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Acute myeloid leukemia (AML) is a heterogeneous hematopoietic disorder with a poor prognosis. The clinical significance of Leukemia stem cells (LSCs) plays an important role in the generation of AML and is the main cause of the recurrence after remission. Osteopontin (OPN), an extracellular matrix protein, has been implicated in hematopoietic malignancies. However, the specific role and the underlying mechanism of AML cell autocrined OPN in leukemia maintenance remain unknown. Here, we showed that knockdown of Opn expression significantly prolonged the survival of mice with MLL-AF9 cell-induced AML and markedly reduced the tumor burden. The LSCs from the Opn-knockdown groups exhibited decreased numbers and impaired function as determined by immunophenotype, colony-forming and limiting dilution assays. Further analysis revealed that Opn prevents LSCs from undergoing apoptosis and cell cycle arrest. Repression of OPN in human AML cell lines in vitro mimics the phenotypes observed in the mouse model. Overall, our data indicated that OPN is a potent therapeutic target for eradicating LSCs in AML.

Epigenetic regulations play crucial roles in leukemogenesis and leukemia progression. SUV39H1 is the dominant H3K9 methyltransferase in the hematopoietic system, and its expression declines with aging. However, the role of SUV39H1 via its-mediated repressive modification H3K9me3 in leukemogenesis/leukemia progression remains to be explored. We found that SUV39H1 was down-regulated in a variety of leukemias, including MLL-r AML, as compared with normal individuals. Decreased levels of Suv39h1 expression and genomic H3K9me3 occupancy were observed in LSCs from MLL-r-induced AML mouse models in comparison with that of hematopoietic stem/progenitor cells. Suv39h1 overexpression increased leukemia latency and decreased the frequency of LSCs in MLL-r AML mouse models, while Suv39h1 knockdown accelerated disease progression with increased number of LSCs. Increased Suv39h1 expression led to the inactivation of Hoxb13 and Six1, as well as reversion of Hoxa9/Meis1 downstream target genes, which in turn decelerated leukemia progression. Interestingly, Hoxb13 expression is up-regulated in MLL-AF9-induced AML cells, while knockdown of Hoxb13 in MLL-AF9 leukemic cells significantly prolonged the survival of leukemic mice with reduced LSC frequencies. Our data revealed that SUV39H1 functions as a tumor suppressor in MLL-AF9-induced AML progression. These findings provide the direct link of SUV39H1 to AML development and progression.

PRDM family proteins are dysregulated in many human diseases, especially hematological malignancies and solid cancers, and share a unique N‐terminal PR domain followed by zinc fingers toward the C terminus. With a high frequency of DNA promoter hypermethylation, PRDM5 is primarily considered as a tumor suppressor in solid tumors. However, little is known about the function of PRDM5 in blood malignancies, especially acute myeloid leukemia (AML). In this study, we showed that high PRDM5 expression levels were independently correlated with poor overall survival in AML patients. PRDM5 overexpression promoted cell proliferation, colony formation, and migration in vitro and enhanced tumorigenesis in an in vivo xenograft model. Furthermore, we found that PRDM5 overexpression promoted cell cycle progression with the decreased level of cell cycle inhibitors such as p16 and p21, and regulated the expression of epithelial‐mesenchymal transition markers ZO‐1 and Vimentin to promote migration. Moreover, we observed that PRDM5 upregulated the Jun N‐terminal kinase (JNK) signaling pathway and downregulated c‐Myc expression. Pharmacological inhibition of JNK by SP600125 partially abrogated PRDM5‐induced cell proliferation and migration. Taken together, our findings demonstrate that PRDM5 functions as an oncogenic driver in AML via JNK pathway, suggesting that PRDM5 is a potential therapeutic target for AML.

