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Abstract Myelodysplastic syndromes (MDS) with mutated SF3B1 gene present features including a favourable outcome distinct from MDS with mutations in other splicing factor genes SRSF2 or U2AF1. Molecular bases of these divergences are poorly understood. Here we find that SF3B1-mutated MDS show reduced R-loop formation predominating in gene bodies associated with intron retention reduction, not found in U2AF1- or SRSF2-mutated MDS. Compared to erythroblasts from SRSF2- or U2AF1-mutated patients, SF3B1-mutated erythroblasts exhibit augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation. Importantly, histone deacetylase inhibition using vorinostat restores R-loop formation, slows down DNA replication forks and improves SF3B1-mutated erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress represents a hallmark of SF3B1-mutated MDS ineffective erythropoiesis, which could be used as a therapeutic target.

Abstract Background Pregnant women and their fetuses are particularly susceptible to respiratory pathogens. How they respond to SARS-CoV-2 infection is still under investigation. Methods We studied the transcriptome and phenotype of umbilical cord blood cells in pregnant women infected or not with SARS-CoV-2. Results Here we show that symptomatic maternal COVID-19 is associated with a transcriptional erythroid cell signature as compared with asymptomatic and uninfected mothers. We observe an expansion of fetal hematopoietic multipotent progenitors skewed towards erythroid differentiation that display increased clonogenicity. There was no difference in inflammatory cytokines levels in the cord blood upon maternal SARS-CoV-2 infection. Interestingly, we show an activation of hypoxia pathway in cord blood cells from symptomatic COVID-19 mothers, suggesting that maternal hypoxia may be triggering this fetal stress hematopoiesis. Conclusions Overall, these results show a fetal hematopoietic response to symptomatic COVID-19 in pregnant mothers in the absence of vertically transmitted SARS-CoV-2 infection which is likely to be a mechanism of fetal adaptation to the maternal infection and reduced oxygen supply.

Abstract Advancements in comprehending myelodysplastic neoplasms (MDS) have unfolded significantly in recent years, elucidating a myriad of cellular and molecular underpinnings integral to disease progression. While molecular inclusions into prognostic models have substantively advanced risk stratification, recent revelations have emphasized the pivotal role of immune dysregulation within the bone marrow milieu during MDS evolution. Nonetheless, immunotherapy for MDS has not experienced breakthroughs seen in other malignancies, partly attributable to the absence of an immune classification that could stratify patients toward optimally targeted immunotherapeutic approaches. A pivotal obstacle to establishing “immune classes” among MDS patients is the absence of validated accepted immune panels suitable for routine application in clinical laboratories. In response, we formed International Integrative Innovative Immunology for MDS (i4MDS), a consortium of multidisciplinary experts, and created the following recommendations for standardized methodologies to monitor immune responses in MDS. A central goal of i4MDS is the development of an immune score that could be incorporated into current clinical risk stratification models. This position paper first consolidates current knowledge on MDS immunology. Subsequently, in collaboration with clinical and laboratory specialists, we introduce flow cytometry panels and cytokine assays, meticulously devised for clinical laboratories, aiming to monitor the immune status of MDS patients, evaluating both immune fitness and identifying potential immune “risk factors.” By amalgamating this immunological characterization data and molecular data, we aim to enhance patient stratification, identify predictive markers for treatment responsiveness, and accelerate the development of systems immunology tools and innovative immunotherapies.

