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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.

Radiotherapy uses high-energy X-rays or other particles to destroy cancer cells and medical practitioners have used this approach extensively for cancer treatment (Hachadorian et al., 2020). However, it is accompanied by risks because it seriously harms normal cells while killing cancer cells. The side effects can lower cancer patients' quality of life and are very unpredictable due to individual differences (Bentzen, 2006). Therefore, it is essential to assess a patient's body damage after radiotherapy to formulate an individualized recovery treatment plan. Exhaled volatile organic compounds (VOCs) can be changed by radiotherapy and thus used for medical diagnosis (Vaks et al., 2012). During treatment, high-energy X-rays can induce apoptosis; meanwhile, cell membranes are damaged due to lipid peroxidation, converting unsaturated fatty acids into volatile metabolites (Losada-Barreiro and Bravo-Díaz, 2017). At the same time, radiotherapy oxidizes water, resulting in reactive oxygen species (ROS) that can increase the epithelial permeability of pulmonary alveoli, enabling the respiratory system to exhale volatile metabolites (Davidovich et al., 2013; Popa et al., 2020). These exhaled VOCs can be used to monitor body damage caused by radiotherapy.

Colony-stimulating factor 3 receptor (CSF3R) mutations have been identified in a variety of myeloid disorders. Although CSF3R point mutations (eg, T618I) are emerging as key players in chronic neutrophilic leukemia/atypical chronic myelogenous leukemia , the significance of rarer CSF3R mutations is unknown. Here, we report a 32-year-old female who was diagnosed as Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) with the CSF3R M696T mutation and was undergone unrelated donor hematopoietic stem cell transplantation. The patient achieved complete remission with chemotherapy in combination with tyrosine kinase inhibitor (TKI) and long-term survival by unrelated donor transplantation. Meanwhile, we performed a series of experiments using murine interleukin 3 (IL-3)-dependent Ba/F3 cell line to evaluate the transforming capacity of the CSF3R M696T mutation. We confirmed the presence of a CSF3R M696T germline mutation in this patient which was inherited from her mother. The in vitro experiment results showed that the CSF3R M696T mutation contributes marginally to the tumor transformation of Ba/F3 cells, indicating that CSF3R M696T mutation was neutral in tumor transformation ability. We concluded that TKI is effective in patients with the CSF3R M696T mutation in Ph+ ALL and donors with CSF3R M696T mutation might still be selected as the candidate for transplantation.

Aim Preoperative anaemia is common in colorectal cancer patients. Little attention has been given to the prevalence and consequences of postoperative anaemia. The aim of this study was to systematically review the published literature and determine the knowledge of the prevalence and impact of postoperative anaemia in colorectal cancer patients. Methods The databases Cumulative Index of Nursing and Allied Health Literature (CINAHL) and Medline, via EBSCOhost, were systematically searched to identify suitable articles published between 2004 and 2020. After an initial search, articles were screened and all eligible articles reporting on the prevalence of postoperative anaemia and clinical and long-term outcome data in colorectal cancer patients undergoing surgery were included. The Risk of Bias 2.0 tool for the assessment of randomized controlled trials and the Risk of Bias 1.0 tool for non-randomized studies were used for the assessment of bias in the studies selected in our review. Results Six studies, one randomized control trial and five cohort studies, were included with a total population size of 1714. The prevalence of anaemia at discharge of 76.6% was reported as the primary end-point in only one study. The rate of red blood cell transfusion and length of hospital stay were found to be significantly increased in anaemic patients, while postoperative infection rate results were variable. Quality of life scores and overall survival at 5 years were significantly affected among anaemic patients as reported in two papers. Conclusion The available limited evidence on postoperative anaemia indicates its high prevalence with negative impact on clinical and long-term outcomes. Further research is required to standardize the measurement and address the true impact of correcting postoperative anaemia on functional and oncological outcomes.

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.

Ten-eleven translocation 2 (TET2) functions as a methylcytosine dioxygenase that catalyzes the iterative oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. TET2 has been shown to be crucial for the maintenance and differentiation of hematopoietic stem cells, and its deletion and/or mutations results in the expansion of HSPCs, and leads to hematological malignancies. TET2 mutations were found in a variety of hematological disorders such as CMML (60%), MDS (30%), MPN (13%) and AML (20%). Interestingly, it was shown that CMML patients with TET2 mutation exhibited fewer platelets than CMML patients without TET2 mutation. However, the role and function of TET2 in platelet hemostasis and thrombogenesis is not well defined. Here in this study, using a genetically engineered Tet2 deletion mouse model, we found that the absence of Tet2 caused a decrease in the proportion of MEP cells and hyperploid megakaryocytes. Additionally, Tet2-deficient mice displayed impaired platelet activation and aggregation under stimulation of ADP and low concentrations of thrombin, although the modestly compromised platelet function and MEP differentiation in Tet2-deficient mice could be compensated without affecting blood coagulation function. Our study indicate that Tet2 deficiency leads to mild impairment of platelet function and thrombopoiesis in mice.

