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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.

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.

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.

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.

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.

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.

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.

Acute myeloid leukemia (AML) is the most frequent and heterogeneous malignancy in adult leukemic patients. Genome-wide analyses revealed that genes involved in epigenetic modifications are among the most often re-occurring mutations in AML, suggesting a crucial role of epigenetic regulation in leukemogenesis and leukemia relapse. As a mammalian lysine methyltransferase, SUV39H1 catalyzes di- and tri-methylation of histone 3 lysine 9, and is the predominant H3K9 methyltransferase expressed in hematopoietic stem cells (HSCs). Previous studies have shown that in MLL-rearranged leukemic cells, the normal localization of Suv39h1 and Sirt1 was interrupted due to the DNA binding of Dot1L to DNA. However, the biological role of SUV39H1 in MLL-rearranged leukemia remains unexplored. In this study, we investigated the role and the underlying mechanism of Suv39h1 during leukemia progression. By analyzing the clinical databases, we found a significantly reduced expression of SUV39H1 in AML cells in comparison with normal bone marrow (BM) cells. More importantly, we found that low expression of SUV39H1 predicts poorer survival in AML patients. In MLL-fusion induced AML mouse models (MLL-AF9/MA9 and MLL-NRIP3/MN3), Suv39h1 also exhibited lower expression in leukemia stem cells (LSCs, defined as c-Kit+ or Lin-Sca1-IL-7R-c-Kit+CD34+CD16/32+ L-GMP cells) when compared with normal HSPCs. These data suggest a potential role of SUV39H1 in leukemic progression and/or maintenance. To explore if Suv39h1 functions as a tumor suppressor in MLL-fusion driven leukemogenesis, we overexpressed Suv39h1 in MA9 BM AML cells. Western blotting analysis confirmed the overexpression of Suv39h1 with a moderate increase in global H3K9me3 levels in Suv39h1-overexpressed (SUV-OE) MA9 AML cells. Interestingly, Suv39h1 overexpression prolonged the survival of recipient AML mice in both secondary and tertiary transplantation groups. Both the frequency and the absolute number of phenotypic LSCs in BM and SP were significantly reduced in SUV-OE groups as manifested by flow cytometry. Furthermore, limiting dilution assays revealed a significant six-fold decrease of functional LSCs in SUV-OE AML cells (1/314 LSCs in SUV-OE AML cells vs 1/56 in controls). Cell cycle analysis of control and SUV-OE LSCs from BM revealed a significantly decreased proportion of SUV-OE cells in the S/G2/M phase concordant by an increased proportion of G0/G1 phases when compared with control cells. In contrast, a similar apoptotic ratio of L-GMPs in BM was observed between control and SUV-OE groups. Taken together, these data demonstrated that overexpressing Suv39h1 in AML cells reduces the frequency of functional LSCs by suppression its proliferation. To explore the underlying mechanisms, gene expression profiles were assessed by RNA-Seq of SUV-OE and control mouse AML c-Kit+ cells. A total of 69 genes were differentially expressed with fold change ≥ 4. Among these genes, Hoxb13 was of particular interesting since it was reported to be recurrently mutated in several types of cancers including leukemia. ChIP-qPCR revealed a two-fold increase of H3K9m3 distribution at the promoter of Hoxb13 in SUV-OE groups, indicating Hoxb13 may be a direct downstream target of Suv39h1. Restoring the expression of Hoxb13 in SUV-OE AML cells diminished the effect of SUV-OE-mediated prolonged survival of SUV-OE AML mice. Interestingly, overexpression of Hoxb13 alone in MA9 cells had no significant effect on the survival of MA9 AML mice, indicating that Hoxb13 is a downstream effector of Suv39h1, rather than MA9, and Suv39h1 itself is a downstream mediator of MA9. To summarize, we here for the first time, demonstrate that Suv39h1 is significantly down-regulated in AMLs and could function as a tumor suppressor in MLL-rearranged leukemia by epigenetically inhibiting the Hoxb13 expression. The molecular mechanism mediated by Suv39h1-Hoxb13 axis in tumor suppression could potentially provide us novel therapeutic strategies for MLL-rearranged leukemia. YJ.C, YP.C and HD.G contributed equally to this work. Corresponding authors: WP.Y and MJ.X. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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.

