Defective Hematopoietic Stem Cells Research Articles

Benzalkonium chloride induces hematopoietic stem cell reduction and immunotoxicity in zebrafish larvae

Benzalkonium chloride (BAC) is a broad-spectrum antibacterial agent that possesses cleaning and bactericidal properties, but impact of BAC on wellbeing of aquatic organisms remains uncertain. Consequently, in this current study, we have examined the immunotoxic potential of BAC in zebrafish embryos, thus marking it as the pioneering effort in this field. According to the findings, zebrafish embryos exposed to BAC exhibited a decline in yolk area that varied with the concentration, along with a significant decrease in the count of neutrophils, macrophages, red blood cells, and thymus T-cells. We observed significantly up-regulated expression of immune-related signaling genes such as cxcl-c1c, il-8, tir4 and inf-γ, but expression of nf-κb was downregulated. In addition, we observed a marked reduction in the number of hematopoietic stem cells in zebrafish larvae after BAC exposure, which could be the result of oxidative stress-mediated apoptosis. We found that compared with the control group, the number of red blood cells in juvenile zebrafish in BAC-exposure group was significantly down-regulated, which could be attributed to hematopoietic stem cell defect. Astaxanthin restored immune cells and hematopoietic stem cells after BAC exposure, whereas Inhibitor of Wnt Response-1(IWR-1) restored neutrophils after BAC exposure. The research findings demonstrated that exposure to BAC displayed harmful effects on the development and immune system of zebrafish embryos. These effects might be associated with alterations in reactive oxygen species(ROS) levels and activation of the Wnt signaling pathway caused by BAC.

Restoration of T and B Cell Differentiation after RAG1 Gene Transfer in Human RAG1 Defective Hematopoietic Stem Cells.

Recombinase-activating gene (RAG)-deficient SCID patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. The two RAG genes act as a required dimer to initiate gene recombination. Gene therapy is a valid treatment alternative for RAG-SCID patients who lack a suitable bone marrow donor, but developing such therapy for RAG1/2 has proven challenging. Using a clinically approved lentiviral vector with a codon-optimized RAG1 gene, we report here preclinical studies using CD34+ cells from four RAG1-SCID patients. We used in vitro T cell developmental assays and in vivo assays in xenografted NSG mice. The RAG1-SCID patient CD34+ cells transduced with the RAG1 vector and transplanted into NSG mice led to restored human B and T cell development. Together with favorable safety data on integration sites, these results substantiate an ongoing phase I/II clinical trial for RAG1-SCID.

STAT3 protects hematopoietic stem cells by preventing activation of a deleterious autocrine type-I interferon response.

Hematopoietic stem and progenitor cells (HSPCs) maintain blood-forming and immune activity, yet intrinsic regulators of HSPCs remain elusive. STAT3 function in HSPCs has been difficult to dissect as Stat3-deficiency in the hematopoietic compartment induces systemic inflammation, which can impact HSPC activity. Here, we developed mixed bone marrow (BM) chimeric mice with inducible Stat3 deletion in 20% of the hematopoietic compartment to avoid systemic inflammation. Stat3-deficient HSPCs were significantly impaired in reconstitution ability following primary or secondary bone marrow transplantation, indicating hematopoietic stem cell (HSC) defects. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells (LSKs) revealed aberrant activation of cell cycle, p53, and interferon (IFN) pathways in Stat3-deficient HSPCs. Stat3-deficient LSKs accumulated γH2AX and showed increased expression of DNA sensors and type-I IFN (IFN-I), while treatment with A151-ODN inhibited expression of IFN-I and IFN-responsive genes. Further, the blockade of IFN-I receptor signaling suppressed aberrant cell cycling, STAT1 activation, and nuclear p53 accumulation. Collectively, our results show that STAT3 inhibits a deleterious autocrine IFN response in HSCs to maintain long-term HSC function. These data signify the importance of ensuring therapeutic STAT3 inhibitors are targeted specifically to diseased cells to avoid off-target loss of healthy HSPCs.

Tgfb1 deficiency impairs the self-renewal capacity of murine hematopoietic stem/progenitor cells in vivo

Transforming growth factor β1 (TGFB1) refers to a pleiotropic cytokine exerting contrasting roles in hematopoietic stem cells (HSCs) functions in vitro and in vivo. However, the understanding of hematopoiesis in vivo, when TGFB1 is constantly deactivated, is still unclear, mainly due to significant embryonic lethality and the emergence of a fatal inflammatory condition, which makes doing these investigations challenging. Our study aims to find the specific role of TGFB1 in regulating hematopoiesis in vivo. We engineered mice strains (Vav1 or Mx1 promoter-driven TGFB1 knockout) with conditional knockout of TGFB1 to study its role in hematopoiesis in vivo. In fetal and adult hematopoiesis, TGFB1 KO mice displayed deficiency and decreased self-renewal capacity of HSCs with myeloid-biased differentiation. The results were different from the regulating role of TGFB1 in vitro. Additionally, our results showed that TGFB1 deficiency from fetal hematopoiesis stage caused more severe defect of HSCs than in the adult stage. Mechanistically, our findings identified TGFB1-SOX9-FOS/JUNB/TWIST1 signal axis as an essential regulating pathway in HSCs homeostasis. Our study may provide a scientific basis for clinical HSC transplantation and expansion.

