技术资料

Angiotensin (Ang)-(1–7) stimulates vasoprotective functions of diabetic (DB) CD34 + hematopoietic stem/progenitor cells partly by decreasing reactive oxygen species (ROS),increasing nitric oxide (NO) levels and decreasing TGFβ1 secretion. Telomerase reverse transcriptase (TERT) translocates to mitochondria and regulates ROS generation. Alternative splicing of TERT results in variants α-,β- and α-β-TERT,which may oppose functions of full-length (FL) TERT. This study tested if the protective functions of Ang-(1–7) or TGFβ1-silencing are mediated by mitoTERT and that diabetes decreases FL-TERT expression by inducing splicing. CD34 + cells were isolated from the peripheral blood mononuclear cells of nondiabetic (ND,n = 68) or DB (n = 74) subjects. NO and mitoROS levels were evaluated by flow cytometry. TERT splice variants and mitoDNA-lesions were characterized by qPCR. TRAP assay was used for telomerase activity. Decoy peptide was used to block mitochondrial translocation (mitoXTERT). TERT inhibitor or mitoXTERT prevented the effects of Ang-(1–7) on NO or mitoROS levels in DB-CD34 + cells. FL-TERT expression and telomerase activity were lower and mitoDNA-lesions were higher in DB cells compared to ND and were reversed by Ang-(1–7) or TGFβ1-silencing. The prevalence of TERT splice variants,with predominant β-TERT expression,was higher and the expression of FL-TERT was lower in DB cells (n = 25) compared to ND (n = 30). Ang-(1–7) or TGFβ1-silencing decreased TERT-splicing and increased FL-TERT. Blocking of β-splicing increased FL-TERT and protected mitoDNA in DB-cells. The findings suggest that diabetes induces TERT-splicing in CD34 + cells and that β-TERT splice variant largely contributes to the mitoDNA oxidative damage. View Publication

Pseudouridylation plays a regulatory role in various physiological and pathological processes. A prime example is the mitochondrial myopathy,lactic acidosis,and sideroblastic anemia syndrome (MLASA),characterized by defective pseudouridylation resulting from genetic mutations in pseudouridine synthase 1 (PUS1). However,the roles and mechanisms of pseudouridylation in normal erythropoiesis and MLASA-related anemia remain elusive. We established a mouse model carrying a point mutation (R110W) in the enzymatic domain of PUS1,mimicking the common mutation in human MLASA. Pus1 -mutant mice exhibited anemia at 4 weeks old. Impaired mitochondrial oxidative phosphorylation was also observed in mutant erythroblasts. Mechanistically,mutant erythroblasts showed defective pseudouridylation of targeted tRNAs,altered tRNA profiles,decreased translation efficiency of ribosomal protein genes,and reduced globin synthesis,culminating in ineffective erythropoiesis. Our study thus provided direct evidence that pseudouridylation participates in erythropoiesis in vivo. We demonstrated the critical role of pseudouridylation in regulating tRNA homeostasis,cytoplasmic translation,and erythropoiesis. Subject areas: Molecular biology,Cell biology View Publication

