Phenotypic Alterations in Erythroid Nucleated Cells of Spleen and Bone Marrow in Acute Hypoxia.

Hypoxia leads to metabolic changes at the cellular, tissue, and organismal levels. The molecular mechanisms for controlling physiological changes during hypoxia have not yet been fully studied. Erythroid cells are essential for adjusting the rate of erythropoiesis and can influence the development and differentiation of immune cells under normal and pathological conditions. We simulated high-altitude hypoxia conditions for mice and assessed the content of erythroid nucleated cells in the spleen and bone marrow under the existing microenvironment. For a pure population of CD71+ erythroid cells, we assessed the production of cytokines and the expression of genes that regulate the immune response. Our findings show changes in the cellular composition of the bone marrow and spleen during hypoxia, as well as changes in the composition of the erythroid cell subpopulations during acute hypoxic exposure in the form of a decrease in orthochromatophilic erythroid cells that are ready for rapid enucleation and the accumulation of their precursors. Cytokine production normally differs only between organs; this effect persists during hypoxia. In the bone marrow, during hypoxia, genes of the C-lectin pathway are activated. Thus, hypoxia triggers the activation of various adaptive and compensatory mechanisms in order to limit inflammatory processes and modify metabolism.

CD71+ erythroid cells suppress T-cell effector functions and predict immunotherapy outcomes in patients with virus-associated solid tumors.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer. However, only a portion of patients respond to such treatments. Therefore, it remains a prevailing clinical need to identify factors associated with acquired resistance or lack of response to ICIs. We hypothesized that the immunosuppressive CD71+ erythroid cells (CECs) within the tumor and/or distant 'out-of-field' may impair antitumor response. METHODS: We studied 38 patients with cancer through a phase II clinical trial investigating the effects of oral valproate combined with avelumab (anti-programmed death-ligand 1 (PD-L1)) in virus-associated solid tumors (VASTs). We quantified the frequency/functionality of CECs in blood and biopsies of patients. Also, we established an animal model of melanoma (B16-F10) to investigate the possible effects of erythropoietin (EPO) treatment on anti-PD-L1 therapy. RESULTS: We found a substantial expansion of CECs in the blood of patients with VAST compared with healthy controls. We noted that the frequency of CECs in circulation was significantly higher at the baseline and throughout the study in non-responders versus responders to PD-L1 therapy. Moreover, we observed that CECs in a dose-dependent manner suppress effector functions of autologous T cells in vitro. The subpopulation of CD45+CECs appears to have a more robust immunosuppressive property compared with their CD45- counterparts. This was illustrated by a stronger expression of reactive oxygen species, PD-L1/PD-L2, and V-domain Ig suppressor of T-cell activation in this subpopulation. Lastly, we found a higher frequency of CECs in the blood circulation at the later cancer stage and their abundance was associated with anemia, and a poor response to immunotherapy. Finally, we report the expansion of CECs in the spleen and tumor microenvironment of mice with melanoma. We found that although CECs in tumor-bearing mice secret artemin, this was not the case for VAST-derived CECs in humans. Notably, our results imply that EPO, a frequently used drug for anemia treatment in patients with cancer, may promote the generation of CECs and subsequently abrogates the therapeutic effects of ICIs (eg, anti-PD-L1). CONCLUSIONS: Our results demonstrate that anemia by the expansion of CECs may enhance cancer progression. Notably, measuring the frequency of CECs may serve as a valuable biomarker to predict immunotherapy outcomes.

Tipping the balance between erythroid cell differentiation and induction of anemia in response to the inflammatory pathology associated with chronic trypanosome infections.