Abstract Background Mature blood cells arise from hematopoietic stem cells in the bone marrow by a process of differentiation along one of several different lineage trajectories. This is often represented as a series of discrete steps of increasing progenitor cell commitment to a given lineage, but as for differentiation in general, whether the process is instructive or stochastic remains controversial. Here, we examine this question by analyzing single-cell transcriptomic data from human bone marrow cells, assessing cell-to-cell variability along the trajectories of hematopoietic differentiation into four different types of mature blood cells. The instructive model predicts that cells will be following the same sequence of instructions and that there will be minimal variability of gene expression between them throughout the process, while the stochastic model predicts a role for cell-to-cell variability when lineage commitments are being made. Results Applying Shannon entropy to measure cell-to-cell variability among human hematopoietic bone marrow cells at the same stage of differentiation, we observed a transient peak of gene expression variability occurring at characteristic points in all hematopoietic differentiation pathways. Strikingly, the genes whose cell-to-cell variation of expression fluctuated the most over the course of a given differentiation trajectory are pathway-specific genes, whereas genes which showed the greatest variation of mean expression are common to all pathways. Finally, we showed that the level of cell-to-cell variation is increased in the most immature compartment of hematopoiesis in myelodysplastic syndromes. Conclusions These data suggest that human hematopoietic differentiation could be better conceptualized as a dynamical stochastic process with a transient stage of cellular indetermination, and strongly support the stochastic view of differentiation. They also highlight the need to consider the role of stochastic gene expression in complex physiological processes and pathologies such as cancers, paving the way for possible noise-based therapies through epigenetic regulation.

Ineffective hematopoiesis is the major characteristic of early myelodysplastic syndromes. Its pathophysiology relies on a diversity of mechanisms supported by genetic events that develop in aging hematopoietic stem cells. Deletion and mutations trigger epigenetic modifications, and co-transcriptional and post-transcriptional deregulations of gene expression. Epistatic interactions between mutants may aggravate the phenotype. Amplification of minor subclones containing mutations that promote their growth and suppress the others drives the clonal evolution. Aging also participates in reprogramming the immune microenvironment towards an inflammatory state, which precedes the expansion of immunosuppressive cells such as Tregs and myeloid-derived suppressive cells that alters the anti-tumor response of effector cells. Integrating biomarkers of transcription/translation deregulation and immune contexture will help the design of personalized treatments.

Summary: Background: Severe COVID-19 is associated with a high circulating level of calprotectin, the S100A8/S100A9 alarmin heterodimer. Baseline calprotectin amount measured in peripheral blood at diagnosis correlates with disease severity. The optimal use of this biomarker along COVID-19 course remains to be delineated. Methods: We focused on patients with a WHO-defined moderate COVID-19 requiring hospitalization in a medical ward. We collected plasma and serum from three independent cohorts (N = 626 patients) and measured calprotectin amount at admission. We performed longitudinal measures of calprotectin in 457 of these patients (1461 samples) and used a joint latent class mixture model in which classes were defined by age, body mass index and comorbidities to identify calprotectin trajectories predicting the risk of transfer into an intensive care unit or death. Findings: After adjustment for age, sex, body mass index and comorbidities, the predictive value of baseline calprotectin in patients with moderate COVID19 could be refined by serial monitoring of the biomarker. We discriminated three calprotectin trajectories associated with low, moderate, and high risk of poor outcome, and we designed an algorithm available as online software (https://calpla.gustaveroussy.fr:8443/) to monitor the probability of a poor outcome in individual patients with moderate COVID-19. Interpretation: These results emphasize the clinical interest of serial monitoring of calprotectin amount in the peripheral blood to anticipate the risk of poor outcomes in patients with moderate COVID-19 hospitalized in a standard care unit. Funding: The study received support (research grants) from ThermoFisher immunodiagnostics (France) and Gustave Roussy Foundation.

Abstract The circulating metabolome provides a snapshot of the physiological state of the organism responding to pathogenic challenges. Here we report alterations in the plasma metabolome reflecting the clinical presentation of COVID-19 patients with mild (ambulatory) diseases, moderate disease (radiologically confirmed pneumonitis, hospitalization and oxygen therapy), and critical disease (in intensive care). This analysis revealed major disease- and stage-associated shifts in the metabolome, meaning that at least 77 metabolites including amino acids, lipids, polyamines and sugars, as well as their derivatives, were altered in critical COVID-19 patient’s plasma as compared to mild COVID-19 patients. Among a uniformly moderate cohort of patients who received tocilizumab, only 10 metabolites were different among individuals with a favorable evolution as compared to those who required transfer into the intensive care unit. The elevation of one single metabolite, anthranilic acid, had a poor prognostic value, correlating with the maintenance of high interleukin-10 and -18 levels. Given that products of the kynurenine pathway including anthranilic acid have immunosuppressive properties, we speculate on the therapeutic utility to inhibit the rate-limiting enzymes of this pathway including indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase.