Acute myeloid leukemia (AML) is a highly aggressive hematologic cancer with poor survival across a broad range of molecular subtypes. Development of efficacious and well-tolerable therapies encompassing the range of mutations that can arise in AML remains an unmet need. The bromo- and extra-terminal domain (BET) family of proteins represents an attractive therapeutic target in AML due to their crucial roles in many cellular functions,regardless of any specific mutation. Many BET inhibitors (BETi) are currently in pre-clinical and early clinical development,but acquisition of resistance continues to remain an obstacle for the drug class. Novel methods to circumvent this development of resistance could be instrumental for the future use of BET inhibitors in AML,both as monotherapy and in combination. To date,many investigations into possible drug combinations of BETi with CDK inhibitors have focused on CDK9,which has a known physical and functional interaction with the BET protein BRD4. Therefore,we wished to investigate possible synergy and additive effects between inhibitors of these targets in AML. Here,we describe combination therapy with the multi-CDK inhibitor dinaciclib and the BETi PLX51107 in pre-clinical models of AML. Dinaciclib and PLX51107 demonstrate additive effects in AML cell lines,primary AML samples,and in vivo. Further,we demonstrate novel activity of dinaciclib through inhibition of the canonical/β-catenin dependent Wnt signaling pathway,a known resistance mechanism to BETi in AML. We show dinaciclib inhibits Wnt signaling at multiple levels,including downregulation of β-catenin,the Wnt co-receptor LRP6,as well as many Wnt pathway components and targets. Moreover,dinaciclib sensitivity remains unaffected in a setting of BET resistance,demonstrating similar inhibitory effects on Wnt signaling when compared to BET-sensitive cells. Ultimately,our results demonstrate rationale for combination CDKi and BETi in AML. In addition,our novel finding of Wnt signaling inhibition could have potential implications in other cancers where Wnt signaling is dysregulated and demonstrates one possible approach to circumvent development of BET resistance in AML. The online version contains supplementary material available at 10.1186/s40164-024-00483-w. View Publication

In the adult bone marrow (BM),endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche,which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM,distinct vascular arteriole,transitional,and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However,the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover,constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this,we describe two tamoxifen-inducible cre -expressing lines,Vegfr3-creER T2 and Cx40-creER T2,that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow,without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre -dependent recombination constitutively-activates MAPK signaling within adult endothelium,we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creER T2 -expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM,providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis. The online version contains supplementary material available at 10.1007/s12015-024-10703-9. View Publication

The heart is a metabolic “omnivore” and adjusts its energy source depending on the circulating metabolites. Human cardiac organoids,a three-dimensional in vitro model of the heart wall,are a useful tool to study cardiac physiology and pathology. However,cardiac tissue naturally experiences shear stress and nutrient fluctuations via blood flow in vivo,whilst in vitro models are conventionally cultivated in a static medium. This necessitates the regular refreshing of culture media,which creates acute cellular disturbances and large metabolic fluxes. To culture human cardiac organoids in a more physiological manner,we have developed a perfused bioreactor for cultures in a 96-well plate format. The designed bioreactor is easy to fabricate using a common culture plate and a 3D printer. Its open system allows for the use of traditional molecular biology techniques,prevents flow blockage issues,and provides easy access for sampling and cell assays. We hypothesized that a perfused culture would create more stable environment improving cardiac function and maturation. We found that lactate is rapidly produced by human cardiac organoids,resulting in large fluctuations in this metabolite under static culture. Despite this,neither medium perfusion in bioreactor culture nor lactate supplementation improved cardiac function or maturation. In fact,RNA sequencing revealed little change across the transcriptome. This demonstrates that cardiac organoids are robust in response to fluctuating environmental conditions under normal physiological conditions. Together,we provide a framework for establishing an easily accessible perfusion system that can be adapted to a range of miniaturized cell culture systems. View Publication

The human WDR33 gene encodes three major isoforms. The canonical isoform WDR33v1 (V1) is a well-characterized nuclear mRNA polyadenylation factor,while the other two,WDR33v2 (V2) and WDR33v3 (V3),have not been studied. Here,we report that V2 and V3 are generated by alternative polyadenylation,and neither protein contains all seven WD (tryptophan-aspartic acid) repeats that characterize V1. Surprisingly,V2 and V3 are not polyadenylation factors but localize to the endoplasmic reticulum and interact with stimulator of interferon genes (STING),the immune factor that induces the cellular response to cytosolic double-stranded DNA. V2 suppresses interferon-β induction by preventing STING disulfide oligomerization but promotes autophagy,likely by recruiting WIPI2 isoforms. V3,on the other hand,functions to ncrease STING protein levels. Our study has not only provided mechanistic insights into STING regulation but also revealed that protein isoforms can be functionally completely unrelated,indicating that alternative mRNA processing is a more powerful mechanism than previously appreciated. View Publication