Minimal intensity conditioning strategies for bone marrow failure: is it time for "preventative" transplants?

Hematopoietic cell transplantation (HCT) can cure blood dyscrasias and reduce the risk of hematologic cancers in patients with inherited bone marrow failure syndromes (IBMFS). However, because of its high mortality rate, HCT is generally reserved until patients with IBMFS manifest life-threatening cytopenias or myeloid malignancy, at which point outcomes are poor. Screening tests that accurately predict transformation and enable timely intervention are lacking. These unknowns and risks limit the use of HCT in patients with IBMFS, sometimes until significant disease-related sequelae have occurred. A major goal for IBMFS is to reduce cellular therapy-related complications to the point that earlier intervention can be considered before significant transfusion exposure, occurrence of comorbidities, or malignant transformation. In recent decades, disease-specific allogeneic HCT trials have yielded significant improvements in outcomes in IBMFS conditions, including Fanconi anemia and dyskeratosis congenita. This is in large part due to marked reductions in conditioning intensity to address the increased sensitivity of these patients to cytotoxic chemotherapy and radiation. The success of these approaches may also indicate an ability to leverage intrinsic fitness defects of hematopoietic stem and progenitor cells across IBMFS disorders. Now with advances in tracking somatic genetic evolution in hematopoiesis and tailored minimal intensity conditioning regimens, this question arises: is it time for preventative HCT for IBMFS?

Chromatin Remodeler DEK Is a Sensor to Replication Stress and Contributes to HSC Defects in Fanconi Anemia

Hematopoietic stem cells (HSCs) have self-renewal capacity and response for life-long hematopoiesis. Any interference with normal DNA replication leads to replication stress, which drives function decline in HSCs. However, the response of HSCs to replication stress remains largely unknown. Here, draw a transcriptional and epigenetic landscape of HSCs under replication stress. We found that HSCs required opened chromatin to counteract replication stress, along with altered H3K27 acetylation and H3K4 trimethylation. The increased chromatin accessibility was mainly attributed to the reduced chromatin remodelers DEK, while DEK overexpressed HSCs confronted with a strong replication challenge, shown by increased γ-H2AX. Excessive DEK protein impeded proliferation and impaired HSC maintenance (>50% reduction), along with reduced oxidative phosphorylation and protein synthesis. Notably, we found that DEK protein was abnormally accumulated in BM CD34 + cells in Fanconi anemia, which arises from mutations in FA genes essential for replication stress tolerance. Inhibition of DEK substantially recovered HSC proliferation capacity in vitro and engraftment in vivo. At the molecular level, we identified that ATF2 directly promoted the transcription of DEK, especially relying on phosphorylated ATF2 (ser69/71) rather than phosphorylated ATF2 (ser490/498). Rad3-related (ATR) induced the phosphorylation of ATF2 at ser490/498 and mediated the reduction of DEK under replication stress. Nonetheless, knock-down of FA genes (FANCD2) in HSCs resulted in increased phosphorylated ATF2 (ser69/71), which might contribute to DEK accumulation in FA. Collectively, our findings uncover the landscape of HSC response to replication stress, reveal a novel ATR-ATF2-DEK axis helping HSCs counteract replication stress, and suggest inhibition of DEK as a therapeutic utility in FA.

