ALBERT

Description: Key Points This study identifies a calmodulin-binding sequence in Sema4D and shows that calmodulin binds to Sema4D in resting platelets. Dissociation of the Sema4D:calmodulin complex is sufficient to trigger Sema4D cleavage and shedding of the extracellular domain.

Description: Abstract 1131 Humanin (HN), a 24-amino acid endogenous antiapoptotic peptide, was initially shown to protect against neuronal cell death by Alzheimer's disease-related insults. It has recently been found that an exogenous analog of HN (HNG) in which the 14th amino acid serine is replaced with glycine protected against cerebral and cardiac ischemia reperfusion (I/R) injury in cortical neurons and cardiomyocytes, respectively. Platelet activation and thrombus formation has been shown to play an important role during I/R injury by exacerbating the extent of the infarct size. However, it is presently unknown whether HNG affects platelet function and the subsequent arterial thrombus formation. We thus examined whether HNG affects platelet activation and thrombus formation both in vitro and in vivo. Human platelets were isolated from healthy adults. Preincubation of washed human platelets with HNG (4μM) reduced collagen- or convulxin-induced platelet aggregation by 56.8% (P

Description: Adult hematopoietic stem cells (HSCs) primarily reside in the hypoxic bone marrow microenvironment, and preferentially utilize anaerobic glycolysis to obtain energy. Cited2 is a cytokine-inducible gene, which plays various roles during mouse development. Our previous studies showed that deletion of Cited2 in adult mouse results in loss of HSC quiescence, increased apoptosis, and impaired HSC reconstitution capacity (Blood 2012, 119:2789-2798). In this study, we conditionally deleted Cited2 in Cited2fl/fl;Mx1-Cre mice and investigated the role of Cited2 in the metabolic regulation of HSCs. First, we examined mitochondrial alterations in Cited2 knockout (KO) long-term (LT-) and short-term (ST-) HSCs defined as “Flt3-CD34- LSK” and “Flt3-CD34+ LSK”, respectively. Staining with MitoTracker Green revealed that deletion of Cited2 resulted in a significant increase in mitochondrial mass in both LT- and ST-HSCs but not in the whole bone marrow cells. To explore the morphological changes of mitochondria in Cited2 KO HSCs, we sorted Flt3-LSK cells (containing LT- and ST- HSCs) and performed electron microscopy ultrastructural analysis. The mitochondria in wild type (WT) HSCs were mostly small, round or oval, and dark (Figure 1). However, Cited2 KO HSCs displayed markedly elongated and brighter mitochondria, similar to those observed in aged WT HSCs (20–24 months old mice) by others. The frequency of Cited2 KO LT-HSCs with high mitochondrial membrane potential was significantly increased (8.5% in WT versus 15.1% in KO). Furthermore, the reactive oxygen species (ROS) levels in Cited2 KO HSCs were significantly higher than those in WT controls. To further understand the metabolic changes in Cited2 KO HSCs, we measured glucose uptake using fluorescent indicator 2-NBDG. Glucose uptake was unchanged in the Cited2 KO LT- and ST- HSCs. Also, intracellular ATP content was maintained at the normal levels in Cited2 KO LT-HSCs, although slightly increased in ST-HSCs compared with WT controls. To assess the utilization of glycolysis in Cited2 KO HSCs, glycolytic flux was determined by glucose-derived 13C-lactate production using Gas Chromatography–Mass Spectrometry (GC-MS). We found that the rate of 13C-lactate production was significantly lower in both LT- and ST-HSCs lacking Cited2 than in WT controls. To further confirm this finding, we treated HSCs with antimycin A (AMA), a specific inhibitor of mitochondrial electron transport chain. We found that Cited2 KO HSCs displayed increased NADH after AMA treatment, compared with the WT control, indicating that mitochondrial respiration was increased in KO HSCs and produced more NADH. At the molecular level, deletion of Cited2 significantly reduced the expression of metabolism related genes in HSCs, such as lactate dehydrogenase (LDH) B and LDHD, pyruvate dehydrogenase kinase (Pdk) 2 and Pdk4, PYGL (phosphorylase, glycogen, liver), and GPX1 (glutathione peroxidase 1). Notably, Pdk2 and Pdk4 were recently shown to be critical controllers of glycolysis and checkpoint for cell cycle in HSCs. Consistent with reduced expression of Pdk, the phosphorylation of PDH-E1α was significantly decreased in Cited2 KO HSCs. Akt, mTOR, and FoxOs are known