Abstract: Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders with an inherent tendency for transformation in secondary acute myeloid leukemia. This study focused on the redox metabolism of bone marrow (BM) cells from 97 patients compared with 25 healthy controls. The level of reactive oxygen species (ROS) was quantified by flow cytometry in BM cell subsets as well as the expression level of 28 transcripts encoding for major enzymes involved in the antioxidant cellular response. Our results highlight increased ROS levels in BM nonlymphoid cells and especially in primitive CD34posCD38low progenitor cells. Moreover, we identified a specific antioxidant signature, dubbed “antioxidogram,” for the different MDS subgroups or secondary acute myeloblastic leukemia (sAML). Our results suggest that progression from MDS toward sAML could be characterized by 3 successive molecular steps: (1) overexpression of enzymes reducing proteic disulfide bonds (MDS with

Background: Direct oral factor Xa (FXa) inhibitors interfere with lupus anticoagulant (LA) assays challenging antiphospholipid syndrome diagnosis in treated patients. We evaluated a new device, called DOAC Filter, and its usefulness in this setting. It is a single-use filtration cartridge in which FXa inhibitor compounds are trapped by non-covalent binding while plasma is filtered through a solid phase. Patient samples were analyzed before and after filtration: 38 rivaroxaban, 41 apixaban, and 68 none. Anticoagulant plasma concentrations were measured using specific anti-Xa assays and HPLC-MS/MS. LA testing was performed using dilute Russell Viper Venom Time (dRVVT) and Silica Clotting Time (SCT). Baseline median [min–max] concentrations were 64.8 [17.6; 311.4] for rivaroxaban and 92.1 ng/mL [37.1; 390.7] for apixaban (HPLC-MS/MS). They were significantly correlated with anti-Xa assay results (r = 0.98 and r = 0.94, respectively). dRVVT was positive in 92% rivaroxaban and 72% apixaban and SCT in 28 and 41% of samples, respectively. Post-filtration, median % of neutralization was 100% with rivaroxaban and apixaban concentrations of, respectively,

High-dimensional cytometry is an innovative tool for immune monitoring in health and disease, and it has provided novel insight into the underlying biology as well as biomarkers for a variety of diseases. However, the analysis of large multiparametric datasets usually requires specialist computational knowledge. Here, we describe ImmunoCluster (https://github.com/kordastilab/ImmunoCluster), an R package for immune profiling cellular heterogeneity in high-dimensional liquid and imaging mass cytometry, and flow cytometry data, designed to facilitate computational analysis by a nonspecialist. The analysis framework implemented within ImmunoCluster is readily scalable to millions of cells and provides a variety of visualization and analytical approaches, as well as a rich array of plotting tools that can be tailored to users’ needs. The protocol consists of three core computational stages: (1) data import and quality control; (2) dimensionality reduction and unsupervised clustering; and (3) annotation and differential testing, all contained within an R-based open-source framework.

APR-246 is a promising new therapeutic agent that targets p53 mutated proteins in myelodysplastic syndromes and in acute myeloid leukemia (AML). APR-246 reactivates the transcriptional activity of p53 mutants by facilitating their binding to DNA target sites. Recent studies in solid cancers have found that APR-246 can also induce p53-independent cell death. In this study, we demonstrate that AML cell death occurring early after APR-246 exposure is suppressed by iron chelators, lipophilic antioxidants and inhibitors of lipid peroxidation, and correlates with the accumulation of markers of lipid peroxidation, thus fulfilling the definition of ferroptosis, a recently described cell death process. The capacity of AML cells to detoxify lipid peroxides by increasing their cystine uptake to maintain major antioxidant molecule glutathione biosynthesis after exposure to APR-246 may be a key determinant of sensitivity to this compound. The association of APR-246 with induction of ferroptosis (either by pharmacological compounds, or genetic inactivation of SLC7A11 or GPX4) had a synergistic effect on the promotion of cell death, both in vivo and ex vivo.

