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1
Yang, Liang En, Xuan Ze Wang, and Lin Zheng. "Displacement Sensor with Controlled Measuring Force and its Characteristics Analysis." Applied Mechanics and Materials 103 (September 2011): 309–13. http://dx.doi.org/10.4028/www.scientific.net/amm.103.309.
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A displacement sensor with controlled measuring force and its characteristics analysis are discussed in this paper. The displacement sensor consists of an electric induction transducer with high resolution and a voice coil motor (VCM). The measuring principles, structure of the sensor are discussed. Theory model, dynamic and static characteristics of VCM are analysed. A measuring system for surface topography with large measuring range is constructed based on the displacement sensor and 2D moving platform. Measuring force of the sensor in measurement process of surface topography can be controlled at μN level and hardly changes. It has been used in measurement of bearing ball，bullet mark, etc.
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DEVADASON, JOATHAN, PAUL S. MOSES, and MOHAMMAD A. S. MASOUM. "Multiparameter Stability Analysis of Systems with Induction Motor Loads, Weak Interconnections and Series Compensation." WSEAS TRANSACTIONS ON CIRCUITS AND SYSTEMS 20 (June 28, 2021): 128–38. http://dx.doi.org/10.37394/23201.2021.20.16.
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Dynamic modeling and stability domain analysis of a system consisting of a synchronous generator sup-plying an induction motor load through a series compensated weak network has been carried out in this paper. The impact of X/Rratio of the feeder and generation control system parameters on the stability domain with respect to series compensation has been examined through eigenvalue calculations and time domain simulations. From the studies conducted, it was observed that the stability domain of the system with respect to series compensation depends on the grid strength in addition to the excitation system parameters. Eigenvalue analysis shows that there is a strong correlation between the exciter gain, time constants of the measurement transducer and exciter, and the series compensation level. The main contribution of this work is to reveal new bifurcations which arise in these systems which has been studied through eigenvalue analysis and time domain simulations for various combinations of system parameters.
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Liu, Xuan, Carolina Ciumas, Yu-Min Huang, Knut R. Steffensen, Hong Lian, Hans Link, and Bao-Guo Xiao. "Autoantigen-pulsed dendritic cells constitute a beneficial cytokine and growth factor network in ameliorating experimental allergic encephalomyelitis." Multiple Sclerosis Journal 11, no. 4 (August 2005): 381–89. http://dx.doi.org/10.1191/1352458505ms1180oa.
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Injection of myelin basic protein (MBP)-pulsed dendritic cells (DC) into healthy rats, as we reported before and observed in this study, did not induce clinical experimental allergic encephalomyelitis (EAE), but effectively protected the rats from subsequent EAE induction. The mechanisms by which MBP-pulsed DC mediate immune protection are not completely understood. In the present study, we mainly explored the dynamic change of cytokine and growth factor mRNA expression in spinal cords after subcutaneous injection of MBP-pulsed and unpulsed DC. The expression of interleukin (IL)-1, interferon-g and tumour necrosis factor-a as well as programmed death ligand (PDL)-1, PDL-2, signal transducer and activator of transcription (STAT)4, STAT6, matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinases (TIMP)-2 was increased on day 0 postimmunization (p.i.). The increase of IL-12 expression was observed on day 7 p.i., while the increase of IL-10 expression mainly occurred on day 14 p.i. Except downregulation of insulin-like growth factor-1, the expression of brain-derived neurotrophic factor, ciliary neurotrophic factor, fibroblast growth factor (FGF)-2 and platelet-derived growth factor (PDGF)-B/C as well as nerve growth factor receptor (NGF-R), FGF receptor, PDGF-R-a and b was elevated on day 0 p.i., while the increase of TIMP and NGF was observed on days 0 and 7 p.i. There were no significant differences on MMP-2, spinal cord-derived growth factor and PDGF-A mRNA expression. In line with the suppression of EAE induced by MBP-pulsed DC, the dynamic change of cytokines and growth factors in spinal cords should constitute a beneficial microenvironment against EAE.
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Bobkov, Yurii. "Classification and analysis of measuring transducers of intensity (induction) of alternating magnetic fields." MECHANICS OF GYROSCOPIC SYSTEMS, no. 40 (December 26, 2021): 82–93. http://dx.doi.org/10.20535/0203-3771402020249113.
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The current state of technology is characterized by the mass use of electricity, the use of various electrical, electronic and radio devices. This causes expansion of magnetic measurements and the need to develop new highly sensitive measuring equipment for a wide range of frequencies. One of its main elements, that largely determines the accuracy, frequency and dynamic ranges, are the primary measuring sensors of strength (induction) of alternating magnetic fields.
Many works have been devoted to the analysis and development of various sensors of strength (induction) of magnetic fields. At the same time, it can be noted the lack of a systematic approach to the measurement of alternating magnetic fields. The problem of the general classification of methods of measurement of alternating magnetic fields and, accordingly, primary measuring sensors of strength (induction) of alternating magnetic fields is not solved. In most cases, separate issues of measuring alternating magnetic fields and certain types of sensors are considered. That does not allow obtaining a holistic picture in this area and make the right choice of direction for solving assigned tasks.
The comprehensive analysis of methods of measuring alternating magnetic fields was carried out in this work. Based on it, the classification of primary measuring sensors of strength (induction) of alternating magnetic fields, on the physical principles of transformation was proposed.
Accordingly, the available measuring sensors of alternating magnetic fields following to the group of used physical phenomena can be divided into: magnetomechanical, induction, galvanomagnetic, quantum, magneto-optical and photomagnetic. Depending on the characteristics of each of these phenomena, separate measurement methods and types of measuring sensors were highlighted.
The current state of development of each of the types of measuring sensors of strength of alternating magnetic fields was analyzed, their advantages and disadvantages were determined, the limits of dynamic and frequency ranges, the maximum values of errors were outlined.
The obtained results allow to significantly simplify and reduce the time of choosing the necessary method of strength (induction) of alternating magnetic fields measuring and to choose the necessary type of measuring sensor to effectively solve the tasks.
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Liu, Xiangyang, Sanjan T. P. Gupta, Devesh Bhimsaria, Jennifer L. Reed, José A. Rodríguez-Martínez, Aseem Z. Ansari, and Srivatsan Raman. "De novo design of programmable inducible promoters." Nucleic Acids Research 47, no. 19 (September 25, 2019): 10452–63. http://dx.doi.org/10.1093/nar/gkz772.
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Abstract Ligand-responsive allosteric transcription factors (aTF) play a vital role in genetic circuits and high-throughput screening because they transduce biochemical signals into gene expression changes. Programmable control of gene expression from aTF-regulated promoter is important because different downstream effector genes function optimally at different expression levels. However, tuning gene expression of native promoters is difficult due to complex layers of homeostatic regulation encoded within them. We engineered synthetic promoters de novo by embedding operator sites with varying affinities and radically reshaped binding preferences within a minimal, constitutive Escherichia coli promoter. Multiplexed cell-based screening of promoters for three TetR-like aTFs generated with this approach gave rich diversity of gene expression levels, dynamic ranges and ligand sensitivities and were 50- to 100-fold more active over their respective native promoters. Machine learning on our dataset revealed that relative position of the core motif and bases flanking the core motif play an important role in modulating induction response. Our generalized approach yields customizable and programmable aTF-regulated promoters for engineering cellular pathways and enables the discovery of new small molecule biosensors.
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Kramer, Anne Marijn, Mengyong Yan, Karl S. Peggs, John Anderson, and Kenth Gustafsson. "Tumor-Associated Antigen Presentation by γδ T-Cells in Cancer Immunotherapy." Blood 124, no. 21 (December 6, 2014): 1411. http://dx.doi.org/10.1182/blood.v124.21.1411.1411.