Infection caused by extracellular single-celled trypanosomes triggers a lethal chronic wasting disease in livestock and game animals. Through screening of 10 Trypanosoma evansi field isolates, exhibiting different levels of virulence in mice, the current study identifies an experimental disease model in which infection can last well over 100 days, mimicking the major features of chronic animal trypanosomosis. In this model, despite the well-controlled parasitemia, infection is hallmarked by severe trypanosomosis-associated pathology. An in-depth scRNA-seq analysis of the latter revealed the complexity of the spleen macrophage activation status, highlighting the crucial role of tissue resident macrophages (TRMs) in regulating splenic extramedullary erythropoiesis. These new data show that in the field of experimental trypanosomosis, macrophage activation profiles have so far been oversimplified into a bi-polar paradigm (M1 vs M2). Interestingly, TRMs exert a double-sided effect on erythroid cells. On one hand, these cells express an erythrophagocytosis associated signature. On another hand, TRMs show high levels of Vcam1 expression, known to support their interaction with hematopoietic stem and progenitor cells (HSPCs). During chronic infection, the latter exhibit upregulated expression of Klf1, E2f8, and Gfi1b genes, involved in erythroid differentiation and extramedullary erythropoiesis. This process gives rise to differentiation of stem cells to BFU-e/CFU-e, Pro E, and Baso E subpopulations. However, infection truncates progressing differentiation at the orthochromatic erythrocytes level, as demonstrated by scRNAseq and flow cytometry. As such, these cells are unable to pass to the reticulocyte stage, resulting in reduced number of mature circulating RBCs and the occurrence of chronic anemia. The physiological consequence of these events is the prolonged poor delivery of oxygen to various tissues, triggering lactic acid acidosis and the catabolic breakdown of muscle tissue, reminiscent of the wasting syndrome that is characteristic for the lethal stage of animal trypanosomosis.

Integration of the Ki-67 proliferation index into the Ogata score improves its diagnostic sensitivity for low-grade myelodysplastic syndromes.

BACKGROUND: Although flow cytometric detection of myelodysplastic syndrome (MDS) with the Ogata score has a high specificity, its sensitivity for low-grade MDS is low. Additional markers are needed to improve its diagnostic reliability. Therefore, we investigated the diagnostic performance of the Ki-67 proliferation index in bone marrow (BM) cell populations for detection of MDS. METHODS: BM aspirates from 50 MDS patients and 20 non-clonal cytopenic controls were analyzed with flow cytometry to determine the Ogata score and the Ki-67 proliferation indices in different cell populations. RESULTS: Ki-67 proliferation indices alone could be used to detect MDS with a sensitivity of up to 80 % and specificity of up to 70 %. Combining the Ogata score with the Ki-67 proliferation index of erythroid cells significantly improved its sensitivity for detection of MDS from 66 % to 90 %, while maintaining a specificity of 100 %. Particularly, the sensitivity for detection of low-grade MDS improved from 56 % to 91 %. CONCLUSIONS: This is the first study using Ki-67 proliferation indices to detect MDS and shows their particularly high diagnostic sensitivity for detection of low-grade MDS. Integration of the Ki-67 proliferation index of erythroid cells into the Ogata score significantly improved its sensitivity without loss of the high specificity.

SF3B1 mutant-induced missplicing of MAP3K7 causes anemia in myelodysplastic syndromes.

SF3B1 is the most frequently mutated RNA splicing factor in cancer, including in ∼25% of myelodysplastic syndromes (MDS) patients. SF3B1-mutated MDS, which is strongly associated with ringed sideroblast morphology, is characterized by ineffective erythropoiesis, leading to severe, often fatal anemia. However, functional evidence linking SF3B1 mutations to the anemia described in MDS patients harboring this genetic aberration is weak, and the underlying mechanism is completely unknown. Using isogenic SF3B1 WT and mutant cell lines, normal human CD34 cells, and MDS patient cells, we define a previously unrecognized role of the kinase MAP3K7, encoded by a known mutant SF3B1-targeted transcript, in controlling proper terminal erythroid differentiation, and show how MAP3K7 missplicing leads to the anemia characteristic of SF3B1-mutated MDS, although not to ringed sideroblast formation. We found that p38 MAPK is deactivated in SF3B1 mutant isogenic and patient cells and that MAP3K7 is an upstream positive effector of p38 MAPK. We demonstrate that disruption of this MAP3K7-p38 MAPK pathway leads to premature down-regulation of GATA1, a master regulator of erythroid differentiation, and that this is sufficient to trigger accelerated differentiation, erythroid hyperplasia, and ultimately apoptosis. Our findings thus define the mechanism leading to the severe anemia found in MDS patients harboring SF3B1 mutations.

Meaningful response criteria for myelodysplastic syndromes.

Standardizing response criteria for myelodysplastic syndromes (MDS), a clinically and biologically heterogeneous group of disorders, has been historically challenging. The International Working Group (IWG) response criteria, first proposed in 2000 and modified in 2006 and 2018, represent the best effort by a group of international experts to define a set of clinically meaningful end-points in MDS. These criteria have been adopted in many MDS clinical trials, allowing for comparisons of response across trials. However, clinical experience has also revealed some limitations of these criteria, and most of the end-points proposed by the IWG require further validation. In this review, we present a critical analysis of the current MDS response criteria from both a practical standpoint and based on currently available clinical trial data. Potential areas for improvement in the criteria are highlighted, which may be considered in future iterations of the response criteria.

