSCs: decreased proliferation rate and decreased potential for pan-hematopoietic differentiation and reduced clonogenic capacity of their hematopoietic progeny. These phenotypes are consistent across multiple iPSC lines from different patients and absent from their isogenic normal iPSC controls. They are reminiscent of the behavior of ex vivo cultured primary MDS BM cells and are therefore likely to be relevant to the disease process. Furthermore, they are rescued by spontaneous compensation for chr7q dosage through acquisition of an extra chr7 and recapitulated by the engineering of artificial chr7q deletions in normal iPSCs. To identify critical MDS gene(s) on chr7q, we performed a screen of 62 candidate haploinsufficient genes (with reduced expression by at least 1.5-fold in our del(7q)-iPSCs compared to their isogenic normal iPSCs) for rescue of the proliferation and/or hematopoietic differentiation phenotype. We constructed a lentiviral library of all candidate ORFs (and 14 additional alternative transcripts) linked to eGFP through a P2A peptide and each tagged with a unique 4-nt barcode sequence to its 3’ UTR. The library was packaged as a pool and transduced into two different del(7q)-iPSC lines. The cells were passaged for 16 weeks and gDNA was isolated every 2 weeks. In parallel, the cells were differentiated along the hematopoietic lineage and gDNA was isolated from CD45+ cells FACS-sorted on day 15 of differentiation. High-throughput sequencing of the barcodes identified 5 ORFs that became enriched in undifferentiated iPSCs over time (rescue of proliferation) and 4 ORFs enriched in sorted CD45+hematopoietic progenitors (rescue of hematopoietic differentiation), 2 of which overlapped. All 9 hits are being further validated in more focused screens. Second, we used the same platform for a high-throughput small molecule screen. We optimized the plating conditions and densities for a 384-well format using a luminescent cell viability assay and conducted a screen of 2000 compounds comprising known drugs, natural products, and other bioactives and chemicals, in an MDS-iPSC line (2.13) and its isogenic normal control (2.8). Primary hits were defined as compounds that enhanced the growth of the MDS-iPSC line, but not of the control normal iPSC line in a compound dose-dependent manner. 38 primary hits were retested in a dose-response survival assay in one additional MDS- (2.A1-3), one additional isogenic normal iPSC line (2.12) and one sAML iPSC line over 8 concentrations and 12 compounds were prioritized for further studies. Our data highlight the potential of this new iPSC-based model of MDS for screens to identify genes or compounds that affect cellular phenotypes by acting on previously undefined targets. Disclosures: No relevant conflicts of interest to declare.

Connexins, the gap junction proteins, through their multitude of actions are implicated in a variety of cell processes during animal development and cancer. They allow direct or paracrine/autocrine cell communication through their channel and hemi-channel functions. They enable adhesion and interact with a plethora of signalling molecules. Here, we review the common themes in developmental and pathological processes and we focus in their involvement in cell migration in four different systems: neurons, astrocytes, neural crest and cancer.