The gene CXXC5, encoding a retinoid-inducible nuclear factor (RINF), is located within a region at 5q31.2 commonly deleted in myelodysplastic syndrome and adult acute myeloid leukemia. RINF may act as an epigenetic regulator and has been proposed as a tumor suppressor in hematopoietic malignancies. However, functional studies in normal hematopoiesis are lacking, and its mechanism of action is unknown. Here, we evaluated the consequences of RINF silencing on cytokine-induced erythroid differentiation of human primary CD34+ progenitors. We found that RINF is expressed in immature erythroid cells and that RINF-knockdown accelerated erythropoietin-driven maturation, leading to a significant reduction (~45%) in the number of red blood cells, without affecting cell viability. The phenotype induced by RINF-silencing was dependent on tumor growth factor b (TGFb) and mediated by SMAD7, a TGFb-signaling inhibitor. RINF upregulates SMAD7 expression by direct binding to its promoter and we found a close correlation between RINF and SMAD7 mRNA levels both in CD34+ cells isolated from bone marrow of healthy donors and myelodysplastic syndrome patients with del(5q). Importantly, RINF knockdown attenuated SMAD7 expression in primary cells and ectopic SMAD7 expression was sufficient to prevent the RINF knockdown-dependent erythroid phenotype. Finally, RINF silencing affects 5’-hydroxymethylation of human erythroblasts, in agreement with its recently described role as a TET2-anchoring platform in mouse. Collectively, our data bring insight into how the epigenetic factor RINF, as a transcriptional regulator of SMAD7, may fine-tune cell sensitivity to TGFb superfamily cytokines and thus play an important role in both normal and pathological erythropoiesis.

In ribosomopathies, the Diamond-Blackfan anemia (DBA) or 5q- syndrome, ribosomal protein (RP) genes are affected by mutation or deletion, resulting in bone marrow erythroid hypoplasia. Unbalanced production of ribosomal subunits leading to a limited ribosome cellular content, regulates translation at the expense of the master erythroid transcription factor GATA1. In RPS14-deficient cells mimicking 5q- syndrome erythroid defects, we show that the transcript length, codon bias of the coding sequence (CDS) and 3'UTR structure are the key determinants of translation. In these cells, short transcripts with a structured 3'UTR and high CAI showed a decreased translation efficiency. Quantitative analysis of the whole proteome confirmed that the post-transcriptional changes depended on the transcript characteristics that governed the translation efficiency in conditions of low ribosome availability. In addition, proteins involved in normal erythroid differentiation share most determinants of translation selectivity. Our findings thus indicate that impaired erythroid maturation due to 5q- syndrome may proceed from a translational selectivity at the expense of the erythroid differentiation program and suggest that an interplay between the CDS and UTRs may regulate mRNA translation.

Formyl peptide receptor 1 (FPR1) is a pattern-recognition receptor that detects bacterial as well as endogenous danger-associated molecular patterns to trigger innate immune responses by myeloid cells. A single nucleotide polymorphism, rs867228 (allelic frequency 19–20%), in the gene coding for FPR1 accelerates the manifestation of multiple carcinomas, likely due to reduced anticancer immunosurveillance secondary to a defect in antigen presentation by dendritic cells. Another polymorphism in FPR1, rs5030880 (allelic frequency 12–13%), has been involved in the resistance to plague, correlating with the fact that FPR1 is the receptor for Yersinia pestis. Driven by the reported preclinical effects of FPR1 on lung inflammation and fibrosis, we investigated whether rs867228 or rs5030880 would affect the severity of coronavirus disease-19 (COVID-19). Data obtained on patients from two different hospitals in Paris refute the hypothesis that rs867228 or rs5030880 would affect the severity of COVID-19.