Inner nuclear protein Matrin-3 coordinates cell differentiation by stabilizing chromatin architecture.

Precise control of gene expression during differentiation relies on the interplay of chromatin and nuclear structure. Despite an established contribution of nuclear membrane proteins to developmental gene regulation, little is known regarding the role of inner nuclear proteins. Here we demonstrate that loss of the nuclear scaffolding protein Matrin-3 (Matr3) in erythroid cells leads to morphological and gene expression changes characteristic of accelerated maturation, as well as broad alterations in chromatin organization similar to those accompanying differentiation. Matr3 protein interacts with CTCF and the cohesin complex, and its loss perturbs their occupancy at a subset of sites. Destabilization of CTCF and cohesin binding correlates with altered transcription and accelerated differentiation. This association is conserved in embryonic stem cells. Our findings indicate Matr3 negatively affects cell fate transitions and demonstrate that a critical inner nuclear protein impacts occupancy of architectural factors, culminating in broad effects on chromatin organization and cell differentiation.

Disruption of a GATA2-TAL1-ERG regulatory circuit promotes erythroid transition in healthy and leukemic stem cells.

Changes in gene regulation and expression govern orderly transitions from hematopoietic stem cells to terminally differentiated blood cell types. These transitions are disrupted during leukemic transformation, but knowledge of the gene regulatory changes underpinning this process is elusive. We hypothesized that identifying core gene regulatory networks in healthy hematopoietic and leukemic cells could provide insights into network alterations that perturb cell state transitions. A heptad of transcription factors (LYL1, TAL1, LMO2, FLI1, ERG, GATA2, and RUNX1) bind key hematopoietic genes in human CD34+ hematopoietic stem and progenitor cells (HSPCs) and have prognostic significance in acute myeloid leukemia (AML). These factors also form a densely interconnected circuit by binding combinatorially at their own, and each other's, regulatory elements. However, their mutual regulation during normal hematopoiesis and in AML cells, and how perturbation of their expression levels influences cell fate decisions remains unclear. In this study, we integrated bulk and single-cell data and found that the fully connected heptad circuit identified in healthy HSPCs persists, with only minor alterations in AML, and that chromatin accessibility at key heptad regulatory elements was predictive of cell identity in both healthy progenitors and leukemic cells. The heptad factors GATA2, TAL1, and ERG formed an integrated subcircuit that regulates stem cell-to-erythroid transition in both healthy and leukemic cells. Components of this triad could be manipulated to facilitate erythroid transition providing a proof of concept that such regulatory circuits can be harnessed to promote specific cell-type transitions and overcome dysregulated hematopoiesis.

Erythroid nuclear dysplasia is associated with inferior outcomes for patients with myelodysplastic syndrome undergoing allogeneic hematopoietic cell transplantation.

Disease burden prior to hematopoietic cell transplantation (HCT) is difficult to assess in myelodysplastic syndrome (MDS), particularly in patients without excess blasts. We assessed whether morphologic dysplasia at the time of transplant can be a metric of disease burden that is associated with post-transplant outcomes in MDS patients. We identified 84 MDS patients undergoing allogeneic HCT at our institution between 2010 and 2017 who received a bone marrow evaluation immediately prior to HCT. Dysplasia was independently determined by two hematopathologists blinded to existing pathology reports. Erythroid nuclear dysplasia, but not megakaryocytic or myeloid, was associated with post-HCT outcomes. Presence compared to absence of erythroid nuclear dysplasia was associated with lower 2-year progression-free survival (PFS; 34 % vs 62 %, p = 0.0495) and 2-year overall survival (OS; 34 % vs 62 %, p = 0.042). In a multivariate analysis including age, IPSS-R at the time of transplant, pre-HCT therapy, and donor type as covariates, erythroid nuclear dysplasia remained associated with lower PFS (HR 2.6, p = 0.036) and OS (HR 2.7, p = 0.028). Dysplasia assessment prior to transplant may serve as an estimate of disease burden in MDS and identify high-risk patients who merit additional therapies pre- or post-transplant.

