Massively parallel base editing to map variant effects in human hematopoiesis.

Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.

Deciphering cell states and genealogies of human haematopoiesis.

The human blood system is maintained through the differentiation and massive amplification of a limited number of long-lived haematopoietic stem cells (HSCs)1. Perturbations to this process underlie diverse diseases, but the clonal contributions to human haematopoiesis and how this changes with age remain incompletely understood. Although recent insights have emerged from barcoding studies in model systems2-5, simultaneous detection of cell states and phylogenies from natural barcodes in humans remains challenging. Here we introduce an improved, single-cell lineage-tracing system based on deep detection of naturally occurring mitochondrial DNA mutations with simultaneous readout of transcriptional states and chromatin accessibility. We use this system to define the clonal architecture of HSCs and map the physiological state and output of clones. We uncover functional heterogeneity in HSC clones, which is stable over months and manifests as both differences in total HSC output and biases towards the production of different mature cell types. We also find that the diversity of HSC clones decreases markedly with age, leading to an oligoclonal structure with multiple distinct clonal expansions. Our study thus provides a clonally resolved and cell-state-aware atlas of human haematopoiesis at single-cell resolution, showing an unappreciated functional diversity of human HSC clones and, more broadly, paving the way for refined studies of clonal dynamics across a range of tissues in human health and disease.

Molecular and cellular mechanisms that regulate human erythropoiesis.

To enable effective oxygen transport, ∼200 billion red blood cells (RBCs) need to be produced every day in the bone marrow through the fine-tuned process of erythropoiesis. Erythropoiesis is regulated at multiple levels to ensure that defective RBC maturation or overproduction can be avoided. Here, we provide an overview of different layers of this control, ranging from cytokine signaling mechanisms that enable extrinsic regulation of RBC production to intrinsic transcriptional pathways necessary for effective erythropoiesis. Recent studies have also elucidated the importance of posttranscriptional regulation and highlighted additional gatekeeping mechanisms necessary for effective erythropoiesis. We additionally discuss the insights gained by studying human genetic variation affecting erythropoiesis and highlight the discovery of BCL11A as a regulator of hemoglobin switching through genetic studies. Finally, we provide an outlook of how our ability to measure multiple facets of this process at single-cell resolution, while accounting for the impact of human variation, will continue to refine our knowledge of erythropoiesis and how this process is perturbed in disease. As we learn more about this intricate and important process, additional opportunities to modulate erythropoiesis for therapeutic purposes will undoubtedly emerge.

HDAC6 regulates human erythroid differentiation through modulation of JAK2 signalling.

Among histone deacetylases, HDAC6 is unusual in its cytoplasmic localization. Its inhibition leads to hyperacetylation of non-histone proteins, inhibiting cell cycle, proliferation and apoptosis. Ricolinostat (ACY-1215) is a selective inhibitor of the histone deacetylase HDAC6 with proven efficacy in the treatment of malignant diseases, but anaemia is one of the most frequent side effects. We investigated here the underlying mechanisms of this erythroid toxicity. We first confirmed that HDAC6 was strongly expressed at both RNA and protein levels in CD34+ -cells-derived erythroid progenitors. ACY-1215 exposure on CD34+ -cells driven in vitro towards the erythroid lineage led to a decreased cell count, an increased apoptotic rate and a delayed erythroid differentiation with accumulation of weakly hemoglobinized immature erythroblasts. This was accompanied by drastic changes in the transcriptomic profile of primary cells as shown by RNAseq. In erythroid cells, ACY-1215 and shRNA-mediated HDAC6 knockdown inhibited the EPO-dependent JAK2 phosphorylation. Using acetylome, we identified 14-3-3ζ, known to interact directly with the JAK2 negative regulator LNK, as a potential HDAC6 target in erythroid cells. We confirmed that 14-3-3ζ was hyperacetylated after ACY-1215 exposure, which decreased the 14-3-3ζ/LNK interaction while increased LNK ability to interact with JAK2. Thus, in addition to its previously described role in the enucleation of mouse fetal liver erythroblasts, we identified here a new mechanism of HDAC6-dependent control of erythropoiesis through 14-3-3ζ acetylation level, LNK availability and finally JAK2 activation in response to EPO, which is crucial downstream of EPO-R activation for human erythroid cell survival, proliferation and differentiation.