regulators of mitochondrial functions in HSCs. We found that Akt-mTOR signaling activity was increased in Cited2 KO HSCs, as indicated by increased phosphorylation of Akt and S6 ribosomal protein. However, in vitro treatment of LT-HSCs with mTORC1 inhibitor rapamycin did not resume decreased expression of LDHB, LDHD, Pdk2, and Pdk4, suggesting that elevated mTORC1 activity may not be the major contributor to the downregulation of glycolysis related genes. Meanwhile, we also found that in Cited2 KO LT-HSCs, phosphorylation of FoxO1 and FoxO3 was increased, both of which are known regulators of Pdk4 expression. Interestingly, in vitro treatment of LT-HSCs with PI3/Akt inhibitor LY294002, partially rescued the expression of Pdk4. Together, these findings suggest that the downregulation of Pdk4 in Cited2 KO HSCs is likely mediated by the inactivation of FoxOs caused by the elevated Akt activity. In summary, these results show that loss of Cited2 attenuates HSCs' glycolytic metabolism while simultaneously enhancing their overall mitochondrial oxidative phosphorylation, thus suggesting a critical role of Cited2 in the maintenance of adult HSC glycolytic metabolism likely through regulating LDH, Pdk, and Akt activity. Disclosures: No relevant conflicts of interest to declare.

Description: The clinical value of plasma Epstein-Barr virus (EBV) DNA has not been evaluated in patients with early-stage extranodal nasal-type NK/T-cell lymphoma (NKTCL) receiving primary radiotherapy. Fifty-eight patients with stage I disease and 11 with stage II disease were recruited. High pretreatment EBV-DNA concentrations were associated with B-symptoms, elevated lactate dehydrogenase levels, and a high International Prognostic Index score. EBV-DNA levels significantly decreased after treatment. The 3-year overall survival (OS) rate was 82.6% for all patients. Stage I or II patients with a pretreatment EBV-DNA level of ≤ 500 copies/mL had 3-year OS and progression-free survival (PFS) rates of 97.1% and 79.0%, respectively, compared with 66.3% (P = .002) and 52.2% (P = .045) in patients with EBV-DNA levels of 〉 500 copies/mL. The 3-year OS and PFS rates for patients with undetectable EBV-DNA after treatment was significantly higher than patients with detectable EBV-DNA (OS, 92.0% vs 69.8%, P = .031; PFS, 77.5% vs 50.7%, P = .028). Similar results were observed in stage I patients. EBV-DNA levels correlate with tumor load and a poorer prognosis in early-stage NKTCL. The circulating EBV-DNA level could serve both as a valuable biomarker of tumor load for the accurate classification of early-stage NKTCL and as a prognostic factor.

Description: Aberrant transcriptional programs play a critical role in the development of acute myeloid leukemias (AMLs). Although persistent over-expression of MEIS1 and HOXA9 has been shown to be essential for the initiation and maintenance of MLL-associated leukemia, it is still poorly understood what additional transcriptional regulators, independent of the MLL fusion-driven MEIS/HOX pathway, dictate the development of MLL leukemia. Considering that AMLs with MLL translocation are typically associated with the monocytic lineage (FAB M4 and M5), we explored the potential role of the monocytic lineage-specific transcriptional program in MLL leukemia. Using 97 genome-wide expression profiles of human MLL leukemias, we constructed an MLL distinctive transcriptional regulatory network. In addition to well-known transcriptional factors in leukemia development such as MEIS1 and HOXA family genes, we identified a highly active monocyte-specific gene signature that includes transcription factor PU.1. In our effort to determine the functional role of PU.1 in MLL leukemia, we found that lower PU.1 expression significantly delayed the onset of MLL- AF9 induced leukemia in primary bone marrow transplantation assay. MLL leukemia failed to maintain in vivo upon induced deletion of the PU.1 gene. To examine the clinical relevance of the PU.1 in AML patients, we further performed multivariate Cox proportional-hazards regression analysis in four published datasets of patients with AML, for whom gene expression and time-to-event data were available. We found that a PU.1-regulated 40-gene signature showed profound concordance with prognosis in segregating high-risk and low-risk AML patients. When specific subgroups of AMLs were examined, the PU.1 expression signature could predict patient outcome for MLL patients, but not in other major AMLs, such as