SETD5 mutations were identified as the genetic causes of neurodevelopmental disorders. While the whole-body knockout of Setd5 in mice leads to embryonic lethality, the role of SETD5 in adult stem cell remains unexplored. Here, a critical role of Setd5 in hematopoietic stem cells (HSCs) is identified. Specific deletion of Setd5 in hematopoietic system significantly increased the number of immunophenotypic HSCs by promoting HSC proliferation. Setd5-deficient HSCs exhibited impaired long-term self-renewal capacity and multiple-lineage differentiation potentials under transplantation pressure. Transcriptome analysis of Setd5-deficient HSCs revealed a disruption of quiescence state of long-term HSCs, a cause of the exhaustion of functional HSCs. Mechanistically, SETD5 was shown to regulate HSC quiescence by mediating the release of promoter-proximal paused RNA polymerase II (Pol II) on E2F targets in cooperation with HCF-1 and PAF1 complex. Taken together, these findings reveal an essential role of SETD5 in regulating Pol II pausing-mediated maintenance of adult stem cells.

Obesity is a complex disorder where the genome interacts with diet and environmental factors to ultimately influence body mass, composition, and shape. Numerous studies have investigated how bulk lipid metabolism of adipose tissue changes with obesity and, in particular, how the composition of triglycerides (TGs) changes with increased adipocyte expansion. However, reflecting the analytical challenge posed by examining non-TG lipids in extracts dominated by TGs, the glycerophospholipid composition of cell membranes has been seldom investigated. Phospholipids (PLs) contribute to a variety of cellular processes including maintaining organelle functionality, providing an optimized environment for membrane-associated proteins, and acting as pools for metabolites (e.g. choline for one-carbon metabolism and for methylation of DNA). We have conducted a comprehensive lipidomic study of white adipose tissue in mice which become obese either through genetic modification (ob/ob), diet (high fat diet), or a combination of the two, using both solid phase extraction and ion mobility to increase coverage of the lipidome. Composition changes in seven classes of lipids (free fatty acids, diglycerides, TGs, phosphatidylcholines, lyso-phosphatidylcholines, phosphatidylethanolamines, and phosphatidylserines) correlated with perturbations in one-carbon metabolism and transcriptional changes in adipose tissue. We demonstrate that changes in TGs that dominate the overall lipid composition of white adipose tissue are distinct from diet-induced alterations of PLs, the predominant components of the cell membranes. PLs correlate better with transcriptional and one-carbon metabolism changes within the cell, suggesting that the compositional changes that occur in cell membranes during adipocyte expansion have far-reaching functional consequences. Data are available at MetaboLights under the submission number: MTBLS1775.

Hematopoietic stem cells constitute a unique subpopulation of blood cells that can give rise to all types of mature cells in response to physiological demands. However, the intrinsic molecular machinery that regulates this transformative property remains elusive. In this paper, we demonstrate that small GTPase Rheb1 is a critical regulator of proliferation and differentiation of hematopoietic stem cells in vivo. Rheb1 deletion led to increased phenotypic hematopoietic stem cell/hematopoietic progenitor cell proliferation under a steady state condition. Over-proliferating Rheb1-deficient hematopoietic stem cells were severely impaired in functional repopulation assays, and they failed to regenerate the blood system when challenged with hematopoietic ablation by sublethal irradiation. In addition, it was discovered that Rheb1 loss resulted in a lack of maturation of neutrophils / caused neutrophil immaturation by reducing mTORC1 activity, and that activation of the mTORC1 signaling pathway by mTOR activator 3BDO partially restored the maturation of Rheb1-deficient neutrophils. Rheb1 deficiency led to a progressive enlargement of the hematopoietic stem cell population and an eventual excessive myeloproliferation in vivo, including an overproduction of peripheral neutrophils and an excessive expansion of extramedullary hematopoiesis. Moreover, low RHEB expression was correlated with poor survival in acute myeloid leukemia patients with normal karyotype. Our results, therefore, demonstrate a critical and unique role for Rheb1 in maintaining proper hematopoiesis and myeloid differentiation.

