Maintenance Of Hematopoietic Stem Cells Research Articles

A Transient Induction of Blood-Circulating Bile Acid Is a Therapeutic Marker and Target of Chemotherapy-Oriented Myelosuppression

Rapid recovery of normal blood production is essential for the completion of chemotherapy for different types of solid tumors and leukemia's. However, chemotherapeutic agents can damage bone marrow cells and cause myelosuppression, which is one of the serious side effects of the cancer treatment. It is hard to predict in which patients this will be problematic but this results in a delay in the completion of the treatment protocol. An early, reliable, marker of successful recovery of blood production during cycles of chemotherapy would thus be highly beneficial.Under normal steady state conditions concentrations of bile acids (BA) in peripheral blood are low. However, in several liver diseases and critically ill patients circulating BA levels rise substantially. Some studies even argue that the levels of BA are correlated to clinical outcome and mortality. In contrast, we have previously reported that a specific BA, tauroursodeoxycholic acid (TUDCA), has a beneficial role as a molecular chaperone in maintenance and expansion of hematopoietic stem and progenitor cells (HSPC). More recently, we have also seen that BA in mouse PB are increased during hematopoietic stress (irradiation, LPS injections and 5-FU treatment). Circulating BA levels may thus have a more clinical relevance in hematopoiesis than previously has been recognized. At present the cause and precise role of increased BA levels during hematopoietic stress are unknown. In light of these findings we hypothesized that in the rebuild of the blood system after stress, circulating BA levels may be an indicator of recovery and they may have an unknown role in the blood recovery process.Initially, we measured levels of BA in pediatric patients in treatment for different types of childhood cancers. These measurements showed clearly that circulating BA levels are upregulalted during recovery after chemotherapy. This was especially seen during the recovery phase from myelosuppression. Furthermore, there was a significant, positive, correlation between increased BA levels and favorable clinical outcome in these patients. Mass spectrometry analyses showed that the main component of the increased BA was glycochenodeoxycholic acid (GCDCA), glycoursodeoxycholic acid (GUDCA) and GUDCA-3S. To replicate these results in a mouse setting we used 5-FU injections and examined PB, plasma and BM cells at different time points. In the nadir (day 3 to day 6) in the mouse, BA levels were increased moderately but during BM recovery (day 6 to day 12) the levels were highly and significantly increased.To assess if ursodeoxycholic acid (UDCA) plays a role during hematopoietic recovery, we injected TUDCA into 5-FU treated mice. PB and BM analyses revealed that TUDCA treated mice showed more rapid and better recovery. Since T/G/UDCA are known for their ability to suppress an induction of endoplasmic reticulum (ER) stress, 5-FU treated mice were also treated with an ER stress inhibitor, Salubrinal, and the mice showed similar or better degree of recovery. These findings strongly indicate that the induction of specific types of BA can serve as a supportive factor in blood recovery possibly through chaperone function and suppression of ER stress.We also tested if the induced BA upon 5-FU treatment has a biological meaning in mice, 5-FU treated mice were injected with a chemical compound, GW4064, which suppresses BA production. However, the suppression of BA levels did not impact PB and BM profiles. This might be due to the fact that the UDCA is rarely detected in rodents even under stress and compositions of BA are largely different between human and rodents.During critical illness glucocorticoids (GC) are known to increase substantially and the induction of circulating BA might be reflecting the GC levels. We have seen that the concentration of GC is increased at similar timing with BA induction after 5-FU treatment. In support of this, injection of corticosterone to mice also increased the level of circulating BA. Our data suggests that the BA increase upon chemotherapy is a consequence of the elevation of GC levels, possibly through the alteration of production and/or reabsorption of BA.To conclude, BA in the circulating blood is a potential marker to assess the recovery timing of chemotherapy-treated patients. Moreover, medication of TUDCA/GUDCA may be a novel optional treatment to boost-up the recovery from myelosuppresive conditions. DisclosuresNo relevant conflicts of interest to declare.