Eltrombopag Improves Erythroid Differentiation in a Human Induced Pluripotent Stem Cell Model of Diamond Blackfan Anemia.

Diamond Blackfan Anemia (DBA) is a congenital macrocytic anemia associated with ribosomal protein haploinsufficiency. Ribosomal dysfunction delays globin synthesis, resulting in excess toxic free heme in erythroid progenitors, early differentiation arrest, and pure red cell aplasia. In this study, DBA induced pluripotent stem cell (iPSC) lines were generated from blood mononuclear cells of DBA patients with inactivating mutations in RPS19 and subjected to hematopoietic differentiation to model disease phenotypes. In vitro differentiated hematopoietic cells were used to investigate whether eltrombopag, an FDA-approved mimetic of thrombopoietin with robust intracellular iron chelating properties, could rescue erythropoiesis in DBA by restricting the labile iron pool (LIP) derived from excessive free heme. DBA iPSCs exhibited RPS19 haploinsufficiency, reduction in the 40S/60S ribosomal subunit ratio and early erythroid differentiation arrest in the absence of eltrombopag, compared to control isogenic iPSCs established by CRISPR/Cas9-mediated correction of the RPS19 point mutation. Notably, differentiation of DBA iPSCs in the presence of eltrombopag markedly improved erythroid maturation. Consistent with a molecular mechanism based on intracellular iron chelation, we observed that deferasirox, a clinically licensed iron chelator able to permeate into cells, also enhanced erythropoiesis in our DBA iPSC model. In contrast, erythroid maturation did not improve substantially in DBA iPSC differentiation cultures supplemented with deferoxamine, a clinically available iron chelator that poorly accesses LIP within cellular compartments. These findings identify eltrombopag as a promising new therapeutic to improve anemia in DBA.

To bi or not to bi: Acute erythroid leukemias and hematopoietic lineage choice.

Acute erythroid leukemia (AEL) is an acute leukemia characterized by erythroid lineage transformation. The World Health Organization (WHO) 2008 classification recognized two subtypes of AEL: bilineage erythroleukemia (erythroid/myeloid leukemia) and pure erythroid leukemia. The erythroleukemia subtype was removed in the updated 2016 WHO classification, with about half of cases reclassified as myelodysplastic syndrome (MDS) and half as acute myeloid leukemia (AML). Diagnosis and classification are currently based on morphology using standard blast cutoffs, without integration of underlying genomic and other molecular features. Key outstanding questions are therefore whether AEL can be accurately diagnosed based solely on morphology or whether genetic or other molecular criteria should be included in its classification, and whether considering AEL as an entity distinct from AML and MDS is clinically relevant. We discuss recent work on the molecular basis of AEL, including the identification of mutations causative of AEL and of transcriptional and epigenetic features that can be used to distinguish AEL from MDS and nonerythroid AML, and the prognostic value of these molecular features.

Role of c-Myc haploinsufficiency in the maintenance of HSCs in mice.

This study was conducted to determine the dosage effect of c-Myc on hematopoiesis and its distinct role in mediating the Wnt/ß-catenin pathway in hematopoietic stem cell (HSC) and bone marrow niche cells. c-Myc haploinsufficiency led to ineffective hematopoiesis by inhibiting HSC self-renewal and quiescence and by promoting apoptosis. We have identified Nr4a1, Nr4a2, and Jmjd3, which are critical for the maintenance of HSC functions, as previously unrecognized downstream targets of c-Myc in HSCs. c-Myc directly binds to the promoter regions of Nr4a1, Nr4a2, and Jmjd3 and regulates their expression. Our results revealed that Nr4a1 and Nr4a2 mediates the function of c-Myc in regulating HSC quiescence, whereas all 3 genes contribute to the function of c-Myc in the maintenance of HSC survival. Adenomatous polyposis coli (Apc) is a negative regulator of the Wnt/ß-catenin pathway. We have provided the first evidence that Apc haploinsufficiency induces a blockage of erythroid lineage differentiation through promoting secretion of IL6 in bone marrow endothelial cells. We found that c-Myc haploinsufficiency failed to rescue defective function of Apc-deficient HSCs in vivo but it was sufficient to prevent the development of severe anemia in Apc-heterozygous mice and to significantly prolong the survival of those mice. Furthermore, we showed that c-Myc-mediated Apc loss induced IL6 secretion in endothelial cells, and c-Myc haploinsufficiency reversed the negative effect of Apc-deficient endothelial cells on erythroid cell differentiation. Our studies indicate that c-Myc has a context-dependent role in mediating the function of Apc in hematopoiesis.