PDK1 (phosphoinositide dependent kinase-1) plays an important regulatory role in B cells, T cells and platelets. Less is known about how PDK1 acts in hematopoietic stem cells (HSCs), especially in the fetal liver (FL) during embryonic hematopoiesis, as the FL is the primary fetal hematopoietic organ and the main site of HSC expansion and differentiation. Here, we deleted the PDK1 gene in hematopoietic cells by crossing Vav-Cre transgenic mice with PDK1f/f mice. Using a transplantation assay, we found that HSCs from the E15.5 FL of Vav-Cre;PDK1f/f embryos are severely impaired compared when compared with HSCs from PDK1f/f or PDK1f/+ FLs. Additionally, we found that there were more FL HSCs in an apoptotic state and active cell cycle in PDK1-deficient embryos than in control embryos. By comparing the expression profiles of FL-derived LSKs in Vav-Cre;PDK1f/f embryos to the controls, we found that the BH3-only protein PUMA and the cyclin family proteins were expressed higher in the Vav-Cre;PDK1f/f group, which may account for the increased apoptosis and activated cell cycle in the deficient HSCs. Furthermore, we demonstrated that the expression of FoxO3a was higher in PDK1-deficient LSKs, indicating that the Akt-FoxO3a-PUMA axis may participate in regulating LSKs apoptosis in the E15.5 FL. In contrast, FoxO1 expression was lower in PDK1-deficient LSK cells, suggesting that Akt-FoxO1-CCND may regulate the HSC cell cycle. Taken together, our findings support a critical role for PDK1 in maintaining FL hematopoiesis via regulating apoptosis and cell cycle of definitive hematopoiesis by the Akt-FOXO signaling pathways. Keywords: PDK1, Akt, HSC, FOXO, Fetal liver, Embryonic hematopoiesis

As a dioxygenase, Ten-Eleven Translocation 2 (TET2) catalyzes subsequent steps of 5-methylcytosine (5mC) oxidation. TET2 plays a critical role in the self-renewal, proliferation, and differentiation of hematopoietic stem cells, but its impact on mature hematopoietic cells is not well-characterized. Here we show that Tet2 plays an essential role in osteoclastogenesis. Deletion of Tet2 impairs the differentiation of osteoclast precursor cells (macrophages) and their maturation into bone-resorbing osteoclasts in vitro. Furthermore, Tet2−/− mice exhibit mild osteopetrosis, accompanied by decreased number of osteoclasts in vivo. Tet2 loss in macrophages results in the altered expression of a set of genes implicated in osteoclast differentiation, such as Cebpa, Mafb, and Nfkbiz. Tet2 deletion also leads to a genome-wide alteration in the level of 5-hydroxymethylcytosine (5hmC) and altered expression of a specific subset of macrophage genes associated with osteoclast differentiation. Furthermore, Tet2 interacts with Runx1 and negatively modulates its transcriptional activity. Our studies demonstrate a novel molecular mechanism controlling osteoclast differentiation and function by Tet2, that is, through interactions with Runx1 and the maintenance of genomic 5hmC. Targeting Tet2 and its pathway could be a potential therapeutic strategy for the prevention and treatment of abnormal bone mass caused by the deregulation of osteoclast activities. Keywords: Tet2, 5hmC, Macrophage, Osteoclast, Runx1

