NF-E2 Expression Research Articles

Identification of Nfel1a and Nfel3 as novel regulators for zebrafish thrombopoiesis

In mammalian hematopoiesis, megakaryocytes mature and become polyploid in the bone marrow before releasing platelets into circulation. In contrast, fish produce thrombocytes in kidney marrow, where young thrombocytes undergo maturation in circulation, akin to platelets. Despite morphological differences, our single-cell sequencing revealed significant gene expression similarities between fish thrombocytes and mammalian megakaryocytes, including Nfe2, which is crucial in platelet production. In addition to nfe2 expression, we found four nfe2-related genes, nfe2I1a, nfe2I1b, nfe2I2a, and nfe2I3, were expressed in mature thrombocytes. However, only nfe2, nfe2l2a, and nfe2l3 are expressed in young thrombocytes. Thus, we hypothesized that Nfe2-related factors may also be involved in thrombocyte production. To address this, we knocked down the four novel nfe2-related genes by the piggyback method and found both young and mature thrombocytes reduced when nfe2, nfe2l1a, and nfe2l3 were knocked down. They also exhibited greater gill bleeding and prolonged time to occlusion (TTO) compared to controls. In summary, our study shows Nfe2I1a and Nfe2l3 as novel transcription factors that are positive regulators influencing adult zebrafish thrombopoiesis.

Genistein in combination with Chlorogenic acid ameliorates insulin resistance and oxidative stress in skeletal muscle of high-fat diet fed mice

Objectives: Increasing evidence has demonstrated that oxidative stress plays a significant role in the pathogenesis of muscle insulin resistance. The beneficial impacts of genistein (GEN) and chlorogenic acid (CGA) on insulin resistance have already been revealed. However, their combined effects on skeletal muscle oxidative stress and insulin resistance have not been completely understood. The aim of the present study was to determine the effect of GEN in combination with CGA on skeletal muscle insulin resistance in high-fat diet (HFD) fed C57BL/6 mice.
 Methods: C57BL/6 male mice were fed an HFD for 15 weeks. The mice were then divided into five groups: standard chow diet (SCD), HFD, HFD + GEN, HFD + CGA, and HFD + GEN + CGA for 10 weeks.
 Results: The findings indicated that single treatment with GEN or CGA, and with a stronger effect of GEN+CGA combined treatment, decreased body weight gain and improved glucose intolerance. Moreover, following treatment with GEN and CGA alone or in combination with further effect, the level of plasma and muscle triglyceride (TG) were reduced. Furthermore, the combination therapy of GEN and CGA with greater efficacy than the single treatments, could decrease several oxidative stress markers in the skeletal muscle. Treatment with GEN and CGA alone or in combination could increase the expression of NF-E2–related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NAD(P): Quinone Oxidoreductase 1 (NQO1).
 Conclusion: The data of this study suggest that the combination of GEN and CGA might ameliorate insulin resistance and reduce oxidative stress in the skeletal muscle of mice fed HFD.

Identification of transcription factors related to diabetic tubulointerstitial injury

BackgroundDiabetic nephropathy (DN) is a main cause of chronic renal failure. Despite decades of extensive study, the molecular mechanisms underlying diabetic tubulointerstitial injury remain unclear. We aim to identify key transcription factor genes involved in diabetic tubulointerstitial injury.MethodsA microarray dataset (GSE30122) from Gene Expression Omnibus (GEO) was downloaded. A total of 38 transcription factor genes based on 166 differentially expressed genes (DEGs) were identified by UCSC_TFBS.ResultsThe regulatory network showed connections between the top 10 transcription factors and their target DEGs. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of targeted DEGs indicated that extracellular space, extracellular exosome, cell surface and complement and coagulation cascades were most significantly enriched. Utilizing Nephroseq v5 online platform, the mRNA expression pattern analysis of transcription factor genes demonstrated that mRNA expression of CDC5, CEBPA, FAC1, HFH1, IRF1, NFE2 and TGIF1 increased in renal tubulointerstitium of DN patients compared with normal controls while that of CEBPB and FOXO4 decreased in renal tubulointerstitium of DN patients compared with normal controls. Correlation analysis between mRNA expression of transcription factor genes in renal tubulointerstitium and clinical features showed that AP1, BACH1, CDC5, FAC1, FOXD1, FOXJ2, FOXO1, FOXO4, HFH1, IRF1, POU3F2, SOX5, SOX9, RSRFC4, S8 and TGIF1 may be related to diabetic tubulointerstitial injury.Conclusions(1) CDC5, FAC1, FOXO4, HFH1, IRF1 and TGIF1 may be key transcription factor genes. (2)Transcription factors involved in diabetic tubulointerstitial injury may become prospective targets for diagnosis and treatment of DN.

Weighted gene co-expression network analysis identifies key hub genes and pathways in acute myeloid leukemia.

