Search

Treatment options for patients with relapsed/refractory acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are scarce. Recurring mutations, such as mutations in isocitrate dehydrogenase-1 and -2 (IDH1/2) are found in subsets of AML and MDS, are therapeutically targeted by mutant enzyme-specific small molecule inhibitors (IDH

The last decade has witnessed massive advancements in high-throughput techniques capable of producing quantifications of transcript and protein levels across time and space, and at high resolution. Yet, the large volume of big data available and the complexity of experimental designs hamper an easy understanding and effective communication of the results.Here we present expressyouRcell, a unique and easy-to-use R package to map the multi-dimensional variations of transcript and protein levels in cell-pictographs. These variations are outcomes of differential and gene set enrichment analysis across space and time. Our tool directly associates these results with up to twenty specific cellular compartments, visualising them as pictographic representations of four different cellular thematic maps. expressyouRcell visually reduces the complexity of displaying gene expression and protein level changes across multiple time-points by generating dynamic representations of cellular pictographs.We applied expressyouRcell to six datasets, demonstrating its flexibility and usability in the visualization of simple and highly effective static and dynamic representations of time course variations in gene expression. Our approach complements classical plot-based methods for visualization and exploitation of biological data, improving the standard quantitative interpretation and communication of relevant results.

The essential pre-mRNA splicing factor U2AF2 (also called U2AF65) identifies polypyrimidine (Py) tract signals of nascent transcripts, despite length and sequence variations. Previous studies have shown that the U2AF2 RNA recognition motifs (RRM1 and RRM2) preferentially bind uridine-rich RNAs. Nonetheless, the specificity of the RRM1/RRM2 interface for the central Py tract nucleotide has yet to be investigated. We addressed this question by determining crystal structures of U2AF2 bound to a cytidine, guanosine, or adenosine at the central position of the Py tract, and compared U2AF2-bound uridine structures. Local movements of the RNA site accommodated the different nucleotides, whereas the polypeptide backbone remained similar among the structures. Accordingly, molecular dynamics simulations revealed flexible conformations of the central, U2AF2-bound nucleotide. The RNA binding affinities and splicing efficiencies of structure-guided mutants demonstrated that U2AF2 tolerates nucleotide substitutions at the central position of the Py tract. Moreover, enhanced UV-crosslinking and immunoprecipitation of endogenous U2AF2 in human erythroleukemia cells showed uridine-sensitive binding sites, with lower sequence conservation at the central nucleotide positions of otherwise uridine-rich, U2AF2-bound splice sites. Altogether, these results highlight the importance of RNA flexibility for protein recognition and take a step towards relating splice site motifs to pre-mRNA splicing efficiencies.

A red-letter day for RBC research The study of primary human red blood cell (huRBC) disorders such as sickle cell disease (SCD) and infectious diseases such as malaria has been hampered by a lack of in vivo models of human erythropoiesis. Song et al. transferred human fetal liver cells into MISTRG mice, which are immunodeficient and are genetically engineered with several human genes involved in hematopoiesis. This approach was unsuccessful because mature huRBCs are rapidly destroyed in the mouse liver. They then used CRISPR-Cas9 to mutate these mice into a fumarylacetoacetate hydrolase–deficient strain, allowing them to replace the mouse liver with engrafted human hepatocytes. These mice exhibited enhanced human erythropoiesis and circulating huRBC survival and could recapitulate SCD pathology when reconstituted with SCD-derived HSCs. Science , this issue p. 1019

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.

Argonaute 2 (AGO2) is a ubiquitously expressed protein critical for regulation of mRNA translation and vital to animal development. AGO2 protein is found in both cytoplasmic and nuclear compartments, and although its cytoplasmic role is well studied, the biological relevance of nuclear AGO2 is unclear. Here, we address this problem in vivo using spermatogenic cells as a model. We find that AGO2 transiently binds both chromatin and nucleus-specific mRNA transcripts of hundreds of genes required for sperm production during male meiosis in mice, and that germline conditional knockout (cKO) of Ago2 causes depletion of the encoded proteins. Correspondingly, Ago2 cKO males show abnormal sperm head morphology and reduced sperm count, along with reduced postnatal viability of offspring. Together, our data reveal an unexpected nuclear role for AGO2 in enhancing expression of developmentally important genes during mammalian male reproduction.