Tet2 (member 2 of the Tet family) plays an important role in DNA demethylation modification, epigenetic regulation, and hematopoiesis. In our previous study, we found that Tet2 knockout mice progressively developed lymphocytic leukemia and myeloid leukemia with aging. However，the role of Tet2 in bone marrow microenvironment is unclear. Here in this study, we found that more Tet2-/- mesenchymal stem cells (MSCs) from bone marrow were in the G2/M cell cycle stages. The division time of Tet2-/- MSCs was shorter than that of the control cells. The growth rate of Tet2-/- MSCs was accelerated. The cobblestone area-forming cells assay (CAFC) showed that Tet2 knockout MSCs supported the expansion of hematopoietic stem cells (HSCs) and the differentiation of HSCs was skewed towards myeloid cells. Through the dot blotting experiment, we found that the total methylation level was increased in Tet2-/- bone marrow cells (BM). We used the methylation-chip to analyze the methylation level of Tet2-/- bone marrow cells and found that the level of methylation was increased in the transcriptional starting area (TSS), exons (EXONS) and 3' untranslated region (3' UTR). Moreover, we found that the cytokines secreted by Tet2-/- MSCs, such as IL-8 and IL-18, were decreased. While the expressions of GM-CSF and CCL-3, which supported hematopoietic stem cells to differentiate to myeloid cells, were increased in Tet2-/- MSCs. Our results demonstrated that Tet2 regulates MSCs to support hematopoiesis.Tet2 (Tet 家族成员2) 在DNA 去甲基化修饰、表观遗传调控及骨髓造血中起着重要作用。笔者课题组前期研究发现，随着年龄增长，Tet2 敲除小鼠逐步发展为淋系白血病和髓系白血病。但Tet2 在骨髓微环境中的作用仍不清楚。进一步研究发现，Tet2 敲除的骨髓间充质干细胞 (Mesenchymal stem cells，MSC)更多处于G2/M 分裂期，其细胞分裂时间缩短，生长速度加快。长周期培养-起始细胞实验表明，Tet2 敲除的MSC 支持造血干细胞扩增和髓系分化的能力增强。通过点杂交实验发现，Tet2 敲除后，骨髓细胞DNA总甲基化水平升高。对Tet2 缺失的骨髓细胞进行甲基化测序，结果表明：基因组转录调控区域等多个功能性结构域的甲基化水平明显升高。同时，敲除Tet2 的MSC 分泌IL-8、IL-18 等炎性细胞因子的能力下降；敲除Tet2 的MSC 更多分泌促进造血干细胞髓系分化的GM-CSF 和CCL-3 等细胞因子。Tet2 可以影响间充质干细胞造血支持作用，进而调节造血。.

Molecular genetic changes in acute myeloid leukemia (AML) play crucial roles in leukemogenesis, including recurrent chromosome translocations, epigenetic/spliceosome mutations and transcription factor aberrations. Six1, a transcription factor of the Sine oculis homeobox (Six) family, has been shown to transform normal hematopoietic progenitors into leukemia in cooperation with Eya. However, the specific role and the underlying mechanism of Six1 in leukemia maintenance remain unexplored. Here, we showed increased expression of SIX1 in AML patients and murine leukemia stem cells (c‐Kit+ cells, LSCs). Importantly, we also observed that a higher level of Six1 in human patients predicts a worse prognosis. Notably, knockdown of Six1 significantly prolonged the survival of MLL‐AF9‐induced AML mice with reduced peripheral infiltration and tumor burden. AML cells from Six1‐knockdown (KD) mice displayed a significantly decreased number and function of LSC, as assessed by the immunophenotype, colony‐forming ability and limiting dilution assay. Further analysis revealed the augmented apoptosis of LSC and decreased expression of glycolytic genes in Six1 KD mice. Overall, our data showed that Six1 is essential for the progression of MLL‐AF9‐induced AML via maintaining the pool of LSC.

3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a pivotal regulator in the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway that have been shown to play key roles in the functional development of B and T cells via activation of AGC protein kinases during hematopoiesis. However, the role of PDK1 in HSCs has not been fully defined. Here we specifically deleted the PDK1 gene in the hematopoietic system and found that PDK1-deficient HSCs exhibited impaired function and defective lineage commitment abilities. Lack of PDK1 caused HSCs to be less quiescent and to produce a higher number of phenotypic HSCs and fewer progenitors. PDK1-deficient HSCs were also unable to reconstitute the hematopoietic system. Notably, HSC function was more dependent on PDK1 than on mTORC2, which indicates that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function. PDK1-deficient HSCs also exhibited reduced ROS levels, and treatment of PDK1-deficient HSCs with L-butathioninesulfoximine in vitro elevated the low ROS level and promoted colony formation. Therefore, PDK1 appears to contribute to HSC function partially via regulating ROS levels.

