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Hematopoietic stem cell (HSC) aging is accompanied by hematopoietic reconstitution dysfunction, including loss of regenerative and engraftment ability, myeloid differentiation bias, and elevated risks of hematopoietic malignancies. Gut microbiota, a key regulator of host health and immunity, has recently been reported to affect hematopoiesis. However, there is currently limited empirical evidence explaining the direct impact of gut microbiome on aging hematopoiesis. In this study, we performed fecal microbiota transplantation (FMT) from young mice to aged mice and observed a significant increment in lymphoid differentiation and decrease in myeloid differentiation in aged recipient mice. Furthermore, FMT from young mice rejuvenated aged HSCs with enhanced short-term and long-term hematopoietic repopulation capacity. Mechanistically, single-cell RNA sequencing deciphered that FMT from young mice mitigated inflammatory signals, upregulated the FoxO signaling pathway, and promoted lymphoid differentiation of HSCs during aging. Finally, integrated microbiome and metabolome analyses uncovered that FMT reshaped gut microbiota composition and metabolite landscape, and Lachnospiraceae and tryptophan-associated metabolites promoted the recovery of hematopoiesis and rejuvenated aged HSCs. Together, our study highlights the paramount importance of the gut microbiota in HSC aging and provides insights into therapeutic strategies for aging-related hematologic disorders.

Hematopoietic stem cells (HSCs) have reduced capacities to properly maintain and replenish the hematopoietic system during myelosuppressive injury or aging. Expanding and rejuvenating HSCs for therapeutic purposes has been a long-sought goal with limited progress. Here, we show that the enzyme Sphk2 (sphingosine kinase 2), which generates the lipid metabolite sphingosine-1-phosphate, is highly expressed in HSCs. The deletion of Sphk2 markedly promotes self-renewal and increases the regenerative potential of HSCs. More importantly, Sphk2 deletion globally preserves the young HSC gene expression pattern, improves the function, and sustains the multilineage potential of HSCs during aging. Mechanistically, Sphk2 interacts with prolyl hydroxylase 2 and the Von Hippel-Lindau protein to facilitate HIF1α ubiquitination in the nucleus independent of the Sphk2 catalytic activity. Deletion of Sphk2 increases hypoxic responses by stabilizing the HIF1α protein to upregulate PDK3, a glycolysis checkpoint protein for HSC quiescence, which subsequently enhances the function of HSCs by improving their metabolic fitness; specifically, it enhances anaerobic glycolysis but suppresses mitochondrial oxidative phosphorylation and generation of reactive oxygen species. Overall, targeting Sphk2 to enhance the metabolic fitness of HSCs is a promising strategy to expand and rejuvenate functional HSCs.

Chimeric antigen receptor T cells (CAR-T) therapy has achieved remarkable therapeutic success in treating a variety of hematopoietic malignancies. However, the high relapse rate and poor in vivo persistence, partially caused by CAR-T cell exhaustion, are still important barriers against CAR-T therapy. It remains largely elusive on the mechanisms of CAR-T exhaustion and how to attenuate exhaustion to achieve better therapeutic efficacy. In this study, we initially observed that CAR-T cells showed rapid differentiation and increased exhaustion after co-culture with tumor cells in vitro, and then performed single-cell ATAC-seq to depict the comprehensive and dynamic landscape of chromatin accessibility of CAR-T cells during tumor cell stimulation. Analyses of differential chromatin accessible regions and motif accessibility revealed that TFs were distinct in each cell type and reconstituted a coordinated regulatory network to drive CAR-T exhaustion. Furthermore, we performed scATAC-seq in patient-derived CAR-T cells and identified BATF and IRF4 as pivotal regulators in CAR-T cell exhaustion. Finally, knockdown of BATF or IRF4 enhanced the killing ability, inhibited exhaustion, and prolonged the persistence of CAR-T cells in vivo. Together, our study unraveled the epigenetic regulatory mechanisms of CAR-T exhaustion and provided new insights into CAR-T engineering to achieve better clinical treatment benefits.

The success of allogeneic hematopoietic stem cell transplant for hematological malignancies is heavily dependent on the availability of suitable donors. Haploidentical donor (HID) and matched sibling donor (MSD) are two important donor options providing faster and easier sources of stem cells, however, due to confounding factors present in most retrospective studies, the validity of comparing outcomes between these two donor types remains uncertain. We conducted a post-hoc analysis of a prospective clinical trial (trial registration: Chinese Clinical Trial Registry; #ChiCTR-OCH-12002490; registered 22 February 2012; https://www.chictr.org.cn/showproj.aspx?proj=7061) to compare outcomes of HID versus MSD peripheral blood stem cell-derived transplants in patients with hematologic malignancies between 2015 and 2022. All HID-receiving patients had antithymocyte globulin-based conditioning. Propensity score matching was employed to minimize potential confounding factors between the two cohorts. A total of 1060 patients were initially reviewed and then 663 patients were ultimately included in the analysis after propensity score matching. The overall survival, relapse-free survival, non-relapse mortality rate and cumulative incidence of relapse were similar between HID and MSD cohorts. Subgroup analysis revealed that patients with positive measurable residual disease in first complete remission may have better overall survival with an HID transplant. The present demonstrated that haploidentical transplants can provide outcomes comparable to conventional MSD transplants, and HID should be recommended as one of the optimal donor choices for patients with positive measurable residual disease in first complete remission.