Wiskott-Aldrich syndrome (WAS) is a severe X-linked primary immunodeficiency resulting from a diversity of mutations distributed across all 12 exons of the WAS gene. WAS encodes a hematopoietic-specific and developmentally regulated cytoplasmic protein (WASp). The objective of this study was to develop a gene correction strategy potentially applicable to most WAS patients by employing nuclease-mediated,site-specific integration of a corrective WAS gene sequence into the endogenous WAS chromosomal locus. In this study,we demonstrate the ability to target the integration of WAS 2-12 -containing constructs into intron 1 of the endogenous WAS gene of primary CD34 + hematopoietic stem and progenitor cells (HSPCs),as well as WASp-deficient B cell lines and WASp-deficient primary T cells. This intron 1 targeted integration (TI) approach proved to be quite efficient and restored WASp expression in treated cells. Furthermore,TI restored WASp-dependent function to WAS patient T cells. Edited CD34 + HSPCs exhibited the capacity for multipotent differentiation to various hematopoietic lineages in vitro and in transplanted immunodeficient mice. This methodology offers a potential editing approach for treatment of WAS using patient’s CD34 + cells. View Publication

Current anticancer therapies cannot eliminate all cancer cells,which hijack normal arginine methylation as a means to promote their maintenance via unknown mechanisms. Here we show that targeting protein arginine N -methyltransferase 9 (PRMT9),whose activities are elevated in blasts and leukemia stem cells (LSCs) from patients with acute myeloid leukemia (AML),eliminates disease via cancer-intrinsic mechanisms and cancer-extrinsic type I interferon (IFN)-associated immunity. PRMT9 ablation in AML cells decreased the arginine methylation of regulators of RNA translation and the DNA damage response,suppressing cell survival. Notably,PRMT9 inhibition promoted DNA damage and activated cyclic GMP-AMP synthase,which underlies the type I IFN response. Genetically activating cyclic GMP-AMP synthase in AML cells blocked leukemogenesis. We also report synergy of a PRMT9 inhibitor with anti-programmed cell death protein 1 in eradicating AML. Overall,we conclude that PRMT9 functions in survival and immune evasion of both LSCs and non-LSCs; targeting PRMT9 may represent a potential anticancer strategy. Subject terms: Cancer,Tumour immunology View Publication

Hematopoietic stem cells (HSCs) produce all essential cellular components of the blood. Stromal cell lines supporting HSCs follow a vascular smooth muscle cell (vSMC) differentiation pathway,suggesting that some hematopoiesis-supporting cells originate from vSMC precursors. These pericyte-like precursors were recently identified in the aorta-gonad-mesonephros (AGM) region; however,their role in the hematopoietic development in vivo remains unknown. Here,we identify a subpopulation of NG2 + Runx1 + perivascular cells that display a sclerotome-derived vSMC transcriptomic profile. We show that deleting Runx1 in NG2 + cells impairs the hematopoietic development in vivo and causes transcriptional changes in pericytes/vSMCs,endothelial cells and hematopoietic cells in the murine AGM. Importantly,this deletion leads also to a significant reduction of HSC reconstitution potential in the bone marrow in vivo. This defect is developmental,as NG2 + Runx1 + cells were not detected in the adult bone marrow,demonstrating the existence of a specialised pericyte population in the HSC-generating niche,unique to the embryo. Subject terms: Cell biology,Haematopoiesis,Cardiovascular biology View Publication

Here,we present a protocol for isolating and culturing mouse photoreceptors in a minimal,chemically defined medium free from serum. We describe steps for retina dissection,enzymatic dissociation,photoreceptor enrichment,cell culture,extracellular vesicles (EVs) enrichment,and EV ultrastructural analysis. This protocol,which has been verified for cultured cells derived from multiple murine strains,allows for the study of several aspects of photoreceptor biology,including EV isolation and nanotube formation. For complete details on the use and execution of this protocol,please refer to Kalargyrou et al. (2021). 1 Subject areas: Cell Biology,Molecular Biology,Neuroscience View Publication

