Phase separation-competent FBL promotes early pre-rRNA processing and translation in acute myeloid leukaemia.

RNA-binding proteins (RBPs) are pivotal in acute myeloid leukaemia (AML), a lethal disease. Although specific phase separation-competent RBPs are recognized in AML, the effect of their condensate formation on AML leukaemogenesis, and the therapeutic potential of inhibition of phase separation are underexplored. In our in vivo CRISPR RBP screen, fibrillarin (FBL) emerges as a crucial nucleolar protein that regulates AML cell survival, primarily through its phase separation domains rather than methyltransferase or acetylation domains. These phase separation domains, with specific features, coordinately drive nucleoli formation and early processing of pre-rRNA (including efflux, cleavage and methylation), eventually enhancing the translation of oncogenes such as MYC. Targeting the phase separation capability of FBL with CGX-635 leads to elimination of AML cells, suggesting an additional mechanism of action for CGX-635 that complements its established therapeutic effects. We highlight the potential of PS modulation of critical proteins as a possible therapeutic strategy for AML.

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

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

Inhibition of CD38 enzymatic activity enhances CAR-T cell immune-therapeutic efficacy by repressing glycolytic metabolism.

Chimeric antigen receptor (CAR)-T therapy has shown superior efficacy against hematopoietic malignancies. However, many patients failed to achieve sustainable tumor control partially due to CAR-T cell exhaustion and limited persistence. In this study, by performing single-cell multi-omics data analysis on patient-derived CAR-T cells, we identify CD38 as a potential hallmark of exhausted CAR-T cells, which is positively correlated with exhaustion-related transcription factors and further confirmed with in vitro exhaustion models. Moreover, inhibiting CD38 activity reverses tonic signaling- or tumor antigen-induced exhaustion independent of single-chain variable fragment design or costimulatory domain, resulting in improved CAR-T cell cytotoxicity and antitumor response. Mechanistically, CD38 inhibition synergizes the downregulation of CD38-cADPR -Ca2+ signaling and activation of the CD38-NAD+-SIRT1 axis to suppress glycolysis. Collectively, our findings shed light on the role of CD38 in CAR-T cell exhaustion and suggest potential clinical applications of CD38 inhibition in enhancing the efficacy and persistence of CAR-T cell therapy.

Decoding leukemia at the single-cell level: clonal architecture, classification, microenvironment, and drug resistance.

Leukemias are refractory hematological malignancies, characterized by marked intrinsic heterogeneity which poses significant obstacles to effective treatment. However, traditional bulk sequencing techniques have not been able to effectively unravel the heterogeneity among individual tumor cells. With the emergence of single-cell sequencing technology, it has bestowed upon us an unprecedented resolution to comprehend the mechanisms underlying leukemogenesis and drug resistance across various levels, including the genome, epigenome, transcriptome and proteome. Here, we provide an overview of the currently prevalent single-cell sequencing technologies and a detailed summary of single-cell studies conducted on leukemia, with a specific focus on four key aspects: (1) leukemia's clonal architecture, (2) frameworks to determine leukemia subtypes, (3) tumor microenvironment (TME) and (4) the drug-resistant mechanisms of leukemia. This review provides a comprehensive summary of current single-cell studies on leukemia and highlights the markers and mechanisms that show promising clinical implications for the diagnosis and treatment of leukemia.

Crohn's disease as the intestinal manifestation of pan-lymphatic dysfunction: An exploratory proposal based on basic and clinical data.

Crohn's disease (CD) is caused by immune, environmental, and genetic factors. It can involve the entire gastrointestinal tract, and although its prevalence is rapidly increasing its etiology remains unclear. Emerging biological and small-molecule drugs have advanced the treatment of CD; however, a considerable proportion of patients are non-responsive to all known drugs. To achieve a breakthrough in this field, innovations that could guide the further development of effective therapies are of utmost urgency. In this review, we first propose the innovative concept of pan-lymphatic dysfunction for the general distribution of lymphatic dysfunction in various diseases, and suggest that CD is the intestinal manifestation of pan-lymphatic dysfunction based on basic and clinical preliminary data. The supporting evidence is fully summarized, including the existence of lymphatic system dysfunction, recognition of the inside-out model, disorders of immune cells, changes in cell plasticity, partial overlap of the underlying mechanisms, and common gut-derived fatty and bile acid metabolism. Another benefit of this novel concept is that it proposes adopting the zebrafish model for studying intestinal diseases, especially CD, as this model is good at presenting and mimicking lymphatic dysfunction. More importantly, the ensuing focus on improving lymphatic function may lead to novel and promising therapeutic strategies for CD.

Single-cell RNA sequencing reveals the immunoregulatory roles of PegIFN-α in patients with chronic hepatitis B.