Loss of ASXL1 in the Bone Marrow Niche Dysregulates Hematopoietic Stem Cell Fates through RNA Polymerase II Complex

Introduction: Somatic or de novo mutations of Additional Sex Combs-like 1 (ASXL1) frequently occur in patients with myeloid malignancies or Bohring-Opitz syndrome, respectively. Conditional deletion of Asxl1 in hematopoietic system (Asxl1Δ/Δ, Abdel-Wahab et al. J Exp Med 2013) or global loss of Asxl1 (Asxl1-/-, Wang et al. Blood 2014) resulted in myeloid malignancies in mice. Interestingly, hematopoietic disease phenotypes are severer in Asxl1-/- mice than that in Asxl1Δ/Δ mice. The difference in disease severity between the two different mouse models led us to hypothesize that ASXL1 plays an important role in the function of marrow niche for normal hematopoiesis and deletion of Asxl1 in the niche contributes to the hematopoietic defect in vivo.Methods: In the current study, we performed a serial hematopoietic phenotypic analyses in OsxCre;Asxl1fl/fl mice, in which Asxl1 is only deleted in the mesenchymal stromal cells (MSCs) and preosteoblasts. Bone marrow (BM) transplantation was performed to identify the biological effects of ASXL1 loss in the niche on hematopoietic stem cell (HSC) functions. Convergent analyses of RNA-seq and ChIP-seq data were performed to survey ASXL1 target genes that are critical for HSC maintenance.Results:OsxCre;Asxl1fl/fl mice had a reduced LT-HSC pool and skewed cell differentiation favoring granulocytic/monocytic lineages in the BM by flow cytometric analyses. Co-culture of MSC with Lin-cKit+ (LK) cells revealed that Asxl1-/- MSCs induced a preferential myeloid differentiation of LK cells compared with WT MSCs. BM transplantation assay further confirmed that deletion of Asxl1 in MSCs/preosteoblasts resulted in a reduced HSC pool and a preferential myeloid lineage increment. Gene set enrichment analysis of RNA-seq data showed that downregulated genes in Asxl1-/- MSCs were enriched in cell cycle, stem cell division/proliferation, and cell surface receptor signaling pathway. Gene ontology analysis revealed that some of the differentially expressed genes were associated with cell cycle, cell division/proliferation, extracellular matrix organization, and cell adhesion. Protein mass spectrometry analysis and immunoprecipitation demonstrated a novel interaction of ASXL1 with multiple core subunits of RNA polymerase II (RNAPII) complex. Gel filtration chromatography assay verified the interaction between ASXL1 and RNAPII. ChIP-seq analysis using ASXL1 and RNAPII antibodies showed a high degree of the overlap peaks between ASXL1 and RNAPII bound genes. Convergent analyses of RNA-seq and ChIP-seq data revealed that loss of Asxl1 deregulated RNAPII transcriptional function and altered the expression of genes critical for HSC maintenance, such as Cxcl1, Cxcl2, and Vcam1 . Importantly, ASXL1 expression decreased markedly in MSCs of patients with chronic myelomonocytic leukemia (CMML-MSCs) compared with that in healthy donors. Consistently, co-culture of CMML-MSCs with cord blood CD34+ cells revealed that CMML-MSCs had a reduced hematopoietic supportive activity and induced a myeloid differentiation bias of CD34+ cells.Conclusion: Our in vivo studies demonstrate that Asxl1 in MSCs/preosteoblasts is required for normal hematopoiesis through regulating RNAPII transcriptional function. This study provides a mechanistic insight into the function of ASXL1 in the niche to maintain normal hematopoiesis; and ASXL1 alteration in, at least, a subset of the niche cells induces myeloid differentiation bias, thus, contributes the progression of myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

TGF-β Signaling in Mesenchymal Progenitor Cells during Development Plays an Essential Role in the Emergence of the Bone Marrow Niche

