Regulation Of Hematopoiesis Research Articles

Cohesin RAD21 Gene Promoter Methylation in Patients with Acute Myeloid Leukemia.

Aberrant gene promoter methylation is one of the hallmarks of Acute Myeloid Leukemia (AML). RAD21 is an important gene, implicated in sister chromatids cohesion, DNA repair, the regulation of gene transcription, apoptosis and hematopoiesis. In this study, we investigate the possible implication of RAD21 promoter methylation in AML pathogenesis using a cohort of AML patients and a cohort of healthy individuals. RAD21 promoter methylation was found in 24% of patients and in none of the controls (p = 0.023), indicating a possible contribution to AML development. Interestingly, a statistically higher frequency of RAD21 methylation was observed in patients with trisomy 8 (9/21, 42.9%, p = 0.021), while none of the patients with aberrations of chromosome 11 had RAD21 gene promoter methylation (0%, 0/11, p = 0.048). Patients with monosomal and complex karyotypes showed low frequencies of RAD21 methylation (7.7% and 15.4%, respectively) without reaching statistical significance. Moreover, ASXL1 mutations were not found to be associated with RAD21 methylation. This is the first study which provides evidence for a possible pathogenetic role of RAD21 promoter methylation in AML development and especially in AML with trisomy 8. Further studies of RAD21 promoter methylation in large series of different AML genetic subgroups may contribute to the elucidation of AML pathogenesis and to the identification of new epigenetic biomarkers with diagnostic and prognostic value.

A CXCR4 partial agonist improves immunotherapy by targeting polymorphonuclear myeloid-derived suppressor cells and cancer-driven granulopoiesis.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are pathologically activated neutrophils that potently impair immunotherapy responses. The chemokine receptor CXCR4, a central regulator of hematopoiesis, represents an attractive PMN-MDSC target1. Here, we fused a secreted CXCR4 partial agonist TFF2 to mouse serum albumin (MSA) and demonstrated that TFF2-MSA peptide synergized with anti-PD-1 to induce tumor regression or eradication, inhibited distant metastases, and prolonged survival in multiple gastric cancer (GC) models. Using histidine decarboxylase (Hdc)-GFP transgenic mice to track PMN-MDSC in vivo , we found TFF2-MSA selectively reduced the immunosuppressive Hdc-GFP + CXCR4 hi tumor PMN-MDSCs while preserving proinflammatory neutrophils, thereby boosting CD8 + T cell-mediated anti-tumor response together with anti-PD-1. Furthermore, TFF2-MSA systemically reduced PMN-MDSCs and bone marrow granulopoiesis. In contrast, CXCR4 antagonism plus anti-PD-1 failed to provide a similar therapeutic benefit. In GC patients, expanded PMN-MDSCs containing a prominent CXCR4 + LOX-1 + subset are inversely correlated with the TFF2 level and CD8 + T cells in circulation. Collectively, our studies introduce a strategy of using CXCR4 partial agonism to restore anti-PD-1 sensitivity in GC by targeting PMN-MDSCs and granulopoiesis.

7260 Nuclear Corepressor 1 (NCoR1) plays important role in the regulation of insulin gene transcription in mouse Min6 pancreatic beta cells

Abstract Disclosure: H. Shimizu: None. Y. Horibata: None. I. Amano: None. M.J. Ritter: None. M. Ohyama: None. M. Domae: None. H. Sugimoto: None. A.N. Hollenberg: None. Background: Nuclear Corepressor 1 (NCoR1) is a transcriptional corepressor which has been recognized as an important player in the regulation of hepatic lipogenesis and hematopoiesis in mouse embryo. NCoR1 is also widely expressed in mouse insulin sensitive organs, for instance adipocyte, skeletal muscle, and hepatocytes. Mouse genetic deletion strategies of either NCoR1 or SMRT(NCoR2; an ortholog corepressor of NCoR1) demonstrated that each corepressor effects the insulin sensitivity in the tissues. However, the role of each corepressor in pancreatic beta cell is not still elucidated. Methods: To clarify this, we took the gene-targeting strategy for NCoR1 using CRISPR Cas-9 and made the pancreatic beta Min6 clone which lacks NCoR1 protein (Min6-NCoR1KO; NKO cells). For this study, wild type Min6 cells (WT) and NKO cells were cultured in the Dulbecco's DMEM medium with 10% fetal bovine serum for 4-5 days, and the expression analyses compared between two cell types were performed. Results: Both comprehensive proteomics and following expression analyses compared between WT and NKO cells demonstrated that NKO cells have significantly lower expression level of insulin (INS-1 and INS-2), accompanied with high expression of histone deacetylase 6 (HDAC6). For further analyses, we also made the Min6 clone which lacks SMRT(Min6-SMRTKO; SKO cells). Interestingly, SKO cell had higher insulin and lower HDAC6 levels, accompanied with significant up-regulation of NCoR1. Conclusion: Taken together, these data indicate that NCoR1 has dominant effects in the regulation of both histone deacetylation and insulin gene transcription in Min6 cells. Further investigations for the function of NCoR1 in the insulin gene transcription observed in Min6 cells will be needed. Presentation: 6/2/2024