BACKGROUND AND AIMS: Chronic hepatitis B (CHB) is caused by HBV infection and affects the lives of millions of people worldwide by causing liver inflammation, cirrhosis, and liver cancer. Interferon-alpha (IFN-α) therapy is a conventional immunotherapy that has been widely used in CHB treatment and achieved promising therapeutic outcomes by activating viral sensors and interferon-stimulated genes (ISGs) suppressed by HBV. However, the longitudinal landscape of immune cells of CHB patients and the effect of IFN-α on the immune system are not fully understood. APPROACH AND RESULTS: Here, we applied single-cell RNA sequencing (scRNA-seq) to delineate the transcriptomic landscape of peripheral immune cells in CHB patients before and after PegIFN-α therapy. Notably, we identified three CHB-specific cell subsets, pro-inflammatory (Pro-infla) CD14+ monocytes, Pro-infla CD16+ monocytes and IFNG+ CX3CR1- NK cells, which highly expressed proinflammatory genes and positively correlated with HBsAg. Furthermore, PegIFN-α treatment attenuated percentages of hyperactivated monocytes, increased ratios of long-lived naive/memory T cells and enhanced effector T cell cytotoxicity. Finally, PegIFN-α treatment switched the transcriptional profiles of entire immune cells from TNF-driven to IFN-α-driven pattern and enhanced innate antiviral response, including virus sensing and antigen presentation. CONCLUSIONS: Collectively, our study expands the understanding of the pathological characteristics of CHB and the immunoregulatory roles of PegIFN-α, which provides a new powerful reference for the clinical diagnosis and treatment of CHB.

AMLnet, A deep-learning pipeline for the differential diagnosis of acute myeloid leukemia from bone marrow smears.

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.

Dlk1 maintains adult mice long-term HSCs by activating Notch signaling to restrict mitochondrial metabolism.

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.

Graphdiyne oxide nanosheets display selective anti-leukemia efficacy against DNMT3A-mutant AML cells.

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 ß2 (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.

Single-cell ATAC-seq maps the comprehensive and dynamic chromatin accessibility landscape of CAR-T cell dysfunction.

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.

Dasatinib enhances anti-leukemia efficacy of chimeric antigen receptor T cells by inhibiting cell differentiation and exhaustion.

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.

PHC1 maintains pluripotency by organizing genome-wide chromatin interactions of the Nanog locus.

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.

Regulation of MYB by distal enhancer elements in human myeloid leukemia.

MYB plays vital roles in regulating proliferation and differentiation of hematopoietic progenitor cells, dysregulation of MYB has been implicated in the pathogenesis of leukemia. Although the transcription of MYB has been well studied, its detailed underlying regulatory mechanisms still remain elusive. Here, we detected the long-range interaction between the upstream regions, -34k and -88k, and the MYB promoter in K562, U937, and HL-60 cells using circularized chromosome conformation capture (4C) assay, which declined when MYB was downregulated during chemical-induced differentiation. The enrichment of enhancer markers, H3K4me1 and H3K27ac, and enhancer activity at the -34k and -88k regions were confirmed by ChIP-qPCR and luciferase assay respectively. ChIP-qPCR showed the dynamic binding of GATA1, TAL1, and CCAAT/enhancer-binding protein (C/EBPß) at -34k and -88k during differentiation of K562 cells. Epigenome editing by a CRISPR-Cas9-based method showed that H3K27ac at -34k enhanced TF binding and MYB expression, while DNA methylation inhibited MYB expression. Taken together, our data revealed that enhancer elements at -34k are required for MYB expression, TF binding, and epigenetic modification are closely involved in this process in human myeloid leukemia cells.

Risk SNP-induced lncRNA-SLCC1 drives colorectal cancer through activating glycolysis signaling.

Long non-coding RNAs (lncRNAs) play key roles in colorectal carcinogenesis. Here, we aimed to identify the risk SNP-induced lncRNAs and to investigate their roles in colorectal carcinogenesis. First, we identified rs6695584 as the causative SNP in 1q41 locus. The A>G mutation of rs6695584 created a protein-binding motif of BATF, altered the enhancer activity, and subsequently activated lncSLCC1 expression. Further validation in two independent CRC cohorts confirmed the upregulation of lncSLCC1 in CRC tissues, and revealed that increased lncSLCC1 expression was associated with poor survival in CRC patients. Mechanistically, lncRNA-SLCC1 interacted with AHR and transcriptionally activated HK2 expression, the crucial enzyme in glucose metabolism, thereby driving the glycolysis pathway and accelerating CRC tumor growth. The functional assays revealed that lncSLCC1 induced glycolysis activation and tumor growth in CRC mediated by HK2. In addition, HK2 was upregulated in colorectal cancer tissues and positively correlated with lncSLCC1 expression and patient survival. Taken together, our findings reveal a risk SNP-mediated oncogene lncRNA-SLCC1 promotes CRC through activating the glycolysis pathway.

Inhibition of Embryonic HSP 90 Function Promotes Variation of Cold Tolerance in Zebrafish.