Epidemiological landscape of young patients with multiple myeloma diagnosed before 40 years of age: the French experience.

Multiple myeloma (MM) is rare in young patients, especially before age 40 years at diagnosis, representing <2% of all patients with MM. Little is known about the disease characteristics and prognosis of these patients. In this study, we examined 214 patients diagnosed with MM at age ≤40 years over 15 years, in the era of modern treatments. Among them, 189 patients had symptomatic MM. Disease characteristics were similar to older patients: 35% had anemia, 17% had renal impairment, and 13% had hypercalcemia. The staging was ISS-1 in 52.4%, ISS-2 in 27.5%, and ISS-3 in 20.1%. Overall, 18% of patients had high-risk cytogenetics [del 17p and/or t(4;14)]. Ninety percent of patients received intensive chemotherapy followed by autologous stem cell transplant, and 25% of patients had allogeneic stem cell transplant predominantly at time of relapse. The median follow-up was 76 months, the estimated median overall survival was 14.5 years, and the median progression free-survival was 41 months. In multivariate analysis, bone lesions (hazard ratio [HR], 3.95; P = .01), high ISS score (HR, 2.14; P = .03), and high-risk cytogenetics (HR, 4.54; P < .0001) were significant risk factors for poor outcomes. Among predefined time-dependent covariables, onset of progression (HR, 13.2; P < .0001) significantly shortened overall survival. At 5 years, relative survival compared with same age- and sex-matched individuals was 83.5%, and estimated standardized mortality ratio was 69.9 (95% confidence interval, 52.7-91.1), confirming that MM dramatically shortens the survival of young patients despite an extended survival after diagnosis.

Artificial intelligence to empower diagnosis of myelodysplastic syndromes by multiparametric flow cytometry.

The diagnosis of myelodysplastic syndromes (MDS) might be challenging and relies on the convergence of cytological, cytogenetic, and molecular factors. Multiparametric flow cytometry (MFC) helps diagnose MDS, especially when other features do not contribute to the decision-making process, but its usefulness remains underestimated, mostly due to a lack of standardization of cytometers. We present here an innovative model integrating artificial intelligence (AI) with MFC to improve the diagnosis and the classification of MDS. We develop a machine learning model through an elasticnet algorithm directed on a cohort of 191 patients, only based on flow cytometry parameters selected by the Boruta algorithm, to build a simple but reliable prediction score with five parameters. Our AI-assisted MDS prediction score greatly improves the sensitivity of the Ogata score while keeping an excellent specificity validated on an external cohort of 89 patients with an Area Under the Curve of 0.935. This model allows the diagnosis of both high- and low-risk MDS with 91.8% sensitivity and 92.5% specificity. Interestingly, it highlights a progressive evolution of the score from clonal hematopoiesis of indeterminate potential (CHIP) to highrisk MDS, suggesting a linear evolution between these different stages. By significantly decreasing the overall misclassification of 52% for patients with MDS and of 31.3% for those without MDS (P=0.02), our AI-assisted prediction score outperforms the Ogata score and positions itself as a reliable tool to help diagnose MDS.

PIEZO1, sensing the touch during erythropoiesis.

PURPOSE OF REVIEW: Awarding the 2021 Nobel to Ardem Patapoutian for the discovery of the PIEZO mechanotransducers has emphasized the importance of touch-sensing mechanisms in cell physiology. It is well known that PIEZO1 is expressed at the surface of red blood cells where it adjusts their hydration status under mechanical constraints. Besides this, recent findings suggest that PIEZO1 plays a broader role in erythroid lineage. This review aims to actualize the knowledge on PIEZO1 functions all along erythropoiesis. RECENT FINDINGS: PIEZO1 is expressed in erythroid progenitors, and controls proliferation and differentiation of nucleated cells, as well as maturation of reticulocytes. As PIEZO1 detects displacements in the range of cell-cell interactions, it might mediate the interaction between the differentiating cells and their microenvironment through an inside-out activation of integrins on human erythroblasts as suggested by in-vitro data. Moreover, PIEZO1 is also expressed at the surface of macrophages where it regulates red blood cells clearance through erythrophagocytosis. SUMMARY: These new findings on PIEZO1 suggest a continuous effect of mechanotransduction all over erythropoiesis from progenitors to clearance of red blood cells. Therefore, they open a new era in the understanding of hereditary xerocytosis pathophysiology, helping identify new potential therapeutic targets for the future.