Genome-Wide Methylation Profiling of Peripheral T-Cell Lymphomas Identifies TRIP13 as a Critical Driver of Tumor Proliferation and Survival.

Cytosine methylation contributes to the regulation of gene expression and normal hematopoiesis in mammals. It is catalyzed by the family of DNA methyltransferases that include DNMT1, DNMT3A, and DNMT3B. Peripheral T-cell lymphomas (PTCLs) represent aggressive mature T-cell malignancies exhibiting a broad spectrum of clinical features with poor prognosis and inadequately understood molecular pathobiology. To better understand the molecular landscape and identify candidate genes involved in disease maintenance, we profiled DNA methylation and gene expression of PTCLs. We found that the methylation patterns in PTCLs are deregulated and heterogeneous but share 767 hypo- and 567 hypermethylated differentially methylated regions (DMRs) along with 231 genes up- and 91 genes downregulated in all samples, suggesting a potential association with tumor development. We further identified 39 hypomethylated promoters associated with increased gene expression in the majority of PTCLs. This putative oncogenic signature included the TRIP13 (thyroid hormone receptor interactor 13) gene whose genetic and pharmacologic inactivation inhibited the proliferation of T-cell lines by inducing G2-M arrest and apoptosis. Our data thus show that human PTCLs have a significant number of recurrent methylation alterations that may affect the expression of genes critical for proliferation whose targeting might be beneficial in anti-lymphoma treatments.

Deciphering the role of HLF in idiopathic orbital inflammation: integrative analysis via bioinformatics and machine learning techniques

Idiopathic orbital inflammation, formerly known as NSOI (nonspecific orbital inflammation), is characterized as a spectrum disorder distinguished by the polymorphic infiltration of lymphoid tissue, presenting a complex and poorly understood etiology. Recent advancements have shed light on the HLF (Human lactoferrin), proposing its critical involvement in the regulation of hematopoiesis and the maintenance of innate mucosal immunity. This revelation has generated significant interest in exploring HLF's utility as a biomarker for NSOI, despite the existing gaps in our understanding of its biosynthetic pathways and operational mechanisms. Intersecting multi-omic datasets—specifically, common differentially expressed genes between GSE58331 and GSE105149 from the Gene Expression Omnibus and immune-related gene compendiums from the ImmPort database—we employed sophisticated analytical methodologies, including Lasso regression and support vector machine-recursive feature elimination, to identify HLF. Gene set enrichment analysis and gene set variation analysis disclosed significant immune pathway enrichment within gene sets linked to HLF. The intricate relationship between HLF expression and immunological processes was further dissected through the utilization of CIBERSORT and ESTIMATE algorithms, which assess characteristics of the immune microenvironment, highlighting a noteworthy association between increased HLF expression and enhanced immune cell infiltration. The expression levels of HLF were corroborated using data from the GSE58331 dataset, reinforcing the validity of our findings. Analysis of 218 HLF-related differentially expressed genes revealed statistically significant discrepancies. Fifteen hub genes were distilled using LASSO and SVM-RFE algorithms. Biological functions connected with HLF, such as leukocyte migration, ossification, and the negative regulation of immune processes, were illuminated. Immune cell analysis depicted a positive correlation between HLF and various cells, including resting mast cells, activated NK cells, plasma cells, and CD8 T cells. Conversely, a negative association was observed with gamma delta T cells, naive B cells, M0 and M1 macrophages, and activated mast cells. Diagnostic assessments of HLF in distinguishing NSOI showed promising accuracy. Our investigation delineates HLF as intricately associated with NSOI, casting light on novel biomarkers for diagnosis and progression monitoring of this perplexing condition.