Introduction: Acute myeloid leukemia (AML) is the most common type of leukemia in adults. However, there is a gap in understanding the molecular basis of the disease, partly because key genes associated with AML have not been extensively explored. In the current study, we aimed to identify genes that have strong association with AML based on a cross-species integrative approach. Methods: We used Weighted Gene Co-Expression Network Analysis (WGCNA) to identify co-expressed gene modules significantly correlated with human AML, and further selected the genes exhibiting a significant difference in expression between AML and healthy mouse. Protein-protein interactions, transcription factors, gene function, genetic regulation, and coding sequence variants were integrated to identify key hub genes in AML. Results: The cross-species approach identified a total of 412 genes associated with both human and mouse AML. Enrichment analysis confirmed an association of these genes with hematopoietic and immune-related functions, phenotypes, processes, and pathways. Further, the integrated analysis approach identified a set of important module genes including Nfe2, Trim27, Mef2c, Ets1, Tal1, Foxo1, and Gata1 in AML. Six of these genes (except ETS1) showed significant differential expression between human AML and healthy samples in an independent microarray dataset. All of these genes are known to be involved in immune/hematopoietic functions, and in transcriptional regulation. In addition, Nfe2, Trim27, Mef2c, and Ets1 harbor coding sequence variants, whereas Nfe2 and Trim27 are cis-regulated, making them attractive candidates for validation. Furthermore, subtype-specific analysis of the hub genes in human AML indicated high expression of NFE2 across all the subtypes (M0 through M7) and enriched expression of ETS1, LEF1, GATA1, and TAL1 in M6 and M7 subtypes. A significant correlation between methylation status and expression level was observed for most of these genes in AML patients. Conclusion: Findings from the current study highlight the importance of our cross-species approach in the identification of multiple key candidate genes in AML, which can be further studied to explore their detailed role in leukemia/AML.

Hirsutine, a novel megakaryopoiesis inducer, promotes thrombopoiesis via MEK/ERK/FOG1/TAL1 signaling

BackgroundThrombocytopenia (TP) remains a challenge in clinical hematology. TP may have serious consequences, such as recurrent skin and mucosal bleeding and increased risk of intracranial and internal organ hemorrhage. However, effective and safe therapeutic drugs for the long-term management of TP are still lacking. PurposeThis study aimed to identify more effective active compounds for TP therapy. MethodsLiquid chromatography-mass spectrometry-nuclear magnetic resonance analysis was used to confirm the medicinal species and chemical structure of Hirsutine (HS). The proliferation of HS was examined by Cell Counting Kit (CCK-8) assay on cells lines. The effect of HS on megakaryocyte differentiation was analyzed by evaluating the expression of CD41, CD42b, and DNA ploidy via flow cytometry (FCM). The morphology of megakaryocytes and intermediate cells was observed using an optical microscope. K562 cells were then stained with Giemsa and benzidine. qRT-PCR was used to examine the mRNA expression of GATA-1, GATA-2, FOG-1, TAL-1, RUNX-1, NF-E2, and KLF-1 in K562 cells. Protein levels of the transcription factors were analyzed by western blotting. An MEK inhibitor was used to verify the relationship between the MEK/ERK signaling pathway and CD41/CD42b (FCM), FOG-1, and TAL-1. The Kunming thrombocytopenia mouse model was established by X-ray irradiation (4 Gy) and used to test HS activity and related hematopoietic organ index in vivo. Finally, computer simulations of molecular docking were used to predict the binding energies between HS-MEK and HS-ERK. ResultsWe preliminarily identified HS by screening a plant-sourced compound library for natural compounds with megakaryocytic differentiation and maturation (MKD/MKM)-promoting activity. We found that HS not only enhanced MKD/MKM of K562 and Meg01 cells, but also suppressed the decline of peripheral platelet levels in X-ray-induced myelosuppressive mice. In addition, HS promoted MKD via activation of MEK-ERK-FOG1/TAL1 signaling, which may be the key molecular mechanism of HS action in TP treatment. Molecular docking simulations further verified that HS targets the signaling protein MEK with high-affinity. ConclusionIn this study, we report for the first time that hirsutine boosts MKD/MKM through the MEK/ERK/FOG1/TAL1 signaling pathway and thus represents a promising treatment option for TP.

Brg1 mutation alters oxidative stress responses in glioblastoma

Increasing evidences suggest that the SWI/SNF chromatin remodeling complex involved in the organization of chromatin architecture via ATP hydrolysis, plays an important role in human cancer. As TCGA gene expression analyses revealed signature of enhanced oxidative stress in GBMs harbouring Brg1mutations, we examined the involvement of ATPase subunit of BRG1 in regulating oxidative stress responses in glioma. BRG1-MUT overexpressing glioma cells exhibit intrinsically higher reactive oxygen species (ROS) levels as compared to BRG1-WT. Elevated ROS generation was concomitant with decreased expression of NF-E2– related factor 2 (NRF2), superoxide dismutases (SOD-1,2) and thioredoxins (TrX-1,2). A similar change in redox regulatory genes and ROS production was observed upon siRNA-mediated knockdown of Brg1. Increased sensitivity to temozolomide was observed upon loss of BRG1-ATPase catalytic domain. These findings highlight the role of ATPase domain of BRG1 in regulating redox homeostasis and sensitivity to oxidative stressors in glioma cells. BRG1 mutation created vulnerability to elevated ROS levels can be therapeutically exploited, with ROS stressors as a promising therapeutic target for the treatment of BRG1-mutant cancers.

TMEA, a Polyphenol in Sanguisorba officinalis, Promotes Thrombocytopoiesis by Upregulating PI3K/Akt Signaling.

