The ATF4-RPS19BP1 axis modulates ribosome biogenesis to promote erythropoiesis.

ABSTRACT: Hematopoietic differentiation is controlled by intrinsic regulators and the extrinsic hematopoietic niche. Activating transcription factor 4 (ATF4) plays a crucial role in the function of fetal and adult hematopoietic stem cell maintenance. However, the precise function of ATF4 in the bone marrow (BM) niche and the mechanism by which ATF4 regulates adult hematopoiesis remain largely unknown. Here, we used 4 cell-type-specific mouse Cre lines to achieve conditional knockout of Atf4 in Cdh5+ endothelial cells, Prx1+ BM stromal cells, Osx+ osteoprogenitor cells, and Mx1+ hematopoietic cells and uncovered the role of Atf4 in niche cells and hematopoiesis. Intriguingly, depletion of Atf4 in niche cells did not affect hematopoiesis; however, Atf4-deficient hematopoietic cells exhibited erythroid differentiation defects, leading to hypoplastic anemia. Mechanistically, ATF4 mediated direct regulation of Rps19bp1 transcription, which is, in turn, involved in 40 S ribosomal subunit assembly to coordinate ribosome biogenesis and promote erythropoiesis. Finally, we demonstrate that under conditions of 5-fluorouracil-induced stress, Atf4 depletion impedes the recovery of hematopoietic lineages, which requires efficient ribosome biogenesis. Taken together, our findings highlight the indispensable role of the ATF4-RPS19BP1 axis in the regulation of erythropoiesis.

Small extracellular vesicles derived from acute myeloid leukemia cells promote leukemogenesis by transferring miR-221-3p.

Small extracellular vesicles (sEVs) transfer cargos between cells and participate in various physiological and pathological processes through their autocrine and paracrine effects. However, the pathological mechanisms employed by sEV-encapsulated microRNAs (miRNAs) in acute myeloid leukemia (AML) are still obscure. In this study, we aimed to investigate the effects of AML cells-derived sEVs (AML-sEVs) on AML cells and delineate the underlying mechanisms. We initially used high-throughput sequencing to identify miR-221-3p as the miRNA prominently enriched in AML-sEVs. Our findings revealed that miR-221-3p promoted AML cell proliferation and leukemogenesis by accelerating cell cycle entry and inhibiting apoptosis. Furthermore, Gbp2 was confirmed as a target gene of miR-221-3p by dual luciferase reporter assays and rescue experiments. Additionally, AML-sEVs impaired the clonogenicity, particularly the erythroid differentiation ability, of hematopoietic stem and progenitor cells. Taken together, our findings reveal how sEVs-delivered miRNAs contribute to AML pathogenesis, which can be exploited as a potential therapeutic target to attenuate AML progression.

The bone-liver interaction modulates immune and hematopoietic function through Pinch-Cxcl12-Mbl2 pathway.

Mesenchymal stromal cells (MSCs) are used to treat infectious and immune diseases and disorders; however, its mechanism(s) remain incompletely defined. Here we find that bone marrow stromal cells (BMSCs) lacking Pinch1/2 proteins display dramatically reduced ability to suppress lipopolysaccharide (LPS)-induced acute lung injury and dextran sulfate sodium (DSS)-induced inflammatory bowel disease in mice. Prx1-Cre; Pinch1f/f; Pinch2-/- transgenic mice have severe defects in both immune and hematopoietic functions, resulting in premature death, which can be restored by intravenous injection of wild-type BMSCs. Single cell sequencing analyses reveal dramatic alterations in subpopulations of the BMSCs in Pinch mutant mice. Pinch loss in Prx1+ cells blocks differentiation and maturation of hematopoietic cells in the bone marrow and increases production of pro-inflammatory cytokines TNF-α and IL-1ß in monocytes. We find that Pinch is critical for expression of Cxcl12 in BMSCs; reduced production of Cxcl12 protein from Pinch-deficient BMSCs reduces expression of the Mbl2 complement in hepatocytes, thus impairing the innate immunity and thereby contributing to infection and death. Administration of recombinant Mbl2 protein restores the lethality induced by Pinch loss in mice. Collectively, we demonstrate that the novel Pinch-Cxcl12-Mbl2 signaling pathway promotes the interactions between bone and liver to modulate immunity and hematopoiesis and may provide a useful therapeutic target for immune and infectious diseases.