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Abstract Tumor-Associated Antigen Presentation by γδ T-Cells in Cancer Immunotherapy Human γδ T-cells are considered to represent a link between innate and adaptive immunity. Their innate killing properties display a potent cytotoxic activity against solid tumors as well as lymphoid and myeloid malignancies. Subsequently, by lysing affected target cells and liberating antigen for uptake, they can differentiate into professional antigen presenting cells (pAPCs) for induction of CD4+ and CD8+ T cell responses. The degree of antigen-specific stimulation of responder T cells is increased in the presence of antibody(Ab)-assisted opsonized target cells, involving the low-affinity receptor for IgG CD16 (Fc γRIII), equivalent to that seen with mature antigen-loaded DCs. To elaborate the implications of this combined killing and pAPC function we have studied how freshly isolated as well as expanded and cloned populations of γδ T-cell subsets kill a target tumor cell, and take up and cross-present tumor-associated antigens (TAA). We performed quantitative analysis on the cellular uptake of different sizes of microspheres, analyzing the correlation between opsonization and internalization. All γδ T-cell subtypes were expanded using artificial APC, engineered to express CD86, CD137L and IL-15, and anti- γδ TCR Ab (B1). Short (EAAGIGILTV) and long (GHSYTTAEEAAGIGILTVILGVLLL) MART-1 peptides were used as antigens for γδ T-cell presentation to MART-1 TCR-transduced cytotoxic T-cells. A CFSE assay was performed to assess cytotoxic T-cell proliferation. Target cells and polysterene microspheres were opsonized with human anti-CD20 IgG1, Rituximab (RTX). CD16 function was blocked with a mouse monoclonal IgG1 anti-CD16 blocking Ab (clone LNK16). Imaging flowcytometry allowed us to quantify internalization of FITC-labeled microspheres. The Internalization Score is defined as the ratio of intensity inside the cell to the intensity of the entire cell. Both γδ T-cell lines and expanded γδ T-cell clones cultured long-term, remarkably, retain both tumor cell killing and take up tumor cell lysates or long synthetic TAA peptides and cross-present these on MHC class I to CD8+ cytotoxic T-cells in a dynamic, controllable fashion, dependent on Ab-opsonization. (Figure 1). The Ab-opsonization of 1 µm microspheres correlates with a higher receptor-mediated phagocytic uptake, in a CD16 dependent manner (Figure 2). The opsonization of 0,5 µm microspheres led to clumping of the microspheres, accounting for the lower uptake in this particular subgroup. For a lack of better alternative, moDCs have been widely used in experimental immunotherapy settings. The ease of manipulation of human γδ T-cells, the ability to be expanded ex-vivo combined with antigen presentation makes them a great potential tool for immunotherapy as a complementary or integrative strategy. Ligation of the γδ T-cell receptor at the tumor site will activate their expansion and innate killing. Yet, antigen presentation will only occur after binding of an immunoglobulin to the tumor cell, thereby activating their dual role. Our goal is to define an effective adjuvant vaccine formulation for inducing leukemia-specific cytolytic effects. We are currently investigating whether γδ T-cells can directly present and/or cross-present to cytotoxic T-cells in-vivo in a humanized mouse model. We believe that the uptake of microspheres by γδ T-cells has an impact on the development of vaccination strategies for cancer immunotherapy, as the immunization of γδ T-cells is a powerful method for the induction or reactivation of cytotoxic T cell specific responses. FIGURE 1 CFSE assay of γδ T-cell lines cross-presenting short and long MART-1 peptides to MART-1 TCR-transduced cytotoxic T-cells in a dynamic, controllable fashion, dependent on Ab-opsonization FIGURE 1. CFSE assay of γδ T-cell lines cross-presenting short and long MART-1 peptides to MART-1 TCR-transduced cytotoxic T-cells in a dynamic, controllable fashion, dependent on Ab-opsonization FIGURE 2a FIGURE 2a. FIGURE 2b FIGURE 2b. Disclosures No relevant conflicts of interest to declare.
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Dunussi-Joannopoulos, Kyriaki, Kathlene Runyon, Jamie Erickson, Robert G. Schaub, Robert G. Hawley, and John P. Leonard. "Vaccines With Interleukin-12–Transduced Acute Myeloid Leukemia Cells Elicit Very Potent Therapeutic and Long-Lasting Protective Immunity." Blood 94, no. 12 (December 15, 1999): 4263–73. http://dx.doi.org/10.1182/blood.v94.12.4263.
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Abstract Interleukin-12 (IL-12) is a heterodimeric cytokine mediating a dynamic interplay between T cells and antigen-presenting cells (APCs). Preclinical studies have demonstrated that recombinant murine IL-12 (rmIL-12) promotes specific antitumor immunity mediated by T cells in several types of tumors. However, the in vivo antitumor properties of IL-12 in acute myeloid leukemia (AML) have not been previously reported. We show here in a murine AML model that systemic administration of rmIL-12 significantly delays tumor growth but is incapable of rescuing mice from lethal leukemia. In contrast, AML cells genetically modified to express IL-12 (IL12-AML) using murine stem cell virus (MSCV) p40 + p35 elicit very potent antileukemic activity. Vaccines with lethally irradiated IL12-AML cells protect naive mice against challenge with wild-type AML cells and, more importantly, can cure mice bearing a considerable leukemic burden. Immunized mice show no signs of systemic IL-12 toxicity and their spleen histology is comparable with naive mice spleen. In vivo depletion of IL-12, interferon-γ (IFN-γ), or CD8+ T cells after injections with live IL12-AML cells abrogates completely the antileukemia immune responses. Studies on the in vitro effects of IFN-γ on AML cells demonstrate enhanced expression of major histocompatibility complex (MHC) and accessory molecules and induction of the costimulatory molecules B7.1 and B7.2, but no significant direct antiproliferative effect. 51Cr release assays show that rejection of live IL12-AML cells supports the development of long-lasting leukemia-specific cytotoxic T lymphocyte (CTL) activity. In conclusion, our results demonstrate that IL12-AML vaccination is a safe and potent immunotherapeutic approach that has a great potential to eliminate minimal residual disease in patients with AML.
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Dunussi-Joannopoulos, Kyriaki, Kathlene Runyon, Jamie Erickson, Robert G. Schaub, Robert G. Hawley, and John P. Leonard. "Vaccines With Interleukin-12–Transduced Acute Myeloid Leukemia Cells Elicit Very Potent Therapeutic and Long-Lasting Protective Immunity." Blood 94, no. 12 (December 15, 1999): 4263–73. http://dx.doi.org/10.1182/blood.v94.12.4263.424k30_4263_4273.
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Interleukin-12 (IL-12) is a heterodimeric cytokine mediating a dynamic interplay between T cells and antigen-presenting cells (APCs). Preclinical studies have demonstrated that recombinant murine IL-12 (rmIL-12) promotes specific antitumor immunity mediated by T cells in several types of tumors. However, the in vivo antitumor properties of IL-12 in acute myeloid leukemia (AML) have not been previously reported. We show here in a murine AML model that systemic administration of rmIL-12 significantly delays tumor growth but is incapable of rescuing mice from lethal leukemia. In contrast, AML cells genetically modified to express IL-12 (IL12-AML) using murine stem cell virus (MSCV) p40 + p35 elicit very potent antileukemic activity. Vaccines with lethally irradiated IL12-AML cells protect naive mice against challenge with wild-type AML cells and, more importantly, can cure mice bearing a considerable leukemic burden. Immunized mice show no signs of systemic IL-12 toxicity and their spleen histology is comparable with naive mice spleen. In vivo depletion of IL-12, interferon-γ (IFN-γ), or CD8+ T cells after injections with live IL12-AML cells abrogates completely the antileukemia immune responses. Studies on the in vitro effects of IFN-γ on AML cells demonstrate enhanced expression of major histocompatibility complex (MHC) and accessory molecules and induction of the costimulatory molecules B7.1 and B7.2, but no significant direct antiproliferative effect. 51Cr release assays show that rejection of live IL12-AML cells supports the development of long-lasting leukemia-specific cytotoxic T lymphocyte (CTL) activity. In conclusion, our results demonstrate that IL12-AML vaccination is a safe and potent immunotherapeutic approach that has a great potential to eliminate minimal residual disease in patients with AML.
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Salari, Azam, Kathrin Thomay, Andrea Schienke, Maike Hagedorn, Juliane Ebersold, Andreas Krueger, Axel Schambach, Brigitte Schlegelberger, and Gudrun Göhring. "Dynamic Telomere Shortening and Chromosomal Instability in Irradiated CD34+ Cells Transduced with TP53 Hotspot Mutations R175H, R248W and R249S." Blood 126, no. 23 (December 3, 2015): 4832. http://dx.doi.org/10.1182/blood.v126.23.4832.4832.