The Association of SEPTIN6 Mutations with Pediatric Myelodysplasia

Septin family proteins belong to the RAS-like GTPase superclass and are considered as a structural element of the cytoskeleton. They are evolutionarily conserved and are important in cytokinesis, polarity, cell cycle, vesicle trafficking, and exocytosis. They are implicated in T and B cell lymphomas; and acute myeloid leukemias, where they frequently act as fusion partners with the Mixed-Lineage Leukemia (MLL) gene. Previously, we described two non-syndromic patients with severe neutropenia which progressed to myelodysplastic syndrome (MDS) (Renella et al. AJH, 2022, Mohamad et al. ASH, 2022). Next-generation sequencing revealed both patients (P1 and P2) to have novel X-linked germline mutations in codon 428 of the SEPTIN6 gene (P1: SEPTIN6 c.1282T>C; P2: SEPTIN6 c.1282T>A). One of these patients (P1) had an additional somatic stop-gain mutation (P1: SEPTIN6 c.43C>T) of unclear function. Both patient germline mutations were predicted to add 9 amino acids to the C-terminal end creating a novel mutated SEPTIN6 protein. Since SEPTIN6 is located on the X-chromosome and expression of these mutated proteins occurred in males, no wild-type protein was present in the patient cells. To further explore the biological relevance of these mutations, single base-pair gene editing was used to introduce the (P1: T>C) mutation into normal male donor CD34+ cells. Morphological studies demonstrated that SEPTIN6 edited cells had enlarged, multi-nucleated and dysplastic nuclei; and reduced clonogenic activity. To further assess the effect of the T>C mutation on hematopoietic stem cell (HSC) function, the SEPTIN6 edited cells were transplanted into immunocompromised NSGW mice. Analysis of peripheral blood demonstrated reduced chimerism in edited recipients (4.27% +/-3.4%) compared to controls (20.56% +/-6.2%, mean+/- SD, p= 0.0008) indicating a functional HSC defect upon mutation acquisition. Bulk mRNA sequencing demonstrated changes in ribosome, DNA replication, homologous recombination and mismatch repair pathways which imply DNA damage and cell cycle defects (Figure 1). To examine the potential dominant negative vs haploinsufficiency role of these mutations, we introduced the mutated SEPTIN6 (P1: SEPTIN6 c.1282T>C) cDNA into wild type human male CD34+ cells. Morphological studies demonstrated enlarged cells with multi-nucleated and dysplastic nuclei. Moreover, clonogenic assays demonstrated that cells containing both the mutated and wild type protein had reduced number of colonies (17.7+/- 5.5) compared to controls (127+/- 13.7, mean+/- SD, p <0.0001). This indicates that the mutated SEPTIN6 protein has a dominant negative effect over the wild type protein. We hypothesized that the somatic stop-gain mutation in the patient (P1) genome may mitigate the deleterious effects of the primary germline SEPTIN6 c.1282T>C mutation. To test this hypothesis, and determine the effect of each individual mutation, we created lentivirus vectors which contained a shmiR against the native SEPTIN6 mRNA and also contained (a) the germline P1: SEPTIN6 c.1282T>C mutation (M2 mutation) alone, or (b) the germline M2 mutation and the somatic stop-gain mutation (P1: M1+M2 mutation) in tandem, or (c) germline P2: SEPTIN6 c.1282T>A (M3 mutation). The wild type SEPTIN6 gene was reintroduced as a control. Clonogenic assays demonstrated that SEPTIN6 knockdown in human CD34+ cells caused no significant change in progenitor activity (Table 1). Furthermore, introduction of the M2 or the M3 germline mutants in SEPTIN6 knocked down cells caused a reduction in the total number of colonies, especially the number of GM colonies with no change in the number of BFU-E colonies (Table 1). This data validates our previous experiments performed with single base pair gene editing in human CD34+ cells. Interestingly, introduction of the stop-gain M1 mutation along with the M2 mutation (M1+M2) reversed the reduced clonogenic activity seen with expression of the M2 mutation (Table 1). This indicates that the M1 mutation by introducing an early stop codon creates a SEPTIN6 knockdown effect and rescues hematopoietic function from the deleterious M2 mutation. Overall, our data demonstrates the acquisition of de novo gain of function mutations in the SEPTIN6 gene in myelopoiesis and suggests deleterious effects on engrafting hematopoietic stem cells. These mutations are associated with the development of MDS in two pediatric patients.

Dysregulated Proteostasis Restrains the Developmental HSC Expansion in Fanconi Anemia

Hematopoietic stem cells (HSCs) require precise regulation of protein synthesis, processing, and degradation. Protein homeostasis (Proteostasis) is particularly critical during development, relying on chaperone proteins to maintain folding capacity in a highly proliferative HSC pool. Even modest changes in synthesis or degradation lead to the accumulation of unfolded proteins and compromise fetal liver HSC pool formation. Fanconi anemia (FA) is a multisystem disorder that manifests with bone marrow failure (BMF) early in life. Previous reports from our lab and others suggest that pre-natal HSC depletion in FA is a critical factor for the early onset of clinical BMF. To identify the cellular and molecular mechanisms underlying prenatal HSC defects in FA, we performed single-cell transcriptome profiling in both Fancd2 +/+and Fancd2 -/- FL samples. Gene set enrichment analysis (GSEA) of differentially expressed genes revealed significantly increased expression of c-Myc target genes in Fancd2 -/ - HSCs (normalized enrichment score, NES: 2.04, p<10 -5 FDR<10 -5), matched by an increase in c-Myc in Fancd2 -/- LT-HSC (CD150 + CD48 - Lineage - Sca-1 + c-KIT +) at the protein level (1.7-fold (p<0.05). Our sc-RNA Seq data further showed an increase in ribosome biogenesis (NES: 1.8, p<10 -5, FDR<10 -3) and translation (NES: 2.2, p<10 -5, FDR<10 -5) in Fancd2 -/- HSC, functionally corroborated by increased protein synthesis rates across Fancd2 -/- FL HSPC subpopulations (1.3-fold in LT- HSCs, p<0.01) using the in vivo O-Propargyl-puromycin (OPP) assay. Not surprisingly, the increased protein synthesis rates exceed the physiologic protein folding capacity, leading to elevated levels of unfolded proteins in Fancd2 -/- FL HSPCs by Tetraphenylethene maleimide (TPE-MI) stain. The aggregated misfolded proteins (aggresomes) in Fancd2 -/- FL HSPCs were also observed by immunofluorescence microscopy. Revisiting our scRNA Seq data, the observed proteotoxic stress was also reflected in the GSEA enrichment for unfolded protein response (UPR; NES: 1.30, p=0.01, FDR=0.01) as well as heat shock stress response (NES:1.7,p<0.0001, FDR=0.09), and coincided with upregulation of response to heat shock factor-1 ( Hsf-1; NES:1.86,p<0.0001, FDR=0.02) in Fancd2 -/- FL HSCs. Because chaperone supplementation has been shown to enhance HSC protein folding capacity, we serially injected the bile acid TUDCA (a chemical chaperone) into the pregnant dam. This not only significantly reduced unfolded protein levels, but indeed rescued FL HSC numbers in Fancd2 -/- littermates. Our observations in aggregate indicate that dysregulated proteostasis is a previously unrecognized pathogenic process constraining fetal Fancd2 -/- hematopoiesis. Protection of the HSPC proteome appears to be a new, non-canonical function of FANCD2, without immediate connection to the FA pathway and DNA crosslink repair. Importantly, we demonstrate the successful pharmacological rescue of the fetal Fancd2 -/- HSPC pool.