Background In vertebrates, hematopoietic stem and progenitor cells (HSPCs) emerge from hemogenic endothelium in the floor of the dorsal aorta and subsequently migrate to secondary niches where they expand and differentiate into committed lineages. Glia maturation factor γ (gmfg) is a key regulator of actin dynamics that was shown to be highly expressed in hematopoietic tissue. Our goal is to investigate the role and mechanism of gmfg in embryonic HSPC development. Methods In-depth bioinformatics analysis of our published RNA-seq data identified gmfg as a cogent candidate gene implicated in HSPC development. Loss and gain-of-function strategies were applied to study the biological function of gmfg. Whole-mount in situ hybridization, confocal microscopy, flow cytometry, and western blotting were used to evaluate changes in the number of various hematopoietic cells and expression levels of cell proliferation, cell apoptosis and hematopoietic-related markers. RNA-seq was performed to screen signaling pathways responsible for gmfg deficiency-induced defects in HSPC initiation. The effect of gmfg on YAP sublocalization was assessed in vitro by utilizing HUVEC cell line. Results We took advantage of zebrafish embryos to illustrate that loss of gmfg impaired HSPC initiation and maintenance. In gmfg-deficient embryos, the number of hemogenic endothelium and HSPCs was significantly reduced, with the accompanying decreased number of erythrocytes, myelocytes and lymphocytes. We found that blood flow modulates gmfg expression and gmfg overexpression could partially rescue the reduction of HSPCs in the absence of blood flow. Assays in zebrafish and HUVEC showed that gmfg deficiency suppressed the activity of YAP, a well-established blood flow mediator, by preventing its shuttling from cytoplasm to nucleus. During HSPC initiation, loss of gmfg resulted in Notch inactivation and the induction of Notch intracellular domain could partially restore the HSPC loss in gmfg-deficient embryos. Conclusions We conclude that gmfg mediates blood flow-induced HSPC maintenance via regulation of YAP, and contributes to HSPC initiation through the modulation of Notch signaling. Our findings reveal a brand-new aspect of gmfg function and highlight a novel mechanism for embryonic HSPC development.

Although widely applied in treating hematopoietic malignancies, transplantation of hematopoietic stem/progenitor cells (HSPCs) is impeded by HSPC shortage. Whether circulating HSPCs (cHSPCs) in steady-state blood could be used as an alternative source remains largely elusive. Here we develop a three-dimensional culture system (3DCS) including arginine, glycine, aspartate, and a series of factors. Fourteen-day culture of peripheral blood mononuclear cells (PBMNCs) in 3DCS led to 125- and 70-fold increase of the frequency and number of CD34+ cells. Further, 3DCS-expanded cHSPCs exhibited the similar reconstitution rate compared to CD34+ HSPCs in bone marrow. Mechanistically, 3DCS fabricated an immunomodulatory niche, secreting cytokines as TNF to support cHSPC survival and proliferation. Finally, 3DCS could also promote the expansion of cHSPCs in patients who failed in HSPC mobilization. Our 3DCS successfully expands rare cHSPCs, providing an alternative source for the HSPC therapy, particularly for the patients/donors who have failed in HSPC mobilization.

The dysregulation of miRNAs has been increasingly recognized as a critical mediator of cancer development and progression. Here, we show that frequent deletion of the MIR135A1 locus is associated with poor prognosis in primary breast cancer. Forced expression of miR-135a decreased breast cancer progression, while inhibition of miR-135a with a specific miRNA sponge elicited opposing effects, suggestive of a tumor suppressive role of miR-135a in breast cancer. Estrogen receptor alpha (ERα) bound the promoter of MIR135A1 for its transcriptional activation, whereas tamoxifen treatment inhibited expression of miR-135a in ERα+ breast cancer cells. miR-135a directly targeted ESR1, ESRRA, and NCOA1, forming a negative feedback loop to inhibit ERα signaling. This regulatory feedback between miR-135a and ERα demonstrated that miR-135a regulated the response to tamoxifen. The tamoxifen-mediated decrease in miR-135a expression increased the expression of miR-135a targets to reduce tamoxifen sensitivity. Consistently, miR-135a expression was downregulated in ERα+ breast cancer cells with acquired tamoxifen resistance, while forced expression of miR-135a partially resensitized these cells to tamoxifen. Tamoxifen resistance mediated by the loss of miR-135a was shown to be partially dependent on the activation of the ERK1/2 and AKT pathways by miR-135a–targeted genes. Taken together, these results indicate that deletion of the MIR135A1 locus and decreased miR-135a expression promote ERα+ breast cancer progression and tamoxifen resistance.Significance: Loss of miR-135a in breast cancer disrupts an estrogen receptor-induced negative feedback loop, perpetuating disease progression and resistance to therapy.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/17/4915/F1.large.jpg. Cancer Res; 78(17); 4915–28. ©2018 AACR.

Hematopoietic stem cells (HSCs) developing from mesoderm during embryogenesis are important for the blood circulatory system and immune system. Many factors such as genetic factors, chemical exposure, physical radiation, and viral infection, can lead to the dysfunction of HSCs. Hematological malignancies (involving leukemia, lymphoma, and myeloma) were diagnosed in more than 1.3 million people globally in 2021, taking up 7% of total newly-diagnosed cancer patients. Although many treatments like chemotherapy, bone marrow transplantation, and stem cell transplantation have been applied in clinical therapeutics, the average 5-year survival rate for leukemia, lymphoma, and myeloma is about 65%, 72%, and 54% respectively. Small non-coding RNAs play key roles in a variety of biological processes, including cell division and proliferation, immunological response and cell death. With the development of technologies in high-throughput sequencing and bioinformatic analysis, there is emerging research about modifications on small non-coding RNAs, as well as their functions in hematopoiesis and related diseases. In this study, we summarize the updated information of small non-coding RNAs and RNA modifications in normal and malignant hematopoiesis, which sheds lights into the future application of HSCs into the treatment of blood diseases.