Thrombocytopenia is closely linked with hemorrhagic diseases, for which induction of thrombopoiesis shows promise as an effective treatment. Polyphenols widely exist in plants and manifest antioxidation and antitumour activities. In this study, we investigated the thrombopoietic effect and mechanism of 3,3′,4′-trimethylellagic acid (TMEA, a polyphenol in Sanguisorba officinalis L.) using in silico prediction and experimental validation. A KEGG analysis indicated that PI3K/Akt signalling functioned as a crucial pathway. Furthermore, the virtual molecular docking results showed high-affinity binding (a docking score of 6.65) between TMEA and mTOR, suggesting that TMEA might target the mTOR protein to modulate signalling activity. After isolation of TMEA, in vitro and in vivo validation revealed that this compound could promote megakaryocyte differentiation/maturation and platelet formation. In addition, it enhanced the phosphorylation of PI3K, Akt, mTOR, and P70S6K and increased the expression of GATA-1 and NF-E2, which confirmed the mechanism prediction. In conclusion, our findings are the first to demonstrate that TMEA may provide a novel therapeutic strategy that relies on the PI3K/Akt/mTOR pathway to facilitate megakaryocyte differentiation and platelet production.

Pro-Inflammatory Cytokine IL-1b on Megakaryopoiesis: A Possible Explanation of Thrombocytosis in Inflammation

Pro-inflammatory cytokine interleukin-1b (IL-1b) is multifunctional and exerts its effects on a wide variety of cell types. Among many functions, IL-1b enhances the hematopoietic system by inducing T and B cell proliferation and works in combination with other cytokines on early stem and progenitor cell expansion and differentiation. Megakaryopoiesis and platelet production is maily controlled by thrombopoietin (TPO, Mpl-ligand), which is a growth factor for the megakaryocyte/ platelet lineage. Other early acting cytokines including IL-1, IL-3, IL-6 and IL-11 are also involved in the regulation of this lineage at different degree. Thrombocytosis is observed in various inflammation diseases. IL-1b, as a highly pro-inflammatory cytokine, induces thrombocytosis in mice. However, its mechanism on megakaryopoiesis is still not well understood. Our hypothesis is that IL-1b possesses a direct stimulatory effect on megakaryocytes via its receptors and the transcription factor NF-E2. The expression of IL-1 type I (IL-1 RI) and type II receptors was studied in megakaryotic cell lines: Meg-01, Dami, CHRF-288-11 and M-07e by RT-PCR, southern blot hybridization, flow cytometry or immuno-fluorescent staining. The effect of IL-1b on the formation of megakaryocytic colonies from human and murine stem and progenitor cells was investigated by the plasma clot culture method. The expression of the transcription factor p45 NF-E2, in response to IL-1b, was analyzed by Western blot. Our results show that IL-1 RI and IL-1 RII were expressed in the four human megakaryocytic cells. IL-1b alone or in combination with TPO induced megakaryocyte colony forming units (CFU-MK) in the culture system. The mitogenic effect of IL-1b on the enhancement of TPO-induced megakaryopoiesis was stronger than that of IL-3, IL-6, Flt-3 ligand and stem cell factor. The expression of NF-E2 was demonstrated in all four cell lines. In Meg-01 cells, the expression of NF-E2 was increased at both transcriptional and translational level after treatment with IL-1b for 4 hours. These observations strengthen the evidence of the direct effect of IL-1b on megakaryopoiesis. Our findings also provide a possible explanation of thrombocytosis during inflammation conditions. DisclosuresNo relevant conflicts of interest to declare.

Aberrant Level of Estrogen Correlates with Defective Thrombopoiesis during Pregnancy in Patients with Immune Thrombocytopenia

AbstractPregnancy seems to be a high-risk event in immune thrombocytopenia(ITP)because disease flares are frequently observed during gestation clinically, and the treatment with glucocorticoid is often ineffective. Sex hormones are changed dramatically during pregnancy to maintain a stable environment both for the mother and for the fetus. To determine the sex hormone levels during pregnancy in ITP, sex hormones were analyzed in patients with ITP and matched healthy women before, during, and after pregnancy. We demonstrated that there is a relationship between estrogen level and thrombopoiesis in ITP pregnancy. Moreover, we showed that estrogen plays a role in glucocorticoid resistance which is common in ITP patients who are pregnant.Thirty consecutive successful pregnancies in 30 patients with ITP, 21 pregnancies in 21 healthy controls, twenty patients with primary ITP and 19 healthy subjects were studied. Serum samples for sex hormone testing, including estrogen, progesterone, testosterone, prolactin, LH and FSH, were collected and analyzed. We confirmed that serum levels of all sex hormones varied significantly during pregnancy and postpartum, both in the patients with ITP and in healthy controls, as expected. However, the concentration of estrogen was significantly lower in pregnant ITP patients, compared with healthy pregnant subjects, leading to a completely different profile.To clarify whether the different estrogen concentration has a direct effect on megakaryocyte(MK) maturation and thrombopoiesis during pregnancy in ITP, BM-derived CD34+ cells from ITP patients and healthy controls in pregnancy, were treated with different doses of estrogen. We found that estrogen had the ability to promote MK polyploidization, maturation and proplatelet formation via estrogen receptor(ER) in healthy controls, but the concentration of estrogen, which was lower than that of the normal-pregnancy group, could not play the above role in CD34+ cells of pregnancy-ITP group. To determine the differences in MKs treated with estrogen of different doses in each group, we screened some essential transcription factors that may participate in the regulation of MK polyploidization and maturation. The expression levels of GATA1, FLI1, FOG-1, KLF-1, and NF-E2, were detected by RT-PCR and Western blot. We found that, in the pregnancy-ITP group treated with lower estrogen, the expression of GATA1 and NF-E2 was significantly reduced compared with the healthy control. We obtained similar results with the bone marrow samples, indicating that pregnant ITP patients showed more obvious detects in megakaryocyte maturation defect and lower expression of transcription factors. Further investigation is needed to evaluate the expression of ER and its nuclear translocation in MKs of the pregnancy-ITP group treated with the relatively lower level of estrogen.We also explored if the relatively reduced estrogen level plays a role in glucocorticoid resistance which is commonly observed in ITP patients during pregnancy. We evaluated the inhibitory effects of dexamethasone, with or without different concentrations of estrogen, on the proliferation of PBMCs from the pregnancy-ITP group induced by a mitogen. We found that estrogen impaired the inhibitory effects of dexamethasone. Further study with immunofluorescence demonstrated that estrogen upregulated the expression of protein phosphatase, which affected the glucocorticoid receptor activation.In conclusion, an unexpected lack of estrogen serum level increase occurs in patients with ITP during pregnancy. The relatively reduced level of estrogen cannot induce the expression of GATA1 and NF-E2 via ER in pregnant ITP patients, which is required for MK maturation and thrombopoiesis. Meanwhile, setrogen inhibits glucocorticoid action via protein phosphatase, which may account for the glucocorticoid resistance observed in pregnant ITP patients. DisclosuresNo relevant conflicts of interest to declare.