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Abstract Patients with MDS and a complex karyotype have a very short median survival and a high risk of transformation into AML. We showed earlier that TP53 mutations are associated with complex karyotype and disease progression. However, it is poorly understood how TP53 mutations contribute to the induction of chromosomal instability in hematopoietic stem and progenitor cells. We therefore established a long-term cell culture (LTC) model and investigated the role of TP53 in human CD34+ hematopoietic stem and progenitor cells (HSCs) isolated from cord blood and in HT1080 cells over 6 weeks. We chose 3 different modifications: 1) TP53-deficient HSCs via shRNA knockdown, 2) HSCs with different lentivirally introduced TP53 hotspot mutations (R248W, R175H, R273H, R249S) and 3) HT1080 cells with different lentivirally introduced TP53 hotspot mutations (R248W, R175H, R273H, R249S). We performed each LTC at least three times. In order to stress the cells and induce chromosomal instability, we irradiated half of the cells. Besides functional assays in the first week, we performed detailed cytogenetic analysis including telomere length measurement at weeks 1, 3 and 6. TP53 mutations and the downregulation of TP53 led to impaired hematopoiesis with decreased erythroid differentiation, increased apoptosis and decreased proliferation. None of the modifications induced chromosomal instability in cells without irradiation. In the irradiated cells, all cells carrying a TP53 mutation or a TP53 downregulation developed a chromosomal instability in comparison to the cells transduced with control vectors. However, no stable complex clones developed. Telomeres shortened during follow-up in HSCs carrying the mutations R175H, R248W and R249S. No other cells showed a dynamic response in telomere length. In order to analyze the DNA repair capacity, we performed yH2AX foci assay. Surprisingly, the same cells which showed a telomere shortening showed a lower amount of foci. This could be due to a lower amount of double-strand breaks or to a lower ability to form foci. In summary, TP53 mutations and the downregulation of TP53 led to an increased chromosomal instability in irradiated cells only. Modifications alone did not lead to the development of complex karyotypes. Furthermore, only the mutations R175H, R248W and R249S led to a telomere shortening. In conclusion, a TP53 mutation seems to require additional passenger mutations in order to lead to MDS or AML with complex karyotype. Disclosures No relevant conflicts of interest to declare.
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Taylor, Cormac T. "Mitochondria and cellular oxygen sensing in the HIF pathway." Biochemical Journal 409, no. 1 (December 11, 2007): 19–26. http://dx.doi.org/10.1042/bj20071249.
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Mitochondrial respiration is responsible for more than 90% of oxygen consumption in humans. Cells utilize oxygen as the final electron acceptor in the aerobic metabolism of glucose to generate ATP which fuels most active cellular processes. Consequently, a drop in tissue oxygen levels to the point where oxygen demand exceeds supply (termed hypoxia) leads rapidly to metabolic crisis and represents a severe threat to ongoing physiological function and ultimately, viability. Because of the central role of oxygen in metabolism, it is perhaps not surprising that we have evolved an efficient and rapid molecular response system which senses hypoxia in cells, leading to the induction of an array of adaptive genes which facilitate increased oxygen supply and support anaerobic ATP generation. This response is governed by HIF (hypoxia-inducible factor). The oxygen sensitivity of this pathway is conferred by a family of hydroxylases which repress HIF activity in normoxia allowing its rapid activation in hypoxia. Because of its importance in a diverse range of disease states, the mechanism by which cells sense hypoxia and transduce a signal to the HIF pathway is an area of intense investigation. Inhibition of mitochondrial function reverses hypoxia-induced HIF leading to speculation of a role for mitochondria in cellular oxygen sensing. However, the nature of the signal between mitochondria and oxygen-sensing hydroxylase enzymes has remained controversial. In the present review, two models of the role for mitochondria in oxygen sensing will be discussed and recent evidence will be presented which raises the possibility that these two models which implicate ROS (reactive oxygen species) and oxygen redistribution respectively may complement each other and facilitate rapid and dynamic activation of the HIF pathway in hypoxia.
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Reiss, Kim, Yuan Yuan, Debora Barton, Amy Ronczka, Daniel Cushing, Michael Klichinsky, Sascha Abramson, Rehman Qureshi, Thomas Condamine, and Elizabeth Dees. "951 A phase 1 first in human study of adenovirally transduced anti-HER2 CAR macrophages in subjects with HER2 overexpressing solid tumors: preliminary safety, pharmacokinetics, and TME reprogramming data." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A1000. http://dx.doi.org/10.1136/jitc-2021-sitc2021.951.
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BackgroundCT-0508 is an autologous monocyte-derived pro-inflammatory macrophage cell product engineered with Ad5f35 to express an anti-HER2 CAR. In pre-clinical studies CT-0508 was safe and effective. This abstract contains preliminary results from the first-in-human experience with CAR macrophages (CAR-M).MethodsThis First-In-Human Phase 1, multi-center, open-label study is evaluating the safety, tolerability, manufacturing feasibility, pharmacokinetics and mechanism of action of CT-0508 in 18 subjects with advanced solid tumors overexpressing HER2 who have progressed on prior therapies, including HER2 targeted therapies if indicated.Patients receive four doses of filgrastim for monocyte mobilization prior to apheresis. CT-0508 CAR-M is manufactured from autologous apheresis products and delivered as a cryopreserved cell product. Group 1 subjects enter an intra-patient fractionated dose escalation regimen, receiving CT-0508 on D1, D3 and D5, followed by Group 2 subjects who receive CT-0508 on D1. There is no preparative chemotherapy prior to CT-0508 infusion.Pre and post treatment biopsies and blood samples are collected to investigate correlates of safety, serum cytokines and chemokines, pharmacokinetics, TME modulation, and induction of an adaptive anti-tumor immune response.ResultsTo date, two subjects have been treated with CT-0508 (esophageal adenocarcinoma and extrahepatic cholangiocarcinoma). Patient product was successfully manufactured, CT-0508 treatment was well tolerated, with no dose limiting and no major organ toxicities observed.One subject experienced Grade 2 CRS on Day 3 which resolved on the same day.Grade 3 AEs included anemia (present at baseline for both subjects) and lymphopenia (present at baseline in one subject). One subject experienced one SAE of Grade 4 tumor bleeding which was unrelated to CT-0508, 88 days after the last infusion.CAR-M were transiently detected in the peripheral blood following each infusion, demonstrating rapid egress from the periphery into tissues within hours. Transient cytokine/chemokine elevations were observed (peak: 2 hours, back to baseline at 48 hours). Single cell RNAseq analysis of dissociated tumor tissue samples (pre-treatment, day 8 and week 4) demonstrated dynamic TME reprogramming, with recruitment of inflammatory innate immune cells and naïve T cells at day 8, and significant CD8+ T cell infiltration, activation, and proliferation at week 4.ConclusionsCT-0508 has been administered to two subjects thus far, exhibiting safety, good tolerability, T cell repertoire modulation, and reprogramming of the TME consistent with the induction of anti-tumor immunity. The study continues to recruit patients and updated data will be presented.Trial RegistrationNCT04660929ReferenceKlichinsky M, Ruella M, Shestova O, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 2020;38(8):947–953.Ethics ApprovalEthics approvals have been obtained from the clinical sites enrolling patients: the University of Pennsylvania (844106/IORG0000029), the University of North Carolina and City of Hope Comprehensive Cancer Center (20201732/IORG0000432).
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Sive, Jonathan I., Fernando J. Calero-Nieto, Rebecca Hannah, and Berthold Göttgens. "Genome-Scale Analysis of the Pu.1 Driven Transcriptional Programme That Overcomes the Differentiation Block in a Novel Pu.1-Inducible AML Cell Line." Blood 124, no. 21 (December 6, 2014): 869. http://dx.doi.org/10.1182/blood.v124.21.869.869.