Supplementary table S1 lists all the sequence of miR-135a mimic, siRNAs, shRNAs, and primers for plasmid construction, ChIP assay, and qRT-PCR used in this study. Supplementary table S2 shows the information of antibodies used in this study. Supplementary table S3 and S4 show the correlation of MIR135A1 deletion and low miR-135a-1 with the clinicopathologic features of breast cancer in the TCGA breast tumors, respectively. Supplementary table S5 lists genes including EMT markers, ERα cofactors and interacting proteins, and predicted miR-135a targets which expression levels negatively correlate with miR-135a in breast tumors. Supplementary figure S1 shows that the copy number of MIR135A1 gene and the expression levels of pri-miR-135a-1 are decreased in breast cancer. Supplementary figure S2 shows that miR-135a is a tumor suppressor gene in T47D and MDA-MB-231 cells and inhibits EMT of breast cancer cells. Supplementary figure S3 shows that miR-135a impairs the in vivo tumor growth. Supplementary figure S4 shows that the transcription of MIR135A1 positively correlates with E2-ERα axis. Supplementary figure S5 shows that miR-135a modulates the estrogen independent growth and the response to tamoxifen in ERα+ breast cancer cells. Supplementary figure S6 shows the validation of miR-135a targets in breast cancer cells. Supplementary figure S7 shows that miR-135a modulates ERα-ERK-AKT crosstalk.

PDF file - 1291K, FIGURE S1: Representative data of genomic real-time PCR screening for DNA copy numbers of aberrant microRNAs on human chromosome 3p in breast carcinoma and the correlation between expression levels of miR-128 and ARPP21; FIGURE S2: miR-128 depletion confers non-tumorigenic MCF-10A cells with a malignant phenotype in vitro; FIGURE S3: Forced expression of miR-128 alters the morphology and reduces migration and invasion of MCF-7 cells in vitro, whereas miR-128 depletion confers non-tumorigenic HBL-100 cells with a malignant phenotype in vitro; FIGURE S4: miR-128 depletion confers a malignant phenotype on non-tumorigenic MCF-10A cells in vivo and miR-128 represses the CD44+CD24 Neg/Low population in mammary carcinoma cells; FIGURE S5: Interactions between miR-128 and its binding targets; FIGURE S6: Luciferase assay for promoter activity

PDF file - 226K, TABLE S1: List of miRNAs on human chromosome 3p and the changes of their genome copies determined by genomic real-time PCR; TABLE S2: Clinical significance of the expression of miR-128 in breast cancer patients; TABLE S3: Invariable analysis of clinical outcome according to the expression of miR-128 in breast cancer patients (85 cases); TABLE S4: List of mRNAs predicted to be targeted by miR-128 by all the three programs: MiRanda, Pictar and TargetScan; TABLE S5: qRT-PCR analysis of the effect of forced expression of miR-128 in MDA-MB-231 cells or depleted expression of miR-128 in MCF-10A cells on mRNA levels of several key genes functionally involved in oncogenicity and neoplastic progression; TABLE S6: Sequences of the oligonucleotides for miRNAs, siRNAs, construct-making and ChIP assays; TABLE S7: Sequence of the oligonucleotides for real-time PCR experiments; TABLE S8: List of proteins tested by antibodies and characteristics of the corresponding antibodies used

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Screening of the entire let-7 family of microRNAs (miRNA) by in situ hybridization identified let-7g as the only member, the diminished expression of which was significantly associated with lymph node metastasis and poor survival in breast cancer patients. Abrogation of let-7g expression in otherwise nonmetastatic mammary carcinoma cells elicited rapid metastasis from the orthotopic location, through preferential targets, Grb2-associated binding protein 2 (GAB2) and fibronectin 1 (FN1), and consequent activation of p44/42 mitogen-activated protein kinase (MAPK) and specific matrix metalloproteinases. Treatment with estrogen or epidermal growth factor specifically reduced the expression of mature let-7g through activation of p44/42 MAPK and subsequently stimulated expression of GAB2 and FN1, which, in turn, promoted tumor invasion. We thus identify let-7g as a unique member of the let-7 miRNA family that can serve as a prognostic biomarker in breast cancer and also propose a paradigm used by specific signaling molecules via let-7g to cooperatively promote breast cancer invasion and metastasis. Thus, let-7 family members neither possess equivalent clinicopathologic correlation nor function in breast cancer. Cancer Res; 71(20); 6463–74. ©2011 AACR.

PDF file - 190K, Includes patient and specimen data, reagent information, miRNA and siRNA transfection, and statistical analysis. Also includes supplementary references

Acute myeloid leukemia (AML) is a deadly hematological malignancy. Cellular morphology detection of bone marrow smears based on the French–American–British (FAB) classification system remains an essential criterion in the diagnosis of hematological malignancies. However, the diagnosis and discrimination of distinct FAB subtypes of AML obtained from bone marrow smear images are tedious and time-consuming. In addition, there is considerable variation within and among pathologists, particularly in rural areas, where pathologists may not have relevant expertise. Here, we established a comprehensive database encompassing 8245 bone marrow smear images from 651 patients based on a retrospective dual-center study between 2010 and 2021 for the purpose of training and testing. Furthermore, we developed AMLnet, a deep-learning pipeline based on bone marrow smear images, that can discriminate not only between AML patients and healthy individuals but also accurately identify various AML subtypes. AMLnet achieved an AUC of 0.885 at the image level and 0.921 at the patient level in distinguishing nine AML subtypes on the test dataset. Furthermore, AMLnet outperformed junior human experts and was comparable to senior experts on the test dataset at the patient level. Finally, we provided an interactive demo website to visualize the saliency maps and the results of AMLnet for aiding pathologists’ diagnosis. Collectively, AMLnet has the potential to serve as a fast prescreening and decision support tool for cytomorphological pathologists, especially in areas where pathologists are overburdened by medical demands as well as in rural areas where medical resources are scarce.