Ruxolitinib improves hematopoietic regeneration by restoring mesenchymal stromal cell function in acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is a severe complication of allogeneic hematopoietic stem cell transplantation. Hematopoietic dysfunction accompanied by severe aGVHD, which may be caused by niche impairment, is a long-standing clinical problem. However, how the bone marrow (BM) niche is damaged in aGVHD hosts is poorly defined. To comprehensively address this question, we used a haplo-MHC-matched transplantation aGVHD murine model and performed single-cell RNA-Seq of nonhematopoietic BM cells. Transcriptional analysis showed that BM mesenchymal stromal cells (BMSCs) were severely affected, with a reduction in cell ratio, abnormal metabolism, compromised differentiation potential, and defective hematopoiesis-supportive function, all of which were validated by functional assays. We found that ruxolitinib, a selective JAK1/2 inhibitor, ameliorated aGVHD-related hematopoietic dysfunction through a direct effect on recipient BMSCs, resulting in improved proliferation ability, adipogenesis/osteogenesis potential, mitochondria metabolism capacity, and crosstalk with donor-derived hematopoietic stem/progenitor cells. By inhibiting the JAK2/STAT1 pathway, ruxolitinib maintained long-term improvement of aGVHD BMSC function. Additionally, ruxolitinib pretreatment in vitro primed BMSCs to better support donor-derived hematopoiesis in vivo. These observations in the murine model were validated in patient samples. Overall, our findings suggest that ruxolitinib can directly restore BMSC function via the JAK2/STAT1 pathway and, in turn, improve the hematopoietic dysfunction caused by aGVHD.

Uncovering the emergence of HSCs in the human fetal bone marrow by single-cell RNA-seq analysis.

During fetal development, human hematopoietic stem cells (HSCs) colonize the bone marrow (BM), where they self-renew and sustain hematopoiesis throughout life; however, the precise timepoint at which HSCs seed the BM is unclear. We used single-cell RNA-sequencing to map the transcriptomic landscape of human fetal BM and spleen hematopoietic stem/progenitor cells (HSPCs) and their microenvironment from 10 to 14 post-conception weeks (PCWs). We further demonstrated that functional HSCs capable of reconstituting long-term multi-lineage hematopoiesis in adult NOG mice do not emerge in the BM until 12 PCWs. In contrast, functional HSCs were not detected in the spleen by 14 PCWs. By comparing the niche-HSPC interactions between BM and spleen, we identified ligand-receptor pairs likely to be involved in fetal HSC migration and maintenance. Our work paves the way for research into the mechanisms underlying HSC colonization in human fetal BM and provides invaluable resources for future studies on HSC development.

Emerging roles of extracellular vesicles in normal and malignant hematopoiesis.

Hematopoietic stem cells, regulated by their microenvironment (or "niche"), sustain the production of mature blood and immune cells. Leukemia cells remodel the microenvironment to enhance their survival, which is accompanied by the loss of support for normal hematopoiesis in hematologic malignancies. Extracellular vesicles (EVs) mediate intercellular communication in physiological and pathological conditions, and deciphering their functions in cell-cell interactions in the ecosystem can highlight potential therapeutic targets. In this Review, we illustrate the utility of EVs derived from various cell types, focusing on the biological molecules they contain and the behavioral alterations they can induce in recipient cells. We also discuss the potential for clinical application in hematologic malignancies, including EV-based therapeutic regimens, drug delivery via EVs, and the use of EVs (or their cargoes) as biomarkers.