Background Evidence from several recent metabolomic studies suggests that increased concentrations of triacylglycerols with shorter (14–16 carbon atoms), saturated fatty acids are associated with insulin resistance and the risk of type 2 diabetes. Although causality cannot be inferred from association studies, patients in whom the primary cause of insulin resistance can be genetically defined offer unique opportunities to address this challenge. Methods We compared metabolite profiles in patients with congenital lipodystrophy or loss-of-function insulin resistance (INSR gene) mutations with healthy controls. Results The absence of significant differences in triacylglycerol species in the INSR group suggest that changes previously observed in epidemiological studies are not purely a consequence of insulin resistance. The presence of triacylglycerols with lower carbon numbers and high saturation in patients with lipodystrophy suggests that these metabolite changes may be associated with primary adipose tissue dysfunction. The observed pattern of triacylglycerol species is indicative of increased de novo lipogenesis in the liver. To test this we investigated the distribution of these triacylglycerols in lipoprotein fractions using size exclusion chromatography prior to mass spectrometry. This associated these triacylglycerols with very low-density lipoprotein particles, and hence release of triacylglycerols into the blood from the liver. To test further the hepatic origin of these triacylglycerols we induced de novo lipogenesis in the mouse, comparing ob/ob and wild-type mice on a chow or high fat diet, confirming that de novo lipogenesis induced an increase in relatively shorter, more saturated fatty acids. Conclusions Overall, these studies highlight hepatic de novo lipogenesis in the pathogenesis of metabolic dyslipidaemia in states where energy intake exceeds the capacity of adipose tissue. Electronic supplementary material The online version of this article (doi:10.1186/s13073-015-0179-6) contains supplementary material, which is available to authorized users.

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.