CPT1a Controls the Function of Long-Term Hematopoietic Stem Cells By Regulating the Balance between Fatty Acid Oxidation and Oxphos in Mitochondria

Hematopoietic stem cells (HSCs) rely on the balance in place of quiescence and self-renewal to sustain stem cell potential and differentiation to generate mature blood cells. Appropriate regulation of cellular and mitochondrial metabolic processes such as glycolysis, OXPHOS, fatty acid metabolism, and amino acid metabolism are required for HSC maintenance. However, the mechanisms by which cellular and mitochondrial metabolisms regulate HSC quiescence, proliferation, and differentiation remain to be fully understood. Fatty acid oxidation (FAO) is a metabolic process involving the breakdown of fatty acids into acetyl-CoA, which is then transported into mitochondria by carnitine palmitoyltransferase 1a (CPT1a). It has been reported that inhibiting FAO using non-specific inhibitors of CPT1a impairs HSC maintenance. Interestingly, CPT1a forms a complex with the Fanconi anemia complementation group D2 (FANCD2) protein (T Zhang et al., Scientific Reports, 2017). Given that Fanconi anemia is associated with HSC defects, this CPT1a complex may play a critical role in maintaining fatty acid dependent mitochondrial metabolism that mediates HSC dormancy and stemness. However, the exact role, in HSCs, of mitochondrial CPT1a, the rate-limiting enzyme in FAO, remains unknown. Here, we find that CPT1a is highly enriched in hematopoietic stem cells. Further, we investigated the role of CPT1a in hematopoiesis utilizing a CPT1aconditional knockout in mice driven by Vav1-Cre. The CPT1a knockout ( Cpt1aΔ/Δ) displays profound HSC defects, including loss of HSC self-renewal and accelerated differentiation. Mechanistically, we find that loss of Cpt1a results in increased ATP production, mitochondrial membrane potential (Δφm), and mitochondrial ROS accumulation in long-term HSCs (LT-HSCs), as well as defective LT-HSC function indicated by bone marrow transplantation and 5-FU genotoxic stress assays. Furthermore, utilizing mitochondrial complex formation assays and measurements of mitochondrial complex activity, BM cells of Cpt1aΔ/Δ mice display enhanced mitochondrial complex formation and up-regulation of mitochondrial complex activity. Our results suggest that Cpt1a regulates FAO transport and that loss of Cpt1a deregulates mitochondrial metabolisms including oxidative phosphorylation (OXPHOS). Taken together, our study suggests that CPT1a plays a key role in LT-HSC maintenance by controlling the balance between FAO and OXPHOS in mitochondria. Because of the noncanonical relationship between CPT1a and FANCD2 for the regulation of mitochondrial metabolism, correction of fatty acid metabolism and OXPHOS may present a novel therapeutic avenue for Fanconi anemia.