Persistent neutrophil-dominated lung inflammation contributes to lung damage in cystic fibrosis (CF). However, the mechanisms that drive persistent lung neutrophilia and tissue deterioration in CF are not well characterized. Starting from the observation that, in patients with CF, c-c motif chemokine receptor 2 (CCR2)

The essential pre-mRNA splicing factor U2AF2 (also called U2AF65) identifies polypyrimidine (Py) tract signals of nascent transcripts, despite length and sequence variations. Previous studies have shown that the U2AF2 RNA recognition motifs (RRM1 and RRM2) preferentially bind uridine-rich RNAs. Nonetheless, the specificity of the RRM1/RRM2 interface for the central Py tract nucleotide has yet to be investigated. We addressed this question by determining crystal structures of U2AF2 bound to a cytidine, guanosine, or adenosine at the central position of the Py tract, and compared U2AF2-bound uridine structures. Local movements of the RNA site accommodated the different nucleotides, whereas the polypeptide backbone remained similar among the structures. Accordingly, molecular dynamics simulations revealed flexible conformations of the central, U2AF2-bound nucleotide. The RNA binding affinities and splicing efficiencies of structure-guided mutants demonstrated that U2AF2 tolerates nucleotide substitutions at the central position of the Py tract. Moreover, enhanced crosslinking and immunoprecipitation of endogenous U2AF2 in human erythroleukemia cells showed uridine-sensitive binding sites with lower sequence conservation at the central nucleotide positions of otherwise uridine-rich, U2AF2-bound splice sites. Altogether, these results highlight the importance of RNA flexibility for protein recognition and take a step towards relating splice site motifs to pre-mRNA splicing efficiencies.

Pediatric and adult high-grade gliomas are the most common primary malignant brain tumors, with poor prognosis due to recurrence and tumor infiltration after therapy. Quiescent cells have been implicated in tumor recurrence and treatment resistance, but their direct visualization and targeting remain challenging, precluding their mechanistic study. Here, we identify a population of malignant cells expressing Prominin-1 in a non-proliferating state in pediatric high-grade glioma patients. Using a genetic tool to visualize and ablate quiescent cells in mouse brain cancer and human cancer organoids, we reveal their localization at both the core and the edge of the tumors, and we demonstrate that quiescent cells are involved in infiltration of brain cancer cells. Finally, we find that Harmine, a DYRK1A/B inhibitor, partially decreases the number of quiescent and infiltrating cancer cells. Our data point to a subpopulation of quiescent cells as partially responsible of tumor invasiveness, one of the major causes of brain cancer morbidity.

Somatic mutations in splicing factors are of significant interest in myeloid malignancies and other cancers. U2AF1, together with U2AF2, is essential for 3’ splice site recognition. U2AF1 mutations result in aberrant splicing, but the molecular mechanism and the full spectrum of consequences on RNA biology have not been fully elucidated to date. We performed multi-omics profiling of in vivo RNA binding, splicing and turnover for U2AF1 S34F and Q157R mutants. We dissected specific binding signals of U2AF1 and U2AF2 and showed that U2AF1 mutations individually alter U2AF1-RNA binding, resulting in defective U2AF2 recruitment. We demonstrated a complex relationship between differential binding and splicing, expanding upon the currently accepted loss-of-binding model. Finally, we observed that U2AF1 mutations increase the formation of stress granules in both cell lines and primary acute myeloid leukemia samples. Our results uncover U2AF1 mutation-dependent pathogenic RNA mechanisms and provide the basis for developing targeted therapeutic strategies.

