Regulation Of Hematopoietic Cells Research Articles

Understanding immunoactinopathies: A decade of research on WAS gene defects.

Immunoactinopathies caused by mutations in actin-related proteins are a growing group of inborn errors of immunity (IEI). Immunoactinopathies are caused by a dysregulated actin cytoskeleton and affect hematopoietic cells especially because of their unique capacity to survey the body for invading pathogens and altered self, such as cancer cells. These cell motility and cell-to-cell interaction properties depend on the dynamic nature of the actin cytoskeleton. Wiskott-Aldrich syndrome (WAS) is the archetypical immunoactinopathy and the first described. WAS is caused by loss-of-function and gain-of-function mutations in the actin regulator WASp, uniquely expressed in hematopoietic cells. Mutations in WAS cause a profound disturbance of actin cytoskeleton regulation of hematopoietic cells. Studies during the last 10 years have shed light on the specific effects on different hematopoietic cells, revealing that they are not affected equally by mutations in the WAS gene. Moreover, the mechanistic understanding of how WASp controls nuclear and cytoplasmatic activities may help to find therapeutic alternatives according to the site of the mutation and clinical phenotypes. In this review, we summarize recent findings that have added to the complexity and increased our understanding of WAS-related diseases and immunoactinopathies.

Evaluating Histone Acetylation in Mouse Hematopoietic Stem and Progenitor Cells Using Chromatin Immunoprecipitation.

Epigenetics is the study of how cells control gene activity without changing the DNA sequence. Various epigenetic processes have been identified, including methylation, acetylation, phosphorylation, and ubiquitylation. Epigenetic processes are natural and essential to cell functions; however, when they occur improperly or at the wrong time, adverse effects can occur. A significant epigenetic process is chromatin modification. Chromatin-DNA complexes can be modified by acetylation, altering chromatin structure to influence gene expression. Stresses to hematopoietic stem and progenitor cells, such as ionizing radiation and aging, have significant effects on genomic function. Understanding epigenetic regulation in hematopoietic cells, particularly under stress, offers the potential for therapeutic intervention. We have utilized Chromatin immunoprecipitation (ChIP) in HSPCs to understand epigenetic regulation in response to ionizing radiation. This technique can be applied reliably to rare hematopoietic cells and offers a powerful tool to explore epigenetic regulation in HSPCs.

Characterization of the MYB-inhibitory potential of the Pan-HDAC inhibitor LAQ824

A large body of work has shown that MYB acts as a master transcription regulator in hematopoietic cells and has pinpointed MYB as a potential drug target for acute myeloid leukemia (AML). Here, we have examined the MYB-inhibitory potential of the HDAC inhibitor LAQ824, which was identified in a screen for novel MYB inhibitors. We show that nanomolar concentrations of LAQ824 and the related HDAC inhibitors vorinostat and panobinostat interfere with MYB function in two ways, by inducing its degradation and inhibiting its activity. Reporter assays show that the inhibition of MYB activity by LAQ824 involves the MYB transactivation domain and the cooperation of MYB with co-activator p300, a key MYB interaction partner and driver of MYB activity. In AML cells, LAQ824-induced degradation of MYB is accompanied by expression of myeloid differentiation markers and apoptotic and necrotic cell death. The ability of LAQ824 to inhibit MYB activity is supported by the observation that down-regulation of direct MYB target genes MYC and GFI1 occurs without apparent decrease of MYB expression already after 2 h of treatment with LAQ824. Furthermore, ectopic expression of an activated version of MYB In HL60 cells counteracts the induction of myeloid differentiation by LAQ824. Overall, our data identify LAQ824 and related HDAC inhibitors as potent MYB-inhibitory agents that exert dual effects on MYB expression and activity in AML cells.