Reduced Polycomb Repressor Complex 2 (PRC2) Activity and Increased TP53 Activity Mediate Hematopoietic Stem Cell Dysfunction in RPS19-Mutated Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia caused by heterozygous loss-of-function mutations in one of 23 ribosomal protein (RP) genes, with RPS19 being mutated in approximately 25% of patients. While DBA typically manifests in infancy as isolated anemia with reticulocytopenia, older patients can develop multilineage cytopenias and bone marrow (BM) hypocellularity suggesting impaired hematopoietic stem cell (HSC) function. Defects in HSCs have been observed in Rps19-depleted mice but studies on human DBA HSCs have been inconclusive, largely because patient BM cells are difficult to obtain for research. To address this gap, we developed a new, robust, and scalable cellular model using CRISPR/Cas9 to create heterozygous RPS19 loss-of-function mutations in healthy donor CD34 + hematopoietic stem and progenitor cells (HSPCs). Transplantation of RPS19+/- HSPCs into NBSGW immunodeficient mice revealed a defect in BM repopulation that was partially rescued by co-disruption of the TP53 gene (Bhoopalan et al, JCI Insight, 2023). Here we provide further mechanistic insight into this HSC defect by showing that RP imbalance caused by RPS19 deficiency leads to the activation of TP53 and inhibition of Polycomb Repressor Complex 2 (PRC2), which has been previously shown to be required for HSC maintenance. Healthy donor CD34 + HSPC were electroporated with ribonucleoprotein (RNP) complex consisting of Cas9 and guide RNAs (gRNAs) targeting RPS19 or the AAVS1 safe harbor locus as a negative control. RPS19 disruption was verified by next-generation sequencing of the targeted region and by Western blot analysis showing reduction of RPS19 protein. RNA-Seq at 3 days after electroporation showed enrichment of TP53 pathway genes in RPS19-edited HSPCs compared to control cells, confirming our previous observations. Additionally, we noted an enrichment of HSPC genes that were previously shown to harbor the repressive histone mark H3K27me3 in normal HSPCs. Methylation of H3K27 is catalyzed by EZH2, the catalytic component of PRC2, which maintains HSCs by inhibiting transcription of genes that promote differentiation. Gene set enrichment analysis (GSEA) revealed significant induction of PRC2 target genes in RPS19-disrupted HSPCs and in induced pluripotent stem cells (iPSCs) generated from two different DBA patients with heterozygous RPS19 mutations. The “Cleavage under targets and release using nuclease (CUT&RUN)” assay indicated globally reduced EZH2 binding to chromatin in RPS19-disrupted HSPCs. These findings suggest that PRC2 activity is inhibited by RPS19 haploinsufficiency. Previous studies have shown that RPS19 haploinsufficiency can cause accumulation of large RP subunits, RPL5 and RPL11, which bind 5S ribosomal RNA to form 5S RNP. This complex can sequester the ubiquitin ligase MDM2 to inhibit its function. MDM2 ubiquitinates TP53, targeting it for proteasomal degradation. Additionally, MDM2 binds PRC2 to augment its activity. Thus, we theorized that RPS19 haploinsufficiency leads to the accumulation of 5S RNP complex which sequesters MDM2, causing increased TP53 activity and suppression of PRC2 activity, both of which can inhibit HSC maintenance. To test this, we inhibited formation of 5S RNP complex by co-disrupting RPL5 or RPL11 along with RPS19 in CD34 + HSPCs, followed by RNA-Seq analysis or transplantation into NBSGW mice. Remarkably, haploinsufficiency of either large RP gene rescued the BM repopulation defect of RPS19 +/- HSPC (p<0.05) ( Fig. 1A), reduced TP53 activity and improved PRC2 activity (p<0.001 and FDR q<0.01) ( Fig. 1B). In summary, our data support the model that RPS19 haploinsufficiency leads to increased TP53 activity and reduced PRC2 activity through 5S RNP-mediated sequestration of MDM2, causing impairment of HSC function. These studies provide insights into the molecular mechanisms of DBA and could potentially inform new therapies.

Nynrin Preserves Hematopoietic Stem Cell Function Via the Ppif-Mediated Mitochondrial Permeability Transition

Mitochondria have recently been identified as a critical regulator for homeostasis of hematopoietic stem cells (HSCs). However, the mechanism underlying HSC regulation remains poorly clarified. Here, we identify transcription factor Nynrin as a novel regulator of HSC maintenance through modulation of mitochondrial function. We demonstrate that Nynrin knockout leads to significantly decreased long-term HSC frequency, markedly reduced HSC dormancy and self-renewal capacity in steady-state and stress hematopoiesis. Nynrin-deficient HSCs are more compromised in tolerance of irradiation- and 5-fluorouracil-induced stresses and exhibit typical phenotypes of necrosis. Mechanistically, Nynrin deletion induces transactivation of Ppif, its product cyclophilin D is one of the prominent members of mitochondrial permeability transition (mPT). Overexpression of Ppif exhausts long-term HSC, impairs genotoxic hematopoietic recovery, and enhances mPT, resulting in mitochondrial swelling and reactive oxygen species production. Suppression of Ppif essentially rescues the phenotypic and functional defects in Nynrin-deleted HSCs. Collectively, our data, for the first time, characterize Nynrin as a critical regulator of HSC function acting through the Ppif/ mPT mitochondria axis and highlight the importance of Nynrin in HSC maintenance. These observations provide new insights into the mechanisms for controlling HSC fate.