Involvement of a Transcription factor, Nfe2, in Breast Cancer Metastasis to Bone.

Simple SummaryBreast cancer patients are frequently complicated by bone metastasis, which deteriorates their life expectancy. Bone metastasis is treated with the drugs targeting osteoclast activation, which is mostly observed at the metastasis sites. However, these drugs cannot directly inhibit cancer cell growth and therefore, a novel therapeutic strategy is required to impede cancer cell proliferation at bone metastasis sites. Here, we proved that a transcription factor, NFE2, was expressed selectively in breast cancer cells at bone metastasis sites and contributed crucially to their enhanced proliferation therein, by activating Wnt pathway. Thus, NFE2 can be a novel molecular target to treat breast cancer bone metastasis.Patients with triple negative breast cancer (TNBC) is frequently complicated by bone metastasis, which deteriorates the life expectancy of this patient cohort. In order to develop a novel type of therapy for bone metastasis, we established 4T1.3 clone with a high capacity to metastasize to bone after orthotopic injection, from a murine TNBC cell line, 4T1.0. To elucidate the molecular mechanism underlying a high growth ability of 4T1.3 in a bone cavity, we searched for a novel candidate molecule with a focus on a transcription factor whose expression was selectively enhanced in a bone cavity. Comprehensive gene expression analysis detected enhanced Nfe2 mRNA expression in 4T1.3 grown in a bone cavity, compared with in vitro culture conditions. Moreover, Nfe2 gene transduction into 4T1.0 cells enhanced their capability to form intraosseous tumors. Moreover, Nfe2 shRNA treatment reduced tumor formation arising from intraosseous injection of 4T1.3 clone as well as another mouse TNBC-derived TS/A.3 clone with an augmented intraosseous tumor formation ability. Furthermore, NFE2 expression was associated with in vitro growth advantages of these TNBC cell lines under hypoxic condition, which mimics the bone microenvironment, as well as Wnt pathway activation. These observations suggest that NFE2 can potentially contribute to breast cancer cell survival in the bone microenvironment.

Abstract 1742: Inhibition of BET signaling leads to reversible GATA1-associated repression of hematopoietic progenitors: translation from preclinical assessment to clinical development

Abstract Bromodomain and extraterminal (BET) proteins recognize acetylated lysine residues for the purpose of transcriptional regulation of genes, including those involved in stem cell renewal and oncogenes such as MYC. Inhibition of BET proteins BRD2, BRD3, BRD4, and BRDT represents a promising treatment option for patients with cancer. However, little is known about the consequence of BET inhibition on normal stem cell renewal processes, such as hematopoiesis. This study explored the mechanistic effects of BET inhibition on bone marrow (BM) hematopoiesis. Rats treated with BET inhibitors JQ1, BMS-X, or BMS-986158 for 4 days in vivo showed dose-dependent pan-cellular BM atrophy and reduction of hematopoietic progenitors of myeloid and erythroid lineage (combination of anti-rat CD45, CD11b, anti-granulocyte, CD71, anti-erythroid, and CD90 via flow cytometry) and consequential reductions in circulating platelet and reticulocyte counts, with complete reversibility within 10 days of stopping treatment with BET inhibitors. Primary rat BM stem and progenitor cells treated with BET inhibitors in vitro were evaluated with the colony-forming unit assay and resulted in dose-dependent reduction of multiple lineage progenitor colonies, especially the erythroid and megakaryocyte lineages. To further elucidate pathways involved in BET-related BM atrophy, erythropoiesis and thrombopoiesis genes regulated by GATA1, a BRD-associated transcription factor, from rat BM, as well as rat and human whole-blood samples exposed to BET inhibitor(s) were evaluated via RNAseq and RT-PCR. Dose-dependent responses in genes involved in erythropoiesis (Alas2, ABCme, PBG-D, HMBS) and thrombopoiesis (NFE2, PF4, GP1Bb, MPL) were observed after 4 days of treatment with BMS-986158. In a clinical trial (NCT02419417), patients with solid tumors treated with BMS-986158 demonstrated reversible thrombocytopenia and downregulation of NFE2, PF4, and HMBS expression, similar to that observed in rats. GATA1 was also downregulated in rat BM, with target engagement in rat and human demonstrated by the induction of HEXIM1 transcription, a pharmacodynamic (PD) biomarker of growth inhibition and apoptosis induced by BRD4 inhibition. Overall, our results suggest inhibition of BET signaling causes target-related, dose-dependent repression of hematopoietic progenitors through alterations of GATA1-associated erythropoiesis and thrombopoiesis regulation in rat BM and human blood samples, and these effects are reversible on cessation of BET inhibitor treatment. This is the first in vivo study demonstrating the mechanism of BET inhibition resulting in GATA1-associated repression of hematopoietic progenitors that is correlated to clinical pharmacokinetics and PD (Chen X, et al. AACR 2020) and is translatable from preclinical evaluation to clinical experience. Citation Format: Cindy Zhang, Ke Xu, Julie Panzica-Kelly, Jennifer Price, Denise Bounous, Shodeinde Coker, Kezi Unsal-Kacmaz, Danielle Greenawalt, Ashvinikumar Gavai, Ronald Fleming, Karen Augustine-Rauch, Richard Westhouse. Inhibition of BET signaling leads to reversible GATA1-associated repression of hematopoietic progenitors: translation from preclinical assessment to clinical development [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1742.