Hematopoietic stem cell (HSC) quiescence and function are regulated, in part, by signals generated by stromal cells in the bone marrow. Mesenchymal stromal cells implicated in HSC maintenance include CXCL12-abudndant reticular (CAR) cells, osteolineage cells, and adipocytes, all of which, are derived from mesenchymal stem cells. The factors that regulate the growth, differentiation, and function of mesenchymal stems in the bone marrow are largely unknown. Here, we focus on transforming growth factor beta (TGF-β), which regulates osteoblast differentiation and has been implicated in the development of myelofibrosis in myeloproliferative neoplasms. We previously reported that constitutive deletion of Tgfbr2 (which abrogates all canonical TGF-β signaling) in mesenchymal stromal cells targeted by Osx-Cre results in marked alterations in the bone marrow microenvironment. These alterations include the following: 1) a 85-fold increase in adipocytes; 2) a 5-fold increase in CXCL12-abundant reticular (CAR) cells; and 3) the near absence of osteocalcin+ osteoblasts. Surprisingly, hematopoietic stem cells were only modestly reduced and had normal long-term repopulating and self-renewal capacity. These data suggest that TGF-β signaling plays a crucial role in determining the lineage commitment of mesenchymal progenitors.To investigate mechanisms by which TGF-β signaling regulates mesenchymal stromal cells, we first generated mice in which Tgfbr2 deletion is limited to Dmp1-Cre targeted stromal cells, which is mostly restricted to osteolineage cells. No alteration in stromal cells or hematopoiesis was observed. We next used a conditional Osx-Cre transgene to post-natally delete Tgfbr2 starting on P1. Surprisingly, these mice also had no alteration in osteoblasts, CAR cells, or adipocytes. Together, these experiments suggested the hypothesis that TGF-β signaling in mesenchymal progenitors during development may determine their lineage specification. To test this hypothesis, we analyzed Osx-Cre Tgfbr2fl/fl Ai9 mice during development; in these mice, Osx-Cre targeted cells express TdTomato. On E14.5 the nascent long-bones of control and Tgfbr2 -deleted mice appeared similar. Specifically, the primary ossification center contained a similar number and distribution of aggrecan-positive chondrocytes. Likewise, the number of TdTomato+ cells in the surrounding perichondrial “bone collar” was similar. However, on E16.5, a striking accumulation of perilipin-positive adipocytes was observed in Tgfbr2 -deleted but not control mice. Consistent with this finding, RNA expression profiling of Osx-Cre targeted (TdTomato-positive) cells sorted from the E16.5 long bones of Tgfbr2 -deleted mice showed a significant increase in adipocyte marker genes compared to control mice. Expression of peroxisome proliferator-activated receptor-gamma PPARG), a key transcription factor regulating adipogenesis, was increased 2-fold in Tgfbr2 -deleted cells. This is consistent with prior studies suggesting that TGF-β signaling negatively regulates PPARG expression.Collectively, these data show that TGF-β plays a key and non-redundant role in specifying the lineage commitment of mesenchymal progenitors in the bone marrow during development. Specifically, TGF-β suppresses the adipogenic potential of mesenchymal progenitors, possibly by regulating PPARG expression. Whether TGF-β signaling regulates bone marrow adiposity in adults, especially in response to aging or inflammation, is an open and interesting question. DisclosuresNo relevant conflicts of interest to declare.

C/EBP homologous protein deficiency enhances hematopoietic stem cell function via reducing ATF3/ROS-induced cell apoptosis.

Hematopoietic stem cells (HSCs) reside in a quiescent niche to reserve their capacity of self‐renewal. Upon hematopoietic injuries, HSCs enter the cell cycle and encounter protein homeostasis problems caused by accumulation of misfolded proteins. However, the mechanism by which protein homeostasis influences HSC function and maintenance remains poorly understood. Here, we show that C/EBP homologous protein (CHOP), demonstrated previously to induces cell death upon unfolded protein response (UPR), plays an important role in HSCs regeneration. CHOP−/− mice showed normal hematopoietic stem and progenitor cell frequencies in steady state. However, when treated with 5‐FU, CHOP deficiency resulted in higher survival rates, associated with an increased number of HSCs and reduced level of apoptosis. In serial competitive transplantation experiments, CHOP−/− HSCs showed a dramatic enhancement of repopulation ability and a reduction of protein aggresomes. Mechanistically, CHOP deletion causes reduced ATF3 expression and further leads to decreased protein aggregation and ROS. In addition, CHOP−/− HSCs exhibited an increased resistance to IR‐induced DNA damage and improved HSCs homeostasis and function in telomere dysfunctional (G3Terc −/−) mice. In summary, these findings disclose a new role of CHOP in the regulation of the HSCs function and homeostasis through reducing ATF3 and ROS signaling.

Single-cell analysis of ploidy and the transcriptome reveals functional and spatial divergency in murine megakaryopoiesis