LepR+ niche cell–derived AREG compromises hematopoietic stem cell maintenance under conditions of DNA repair deficiency and aging

AbstractThe cross talk between extrinsic niche-derived and intrinsic hematopoietic stem cell (HSC) factors controlling HSC maintenance remains elusive. Here, we demonstrated that amphiregulin (AREG) from bone marrow (BM) leptin receptor (LepR+) niche cells is an important factor that mediates the cross talk between the BM niche and HSCs in stem cell maintenance. Mice deficient of the DNA repair gene Brca2, specifically in LepR+ cells (LepR-Cre;Brca2fl/fl), exhibited increased frequencies of total and myeloid-biased HSCs. Furthermore, HSCs from LepR-Cre;Brca2fl/fl mice showed compromised repopulation, increased expansion of donor-derived, myeloid-biased HSCs, and increased myeloid output. Brca2-deficient BM LepR+ cells exhibited persistent DNA damage–inducible overproduction of AREG. Ex vivo treatment of wild-type HSCs or systemic treatment of C57BL/6 mice with recombinant AREG impaired repopulation, leading to HSC exhaustion. Conversely, inhibition of AREG by an anti–AREG-neutralizing antibody or deletion of the Areg gene in LepR-Cre;Brca2fl/fl mice rescued HSC defects caused by AREG. Mechanistically, AREG activated the phosphoinositide 3-kinases (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, promoted HSC cycling, and compromised HSC quiescence. Finally, we demonstrated that BM LepR+ niche cells from other DNA repair–deficient and aged mice also showed persistent DNA damage–associated overexpression of AREG, which exerts similar negative effects on HSC maintenance. Therefore, we identified an important factor that regulates HSCs function under conditions of DNA repair deficiency and aging.

Modulating the immune system as a therapeutic target for myelodysplastic syndromes and acute myeloid leukemia.

Modulating the immune system to treat diseases, including myeloid malignancies, has resulted in the development of a multitude of novel therapeutics in recent years. Myelodysplastic syndromes or neoplasms (MDS) and acute myeloid leukemia (AML) are hematologic malignancies that arise from defects in hematopoietic stem and progenitor cells (HSPCs). Dysregulated immune responses, especially in innate immune and inflammatory pathways, are highly associated with the acquisition of HSPC defects in MDS and AML pathogenesis. In addition to utilizing the immune system in immunotherapeutic interventions such as chimeric antigen receptor T cell therapy, vaccines, and immune checkpoint inhibitors, mitigating dysregulation of innate immune and inflammatory responses in MDS and AML remains a priority in slowing the initiation and progression of these myeloid malignancies. This review provides a comprehensive summary of the current progress of diverse strategies to utilize or modulate the immune system in the treatment of MDS and AML.

RNA binding protein SYNCRIP maintains proteostasis and self-renewal of hematopoietic stem and progenitor cells

Tissue homeostasis is maintained after stress by engaging and activating the hematopoietic stem and progenitor compartments in the blood. Hematopoietic stem cells (HSCs) are essential for long-term repopulation after secondary transplantation. Here, using a conditional knockout mouse model, we revealed that the RNA-binding protein SYNCRIP is required for maintenance of blood homeostasis especially after regenerative stress due to defects in HSCs and progenitors. Mechanistically, we find that SYNCRIP loss results in a failure to maintain proteome homeostasis that is essential for HSC maintenance. SYNCRIP depletion results in increased protein synthesis, a dysregulated epichaperome, an accumulation of misfolded proteins and induces endoplasmic reticulum stress. Additionally, we find that SYNCRIP is required for translation of CDC42 RHO-GTPase, and loss of SYNCRIP results in defects in polarity, asymmetric segregation, and dilution of unfolded proteins. Forced expression of CDC42 recovers polarity and in vitro replating activities of HSCs. Taken together, we uncovered a post-transcriptional regulatory program that safeguards HSC self-renewal capacity and blood homeostasis.

Epigenetic regulation by ASXL1 in myeloid malignancies.

Myeloid malignancies areclonal hematopoietic disorders that are comprised of a spectrum of genetically heterogeneous disorders, including myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), and acute myeloid leukemia (AML). Myeloid malignancies are characterized by excessive proliferation, abnormal self-renewal, and/or differentiation defects of hematopoietic stem cells (HSCs) and myeloid progenitor cells hematopoietic stem/progenitor cells (HSPCs). Myeloid malignancies can be caused by genetic and epigenetic alterations that provoke key cellular functions, such as self-renewal, proliferation, biased lineage commitment, and differentiation. Advances in next-generation sequencing led to the identification of multiple mutations in myeloid neoplasms, and many new gene mutations were identified as key factors in driving the pathogenesis of myeloid malignancies. The polycomb protein ASXL1 was identified to be frequently mutated in all forms of myeloid malignancies, with mutational frequencies of 20%, 43%, 10%, and 20% in MDS, CMML, MPN, and AML, respectively. Significantly, ASXL1 mutations are associated with a poor prognosis in all forms of myeloid malignancies. The fact that ASXL1 mutations are associated with poor prognosis in patients with CMML, MDS, and AML, points to the possibility that ASXL1 mutation is a key factor in the development of myeloid malignancies. This review summarizes the recent advances in understanding myeloid malignancies with a specific focus on ASXL1 mutations.