473-P: HDAC Inhibition Augments TGF-ß-Induced JNK Activation, CTGF Expression, and NF-E2 Degradation Promoting Profibrotic Signaling in Renal Proximal Tubules

The role of TGF-β in diabetes-induced renal fibrosis remains incompletely understood. We showed decreased NFE2 expression in TGFβ-treated renal proximal tubule cells and in diabetic mouse kidneys. TGF-β treatment increased NFE2 mRNA level in HK11 cells suggesting a role for NFE2 degradation. ERK and p38 MAPKs promoted TGFβ-induced NFE2 degradation, CTGF and FN expression. NFE2 over-expression/silencing inhibited exacerbated TGFβ-induced CTGF. Proteasome inhibitor MG132 preserved TGFβ-induced NFE2 expression and inhibited CTGF and FN expression. Current study examined role of JNK MAPK and HDAC inhibition on NFE2 and CTGF expression in TGF-β treated HK-11 cells and effects of MG132 on JNK activation in OVE26 mouse kidneys and in TGFβ-treated HK-11 cells.HK11 cells were pre-treated with/without JNK inhibitor SP600125, HDAC inhibitor trichostatin A, and MG132, for an hour prior to treatment with TGFβ for 24 h.JNK blockade inhibited TGFβ-induced CTGF expression but not NF-E2 expression. MG132 blocked TGFβ-induced JNK activation in HK11 cells and in OVE26 mice. HDAC inhibition exacerbated TGFβ-induced NFE2 degradation and induced CTGF expression. ERK, p38 and JNK pathways contribute to enhanced CTGF expression but only p38 and ERK MAPK pathways contribute to NFE2 degradation. MG132 blocked TGFβ-induced JNK activation, suggesting that proteasome function is necessary for JNK activation and therefore, blocking JNK had no effect on preserving NF-E2 expression. Blocking HDAC activation possibly promoted HAT activity, NFE2 acetylation and enhanced degradation. Thus, proteasome inhibition plus TGFβ preserved NFE2 expression and prevented JNK activation and CTGF. HDAC inhibition plus TGFβ exacerbated NFE2 degradation and enhanced JNK activation and CTGF. We identified NFE2 as a potential anti-fibrotic protein in the kidney. Disclosure J. Li: None. M. Rane: None. L. Cai: None. Y. Tan: None.

Defective Platelet Thromboxane A2 Signaling and Serotonin Release in the Pathogenesis of Bleeding during Viral Infection

Viral hemorrhagic fever (VHF) refers to a group of diverse acute human diseases caused by single stranded RNA viruses typically associated with fever, vascular damage and abnormal vascular barrier function. These infections are a continuing threat to public health due to the high case-fatality rate and potential bioterrorism threats. Critically infected VHF patients with unfavorable prognosis often present bleeding symptoms and shock. Thrombocytopenia associated with endothelial, coagulation and platelet dysfunction has been reported during these infections, but their relationship to bleeding is still poorly defined. We have previously established that infection by lymphocytic choriomeningitis virus (LCMV) in extremely thrombocytopenic mice causes severe mucocutaneous bleeding and death mediated by interferon α/β receptor (IFNAR) signaling. We now show in vivo that IFNAR is expressed on megakaryocytes but not on circulating platelets, contrary to previously reported evidence. To explain how increased IFN-I in LCMV-infected mice alters the function of platelets lacking IFNAR, we cross-transplanted WT or IFNAR-/- bone marrow (BM) cells into IFNAR-/- and WT mice. Upon platelet depletion and LCMV infection, only chimeras with WT bone marrow died, suggest that IFN-I-sensitive bone marrow-derived cells are responsible for the mucocutaneous bleeding observed in conditions of extreme thrombocytopenia. Moreover, the results of BM transplantation studies prove that endothelial cell alterations and infection-related inflammatory conditions are not alone sufficient to cause hemorrhage. Circulating platelets during LCMV infection show a generalized aggregation disfunction that persists longer for the arachidonic acid/thromboxane A2 (AA/TBXA2) activation pathway. These aggregation defects are associated, and possibly linked to an impaired secretory ability of both α- and δ-granule. Since circulating platelets are unresponsive to IFN-I, these findings are consistent with the possibility that bone marrow-resident megakaryocytes are targets of IFN-I stimulation during LCMV infection and are the origin of structural and functional platelet abnormalities observed in the circulation. Indeed, megakaryocytes from LCMV-infected mice exhibited fewer granules limited to the perinuclear zone rather than normally diffused throughout the demarcation membrane system; the latter was also underdeveloped. Moreover, platelet transcriptome evaluation of genes involved in platelet production and function showed that IFN-I downregulates bone marrow expression of NF-E2 - a key transcription factor for thrombocytopoiesis - as well as of cyclooxygenase-1 and thromboxane-A synthase. The latter enzymes are required for TBXA2 synthesis from AA amplifying platelet activation. Thus, in addition to being decreased in number, circulating platelets of LCMV-infected animals have impaired activation-induced cargo release from storage granules supporting hemostasis. Experimentally, we observed that bleeding during LCMV infection is increased following aspirin, but not clopidogrel, administration. This suggested that serotonin secretion may be the final effector required to control erythrocyte extravasation. Accordingly, bleeding associated with LCMV infection was markedly enhanced in mice lacking platelet serotonin (TPH1-/-mice), and this was ameliorated by transfusion of normal platelets. In conclusion, we have identified a IFN-I targeting of megakaryocytes leading to reduced production of dysfunctional platelets with abnormal serotonin release as the cause of potentially lethal bleeding in a mouse model of arenavirus infection. Our demonstration that infusion of viable platelets can prevent lethal anemia in LCMV-infected susceptible mouse strains indicates that the same therapeutic intervention monitored by platelet response to AA stimulation may aid in treating severe VHF cases in human patients. Disclosures Aiolfi: MERU-VasImmune, Inc: Other: Stock option. Ruggeri:MERU-VasImmune Inc.: Equity Ownership, Other: CEO and Founder.