Accurate classification of plasma cell dyscrasias is achieved by combining artificial intelligence and flow cytometry.

Monoclonal gammopathy of unknown significance (MGUS), smouldering multiple myeloma (SMM), and multiple myeloma (MM) are very common neoplasms. However, it is often difficult to distinguish between these entities. In the present study, we aimed to classify the most powerful markers that could improve diagnosis by multiparametric flow cytometry (MFC). The present study included 348 patients based on two independent cohorts. We first assessed how representative the data were in the discovery cohort (123 MM, 97 MGUS) and then analysed their respective plasma cell (PC) phenotype in order to obtain a set of correlations with a hypersphere visualisation. Cluster of differentiation (CD)27 and CD38 were differentially expressed in MGUS and MM (P < 0·001). We found by a gradient boosting machine method that the percentage of abnormal PCs and the ratio PC/CD117 positive precursors were the most influential parameters at diagnosis to distinguish MGUS and MM. Finally, we designed a decisional algorithm allowing a predictive classification ≥95% when PC dyscrasias were suspected, without any misclassification between MGUS and SMM. We validated this algorithm in an independent cohort of PC dyscrasias (n = 87 MM, n = 41 MGUS). This artificial intelligence model is freely available online as a diagnostic tool application website for all MFC centers worldwide (https://aihematology.shinyapps.io/PCdyscrasiasToolDg/).

Human erythroleukemia genetics and transcriptomes identify master transcription factors as functional disease drivers.

Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.

Progressive multifocal leukoencephalopathy: MRI findings in HIV-infected patients are closer to rituximab- than natalizumab-associated PML.

OBJECTIVES: To compare brain MRI findings in progressive multifocal leukoencephalopathy (PML) associated to rituximab and natalizumab treatments and HIV infection. MATERIALS AND METHODS: In this retrospective, multicentric study, we analyzed brain MRI exams from 72 patients diagnosed with definite PML: 32 after natalizumab treatment, 20 after rituximab treatment, and 20 HIV patients. We compared T2- or FLAIR-weighted images, diffusion-weighted images, T2*-weighted images, and contrast enhancement features, as well as lesion distribution, especially gray matter involvement. RESULTS: The three PML entities affect U-fibers associated with low signal intensities on T2*-weighted sequences. Natalizumab-associated PML showed a punctuate microcystic appearance in or in the vicinity of the main PML lesions, a potential involvement of the cortex, and contrast enhancement. HIV and rituximab-associated PML showed only mild contrast enhancement, punctuate appearance, and cortical involvement. The CD4/CD8 ratio showed a trend to be higher in the natalizumab group, possibly mirroring a more efficient immune response. CONCLUSION: Imaging features of rituximab-associated PML are different from those of natalizumab-associated PML and are closer to those observed in HIV-associated PML. KEY POINTS: • Nowadays, PML is emerging as a complication of new effective therapies based on monoclonal antibodies. • Natalizumab-associated PML shows more inflammatory signs, a perivascular distribution "the milky way," and more cortex involvement than rituximab- and HIV-associated PML. • MRI differences are probably related to higher levels of immunosuppression in HIV patients and those under rituximab therapy.

Recent advances in the pathophysiology of PIEZO1-related hereditary xerocytosis.

Hereditary xerocytosis is a rare red blood cell disease related to gain-of-function mutations in the FAM38A gene, encoding PIEZO1, in 90% of cases; PIEZO1 is a broadly expressed mechano-transducer that plays a major role in many cell systems and tissues that respond to mechanical stress. In erythrocytes, PIEZO1 adapts the intracellular ionic content and cell hydration status to the mechanical constraints induced by the environment. Until recently, the pathophysiology of hereditary xerocytosis was mainly believed to be based on the "PIEZO1-Gardos channel axis" in erythrocytes, according to which PIEZO1-activating mutations induce a calcium influx that secondarily activates the Gardos channel, leading to potassium and water efflux and subsequently to red blood cell dehydration. However, recent studies have demonstrated additional roles for PIEZO1 during early erythropoiesis and reticulocyte maturation, as well as roles in other tissues and cells such as lymphatic vessels, hepatocytes, macrophages and platelets that may affect the pathophysiology of the disease. These findings, presented and discussed in this review, broaden our understanding of hereditary xerocytosis beyond that of primarily being a red blood cell disease and identify potential therapeutic targets.