The early stages of nonalcoholic fatty liver disease (NAFLD) are characterized by the accumulation of fat in the liver (steatosis). This can lead to cell injury and inflammation resulting in nonalcoholic steatohepatitis (NASH). To determine whether lipid profiling of liver tissue can identify metabolic signatures associated with disease presence and severity, we explored liquid extraction surface analysis mass spectrometry (LESA-MS) as a novel sampling tool. Using LESA-MS, lipids were extracted directly from the surface of ultrathin slices of liver tissue prior to detection by high-resolution mass spectrometry (MS). An isotopically labeled internal standard mix was incorporated into the extraction solvent to attain semiquantitative data. Data mining and multivariate statistics were employed to evaluate the generated lipid profiles and abundances. With this approach, we were able to differentiate healthy and NAFLD liver in mouse and human tissue samples, finding several triacylglyceride (TAG) and free fatty acid (FFA) species to be significantly increased. Furthermore, LESA-MS was able to successfully differentiate between simple steatosis and more severe NASH, based on a set of short-chain TAGs and FFAs. We compared the data obtained by LESA-MS to that from liquid chromatography (LC)-MS and matrix-assisted laser desorption ionization MS. Advantages of LESA-MS include rapid analysis, minimal sample preparation, and high lipid coverage. Furthermore, since tissue slices are routinely used for diagnostics in clinical settings, LESA-MS is ideally placed to complement traditional histology. Overall LESA-MS is found to be a robust, fast, and discriminating approach for determining NAFLD presence and severity in clinical samples.

Mediator complex is a molecular hub integrating signaling, transcription factors, and RNA polymerase II (RNAPII) machinery. Mediator MED23 is involved in adipogenesis and smooth muscle cell differentiation, suggesting its role in energy homeostasis. Here, through the generation and analysis of a liver-specific Med23-knockout mouse, we found that liver Med23 deletion improved glucose and lipid metabolism, as well as insulin responsiveness, and prevented diet-induced obesity. Remarkably, acute hepatic Med23 knockdown in db/db mice significantly improved the lipid profile and glucose tolerance. Mechanistically, MED23 participates in gluconeogenesis and cholesterol synthesis through modulating the transcriptional activity of FOXO1, a key metabolic transcription factor. Indeed, hepatic Med23 deletion impaired the Mediator and RNAPII recruitment and attenuated the expression of FOXO1 target genes. Moreover, this functional interaction between FOXO1 and MED23 is evolutionarily conserved, as the in vivo activities of dFOXO in larval fat body and in adult wing can be partially blocked by Med23 knockdown in Drosophila. Collectively, our data revealed Mediator MED23 as a novel regulator for energy homeostasis, suggesting potential therapeutic strategies against metabolic diseases.