Background Adult hematopoietic stem cells (HSCs) homeostasis is critically important in maintaining lifelong hematopoiesis. However, how adult HSCs orchestrate its homeostasis remains not fully understood. Imprinted gene Dlk1 has been shown to play critical role in mouse embryonic hematopoiesis and in regulation of stem cells, but its physiological roles in adult HSCs are unknown. Methods We performed gene expression analysis of Dlk1, and constructed conditional Dlk1 knockout (KO) mice by crossing Mx1 cre mice with Dlkflox/flox mice. Western blot and quantitative PCR were used to detect Dlk1 KO efficiency. Flow cytometry was performed to investigate the effects of Dlk1 KO on HSCs, progenitors and linage cells in primary mice. Competitive HSCs transplantation and secondary transplantation was used to examine the effects of Dlk1 KO on long-term hematopoietic repopulation potential of HSCs. RNA-Seq and cell metabolism assays was used to determine the underlying mechanisms. Results Dlk1 was highly expressed in adult mice long-term HSCs (LT-HSCs) relative to progenitors and mature lineage cells. Dlk1 KO in adult mice HSCs drove HSCs enter active cell cycle, and expanded phenotypical LT-HSCs, but undermined its long-term hematopoietic repopulation potential. Dlk1 KO resulted in an increase in HSCs’ metabolic activity, including glucose uptake, ribosomal translation, mitochondrial metabolism and ROS production, which impaired HSCs function. Further, Dlk1 KO in adult mice HSCs attenuated Notch signaling, and re-activation of Notch signaling under Dlk1 KO decreased the mitochondrial activity and ROS production, and rescued the changes in frequency and absolute number of HSCs. Scavenging ROS by antioxidant N-acetylcysteine could inhibit mitochondrial metabolic activity, and rescue the changes in HSCs caused by Dlk1 KO. Conclusion Our study showed that Dlk1 played an essential role in maintaining HSC homeostasis, which is realized by governing cell cycle and restricting mitochondrial metabolic activity.

The nucleolus is the organelle for ribosome biogenesis and sensing various types of stress. However, its role in regulating stem cell fate remains unclear. Here, we present evidence that nucleolar stress induced by interfering rRNA biogenesis can drive the 2-cell stage embryo-like (2C-like) program and induce an expanded 2C-like cell population in mouse embryonic stem (mES) cells. Mechanistically, nucleolar integrity maintains normal liquid-liquid phase separation (LLPS) of the nucleolus and the formation of peri-nucleolar heterochromatin (PNH). Upon defects in rRNA biogenesis, the natural state of nucleolus LLPS is disrupted, causing dissociation of the NCL/TRIM28 complex from PNH and changes in epigenetic state and reorganization of the 3D structure of PNH, which leads to release of Dux, a 2C program transcription factor, from PNH to activate a 2C-like program. Correspondingly, embryos with rRNA biogenesis defect are unable to develop from 2-cell (2C) to 4-cell embryos, with delayed repression of 2C/ERV genes and a transcriptome skewed toward earlier cleavage embryo signatures. Our results highlight that rRNA-mediated nucleolar integrity and 3D structure reshaping of the PNH compartment regulates the fate transition of mES cells to 2C-like cells, and that rRNA biogenesis is a critical regulator during the 2-cell to 4-cell transition of murine pre-implantation embryo development., The role of the nucleolus in cell fate is not well known. Here the authors show nucleolar integrity maintained by rRNA-mediated liquid-liquid phase separation and 3D chromatin structure of perinucleolar heterochromatin regulates the fate transition of mouse embryonic stem cells to 2 cell-stage embryo-like cells, and that rRNA biogenesis regulates the 2-cell-to-4-cell transition of development.

DNA methyltransferase 3 A (DNMT3A) is the most frequently mutated gene in acute myeloid leukemia (AML). Although chemotherapy agents have improved outcomes for DNMT3A-mutant AML patients, there is still no targeted therapy highlighting the need for further study of how DNMT3A mutations affect AML phenotype. Here, we demonstrate that cell adhesion-related genes are predominantly enriched in DNMT3A-mutant AML cells and identify that graphdiyne oxide (GDYO) display an anti-leukemia effect specifically against these mutated cells. Mechanistically, GDYO directly interacts with integrin β2 (ITGB2) and c-type mannose receptor (MRC2), which facilitate the attachment and cellular uptake of GDYO. Furthermore, GDYO binds to actin and prevents actin polymerization, thus disrupting the actin cytoskeleton and eventually leading to cell apoptosis. Finally, we validate the in vivo safety and therapeutic potential of GDYO against DNMT3A-mutant AML cells. Collectively, these findings demonstrate that GDYO is an efficient and specific drug candidate against DNMT3A-mutant AML.

Small nucleolar RNAs (snoRNAs) belong to a family of noncoding RNAs that are 60-300 nucleotides in length, and they are classified into two classes according to their structure and function: C/D box snoRNAs, playing an essential role in 2’-O-methylation modification on ribosomal RNA; H/ACA box snoRNAs, involved in the pseudouridylation of rRNA. SnoRNAs with unclear functions, no predictable targets, and unusual subcellular locations are called orphan snoRNAs. Recent studies have revealed abnormal expression and demonstrated the pivotal roles of snoRNAs and their host genes in various types of hematological malignancies. This review discusses recent discoveries concerning snoRNAs in a variety of hematological malignancies, including multiple myeloma, lymphoma and leukemia, and sheds light on the application of snoRNAs as diagnostic and prognostic markers as well as therapeutic targets of hematological malignancies in the future.

Chimeric Antigen-Receptor (CAR) T-cell therapies have shown dramatic efficacy in treating relapsed and refractory cancers, especially B cell malignancies. However, these innovative therapies cause adverse toxicities that limit the broad application in clinical settings. Hematologic cytopenias, one frequently reported adverse event following CAR T cell treatment, are manifested as a disorder of hematopoiesis with decreased number of mature blood cells and subdivided into anemia, thrombocytopenia, leukopenia, and neutropenia, which increase the risk of infections, fatigue, bleeding, fever, and even fatality. Herein, we initially summarized the symptoms, etiology, risk factors and management of cytopenias. Further, we elaborated the cellular and molecular mechanisms underlying the initiation and progression of cytopenias following CAR T cell therapy based on previous studies about acquired cytopenias. Overall, this review will facilitate our understanding of the etiology of cytopenias and shed lights into developing new therapies against CAR T cell-induced cytopenias.