PURE-seq identifies Egr1 as a Potential Master Regulator in Murine Aging by Sequencing Long-Term Hematopoietic Stem Cells.

Single-cell transcriptomics is valuable for uncovering individual cell properties, particularly in highly heterogeneous systems. However, this technique often results in the analysis of many well-characterized cells, increasing costs and diluting rare cell populations. To address this, we developed PURE-seq (PIP-seq for Rare-cell Enrichment and Sequencing) for scalable sequencing of rare cells. PURE-seq allows direct cell loading from FACS into PIP-seq reactions, minimizing handling and reducing cell loss. PURE-seq reliably captures rare cells, with 60 minutes of sorting capturing tens of cells at a rarity of 1 in 1,000,000. Using PURE-seq, we investigated murine long-term hematopoietic stem cells and their transcriptomes in the context of hematopoietic aging, identifying Egr1 as a potential master regulator of hematopoiesis in the aging context. PURE-seq offers an accessible and reliable method for isolating and sequencing cells that are currently too rare to capture successfully with existing methods.

Loss of Dnajc21 leads to cytopenia and altered nucleotide metabolism in zebrafish

Mutations in the DNAJC21 gene were recently described in Shwachman–Diamond syndrome (SDS), a bone marrow failure syndrome with high predisposition for myeloid malignancies. To study the underlying biology in hematopoiesis regulation and disease, we generated the first in vivo model of Dnajc21 deficiency using the zebrafish. Zebrafish dnajc21 mutants phenocopy key SDS patient phenotypes such as cytopenia, reduced growth, and defective protein synthesis. We show that cytopenia results from impaired hematopoietic differentiation, accumulation of DNA damage, and reduced cell proliferation. The introduction of a biallelic tp53 mutation in the dnajc21 mutants leads to the development of myelodysplastic neoplasia-like features defined by abnormal erythroid morphology and expansion of hematopoietic progenitors. Using transcriptomic and metabolomic analyses, we uncover a novel role for Dnajc21 in nucleotide metabolism. Exogenous nucleoside supplementation restores neutrophil counts, revealing an association between nucleotide imbalance and neutrophil differentiation, suggesting a novel mechanism in dnajc21-mutant SDS biology.

PURE-seq identifies Egr1 as a Potential Master Regulator in Murine Aging by Sequencing Long-Term Hematopoietic Stem Cells.

Single-cell transcriptomics is valuable for uncovering individual cell properties, particularly in highly heterogeneous systems. However, this technique often results in the analysis of many well-characterized cells, increasing costs and diluting rare cell populations. To address this, we developed PURE-seq (PIP-seq for Rare-cell Enrichment and Sequencing) for scalable sequencing of rare cells. PURE-seq allows direct cell loading from FACS into PIP-seq reactions, minimizing handling and reducing cell loss. PURE-seq reliably captures rare cells, with 60 minutes of sorting capturing tens of cells at a rarity of 1 in 1,000,000. Using PURE-seq, we investigated murine long-term hematopoietic stem cells and their transcriptomes in the context of hematopoietic aging, identifying Egr1 as a potential master regulator of hematopoiesis in the aging context. PURE-seq offers an accessible and reliable method for isolating and sequencing cells that are currently too rare to capture successfully with existing methods.