PIEZO1 activation delays erythroid differentiation of normal and hereditary xerocytosis-derived human progenitor cells.

Hereditary xerocytosis is a dominantly inherited red cell membrane disorder caused in most cases by gain-of-function mutations in PIEZO1, encoding a mechanosensitive ion channel that translates a mechanic stimulus into calcium influx. We found that PIEZO1 was expressed early in erythroid progenitor cells, and investigated whether it could be involved in erythropoiesis, besides having a role in the homeostasis of mature red cell hydration. In UT7 cells, chemical PIEZO1 activation using YODA1 repressed glycophorin A expression by 75%. This effect was PIEZO1-dependent since it was reverted using specific short hairpin-RNA knockdown. The effect of PIEZO1 activation was confirmed in human primary progenitor cells, maintaining cells at an immature stage for longer and modifying the transcriptional balance in favor of genes associated with early erythropoiesis, as shown by a high GATA2/GATA1 ratio and decreased α/ß-globin expression. The cell proliferation rate was also reduced, with accumulation of cells in G0/G1 of the cell cycle. The PIEZO1-mediated effect on UT7 cells required calcium-dependent activation of the NFAT and ERK1/2 pathways. In primary erythroid cells, PIEZO1 activation synergized with erythropoietin to activate STAT5 and ERK, indicating that it may modulate signaling pathways downstream of erythropoietin receptor activation. Finally, we studied the in-vitro erythroid differentiation of primary cells obtained from 14 PIEZO1-mutated patients, from 11 families, carrying ten different mutations. We observed a delay in erythroid differentiation in all cases, ranging from mild (n=3) to marked (n=8). Overall, these data demonstrate a role for PIEZO1 during erythropoiesis, since activation of PIEZO1 - both chemically and through activating mutations - delays erythroid maturation, providing new insights into the pathophysiology of hereditary xerocytosis.

Bortezomib produces high hematological response rates with prolonged renal survival in monoclonal immunoglobulin deposition disease.

Monoclonal immunoglobulin deposition disease (MIDD) is a rare complication of plasma cell disorders, defined by linear Congo red-negative deposits of monoclonal light chain, heavy chain, or both along basement membranes. While renal involvement is prominent, treatment strategies, such as the impact of novel anti-myeloma agents, remain poorly defined. Here we retrospectively studied 49 patients with MIDD who received a median of 4.5 cycles of intravenous bortezomib plus dexamethasone. Of these, 25 received no additional treatment, 18 also received cyclophosphamide, while 6 also received thalidomide or lenalidomide. The hematological diagnoses identified 38 patients with monoclonal gammopathy of renal significance, 10 with symptomatic multiple myeloma, and 1 with Waldenstrom macroglobulinemia. The overall hematologic response rate, based on the difference between involved and uninvolved serum-free light chains (dFLCs), was 91%. After median follow-up of 54 months, 5 patients died and 10 had reached end-stage renal disease. Renal response was achieved in 26 patients, with a 35% increase in median eGFR and an 86% decrease in median 24-h proteinuria. Predictive factors were pre-treatment eGFR over 30 ml/min per 1.73 m(2) and post-treatment dFLC under 40 mg/l; the latter was the sole predictive factor of renal response by multivariable analysis. Thus, bortezomib-based therapy is a promising treatment strategy in MIDD, mainly when used early in the disease course. dFLC response is a favorable prognostic factor for renal survival.

PIEZO1 is essential for the survival and proliferation of acute myeloid leukemia cells.