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Abstract Transcription factors (TFs) play a key role in determining normal haematopoiesis; haematological cancers often being characterised by TF dysregulation. Pu.1 is a TF that plays a major role in myeloid differentiation, with mutations and down regulation of Pu.1 activity described in human acute myeloid leukemia (AML). To investigate the role of Pu.1 in normal and leukemic myeloid fate, we developed a model of inducible Pu.1 rescue in a murine AML cell line, and utilised it to assess genome-wide Pu.1-DNA-binding profiles and the subsequent effects on histone remodelling, gene expression and binding of the cooperative TF CEBPA. We used a previously described radiation-induced murine AML cell line known to be deficient in functional Pu.1 (Cook et al, Blood 2004; 104:3437-3444) and transduced cells with 4-hydroxy-tamoxifen (OHT)-inducible Pu.1 (PuER) or empty vector (EV). Clonal populations were produced by limiting dilution, and genotype and protein phenotype confirmed by PCR and Western blotting respectively. Incubation in 100nM OHT caused cessation of proliferation, and increased expression of the maturation markers CD11b and Gr-1. To assess the effects of Pu.1 binding, cells were incubated for 2 hours in 100nM OHT and cross-linked chromatin harvested for ChIP with antibodies against Pu.1, CEBPA and H3K27Ac. ChIP samples were amplified and sequenced, reads were mapped to the mouse reference genome, binding peaks identified, and binding levels normalised for peak length and total read count. RNA from was extracted from the same samples and analysed by microarray expression profiling. Binding peak analysis revealed an increase in Pu.1 binding sites from 5283 to 15675 following Pu.1 induction, showing that the mutant Pu.1 is able to perform limited DNA-binding, but not in a manner sufficient to induce transcription of genes necessary for normal differentiation. To investigate the effect of Pu.1 binding on histone remodelling, we selected a set of 613 peaks with ≥4-fold increase in Pu.1 read count, ≥4fold increase in H3K27Ac read count, and post-induction minimal H3K27Ac read count of ≥50 reads/region. Interestingly, these highly “dynamic” Pu.1 binding peaks were restricted to non-promoter regions, suggesting increased binding at distal enhancer sites. These 613 peaks mapped to 553 genes, which showed a small but significant overexpression post-Pu.1 induction, compared to genes adjacent to peaks with no change in Pu.1 (p = 5.357e-16). These genes were intersected with the 284 genes overexpressed in the gene expression array post Pu.1-induction, producing a list of 46 genes of relevant Pu.1 targets. Using Gene Ontology and Functional Term Enrichment analysis, we discovered a significant enrichment for genes overexpressed in bone marrow macrophages post-lipopolysaccharide stimulation (z score = -2.19). Investigating the effect of Pu.1 induction on CEBPA binding, we found that increased Pu.1 binding was enriched in CEBPA peaks which were ≥4fold increased post-Pu.1 induction (p< 0.001), suggesting recruitment of CEBPA in a significant proportion of regions. Further evidence of the relationship between Pu.1 and CEBPA comes from motif analysis of the ≥4fold Pu.1 peaks which showed CEBP motifs present in 30.0% of targets. Similarly, analysis of ≥4fold CEBPA peaks showed Ets motifs in 34.5% of these peaks with ≥ 4fold Pu.1 binding, but only 7.5% in those without. In conclusion, we present a novel model system where we have examined at genome-scale the effects of Pu.1-DNA binding to overcome the differentiation block in a Pu.1-deficient AML model. Functional Pu.1 binding rapidly induces changes in H3K27Ac profile at key enhancer sites, which subsequently drives the cell into a maturation pathway. The interaction between Pu.1 and CEBPA is also confirmed, with evidence of Pu.1 recruiting CEBPA to DNA-binding sites. Disclosures No relevant conflicts of interest to declare.
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Huang, Huilin, Hengyou Weng, Xi Qin, Boxuan Simen Zhao, Lou Dore, Jennifer Strong, Rui Su, et al. "The N6-Adenine Methyltransferase METTL14 Plays an Oncogenic Role in Acute Myeloid Leukemia." Blood 128, no. 22 (December 2, 2016): 1536. http://dx.doi.org/10.1182/blood.v128.22.1536.1536.
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Abstract N 6-methyladenosine (m6A) modification is the most abundant internal RNA modification in eukaryotes. Recent studies have shown that the dynamic and reversible regulation of m6A modifications in mRNAs or non-coding RNAs plays critical roles in tissue development, stem cell self-renewal and differentiation, control of heat shock response, and circadian clock controlling, as well as in RNA metabolism and processing. However, little is known about the functions of m6A and m6A regulators in malignant hematopoiesis. METTL14 is a major m6A writer which together with METTL3 forms the core of the methyltransferase complex that catalyzes the conversion of adenosine (A) to m6A. Through qPCR assays, we found that METTL14 was aberrantly up-regulated in mononuclear cells (MNC) from acute myeloid leukemia (AML) patients with t(11q23), t(15;17), or t(8;21) relative to those from healthy donors. To investigate the pathological role of METTL14 in AML, we transduced lineage negative (Lin-) bone marrow (BM) progenitor cells from Mettl14fl/flCreERT mice with MLL-AF9, AML1-ETO9a, or PML-RARa fusion genes and performed colony-forming/replating assays with or without addition of 4-hydroxytamoxifen (4-OHT). Induction of genetic knockout of Mettl14 by 4-OHT treatment remarkably impaired the colony-forming ability of all these AML-related fusion genes after replating. After the first round of plating, we harvested MLL-AF9-transduced cells that were not treated with 4-OHT and transplanted them into lethally irradiated recipient mice. As expected, tamoxifen (TAM) treatment of transplanted mice significantly delayed leukemogenesis compared to mice treated with vehicle (MLL-AF9+TAM, with median survival of 91 days; MLL-AF9+vehicle, with median survival of 71 days; P=0.0012) (Fig.1A). In addition, specific knockdown of Mettl14 with shRNAs showed similar patterns to Mettl14 knockout. Thus our data demonstrate that Mettl14 is crucial for cell transformation and leukemogenesis. Further, to determine the role of Mettl14 in the maintenance of leukemia, we transduced leukemic BM cells from primary MLL-AF9 leukemic mice with shRNAs against Mettl14 or scramble shRNA and transplanted these cells into lethally irradiated recipient mice. Again, a significantly prolonged survival was observed in Mettl14 knockdown groups compared to that in the control group (MLL-AF9+shRNA1, with median survival of 32 days; MLL-AF9+shRNA2, with median survival of 32 days; MLL-AF9+shScramble, with median survival of 23.5 days; P< 0.001 for both knockdown groups) (Fig.1B). Noticeable, mice in Mettl14 knockdown groups showed less c-kit+ cells in BM than mice in the control group (Fig.1C). In addition to the mouse model, we used human leukemia cell lines to investigate the function of METTL14 in human AML cells. Silencing of METTL14 with shRNAs significantly inhibited cell viability, induced apoptosis as well as terminal differentiation of MONOMAC6 and NB4 cell lines (Fig.1D, E, F). Moreover, xenograft model showed that repression of METTL14 significantly inhibited the engraftment of MONOMAC6 cells and thus delayed the onset of leukemia in NSG-SGM3 (NSGS) immunodeficient mice (Fig.1G). Furthermore, knockdown of METTL14 sensitized MONOMAC cells to ATRA or PMA-induced differentiation. Taken together, our results support the oncogenic role of METTL14 in AML and highlight METTL14 as a novel therapeutic target in AML. Figure 1 Oncogenic roles of METTL14 in AML. Figure 1. Oncogenic roles of METTL14 in AML. Disclosures No relevant conflicts of interest to declare.
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Stokes, Nicole, David Dominguez-Sola, and Eirini P. Papapetrou. "Functional Analysis of MYC Deregulation By Diverse Genetic Mechanisms during Hematopoiesis." Blood 132, Supplement 1 (November 29, 2018): 1323. http://dx.doi.org/10.1182/blood-2018-99-110829.