AbstractMegakaryocytes (MKs), the platelet progenitor cells, play important roles in hematopoietic stem cell (HSC) maintenance and immunity. However, it is not known whether these diverse programs are executed by a single population or by distinct subsets of cells. Here, we manually isolated primary CD41+ MKs from the bone marrow (BM) of mice and human donors based on ploidy (2N-32N) and performed single-cell RNA sequencing analysis. We found that cellular heterogeneity existed within 3 distinct subpopulations that possess gene signatures related to platelet generation, HSC niche interaction, and inflammatory responses. In situ immunostaining of mouse BM demonstrated that platelet generation and the HSC niche–related MKs were in close physical proximity to blood vessels and HSCs, respectively. Proplatelets, which could give rise to platelets under blood shear forces, were predominantly formed on a platelet generation subset. Remarkably, the inflammatory responses subpopulation, consisting generally of low-ploidy LSP1+ and CD53+ MKs (≤8N), represented ∼5% of total MKs in the BM. These MKs could specifically respond to pathogenic infections in mice. Rapid expansion of this population was accompanied by strong upregulation of a preexisting PU.1- and IRF-8–associated monocytic-like transcriptional program involved in pathogen recognition and clearance as well as antigen presentation. Consistently, isolated primary CD53+ cells were capable of engulfing and digesting bacteria and stimulating T cells in vitro. Together, our findings uncover new molecular, spatial, and functional heterogeneity within MKs in vivo and demonstrate the existence of a specialized MK subpopulation that may act as a new type of immune cell.

The thrombopoietin receptor: revisiting the master regulator of platelet production

Thrombopoietin (TPO) and its receptor, MPL, are the primary regulators of platelet production and critical for hematopoietic stem cell (HSC) maintenance. Since TPO was first cloned in 1994, the physiological and pathological roles of TPO and MPL have been well characterized, culminating in the first MPL agonists being approved for the treatment of chronic immune thrombocytopenia in 2008. Dysregulation of the TPO-MPL signaling axis contributes to the pathogenesis of hematological disorders: decreased expression or function results in severe thrombocytopenia progressing to bone marrow failure, while hyperactivation of MPL signaling, either by mutations in the receptor or associated Janus kinase 2 (JAK2), results in pathological myeloproliferation. Despite its importance, it was only recently that the long-running debate over the mechanism by which TPO binding activates MPL has been resolved. This review will cover key aspects of TPO and MPL structure and function and their importance in receptor activation, discuss how these are altered in hematological disorders and consider how a greater understanding could lead to the development of better-targeted and more efficacious therapies.

Small molecule-mediated modulation of ubiquitination and neddylation improves HSC function ex vivo.

Hematopoietic stem cells (HSCs) are particularly characterized by their quiescence and self-renewal. Cell cycle regulators tightly control quiescence and self-renewal capacity. Studies suggest that modulation of ubiquitination and neddylation could contribute to HSC function via cyclin-dependent kinase inhibitors (CDKIs). S-phase kinase-associated protein 2 (SKP2) is responsible for ubiquitin-mediated proteolysis of CDKIs. Here, we modulated overall neddylation and SKP2-associated ubiquitination in HSCs by using SKP2-C25, an SKP2 inhibitor, and MLN4924 (Pevonedistat) as an inhibitor of the NEDD8 system. Treatments of SKP2-C25 and MLN4924 increased both murine and human stem and progenitor cell (HSPC) compartments. This is associated with the improved quiescence of murine HSC by upregulation of p27 and p57 CDKIs. A colony-forming unit assay showed an enhanced in vitro self-renewal potential post inhibition of ubiquitination and neddylation. In addition, MLN4924 triggered the mobilization of bone marrow HSPCs to peripheral blood. Intriguingly, MLN4924 treatment could decrease the proliferation of murine bone marrow mesenchymal stem cells or endothelial cells. These findings shed light on the contribution of SKP2, and associated ubiquitination and neddylation in HSC maintenance, self-renewal, and expansion.

Inflammation, Aging and Hematopoiesis: A Complex Relationship.

All vertebrate blood cells descend from multipotent hematopoietic stem cells (HSCs), whose activity and differentiation depend on a complex and incompletely understood relationship with inflammatory signals. Although homeostatic levels of inflammatory signaling play an intricate role in HSC maintenance, activation, proliferation, and differentiation, acute or chronic exposure to inflammation can have deleterious effects on HSC function and self-renewal capacity, and bias their differentiation program. Increased levels of inflammatory signaling are observed during aging, affecting HSCs either directly or indirectly via the bone marrow niche and contributing to their loss of self-renewal capacity, diminished overall functionality, and myeloid differentiation skewing. These changes can have significant pathological consequences. Here, we provide an overview of the current literature on the complex interplay between HSCs and inflammatory signaling, and how this relationship contributes to age-related phenotypes. Understanding the mechanisms and outcomes of this interaction during different life stages will have significant implications in the modulation and restoration of the hematopoietic system in human disease, recovery from cancer and chemotherapeutic treatments, stem cell transplantation, and aging.