Acute myeloid leukemia (AML) with MLL translocation is recognized as a group of aggressive hematopoietic malignancies with poor prognosis. An atypical homeodomain protein family (TALE family, three-amino-acid loop extension) has been reported to be involved in leukemogenesis and leukemia progression. For example, MEIS1 and PBX3 were abnormally expressed in examined AML patient samples when compared with their normal counterparts. While MEIS1 has been studied extensively, the precise role of PBX3 in leukemia development is still largely unknown. In this study, we explored the epigenetic regulatory network of PBX3 and the specific role of PBX3 in leukemia progression. By analyzing the clinical database, we found that the high expression of PBX3 iscorrelated with poor prognosis of AML patients. In MLL-fusion induced AML mouse models that were established by retrovirus-mediated ectopic expression of MLL-AF9 or MLL-NRIP3 genes in murine BM progenitor cells (c-Kit+). PBX3 also showed high expression level in leukemia stem cells (LSC) compared with normal HSPCs. Unlike other homeobox genes such as HOXA9 and MEIS1 that were both highly expressed in LSKs and LSCs, PBX3 only showed extremely high expression in LSCs in comparison with HSPCs or other normal hematopoietic cells. To explore the role of PBX3 in leukemia, we analyzed the epigenetic modification patterns along PBX3 gene. By analyzing the public ChIP-seq database, we found that increased activation mark H3K79me2 and decreased repressive mark H3K27me3 exhibited on PBX3 in LSCs. Additionally, transcription activating modifications H3K79 hypermethylation of PBX3 was only observed in LSCs but not LSKs or GMPs, consistent with increased PBX3 expression level in LSCs compared with HSPCs. To further confirm this, we assessed H3K79me2 level of PBX3 in purified c-Kit+ cells which are enriched with stem cells from leukemic or control mice by ChIP-qPCR. Consistently, ChIP-qPCR results revealed the similar trend with dataset analysis. Moreover, we also detected the transcription repressive mark H3K9me3 on PBX3 in c-Kit+ cells and found a dramatic decrease of H3K9me3 in LSCs in comparison with HSPCs. Thus, our results suggested that high expression level of PBX3 in LSCs might be caused by elevated activation mark H3K79me2 and depressed repressive mark H3K27me3 and H3K9me3. Considering that high expression level of PBX3 in LSCs may contribute to leukemic progression, we used the CRISPR/Cas9 system to delete PBX3 to examine its potential role in MLL-AF9 mediated AML development. We found that PBX3 inactivation significantly prolonged the survival of leukemic mice (median survival was 25 days of control, 31 days of SgPBX3-a, 30 days of SgPBX3-b, 10 mice per group). Morphological analysis revealed more leukemic blast cells with segment nuclei exhibited in PBX3 inactivation group, suggesting that PBX3 deficiency is associated with increased leukemic cell differentiation. Since LSC was considered to be sufficient to drive leukemogenesis and maintain leukemia progression, we investigated the impact of PBX3 inactivation on LSC capability. In vitro colony forming assay showed that PBX3 inactivation group formed significantly fewer colonies of total numbers and type A clones. Flow cytometric analysis showed decreased LSCs frequency in murine leukemia cells of PBX3 inactivation group. Moreover, limiting dilution transplantation assays revealed 4-7 fold decreased of functional LSCs after PBX3 inactivation with the 1/102 of LSCs in control leukemic bone marrow in comparison with 1/442 in SgPBX3-a and 1/741 in SgPBX3-b respectively. Further analysis suggested that depletion of functional LSCs in PBX3 inactivation group mainly through accelerating apoptosis of LSCs, evidenced by both increased Annexin V positive cell frequency of whole BM blasts and LSCs in PBX3 inactivation group. In conclusion, we found PBX3 is epigenetically dis-regulated in LSCs of MLL-r AMLs and is essential for leukemia development. Inactivation of PBX3 could suppress leukemia progression by reducing LSCs frequency and impairing its function. More importantly, the differential expression of PBX3 in normal and malignant hematopoietic cells provides itself potentially as a prognostic marker and therapeutic target for MLL-rearranged leukemia. HD.G and YJ.C contributed equally to this work Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem cells (HSCs) exist as a rare population with two essential properties of self-renewal and differentiation. HSCs can give rise to all hematopoietic progenitor and mature cells. While critical for a full understanding of the hematopoietic process and HSC-related clinical applications, the mechanisms of self-renewal and differentiation of HSCs remain elusive. The PI3K-Akt signaling pathway plays essential roles in the regulation of hematopoiesis. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) activates multiple AGC kinases including Akt and is a pivotal regulator in this pathway. PDK1 phosphorylates Akt at its T308 residue and regulates the functional development of B and T cells during hematopoiesis. However, the role of PDK1 in HSCs has not been fully defined. In this study, we generated PDK1 conditional knockout mice Vav-Cre;PDK1fl/fl (PDK1Δ/Δ) to explore the roles of PDK1 in HSCs. While PDK1Δ/Δ mice have reduced B and T cell counts as previously described, their LT-HSCs and ST-HSCs were significantly increased in comparison with WT mice while MPPs and CMPs were decreased after PDK1 deletion, indicating that the loss of PDK1 perturbed the steady-state hematopoiesis. Furthermore, although deletion of PDK1 increased the frequency of HSCs, PDK1-deficient HSCs fail to reconstitute the hematopoietic system when PDK1-deficient HSCs were used in bone marrow transplantation and competitive transplantation experiments in comparison to the WT HSCs, indicating that PDK1 is vital for hematopoiesis. To explore the mechanisms by which PDK1 regulates HSC function, we examined the cell cycle status and found the percentage of PDK1Δ/Δ HSCs was decreased significantly in G0 stage while increased in G1 and S/G2/M phases. This suggests an increase in HSC exit from a quiescent state. Since MPPs were significantly decreased in bone marrow, we examined the percentage of Annexin V+ DAPI- PDK1Δ/Δ and WT MPPs and found that they are comparable. This indicates that apoptosis did not cause the decrease in MPPs. In addition, a total of 300 LT-HSCs from PDK1Δ/Δ or WT mice and competitor cells were transplanted into lethally irradiated recipient mice to examine whether the decrease in MPPs is due to a defect in HSC differentiation. We found that less than 1% of MPPs arose from PDK1Δ/Δ HSCs 12 weeks after transplantation, indicating that PDK1 is required for the differentiation from LT-HSCs to MPPs. Because the full activation of Akt requires cooperative phosphorylation at its S473 and T308 residues by mTORC2 and PDK1, respectively, we also investigated the function of HSCs in RictorΔ/Δ PDK1Δ/Δ (DKO) mice in conjunction with RictorΔ/Δ or PDK1Δ/Δ mice to explore how mTORC2 and/or PDK1 influence Akt function in HSCs. The flow cytometric analyses of peripheral blood and bone marrow samples revealed very similar parameters of RictorΔ/Δ PDK1Δ/Δ and PDK1Δ/Δ mice. Interestingly, Rictor seemed to exert a minimal impact on HSCs and MPPs. More importantly, in contrast to RictorΔ/Δ, RictorΔ/Δ PDK1Δ/Δ HSCs failed to reconstitute the hematopoietic system after transplantation as PDK1Δ/Δ HSCs, suggesting that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function. To explore the mechanism that leads to the defect in HSCs due to loss of PDK1, we assessed ROS levels in PDK1-deficient HSCs and found that PDK1-deficient LSKs and HSCs exhibit greatly reduced ROS levels when compared with the control HSCs. Treating PDK1-deficient BM cells with BSO in vitro increased cellular ROS levels and the colony counts of PDK1-deficient BM cells significantly. Notably, the recovery effect was only observed with BSO concentrations lower than 0.03 mM. This suggests that ROS levels are precisely controlled in HSCs. Higher or lower ROS levels beyond the normal range are both harmful to normal HSC functions. Since increased SDFα expression is associated with cellular ROS levels in various cells including hematopoietic cells, we also treated PDK1Δ/Δ mice with SDFα and found that it couldpartially rescue the defective differentiation ability of PDK1-deficient HSCs. In addition, we found that PDK1 deletion could significantly prolong the life span and inhibit the leukemia development in murine T-ALL model via altering leukemic cell differentiation and proliferation. Taken together, PDK1 controls HSC differentiation via regulating cellular ROS levels and regulates malignant hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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.