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Abstract Alterations of the c-MYC (MYC) locus, mainly in the form of translocations or amplifications, are common in hematologic malignancies and result in deregulated MYC expression, disrupting cellular proliferation and differentiation and promoting leukemogenesis. While amplified MYC loci typically maintain all native cis-regulatory elements, translocated loci are instead driven by heterologous regulatory regions and are therefore expected to be unresponsive to external signals. However, no studies have directly compared the effects of different modes of MYC deregulation in cellular phenotypes and functions. Consistent with the idea that different modes of MYC deregulation may respond differentially to external mitogenic signals, we found that treatment with DMSO resulted in rapid downregulation of MYC protein expression in hematopoietic cell lines with amplified MYC loci (HL-60, BJAB), but failed to do so in cell lines harboring a t(8;14) MYC translocation (Ramos, LY8) despite the induction of cell cycle arrest in all lines. To precisely model these two genetic mechanisms of MYC deregulation during hematopoiesis and study their effects in phenotypes relevant to oncogenesis (differentiation and proliferation), we used genetic engineering of human embryonic stem cells (hESCs). First, MYC deregulation upon translocation was modeled using a doxycycline (DOX)-inducible lentiviral construct to ectopically express MYC with GFP (MYC-DoxOE). Transduced single cell clones of the H1 hESC line were selected to ensure uniform MYC expression. Second, we created a conditional MYC gene amplification model in H1 hESCs by using CRISPR/Cas9 to insert a floxed cassette consisting of three tandem MYC cDNA copies and GFP in the MYC locus (MYC-Amp). Correctly edited clones were identified via PCR and Southern Blot, and one clone with bi-allelic targeting (yielding 8 inducible MYC copies) was selected. All clones were confirmed to be karyotypically normal. Upon in vitro hematopoietic differentiation of unmodified H1 hESCs, we identified a peak of MYC expression (day 12) coinciding with the emergence of CD34+/CD45+ hematopoietic progenitor cells (HPCs). MYC expression subsequently declined as cells matured further down the myeloid lineage and lost CD34 expression (days 14-20). To test the effects of MYC deregulation in the two models we induced MYC expression by doxycycline (MYC-DoxOE) or cell-permeant TAT-Cre recombinase (MYC-Amp) on day 10 of hematopoietic differentiation. Induction of MYC expression above normal levels was found to be comparable between the two models during peak MYC expression (day 12). However, while ectopically expressed MYC in the MYC-DoxOE model maintained high levels throughout the culture period, amplified MYC in the MYC-Amp model - which is subject to regulation by the native MYC locus cis regulatory elements - was downregulated after day 12, in a pattern similar to that observed in uninduced H1 cells. MYC induction in the MYC-DoxOE model caused a block in differentiation with accumulation of CD34+ HPCs and reduction of more mature CD34-/CD45+ hematopoietic cells. Wright-Giemsa staining corroborated these results showing a predominance of morphologically immature myeloid cells. Strikingly, MYC-DoxOE HPCs could be maintained in culture for over 90 days, while H1-derived HPCs without doxycycline treatment completely arrested their proliferation after approximately 28 days of culture. This phenotype is dramatically distinct from the finite growth potential of HPCs derived from normal human pluripotent stem cells, documented in multiple hESC and induced pluripotent stem cell (iPSC) lines, and reminiscent of the extended proliferation we previously reported in HPCs derived from iPSCs generated from patients with acute myeloid leukemia (AML) (Kotini et al. Cell Stem Cell 2017). Induction of MYC in the MYC-Amp model had similar effects in differentiation and proliferation, albeit much less pronounced, extending the growth of HPCs to approximately 50 days. This milder phenotypic perturbation is likely due to the downregulation of amplified MYC, as opposed to the continuous high expression of the ectopically expressed MYC in the MYC-DoxOE model. In summary, we have generated a novel model that offers for the first time the opportunity to study the effects of MYC deregulation by different genetic mechanisms in human hematopoiesis in a dynamic fashion. Disclosures No relevant conflicts of interest to declare.
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Yoshihara, Hiroki, Michelle L. Churchman, Jennifer L. Peters, David B. Finkelstein, Elisabeth M. Paietta, Mark R. Litzow, and Charles G. Mullighan. "Functional and Genomic Characterization of the Interaction between Acute Lymphoblastic Leukemia Cells and the Microenvironment Identifies Pathways for Therapeutic Intervention." Blood 132, Supplement 1 (November 29, 2018): 1550. http://dx.doi.org/10.1182/blood-2018-99-117894.
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Abstract Introduction Residence and interaction with a specialized bone marrow microenvironment is important for normal hematopoietic stem cells and for initiation and progression of myeloid malignancies, but the role of the microenvironment in propagation and therapeutic response of acute lymphoblastic leukemia (ALL) is not well known. Prior work has identified the efficacy of inhibiting FAK signaling, which is deregulated by IKZF1 alterations resulting in induction of THY1-Integrin alpha 5 adhesion in Ph-positive (Ph+) ALL. Here, we hypothesized that this mechanism may be more broadly important in ALL. We applied a systematic integrated genomic/imaging/functional approach to define the nature of interaction and identify changes in leukemic cells upon interaction that may be targetable. Materials and methods Time-lapse confocal imaging was performed to examine how leukemia cells migrate and adhere to mesenchymal stem cells (MSCs). NALM6 (DUX4/ERG), MHH-CALL2 (hypodiploid), 697 (TCF3-PBX1), Reh (ETV6-RUNX1) and SUP-B15 (Ph+) cell lines were cultured with immortalized human bone marrow MSCs transduced with telomerase reverse transcriptase (hTERT) (Mihara, Br J Haematol. 2003;120:846). For RNA-sequencing, non-adherent cell line cells were collected after two days of coculture with hTERT while adherent cells were trypsinized and collected. Both samples were sorted for CD19 positive population. Fresh primary ALL samples were cultured on bone marrow MSCs derived from patients with no hematological disease and collected with the same procedure for RT-PCR. Multicolor immunofluorescence imaging was utilized to observe expression of multiple molecules involved in adherence. Results Time-lapse imaging showed that leukemia cells have a dynamic interaction with MSC monolayers, with temporary adherence, accompanied by dynamic change in their shape. NALM6 cells adherent to MSCs reduced cell cycling, with an increase in the ratio of G0/G1 cells (26.7% to 48.0%) and decrease in S phase (60.7 to 41.8%). Analysis of gene expression showed 138 upregulated genes (log2FC >2 and FDR <0.05) in adherent cells which were common in all five cell lines, with striking upregulation leading to gene expression associated with gene ontology of extracellular matrix organization and collagen fibril organization. Representative genes were validated in adherent NALM6 cells by immunoblotting (FN1, TIMP1, and LGALS1). Pathway analysis showed that transforming growth factor beta 1 (TGFB1) was the top ranked upstream regulator (p-value of overlap 5.26E-35) in that 44 of 65 genes had measurement direction consistent with activation of TGFB1 signaling. In addition, other upstream regulators that may be involved were beta-estradiol, fibroblast growth factor 2, and tumor necrosis factor. Adhesion of leukemia cells to stroma may induce integrin expression and downstream signaling. Our transcriptomic analysis showed that integrins (A5, B1, A3 and B5) and caveolin 1 (CAV1), a main component of the caveolae plasma membranes, are highly transcribed in adherent leukemia cells. As hTERT cells showed unexpectedly low expression of THY1, a common MSC marker, we utilized primary bone marrow MSC for subsequent analysis. Immunoblotting assay showed enhanced expression of CAV1 in all cell lines adhered to MSCs. Multicolor immunofluorescence imaging demonstrated CAV1 and ITGB1 expression on leukemia cells that localized adjacent to stromal cells, which confirms that these molecules were upregulated upon adhesion to MSCs. Moreover, primary ALL cells showed remarkable upregulation of CAV1, ITGB1, ITGA3, and ITGB5 when the cells were adherent to MSCs. Conclusions Our results demonstrate that ALL cells dynamically interact with microenvironment cells, inducing changes in cell morphology, cell cycling, and adhesion, which may facilitate altered responsiveness to therapy. Transcriptional results suggest that TGFβ signaling is an upstream regulator after cell adhesion to MSCs. While integrins and CAV1 mediate the signaling, these pathway and molecules will be candidates for exploring inhibitors of signaling, which may affect their interaction and make them novel therapeutic targets. Figure. Figure. Disclosures Mullighan: Loxo Oncology: Research Funding; Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau.
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Dörr, Jan, Selina Keppler, Maja Milanovic, Simone Spieckermann, Peter Aichele, Bernd Dörken, and Clemens A. Schmitt. "Therapy-Inducible Senescence Activates Both Innate and Adaptive Immune Mechanisms That Control Lymphoma Growth In Vivo." Blood 118, no. 21 (November 18, 2011): 107. http://dx.doi.org/10.1182/blood.v118.21.107.107.