Immune cells such as T cells, macrophages, dendritic cells, and other immunoregulatory cells undergo metabolic reprogramming in cancer and inflammation‐derived microenvironment to meet specific physiologic and functional demands. Myeloid‐derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that are characterized by immunosuppressive activity, which plays a key role in host immune homeostasis. In this review, we have discussed the core metabolic pathways, including glycolysis, lipid and fatty acid biosynthesis, and amino acid metabolism in the MDSCs under various pathologic situations. Metabolic reprogramming is a determinant of the phenotype and functions of MDSCs, and is therefore a novel therapeutic possibility in various diseases., Graphical Abstract This work intends to declare what metabolic alterations happen in MDSCs to support their physiologic and functional demands under a variety of pathologic situations.

Acute graft-versus-host disease (aGVHD) is the most severe complication and the major cause of morbidity and mortality in patients who have received hematopoietic stem cell transplantation. Inflammation mediated by donor T cells and neighboring recipient cells is considered to be responsible for intestinal aGVHD. Interleukin (IL)-17A-producing γδT (γδT17) cells have been investigated as key players in cancer, immunity, and inflammatory responses because of their phenotypic plasticity, memory-like activity, and unique migratory features. However, the precise roles and underlying mechanisms of γδT17 cells in aGVHD immunopathogenesis remain elusive. Here, we found that γδT17 cells constituted the major resident γδT population in the intestinal lamina propria of aGVHD mice. Adoptive infusion of induced γδT17 cells markedly attenuated murine aGVHD and increased infiltration of Gr-1+CD11b+ myeloid-derived suppressor cells (MDSCs) into the inflammatory intestine, and did not affect graft-versus-leukemia(GVL) effect. Further experiments indicated that γδT17 cells enhanced both migration ability and immunosuppressive activity of MDSCs. γδT17 cell-mediated protection in aGVHD was blunted by depletion of IL-17A or MDSCs. Our study clarifies an immune pathway where γδT17 cells play a protective role in murine aGVHD by recruiting MDSCs to the inflammatory intestine.

Technologies for gene activation are valuable tools for the study of gene functions and have a wide range of potential applications in bioengineering and medicine. In contrast to existing methods based on recruiting transcriptional modulators via DNA-binding proteins, we developed a strategy termed Narta (nascent RNA-guided transcriptional activation) to achieve gene activation by recruiting artificial transcription factors (aTFs) to transcription sites through nascent RNAs of the target gene. Using Narta, we demonstrate robust activation of a broad range of exogenous and endogenous genes in various cell types, including zebrafish embryos, mouse and human cells. Importantly, the activation is reversible, tunable and specific. Moreover, Narta provides better activation potency of some expressed genes than CRISPRa and, when used in combination with CRISPRa, has an enhancing effect on gene activation. Quantitative imaging illustrated that nascent RNA-directed aTFs could induce the high-density assembly of coactivators at transcription sites, which may explain the larger transcriptional burst size induced by Narta. Overall, our work expands the gene activation toolbox for biomedical research.

The maintenance of the mammalian blood system depends on hematopoietic stem cells (HSCs), which are a rare class of adult stem cells with self-renewal and multilineage differentiation capacities. The homeostasis of hematopoietic stem cells is finely tuned by a variety of endogenous and exogenous regulatory factors, and disrupted balance will lead to hematological diseases including leukemia and anemia. Recently, emerging studies have illustrated the cellular and molecular mechanisms underlying the regulation of HSC homeostasis. Particularly, the rapid development of second-generation sequencing technologies has uncovered that many small noncoding RNAs (ncRNAs) are highly expressed in HSCs, including snoRNAs, miRNAs, tsRNAs, circular RNAs, etc. In this study, we will summarize the essential roles and regulatory mechanisms of these small ncRNAs in maintaining HSC homeostasis. Overall, this review provides up-to-date information in the regulation of HSC homeostasis by small ncRNAs, which sheds light into the development of therapeutic strategies against hematopoietic malignancies.

Evidence on the importance of lymph node (LN) dissection during resection for small cell lung cancer (SCLC) is scarce. This study sought to investigate the clinical impact of the extent of lymphadenectomy on the survival of patients with SCLC.Patients who underwent resection for primary SCLC between 2000 and 2016 were identified from the Surveillance, Epidemiology, and End Results (SEER) cancer registry. The patients were stratified based on the number of LNs dissected (0, 1-3, 4-11, and ≥12) via an X-Tile software analysis, and lung cancer-specific survival (LCSS) and overall survival (OS) were compared between these stratified groups using Kaplan-Meier curves. A propensity score-matched analysis and a Cox regression model were used to adjust for potential confounders.A total of 1,883 patients with SCLC met our criteria and were enrolled in the study. The LCSS and OS analyses revealed that patients who underwent LN dissection during surgery had longer survival times significantly than patients who did not. Similarly, patients who underwent more extensive LN dissection (≥4 LNs) had longer survival times than those who underwent less extensive LN dissection (1-3 LNs). However, no significant increase in survival time was found for patients who underwent the dissection of ≥12 LNs compared to those who underwent the dissection of 4-11 LNs. These results were confirmed in our propensity-matched and Cox regression analyses.Our study revealed that patient survival after surgical resection for SCLC is associated with the number of dissected LNs, and the number of LNs for dissection ranges from 4 to 11 achieve the best survival outcome.