WDR82-binding long noncoding RNA lncEry controls mouse erythroid differentiation and maturation.

Hematopoietic differentiation is controlled by both genetic and epigenetic regulators. Long noncoding RNAs (lncRNAs) have been demonstrated to be important for normal hematopoiesis, but their function in erythropoiesis needs to be further explored. We profiled the transcriptomes of 16 murine hematopoietic cell populations by deep RNA sequencing and identified a novel lncRNA, Gm15915, that was highly expressed in erythroid-related progenitors and erythrocytes. For this reason, we named it lncEry. We also identified a novel lncEry isoform, which was the principal transcript that has not been reported before. lncEry depletion impaired erythropoiesis, indicating the important role of the lncRNA in regulating erythroid differentiation and maturation. Mechanistically, we found that lncEry interacted with WD repeat-containing protein 82 (WDR82) to promote the transcription of Klf1 and globin genes and thus control the early and late stages of erythropoiesis, respectively. These findings identified lncEry as an important player in the transcriptional regulation of erythropoiesis.

Corrigendum: RUNX1 Upregulates CENPE to Promote Leukemic Cell Proliferation.

[This corrects the article DOI: 10.3389/10.3389/fmolb.2021.692880.].

Comprehensive understanding of Tn5 insertion preference improves transcription regulatory element identification.

Tn5 transposase, which can efficiently tagment the genome, has been widely adopted as a molecular tool in next-generation sequencing, from short-read sequencing to more complex methods such as assay for transposase-accessible chromatin using sequencing (ATAC-seq). Here, we systematically map Tn5 insertion characteristics across several model organisms, finding critical parameters that affect its insertion. On naked genomic DNA, we found that Tn5 insertion is not uniformly distributed or random. To uncover drivers of these biases, we used a machine learning framework, which revealed that DNA shape cooperatively works with DNA motif to affect Tn5 insertion preference. These intrinsic insertion preferences can be modeled using nucleotide dependence information from DNA sequences, and we developed a computational pipeline to correct for these biases in ATAC-seq data. Using our pipeline, we show that bias correction improves the overall performance of ATAC-seq peak detection, recovering many potential false-negative peaks. Furthermore, we found that these peaks are bound by transcription factors, underscoring the biological relevance of capturing this additional information. These findings highlight the benefits of an improved understanding and precise correction of Tn5 insertion preference.

RUNX1 Upregulates CENPE to Promote Leukemic Cell Proliferation.

RUNX1 is a Runt family transcription factor that plays a critical role in normal hematopoiesis, including the differentiation and proliferation of hematopoietic cells. RUNX1 mutations, including chromosomal translocations, cause abnormal cell differentiation, but the mutation alone is not sufficient to cause leukemia. In MLL-fusion-induced leukemia, dysregulated wild-type RUNX1 can promote leukemia survival. Nevertheless, the underlying mechanisms of dysregulated wild-type RUNX1 in leukemia development have not been fully elucidated. This study overexpressed and knocked down RUNX1 expression in THP-1 human leukemia cells and CD34+ hematopoietic stem/progenitor cells to investigate the biological functions affected by dysregulated RUNX1. Our data indicated RUNX1 facilitated proliferation to promote leukemia cell growth. Furthermore, we demonstrated that RUNX1 knockdown in leukemia cells drastically diminished colony-forming ability. Finally, the RUNX1-knocked down cell depletion phenotype could be rescued by overexpression of CENPE, a cell proliferation gene and a RUNX1 direct target gene. Our results indicate a possible mechanism involving the RUNX1-CENPE axis on promoting leukemic cell growth.

Understanding the "SMART" features of hematopoietic stem cells and beyond.