The mechanisms underlying thrombocytosis in patients with iron deficiency anemia remain unknown. Here, we present findings that support the hypothesis that low iron biases the commitment of megakaryocytic (Mk)-erythroid progenitors (MEPs) toward the Mk lineage in both human and mouse. In MEPs of transmembrane serine protease 6 knockout (Tmprss6-/-) mice, which exhibit iron deficiency anemia and thrombocytosis, we observed a Mk bias, decreased labile iron, and decreased proliferation relative to wild-type (WT) MEPs. Bone marrow transplantation assays suggest that systemic iron deficiency, rather than a local role for Tmprss6-/- in hematopoietic cells, contributes to the MEP lineage commitment bias observed in Tmprss6-/- mice. Nontransgenic mice with acquired iron deficiency anemia also show thrombocytosis and Mk-biased MEPs. Gene expression analysis reveals that messenger RNAs encoding genes involved in metabolic, vascular endothelial growth factor, and extracellular signal-regulated kinase (ERK) pathways are enriched in Tmprss6-/- vs WT MEPs. Corroborating our findings from the murine models of iron deficiency anemia, primary human MEPs exhibit decreased proliferation and Mk-biased commitment after knockdown of transferrin receptor 2, a putative iron sensor. Signal transduction analyses reveal that both human and murine MEP have lower levels of phospho-ERK1/2 in iron-deficient conditions compared with controls. These data are consistent with a model in which low iron in the marrow environment affects MEP metabolism, attenuates ERK signaling, slows proliferation, and biases MEPs toward Mk lineage commitment.

Pediatric and adult high-grade gliomas are the most common malignant brain tumors, with poor prognosis due to recurrence and tumor infiltration after surgical removal and chemotherapy. Quiescent/slow cycling stem cells have been proposed to be one of the main players of tumor relapse but their involvement in in the infiltration remain unclear. Despite they have been described in mouse models or after transcriptional profiling of human tumor samples, their direct visualization, targeting and ablation remains a challenge. Here, we identified a population of malignant cells expressing Prominin-1 in a non-proliferating state in pediatric high-grade glioma patients. We next used a fluorescent cell cycle sensor to visualize quiescence tumor cells in mouse brain cancer and human cancer organoids. In particular, we characterized them within the tumor revealing the invasiveness capacity of slow cycling tumor cells. Furthermore, we generated a new system to specifically trace and ablate such cells. Indeed, lineage tracing experiments allowed to trace quiescent Prom1 progeny in the tumors after temozolomide treatment. In addition, the selective ablation of quiescent Prom1+ cells in mouse brain cancer reduced tumor infiltration and improved survival. Furthermore, time-lapse experiments showed that slow cycling cells are also able to infiltrate co-cultured human brain cancer organoids. Finally, using our new cancer organoid- and GBM spheroids-based models we identified a drug acting on quiescent cells leading to a reduction in cell invasion. Overall, our data show that quiescence/slow cycling cells are key driver of tumor invasiveness, the major malignant feature of high-grade brain cancer.

Ribosome profiling is based on the deep sequencing of RNA fragments protected by ribosomes from nuclease digestion. This technique has been extensively used to study translation, with the unique ability to provide information about ribosomes positioning along transcripts at single-nucleotide resolution. Classical ribosome profiling approaches do not distinguish between fragments protected by either actively translating or inactive ribosomes. Here we describe an original method, called active ribosome profiling or RiboLace, which is based on a unique puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. This method allows reliable estimates of the translational state of any biological system, in high concordance with protein levels. RiboLace can be applied both in vitro and in vivo and generates snapshots of active ribosome footprints at single-nucleotide resolution and genome-wide level. RiboLace data are suitable for the analysis of translated genes, codon-specific translation rates, and local changes in ribosome occupancy profiles.

International audience; Bacterial genotoxins cause DNA damage in eukaryotic cells, resulting in activation of the DNA damage response (DDR) in vitro. These toxins are produced by Gram-negative bacteria, enriched in the microbiota of inflammatory bowel disease (IBD) and colorectal cancer (CRC) patients. However, their role in infection remains poorly characterized. We address the role of typhoid toxin in modulation of the host-microbial interaction in health and disease. Infection with a genotoxigenic Salmonella protects mice from intestinal inflammation. We show that the presence of an active genotoxin promotes DNA fragmentation and senescence in vivo, which is uncoupled from an inflammatory response and unexpectedly associated with induction of an anti-inflammatory environment. The anti-inflammatory response is lost when infection occurs in mice with acute colitis. These data highlight a complex context-dependent crosstalk between bacterial-genotoxin-induced DDR and the host immune response, underlining an unexpected role for bacterial genotoxins.