C/EBP\u03b2 is a MYB- and p300-cooperating pro-leukemogenic factor and promising drug target in acute myeloid leukemia

Transcription factor MYB has recently emerged as a promising drug target for the treatment of acute myeloid leukemia (AML). Here, we have characterized a group of natural sesquiterpene lactones (STLs), previously shown to suppress MYB activity, for their potential to decrease AML cell proliferation. Unlike what was initially thought, these compounds inhibit MYB indirectly via its cooperation partner C/EBPβ. C/EBPβ-inhibitory STLs affect the expression of a large number of MYB-regulated genes, suggesting that the cooperation of MYB and C/EBPβ broadly shapes the transcriptional program of AML cells. We show that expression of GFI1, a direct MYB target gene, is controlled cooperatively by MYB, C/EBPβ, and co-activator p300, and is down-regulated by C/EBPβ-inhibitory STLs, exemplifying that they target the activity of composite MYB-C/EBPβ-p300 transcriptional modules. Ectopic expression of GFI1, a zinc-finger protein that is required for the maintenance of hematopoietic stem and progenitor cells, partially abrogated STL-induced myelomonocytic differentiation, implicating GFI1 as a relevant target of C/EBPβ-inhibitory STLs. Overall, our data identify C/EBPβ as a pro-leukemogenic factor in AML and suggest that targeting of C/EBPβ may have therapeutic potential against AML.

MAEA is an E3 ubiquitin ligase promoting autophagy and maintenance of haematopoietic stem cells

Haematopoietic stem cells (HSCs) tightly regulate their quiescence, proliferation, and differentiation to generate blood cells during the entire lifetime. The mechanisms by which these critical activities are balanced are still unclear. Here, we report that Macrophage-Erythroblast Attacher (MAEA, also known as EMP), a receptor thus far only identified in erythroblastic island, is a membrane-associated E3 ubiquitin ligase subunit essential for HSC maintenance and lymphoid potential. Maea is highly expressed in HSCs and its deletion in mice severely impairs HSC quiescence and leads to a lethal myeloproliferative syndrome. Mechanistically, we have found that the surface expression of several haematopoietic cytokine receptors (e.g. MPL, FLT3) is stabilised in the absence of Maea, thereby prolonging their intracellular signalling. This is associated with impaired autophagy flux in HSCs but not in mature haematopoietic cells. Administration of receptor kinase inhibitor or autophagy-inducing compounds rescues the functional defects of Maea-deficient HSCs. Our results suggest that MAEA provides E3 ubiquitin ligase activity, guarding HSC function by restricting cytokine receptor signalling via autophagy.

A mitophagy inhibitor targeting p62 attenuates the leukemia-initiation potential of acute myeloid leukemia cells.

There has been an increasing focus on the tumorigenic potential of leukemia initiating cells (LICs) in acute myeloid leukemia (AML). Despite the important role of selective autophagy in the life-long maintenance of hematopoietic stem cells (HSCs), cancer progression, and chemoresistance, the relationship between LICs and selective autophagy remains to be fully elucidated. Sequestosome 1 (SQSTM1), also known as p62, is a selective autophagy receptor for the degradation of ubiquitinated substrates, and its loss impairs leukemia progression in AML mouse models. In this study, we evaluated the underlying mechanisms of mitophagy in the survival of LICs with XRK3F2, a p62-ZZ inhibitor. We demonstrated that XRK3F2 selectively impaired LICs but spared normal HSCs in both mouse and patient-derived tumor xenograft (PDX) AML models. Mechanistically, we observed that XRK3F2 blocked mitophagy by inhibiting the binding of p62 with defective mitochondria. Our study not only evaluated the effectiveness and safety of XRK3F2 in LICs, but also demonstrated that mitophagy plays an indispensable role in the survival of LICs during AML development and progression, which can be impaired by blocking p62.

α-Tocopherol Attenuates Oxidative Phosphorylation of CD34+ Cells, Enhances Their G0 Phase Fraction and Promotes Hematopoietic Stem and Primitive Progenitor Cell Maintenance.