Role of Sclerostin in Multiple Myeloma Progression

Multiple Myeloma (MM) is a tumor of plasma cells that originates and expands within bone marrow (BM). The BM microenvironment, including its constituent stromal cells and cytokines contribute to disease progression. The BM stromal cells include osteolineage cells, osteoclasts, adipocytes, endothelial cells etc. Among these, cells of the osteoblast lineage have been identified as the most versatile regulators of hematopoietic cells. The osteolineage cells include preosteoblasts, committed osteoblasts, mature osteoblasts, and the terminally differentiated osteocytes, each with its own characteristic biological activity. The osteolineage cells have been shown to regulate commitment and differentiation of hematopoietic lineage cells including hematopoietic stem cells, B-cells, and myeloid lineage cells.Previously published data have shown that loss of parathyroid hormone (PTH) signaling in osteolineage cells adversely affects B-cell development (Panaroni et al., 2016). PTH is also a strong inhibitor of sclerostin, a Wnt-signaling inhibitor secreted by osteocytes. Moreover, mice lacking osteocytes show decreased lymphopoiesis, which may in part be regulated by sclerostin. A sclerostin-neutralizing antibody has shown promise for treatment for age-induced osteoporosis. We have recently demonstrated that MM patients have increased levels of sclerostin and that treatment with a SOST neutralizing antibody resulted in increased osteoblastic differentiation (Eda et al., 2015) and decreased tumor burden. Based upon these findings, we hypothesized that the osteolineage cells contribute to MM progression, and that this may in part occur via sclerostin. To test this hypothesis, we have used genetic mouse models of osteolineage or sclerostin deficiency. Mouse MM cell line 5TGM1 was used to examine MM progression in these mouse models.Global sclerostin deficient (SOST KO) mice were generated as described previously. Given that sclerostin acts as a negative regulator of bone formation, SOST KO mice displayed robust increase in trabecular and cortical bone density as analyzed by micro-computed tomography. Co-culture with 5TGM1 MM cells showed that the BM stromal cells from SOST KO mice significantly decreased proliferation of MM cells raising the possibility that sclerostin deficiency may decrease MM progression in vivo. The SOST KO mice showed significantly reduced BM cellularity compared to the controls. However, relevant to MM progression, overall B-lymphopoiesis, proliferation or apoptosis was unchanged during any stages of B-cell development in BM of 3-month old SOST KO mice.To determine if sclerostin deficiency alters MM engraftment in vivo, 5TGM1 cells were injected into the tibia of 3-month old control or SOST KO mice and engraftment was followed for 2-months. Complete blood counts (CBC) showed no significant changes between the control and SOST KO mice injected with the MM cells. The 5TGM1 cells were fluorescently tagged before in vivo injections allowing us to track the cells by whole body bioluminescence imaging and by contralateral tibia BM flow cytometry analysis. Both the analyses showed no changes in the distant MM engraftment between the control and the SOST KO mice. Ongoing work is examining local MM engraftment in the tibia with injected MM cells.These data suggest that the sclerostin deficiency does not alter systemic MM engraftment after 2 months of follow-up observations. It is possible, however, that the local progression of MM cells may have altered. We are investigating this possibility. We are also examining MM engraftment in mice lacking mature osteoblasts and osteocytes to identify sclerostin-dependent and independent factors controlling MM progression. DisclosuresRaje:Millenium: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Onyx: Consultancy.

Adhesion of Flk1-expressing cells under shear flow in phospholipid polymer-coated immunoaffinity channels

Label-free cell separation is a promising method in the field of stem-cell research to obtain desired cell populations. Here, we report on phospholipid polymer-coated microfluidic channels with immobilized antibodies as devices for the capture of cells expressing target antigens in a label-free manner. We fabricated a microfluidic channel containing immobilized antibodies against vascular endothelial growth factor receptor 2 (Flk1), a potential marker for cardiac, angiogenic, and hematopoietic cell regeneration. A series of investigations was carried out to elucidate the effect of the immobilized antibodies on the adhesion behavior of the Flk1-expressing cell subpopulation derived from induced pluripotent stem cells. Increasing the immobilized antibody density (0.18–5.0 × 109 ligands mm−2) led to an increased number of cells adhering to the channel. The antibody-immobilized polymer-coated surface suppressed nonspecific cell adhesion, which was swept away by a weak shear flow, and captured Flk1-expressing cells under a wall shear stress of 1.7 Pa. Flk1 expression was 2.8-fold higher in the cells that adhered than in those that did not adhere. Therefore, an optimal antibody density and sweeping flow are required for effective label-free separation of Flk1-positive cells.

Radioprotective Potential of Nutraceuticals and their Underlying Mechanism of Action.

Radiations are an efficient treatment modality in cancer therapy. Besides the treatment effects of radiations, the ionizing radiations interact with biological systems and generate reactive oxygen species that interfere with the normal cellular process. Previous investigations have been conducted only on few synthetic radioprotectors, mainly owing to some limiting effects. The nutraceuticals act as efficient radioprotectors to protect the tissues from the deleterious effects of radiation. The main radioprotection mechanism of nutraceuticals is the scavenging of free radicals while other strategies involve modulation of signaling transduction pathways like MAPK (JNK, ERK1/2, ERK5, and P38), NF-kB, cytokines, and their protein regulatory gene expression. The current review is focused on the radioprotective effects of nutraceuticals including vitamin E, -C, organosulphur compounds, phenylpropanoids, and polysaccharides. These natural entities protect against radiation-induced DNA damage. The review mainly entails the antioxidant perspective and radioprotective molecular mechanism of nutraceuticals, DNA repair pathway, anti-inflammation, immunomodulatory effects and regeneration of hematopoietic cells.