Since the huge success of bone marrow transplantation technology in clinical practice, hematopoietic stem cells (HSCs) have become the gold standard for defining the properties of adult stem cells (ASCs). Here, we describe the "self-renewal, multi-lineage differentiation, apoptosis, rest, and trafficking" or "SMART" model, which has been developed based on data derived from studies of HSCs as the most well-characterized stem cell type. Given the potential therapeutic applications of ASCs, we delineate the key characteristics of HSCs using this model and speculate on the physiological relevance of stem cells identified in other tissues. Great strides are being made in understanding the biology of ASCs, and efforts are now underway to develop safe and effective ASC-based therapies in this emerging area.

A comprehensive RNA editome reveals that edited Azin1 partners with DDX1 to enable hematopoietic stem cell differentiation.

Adenosine-to-inosine RNA editing and the catalyzing enzyme adenosine deaminase are both essential for hematopoietic development and differentiation. However, the RNA editome during hematopoiesis and the underlying mechanisms are poorly defined. Here, we sorted 12 murine adult hematopoietic cell populations at different stages and identified 30 796 editing sites through RNA sequencing. The dynamic landscape of the RNA editome comprises stage- and group-specific and stable editing patterns, but undergoes significant changes during lineage commitment. Notably, we found that antizyme inhibitor 1 (Azin1) was highly edited in hematopoietic stem and progenitor cells (HSPCs). Azin1 editing results in an amino acid change to induce Azin1 protein (AZI) translocation to the nucleus, enhanced AZI binding affinity for DEAD box polypeptide 1 to alter the chromatin distribution of the latter, and altered expression of multiple hematopoietic regulators that ultimately promote HSPC differentiation. Our findings have delineated an essential role for Azin1 RNA editing in hematopoietic cells, and our data set is a valuable resource for studying RNA editing on a more general basis.

ANGPTL2-containing small extracellular vesicles from vascular endothelial cells accelerate leukemia progression.

Small extracellular vesicles (SEVs) are functional messengers of certain cellular niches that permit noncontact cell communications. Whether niche-specific SEVs fulfill this role in cancer is unclear. Here, we used 7 cell type-specific mouse Cre lines to conditionally knock out Vps33b in Cdh5+ or Tie2+ endothelial cells (ECs), Lepr+ BM perivascular cells, Osx+ osteoprogenitor cells, Pf4+ megakaryocytes, and Tcf21+ spleen stromal cells. We then examined the effects of reduced SEV secretion on progression of MLL-AF9-induced acute myeloid leukemia (AML), as well as normal hematopoiesis. Blocking SEV secretion from ECs, but not perivascular cells, megakaryocytes, or spleen stromal cells, markedly delayed the leukemia progression. Notably, reducing SEV production from ECs had no effect on normal hematopoiesis. Protein analysis showed that EC-derived SEVs contained a high level of ANGPTL2, which accelerated leukemia progression via binding to the LILRB2 receptor. Moreover, ANGPTL2-SEVs released from ECs were governed by VPS33B. Importantly, ANGPTL2-SEVs were also required for primary human AML cell maintenance. These findings demonstrate a role of niche-specific SEVs in cancer development and suggest targeting of ANGPTL2-SEVs from ECs as a potential strategy to interfere with certain types of AML.

tagHi-C Reveals 3D Chromatin Architecture Dynamics during Mouse Hematopoiesis.

Spatiotemporal chromatin reorganization during hematopoietic differentiation has not been comprehensively characterized, mainly because of the large numbers of starting cells required for current chromatin conformation capture approaches. Here, we introduce a low-input tagmentation-based Hi-C (tagHi-C) method to capture the chromatin structures of hundreds of cells. Using tagHi-C, we are able to map the spatiotemporal dynamics of chromatin structure in ten primary hematopoietic stem, progenitor, and differentiated cell populations from mouse bone marrow. Our results reveal that changes in compartment dynamics and the Rabl configuration occur during hematopoietic cell differentiation. We identify gene-body-associating domains (GADs) as general structures for highly expressed genes. Moreover, we extend the body of knowledge regarding genes influenced by genome-wide association study (GWAS) loci through spatial chromatin looping. Our study provides the tagHi-C method for studying the three-dimensional (3D) genome of a small number of cells and maps the comprehensive 3D chromatin landscape of bone marrow hematopoietic cells.