Summary Mouse embryonic stem cells (ESCs) cultured in defined medium resemble the pre-implantation epiblast in the ground state, with full developmental capacity including the germline. β-Catenin is required to maintain ground state pluripotency in mouse ESCs, but its exact role is controversial. Here, we reveal a Tcf3-independent role of β-catenin in restraining germline and somatic lineage differentiation genes. We show that β-catenin binds target genes with E2F6 and forms a complex with E2F6 and HMGA2 or E2F6 and HP1γ. Our data indicate that these complexes help β-catenin restrain and fine-tune germ cell and neural developmental potential. Overall, our data reveal a previously unappreciated role of β-catenin in preserving lineage differentiation integrity in ground state ESCs., Highlights • β-Catenin depletion irreversibly compromised lineage development of ground state ESCs • TCF3-independent role of β-catenin in determining lineage differentiation potential • E2F6, HP1γ, and HMGA2 are β-catenin interaction partners and co-bound to target genes • β-Catenin and protein partners fine-tune germline and neural development potential, In this article, Tao and colleagues show that β-catenin functions beyond derepressing TCF3 targeted pluripotency network in ESCs. β-Catenin physically interacts with protein partners, including transcription factor and chromatin modulators. Together, they preserve the integrity of lineage differentiation of ground state ESCs by restraining and fine-tuning their germline and neural development potential.

Leukaemia stem cells (LSCs) underlie cancer therapy resistance but targeting these cells remains difficult. The Wnt-β-catenin and PI3K-Akt pathways cooperate to promote tumorigenesis and resistance to therapy. In a mouse model in which both pathways are activated in stem and progenitor cells, LSCs expanded under chemotherapy-induced stress. Since Akt can activate β-catenin, inhibiting this interaction might target therapy-resistant LSCs. High-throughput screening identified doxorubicin (DXR) as an inhibitor of the Akt-β-catenin interaction at low doses. Here we repurposed DXR as a targeted inhibitor rather than a broadly cytotoxic chemotherapy. Targeted DXR reduced Akt-activated β-catenin levels in chemoresistant LSCs and reduced LSC tumorigenic activity. Mechanistically, β-catenin binds multiple immune-checkpoint gene loci, and targeted DXR treatment inhibited expression of multiple immune checkpoints specifically in LSCs, including PD-L1, TIM3 and CD24. Overall, LSCs exhibit distinct properties of immune resistance that are reduced by inhibiting Akt-activated β-catenin. These findings suggest a strategy for overcoming cancer therapy resistance and immune escape.

Adults with acute myeloid leukaemia have unsatisfactory clinical outcomes and rates of complete remission. Venetoclax combined with azacytidine or low-dose cytarabine has shown efficacy in adults aged 75 years or older (or 18-74 years with comorbidities precluding intensive chemotherapy) with acute myeloid leukaemia. We aimed to investigate the activity and safety of venetoclax plus 3+7 daunorubicin and cytarabine chemotherapy in adults with acute myeloid leukaemia.We conducted a two-stage, single-arm, phase 2 trial at three public hospitals in China. We enrolled patients aged 18-60 years with previously untreated de novo acute myeloid leukaemia and an Eastern Cooperative Oncology Group performance status of 0-2. Patients received induction treatment with intravenous daunorubicin (60 mg/mBetween Dec 25, 2020, and July 7, 2021, 36 patients were assessed for eligibility and 33 were enrolled. 15 (45%) patients were men and 18 (55%) were women, and all were Asian. The composite complete remission rate after one cycle of DAV regimen was 91% (95% CI 76-98; 30 of 33 patients) in the entire cohort. 29 (97%) of 30 patients who reached complete remission had undetectable measurable residual disease (ie,0·1%). Grade 3 or worse adverse events included neutropenia in 33 (100%) of 33 patients, thrombocytopenia in 33 (100%), anaemia in 33 (100%), febrile neutropenia in 18 (55%), pneumonia in seven (21%), and sepsis in four (12%). No treatment-related deaths occurred. With a median follow-up of 11 months (IQR 9-12), estimated 1-year overall survival was 97% (95% CI 91-100) and 1-year event-free survival was 72% (56-94).The DAV regimen represents an effective induction therapy for newly diagnosed adults with acute myeloid leukaemia, which resulted in a high rate of complete remission. These findings are an important contribution to the field, showing a safe strategy to incorporate venetoclax into the most common induction regimen used to treat newly diagnosed acute myeloid leukaemia internationally.Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang, National Natural Science Foundation of China, Key Research and Development Program of Zhejiang.For the Chinese translation of the abstract see Supplementary Materials section.

The gut microbiota and metabolites play pivotal roles in the pathobiology of various diseases. Here, we describe a protocol to profile the gut microbiome and meta-metabolome of a mouse disease model for acute graft-versus-host disease. We describe steps for fecal sample collection and processing for 16S sequencing and UPLC-MS. Finally, we detail the steps for data analysis and exhibit multi-omic associations to correlate with pathology. For complete details on the use and execution of this protocol, please refer to Li et al. (2020).

Myeloid-derived suppressor cells (MDSCs), a population of myeloid lineage cells with immunosuppressive capacity, can mitigate acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We previously found that the immunosuppressive function of polymorphonuclear population (PMN-MDSCs) was impaired in aGVHD milieu. The aim of this study was to explore the intrinsic mechanism regulating the fate and function of donor-derived PMN-MDSCs during allo-HSCT. We firstly found that mitochondrial permeability transition pore (MPTP) opened in the PMN-MDSCs in response to the intense inflammatory environment of aGVHD, which induced mitochondrial damage, oxidative stress, and apoptosis of PMN-MDSCs. Inhibiting MPTP opening by a traditional immunosuppressant, cyclosporine A (CsA), could restore the immunosuppressive function and viability of PMN-MDSCs in vitro and in vivo, which reveals a new mechanism of CsA application.