Alpha tocopherol acetate (αTOA) is an analogue of alpha tocopherol (αTOC) that exists in the form of an injectable drug. In the context of the metabolic hypothesis of stem cells, we studied the impact of αTOA on the metabolic energetic profile and functional properties of hematopoietic stem and progenitor cells. In ex vivo experiments performed on cord blood CD34+ cells, we found that αTOA effectively attenuates oxidative phosphorylation without affecting the glycolysis rate. This effect concerns complex I and complex II of the mitochondrial respiratory chain and is related to the relatively late increase (3 days) in ROS (Reactive Oxygen Species). The most interesting effect was the inhibition of Hypoxia-Inducible Factor (HIF)-2α (Hexpression, which is a determinant of the most pronounced biological effect—the accumulation of CD34+ cells in the G0 phase of the cell cycle. In parallel, better maintenance of the primitive stem cell activity was revealed by the expansion seen in secondary cultures (higher production of colony forming cells (CFC) and Severe Combined Immunodeficiency-mice (scid)-repopulating cells (SRC)). While the presence of αTOA enhanced the maintenance of Hematopoietic Stem Cells (HSC) and contained their proliferation ex vivo, whether it could play the same role in vivo remained unknown. Creating αTOC deficiency via a vitamin E-free diet in mice, we found an accelerated proliferation of CFC and an expanded compartment of LSK (lineagenegative Sca-1+cKit+) and SLAM (cells expressing Signaling Lymphocytic Activation Molecule family receptors) bone marrow cell populations whose in vivo repopulating capacity was decreased. These in vivo data are in favor of our hypothesis that αTOC may have a physiological role in the maintenance of stem cells. Taking into account that αTOC also exhibits an effect on proliferative capacity, it may also be relevant for the ex vivo manipulation of hematopoietic stem cells. For this purpose, low non-toxic doses of αTOA should be used.

Identification of CXCL12‐abundant reticular cells in human adult bone marrow

SummaryA population of mesenchymal stem cells, termed CXC chemokine ligand (CXCL)12‐abundant reticular (CAR) cells or leptin receptor‐expressing cells, are the major cellular component of niches for haematopoietic stem cells (HSCs) in murine bone marrow. CAR cells are characterized by several salient features, including much higher expression of CXCL12, stem cell factor (SCF), forkhead box C1 (FOXC1) and early B‐cell factor 3 (EBF3), which are essential for HSC maintenance, than other cells. However, the human counterpart of CAR cells has not been fully described. Here, we show the presence of cells expressing much higher CXCL12 than other cells in human adult bone marrow using a flow cytometry‐based in situ technique that enables high‐throughput detection of mRNA at single‐cell resolution. Most CXCL12hi cells expressed high levels of SCF, FOXC1 and EBF3 and had the potential to differentiate into adipocytes and osteoblasts. Histologically, the nuclei of CXCL12hi cells were identified and quantified by EBF3 expression in fixed marrow sections. CXCL12hi cells sorted from residual bone marrow aspirates of chronic myeloid leukaemia patients expressed reduced levels of CXCL12, SCF, FOXC1 and EBF3 in correlation with increased leukaemic burden. Together, we identified the human counterpart of CAR cells, enabling the evaluation of their alterations in various haematological disorders by flow cytometric and histological analyses.

Inhibition of STAT3 prevents bone metastatic progression of prostate cancer in vivo.

Prostate cancer (PC) metastasizes to the skeleton forming predominantly sclerotic lesions, and there is currently no cure for bone metastatic disease. The transcription factor signal transducer and activator of transcription 3 (STAT3) is implicated as a metastatic driver, but its potential as therapeutic target in bone metastasis has not been investigated. In this study, we evaluated for the first time a STAT3 inhibitor, Napabucasin, as a therapeutic option for bone metastatic PC. Effects of STAT3 inhibitors, Stattic and Napabucasin, on metastatic potential in PC cells were studied in vitro by assessment of migration capacity, self-renewal potential, and tumorsphere formation. For evaluation of the role of STAT3 in initial skeletal establishment of PC cells as well as in progressed castration-resistant PC (CRPC) in bone, human VCaP prostate cancer cells were inoculated in the tibia of mice which subsequently were treated with the STAT3 inhibitor Napabucasin. Bone specimens were analyzed using computed tomography (CT), immunohistochemistry, and quantitative polymerase chain reaction. The small molecule STAT3 inhibitors Stattic and Napabucasin both effectively impaired metastatic potential of PC cells in vitro. Furthermore, treatment with Napabucasin prevented metastatic establishment in tibial bones in vivo and thereby also the tumor-induced sclerotic bone response seen in vehicle-treated VCaP xenografts. In addition, treatment with Napabucasin of established bone CRPC significantly decreased both tumor burden and tumor-induced trabecular bone volume compared with effects seen in vehicle-treated animals. Anti-mitotic effects were confirmed by decreased Ki67 staining in Napabucasin-treated xenografts compared with vehicle-treated xenografts. Alterations of gene expression in the femoral bone marrow (BM) niche towardthe maintenance of hematopoietic stem cells and the myeloid lineage were demonstrated by quantitative real-time polymerase chain reaction and were further reflected by a substantial increase in the number of erythrocytes in BM of Napabucasin-treated mice. Furthermore, a unique pattern of STAT3 phosphorylation in osteoblasts/stromal cells surrounding the areas of tumor cells was demonstrated immunohistochemically in bone xenograft models using several different PC cell lines. Inhibition of STAT3 activity disrupts the bone metastatic niche and targets both the skeletal establishment of PC and advanced bone metastatic CRPC in mice, suggesting STAT3 as a candidate for molecular targeted therapies of skeletal metastatic disease.