t(8;21), t(15;17) and inv(16). We further explored the molecular mechanisms underlying the critical role of the PU.1 program in MLL leukemia. Functional annotation of this PU.1 expression signature identified the MEIS/HOX pathway (MEIS1, FLT3, KIT), as well as key genes in the inflammatory response (AIF1, NF-KB1 and CD180). We showed that PU.1 is required to maintain high expression of Meis1 and Pbx3 and also important downstream genes in the MEIS/HOX pathway that includes known MEIS/HOX targets c-Kit and Flt3. Using ChIP-sequencing, we demonstrated that PU.1 interacts with the MEIS/HOX regulatory program through co-binding with MEIS1 at the target genomic regions in a MLL-ENL cell line. In our effort to determine the role of PU.1-controlled inflammatory response genes, we found that the growth inhibition in PU.1 knockdown MLL leukemic cells was partially rescued by addition of the monocytic inflammatory cytokine AIF1. AIF1 provides an anti-apoptotic effect through activation of the NF-ƒÛB pathway and additional known apoptosis regulators. Interestingly, AML patients with higher expression of both AIF1 and MEIS1 had a significantly shorter overall survival time than those with lower expression of both genes. Patients with high expression of either MEIS1 or AIF1 had medium survival possibilities. Notably, the prognostic value of AIF1 and MEIS1 remained in those with monocytic AMLs (P=0.00079), but not in the non- monocytic group of patients (p=0.105). Collectively, these results strongly suggest that the monocyte-specific inflammatory cytokine AIF1 is an MEIS/HOX independent essential regulator in monocytic AMLs such as MLL leukemia. Loss of function PU.1 is leukemogenic in mouse models. Suppression of PU.1 activity is also required for the development of human myelocytic M2/M3 leukemia. Here we reveal a converse role for PU.1 as an essential positive regulator in the development of MLL myeloid leukemia, mostly M4/M5 monocytic AMLs. Our study demonstrats that the monocyte-specific PU.1-driven transcriptional program independently contributes to the development of myeloid MLL leukemia, in parallel with the MLL fusion pathway. PU.1 and downstream macrophage specific inflammatory cytokine AIF1 have important prognostic value and may serve as novel therapeutic targets for MLL leukemias. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3249 We have previously shown that the 150 kDa semaphorin family member, Sema4D (or CD100), is expressed on the surface of human and mouse platelets, where it is able to selectively reinforce collagen-initiated platelet activation by engaging receptors in trans on adjoining platelets in a contact-dependent manner. Key to this effect is the Sema4D extracellular domain, which in addition to being a ligand for Sema4D receptors, is a substrate for the metalloprotease, ADAM17 in platelets. Our previous studies suggest that ADAM17 cleaves Sema4D and other platelet surface proteins close to the platelet plasma membrane gradually producing, in the case of Sema4D, a single large (≈120 kDa) exodomain fragment and a smaller (≈28 kDa) fragment that includes the transmembrane domain and the cytoplasmic domain and remains associated with the platelet (Zhu, et al., PNAS 2007). Exodomain shedding in platelets can be triggered by the phorbol ester, PMA, and by physiologic agonists such as thrombin that raise the cytoplasmic Ca++ concentration, but the mechanisms that regulate the shedding of Sema4D have not been defined. Here we have studied the potential role of an interaction between calmodulin and the Sema4D cytoplasmic domain. Using a public resource (http://calcium.uhnres.utoronto.ca/ctdb/ctdb/home.html), we identified a potential calmodulin-binding sequence (GYLPRQCLKFRSALLIGKKKPKS-COOH, Gly758–Ser780) within the membrane-proximal region of the Sema4D cytoplasmic domain. To test whether this region binds calmodulin, a 23 amino acid peptide corresponding to the predicted Sema4D calmodulin binding sequence (SCBP) was synthesized, as was a scrambled control peptide (RLIKACRQPKPKYKLLGFGSSKL or scrambled SCBP), which is not predicted to bind calmodulin. The results show that SCBP, but not scrambled SCBP, was able to bind to calmodulin-agarose and retrieve calmodulin from platelet lysates. As constitutive association of calmodulin with glycoprotein (GP) Ib has been shown prevent ADAM17-dependent GPIb alpha shedding in platelets, we incubated human platelets with the calmodulin inhibitor, W7. The inhibitor induced gradual Sema4D shedding