Shared roles for Scl and Lyl1 in murine platelet production and function

AbstractThe stem cell leukemia (Scl or Tal1) protein forms part of a multimeric transcription factor complex required for normal megakaryopoiesis. However, unlike other members of this complex such as Gata1, Fli1, and Runx1, mutations of Scl have not been observed as a cause of inherited thrombocytopenia. We postulated that functional redundancy with its closely related family member, lymphoblastic leukemia 1 (Lyl1) might explain this observation. To determine whether Lyl1 can substitute for Scl in megakaryopoiesis, we examined the platelet phenotype of mice lacking 1 or both factors in megakaryocytes. Conditional Scl knockout (KO) mice crossed with transgenic mice expressing Cre recombinase under the control of the mouse platelet factor 4 (Pf4) promoter generated megakaryocytes with markedly reduced but not absent Scl. These Pf4Sclc-KO mice had mild thrombocytopenia and subtle defects in platelet aggregation. However, Pf4Sclc-KO mice generated on an Lyl1-null background (double knockout [DKO] mice) had severe macrothrombocytopenia, abnormal megakaryocyte morphology, defective pro-platelet formation, and markedly impaired platelet aggregation. DKO megakaryocytes, but not single-knockout megakaryocytes, had reduced expression of Gata1, Fli1, Nfe2, and many other genes that cause inherited thrombocytopenia. These gene expression changes were significantly associated with shared Scl and Lyl1 E-box binding sites that were also enriched for Gata1, Ets, and Runx1 motifs. Thus, Scl and Lyl1 share functional roles in platelet production by regulating expression of partner proteins including Gata1. We propose that this functional redundancy provides one explanation for the absence of Scl and Lyl1 mutations in inherited thrombocytopenia.

GATA‐1: A potential novel biomarker for the differentiation of essential thrombocythemia and myelofibrosis

Essentials The BCR-ABL negative myeloproliferative neoplasms are subjected to unknown phenotypic modifiers. GATA-1 is upregulated in ET patients, regardless of treatment regimen or mutational status. Myelofibrosis (MF) megakaryocytes displayed decreased GATA-1 staining. GATA-1 may have utility as a diagnostic marker in ET and in its differential diagnosis from MF. ABSTRACT: Background The BCR-ABL-negative myeloproliferative neoplasms, i.e., polycythemia vera, essential thrombocythemia (ET), and myelofibrosis (MF), are characterized by mutations in JAK2, CALR, or MPL. However, an as yet unknown factor drives the precise disease phenotype. The hematopoietic transcription factor GATA-1 and its downstream targets NFE2 and FLI1 are responsible for determining erythroid and megakaryocyte lineages during hematopoietic stem cell differentiation. Previous studies have demonstrated a low level of GATA-1 expression in megakaryocytes from patients with MF. Objectives and methods The expression of GATA-1, NFE2 and FLI1 was studied for changes in the peripheral blood (PB) of ET patients. Peripheral blood samples were obtained from 36 ET patients, 14 MF patients, and seven healthy control donors. Total RNA from PB mononuclear cells (PBMCs) was extracted, and quantitative polymerase chain reaction was used to determine relative changes in gene expression. Protein levels of GATA-1 were also determined in bone marrow sections from ET and MF patients. Results GATA-1 mRNA was upregulated in ET patients, regardless of treatment regimen or mutational status. FLI1 expression was significantly downregulated, whereas NFE2 expression was unaffected by changes in GATA-1 mRNA levels. Megakaryocytes from ET patients showed increased protein levels of GATA-1 as compared with those from MF patients. Conclusions Our results confirmed, in PB, our previous data demonstrating elevated levels of GATA-1 mRNA in total bone marrow of ET patients. GATA-1 mRNA levels are independent of cytoreductive therapies, and may have utility as a diagnostic marker in ET and in its differential diagnosis from MF.