Interrelations between iron content and immune dysfunctions in frequently ill children of the Aral Sea Region

The widespread occurrence of immune-dependent diseases and disturbances in iron metabolism, as well as their co-occurrence and the impact of iron deficiency on the immune system, underscore the relevance of this study. Modern data also indicate the involvement of iron in regulating cytokine production and protein synthesis by lymphocytes. In addition to the age-related characteristics of the child’s immune system, a significant factor in the development of inadequate course of the infectious process is the disruption of immune system adaptation, which can be caused by frequent infections, other somatic diseases such as anemia, and environmental factors. The aim of this study is to assess the relationship between iron levels and immune dysfunction in children with frequent acute respiratory diseases living in the Aral Sea region. A total of 127 children, who are native residents living at the epicenter of the ecological crisis, were examined. The children’s ages ranged from 1 to 5 years old. Among them, there were 105 frequently ill children (with 6 or more episodes of acute respiratory infections per year) with iron-deficiency anemia (IDA), and 22 healthy children of the same age. All children underwent clinical examination and standard laboratory tests, including complete blood count, relative content and ratio of T and B lymphocytes, as well as the levels of pro- and anti-inflammatory cytokines (IL-2 and 4) and ferritin in the serum. According to the results of the conducted research, it was found that among frequently ill children with iron-deficiency anemia (IDA), there is a moderate positive correlation between the relative quantity of T lymphocytes and the hemoglobin level (r = +0.62), as well as a strong positive correlation with the serum iron level (r = +0.78). This indicates a significant influence of iron deficiency on the level of cellular immunity. Clinical data also indicate a higher risk of respiratory infections development in children with IDA compared to the control group. Ferritin, hemoglobin, lymphocytes, and cytokines act as positive regulators of hematopoiesis in IDA conditions and contribute to the compensation of anemia during iron deficiency replenishment.

Expression of CD45 in non-hematopoietic cells: implications in regenerative medicine and disease management.

CD45 plays a crucial role in the regulation of hematopoiesis. However, a comprehensive understanding of its role in non-hematopoietic cells is lacking. Several tissue precursors express CD45, indicating its crucial role in tissue regeneration. These precursors would fall prey to the recent therapies involving CD45 as a target. CD45+ double-positive tumor cells contribute to cancer progression, but whether CD45 is involved in the process needs to be investigated. Recently, we showed that aging induces CD45 expression in mesenchymal stromal cells and affects their differentiation potential. In this review, we, for the first time, unravel the important implications of the expression of CD45 in non-hematopoietic cells and provide novel insights into its potential therapeutic target in regenerative medicine and disease management.

Sex-dependent niche responses modulate steady-state and regenerative hematopoiesis

Hematopoietic stem cells (HSCs) adapt to organismal blood production needs by balancing self-renewal and differentiation, adjusting to physiological demands and external stimuli. While sex differences have been implicated in differential hematopoietic function in males vs. females, the mediators responsible for these effects require further study. Here, we characterize hematopoiesis at steady state and during regeneration following hematopoietic stem cell transplantation (HST). RNA sequencing of lineage(-) bone marrow cells from C57/Bl6 mice revealed a broad transcriptional similarity between the sexes. However, we identified distinct sex differences in key biological pathways, with female cells showing reduced expression of signatures involved in inflammation and an enrichment of genes related to glycolysis, hypoxia, and cell cycle regulation, suggesting a more quiescent and less inflammatory profile compared to male cells. To determine the functional impacts of the observed transcriptomic differences, we performed sex-matched and mismatched transplantation studies of lineage(-) donor cells. During short-term 56-day HST recovery we found a male donor cell proliferative advantage, coinciding with elevated serum TNF-α, and a male recipient engraftment advantage, coinciding with increased serum CXCL12. Together, we show that sex-specific cell responses, marked by differing expression of pathways regulating metabolism, hypoxia, and inflammation, shapes normal and regenerative hematopoiesis, with implications for the clinical understanding of hematopoietic function.

HnRNPA0 promotes MYB expression by interacting with enhancer lncRNA MY34UE-AS in human leukemia cells

MYB is a key regulator of hematopoiesis and erythropoiesis, and dysregulation of MYB is closely involved in the development of leukemia, however the mechanism of MYB regulation remains still unclear so far. Our previous study identified a long noncoding RNA (lncRNA) derived from the −34 kb enhancer of the MYB locus, which can promote MYB expression, the proliferation and migration of human leukemia cells, and is therefore termed MY34UE-AS. Then the interacting partner proteins of MY34UE-AS were identified and studied in the present study. hnRNPA0 was identified as a binding partner of MY34UE-AS through RNA pulldown assay, which was further validated through RNA immunoprecipitation (RIP). hnRNPA0 interacted with MY34UE-AS mainly through its RRM2 domain. hnRNPA0 overexpression upregulated MYB and increased the proliferation and migration of K562 cells, whereas hnRNPA0 knockdown showed opposite effects. Rescue experiments showed MY34UE-AS was required for above mentioned functions of hnRNPA0. These results reveal that hnRNPA0 is involved in leukemia through upregulating MYB expression by interacting with MY34UE-AS, suggesting that the hnRNPA0/MY34UE-AS axis could serve as a potential target for leukemia treatment.