that was detectable after 5 min and reached a maximum at 60 min, kinetics that are similar to those we have observed with platelet agonists. However, in contrast to platelet agonists, W7-induced Sema4D shedding generated a smaller retained fragment (≈24 kDa Vs. 28 kDa) suggesting that there is either a second or different site of cleavage. Despite their polybasic sequences, flow cytometry and confocal microscopy showed that FITC-conjugated SCBP and scrambled-SCBP are able to cross the plasma membrane. Addition of SCBP, but not scrambled-SCBP, to platelet caused cleavage of Sema4D, producing the same 28 kDa fragment observed with thrombin and PMA. In all cases cleavage of Sema4D was blocked by the metalloprotease inhibitor, TAPI-2. Combined with our earlier observations, these results suggest that 1) Sema4D is a calmodulin binding protein with a site of interaction in the membrane-proximal cytoplasmic domain and a site of cleavage by ADAM17 in the membrane-proximal exodomain, 2) the detachment of calmodulin from Sema4D may be the trigger for Sema4D cleavage in response to platelet agonists, and 3) in contrast to W7, decoying calmodulin from binding sites on Sema4D and other metalloprotease substrates on the platelet surface, as we have done here with a Sema4D cytoplasmic domain peptide, may trigger the same events seen in activated platelets and provide a tool to understand the underlying mechanisms. Disclosures: No relevant conflicts of interest to declare.

Description: Cited2 is a transcriptional modulator involved in various biologic processes including fetal liver hematopoiesis. In the present study, the function of Cited2 in adult hematopoiesis was investigated in conditional knockout mice. Deletion of Cited2 using Mx1-Cre resulted in increased hematopoietic stem cell (HSC) apoptosis, loss of quiescence, and increased cycling, leading to a severely impaired reconstitution capacity as assessed by 5-fluorouracil treatment and long-term transplantation. Transcriptional profiling revealed that multiple HSC quiescence- and hypoxia-related genes such as Egr1, p57, and Hes1 were affected in Cited2-deficient HSCs. Because Cited2 is a negative regulator of HIF-1, which is essential for maintaining HSC quiescence, and because we demonstrated previously that decreased HIF-1α gene dosage partially rescues both cardiac and lens defects caused by Cited2 deficiency, we generated Cited2 and HIF-1α double-knockout mice. Additional deletion of HIF-1α in Cited2-knockout BM partially rescued impaired HSC quiescence and reconstitution capacity. At the transcriptional level, deletion of HIF-1α restored expression of p57 and Hes1 but not Egr1 to normal levels. Our results suggest that Cited2 regulates HSC quiescence through both HIF-1–dependent and HIF-1–independent pathways.

Description: Abstract 3466 Down-regulation of transcription factor PU.1, a key regulator of hematopoiesis, induces myeloid leukemia in mice, demonstrating a role of PU.1 as tumor suppressor. Recent studies, however, have also suggested that PU.1 is required for repopulation/self-renewal capacity of normal hematopoietic stem cells (HSCs), and presence of PU.1 activity may be necessary to favor growth of myeloid leukemia stem cells. To explore whether PU.1 could possibly act as an oncogene in the development of certain type of myeloid leukemia, we set to look for differential up-regulation of PU.1 among AML patients with distinct cytogenetic and genetic alterations in public databases. Consistent with recent molecular studies showing suppression of PU.1 expression by AML1-ETO and PML-RARa fusion proteins, PU.1 expresses at a significant lower level in AML patients with t(8;21) and t(15;17) translocations. In contrast, PU.1 expression level in MLL leukemia patients is significantly higher than that of other subgroups of AML. In addition, we found that a set of PU.1 direct target genes, as defined by genome wide location analysis of this factor, expresses at higher level in MLL leukemia patients comparing with those with t(8;21) and t(15;17) translocations, supporting an increased PU.1 activity in this subgroup of leukemia. In our effort to characterize the functional consequence of high expression of PU.1 in AML, we found that PU.1 plays an essential role in activation of MEIS1, an oncogene essential for MLL leukemia stem cell potential, and in development of MLL fusion leukemia. MEIS1, as PU.1, is differentially up-regulated in MLL leukemia patients, and expresses at a significant