Ribosome profiling is based on the deep sequencing of RNA fragments protected by ribosomes from nuclease digestion. This technique has been extensively used to study translation, with the unique ability to provide information about ribosomes positioning along transcripts at single-nucleotide resolution. Classical ribosome profiling approaches do not distinguish between fragments protected by either actively translating or inactive ribosomes. Here we describe an original method, called active ribosome profiling or RiboLace, which is based on a unique puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. This method allows reliable estimates of the translational state of any biological system, in high concordance with protein levels. RiboLace can be applied both in vitro and in vivo and generates snapshots of active ribosome footprints at single-nucleotide resolution and genome-wide level. RiboLace data are suitable for the analysis of translated genes, codon-specific translation rates, and local changes in ribosome occupancy profiles. © 2021, Springer Science+Business Media, LLC, part of Springer Nature.

SUMMARYWe present DBiT-seq –Deterministic Barcoding in Tissue for spatial omics sequencing – for co-mapping of mRNAs and proteins in a formaldehyde-fixed tissue slide via NGS sequencing. Parallel microfluidic channels were used to deliver DNA barcodes to the surface of a tissue slide and crossflow of two sets of barcodes A1-50 and B1-50 followed by ligationin situyielded a 2D mosaic of tissue pixels, each containing a unique full barcode AB. Application to mouse embryos revealed major tissue types in early organogenesis as well as fine features like microvasculature in a brain and pigmented epithelium in an eye field. Gene expression profiles in 10μm pixels conformed into the clusters of single-cell transcriptomes, allowing for rapid identification of cell types and spatial distributions. DBiT-seq can be adopted by researchers with no experience in microfluidics and may find applications in a range of fields including developmental biology, cancer biology, neuroscience, and clinical pathology.In BriefMicrofluidic deterministic barcoding of mRNAs and proteins in tissue slides followed by high-throughput sequencing enables the construction of a high-spatial-resolution multi-omics atlas at the genome scale. Application to mouse embryos (E10-12) identified major tissue types in early organogenesis and revealed fine tissue features such as retinal pigmented epithelium and endothelial microvasculature at the cellular level.HighlightsDeterministic barcoding in tissue enables NGS-based spatial multi-omics mapping.DBiT-seq identified spatial patterning of major tissue types in mouse embryos.DBiT-seq revealed fine features such as retinal pigmented epithelium and microvascular endothelium at the cellular level.Direct integration with scRNA-seq data allows for rapid cell type identification.

SummaryBacterial genotoxins cause DNA damage in eukaryotic cells, resulting in activation of the DNA damage response (DDR) in vitro. These toxins are produced by Gram negative bacteria, enriched in the microbiota of Inflammatory Bowel Disease (IBD) and colorectal cancer (CRC) patients. However, their role in infection remains poorly characterized. We have addressed the role of the typhoid toxin in the modulation of the host-microbial interaction in health and disease.Infection with a genotoxigenic Salmonella protected mice from intestinal inflammation. The toxin-induced DNA damage caused senescence in vivo, which was uncoupled from the inflammatory response, and associated with the maintenance of an anti-inflammatory environment. This effect was lost when infection occurred in mice suffering from inflammatory conditions that mimic Ulcerative Colitis, a form of IBD.These data highlight a complex context-dependent crosstalk between bacterial genotoxins-induced DDR and the host immune response, underlining an unexpected role for bacterial genotoxins.

Pediatric myelodysplastic syndrome (PMDS) is a very rare and still poorly characterized disorder. In this work, we identified novel potential targets of PMDS by determining genes with aberrant expression, which can be correlated with PMDS pathogenesis. We identified 291 differentially expressed genes (DEGs) in PMDS patients, comprising genes involved in the regulation of apoptosis and the cell cycle, ribosome biogenesis, inflammation and adaptive immunity. Ten selected DEGs were then validated, confirming the sequencing data. These DEGs will potentially represent new molecular biomarkers and therapeutic targets for PMDS.