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Abstract Abstract 107 Introduction: Premature senescence is a cellular failsafe mechanism which is induced upon various cellular insults, such as oncogene activation or exposure to DNA damaging chemotherapy. It suppresses tumor formation and acts as a barrier to tumor progression in vivo. In contrast to apoptotic cells, senescent cells are viably arrested in the G1 phase of the cell cycle. They continue to take up nutrients and interact with tumor and host cells. To what extent senescent cells alter the tumor environment and tumor-host interactions remains largely unsolved. Here, we analyze lymphoma cells with defined genetic lesions, e.g. deletion of the histone H3 lysine 9 methyltransferase Suv39h1 (controlling senescence) and p53 (mediating both apoptosis and senescence), for their influence on immunological tumor-host interactions as a consequence of therapy-induced senescence (TIS) in the Eμ-myc mouse lymphoma model. Our data demonstrate for the first time a senescence-primed T-cell response against lymphoma cells in vitro and in vivo. Methods: Lymphoma cells (LCs) from different genetic were retrovirally transduced with the bcl2 gene to block apoptosis. Subsequently, they were treated with the DNA damaging anticancer agent adriamycin in vitro or the alkylating agent cyclophosphamide upon lymphoma formation in normal immunocompetent mice in vivo. Therapy-inducible senescence (TIS) was detected based on senescence-associated b-galactosidase activity (SA-b-gal), Ki67 staining and BrdU incorporation. The cytokine profile of senescent LCs was analysed by gene expression and protein arrays. Infiltration and activation of immune cells in TIS lymphomas was analysed by immunohistochemistry and flow cytometry with leukocyte-specific antibodies. Immune responses elicited upon TIS induction in vivo were further analysed in gld (generalized lymphoproliferative disease) mice, which lack functional FasL and by systemic depletion of macrophages after clodronate administration. Pharmaceutical inhibitors of FasL and perforin and IFNg knockout mice were used to analyze T-cell mediated cytotoxity in vitro. Results: TIS lymphoma cells, but not Suv39h1- or p53-deficient LCs, upregulate the secretion of pro-inflammatory cytokines, such as IL6 and IL12, with pro-inflammatory on tumor and bystander cells. In vivo, TIS correlates with the attraction of immune cells, particularly macrophages and T cells, to the tumor site. Senescent LCs became sensitive to both macrophage engulfment and death receptor (Fas)-mediated apoptosis. Activation of both CD4 and CD8 T cells leads to production of IFNg and clearing of senescent cells. Clearance can be attenuated by systemic depletion of macrophages and interference with T cell-mediated programmed cell death. T-cells specifically primed by TIS cells in vivo potently killed both senescent and proliferating LCs after isolation and co-incubation in vitro. In vivo clearance of TIS LCs was attenuated by systemic depletion of macrophages or by interference with T-cell-mediated programmed cell death. Lymphoma-bearing gld mice presented with a reduced overall survival when compared to wild-type host mice. Discussion: This study demonstrates that therapy-induced senescence drives a profound remodeling of the tumor site after therapy and unveils functional interactions of senescent LCs with different immune cell subsets in vitro and in vivo. Senescent cells secrete a cytokine program, which stimulates immune cell attraction and an adaptive and presumably lastingly protective immune response. Thus, TIS is a highly dynamic and interdependent process whose paracrine effects and immune cell interactions account for regression of the senescent mass and present an attractive target network for novel therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.
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Dörr, Jan, Maja Milanovic, Christoph Loddenkemper, Dido Lenze, Yong Yu, Bernd Dörken, and Clemens Schmitt. "Role of Senescence in Dynamic Tumor/Host Responses to Cancer Therapy In Vivo." Blood 114, no. 22 (November 20, 2009): 928. http://dx.doi.org/10.1182/blood.v114.22.928.928.
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Abstract Abstract 928 Introduction: Premature senescence is a terminal G1 growth arrest in response to acute cellular stresses, such as oncogene activation or exposure to DNA damaging chemotherapy, with tumor-suppressive activity in vivo. However, senescent cells remain viable. They are metabolically active and have a distinct senescence-associated secretory profile (SASP). Therefore, the function and the fate of senescent cells within the tumor site, particularly their impact on the tumor microenvironment, are highly complex and not well characterized. Here, we analyze lymphoma cells with defined genetic lesions, e.g. deletion of the histone H3 lysine 9 methyltransferase Suv39h1 (controlling senescence) and p53 (mediating both apoptosis and senescence), for their influence on immunological tumor-host and growth-modulating senescent/non-senescent cell interactions as a consequence of therapy-induced senescence (TIS) in the Eμ-myc mouse lymphoma model. We identify novel cell-autonomous and non-cell-autonomous components of the senescence response in vitro and in vivo, which arise as new targets for lymphoma therapy. Methods: Lymphoma cells (LCs) from different genetic backgrounds (Suv39h1-, p53null, atm-/-, p16INK4a-/-, p19ARF-/-, p21CIP1-/-) were retrovirally transduced with the bcl2 gene to block apoptosis. Subsequently, they were treated with the DNA damaging anticancer agent adriamycin in vitro or the alkylating agent cyclophosphamide upon lymphoma formation in normal immunocompetent mice in vivo. TIS was detected based on senescence-associated β-galactosidase activity (SA-β-gal), Ki67 staining and BrdU incorporation. The SASP of senescent LCs was analysed by gene expression and cytokine arrays. A large pharmacological inhibitor screen was used to identify signalling cascades activated by the SASP. To study tumor-host cell interactions in vitro, freshly isolated spleen cells were co-incubated with proliferating or TIS LCs. Immunophenotyping was carried out with leukocyte-specific antibodies. Immune responses elicited upon TIS induction in vivo were further analysed in gld (generalized lymphoproliferative disease) mice, which lack functional FasL, and by systemic depletion of macrophages after clodronate administration. Results: TIS lymphoma cells, but not Suv39h1- or p53-deficient LCs, exhibit a SASP with pro-inflammatory and pro-senescent action on tumor and bystander cells. We identify novel components of the SASP profile with the potential to induce a secondary senescent arrest in bcl2 protected Eμ-myc lymphoma cells via the MAPK and TGFβ pathway. In vivo, TIS correlates with the attraction of immune cells to the tumor site and subsequent clearing of senescent cells, which can be attenuated by systemic depletion of macrophages and interference with T cell- mediated programmed cell death. Senescent LCs interact with different immune cell subsets in vitro, in particular macrophages, granulocytes and T-cells, and become sensitive to macrophage engulfment and death-receptor mediated apoptosis, for example by Fas-FasL cytotoxicity. Discussion: This study demonstrates that therapy-induced senescence drives a profound remodeling of the tumor site after therapy and unveils functional interactions of senescent LCs with proliferating tumor cells and different immune cell subsets in vitro and in vivo. Senescent cells secrete a cytokine program, which limits tumor growth and stimulates immune cell attraction, hereby promoting their own clearance. Thus, TIS is a highly dynamic and interdependent process whose paracrine effects and immune cell interactions account for regression of the senescent mass and present an attractive target network for novel therapeutic strategies. Disclosures: No relevant conflicts of interest to declare.
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Gaines, Peter, Deepali Gotur, Stephanie Halene, Ada L. Olins, and Donald E. Olins. "Dynamic Expression of the Lamin B Receptor and Associated Proteins during Myeloid Differentiation: Insight into Mechanisms of Neutrophil Vs. Macrophage Morphogenesis." Blood 112, no. 11 (November 16, 2008): 3546. http://dx.doi.org/10.1182/blood.v112.11.3546.3546.
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Abstract Neutrophils and macrophages are professional phagocytes that are essential to the immune response and that have developmental and functional similarities. They derive from a common myeloid progenitor and require common transcription factors for their differentiation, such as PU.1 and the CCAAT/enhancer binding proteins C/EBPα and C/EBPε. However, the morphologic maturation of these two lineages is distinct; neutrophil progenitors undergo a progressive series of nuclear changes that culminate in the formation of lobulated nuclei, whereas macrophage progenitors first form circulating monocytes that contain indented nuclei, and then form mature tissue macrophages that contain small, spherical nuclei. To date, little is known regarding the mechanism that regulates nuclear morphogenesis of either lineage. In recent efforts to further understand neutrophil differentiation, we and others have demonstrated that nuclear lobulation in both human and mouse neutrophils is dependent on lamin B receptor (LBR) expression. LBR is an integral membrane protein of the inner nuclear envelope that interacts with B-type lamins and heterochromatin. We have shown that LBR expression is significantly upregulated during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of murine hematopoietic progenitor EML/EPRO cells, which parallels changes in LBR expression exhibited by ATRA-induced human leukemic HL-60 cells. Both models show decreased expression of lamin A/C during neutrophil maturation, whereas lamin B expression remains constant. Importantly, loss of LBR protein expression causes hypolobulation of neutrophil nuclei that is characteristic of the Pelger-Huët anomaly in both mice and humans. These studies support the notion that dynamic changes in the expression of LBR and perhaps lamin A/C play critical roles in neutrophil nuclear maturation. Studies that examine macrophage nuclear maturation, however, have been limited to those performed on HL-60 cells, which showed that LBR expression increased during phorbol ester (TPA) induction. We have now examined the expression of LBR in two additional stem cell factor (SCF)-dependent murine myeloid cell lines that can be induced toward mature neutrophils, pBIM and SCF ER-Hoxb8, as well as in a GM-CSF-dependent ER-Hoxb8 cell line that differentiates into mature macrophages. The pBIM cell line was generated by insertional mutagenesis of mouse marrow cells with the empty pBabe-puro vector. The ER-Hoxb8 cell lines were generated from mouse marrow that was transduced with an estrogen-regulated Hoxb8, which arrests myeloid differentiation. Similar to our findings in EML/EPRO cells, Lbr transcription was upregulated in pBIM cells upon G-CSF-induced neutrophil differentiation, and LBR protein levels significantly increased in SCF ER-Hoxb8 cells upon estrogen withdrawal. By comparison, Lbr transcription dramatically increased during the initial stages of macrophage differentiation of GM-CSF ER-Hoxb8 progenitors, but then decreased in mature macrophages. Initial increases in Lbr transcription during both neutrophil and macrophage differentiation paralleled upregulated expression of both PU.1 and C/EBPε. We also detected increased expression of Lbr in pBIM cells that were generated from a C/EBPε-knockout mouse. This result was surprising given that very recent studies have demonstrated that C/EBPε can directly activate the Lbr promoter. Finally, lamin A/C expression increased during macrophage differentiation of GM-SCF ER-Hoxb8 cells, but was minimally detected in uninduced or differentiated SCF ER-Hoxb8 cells. Together our data indicate that changes in the expression of LBR and lamin A/C play important roles during the maturation of both granulocyte and macrophage lineages, and that PU.1 and/or C/EBPε drive these changes in expression. However, our analyses of C/EBPε-knockout cells indicate that LBR transcription is not absolutely dependent on C/EBPε expression. Current studies to identify the expression and interplay of nuclear envelope proteins during neutrophil and macrophage differentiation that employ these cell line models as well as primary marrow progenitors will further define the mechanisms that regulate morphologic maturation of two important phagocytes.