Acute myeloid leukemia (AML) is a deadly cancer characterized by an expanded self-renewal capacity that is associated with the accumulation of immature myeloid cells. Emerging evidence shows that methyl-CpG-binding domain protein 2 (MBD2), a DNA methylation reader, often participates in the transcriptional silencing of hypermethylated genes in cancer cells. Nevertheless, the role of MBD2 in AML remains unclear. Herein, by using an MLL-AF9 murine model and a human AML cell line, we observed that loss of MBD2 could delay the initiation and progression of leukemia. MBD2 depletion significantly reduced the leukemia burden by decreasing the proportion of leukemic stem cells (LSCs) and inhibiting leukemia cell proliferation in serial transplantation experiments, thereby allowing leukemic blasts to transition to a more mature state reflecting normal myelopoiesis. Both gene expression analyses and bioinformatic studies revealed that MBD2 negatively modulated genes related to myeloid differentiation, and was necessary to sustain the MLL-AF9 oncogene-induced gene program. We further demonstrated that MBD2 could promote LSC cell cycle progression through epigenetic regulation of CDKN1C transcription probably by binding to its promoter region. Taken together, our data suggest that MBD2 promotes AML development and could be a therapeutic target for myeloid malignancies.

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.

By means of glass bottle sampling followed by solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS) technique, the change characteristics of volatile organic compounds (VOCs) in breaths, between before gargling and after gargling, were investigated, respectively, in 41 healthy subjects and 50 esophageal cancer patients. Using an untargeted strategy, 143 VOC chromatographic peaks were enrolled in the statistical analysis. Based on the orthogonal partial least squares discriminant analysis (OPLS-DA), the VOC variations after gargling for each breath test group were obtained according to the combined criteria of variable importance in projection (VIP1.5), Wilcoxon signed-rank test (P0.05), and fold change (FC2.0). When gargled, the levels of indole, phenol, 1-propanol, and p-cresol in the breath of healthy people decreased; meanwhile, for esophageal cancer patients, the declined VOCs in breath were indole, phenol, dimethyl disulfide, and p-cresol. Particularly, these substances were previously reported as breath biomarkers in some diseases such as esophageal, gastric, thyroid, breast, oral, and lung cancers, as well as certain non-cancer disorders. The present work indicates that expiratory VOCs involve the prominent oral cavity source, and in the breath biomarkers study, the potential impact that originates from oral volatiles should be considered. In view of the present results, it is also proposed that gargle pretreatment could eliminate possible interference from the oral cavity VOCs that might benefit breath biomarker investigation. Gargle pretreatment helps to distinguish oral-source VOCs and control their potential impact on breath biomarkers.

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.

We welcome the interest in our recently published systematic review investigating the impact and outcomes of postoperative anaemia in colorectal cancer patients from Froud et al and are pleased to further the discussion on the risk bias in the systematic review. We appreciate that Froud et al agree with the study conclusion that further research is needed to ascertain the true prevalence of postoperative anaemia in colorectal cancer patients, given its potential to have negative impact on clinical and long-term functional outcome.

Myeloid cells have been identified as hematopoietic stem cell (HSC)-regulating cells. However, the mechanisms by which myeloid cells regulate the function of HSCs are not fully defined. Our previous study indicated that the HSCs are over-expanded in Vav1-Cre;Rheb1fl/fl mice. Here, using in vivo and in vitro models, we found that Rheb1-deficient neutrophils remodeled the bone marrow environment and induced expansion of HSCs in vivo. Further studies showed that loss of Rheb1 impaired neutrophils’ ability to secrete IL-6, led mesenchymal stem cells (MSCs) to produce more SCF, and promote HSC proliferation. We further found that IL-6 suppressed SCF mRNA expression in human MSCs. Interesting, the high level of IL-6 was also related with poor survival of chronic myeloid leukemia (CML) patients, and higher expression of IL-6 in CML cells is associated with the lower expression of SCF in MSCs in patients. Our studies suggested that blocking IL-6 signaling pathway might stimulate MSCs to secrete more SCF, and to support hematopoietic stem/progenitor cells proliferation.