The contribution of ribosome heterogeneity and ribosome-associated proteins to the molecular control of proteomes in health and disease remains unclear. Here, we demonstrate that survival motor neuron (SMN) protein—the loss of which causes the neuromuscular disease spinal muscular atrophy (SMA)—binds to ribosomes and that this interaction is tissue-dependent. SMN-primed ribosomes are preferentially positioned within the first five codons of a set of mRNAs that are enriched for translational enhancer sequences in the 5′ untranslated region (UTR) and rare codons at the beginning of their coding sequence. These SMN-specific mRNAs are associated with neurogenesis, lipid metabolism, ubiquitination, chromatin regulation and translation. Loss of SMN induces ribosome depletion, especially at the beginning of the coding sequence of SMN-specific mRNAs, leading to impairment of proteins that are involved in motor neuron function and stability, including acetylcholinesterase. Thus, SMN plays a crucial role in the regulation of ribosome fluxes along mRNAs encoding proteins that are relevant to SMA pathogenesis. Lauria et al. show that SMN, the loss of which causes spinal muscular atrophy (SMA), preferentially positions ribosomes within the first five codons of SMA-related mRNAs and enhances their translation.

The automatic discovery of causal relationships among human genes can shed light on gene regulatory processes and guide drug repositioning. To this end, a computationally-heavy method for causal discovery is distributed on a volunteer computing grid and, taking advantage of variable subsetting and stratification, proves to be useful for expanding local gene regulatory networks. The input data are purely observational measures of transcripts expression in human tissues and cell lines collected within the FANTOM project. The system relies on the BOINC platform and on optimized client code. The functional relevance of results, measured by analyzing the annotations of the identified interactions, increases significantly over the simple Pearson correlation between the transcripts. Additionally, in 82% of cases networks significantly overlap with known protein-protein interactions annotated in biological databases. In the two case studies presented, this approach has been used to expand the networks of genes associated with two severe human pathologies: prostate cancer and coronary artery disease. The method identified respectively 22 and 36 genes to be evaluated as novel targets for already approved drugs, demonstrating the effective applicability of the approach in pipelines aimed to drug repositioning.

Somatic mutations in splicing factor genes are drivers of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The splicing factors U2AF1 and U2AF2 form the U2AF heterodimer that is critical in the 3' splice site (3'SS) recognition and in the recruitment of U2 small nuclear ribonucleoproteins for the activation of the spliceosome complex. U2AF1 carries hotspot mutations in its two RNA binding motifs; yet the molecular mechanisms affecting the splicing process and promoting clonal advantage remain unclear, albeit necessary to develop effective targeted therapies. We applied a multi-omics approach comparing the activities of two U2AF1 mutants (S34F and Q157R) in MDS/AML cell lines and primary samples. Using a novel approach of fractionated enhanced crosslinking immunoprecipitation coupled with deep RNA-sequencing (freCLIP-seq), we mapped transcriptome-wide binding at nucleotide resolution and we identified conformational changes in mutant vs wild-type U2AF1 binding. Specifically, we observed an emergent peak in position -3 of the 3'SS for the S34F mutant and in position +1 for the Q157R mutant, matching the critical positions observed by differential splicing analysis on RNA-seq data. Altered U2AF1-RNA binding compromised U2AF2-RNA interactions, resulting predominantly in exon exclusion and intron retention. Combined binding-splicing analysis showed that while the Q157R mutant mainly exhibits loss of binding, the S34F mutant follows a gain-of-binding pattern, where splicing progression appears impaired by increased mutant binding. Functional analysis of genes affected by both binding and splicing alterations revealed that U2AF1 mutants alter RNA granule biology, affecting in particular stress granule-enriched transcripts and proteins. Stress granules are membrane-less cytoplasmic assemblies of RNAs and RNA binding proteins that improve cellular adaptation in response to stress conditions. Increased stress granule formation has been linked to tumorigenesis as a strategy exploited by cancer cells to regulate gene expression and signal transduction, enhancing their fitness under stress. To probe how aberrant binding and splicing of stress granule components affected stress granule biology, we assessed stress granule formation in U2AF1 mutant vs wild-type cells at steady state and after stress induction with sodium arsenite treatment. Immunofluorescent staining followed by confocal imaging demonstrated that U2AF1 mutations enhance stress granule formation upon arsenite stress in both cell lines and primary samples. RNA turnover analysis by TimeLapse-seq confirmed that U2AF1 S34F and Q157R mutations promote stability/synthesis of transcripts that are enriched in stress granules and determine degradation/shutdown of transcripts that are depleted in stress granules, providing a molecular explanation for the increase in stress granules observed by imaging. Finally, we were able to corroborate our observations by single-cell RNA-seq in patient-derived U2AF1-mutant MDS blasts, establishing the causal link between U2AF1 mutations and upregulation of stress granule components. Collectively, this multi-omics analysis identified biological processes directly influenced by mutant U2AF1 binding and splicing, laying the foundation for a new paradigm where splicing factor mutations enhance stress granule formation by acting on the availability of their RNA and protein components. The enhanced formation of stress granules potentially fosters the stress adaptation of U2AF1-mutant cells, contributing to their clonal advantage in MDS/AML. Stress granule perturbations may therefore represent a novel therapeutic vulnerability in U2AF1-mutant MDS/AML patients and possibly in patients carrying other splicing factor mutations. Disclosures Hunck: Boehringer Ingelheim: Other: Fellowship.