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Rodrigues Lopes, Matheus, João Agostinho Machado-Neto, Fabiola Traina, João Kleber Novais Pereira, Irene Lorand-Metze, Fernando Ferreira Costa, Sara Teresinha Olalla Saad, and Patricia Favaro. "FMNL1 Is Downregulated In Myelodysplastic Syndromes and Is Involved In Megakaryocytic Differentiation." Blood 122, no. 21 (November 15, 2013): 1519. http://dx.doi.org/10.1182/blood.v122.21.1519.1519.
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Abstract Background Myelodysplastic syndromes (MDS) are disorders characterized by morphological dysplasia, impaired differentiation and defective cellular functions, resulting in peripheral cytopenias. FMNL1 belongs to a family of formin-related proteins, indispensable for many fundamental actin-dependent processes. Recently, FMNL1 has been described to be upregulated and play a role in the actin cytoskeleton dynamics during monocyte differentiation to macrophages. Aims The aim of this work was to characterize FMNL1 expression in total bone marrow cells of patients with MDS comparing to normal donors. We also analyzed FMNL1 expression in erythrocytic, granulocytic and megakaryocytic differentiation, using cell line models. Finally, we evaluated the impact of inhibition of FMNL1 during megakaryocytic differentiation. Methods A total of 49 patients with a diagnosis of MDS, receiving no treatment, and 18 samples from normal donors were included in the study, which was approved by the National Ethical Committee Board. Samples were submitted to RNA extraction after removal of erythrocytes by hemolysis. FMNL1 expression levels from cell lines or total bone marrow cells were determined by quantitative PCR (q-PCR) or Western blot. KU812 was treated 50 μM hemin and 100 μM hydroxyurea for erythrocytic differentiation. K562 was stimulated with 20nM of PMA for megakaryocytic differentiation. NB4 was treated with 10-6 M of ATRA for granulocytic differentiation. Megakaryocytic differentiation was followed by the increase in megakaryocytic marker (CD61) determined by flow cytometry and cells were also stained with May–Grunwald–Giemsa. K562 cells were transduced with lentivirus-mediated shRNA targeting LacZ or FMNL1. Apoptosis was assessed by Annexin-V/PI staining and cell cycle was evaluated by flow cytometry, both at 24 and 48 hours after induction with PHA. The statistical methods used were the age-adjusted multivariate linear regression analysis, Mann Whitney test or t test. Results FMNL1 expression in bone marrow samples was significantly lower in MDS when compared with normal donor cells (P=0.01), especially in the high risk group (P<0.02). Using cell line models for hematopoietic differentiation, there was a fifteen-fold increase and a five-fold increase in FMNL1 expression for megakaryocytic (P=0.002) and granulocytic differentiation (P=0.05) respectively. Western blot analysis corroborated these findings. There was no difference for erythrocytic differentiation. After PMA treatment, the level of the megakaryocytic markers CD61 was significantly lower in K562 shFMNL1 when compared with shLacZ (P=0.01). The level of CD41a (P=0.5) and CD42b (P=0.1) showed a trend toward a decrease in K562 shFMNL1 when compared with shLacZ, however not statistically significantly. Corroborating these data, K562 shFMNL1 showed abnormal megakaryocytic morphological features (larger cells with polylobulated or polysegmented nuclei and vacuolization) compared with cells shLacZ. There were no statistical differences in the apoptosis levels and cell cycle analysis between K562 shFMNL1 and shLacz cells. Conclusions The family of formin-related proteins has mainly been related to actin-dependent processes although little is known regarding their possible involvement in haematopoiesis. The lower FMNL1 expression in MDS BM could reflect the role of this protein in cell differentiation. We were therefore prompted to study this issue in depth using megakaryocytic differentiation as a system. In this study, we show increased expression of FMNL1 in PMA-induced megakaryocytic differentiation of K562 cells. Furthermore, knockdown of FMNL1 deregulates differentiation, suggesting that FMNL1 is required in order to maintain the effective megakaryocytopoiesis in MDS. Although FMNL1 silencing effectively down-regulated CD61 expression, CD41a and CD42b were reduced to a lesser extent. The effect of FMNL1 cannot be explained by modifications of the cell cycle or apoptosis during differentiation; and is probably due to the effect in changes in the dynamic remodeling of the cytoskeleton. Disclosures: No relevant conflicts of interest to declare.
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Gregory, Georgia L., Beeke Wienert, Marisa Schwab, Billie Rachael Lianoglou, Roger P. Hollis, Donald B. Kohn, Bruce R. Conklin, and Tippi C. MacKenzie. "Investigating Zeta Globin Gene Expression to Develop a Potential Therapy for Alpha Thalassemia Major." Blood 136, Supplement 1 (November 5, 2020): 3–4. http://dx.doi.org/10.1182/blood-2020-142922.
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Introduction: Alpha globin mutations are very common worldwide, and the severity of resulting anemia depends on the number and type of mutated alleles. While the 4 gene mutation (alpha thalassemia major, ATM) was previously deemed fatal except in rare cases, emerging evidence indicates that survival to birth and good postnatal outcomes are possible with in utero transfusions. We hypothesized that the embryonic zeta globin gene, which is expressed early in gestation prior to alpha globin, may compensate for the lack of alpha globin and that induction of zeta globin after it has naturally been silenced may become a new therapy for patients with ATM. Methods: We evaluated mutations in the UCSF international registry of patients with ATM to understand factors related to patient survival with and without in utero transfusions. We then engineered Human Umbilical Cord Derived Erythroid Progenitor Cells (HUDEP-2 cells) carrying the common SEA alpha globin deletion, in which zeta globin expression is preserved (H-SEA), as well as those on which the zeta globin genes were deleted (HBZ-/-) using CRISPR-Cas9. We evaluated the expression of alpha and zeta globins using qPCR, Western blot, and flow cytometry. We generated lentiviral vectors expressing zeta globin under the control of beta-globin promoters to examine changes in both zeta and alpha globin in a dynamic way. Results: None of the registry patients who survived to birth spontaneously (n=11) had a mutation that involves a concomitant deletion in zeta globin (such as the -FIL, -THAI, or -MEDII mutation), while alpha globin mutations extending into the zeta globin gene were found in 14 of 37 (38%) patients who were diagnosed prenatally, suggesting that the presence of zeta globin may play a role in the ability to survive to birth without fetal therapy. Interestingly, we found that H-SEA clones express higher levels of zeta globin than WT cells, as illustrated by quantitative real-time PCR (Fig 1A), Western blot (Fig 1B) and flow cytometry (Fig 1C). These cells also developed beta globin dimers due to excess unpaired beta-globin chains, as demonstrated by Western blotting with and without reducing agents, indicating that they are an appropriate cell model for ATM. We next generated HUDEP-2 clones lacking zeta globin (HBZ KO) and transduced these clones with lentiviral vectors expressing high levels of zeta globin (lenti-zeta) (Fig 1D). Western blotting revealed that increasing the levels of zeta globin in these cells resulted in decreased expression of alpha globin, suggesting reciprocal control between these genes (Fig 1E). Most importantly, we saw a reduction in toxic beta-globin dimers in H-SEA cells expressing high levels of zeta-globin after transduction with lenti-zeta, suggesting that zeta globin could functionally replace the missing alpha-globin (Fig 1 F,G). To understand transcriptomic differences in H-SEA cells that may result in increased zeta globin expression, we performed bulk RNA sequencing of wild type and H-SEA clones. We confirmed that zeta expression is significantly upregulated in H-SEA compared to wild type (log2 fold change of 4.25, p=2.24E-38). Pathway analysis of differentially expressed genes is ongoing. Conclusions: Our international patient registry suggests that expression of zeta globin may play a role in the spontaneous survival to birth in a subset of patients. Zeta globin expression is increased in the setting of H-SEA cells in vitro, and restoration of zeta expression by lentivirus results in a reduction of toxic beta globin dimers in these ATM cells. Furthermore, expressing zeta globin at high levels in H-WT cells decreased alpha globin expression, suggesting a reciprocal regulation of these two genes. This concept is similar to the relationship between fetal gamma and adult beta globins which has been exploited for therapeutic editing approaches in patients with beta-thalassemia. At this point, the natural repressor of zeta globin is not yet known, but our data suggests that a strategy of upregulating zeta globin could potentially be developed to mimic the ongoing trials of using the BCL11A repressor to induce gamma globin in patients with beta thalassemia and sickle cell disease. Disclosures Wienert: Integral Medicines: Current Employment. Kohn:Allogene Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Patents & Royalties, Research Funding. MacKenzie:Acrigen: Membership on an entity's Board of Directors or advisory committees; Ultragenyx: Research Funding.