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.

Ten-eleven translocation 2 (TET2) functions as a methylcytosine dioxygenase that catalyzes the iterative oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. TET2 has been shown to be crucial for the maintenance and differentiation of hematopoietic stem cells, and its deletion and/or mutations results in the expansion of HSPCs, and leads to hematological malignancies.

In order to find out cancer markers in human breath, in vitro cell culture is often used to study the characteristic volatile organic compounds (VOCs). In the cell culture process, disposable vessels are frequently adopted. However, these vessels are normally made of plastic, and they have the possibility to release some VOCs, which may interfere with the cell-specific volatiles and even can result in an incorrect conclusion. In this study, by using glass cell culture flasks as control, the headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) analyses of the VOCs in plastic cell culture flasks were systematically carried out for the first time. A total of 35 VOCs were detected in five brands of flasks. In each flask, there were between 13 and 25 volatile compounds. Furthermore, the components and packaging bag of each flask were also sampled and analyzed by HS-SPME-GC-MS. The results show that the flask cap, septum, flask body, and packaging bag exhibit respectively different volatile behaviors. The former two parts release the most volatiles which have obvious contributions to the headspace gases in the flasks, while the flask body mainly liberates styrene. For different flasks packed within the same bag, the headspace analyses show that their residual VOCs are inconsistent with each other. Moreover, the residual VOCs in the same flask are variable in three consecutive days. These results indicate that the multiple flasks in parallel cell culture experiments, or the same flask with different cell culture durations, will produce an indelible disturbance to the cell-specific VOCs. In addition, among the 35 VOCs detectable in five brands of empty plastic flasks, 15 VOCs were previously reported as characteristic VOCs from lung cancer, melanoma, cervical cancer cells, or normal cells. This is an alert that, when using plastic flasks, it must be careful to treat the possible interference from the background VOCs in the flasks. This study demonstrates that the cell culture tool needs to be standardized, and the clean glass or metal vessels are strongly recommended for usage when studying cell volatile biomarkers. Graphical abstract.

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

T cell acute lymphoblastic leukemia (T-ALL) is one of the most frequent hematologic malignancies resulted from gene mutations and/or genomic rearrangements that occur in T cell progenitors. The 5-year survival rate of T-ALL patients is less than 50%. Much efforts have been dedicated to decipher the molecular events underlying TALL transformation, with the goals to identify specific therapeutic targets and develop new and more effective drugs. As a member of JAK kinase family, JAK3 mutations can be identified in 16.1% of T-ALL cases, and JAK3 M511I mutation is the most common one within all JAK3 mutations. Activating JAK3 M511I mutation induced a lympho-proliferative disorder, that followed by a T-ALL-like disease. PHF6 mutation is one of the most common co-existing gene mutations with JAK3 in T-ALL patients. Co-mutation events of JAK3 and PHF6 account for 1.89%-10.0% in T-ALL cases. However, the role(s) of PHF6 and JAK3 co-mutations in tumorigenesis is unknown. Here in this study, we first analyzed the genetic data of 449 T-ALL cases from multiple clinical centers in which JAK/STAT mutations is about 21% of the total cases. Interestingly, we found that PHF6 mutations were significantly associated with JAK3 mutations in these T-ALL cases (P To investigate the underlying molecular mechanisms of Phf6 in accelerating T-ALL development,we performed RNAseq to analyze the transcriptome programing changes associated with Phf6 in isogenic Phf6WT +JAK3M511I or Phf6KO +JAK3M511IT-ALL cells. Gene set enrichment analysis (GSEA) showed up-regulated cell cycle in Phf6 KO +JAK3 M511I leukemia cells. We then performed Chromatin immunoprecipitation sequencing (ChIP-seq) and found that PHF6 associated with BAI1 gene. qPCR and Western blot showed that the mRNA and protein expression of Bai1/BAI1 were significantly decreased in Phf6KO +JAK3M511Icells in comparison with Phf6 WT+JAK3M511I cells. BAI1 has been reported to prevent MDM2-mediated P53 ubiquitination, and loss of BAI1 reduces P53 level. We found that the protein expression of BAI1 and P53 was decreased, and the ubiquitination of P53 was significantly increased in PHF6 knockdown (KD) MOLT-4 cells (T-ALL) when compared with the control cells. BAI1 overexpression in PHF6 KD MOLT-4 cells significantly increased P53 expression in PHF6 KD+BAI1 OE MOLT-4 cells when compared with that of PHF6 KD cells. It suggested that Phf6 deficiency increased BAI1-mediated P53 degradation. To confirm this, we treated Phf6 KO + JAK3M511I mice with a JAK3 inhibitor (ruxolitinib) in combination with a MDM2 inhibitor (idasanutlin), and found a synergistic response of the treatment within a significant attenuation in leukemia burden in vivo. In conclusion, we found that PHF6 mutation frequently co-existed with JAK3 mutation in T-ALLs, and together they can drive an aggressive leukemia in mice model. Notably, PHF6 deficiency promoted JAK3-induced T-ALL progression by inhibiting BAI1-MDM2-P53 signaling pathway that is independent of JAK3-STAT signaling pathway. We further demonstrated that combination therapy by tofacitinib and idasanutlin can reduce the Phf6 KO and JAK3 M511I leukemia burden in vivo. Our study suggested that combinational usage of JAK3 inhibitors and MDM2 inhibitors may increase the drug benefit for T-ALL patients with PHF6 and JAK3 co-mutations. Disclosures No relevant conflicts of interest to declare.