Over the past 16 years, three coronaviruses (CoVs), severe acute respiratory syndrome CoV (SARS-CoV) in 2002, Middle East respiratory syndrome CoV (MERS-CoV) in 2012 and 2015, and SARS-CoV-2 in 2020, have been causing severe and fatal human epidemics. The unpredictability of coronavirus disease-19 (COVID-19) poses a major burden on health care and economic systems across the world. This is caused by the paucity of in-depth knowledge of the risk factors for severe COVID-19, insufficient diagnostic tools for the detection of SARS-CoV-2, as well as the absence of specific and effective drug treatments. While protective humoral and cellular immune responses are usually mounted against these betacoronaviruses, immune responses to SARS-CoV2 sometimes derail towards inflammatory tissue damage, leading to rapid admissions to intensive care units. The lack of knowledge on mechanisms that tilt the balance between these two opposite outcomes poses major threats to many ongoing clinical trials dealing with immunostimulatory or immunoregulatory therapeutics. This review will discuss innate and cognate immune responses underlying protective or deleterious immune reactions against these pathogenic coronaviruses.

Somatic mutations contribute to the heterogeneous prognosis of chronic myelomonocytic leukemia (CMML). Hypomethylating agents (HMAs) are active in CMML, but analyses of small series failed to identify mutations predicting response or survival. We analyzed a retrospective multi-center cohort of 174 CMML patients treated with a median of 7 cycles of azacitidine (n = 68) or decitabine (n = 106). Sequencing data before treatment initiation were available for all patients, from Sanger (n = 68) or next generation (n = 106) sequencing. Overall response rate (ORR) was 52%, including complete response (CR) in 28 patients (17%). In multivariate analysis, ASXL1 mutations predicted a lower ORR (Odds Ratio [OR] = 0.85, p = 0.037), whereas TET2mut/ASXL1wt genotype predicted a higher CR rate (OR = 1.18, p = 0.011) independently of clinical parameters. With a median follow-up of 36.7 months, overall survival (OS) was 23.0 months. In multivariate analysis, RUNX1mut (Hazard Ratio [HR] = 2.00, p = .011), CBLmut (HR = 1.90, p = 0.03) genotypes and higher WBC (log10(WBC) HR = 2.30, p = .005) independently predicted worse OS while the TET2mut/ASXL1wt predicted better OS (HR = 0.60, p = 0.05). CMML-specific scores CPSS and GFM had limited predictive power. Our results stress the need for robust biomarkers of HMA activity in CMML and for novel treatment strategies in patients with myeloproliferative features and RUNX1 mutations. Keywords: Chronic myelomonocytic leukemia, Hypomethylating agents, Somatic mutations, Prognosis

Abstract: The TET2 gene encodes an α-ketoglutarate–dependent dioxygenase able to oxidize 5-methylcytosine into 5-hydroxymethylcytosine, which is a step toward active DNA demethylation. TET2 is frequently mutated in myeloid malignancies but also in B- and T-cell malignancies. TET2 somatic mutations are also identified in healthy elderly individuals with clonal hematopoiesis. Tet2-deficient mouse models showed widespread hematological differentiation abnormalities, including myeloid, T-cell, and B-cell malignancies. We show here that, similar to what is observed with constitutive Tet2-deficient mice, B-cell–specific Tet2 knockout leads to abnormalities in the B1-cell subset and a development of B-cell malignancies after long latency. Aging Tet2-deficient mice accumulate clonal CD19+ B220low immunoglobulin M+ B-cell populations with transplantable ability showing similarities to human chronic lymphocytic leukemia, including CD5 expression and sensitivity to ibrutinib-mediated B-cell receptor (BCR) signaling inhibition. Exome sequencing of Tet2−/− malignant B cells reveals C-to-T and G-to-A mutations that lie within single-stranded DNA–specific activation-induced deaminase (AID)/APOBEC (apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like) cytidine deaminases targeted motif, as confirmed by the lack of a B-cell tumor in compound Tet2-Aicda–deficient mice. Finally, we show that Tet2 deficiency accelerates and exacerbates T-cell leukemia/lymphoma 1A–induced leukemogenesis. Together, our data establish that Tet2 deficiency predisposes to mature B-cell malignancies, which development might be attributed in part to AID-mediated accumulating mutations and BCR-mediated signaling.