Chimeric antigen receptor (CAR) T cells are potent agents for recognizing and eliminating tumors, and have achieved remarkable success in the treatment of patients with refractory leukemia and lymphoma. However, dysfunction of T cells, including exhaustion, is an inevitable obstacle for persistent curative effects. Here, the authors initially found that calcium signaling is hyperactivated via sustained tonic signaling in CAR-T cells. Next, it is revealed that the store-operated calcium entry (SOCE) inhibitor BTP-2, but not the calcium chelator BAPTA-AM, markedly diminishes CAR-T cell exhaustion and terminal differentiation of CAR-T cells in both tonic signaling and tumor antigen exposure models. Furthermore, BTP-2 pretreated CAR-T cells show improved antitumor potency and prolonged survival in vivo. Mechanistically, transcriptome and metabolite analyses reveal that treatment with BTP-2 significantly downregulate SOCE-calcineurin-nuclear factor of activated T-cells (NFAT) and glycolysis pathways. Together, the results indicate that modulating the SOCE-calcineurin-NFAT pathway in CAR-T cells renders them resistant to exhaustion, thereby yielding CAR products with enhanced antitumor potency.

Myeloid-derived suppressor cells (MDSCs) represent a population of heterogeneous myeloid cells, which are characterized by their remarkable ability to suppress T cells and natural killer cells. MDSCs have been proven to play a positive role in protecting acute graft-versus-host disease (aGVHD). Here, we aimed to describe the mechanism behind how mTOR signaling regulates MDSCs’ generation and explore its prophylactic and therapeutic potential in aGVHD. Reducing mTOR expression retains myeloid cells with immature characteristics and promotes polymorphonuclear MDSC (PMN-MDSC) immunosuppressive function through STAT3-C/EBPβ pathway. Prophylactic transfusion of mTORKO PMN-MDSCs could alleviate aGVHD while maintaining the graft-versus-leukemia (GVL) effect, which could downregulate the Th1/Th2 ratio, decrease serum proinflammatory cytokines, and increase the proportion of regulatory T cells (Tregs) in aGVHD models at the early stage after transplantation. Moreover, transfusion therapy could promote the reconstruction and function of donor-derived PMN-MDSCs. Not only the percentage and the absolute number of donor-derived PMN-MDSCs significantly increased but also the immunosuppressive ability was much more robust compared to other groups. Altogether, these findings indicated that mTOR is an intrinsic regulator for PMN-MDSCs’ differentiation and immunosuppressive function. Together, mTORKO PMN-MDSC transfusion can play a protective role in alleviating cytokine storm at the initial stage and promoting the quantitative and functional recoveries of donor-derived PMN-MDSCs in aGVHD.

Hematopoietic stem cell (HSC) aging, which is accompanied by loss of self-renewal capacity, myeloid-biased differentiation and increased risks of hematopoietic malignancies, is an important focus in stem cell research. However, the mechanisms underlying HSC aging have not been fully elucidated. In the present study, we integrated 3 independent single-cell transcriptome datasets of HSCs together and identified Il10ra and Tnfsf14 as two markers of inflammatory and apoptosis-biased aged HSCs. Besides, common differentially expressed genes (DEGs) between young and aged HSCs were identified and further validated by quantitative RT-PCR. Functional enrichment analysis revealed that these DEGs were predominantly involved in the cell cycle and the tumor necrosis factor (TNF) signaling pathway. We further found that the Skp2-induced signaling pathway (Skp2→Cip1→CycA/CDK2→DP-1) contributed to a rapid transition through G1 phase in aged HSCs. In addition, analysis of the extrinsic alterations on HSC aging revealed the increased expression levels of inflammation genes in bone marrow microenvironment. Colony formation unit assays showed that inflammatory cytokines promoted cellular senescence and that blockade of inflammatory pathway markedly rejuvenated aged HSC functions and increased B cell output. Collectively, our study elucidated the biological characteristics of HSC aging, and the genes and pathways we identified could be potential biomarkers and targets for the identification and rejuvenation of aged HSCs.

Cancer cells display phenotypic equilibrium between the stem-like and differentiated states during neoplastic homeostasis. The functional and mechanistic implications of this subpopulation plasticity remain largely unknown. Herein, it was demonstrated that the breast cancer stem cell (BCSC) secretome autonomously compressed the stem cell population. Co-implantation with BCSCs decreased the tumor-initiating capacity yet increased metastasis of accompanying cancer cells, wherein DKK1 was identified as a pivotal factor secreted by BCSCs for such functions. DKK1-promoted differentiation was indispensable for disseminated tumor cell metastatic outgrowth. In contrast, DKK1 inhibitors substantially relieved the metastatic burden by restraining metastatic cells in the dormant state. DKK1 increased the expression of SLC7A11 to protect metastasizing cancer cells from lipid peroxidation and ferroptosis. Combined treatment with ferroptosis inducer and DKK1 inhibitor exhibited synergistic effects in diminishing metastasis. Hence, this study deciphers the contribution of CSC-regulated phenotypic plasticity in metastatic colonization and provides therapeutic approaches to limit metastatic outgrowth.

Relapses of CD19-expressing leukemia in patients who achieved initial remission after CART cell treatment have been reported to correlate with poor CART cells persistence. Sustained tonic signaling or strong activation drives CART cell differentiation and exhaustion, which limit the therapeutic efficacy and persistence of CART cells. Here, we identified dasatinib as the optimal candidate to prevent or reverse both CD28/CART and 4-1BB/CART cell differentiation and exhaustion during ex vivo expansion, which profoundly enhanced the therapeutic efficacy and in vivo persistence. Moreover, strong activation-induced CART cells differentiation, exhaustion and apoptosis driven by CD3/CD28 stimulation or antigen exposure were dramatically prevented or reversed by dasatinib treatment. Mechanistically, dasatinib markedly reduced the phosphorylation of Src and Lck, and downregulated the expression of genes involved in CAR signaling pathways, which resulted in the optimization of cell differentiation, exhaustion and apoptosis-related gene expression. Our study proposes a promising pharmacological approach for optimizing CART cells manufacture, and provides an experimental basis for reinvigorating CART cells in clinical application. Supplementary Information The online version contains supplementary material available at 10.1186/s13045-021-01117-y.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13 has drawn broad interest to control gene expression and cell fate at the RNA level in general. Apart from RNA interference mediated by its endonuclease activity, the nuclease-deactivated form of Cas13 further provides a versatile RNA-guided RNA-targeting platform for manipulating kinds of RNA modifications post-transcriptionally. Chemical modifications modulate various aspects of RNA fate, including translation efficiency, alternative splicing, RNA–protein affinity, RNA–RNA interaction, RNA stability and RNA translocation, which ultimately orchestrate cellular biologic activities. This review summarizes the history of the CRISPR-Cas13 system, fundamental components of RNA modifications and the related physiological and pathological functions. We focus on the development of epi-transcriptional editing toolkits based on catalytically inactive Cas13, including RNA Editing for Programmable A to I Replacement (REPAIR) and xABE (adenosine base editor) for adenosine deamination, RNA Editing for Specific C-to-U Exchange (RESCUE) and xCBE (cytidine base editor) for cytidine deamination and dm6ACRISPR, as well as the targeted RNA methylation (TRM) and photoactivatable RNA m6A editing system using CRISPR-dCas13 (PAMEC) for m6A editing. We further highlight the emerging applications of these useful toolkits in cell biology, disease and imaging. Finally, we discuss the potential limitations, such as off-target editing, low editing efficiency and limitation for AAV delivery, and provide possible optimization strategies.