Requirement for antiapoptotic MCL-1 during early erythropoiesis.

Although BCL-xL is critical to the survival of mature erythrocytes, it is still unclear whether other antiapoptotic molecules mediate survival during earlier stages of erythropoiesis. Here, we demonstrate that erythroid-specific Mcl1 deletion results in embryonic lethality beyond embryonic day 13.5 as a result of severe anemia caused by a lack of mature red blood cells (RBCs). Mcl1-deleted embryos exhibit stunted growth, ischemic necrosis, and decreased RBCs in the blood. Furthermore, we demonstrate that MCL-1 is only required during early definitive erythropoiesis; during later stages, developing erythrocytes become MCL-1 independent and upregulate the expression of BCL-xL. Functionally, MCL-1 relies upon its ability to prevent apoptosis to promote erythroid development because codeletion of the proapoptotic effectors Bax and Bak can overcome the requirement for MCL-1 expression. Furthermore, ectopic expression of human BCL2 in erythroid progenitors can compensate for Mcl1 deletion, indicating redundancy between these 2 antiapoptotic family members. These data clearly demonstrate a requirement for MCL-1 in promoting survival of early erythroid progenitors.

Genome-wide analysis of pseudogenes reveals HBBP1's human-specific essentiality in erythropoiesis and implication in ß-thalassemia.

The human genome harbors 14,000 duplicated or retroposed pseudogenes. Given their functionality as regulatory RNAs and low conservation, we hypothesized that pseudogenes could shape human-specific phenotypes. To test this, we performed co-expression analyses and found that pseudogene exhibited tissue-specific expression, especially in the bone marrow. By incorporating genetic data, we identified a bone-marrow-specific duplicated pseudogene, HBBP1 (η-globin), which has been implicated in ß-thalassemia. Extensive functional assays demonstrated that HBBP1 is essential for erythropoiesis by binding the RNA-binding protein (RBP), HNRNPA1, to upregulate TAL1, a key regulator of erythropoiesis. The HBBP1/TAL1 interaction contributes to a milder symptom in ß-thalassemia patients. Comparative studies further indicated that the HBBP1/TAL1 interaction is human-specific. Genome-wide analyses showed that duplicated pseudogenes are often bound by RBPs and less commonly bound by microRNAs compared with retropseudogenes. Taken together, we not only demonstrate that pseudogenes can drive human evolution but also provide insights on their functional landscapes.

Histidine decarboxylase deficiency inhibits NBP-induced extramedullary hematopoiesis by modifying bone marrow and spleen microenvironments.

Histidine decarboxylase (HDC), a histamine synthase, is expressed in various hematopoietic cells and is induced by hematopoietic cytokines such as granulocyte colony-stimulating factor (G-CSF). We previously showed that nitrogen-containing bisphosphonate (NBP)-treatment induces extramedullary hematopoiesis via G-CSF stimulation. However, the function of HDC in NBP-induced medullary and extramedullary hematopoiesis remains unclear. Here, we investigated changes in hematopoiesis in wild-type and HDC-deficient (HDC-KO) mice. NBP treatment did not induce anemia in wild-type or HDC-KO mice, but did produce a gradual increase in serum G-CSF levels in wild-type mice. NBP treatment also enhanced Hdc mRNA expression and erythropoiesis in the spleen and reduced erythropoiesis in bone marrow and the number of vascular adhesion molecule 1 (VCAM-1)-positive macrophages in wild-type mice, as well as increased the levels of hematopoietic progenitor cells and proliferating cells in the spleen and enhanced expression of bone morphogenetic protein 4 (Bmp4), CXC chemokine ligand 12 (Cxcl12), and hypoxia inducible factor 1 (Hif1) in the spleen. However, such changes were not observed in HDC-KO mice. These results suggest that histamine may affect hematopoietic microenvironments of the bone marrow and spleen by changing hematopoiesis-related factors in NBP-induced extramedullary hematopoiesis.

Altered Ca2+ Homeostasis in Red Blood Cells of Polycythemia Vera Patients Following Disturbed Organelle Sorting during Terminal Erythropoiesis.

Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2V617F, with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.