Domain-based analysis of RUNX1somatic mutations in myeloid neoplasms.

6542 Background: RUNX1is a master regulator of hematopoiesis. ELN2022 risk stratification categorizes RUNX1 as an AML/MDS gene associated with poor prognosis. Functional domains in RUNX1are the Runt homology domain (RHD) and a transactivation domain (TAD), with DNA binding and co-activator recruitment functions, respectively. Domain-based analysis can elucidate the functional and biological impact of somatic RUNX1mutations, which have not yet been reported in the literature. Methods: Sequencing data from Karmanos Cancer Institute and a publicly available meta-analytic cohort [Awada et al., Blood 2021, Kewan et al., Nature Communications 2023, cBioPortal (Cerami et al., 2012) and AACR GENIE (v 13.1)] were used to construct a larger dataset of 16,565 patients with myeloid neoplasms. Baseline clinical and molecular characteristics were noted. The chi-square test was used to study various parameters described, and Kaplan-Meier curves were used to estimate survival. Results: RUNX1was mutated in 10.8% (1804/16,565) of patients. Median OS (mOS) for RUNX1MT vs. RUNX1WT was significantly worse at 16.4 vs 23.4 mos. (p < 0.0001). 990 patients with somatic mutations were studied; germline variants were screened and excluded if VAF was between 40-60%. Among somatic RUNX1MT patients, primary AML was the most frequent at 52.8%, followed by secondary AML-35.7% and MDS-9%. Most patients had mutations in the RHD (53.8%), followed by TAD (22%), and the remaining in non-RHD, non-TAD zones (NRNT). There was no statistically significant difference in OS by domain stratification; TAD had the numerically least mOS at 13.6 mos, RHD at 16.1 mos, and NRNT at 20.4 mos. The median age of patients was 70.9 (62-76) years, similar across domains. TAD patients were less likely to be male when compared to RHD (47% vs. 56%, p=0.04). No specific enrichment of disease type or abnormal karyotype was observed across domains. Co-mutational profiles across RHD and TAD domains among all diseases showed spliceosome co-signature enriched in RHD vs. TAD (59% vs. 46%, p=0.01). NPM1was noted to be inversely correlated with RUNX1 in general, with 1% and 3.5% in RHD and TAD. ASXL1was the most frequently distributed co-mutation in both groups, but not statistically different (41% vs. 33%, p=0.11). TAD patients with spliceosome co-mutation had worse mOS (11.4 mos) than those without spliceosome mutation (16.2 mos, p=0.02) or RHD patients with spliceosome co-mutation (16 mos, p=0.001). Missense mutations were strongly associated with RHD (55.3% vs. 7.9%); p <0.00001, while frameshift mutations were enriched in TAD (24.1% vs. 69.8%); p<0.0001. Conclusions: Domain-based analysis of somatic RUNX1MT myeloid neoplasms reveals TAD mutation to be common in females, frequently a frameshift mutation, and numerically has the worst survival. There appears to be a unique spliceosome signature enrichment in RHD compared to TAD, with worse OS in TAD patients with spliceosome co-mutations overall.

Role of joint adipose tissues in osteoarthritis

Osteoarthritis (OA) is the most common musculoskeletal disease, without any curative treatment. Obesity being the main modifiable risk factor for OA, much attention focused on the role of adipose tissues (AT). In addition to the involvement of visceral and subcutaneous AT via systemic ways, many arguments also highlight the involvement of local AT, present in joint tissues. Local AT include intra-articular AT (IAAT), which border the synovium, and bone marrow AT (BMAT) localized within marrow cavities in the bones. This review describes the known features and involvement of IAAT and BMAT in joint homeostasis and OA. Recent findings evidence that alteration in magnetic resonance imaging signal intensity of infrapatellar fat pad can be predictive of the development and progression of knee OA. IAAT and synovium are partners of the same functional unit; IAAT playing an early and pivotal role in synovial inflammation and fibrosis and OA pain. BMAT, whose functions have only recently begun to be studied, is in close functional interaction with its microenvironment. The volume and molecular profile of BMAT change according to the pathophysiological context, enabling fine regulation of haematopoiesis and bone metabolism. Although its role in OA has not yet been studied, the localization of BMAT, its functions and the importance of the bone remodelling processes that occur in OA argue in favour of a role for BMAT in OA.