Regulation of gene transcription of B lymphoma Mo-MLV insertion region 1 homolog (Review).

B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) is a core protein component of the polycomb repressive complex 1 that inhibits cell senescence and maintains the self-renewal ability of stem cells via downregulation of p16Ink4a and p19Arf expression. Bmi-1 serves an important role in hematopoietic stem cell maintenance and neurodevelopment during embryonic development, and it has been shown to enhance tumorigenesis by promoting cancer stem cell self-renewal and epithelial to mesenchymal transition. Emerging evidence suggests that Bmi-1 overexpression is closely related to the development and progression of various types of cancer, and that downregulation of Bmi-1 expression can inhibit the proliferation, invasion and metastasis of cancer cells. It is therefore important to elucidate the mechanisms underlying the regulation of Bmi-1 expression both under normal growth conditions and in malignant tissues. In the present review, the current body of knowledge pertaining to the transcriptional and post-transcriptional regulation of the BMI-1 gene is discussed, and the potential mechanisms by which Bmi-1 is dysregulated in various types of cancer are highlighted. Bmi-1 expression is primarily controlled via transcriptional regulation, and is regulated by the transcription https://www.ushuaia.pl/hyphen/?ln=en factors of the Myc family, including Myb, Twist1, SALL4 and E2F-1. Post-transcriptionally, regulation of Bmi-1 expression is inhibited by several microRNAs and certain small-molecule drugs. Thus, regulatory transcriptional factors are potential therapeutic targets to reduce Bmi-1 expression in cancer cells. Thus, the present review provides an up-to-date review on the regulation of BMI-1 gene expression at the transcriptional and post-transcriptional level.

Nuclear DEK preserves hematopoietic stem cells potential via NCoR1/HDAC3-Akt1/2-mTOR axis.

The oncogene DEK is found fused with the NUP214 gene creating oncoprotein DEK-NUP214 that induces acute myeloid leukemia (AML) in patients, and secreted DEK protein functions as a hematopoietic cytokine to regulate hematopoiesis; however, the intrinsic role of nuclear DEK in hematopoietic stem cells (HSCs) remains largely unknown. Here, we show that HSCs lacking DEK display defects in long-term self-renew capacity, eventually resulting in impaired hematopoiesis. DEK deficiency reduces quiescence and accelerates mitochondrial metabolism in HSCs, in part, dependent upon activating mTOR signaling. At the molecular level, DEK recruits the corepressor NCoR1 to repress acetylation of histone 3 at lysine 27 (H3K27ac) and restricts the chromatin accessibility of HSCs, governing the expression of quiescence-associated genes (e.g., Akt1/2, Ccnb2, and p21). Inhibition of mTOR activity largely restores the maintenance and potential of Dek-cKO HSCs. These findings highlight the crucial role of nuclear DEK in preserving HSC potential, uncovering a new link between chromatin remodelers and HSC homeostasis, and have clinical implications.

Autophagy is dispensable for the maintenance of hematopoietic stem cells in neonates

Hematopoietic stem cells (HSCs) undergo self-renewal or differentiation to sustain lifelong hematopoiesis. HSCs are preserved in quiescence with low mitochondrial activity. Recent studies indicate that autophagy contributes to HSC quiescence through suppressing mitochondrial metabolism. However, it remains unclear whether autophagy is involved in the regulation of neonatal HSCs, which proliferate actively. In this study, we clarified the role of autophagy in neonatal HSCs using 2 types of autophagy-related gene 7 (Atg7)-conditional knockout mice: Mx1-Cre inducible system and Vav-Cre system. Atg7-deficient HSCs exhibited excess cell divisions with enhanced mitochondrial metabolism, leading to bone marrow failure at adult stage. However, Atg7 deficiency minimally affected hematopoiesis and metabolic state in HSCs at neonatal stage. In addition, Atg7-deficient neonatal HSCs exhibited long-term reconstructing activity, equivalent to wild-type neonatal HSCs. Taken together, autophagy is dispensable for stem cell function and hematopoietic homeostasis in neonates and provide a novel aspect into the role of autophagy in the HSC regulation.