Interaction of HOXA9/MEIS1/PBX3 is responsible for hematopoietic system transformation in MLL-rearranged (MLL-r) leukemia. Of these genes, HOXA9 has been shown to be critical for leukemia cell survival, while MEIS1 has been identified as an essential regulator for leukemia stem cell (LSC) maintenance. Although significantly high expression of PBX3 was observed in clinical acute myeloid leukemia (AML) samples, the individual role of PBX3 in leukemia development is still largely unknown. In this study, we explored the specific role of PBX3 and its associated regulatory network in leukemia progression. By analyzing the clinical database, we found that the high expression of PBX3 is significantly correlated with a poor prognosis in AML patients. ChIP-Seq/qPCR analysis in MLL-r mouse models revealed aberrant epigenetic modifications with increased H3K79me2, and decreased H3K9me3 and H3K27me3 levels in LSCs, which may account for the high expression levels of Pbx3. To further examine the role of Pbx3 in AML maintenance and progression, we used the CRISPR/Cas9 system to delete Pbx3 in leukemic cells in the MLL-AF9 induced AML mouse model. We found that Pbx3 deletion significantly prolonged the survival of leukemic mice and decreased the leukemia burden by decreasing the capacity of LSCs and promoting LSC apoptosis. In conclusion, we found that PBX3 is epigenetically aberrant in the LSCs of MLL-r AML and is essential for leukemia development. Significantly, the differential expression of PBX3 in normal and malignant hematopoietic cells suggests PBX3 as a potential prognostic marker and therapeutic target for MLL-r leukemia.

Although great efforts have been made to improve available therapies, the mortality rate of acute myeloid leukemia (AML) remains high due to poor treatment response and frequent relapse after chemotherapy. Leukemia stem cells (LSCs) are thought to account for this poor prognosis and relapse. Phosphoinositide-dependent kinase 1 (PDK1) is a critical regulator of the PI3K/Akt pathway and has been shown to be frequently activated in leukemia. However, the role of PDK1 in the regulation of LSCs in AML is still not clear. Using a PDK1 conditional deletion MLL-AF9 murine AML model, we revealed that the deletion of PDK1 prolonged the survival of AML mice by inducing LSC apoptosis. This was accompanied by the increased expression of the pro-apoptotic genes Bax and p53 and the reduced expression of Stat5, which has been shown to be constitutively activated in leukemia. Thus, our findings suggest that PDK1 plays an essential role in maintaining LSCs. Further delineating the function of PDK1 in LSCs may provide a new strategy for the improved treatment of AML relapse.

T-cell activation is critical for successful immune responses and is controlled at multiple levels. Although many changes of T-cell receptor-associated signalling molecules affect T-cell activation, the transcriptional mechanisms that control this process remain largely unknown. Here we find that T cell-specific deletion of the mediator subunit Med23 leads to hyperactivation of T cells and aged Med23-deficient mice exhibit an autoimmune syndrome. Med23 specifically and consistently promotes the transcription of multiple negative regulators of T-cell activation. In the absence of Med23, the T-cell activation threshold is lower, which results in enhanced antitumour T-cell function. Cumulatively, our data suggest that Med23 contributes to controlling T-cell activation at the transcriptional level and prevents the development of autoimmunity.

Obesity increases the risk of developing life-threatening metabolic diseases including cardiovascular disease, fatty liver disease, diabetes, and cancer. Efforts to curb the global obesity epidemic and its impact have proven unsuccessful in part by a limited understanding of these chronic progressive diseases. It is clear that low-grade chronic inflammation, or metaflammation, underlies the pathogenesis of obesity-associated type 2 diabetes and atherosclerosis. However, the mechanisms that maintain chronicity and prevent inflammatory resolution are poorly understood. Here, we show that inhibitor of κB kinase epsilon (IKBKE) is a novel regulator that limits chronic inflammation during metabolic disease and atherosclerosis. The pathogenic relevance of IKBKE was indicated by the colocalization with macrophages in human and murine tissues and in atherosclerotic plaques. Genetic ablation of IKBKE resulted in enhanced and prolonged priming of the NLRP3 inflammasome in cultured macrophages, in hypertrophic adipose tissue, and in livers of hypercholesterolemic mice. This altered profile associated with enhanced acute phase response, deregulated cholesterol metabolism, and steatoheptatitis. Restoring IKBKE only in hematopoietic cells was sufficient to reverse elevated inflammasome priming and these metabolic features. In advanced atherosclerotic plaques, loss of IKBKE and hematopoietic cell restoration altered plaque composition. These studies reveal a new role for hematopoietic IKBKE: to limit inflammasome priming and metaflammation.