lower level in AML patients with t(8;21) and t(15;17) translocations. Among AML patients with higher level MEIS1 expression, a positive correlation was observed between expression of PU.1 and that of MEIS1. Using promoter reporter assay, electro mobility shift assay (EMSA) and chromatin immunoprecipiation (ChIP) analysis, we found that PU.1 directly binds to and activates MEIS1 promoter in vitro and in vivo. Analysis of a hypomorphic PU.1 mouse model indicated that PU.1 is required to maintain Meis1 expression in murine HSCs and progenitors, and knockdown of PU.1 in patient-derived MLL leukemia cell lines resulted in lower enrichment of PU.1 protein at MEIS1 promoter, accompanied by down-regulation of MEIS1 expression and decreased proliferation and survival of these cells. We are now examining whether the ability of MLL-AF9 fusion protein to drive leukemia is compromised in PU.1-deficient mouse HSC/HPCs, and whether introduction of exogenous Meis1 can compensate for the loss of PU.1 in the development of MLL-AF9 leukemia in mouse bone marrow transplantation model. Finally, we are also testing knock-down of PU.1 as a therapeutic approach to primary AMLs isolated from MLL leukemia patients. Collectively, our data indicate that PU.1 is required for the pathogenesis of MLL associated leukemia, at least partially, through direct activation of MEIS1. In veiw of the dependency of MEIS1 in MLL leukemic transformation, targeting PU.1 mediated MEIS1 gene activation could be an alternative or synergistic approach for MLL leukemia therapies aimed at inhibition of DOT1L and HOXA9. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 912 Hematopoietic stem cells (HSCs) are thought to be localized in hypoxic microenvironment of the bone marrow (BM) and can remain quiescent or differentiate into multiple blood cell lineages. A number of factors have been found to regulate HSC quiescence in either cell-intrinsic or cell-extrinsic manner. Cited2 (CBP/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2), a member of a newly identified transcriptional modulator, is a cytokine inducible gene and plays various roles during mouse development. In particular, Cited2 is essential for fetal liver hematopoiesis. In this study, we used conditional knockout strategy to delete Cited2 in order to further investigate its function in adult hematopoiesis. Sequential injection of poly(I)-poly(C) (pI-pC) efficiently deleted the Cited2 gene in Cited2fl/fl;Mx1-Cre mice. In this mouse model (Cited2−/− mice), the white blood cell (WBC) count, BM cellularity and Lin−Sca-1+c-Kit+ (LSK) cell number were within the normal range. However, the long-term HSC (LT-HSC; defined as Flt3−CD34−LSK or CD48−CD150+LSK) frequency was significantly decreased. Cited2−/− mice also exhibited increased apoptosis in both LSK and CD34−LSK cells. In addition, Cited2 deficiency led to loss of HSC quiescence evidenced by cell cycle analysis and BrdU incorporation assay. HSC reconstitution capacity was significantly impaired assessed by 5-fluorouracil (5-FU) treatment and transplantation experiments. Transcriptional profiling revealed that multiple HSC quiescence and hypoxia related genes were affected, including p57, Hes1 and Egr1. Recent studies have shown that both HIF-1α-deficient and HIF-1α-stabilized HSCs result in impaired hematopoietic reconstitution, suggesting that precise regulation of HIF-1α level is essential for maintaining HSC quiescence and transplantation activity. Cited2 has been shown to be a negative regulator for HIF-1α through competitive binding to CBP/p300 with higher affinity. In addition, we previously showed that HIF-1α haploinsufficiency (HIF-1α+/−) partially rescues the heart defects in Cited2−/− embryos and HIF-1α deletion (HIF-1α−/−) rescues aberrant vasculature in Cited2−/− embryonic lens. These findings prompted us to explore whether defects of Cited2−/− HSC in adult mice are mediated by dysregulated HIF-1 activity by generating Cited2fl/flHIF-1αfl/fl;Mx1-Cre mice. Additional deletion of HIF-1α partially rescued impaired HSC quiescence and reconstitution capacity caused by Cited2 deficiency. Cited2−/− HIF-1α−/− HSCs displayed comparable apoptosis to Cited2−/− counterparts. At the transcriptional level, deletion of HIF-1α restored expression of p57 and Hes1 but not Egr1 to a normal level. Taken together, these results suggest that Cited2 regulates HSC quiescence through HIF-1 dependent and independent pathways. Disclosures: No relevant conflicts of interest to declare.