As one of the best characterized adult stem cells, hematopoietic stem cell (HSC) homeostasis is of great importance to hematopoiesis and immunity due to HSC's abilities of self-renewal and multi-lineage differentiation into functional blood cells. However, excessive self-renewal of HSCs can lead to severe hematopoietic malignancies like leukemia, whereas deficient self-renewal of HSCs may result in HSC exhaustion and eventually apoptosis of specialized cells, giving rise to abnormalities such as immunodeficiency or anemia. How HSC homeostasis is maintained has been studied for decades and regulatory factors can be generally categorized into two classes: genetic factors and epigenetic factors. Although genetic factors such as signaling pathways or transcription factors have been well explored, recent studies have emerged the indispensable roles of epigenetic factors. In this review, we have summarized regulatory mechanisms of HSC homeostasis by epigenetic factors, including DNA methylation, histone modification, chromatin remodeling, non-coding RNAs, and RNA modification, which will facilitate applications such as HSC ex vivo expansion and exploration of novel therapeutic approaches for many hematological diseases.

Polycomb group (PcG) proteins maintain cell identity by repressing gene expression during development. Surprisingly, emerging studies have recently reported that a number of PcG proteins directly activate gene expression during cell fate determination process. However, the mechanisms by which they direct gene activation in pluripotency remain poorly understood. Here, we show that Phc1, a subunit of canonical polycomb repressive complex 1 (cPRC1), can exert its function in pluripotency maintenance via a PRC1-independent activation of Nanog. Ablation of Phc1 reduces the expression of Nanog and overexpression of Nanog partially rescues impaired pluripotency caused by Phc1 depletion. We find that Phc1 interacts with Nanog and activates Nanog transcription by stabilizing the genome-wide chromatin interactions of the Nanog locus. This adds to the already known canonical function of PRC1 in pluripotency maintenance via a PRC1-dependent repression of differentiation genes. Overall, our study reveals a function of Phc1 to activate Nanog transcription through regulating chromatin architecture and proposes a paradigm for PcG proteins to maintain pluripotency., Phc1 is a subunit of the polycomb repressive complex 1 (PRC1), which represses gene expression during development. Here the authors show that Phc1 acts independently from PRC1 to activate Nanog transcription by stabilizing genome-wide chromatin interactions of the Nanog locus, and in turn stabilize pluripotency.

Fat mass and obesity-associated protein (FTO) is the first protein found to have the activity of N6-methyladenosine (m6A) demethylation. It has been reported that FTO was involved in different physiological and pathological processes, including stem cell differentiation, sex determination, tumorigenesis, and progression. To further understand the exact role of FTO in these processes, we generated a FTO knockout human embryonic stem cell (hESC) line by CRISPR/Cas9 mediated gene editing method. This cell line maintained normal karyotype, pluripotency, and trilineage differentiation potential, which are considered as a model for function studies of the FTO protein in hESC self-renewal and differentiation.

Background Decades of efforts have attempted to differentiate the pluripotent stem cells (PSCs) into truly functional hematopoietic stem cells (HSCs), yet the problems of low differentiation efficiency in vitro and poor hematopoiesis reconstitution in vivo still exist, mainly attributing to the lack of solid, reproduced, or pursued differentiation system. Methods In this study, we established an in vitro differentiation system yielding in vivo hematopoietic reconstitution hematopoietic cells from mouse PSCs through a 3D induction system followed by coculture with OP9 stromal cells. The in vivo hematopoietic reconstitution potential of c-kit+ cells derived from the mouse PSCs was evaluated via m-NSG transplantation assay. Flow cytometry analysis, RNA-seq, and cell cycle analysis were used to detect the in vitro hematopoietic ability of endothelial protein C receptor (EPCR, CD201) cells generated in our induction system. Results The c-kit+ cells from 3D self-assembling peptide induction system followed by the OP9 coculture system possessed apparently superiority in terms of in vivo repopulating activity than that of 3D induction system followed by the 0.1% gelatin culture. We interestingly found that our 3D+OP9 system enriched a higher percentage of CD201+c-kit+cells that showed more similar HSC-like features such as transcriptome level and CFU formation ability than CD201-c-kit+cells, which have not been reported in the field of mouse PSCs hematopoietic differentiation. Moreover, CD201+ hematopoietic cells remained in a relatively slow cycling state, consistent with high expression levels of P57 and Ccng2. Further, we innovatively demonstrated that notch signaling pathway is responsible for in vitro CD201+ hematopoietic cell induction from mouse PSCs. Conclusions Altogether, our findings lay a foundation for improving the efficiency of hematopoietic differentiation and generating in vivo functional HSC-like cells from mouse PSCs for clinical application.