Accumulating evidence indicates that heat shock protein 90 (HSP90) plays essential roles in modulation of phenotypic plasticity in vertebrate development, however, the roles of HSP90 in modulation of cold tolerance capacity in fish are still unclear. In the present study, we showed that transient inhibition of embryonic HSP90 function by a chemical inhibitor or low conductivity stress promoted variation of cold tolerance capacity in adult zebrafish. Further work showed that embryonic HSP90 inhibition enhanced cold tolerance in adult zebrafish could be transmitted to their offspring. RNA-seq data showed that embryonic HSP90 inhibition enhanced cold tolerance involves variation of gene expression related to proteasome, lysosome, autophagy, and ribosome. Experiments with zebrafish ZF4 cells showed that two differentially expressed genes atg9b and psmd12 were up-regulated by radicicol treatment and provided protective roles for cells under cold stress, indicating that up-regulation of autophagy and proteasome function contributes to enhanced cold tolerance. The present work sheds a light on the roles of HSP90 in regulation of phenotypic plasticity associated with thermal adaptation in fish.

Virus strain from a mild COVID-19 patient in Hangzhou represents a new trend in SARS-CoV-2 evolution potentially related to Furin cleavage site.

The mutations in the SARS-CoV-2 virus genome during COVID-19 dissemination are unclear. In 788 COVID-19 patients from Zhejiang province, we observed decreased rate of severe/critical cases compared with patients in Wuhan. For mechanisms exploration, we isolated one strain of SARS-CoV-2 (ZJ01) from a mild COVID-19 patient. Thirty-five specific gene mutations were identified. Phylogenetic and relative synonymous codon usage analysis suggested that ZJ01 may be a potential evolutionary branch of SARS-CoV-2. We classified 54 global virus strains based on the base (C or T) at positions 8824 and 28247 while ZJ01 has T at both sites. The prediction of the Furin cleavage site (FCS) and sequence alignment indicated that the FCS may be an important site of coronavirus evolution. ZJ01 mutations identified near the FCS (F1-2) caused changes in the structure and electrostatic distribution of the S surface protein, further affecting the binding capacity of Furin. Single-cell sequencing and ACE2-Furin co-expression results confirmed that the Furin expression was especially higher in glands, liver, kidneys, and colon. The evolutionary pattern of SARS-CoV-2 towards FCS formation may result in its clinical symptom becoming closer to HKU-1 and OC43 caused mild flu-like symptoms, further showing its potential in differentiating into mild COVID-19 subtypes.

Identification and characterization of miRNAs involved in cold acclimation of zebrafish ZF4 cells.

MicroRNAs (miRNAs) play vital roles in various biological processes under multiple stress conditions by leading to mRNA cleavage or translational repression. However, the detailed roles of miRNAs in cold acclimation in fish are still unclear. In the present study, high-throughput sequencing was performed to identify miRNAs from 6 small RNA libraries from the zebrafish embryonic fibroblast ZF4 cells under control (28°C, 30 days) and cold-acclimation (18°C, 30 days) conditions. A total of 414 miRNAs, 349 known and 65 novel, were identified. Among those miRNAs, 24 (19 known and 5 novel) were up-regulated, and 23 (9 known and 14 novel) were down-regulated in cold acclimated cells. The Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses indicated that the target genes of known differentially expressed miRNAs (DE-miRNA) are involved in cold acclimation by regulation of phosphorylation, cell junction, intracellular signal transduction, ECM-receptor interaction and so on. Moreover, both miR-100-3p inhibitor and miR-16b mimics could protect ZF4 cells under cold stress, indicating the involvement of miRNA in cold acclimation. Further study showed that miR-100-3p and miR-16b could regulate inversely the expression of their target gene (atad5a, cyp2ae1, lamp1, rilp, atxn7, tnika, btbd9), and that overexpression of miR-100-3p disturbed the early embryonic development of zebrafish. In summary, the present data show that miRNAs are closely involved in cold acclimation in zebrafish ZF4 cells and provide information for further understanding of the roles of miRNAs in cold acclimation in fish.

A 16q22.1 variant confers susceptibility to colorectal cancer as a distal regulator of ZFP90.

Genome-wide association studies (GWASs) implicate 16q22.1 locus in risk for colorectal cancer (CRC). However, the underlying oncogenic mechanisms remain unknown. Here, through comprehensive filtration, we prioritized rs7198799, a common SNP in the second intron of the CDH1, as the putative causal variant. In addition, we found an association of CRC-risk allele C of rs7198799 with elevated transcript level of biological plausible candidate gene ZFP90 via expression quantitative trait loci analysis. Mechanistically, causal variant rs7198799 resides in an enhancer element and remotely regulate ZFP90 expression by targeting the transcription factor NFATC2. Remarkably, CRISPR/Cas9-guided single-nucleotide editing demonstrated the direct effect of rs7198799 on ZFP90 expression and CRC cellular malignant phenotype. Furthermore, ZFP90 affects several oncogenic pathways, including BMP4, and promotes carcinogenesis in patients and in animal models with ZFP90 specific genetic manipulation. Taken together, these findings reveal a risk SNP-mediated long-range regulation on the NFATC2-ZFP90-BMP4 pathway underlying the initiation of CRC.

Epigenetic regulation of hematopoietic stem cell homeostasis.

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.