Stem cells maintain their self-renewal and differentiation through tight regulation of gene expression patterns at the transcriptional and epigenetic levels. SETD5 is a member of SET domain-containing histone lysine methyltransferase family. Its mutations were identified as the genetic causes of neurodevelopmental disorders, although SETD5 has been shown to lack methyltransferase activity in several studies. Deletion of Setd5 resulted in embryonic lethality at E10.5-11.5 with reduced cell number of CD41 + early hematopoietic cells in the blood island, while the role of SETD5 in adult stem cell, especially in hematopoietic stem cells (HSC) remains unexplored. In this study, by using Vav-Cre and Mx1-Cre mediated conditional knockout murine models, we explored the role of Setd5 in hematopoiesis. We found that Setd5 deficiency led to an enhanced accumulation of HSC in both Setd5 CKO (Vav-Cre; Setd5 fl/fl) and Setd5 IKO (Mx1-Cre; Setd5 fl/fl with pIpC treatment) mice. Cell cycle analysis revealed that higher proportions of SLAM-HSC and LSK + cells underwent active cycling in both Setd5 CKO and Setd5 IKO mice. However, limiting dilution assay revealed a significant ~4-fold decrease of functional HSCs in Setd5 CKO mice, while competitive serial transplantation assays exhibited a progressive decrease in repopulation capacity in Setd5 CKO than that of Setd5 fl/fl. While a progressive decrease in PB chimerism was observed in Setd5 IKO recipients via competitive transplantation assay, myeloid lineage reconstitution was increased, implicating a differentiation bias towards myeloid lineage at the expense of lymphoid lineage of Setd5 IKO HSCs. Thus, our phenotypical studies revealed that Setd5 deficiency impaired the homeostasis of the numbers and function of HSCs. To dissect the underlying mechanism, single cell transcriptome was performed in Setd5 CKO and Setd5 fl/fl control LSK +s using Smart-seq2 method. LSK +s were grouped into 5 clusters with distinct transcriptional features. Significantly, LT stem-like cluster revealed a disruption of LT-HSC and quiescence signatures in Setd5 CKO group, accompanied with an elevated S/G2/M cell cycle signature. When these cells were further projected onto Nestorowa's data (Nestorowa et al, Blood, 2016), Setd5 CKO LSK +s were found to be largely deviated from the core HSC territory toward more differentiated progenitor state. Consistent results were also observed in the bulk RNA-seq of Setd5 CKO and Setd5 IKO SLAM-HSCs. These data indicated that both transcriptional- and immunophenotype-defined LT-HSCs lost the long-term stem cell signatures due to Setd5 deficiency. Although SETD5 has a SET domain, no obvious changes were observed in a majority of histone methylation in Setd5 KO hematopoietic cells. Co-IP assay revealed that SETD5 could interact with HDAC3, PAF1 and HCF-1 complex. SETD5 ChIP-seq revealed that SETD5 could bind to E2F-responsive promoters and regulated the transcription of E2F targets. HCF-1 was reported to interact with E2F and regulate cell cycle entry. HCF-1 and PAF1 was recently found to associate with multiple transcription initiation and elongation complexes to regulate Pol II pausing. We thus investigated whether SETD5 regulates E2F targets via its association with HCF-1, and whether it also regulates E2F targets transcription via modulating Pol II pausing. By analyzing the genome-wide distribution of Pol II in control and Setd5 CKO c-Kit + cells by ChIP-Seq, we found an increase of Pol II occupancy upon SETD5 deficiency. Deletion of Setd5 lead to a significant decrease of pausing index on average which represent a transcription elongation state. Inhibition of the transition of paused Pol II in Setd5 CKO c-Kit + cells with BAY 1143572 (p-TEFb/CDK9 inhibitor) or JQ1 (Brd4 inhibitor) significantly suppressed S phases entry in Setd5 CKO LSK + cells, indicating that increased transition of paused Pol II to elongation mediated by Setd5 depletion could be a main cause for the exit of quiescence stage of Setd5 CKO HSC cells. In summary, we here demonstrate a critical role of Setd5 in HSC maintenance and provide a new insight into the mechanism of Setd5 in regulating HSC quiescence by pausing Pol II-mediated differential expression of cell cycle genes via HCF-1-SETD5-PAF1-Pol II axis. MK.L and YJ.C contributed equally to this work. Corresponding authors: WP.Y and YJ.C. Disclosures: No relevant conflicts of interest to declare. Disclosures No relevant conflicts of interest to declare.