Overlapping Stromal Alterations in Myeloid and Lymphoid Neoplasms.

Myeloid and lymphoid neoplasms share the characteristics of potential bone marrow infiltration as a primary or secondary effect, which readily leads to hematopoietic insufficiency. The mechanisms by which clonal malignant cells inhibit normal hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) have not been unraveled so far. Given the pivotal role of mesenchymal stromal cells (MSCs) in the regulation of hematopoiesis in the BM niche it is assumed that MSCs also play a relevant role in the pathogenesis of hematological neoplasms. We aimed to identify overlapping mechanisms in MSCs derived from myeloid and lymphoid neoplasms contributing to disease progression and suppression of HSPCs to develop interventions that target these mechanisms. MSCs derived from healthy donors (n = 44) and patients diagnosed with myeloproliferative neoplasia (n = 11), myelodysplastic syndromes (n = 16), or acute myeloid leukemia (n = 25) and B-Non-Hodgkin lymphoma (n = 9) with BM infiltration and acute lymphoblastic leukemia (n = 9) were analyzed for their functionality and by RNA sequencing. A reduced growth and differentiation capacity of MSCs was found in all entities. RNA sequencing distinguished both groups but clearly showed overlapping differentially expressed genes, including major players in the BMP/TGF and WNT-signaling pathway which are crucial for growth, osteogenesis, and hematopoiesis. Functional alterations in healthy MSCs were inducible by exposure to supernatants from malignant cells, implicating the involvement of these factors in disease progression. Overall, we were able to identify overlapping factors that pose potential future therapeutic targets.

BRD9 regulates normal human hematopoietic stem cell function and lineage differentiation

Bromodomain containing protein 9 (BRD9), a member of the non-canonical BRG1/BRM-associated factor (ncBAF) chromatin remodeling complex, has been implicated as a synthetic lethal target in AML but its function in normal human hematopoiesis is unknown. In hematopoietic stem and progenitor cells (HSPC) genomic or chemical inhibition of BRD9 led to a proliferative disadvantage and loss of stem cells in vitro. Human HSPCs with reduced BRD9 protein levels produced lower numbers of immature mixed multipotent GEMM colonies in semi-solid media. In lineage-promoting culture conditions, cells with reduced BRD9 levels failed to differentiate into the megakaryocytic lineage and showed delayed differentiation into erythroid cells but enhanced terminal myeloid differentiation. HSPCs with BRD9 knock down (KD) had reduced long-term multilineage engraftment in a xenotransplantation assay. An increased number of downregulated genes in RNAseq analysis after BRD9 KD coupled with a gain in chromatin accessibility at the promoters of several repressive transcription factors (TF) suggest that BRD9 functions in the maintenance of active transcription during HSC differentiation. In particular, the hematopoietic master regulator GATA1 was identified as one of the core TFs regulating the gene networks modulated by BRD9 loss in HSPCs. BRD9 inhibition reduced a GATA1-luciferase reporter signal, further suggesting a role for BRD9 in regulating GATA1 activity. BRD9 is therefore an additional example of epigenetic regulation of human hematopoiesis.