PS1454 CAG PROMOTER DRIVEN C‐MYC OVEREXPRESSION CAUSES RAPIDLY LETHAL CARDIOMYOPATHY

Background:We have previously reported that mice expressing elevated levels of NFE2 display a myeloproliferative phenotype which spontaneously transforms to acute leukemia. Upon leukemic transformation, a high percentage of mice acquire duplications or amplification of the murine c‐myc locus. The CMV early enhancer/chicken‐beta‐actin (CAG) promoter has been reported to direct high level heterologous gene expression specifically to hematopoietic stem cells and myeloid progenitors.Aims:We hypothesized that elevated expression of both NFE2 and c‐myc synergize to induce leukemic transformation. Therefore, we compared the hematological phenotype of mice carrying either augmented NFE2 or c‐myc levels alone to mice overexpressing both genes.Methods:We crossed mice expressing a CAG‐driven tetracycline activator (CAG‐rtTA3) with the tet‐O‐myc mouse strain, in order to achieve high levels of doxycycline‐inducible c‐myc expression in the hematopoietic compartment. Double transgenic CAG‐myc mice were subsequently bred to heterozygous knock‐in mice expressing the NFE2‐D297–300 mutation of the endogenous promoter, generating both CAG‐myc‐NFE2‐wt and CAG‐myc‐NFE2mut mice. C‐myc expression was induced by doxycycline treatment administered through various routes, including drinking water, oral gavage and food pellets.Results:Unexpectedly both CAG‐myc and CAG‐myc‐NFE2 mice died rapidly, between 6 and 7 days following c‐myc induction, independent of the route of doxycycline administration. Autopsy and histological analysis revealed marked cardiomyocyte hypertrophy, with heart volume more than double the wt control (Fig. 1). In addition to hematopoietic stem and progenitor cells, the CAG promoter has been reported to direct expression to cardiomyocytes. C‐myc overexpression in adult cardiomyocytes by tet‐mediated c‐myc induction has previously been shown to cause cell cycle re‐entry that leads to cardiomyopathy and hypertrophy. However, it was not expected that the CAG promoter, seen as a “highly effective tool for probing hematopoietic development and disease”, would have a similarly cardiotoxic effect.Fig. 1: Pathology and histology of hearts following doxycycline induction of CAG‐myc expression. (A) representative autopsy finding from a wt control mouse (left) and a CAG‐myc‐NFE2‐wt mouse. Results are representative of n = 16 CAG‐myc mice. (B) Hematoxylin Eosin (H&E) staining of histological sections from CAG‐myc‐NFE2 wt and CAG‐myc‐NFE2mut mice before and after doxycycline induction. 200x magnification, size bars represent 50 mm.Summary/Conclusion:While our murine model did not allow us to investigate our hypothesized synergistic effect between c‐myc and NFE2 in leukemic transformation, the CAG‐myc mouse strain generated constitutes an excellent model for studying rapidly lethal cardiomyopathy. Moreover, our data serve to alert researchers in the hematopoietic field to possible side effects of using the CAG promoter to drive gene expression in hematopoietic stem and progenitor cells.image

Altered NFE2 activity predisposes to leukemic transformation and myelosarcoma with AML-specific aberrations

AbstractIn acute myeloid leukemia (AML), acquired genetic aberrations carry prognostic implications and guide therapeutic decisions. Clinical algorithms have been improved by the incorporation of novel aberrations. Here, we report the presence and functional characterization of mutations in the transcription factor NFE2 in patients with AML and in a patient with myelosarcoma. We previously described NFE2 mutations in patients with myeloproliferative neoplasms and demonstrated that expression of mutant NFE2 in mice causes a myeloproliferative phenotype. Now, we show that, during follow-up, 34% of these mice transform to leukemia presenting with or without concomitant myelosarcomas, or develop isolated myelosarcomas. These myelosarcomas and leukemias acquired AML-specific alterations, including the murine equivalent of trisomy 8, loss of the AML commonly deleted region on chromosome 5q, and mutations in the tumor suppressor Trp53. Our data show that mutations in NFE2 predispose to the acquisition of secondary changes promoting the development of myelosarcoma and/or AML.

Single Cell RNASeq Demonstrates That Mouse Erythroid Cells Are the Last Lineage to Emerge