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.

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 (PL) composition of cell membranes has been seldom investigated. PLs contribute to a variety of cellular processes including maintaining organelle functionality, providing an optimised environment for membrane-associated proteins and 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 who 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 lipid (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 the compositional changes that occur in cell membranes during adipocyte expansion have far-reaching functional consequences.

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。.

mTOR is a serine/threonine (Ser/Thr) protein kinase that responds to multiple signals, including growth factors, amino acids, energy status, stress, and oxygen, regulates cell survival, cell growth, the cell cycle, and cell metabolism, and maintains homeostasis [1]. Increased or decreased mTORC1 activity can alter HSC function and cause hematological disorders [2, 3]. Therefore, a comprehensive knowledge of mTOR is critical to understanding how HSCs function and maintain homeostasis in the hematopoietic system. In this review, we summarize recent advances in the understanding of the mTOR signaling pathway and its roles in hematopoiesis and leukemia. We also discuss pharmacological approaches to manipulate mTOR activity.

Purpose: Colony-stimulating factor 3 receptor (CSF3R) mutations have been identified in a variety of myeloid disorders, such as severe congenital neutropenia (SCN), chronic neutrophilic leukemia (CNL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and atypical chronic myelogenous leukemia (aCML). Although CSF3R point mutations (e.g., T618I) are emerging as key players in CNL/aCML, the significance of rarer CSF3R mutations is unknown. We report a case of philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) with the M696T mutation in CSF3R gene and assess the pathogenicity of the CSF3R M696T mutation in Ph+ ALL. Experimental Design: Here we report on a 32-year-old female who presented with asthenia. The initial hematological workup revealed white blood cell (WBC) count of 97 x 109/L (normal range 4-10 x 109/L). There was 84% prolymphocyte in the bone marrow. The immunophenotype of the blasts as judged from flow cytometry was in accordance with a B-ALL. The fusion gene for BCR-ABL P210 was positive. Hot mutation closely related to diseases was: CSF3R (nucleotide change c.2087 T>C, amino acid change p.M696T, mutation frequency 50.4%). Cytogenetic analysis showed 46, XX, t (9;22) (q34;q11). The patient was diagnosed as Ph+ ALL with the CSF3R M696T mutation and achieved Long-term survival after unrelated donor hematopoietic stem cell transplantation. Meanwhile we performed a series of experiments using murine interleukin 3 (IL-3)-dependent Ba/F3 cell line to evaluate the transforming capacity of the CSF3R M696T mutation. The phosphorylation of STAT3 was analyzed by G-CSF dependence assays and immunoblot analysis to evaluate the CSF3R M696T mutation contribution to the tumor transformation ability of Ba/F3 cells. Results: This patient achieved complete remission with chemotherapy in combination with tyrosine kinase inhibitor (TKI) and long-term survival by unrelated donor transplantation. We confirmed the presence of a CSF3R M696T germline mutation in this patient, and the mutation was inherited from her mother. The experiments in vitro result showed the CSF3R M696T mutation harbors marginal contribution to the tumor transformation ability of Ba/F3 cells. CSF3R M696T mutation was neutral in tumor transformation ability. Conclusions: We believe that TKI is still effective in patients with the CSF3R M696T mutation in Ph+ ALL. Donor with CSF3R M696T mutation might still be selected. Disclosures No relevant conflicts of interest to declare.

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.