Mesenchymal stem cells suppress leukemia via macrophage-mediated functional restoration of bone marrow microenvironment.

Bone marrow (BM) mesenchymal stem cells (MSCs) are critical components of the BM microenvironment and play an essential role in supporting hematopoiesis. Dysfunction of MSCs is associated with the impaired BM microenvironment that promotes leukemia development. However, whether and how restoration of the impaired BM microenvironment can inhibit leukemia development remain unknown. Using an established leukemia model and the RNA-Seq analysis, we discovered functional degeneration of MSCs during leukemia progression. Importantly, intra-BM instead of systemic transfusion of donor healthy MSCs restored the BM microenvironment, demonstrated by functional recovery of host MSCs, improvement of thrombopoiesis, and rebalance of myelopoiesis. Consequently, intra-BM MSC treatment reduced tumor burden and prolonged survival of the leukemia-bearing mice. Mechanistically, donor MSC treatment restored the function of host MSCs and reprogrammed host macrophages into arginase 1 positive phenotype with tissue-repair features. Transfusion of MSC-reprogrammed macrophages largely recapitulated the therapeutic effects of MSCs. Taken together, our study reveals that donor MSCs reprogram host macrophages to restore the BM microenvironment and inhibit leukemia development.

Targeting of apoptosis gene loci by reprogramming factors leads to selective eradication of leukemia cells.

Applying somatic cell reprogramming strategies in cancer cell biology is a powerful approach to analyze mechanisms of malignancy and develop new therapeutics. Here, we test whether leukemia cells can be reprogrammed in vivo using the canonical reprogramming transcription factors-Oct4, Sox2, Klf4, and c-Myc (termed as OSKM). Unexpectedly, we discover that OSKM can eradicate leukemia cells and dramatically improve survival of leukemia-bearing mice. By contrast, OSKM minimally impact normal hematopoietic cells. Using ATAC-seq, we find OSKM induce chromatin accessibility near genes encoding apoptotic regulators in leukemia cells. Moreover, this selective effect also involves downregulation of H3K9me3 as an early event. Dissection of the functional effects of OSKM shows that Klf4 and Sox2 play dominant roles compared to c-Myc and Oct4 in elimination of leukemia cells. These results reveal an intriguing paradigm by which OSKM-initiated reprogramming induction can be leveraged and diverged to develop novel anti-cancer strategies.

Multicolor Tuning and Temperature-Triggered Anomalous Eu3+-Related Photoemission Enhancement via Interplay of Accelerated Energy Transfer and Release of Defect-Trapped Electrons in the Tb3+,Eu3+-Doped Strontium-Aluminum Chlorites.

So far, a large number of rare earth (RE) and non-RE-doped emission-tunable crystals based on controllable energy transfer have become available, but numerous mechanistic issues, particularly for those that involve temperature-dependent energy transfer between the well-shielded 4f RE ions, lack comprehensive theoretical and experimental investigation, limiting greatly their development and applications in the future. Here, we design and report a type of Tb3+,Eu3+-doped Sr3Al2O5Cl2 phosphors capable of multiemissions upon excitation at 376 nm, through using the orthorhombic Sr3Al2O5Cl2 as the host lattice while the well-shielded 4f Tb3+ and Eu3+ ions as dual luminescent centers. Our results reveal that the energy transfer from Tb3+ to Eu3+ ions, happening via an electric dipole-quadrupole (d-q) interaction, can be controlled by the doping ratio of Tb3+ and Eu3+, leading to the tunable emissions from green (0.3159, 0.5572) to red (0.6579, 0.3046). It is found from time-resolved photoluminescence (PL) spectra that this energy transfer begins at t = 5 µs and gradually ends at t ≥ 200 µs. Moreover, from temperature-dependent PL results, we reveal that the Eu3+ emission features an anomalous intensity enhancement at the earlier heating state. With the density functional theory (DFT) calculations, we have screened the possibilities of site preferential substitution problem. By jointly taking into account the X-ray diffraction Rietveld refinement, DFT findings, and PL and thermoluminescence spectra, a mechanistic profile is proposed for illustrating the PL observations. In particular, our discussions reveal that the temperature-triggered Eu3+ emission enhancement is due to the interplay of the temperature-induced accelerated energy transfer and defect-trapped electrons that are released upon the thermal stimulation. Unlike most of reported phosphor materials that are always suggested for phosphor-converted white light-emitting diodes, we propose new application possibilities for Tb3+,Eu3+-doped Sr3Al2O5Cl2 phosphors, such as anticounterfeiting, temperature-controlled fluorescence sensor, data storage, and security devices.