Humanized mouse model is a powerful tool for the study of human hematopoiesis and hematologic diseases in vivo. However, existing models continue to be limited by absent engraftment of stem cells with inherent growth defects, especially in bone marrow failure (BMF) disorders such as low-grade myelodysplastic syndromes (MDS) and Fanconi Anemia (FA). Previously, we presented a highly efficient MDS/AML PDX model in cytokine-humanized MISTRG mice, that express human M-CSF, IL-3, GM-CSF and THPO in the background of the SIRPα - humanized rag -/- IL2Rg -/- mice. MISTRG mice support efficient, faithful and long-term MDS hematopoietic stem and progenitor cell (HSPC) engraftment with multi-lineage representation. Similar to MDS, FA stem cells are defective and sensitive to toxic stress conferred by inflammation or irradiation. Irradiation, required in conventional mice as pre-conditioning, increases the risk of secondary malignancies, induces activation of inflammatory pathways and may damage the bone marrow microenvironment. In this study, we present MISTRG6kit W41 mice that in addition to the above cytokines express human IL-6, which improves lymphoid development, and that were CRISPR modified to introduce the white spotting 41 mutation in c-Kit (c-kit W41), which results in partial loss of c-kit function in the murine host stem and progenitor cells. To characterize this mouse model, we compare the engraftment capacity between MISTRG6 and MISTRG6kit W41 mice. We find that the HSPCs from both healthy donor and low grade MDS patient can be more efficiently propagated in MISTRG6Kit W41 mice and that they can be serially transplanted. Human cells are detectable at higher levels in PB of MISTRG6Kit W41 mice as early as 8 weeks post transplantation. As previously described, c-kit mutation enhances human erythroid engraftment while multi-lineage engraftment is maintained in engrafted MISTRG6Kit W41 mice. Therefore, MISTRG6Kit W41 mice offer a better host environment that supports human hematopoietic expansion without irradiation. FA is an inherited genetic disease carrying FA gene mutations associated with DNA damage and cancer predisposition and marked by BMF, MDS and AML. FA HSPCs cannot be expanded or grown for sufficient periods in vitro, and existing mouse models have failed to demonstrate engraftment of primary cells from FA patient. We transplanted a MDS sample from a FANCC mutant patient into MISTRG6Kit W41 mice giving rise to a myeloid predominant graft that could be successfully serially transplanted. Moreover, non-transformed FA HSPCs from 2 FANCA and 2 FANCG mutant patients were successfully engrafted in MISTRG6Kit W41 mice. In summary, MISTRG mice with human IL-6 expression and c-Kit W41 mutation provide a more efficient and convenient way for pre-clinical studies of BMFs such as FA and low-grade MDS with maintenance of an intact humanized stem cell niche, and this is the first time we saw primary cells from FA patient propagate in mouse model. We believe that our model would be highly beneficial to the study of additional diseases associated with DNA damage or cancer predisposition, such as dyskeratosis congenita or Schwachman-Diamond syndrome. Disclosures No relevant conflicts of interest to declare.