#1995 Runt-related Transcription Factor 1 (RUNX1) is a mediator of acute kidney injury

Abstract Background and Aims A better understanding of the pathogenesis of acute kidney injury (AKI) may lead to new therapeutic approaches. Kidney transcriptomics analyses of murine folic acid-induced AKI (FA-AKI) identified Runx1 as the most upregulated RUNX family gene. RUNX1 is a key regulator of hematopoiesis, associated to leukemias and other non-immunological cancers and modulates myoblast proliferation during muscle regeneration and cardiac remodelling after myocardial infarction. In kidney diseases, RUNX1 is overexpressed in polycystic kidney disease and kidney cancer and favours fibrosis. Method We examined the expression of RUNX1 in folic acid-AKI (FA-AKI), in bacterial lipopolysaccharide (LPS)-induced cytokine storm-AKI (CS-AKI) and in human AKI. For this, female 12- to 14-week-old C57BL/6J wild type (WT) mice, received a single intraperitoneal (i.p) injection of acid folic (250 mg/kg), LPS (5 mg/kg) or vehicle. In cultured murine tubular MCT cells, we explored the expression and role of RUNX1 in response to the cytokine TWEAK or LPS. The chemical inhibitor of RUNX1 Ro5-3335 and a specific small interfering RNA (siRNA) against RUNX1 were used in cultured cells to explore the role of RUNX1 in TWEAK and LPS-induced inflammation. Ro5-3335 was also used in murine AKI (FA-AKI and CS-AKI) to test its potential as a therapeutic target. In all experimental models, plasma samples were collected to assess renal function. Kidneys were collected for RNA (RT-PCR), protein (Western blot, ELISA, immunohistochemistry) and histopathologic studies. Results Kidney transcriptomics identified Runx1 as the most upregulated Runx gene in FA-AKI at 24 h and the most overactive transcription factor in upstream regulator analysis. RUNX1 overexpression in FA-AKI was validated at mRNA and protein levels and localized mainly to tubular cell nuclei. CS-AKI also upregulated kidney RUNX1 in nuclei. In a transcriptomics array of murine tubular cells stimulated with TWEAK for 6 h, Runx1 was upregulated compared with non-stimulated cells, while Runx2 and Runx3 were not. RT-PCR confirmed an early upregulation of Runx1 (3 h) followed by a progressive return to baseline by 24 h. TWEAK also increased total and nuclear RUNX1 protein levels from 6 h onwards. Since kidney RUNX1 is also increased in CS-AKI, we tested whether LPS regulated RUNX1 in cultured tubular cells. LPS transiently increased Runx1 mRNA expression and it also increased whole cell and nuclear RUNX1 protein levels. Mechanistically, RUNX1 bound to the Il-6 gene promoter in response to TWEAK and LPS, and RUNX1 targeting with the chemical inhibitor Ro5-3335 or a specific siRNA prevented the TWEAK- and LPS-induced upregulation of IL-6. NFκB1 p50 pathway is involved in the RUNX1/IL-6 axis since the specific p50 inhibitor SN50 decreased the nuclear accumulation of RUNX1 and IL-6 expression in TWEAK- or LPS-stimulated MCT cells. In vivo, preventive Ro5-3335 improved kidney function and reduced inflammation in FA-AKI and CS-AKI. Moreover, in FA-AKI, Ro5-3335 also prevented the expression of profibrotic genes. However, Ro5-3335 administration after the insult only improved kidney function in CS-AKI. Kidney transcriptomics identified inflammatory genes and transcription factors such as Yap1 and p53 as key targets of Ro5-3335 in CS-AKI. Conclusion RUNX1 contributes to AKI by driving the expression of genes involved in inflammation and represents a novel therapeutic target in AKI.

Inducible CXCL12/CXCR4–dependent extramedullary hematopoietic niches in the adrenal gland

AbstractAdult hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow (BM) hematopoietic niche, which regulates HSPC quiescence, self-renewal, and commitment in a demand-adapted manner. Although the complex BM niche is responsible for adult hematopoiesis, evidence exists for simpler, albeit functional and more accessible, extramedullary hematopoietic niches. Inspired by the anecdotal description of retroperitoneal hematopoietic masses occurring at higher frequency upon hormonal dysregulation within the adrenal gland, we hypothesized that the adult adrenal gland could be induced into a hematopoietic-supportive environment in a systematic manner, thus revealing mechanisms underlying de novo niche formation in the adult. Here, we show that upon splenectomy and hormonal stimulation, the adult adrenal gland of mice can be induced to recruit and host functional HSPCs, capable of serial transplantation, and that this phenomenon is associated with de novo formation of platelet-derived growth factor receptor α/leptin receptor (PDGFRα+/LEPR+/–)–expressing stromal nodules. We further show in CXCL12–green fluorescent protein reporter mice that adrenal glands contain a stromal population reminiscent of the CXCL12-abundant reticular cells, which compose the BM HSPC niche. Mechanistically, HSPC homing to hormonally induced adrenal glands was found dependent on the CXCR4–CXCL12 axis. Mirroring our findings in mice, we found reticular CXCL12+ cells coexpressing master niche regulator FOXC1 in primary samples from human adrenal myelolipomas, a benign tumor composed of adipose and hematopoietic tissue. Our findings reignite long-standing questions regarding hormonal regulation of hematopoiesis and provide a novel model to facilitate the study of adult-specific inducible hematopoietic niches, which may pave the way to therapeutic applications.