Tespa1 facilitates hematopoietic and leukemic stem cell maintenance by restricting c-Myc degradation

Hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) have robust self-renewal potential, which is responsible for sustaining normal and malignant hematopoiesis, respectively. Although considerable efforts have been made to explore the regulation of HSC and LSC maintenance, the underlying molecular mechanism remains obscure. Here, we observe that the expression of thymocyte-expressed, positive selection-associated 1 (Tespa1) is markedly increased in HSCs after stresses exposure. Of note, deletion of Tespa1 results in short-term expansion but long-term exhaustion of HSCs in mice under stress conditions due to impaired quiescence. Mechanistically, Tespa1 can interact with CSN subunit 6 (CSN6), a subunit of COP9 signalosome, to prevent ubiquitination-mediated degradation of c-Myc protein in HSCs. As a consequence, forcing c-Myc expression improves the functional defect of Tespa1-null HSCs. On the other hand, Tespa1 is identified to be highly enriched in human acute myeloid leukemia (AML) cells and is essential for AML cell growth. Furthermore, using MLL-AF9-induced AML model, we find that Tespa1 deficiency suppresses leukemogenesis and LSC maintenance. In summary, our findings reveal the important role of Tespa1 in promoting HSC and LSC maintenance and therefore provide new insights on the feasibility of hematopoietic regeneration and AML treatment.

Copper overload impairs hematopoietic stem and progenitor cell proliferation via prompting HSF1/SP1 aggregation and the subsequently downregulating FOXM1-Cytoskeleton axis

Unbalanced Cu homeostasis has been suggested to be associated with hematopoietic disease, but the roles of Cu overload in the hematopoietic system and the potential mechanisms are obscure. Here, we report a novel association and the novel potential pathways for Cu overload to induce proliferation defects in zebrafish embryonic hematopoietic stem and progenitor cells (HSPCs) via down-regulating expression of foxm1-cytoskeleton axis, which is conserved from fish to mammals. Mechanistically, we show the direct binding of Cu to transcriptional factors HSF1 and SP1 and that Cu overload induces the cytoplasmic aggregation of proteins HSF1 and SP1. These result in the reduced transcriptional activities of HSF1 and SP1 on their downstream FOXM1 as well as the FOXM1 transcriptional activities on cytoskeletons in HSPCs, which leads to ultimately cell proliferation impairment. These findings unveil the novel linkage of Cu overload with specific signaling transduction as well as the subsequent HSPC proliferation defects.

An RPS19-edited model for Diamond-Blackfan anemia reveals TP53-dependent impairment of hematopoietic stem cell activity.

Diamond-Blackfan anemia (DBA) is a genetic blood disease caused by heterozygous loss-of-function mutations in ribosomal protein (RP) genes, most commonly RPS19. The signature feature of DBA is hypoplastic anemia occurring in infants, although some older patients develop multilineage cytopenias with bone marrow hypocellularity. The mechanism of anemia in DBA is not fully understood and even less is known about the pancytopenia that occurs later in life, in part because patient hematopoietic stem and progenitor cells (HSPCs) are difficult to obtain, and the current experimental models are suboptimal. We modeled DBA by editing healthy human donor CD34+ HSPCs with CRISPR/Cas9 to create RPS19 haploinsufficiency. In vitro differentiation revealed normal myelopoiesis and impaired erythropoiesis, as observed in DBA. After transplantation into immunodeficient mice, bone marrow repopulation by RPS19+/- HSPCs was profoundly reduced, indicating hematopoietic stem cell (HSC) impairment. The erythroid and HSC defects resulting from RPS19 haploinsufficiency were partially corrected by transduction with an RPS19-expressing lentiviral vector or by Cas9 disruption of TP53. Our results define a tractable, biologically relevant experimental model of DBA based on genome editing of primary human HSPCs and they identify an associated HSC defect that emulates the pan-hematopoietic defect of DBA.