The hierarchical model of hematopoiesis posits that hematopoietic stem cells (HSC) give rise to myeloid progenitors (CMP), that can become further restricted to bipotential granulocyte/monocyte progenitors (GMP) or megakaryocyte/erythroid progenitors (MEP). We and others have shown that this model may not accurately depict hematopoiesis. Recent studies have shown that shown that populations of mouse hematopoietic stem and progenitor cells (LSK) have a strong megakaryocyte (Mk) transcriptional profile (Heuston, 2018, Epig. & Chrom.), and single cell studies have identified lineage committed cells in progenitor populations thought to be multipotent. For example, we recently reported that human MEP contain 3 populations: erythroid (Ery) primed, Mk primed, and bipotential (Psaila, 2016; Gen. Bio.).To determine when Mk and Ery cells emerge during mouse hematopoiesis, we performed single cell RNASeq on 10000 LSK, 12000 CMP, 6000 MEP and 8000 GMP cells. Clustering analysis (Satija, 2018, Nat. Biotech.) of all 4 populations identified 33 transcriptionally distinct clusters. In 30 of 33 clusters, 85% of cells were from a single defined population (e.g. MEP). LSK and CMP clusters grouped closely together.We used gene set profiling (Gene Set Enrichment Analysis, GO and KEGG) to correlate transcriptional profiles of clusters with specific hematopoietic lineages and cellular activities. In LSK, the most common transcriptional profiles correlated with active cell cycling. Mk-associated genes (Meis1, Myct1, and Fli1), were co-expressed with lymphoid genes in 56% of all LSK. Consistent with previous studies, we conclude that cells with Mk transcriptional profiles are abundant in LSK. No cells with an Ery RNA signature were observed in LSK.23% of all CMP cells expressed Mk genes (e.g., Pf4, Itga2b, and Fli1) and were enriched for processes involved in platelet biology (p < 3E-18). 12% of CMP had an Ery RNA signature (low expression of Gata1, Klf1, and Nfe2) and decreased Mk gene expression (e.g., Gata2 and Gfi1b, [p < 3E-18]) compared to other CMP clusters. The high ratio of Gata2/Gata1 expression (1.90) suggests that this cluster contained immature Ery cells.More than 94% of all mouse MEP had Ery RNA signatures. Clusters could be distinguished by gene expression (e.g., Gata1, Klf1, Tfrc) and biological processes (ribosome synthesis and heme-biology processes [p < 4 E-10]). Based on the transcriptional profiles, we determined the most mature erythroid cells in MEP were late BFU-E.To compare the differentiation of Mk and Ery cells, we pooled our LSK, CMP, and MEP data for analysis using the Monocle software package. GMP contained only clusters expressing granulocytic or monocytic genes and were excluded from the analysis. Monocle arranges cells into trajectories based on their transcriptional profiles, with more differentiated cells positioned further from a common node (Xiaojie, 2017, bioRxiv). We found that LSK cells near the node had overlapping lymphoid and Mk transcriptional profiles. Closest to the node, we found 38% of CMP expressed a profile similar to LSK. An additional 45% of CMP formed one trajectory with lymphoid and granulocyte RNA signatures. Another 17% of CMP formed a second trajectory, with cells expressing an Mk signature closest to the node, cells with a mixed Ery/Mk signature further along the trajectory, and MEP cells with Ery-only signatures furthest from the node. To clarify the Mk/Ery divergence, we focused our analysis on the CMP populations expressing Mk RNAs (Figure1). We observed cells in G1/S phase with an immature Mk signature to the left of the node where the trajectories diverge. On the right, cells with immature Mk signatures were nearest the node and cells with a mixed Ery/Mk signature were at the end of the trajectory (upper right; Mk/Ery). Along the second trajectory, rapidly cycling G2/M Mk cells with an early endomitosis-associated RNA signature (e.g., Pf4, Gp1bb, Gp9, and Vwf) were located at the end of the trajectory (lower right; Mk early endomitosis).Our data are consistent with a model in which two waves of Mk differentiation begin in LSK and progresses to CMP. The Mk lineage is divided in CMP, producing cells that begin endomitosis and cells that have an Mk-repressing/Ery-activating cell program that gives rise to the Ery lineage. We conclude that the erythroid lineage is derived from an Mk-like precursor and is the last lineage to be specified in mouse hematopoiesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Developmental Regulation of Nuclear Factor Erythroid-2 Related Factors (nrfs) by AHR1b in Zebrafish (Danio rerio).

Interactions between regulatory pathways allow organisms to adapt to their environment and respond to stress. One interaction that has been recently identified occurs between the aryl hydrocarbon receptor (AHR) and the nuclear factor erythroid-2 related factor (NRF) family. Each transcription factor regulates numerous downstream genes involved in the cellular response to toxicants and oxidative stress; they are also implicated in normal developmental pathways. The zebrafish model was used to explore the role of AHR regulation of nrf genes during development and in response to toxicant exposure. To determine if AHR1b is responsible for transcriptional regulation of 6 nrf genes during development, a loss-of-function experiment using morpholino-modified oligonucleotides was conducted followed by a chromatin immunoprecipitation study at the beginning of the pharyngula period (24 h postfertilization). The expression of nrf1a was AHR1b dependent and its expression was directly regulated through specific XREs in its cis-promoter. However, nrf1a expression was not altered by exposure to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), a toxicant and prototypic AHR agonist. The expression of nrf1b, nrf2a, and nfe2 was induced by TCDD, and AHR1b directly regulated their expression by binding to cis-XRE promoter elements. Last, nrf2b and nrf3 were neither induced by TCDD nor regulated by AHR1b. These results show that AHR1b transcriptionally regulates nrf genes under toxicant modulation via binding to specific XREs. These data provide a better understanding of how combinatorial molecular signaling potentially protects embryos from embryotoxic events following toxicant exposure.

Epigenetic regulation of NFE2 overexpression in myeloproliferative neoplasms

AbstractThe transcription factor “nuclear factor erythroid 2” (NFE2) is overexpressed in the majority of patients with myeloproliferative neoplasms (MPNs). In murine models, elevated NFE2 levels cause an MPN phenotype with spontaneous leukemic transformation. However, both the molecular mechanisms leading to NFE2 overexpression and its downstream targets remain incompletely understood. Here, we show that the histone demethylase JMJD1C constitutes a novel NFE2 target gene. JMJD1C levels are significantly elevated in polycythemia vera (PV) and primary myelofibrosis patients; concomitantly, global H3K9me1 and H3K9me2 levels are significantly decreased. JMJD1C binding to the NFE2 promoter is increased in PV patients, decreasing both H3K9me2 levels and binding of the repressive heterochromatin protein-1α (HP1α). Hence, JMJD1C and NFE2 participate in a novel autoregulatory loop. Depleting JMJD1C expression significantly reduced cytokine-independent growth of an MPN cell line. Independently, NFE2 is regulated through the epigenetic JAK2 pathway by phosphorylation of H3Y41. This likewise inhibits HP1α binding. Treatment with decitabine lowered H3Y41ph and augmented H3K9me2 levels at the NFE2 locus in HEL cells, thereby increasing HP1α binding, which normalized NFE2 expression selectively in JAK2V617F-positive cell lines.