Understanding why some triple-negative breast cancer (TNBC) patients respond poorly to existing therapies while others respond well remains a challenge. This study aims to understand the potential underlying mechanisms distinguishing early-stage TNBC tumors that respond to clinical intervention from non-responders,as well as to identify clinically viable therapeutic strategies,specifically for TNBC patients who may not benefit from existing therapies. We conducted retrospective bioinformatics analysis of historical gene expression datasets to identify a group of genes whose expression levels in early-stage tumors predict poor clinical outcomes in TNBC. In vitro small-molecule screening,genetic manipulation,and drug treatment in syngeneic mouse models of TNBC were utilized to investigate potential therapeutic strategies and elucidate mechanisms of drug action. Our bioinformatics analysis reveals a robust association between increased expression of immunosuppressive cytokine S100A8/A9 in early-stage tumors and subsequent disease progression in TNBC. A targeted small-molecule screen identifies PIM kinase inhibitors as capable of decreasing S100A8/A9 expression in multiple cell types,including TNBC and immunosuppressive myeloid cells. Combining PIM inhibition and immune checkpoint blockade induces significant antitumor responses,especially in otherwise resistant S100A8/A9-high PD-1/PD-L1-positive tumors. Notably,serum S100A8/A9 levels mirror those of tumor S100A8/A9 in a syngeneic mouse model of TNBC. Our data propose S100A8/A9 as a potential predictive and pharmacodynamic biomarker in clinical trials evaluating combination therapy targeting PIM and immune checkpoints in TNBC. This work encourages the development of S100A8/A9-based liquid biopsy tests for treatment guidance. Subject terms: Breast cancer,Breast cancer,Prognostic markers View Publication

Ferroptosis is a new form of nonapoptotic and iron-dependent type of cell death. Glutathione peroxidase-4 (GPX4) plays an essential role in anti-ferroptosis by reducing lipid peroxidation. Although acute myeloid leukemia (AML) cells,especially relapsed and refractory (R/R)-AML,present high GPX4 levels and enzyme activities,pharmacological inhibition of GPX4 alone has limited application in AML. Thus,whether inhibition of GPX4 combined with other therapeutic reagents has effective application in AML is largely unknown. Lipid reactive oxygen species (ROS),malondialdehyde (MDA),and glutathione (GSH) assays were used to assess ferroptosis in AML cells treated with the hypomethylating agent (HMA) decitabine (DAC),ferroptosis-inducer (FIN) RAS-selective lethal 3 (RSL3),or their combination. Combination index (CI) analysis was used to assess the synergistic activity of DAC + RSL3 against AML cells. Finally,we evaluated the synergistic activity of DAC + RSL3 in murine AML and a human R/R-AML-xenografted NSG model in vivo. We first assessed GPX4 expression and found that GPX4 levels were higher in AML cells,especially those with MLL rearrangements,than in NCs. Knockdown of GPX4 by shRNA and indirect inhibition of GPX4 enzyme activity by RSL3 robustly induced ferroptosis in AML cells. To reduce the dose of RSL3 and avoid side effects,low doses of DAC (0.5 µM) and RSL3 (0.05 µM) synergistically facilitate ferroptosis by inhibiting the AMP-activated protein kinase (AMPK)-SLC7A11-GPX4 axis. Knockdown of AMPK by shRNA enhanced ferroptosis,and overexpression of SLC7A11 and GPX4 rescued DAC + RSL3-induced anti-leukemogenesis. Mechanistically,DAC increased the expression of MAGEA6 by reducing MAGEA6 promoter hypermethylation. Overexpression of MAGEA6 induced the degradation of AMPK,suggesting that DAC inhibits the AMPK-SLC7A11-GPX4 axis by increasing MAGEA6 expression. In addition,DAC + RSL3 synergistically reduced leukemic burden and extended overall survival compared with either DAC or RSL3 treatment in the MLL-AF9-transformed murine model. Finally,DAC + RSL3 synergistically reduced viability in untreated and R/R-AML cells and extended overall survival in two R/R-AML-xenografted NSG mouse models. Our study first identify vulnerability to ferroptosis by regulating MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway. Combined treatment with HMAs and FINs provides a potential therapeutic choice for AML patients,especially for R/R-AML. The online version contains supplementary material available at 10.1186/s40164-024-00489-4. View Publication