INTRODUCTION: Leukemogenesis is a complex process that interconnects tumoral cells with their microenvironment, but the effect of mechanosensing in acute myeloid leukemia (AML) blasts is poorly known. PIEZO1 perceives and transmits the constraints of the environment to human cells by acting as a non-selective calcium channel, but very little is known about its role in leukemogenesis. RESULTS: For the first time, we show that PIEZO1 is preferentially expressed in healthy hematopoietic stem and progenitor cells in human hematopoiesis, and globally overexpressed in AML cells. In AML subtypes, PIEZO1 expression associates with favorable outcomes as better overall (OS) and disease-free survival (DFS). If PIEZO1 is expressed and functional in THP1 leukemic myeloid cell line, its chemical activation doesn't impact the proliferation, differentiation, nor survival of cells. However, the downregulation of PIEZO1 expression dramatically reduces the proliferation and the survival of THP1 cells. We show that PIEZO1 knock-down blocks the cell cycle in G0/G1 phases of AML cells, impairs the DNA damage response pathways, and critically increases cell death by triggering extrinsic apoptosis pathways. CONCLUSIONS: Altogether, our results reveal a new role for PIEZO1 mechanosensing in the survival and proliferation of leukemic blasts, which could pave the way for new therapeutic strategies to target AML cells.

Ruxolitinib-induced reactivation of cytomegalovirus and Epstein-Barr virus in graft-versus-host disease.

OBJECTIVES: Steroid-refractory graft-versus-host disease (SR-GVHD) is a challenging complication of allogeneic hematopoietic stem cell transplantation, and leads to high morbidity and mortality rates. The orally administered, selective Janus-associated kinase 1/2 inhibitor ruxolitinib gives overall response rates (ORR) of more than 70 % in acute and chronic SR-GVHD. However, several studies have highlighted an elevated risk of cytomegalovirus (CMV) reactivation in patients with ruxolitinib-treated SR-GVHD. METHODS: We therefore analyzed risk of CMV and Epstein-Barr virus (EBV) primary infection or reactivation in 57 patients with ruxolitinib-treated GVHD, while taking account of the competing risk (CR) of death prior to the first reactivation. RESULTS: Initiation of ruxolitinib treatment was a significant adverse prognostic factor for the CR of first CMV reactivation (hazard ratio (HR)= 1.747, 95 % confidence interval (CI): 1.33-2.92, p < 0.0001) and first EBV reactivation (HR=2.657, 95 % CI: 1.82-3.87, p < 0.0001) during GVHD. In our cohort of ruxolitinib-treated patients, the ORR (48 % and 58 % for acute and chronic GVHD, respectively) and the toxicity profile (haematological adverse events in 29.8 % of the patients) were similar to the literature values. CONCLUSION: Given ruxolitinib's efficacy in SR-GVHD, use of this drug should not be limited by the fear of viral reactivation; however, our present results emphasize the importance of monitoring the viral load.

RNA Polymerase II pausing temporally coordinates cell cycle progression and erythroid differentiation.

The controlled release of promoter-proximal paused RNA polymerase II (Pol II) into productive elongation is a major step in gene regulation. However, functional analysis of Pol II pausing is difficult because factors that regulate pause release are almost all essential. In this study, we identified heterozygous loss-of-function mutations in SUPT5H , which encodes SPT5, in individuals with ß-thalassemia unlinked to HBB mutations. During erythropoiesis in healthy human cells, cell cycle genes were highly paused at the transition from progenitors to precursors. When the pathogenic mutations were recapitulated by SUPT5H editing, Pol II pause release was globally disrupted, and the transition from progenitors to precursors was delayed, marked by a transient lag in erythroid-specific gene expression and cell cycle kinetics. Despite this delay, cells terminally differentiate, and cell cycle phase distributions normalize. Therefore, hindering pause release perturbs proliferation and differentiation dynamics at a key transition during erythropoiesis, revealing a role for Pol II pausing in the temporal coordination between the cell cycle and differentiation.

RNA polymerase II pausing temporally coordinates cell cycle progression and erythroid differentiation.