During hematopoietic differentiation from hematopoietic stem cells (HSCs) to mature blood cells, cells undergo a metabolic shift from glycolysis to mitochondrial respiration. The mechanisms by which hematopoietic cells adjust their energy metabolism are still under investigation. N6-mehyladenosine (m 6A) mRNA modification has been reported to regulate numerous fundamental cellular processes through control of RNA stability or translational efficiency. The fat mass and obesity-associated protein (FTO), an m 6A m and m 6A mRNA demethylase, has been reported to affect cellular metabolism in acute myeloid leukemia (AML). ALKBH5, the specific RNA m 6A demethylase, controls oncogene expression in AML. ALKBH5 becomes highly expressed in hematopoietic progenitors during hematopoietic development but the physiological role of RNA m 6A demethylase during hematopoiesis remains unknown. To investigate the function of the RNA m 6A demethylase ALKBH5 in hematopoiesis, we generated Vav-iCre +; Alkbh5fl/fl (vcAlkbh5-/-) mice, resulting in deletion of Alkbh5 specifically in the hematopoietic system. vcAlkbh5-/-mice showed no hematopoietic defects at steady states up to 12 months of age. We applied TimeLapse-seq on lineage-depleted bone marrow cells of WT and vcAlkbh5-/- mice to determine whether loss of ALKBH5 perturbed mRNA stability and/or RNA turnover. Ogdh mRNA was the most destabilized transcript resulting in significantly reduced OGDH protein levels. OGDH is the rate-limiting enzyme in the tricarboxylic acid (TCA) cycle. Inhibition of OGDH subsequently induces production of L-2-hydroxyglutarate (L-2-HG), whose metabolism is closely coupled to energy metabolism through inhibition of oxygen consumption. L-2-HG, the enantiomer of D-2-HG, inhibits the function of a-ketoglutarate (a-KG)-dependent enzymes, including TET and KDM enzymes. We measured L- and D-2-HG in the plasma of WT and vcAlkbh5-/- mice by chiral derivatization to distinguish the two enantiomers. Although D-2-HG levels were similar in the plasma of WT and vcAlkbh5-/- mice, L-2-HG levels were significantly increased in the plasma of vcAlkbh5-/- mice. We therefore determined the function of Jumonji C-domain lysine demethylases (JmjC-KDMs) by measuring histone methylation: H3K9me3, H3K27me3 and H3K36me3 modifications were all significantly increased in Alkbh5-deficient hematopoietic cells. We next sought to understand whether reduction of OGDH expression and resulting increased L-2-HG levels production could impair energy metabolism via perturbation of the TCA cycle and oxidative phosphorylation (OXPHOS) in the mitochondria. We isolated lineage negative hematopoietic stem and progenitor cells (HSPCs) from WT and vcAlkbh5-/- mice and subjected these to the Seahorse ATP Rate Assay. Comparing oxygen consumption rate (OCR) data and the kinetics of the Extracellular Acidification Rate (ECAR) of both groups, we found that less ATP was produced by mitochondria of the vcAlkbh5-/- cells, while ATP produced by glycolysis showed no difference between the two groups. In the meantime, the ultrastructure of mitochondria in the Alkbh5-deficient cells remains normal. We next determined whether the attenuated energy metabolism of Alkbh5-deficient HSPCs was functionally relevant by testing HSPC function in competitive transplantation assays. Interestingly, vcAlkbh5-/- cells showed a significant competitive defect at all differentiation stages except in phenotypic long-term HSCs (LT-HSCs). This suggests that LT-HSCs, thought to preferentially rely on glycolysis as opposed to OXPHOS for their energy source, are protected from loss of ALKBH5 and OGDH. In conclusion, our study demonstrates that ALKBH5 modulates energy metabolism by regulating mRNA stability of metabolic enzymes through its m 6A demethylation activity during hematopoiesis. This finding links Alkbh5 expression kinetics to the metabolic shift from glycolysis to mitochondrial OXPHOS during hematopoietic development. Disclosures No relevant conflicts of interest to declare.