Prolonged maintenance of hematopoietic stem cells that escape from thrombopoietin deprivation

AbstractHematopoietic stem cells (HSC) rarely divide, rest in quiescence, and proliferate only upon stress hematopoiesis. The cytokine thrombopoietin (Thpo) has been perplexingly described to induce quiescence and promote self-renewal divisions in HSCs. To clarify the contradictory effect of Thpo, we conducted a detailed analysis on conventional (Thpo−/−) and liver-specific (Thpofl/fl;AlbCre+/−) Thpo-deletion models. Thpo−/− HSCs exhibited profound loss of quiescence, impaired cell cycle progression, and increased apoptosis. Thpo−/− HSCs also exhibited diminished mitochondrial mass and impaired mitochondrial bioenergetics. Abnormal HSC phenotypes in Thpo−/− mice were reversible after HSC transplantation into wild-type recipients. Moreover, Thpo−/− HSCs acquired quiescence with extended administration of a Thpo receptor agonist, romiplostim, and were prone to subsequent stem cell exhaustion during competitive bone marrow transplantation. Thpofl/fl;AlbCre+/− HSCs exhibited similar stem cell phenotypes but to a lesser degree compared with Thpo−/− HSCs. HSCs that survive Thpo deficiency acquire quiescence in a dose-dependent manner through the modification of their metabolic state.

Neurogenic Heterotopic Ossifications Recapitulate Hematopoietic Stem Cell Niche Development Within an Adult Osteogenic Muscle Environment.

Hematopoiesis and bone interact in various developmental and pathological processes. Neurogenic heterotopic ossifications (NHO) are the formation of ectopic hematopoietic bones in peri-articular muscles that develop following severe lesions of the central nervous system such as traumatic cerebral or spinal injuries or strokes. This review will focus on the hematopoietic facet of NHO. The characterization of NHO demonstrates the presence of hematopoietic marrow in which quiescent hematopoietic stem cells (HSC) are maintained by a functional stromal microenvironment, thus documenting that NHOs are neo-formed ectopic HSC niches. Similarly to adult bone marrow, the NHO permissive environment supports HSC maintenance, proliferation and differentiation through bidirectional signaling with mesenchymal stromal cells and endothelial cells, involving cell adhesion molecules, membrane-bound growth factors, hormones, and secreted matrix proteins. The participation of the nervous system, macrophages and inflammatory cytokines including oncostatin M and transforming growth factor (TGF)-β in this process, reveals how neural circuitry fine-tunes the inflammatory response to generate hematopoietic bones in injured muscles. The localization of NHOs in the peri-articular muscle environment also suggests a role of muscle mesenchymal cells and bone metabolism in development of hematopoiesis in adults. Little is known about the establishment of bone marrow niches and the regulation of HSC cycling during fetal development. Similarities between NHO and development of fetal bones make NHOs an interesting model to study the establishment of bone marrow hematopoiesis during development. Conversely, identification of stage-specific factors that specify HSC developmental state during fetal bone development will give more mechanistic insights into NHO.

Modulation of APLNR Signaling Is Required during the Development and Maintenance of the Hematopoietic System.

SummaryApelin receptor (APLNR/AGTRLl1/APJ) marks a transient cell population during the differentiation of hematopoietic stem and progenitor cells (HSPCs) from pluripotent stem cells, but its function during the production and maintenance of hematopoietic stem cells is not clear. We generated an Aplnr-tdTomato reporter mouse embryonic stem cell (mESC) line and showed that HSPCs are generated exclusively from mesodermal cells that express Aplnr-tdTomato. HSPC production from mESCs was impaired when Aplnr was deleted, implying that this pathway is required for their production. To address the role of APLNR signaling in HSPC maintenance, we added APELIN ligands to ex vivo AGM cultures. Activation of the APLNR pathway in this system impaired the generation of long-term reconstituting HSPCs and appeared to drive myeloid differentiation. Our data suggest that the APLNR signaling is required for the generation of cells that give rise to HSCs, but that its subsequent downregulation is required for their maintenance.