Simultaneous enhancement of photoluminescence and afterglow luminescence through Bi3+ co-doping in the Sr3Al2O5Cl2:Eu2+ phosphor.

In this work, the Sr3Al2O5Cl2:Eu2+ and Sr3Al2O5Cl2:Eu2+,Bi3+ phosphors are synthesized by high temperature solid state reactions. Various characterization techniques, such as X-ray diffraction (XRD), Rietveld refinement, photoluminescence (PL) spectroscopy, afterglow spectroscopy, decay curves and thermoluminescence (TL) spectroscopy, are used to examine the phase purity and PL properties of all samples. The XRD results show that all samples belong to the targeted orthorhombic Sr3Al2O5Cl2 phase with the space group of P212121. Upon excitation with UV light, Eu2+-related reddish photoemission and afterglow luminescence are observed in the Sr3Al2O5Cl2:Eu2+ samples. More remarkably, we find that co-doping with Bi3+ ions can enhance the Eu2+-related photoemission and afterglow intensity as well the afterglow duration. For the optimal Sr3Al2O5Cl2:Eu2+,Bi3+ sample, the afterglow luminescence can continue for nearly 550 min in the dark, which is almost 3-fold the duration of the afterglow luminescence of the optimal Sr3Al2O5Cl2:Eu2+ sample. The TL spectra reveal that co-doping with Bi3+ ions can enhance the defect population that corresponds to trap depths at 63 °C, 75 °C and 150 °C, of which the former two trap depths may help to improve the Eu2+-related luminescence in addition to the afterglow property. Due to an increase in the trap concentration, there is an increase in the re-trapping possibility for the released carriers. This work not only achieves enhanced afterglow luminescence of the Sr3Al2O5Cl2:Eu2+ phosphor by co-doping with the non-rare earth (RE) Bi3+ ions, but also provides new insights into the design of RE and non-RE related enhanced afterglow photonic materials for the future.

Hematopoiesis and microenvironment in hematological malignancies.

Adult hematopoietic stem cells (HSCs) and progenitors (HPCs) reside in the bone marrow, a highly orchestrated architecture. In the bone marrow, the process of how HSCs exert self-renewal and differentiation is tightly regulated by the surrounding microenvironment, or niche. Recent advances in imaging technologies and numerous knockout or knockin mouse models have greatly improved our understanding of the organization of the bone marrow niche. This niche compartment includes a complex network of mesenchymal stem cells (MSC), osteolineage cells, endothelial cells (arterioles and sinusoids), sympathetic nerves, nonmyelinating Schwann cells and megakaryocytes. In addition, different types of mediators, such as cytokines/chemokines, reactive oxygen species (ROS) and exosomes play a pivotal role in regulating the function of hematopoietic cells. Therefore, the niche components and the hematopoietic system make up an ecological environment that maintains the homeostasis and responds to stress, damage or disease conditions. On the other hand, the niche compartment can become a traitor that can do harm to normal hematopoietic cells under pathological conditions. Studies on the diseased bone marrow niche have only recently begun to appear in the extant literature. In this short review, we discuss the most recent advances regarding the behaviors of normal hematopoietic cells and their niche alterations in hematological malignancies.