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Noce, Valeria, Cecilia Battistelli, Angela Maria Cozzolino, Veronica Consalvi, Carla Cicchini, Raffaele Strippoli, Marco Tripodi, Alessandra Marchetti, and Laura Amicone. "YAP integrates the regulatory Snail/HNF4α circuitry controlling epithelial/hepatocyte differentiation." Cell Death & Disease 10, no. 10 (October 2019). http://dx.doi.org/10.1038/s41419-019-2000-8.
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Abstract Yes-associated protein (YAP) is a transcriptional co-factor involved in many cell processes, including development, proliferation, stemness, differentiation, and tumorigenesis. It has been described as a sensor of mechanical and biochemical stimuli that enables cells to integrate environmental signals. Although in the liver the correlation between extracellular matrix elasticity (greatly increased in the most of chronic hepatic diseases), differentiation/functional state of parenchymal cells and subcellular localization/activation of YAP has been previously reported, its role as regulator of the hepatocyte differentiation remains to be clarified. The aim of this study was to evaluate the role of YAP in the regulation of epithelial/hepatocyte differentiation and to clarify how a transducer of general stimuli can integrate tissue-specific molecular mechanisms determining specific cell outcomes. By means of YAP silencing and overexpression we demonstrated that YAP has a functional role in the repression of epithelial/hepatocyte differentiation by inversely modulating the expression of Snail (master regulator of the epithelial-to-mesenchymal transition and liver stemness) and HNF4α (master regulator of hepatocyte differentiation) at transcriptional level, through the direct occupancy of their promoters. Furthermore, we found that Snail, in turn, is able to positively control YAP expression influencing protein level and subcellular localization and that HNF4α stably represses YAP transcription in differentiated hepatocytes both in cell culture and in adult liver. Overall, our data indicate YAP as a new member of the HNF4/Snail epistatic molecular circuitry previously demonstrated to control liver cell state. In this model, the dynamic balance between three main transcriptional regulators, that are able to control reciprocally their expression/activity, is responsible for the induction/maintenance of different liver cell differentiation states and its modulation could be the aim of therapeutic protocols for several chronic liver diseases.
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Suehiro, Jun-ichi, Mai Miura, Tatsuhiko Kodama, and Takashi Minami. "Abstract 1301: NFATc- And SRF-induced Early Growth Response (Egr)-3 Is Essential For VEGF-mediated Endothelial Cell Migration And Tube Formation." Circulation 116, suppl_16 (October 16, 2007). http://dx.doi.org/10.1161/circ.116.suppl_16.ii_266.
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Endothelium is a dynamic cell layer constantly responding to changes in the various extracellular mediators. Responses are usually beneficial to host, but oversustained or dysregulated responses can result in vascular dysfunction, leading to the initiation of atherosclerosis, tumor growth, and inflammation. Such endothelial cell activation and dysfunction are mediated in large part by alterations in gene expression. Here we show, using comprehensive transcriptome analyses, that VEGF, thrombin and TNF-α each induces a dramatic and rapid up-regulation of early growth response (Egr)-3 in human umbilical vein endothelial cells (HUVEC). The effect of VEGF on Egr-3 was similar in human coronary artery, pulmonary artery, and dermal microvascular endothelial cells. In chemical inhibitor studies, VEGF-mediated induction of Egr-3 (mRNA peak 300-fold at 45 min) depended on MEK1/2, JNK, PI3K, PKA, and Ca-calcineurin. Egr-3 promoter luciferase and electrophoretic mobility shift analysis revealed that the 5′-flanking region at −57 to −130 was necessary and sufficient for transducing of VEGF-mediated Egr-3 upregulation and that region could bind NFATc1, c2, and SRF. Co-transfection assays with Egr-3 reporter and either NFATc or SRF expression plasmids resulted in 4 and 2 -fold Egr-3 promoter activation, respectively. In DNA microarray studies, HUVEC treated with VEGF for 1 and 4 hours in the presence of two independent siRNAs against Egr-3, 25 and 70 VEGF-inducible genes were strikingly downregulated, respectively. The pro-angiogenesis factors Ets-1, CXCL1, and tissue factor were among those genes downregu-lated. SiRNA knockdown of Egr-3 markedly impaired VEGF-mediated cell migration and tube formation, as determined by in vitro wound healing, boyden chamber, and collagen gel assays. Moreover in aortic ring assays, VEGF-stimulated neo-angiogenesis from the extracted mice aorta was completely abolished by administration of adenoviral-transferred miRNA against Egr-3. Collectively, these findings suggest that NFATc and SRF cooperatively upregulate Egr-3. Egr-3 might have an important function as a signal transducer in VEGF-mediated angiogenesis in activated endothelium.
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Hershey, David M., Aretha Fiebig, and Sean Crosson. "Flagellar Perturbations Activate Adhesion through Two Distinct Pathways in Caulobacter crescentus." mBio 12, no. 1 (February 9, 2021). http://dx.doi.org/10.1128/mbio.03266-20.
Full textAbstract:
ABSTRACT Bacteria carry out sophisticated developmental programs to colonize exogenous surfaces. The rotary flagellum, a dynamic machine that drives motility, is a key regulator of surface colonization. The specific signals recognized by flagella and the pathways by which those signals are transduced to coordinate adhesion remain subjects of debate. Mutations that disrupt flagellar assembly in the dimorphic bacterium Caulobacter crescentus stimulate the production of a polysaccharide adhesin called the holdfast. Using a genomewide phenotyping approach, we compared surface adhesion profiles in wild-type and flagellar mutant backgrounds of C. crescentus. We identified a diverse set of flagellar mutations that enhance adhesion by inducing a hyperholdfast phenotype and discovered a second set of mutations that suppress this phenotype. Epistasis analysis of the flagellar signaling suppressor (fss) mutations demonstrated that the flagellum stimulates holdfast production via two genetically distinct pathways. The developmental regulator PleD contributes to holdfast induction in mutants disrupted at both early and late stages of flagellar assembly. Mutants disrupted at late stages of flagellar assembly, which assemble an intact rotor complex, induce holdfast production through an additional process that requires the MotAB stator and its associated diguanylate cyclase, DgcB. We have assigned a subset of the fss genes to either the stator- or pleD-dependent networks and characterized two previously unidentified motility genes that regulate holdfast production via the stator complex. We propose a model through which the flagellum integrates mechanical stimuli into the C. crescentus developmental program to coordinate adhesion. IMPORTANCE Understanding how bacteria colonize solid surfaces is of significant clinical, industrial and ecological importance. In this study, we identified genes that are required for Caulobacter crescentus to activate surface attachment in response to signals from a macromolecular machine called the flagellum. Genes involved in transmitting information from the flagellum can be grouped into separate pathways, those that control the C. crescentus morphogenic program and those that are required for flagellar motility. Our results support a model in which a developmental and a mechanical signaling pathway operate in parallel downstream of the flagellum and converge to regulate adhesion. We conclude that the flagellum serves as a signaling hub by integrating internal and external cues to coordinate surface colonization and emphasize the role of signal integration in linking complex sets of environmental stimuli to individual behaviors.