An atlas of spider development at single-cell resolution provides new insights into arthropod embryogenesis

Spiders are a diverse order of chelicerates that diverged from other arthropods over 500 million years ago. Research on spider embryogenesis, particularly studies using the common house spider Parasteatoda tepidariorum, has made important contributions to understanding the evolution of animal development, including axis formation, segmentation, and patterning. However, we lack knowledge about the cells that build spider embryos, their gene expression profiles and fate. Single-cell transcriptomic analyses have been revolutionary in describing these complex landscapes of cellular genetics in a range of animals. Therefore, we carried out single-cell RNA sequencing of P. tepidariorum embryos at stages 7, 8 and 9, which encompass the establishment and patterning of the body plan, and initial differentiation of many tissues and organs. We identified 20 cell clusters, from 18.5 k cells, which were marked by many developmental toolkit genes, as well as a plethora of genes not previously investigated. We found differences in the cell cycle transcriptional signatures, suggestive of different proliferation dynamics, which related to distinctions between endodermal and some mesodermal clusters, compared with ectodermal clusters. We identified many Hox genes as markers of cell clusters, and Hox gene ohnologs were often present in different clusters. This provided additional evidence of sub- and/or neo-functionalisation of these important developmental genes after the whole genome duplication in an arachnopulmonate ancestor (spiders, scorpions, and related orders). We also examined the spatial expression of marker genes for each cluster to generate a comprehensive cell atlas of these embryonic stages. This revealed new insights into the cellular basis and genetic regulation of head patterning, hematopoiesis, limb development, gut development, and posterior segmentation. This atlas will serve as a platform for future analysis of spider cell specification and fate, and studying the evolution of these processes among animals at cellular resolution.

RUNX1 C-terminal mutations impair blood cell differentiation by perturbing specific enhancer-promoter networks

AbstractThe transcription factor RUNX1 is a master regulator of hematopoiesis and is frequently mutated in myeloid malignancies. Mutations in its runt homology domain (RHD) frequently disrupt DNA binding and result in loss of RUNX1 function. However, it is not clearly understood how other RUNX1 mutations contribute to disease development. Here, we characterized RUNX1 mutations outside of the RHD. Our analysis of the patient data sets revealed that mutations within the C-terminus frequently occur in hematopoietic disorders. Remarkably, most of these mutations were nonsense or frameshift mutations and were predicted to be exempt from nonsense-mediated messenger RNA decay. Therefore, this class of mutation is projected to produce DNA-binding proteins that contribute to the pathogenesis in a distinct manner. To model this, we introduced the RUNX1R320∗ mutation into the endogenous gene locus and demonstrated the production of RUNX1R320∗ protein. Expression of RUNX1R320∗ resulted in the disruption of RUNX1 regulated processes such as megakaryocytic differentiation, through a transcriptional signature different from RUNX1 depletion. To understand the underlying mechanisms, we used Global RNA Interactions with DNA by deep sequencing (GRID-seq) to examine enhancer-promoter connections. We identified widespread alterations in the enhancer-promoter networks within RUNX1 mutant cells. Additionally, we uncovered enrichment of RUNX1R320∗ and FOXK2 binding at the MYC super enhancer locus, significantly upregulating MYC transcription and signaling pathways. Together, our study demonstrated that most RUNX1 mutations outside the DNA-binding domain are not subject to nonsense-mediated decay, producing protein products that act in concert with additional cofactors to dysregulate hematopoiesis through mechanisms distinct from those induced by RUNX1 depletion.