Controlled release of promoter-proximal paused RNA polymerase II (RNA Pol II) is crucial for gene regulation. However, studying RNA Pol II pausing is challenging, as pause-release factors are almost all essential. In this study, we identified heterozygous loss-of-function mutations in SUPT5H, which encodes SPT5, in individuals with ß-thalassemia. During erythropoiesis in healthy human cells, cell cycle genes were highly paused as cells transition from progenitors to precursors. When the pathogenic mutations were recapitulated by SUPT5H editing, RNA Pol II pause release was globally disrupted, and as cells began transitioning from progenitors to precursors, differentiation was delayed, accompanied by a transient lag in erythroid-specific gene expression and cell cycle kinetics. Despite this delay, cells terminally differentiate, and cell cycle phase distributions normalize. Therefore, hindering pause release perturbs proliferation and differentiation dynamics at a key transition during erythropoiesis, identifying a role for RNA Pol II pausing in temporally coordinating the cell cycle and erythroid differentiation.

Beneficial outcomes and epidemiologics of atypical electrophoretic profiles arising after allogeneic hematopoietic stem cell transplantation for myeloid malignancies.

PURPOSE OF THE STUDY: Atypical serum protein electrophoresis (SPE) profiles may arise in patients who received allogeneic hematopoietic stem cell transplantation (allo-HSCT), but little is known about their clinical significance. Atypical SPE combine either monoclonal and oligoclonal components, suspected on SPE and confirmed by immunofixation. The aim of the study is to analyze the incidence, the etiology and the clinical significance of atypical SPE profiles in patients who received allo-HSCT. PATIENTS AND METHODS: This retrospective study enrolled 117 patients with myeloid malignancies who received an allo-HSCT between 2012 and 2018. We excluded patients with lymphoid malignancies or multiple myeloma, patients presenting atypical electrophoresis prior to transplantation and patients who died within 100 days post-transplant. RESULTS: Atypical SPE occurred in 42.7% of patients. The cumulative incidence of atypical profiles was significantly higher in patients with acute Graft Versus Host Disease (GVHD, p = 0.019) and in patients with Cytomegalovirus (CMV) reactivation (p = 0.0017). We observed for the first time that atypical SPE profiles mostly occurred in patients transplanted from a CMV+ donor (p = 0.031). CMV reactivation preceded the occurrence of atypical SPE in the majority of patients. We show that atypical SPE delay the relapse of the underlying malignant disease (486 vs 189 days, p = 0.006), and significantly improve overall survival (OS; 33.1 months vs 28.3 months, p = 0.049). In both univariate and multivariate analyzes, the presence of an atypical SPE is the only factor that significantly improves OS. CONCLUSIONS: The occurrence of atypical SPE profiles after allo-HSCT may reflect an adapted post-transplant immune response leading to favourable outcomes.

Red blood cell proteomics reveal remnant protein biosynthesis and folding pathways in PIEZO1-related hereditary xerocytosis.

Hereditary xerocytosis is a dominant red cell membrane disorder characterized by an increased leak of potassium from the inside to outside the red blood cell membrane, associated with loss of water leading to red cell dehydration and chronic hemolysis. 90% of cases are related to heterozygous gain of function mutations in PIEZO1, encoding a mechanotransductor that translates a mechanical stimulus into a biological signaling. Data are still required to understand better PIEZO1-HX pathophysiology. Recent studies identified proteomics as an accurate and high-input tool to study erythroid progenitors and circulating red cell physiology. Here, we isolated red blood cells from 5 controls and 5 HX patients carrying an identified and pathogenic PIEZO1 mutation and performed a comparative deep proteomic analysis. A total of 603 proteins were identified among which 56 were differentially expressed (40 over expressed and 16 under expressed) between controls and HX with a homogenous expression profile within each group. We observed relevant modifications in the protein expression profile related to PIEZO1 mutations, identifying two main "knots". The first contained both proteins of the chaperonin containing TCP1 complex involved in the assembly of unfolded proteins, and proteins involved in translation. The second contained proteins involved in ubiquitination. Deregulation of proteins involved in protein biosynthesis was also observed in in vitro-produced reticulocytes after Yoda1 exposure. Thus, our work identifies significant changes in the protein content of PIEZO1-HX erythrocytes, revealing a "PIEZO1 signature" and identifying potentially targetable pathways in this disease characterized by a heterogeneous clinical expression and contra-indication of splenectomy.