Erythropoiesis-stimulating agents are generally the first line of treatment of anemia in patients with lower-risk myelodysplastic syndrome. We prospectively investigated the predictive value of somatic mutations, and biomarkers of ineffective erythropoiesis including the flow cytometry RED score, serum growth-differentiation factor-15, and hepcidin levels. Inclusion criteria were no prior treatment with erythropoiesis-stimulating agents, low- or intermediate-1-risk myelodysplastic syndrome according to the International Prognostic Scoring System, and a hemoglobin level 4 (P=0.05) and a hepcidin:ferritin ratio 2000 pg/mL and a hepcidin:ferritin ratio

Summary: Tryptophan as the precursor of several active compounds, including kynurenine and serotonin, is critical for numerous important metabolic functions. Enhanced tryptophan metabolism toward the kynurenine pathway has been associated with myelodysplastic syndromes (MDSs), which are preleukemic clonal diseases characterized by dysplastic bone marrow and cytopenias. Here, we reveal a fundamental role for tryptophan metabolized along the serotonin pathway in normal erythropoiesis and in the physiopathology of MDS-related anemia. We identify, both in human and murine erythroid progenitors, a functional cell-autonomous serotonergic network with pro-survival and proliferative functions. In vivo studies demonstrate that pharmacological increase of serotonin levels using fluoxetine, a common antidepressant, has the potential to become an important therapeutic strategy in low-risk MDS anemia refractory to erythropoietin. : Sibon et al. identify a cell-autonomous serotonergic network in human and mouse erythroid progenitors. Reduced levels of serotonin lead to decreased proliferation and survival of erythroid progenitors. Increasing serotonin’s concentration through fluoxetine, commonly used to treat depression, could be a valuable therapeutic intervention to correct myelodysplastic-syndrome-related anemia. Keywords: serotonin, Tph1, erythropoiesis, myelodysplastic syndrome, anemia, SSRI

The stem cell factor receptor (SCF) c-Kit plays a pivotal role in regulating cell proliferation and survival in many cell types. In particular, c-Kit is required for early amplification of erythroid progenitors, while it must disappear from cell surface for the cell entering the final steps of maturation in an erythropoietin-dependent manner. We initially observed that imatinib (IM), an inhibitor targeting the tyrosine kinase activity of c-Kit concomitantly down-regulated the expression of c-Kit and accelerated the Epo-driven differentiation of erythroblasts in the absence of SCF. We investigated the mechanism by which IM or related masitinib (MA) induce c-Kit down-regulation in the human UT-7/Epo cell line. We found that the down-regulation of c-Kit in the presence of IM or MA was inhibited by a pre-incubation with methyl-β-cyclodextrin suggesting that c-Kit was internalized in the absence of ligand. By contrast to SCF, the internalization induced by TKI was independent of the E3 ubiquitin ligase c-Cbl. Furthermore, c-Kit was degraded through lysosomal, but not proteasomal pathway. In pulse-chase experiments, IM did not modulate c-Kit synthesis or maturation. Analysis of phosphotyrosine peptides in UT-7/Epo cells treated or not with IM show that IM did not modify overall tyrosine phosphorylation in these cells. Furthermore, we showed that a T670I mutation preventing the full access of IM to the ATP binding pocket, did not allow the internalization process in the presence of IM. Altogether these data show that TKI-induced internalization of c-Kit is linked to a modification of the integrity of ATP binding pocket.

Zebrafish were proposed as an alternative to mammalian models to assess the efficacy and toxicity of antileukemic drugs. Due to the limited number of transgenic zebrafish leukemia models, we explored human leukemic cell xenograft in zebrafish embryos. Human leukemic cell lines and blast cells sorted from patients with acute myelogenous leukemia were injected 48 hours post-fertilization and remained in the circulation of zebrafish embryos for several days without affecting their development. Imatinib and oxaphorines did not demonstrate any toxicity on normal zebrafish embryos and decreased the leukemic burden in animals xenografted with sensitive leukemic cell lines. Two other molecules, all-trans retinoic acid and the translation inhibitor 4EGI-1, demonstrated teratogenic effects at concentrations shown to be efficient in vitro, which precluded investigation of their antileukemic activity in such models. Altogether, xenografted leukemic cells in zebrafish embryos are a pharmacologically relevant model for screening non-teratogenic drugs.