DELETION OF THROMBOSPONDIN-1 PRESERVES HEMATOPOIETIC STEM CELL HEALTHSPAN DURING AGING

Abstract Aging is associated with defects within blood stem cells, termed hematopoietic stem cells (HSC), including a loss of their self-renewal potential and a skewed differentiation towards myeloid lineages at the expense of lymphoid cells. Collectively, these HSC defects manifest as anemias, poor response to vaccines and an increased incidence of myeloid neoplasms in older adults. Unlike other somatic stem cells, aged HSCs have been shown to be refractory towards established anti-aging interventions including caloric restriction, exercise, parabiosis and plasma transfer. Thrombospondin-1 (TSP1) was initially discovered as an anti-angiogenic molecule, and recent studies have identified that TSP1 promotes age-related pathologies including chronic inflammation, reactive oxygen species (ROS) generation, and mitochondrial dysfunction. Notably, each of these TSP-1 regulated processes have been shown to critically influence HSC biology, particularly in the context of aging. However, whether TSP-1 directly regulates HSC activity remains unexplored. Here, we sought to determine whether TSP-1 is essential for HSC development, and whether blocking TSP1 signaling could ameliorate age-related HSC defects. Utilizing murine models, we demonstrate that TSP-1 is dispensable for normal HSC development and hematopoiesis. We show that deletion of TSP-1 is sufficient to preserve HSC fitness during aging, as evidenced by preservation of youthful self-renewal potential and balanced lineage reconstitution during serial HSC transplantation assays. Mechanistically, we identify that TSP-1 adversely impacts mitochondrial metabolism within HSCs, and show that loss of TSP-1 prevents the age-related decline in HSC mitochondrial membrane potential. Our findings identify TSP-1 as a pro-geronic factor that can be targeted to preserve HSC healthspan.

Endothelial mTOR Preserves Hematopoietic Stem Cell Fitness By Suppressing Thrombospondin1

Hematopoietic stem cells (HSC) reside in close proximity to blood vessels within the bone marrow (BM) termed 'perivascular niches' which provide instructive cues for maintaining HSC self-renewal potential. The perivascular niche is comprised of endothelial cells (ECs) lining the BM blood vessels (BMECs), and an array of perivascular stromal cells that collectively serve to regulate balanced HSC self-renewal and differentiation. While young HSCs are endowed with robust self-renewal potential, aging is associated with a significant decline in HSC fitness including a loss of their self-renewal ability and a biased differentiation towards the myeloid lineages at the expense of lymphopoiesis. These aging related HSC defects result in anemias, impaired response to immunizations, and an increased risk for developing clonal disorders including acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS). However, mechanisms that drive HSC defects during aging remain poorly understood, and represents a critical barrier towards developing strategies for preservation of hematopoietic fitness during aging. While HSC aging was generally assumed as a predominantly cell-intrinsic process, recent studies have identified that aging of the BM niche plays an instructive role in promoting age-related HSC defects. Supporting this, we recently discovered that aging is associated with a significant decline in mTOR activity within BMECs that impairs their capacity to serve as a HSC-supportive niche. Our findings showed that deletion of mTOR specifically within ECs of young (4 month old) mice (Endothelial Cell specific mTOR Knock Out; mTORECKO) is sufficient to recapitulate HSC aging phenotypes including loss of HSC self-renewal, and a myeloid-biased differentiation. Here, we sought to identify the mechanisms by which endothelial mTOR preserves HSC fitness. To identify candidate factors that promote HSC aging in mTORECKO mice, we performed a plasma proteomic analysis that revealed a putative role for Thrombospondin1 (Thbs1) wherein mTORECKO mice displayed elevated levels of Thbs1. Analysis of cultured BMECs revealed that inhibition of mTOR signaling within BMECs was sufficient to increase Thbs1 expression. Thbs1 is a well-defined antiangiogenic factor that has recently been identified to play a critical role in promoting a multitude of age-related cardiovascular and inflammatory disorders. However, whether Thbs1 regulates HSC activity is not known. To this end, we performed ex vivo HSC expansion transplantation assays which revealed that Thbs1 causes a significant decline in long term HSC engraftment. RNA Seq analysis of HSC expansions revealed that Thbs1 activates chronic inflammatory responses within HSCs that have been suggested to promote aging related HSC defects. To determine whether Thbs1 promotes HSC aging in mTORECKO mice, we crossed them with Thbs1 global knockout mice (Thbs1KO) to generate mTORECKOThbs1KO mice. Analysis of mTORECKOThbs1KO mice revealed that deletion of Thbs1 was sufficient to restore hematopoietic defects in mTORECKO mice including restoration of peripheral blood myeloid bias, BM HSC frequency and hematopoietic progenitor activity. Furthermore, serial HSC transplantation analysis confirmed that deletion of Thbs1 was sufficient to restore HSC self-renewal and lymphoid output in mTORECKO mice, identifying a putative role for Thbs1 as a pro-geronic factor for HSCs. To determine whether Thbs1 promotes HSC dysfunction during physiological aging, we aged Thbs1KO mice for 18 months and analyzed their hematopoietic parameters. Our analysis revealed that deletion of Thbs1 was able to prevent the age-associated increase in peripheral blood myeloid bias and BM HSC frequency. Notably, HSCs derived from aged (18 month old) Thbs1KO mice demonstrated a suppression of inflammatory responses by RNA Seq analysis, and a restoration of self-renewal ability and balanced lineage output in serial HSC transplantation assays, that are indistinguishable from HSCs derived from young (3 month old) mice. Our findings identify Thbs1 as a novel pro-geronic factor for HSC aging that can be targeted to preserve hematopoietic healthspan.