Bone Marrow Endothelial Cells Take Up Blood-Borne Immune Complexes via Fcγ Receptor IIb2 in an Erythropoietin-Dependent Manner

Immune complexes (ICs) in blood are efficiently removed mainly by liver reticuloendothelial systems consisting of sinusoidal endothelial cells and Kupffer cells expressing FcγR. The bone marrow (BM) also has sinusoidal vasculatures, and sinusoidal BM endothelial cells (BMECs) bear unique function, including hematopoietic niches and traffic regulation of hematopoietic cells. In this study, we found that sinusoidal BMECs express FcγRIIb2, which is markedly increased in anemic conditions or by the administration of erythropoietin (Epo) in healthy mice. BMECs expressed Epo receptor (EpoR), and the Epo-induced increase in FcγRIIb2 expression was abolished in Epor-/- ::HG1-Epor transgenic mice, which lack EpoR in BMECs except for BM erythroblasts, suggesting the effect was directly mediated via EpoR on BMECs. Further, although BMECs hardly captured i.v.-injected soluble ICs in healthy mice, Epo administration induced a remarkable increase in the uptake of ICs in a FcγRIIb-dependent manner. Enhancement of the IC incorporation capacity by Epo was also observed in cultured BMECs in vitro, suggesting the direct effect of Epo on BMECs. Moreover, we found that i.v.-injected ICs in Epo-treated mice were more rapidly removed from the circulation than in PBS-treated mice. These results reveal a novel function of BMECs to efficiently remove circulating blood-borne ICs in an FcγRIIb2-mediated manner.

Asparagus racemosus Root Extract and Isoprinosine Exhibits Radio-mitigating Activity against Ionizing Radiation-induced Detrimental Effects in Swiss Albino Mice

Objectives: To evaluate the radio – mitigating property of Asparagus racemosus root ethanolic extract and Isoprinosine against ionizing radiationinduced harmful effects in Swiss albino mice. Methods: The experimental animals were exposed to sublethal dose (6 Gy) of whole-body electron beam radiation and administered orally with Asparagus racemosus root ethanolic extract (ARE – 200mg/kg bd. wt) and Isoprinosine (IPR – 400 mg/ kg bd. wt) for 15 consecutive days. After the experimental period, the animals were sacrificed. Blood and liver tissue were collected. Blood sample was used for assessment of hematological parameters, serum was used for biochemical analysis (lipid peroxidation, total antioxidant capacity) and the level of pro-inflammatory cytokines was determined in the liver homogenate. Results: The radiation control group showed a statistically significant reduction in the hematological parameters. IPR post-treatment group showed a substantial increase in the percentage of lymphocytes and monocytes. ARE and IPR post-treatment caused a statistically significant improvement in platelet count. Irradiation of mice resulted in a significant elevation in lipid peroxidation. Post-treatment of mice with ARE and IPR caused significant depletion in lipid peroxidation. The serum total antioxidant capacity (TAC) in the irradiated group decreased significantly. Administration of ARE to the animals after irradiation caused a significant elevation in TAC level. IPR post-treatment did not show any substantial change in the reduced level of TAC. The cytokines level in the liver homogenate of irradiated mice showed that Interleukin -2 (IL-2), Tumor Necrosis Factor alpha (TNF-α) and Interferon gamma (IFN-γ) significantly increased after radiation exposure. Administration of ARE and IPR after irradiation significantly reduced the levels of IL-2 and TNF-α in the posttreatment groups. An elevated level of IFN-γ was reduced considerably in the IPR post-treatment animals. Conclusion: Asparagus racemosus root ethanolic extract and Isoprinosine exhibit radio-mitigating effects against electron beam radiation - induced detrimental effects. This occurs through several mechanisms, such as free radical scavenging, anti-inflammation, facilitation of repair activity, regeneration of hematopoietic cells and affecting to molecular levels. Key words: Asparagus racemosus, Isoprinosine, Radio-mitigation, Ionizing radiation.

An expanded role for dipeptidyl peptidase 4 in cell regulation.

Dipeptidyl peptidase 4 (DPP4) is a serine protease with diverse regulatory functions in healthy and diseased cells. Much remains unknown about the mechanisms and targets of DPP4. Here we discuss new studies exploring DPP4-mediated cellular regulation, provide an updated list of potential targets of DPP4, and discuss clinical implications of each. Recent studies have sought enhanced efficacy of targeting DPP4's role in regulating hematopoietic stem and progenitor cells for improved clinical application. Further studies have identified DPP4 functions in different cellular compartments and have proposed ways to target this protein in malignancy. These findings, together with an expanded list of putative extracellular, cell surface, and intracellular DPP4 targets, provide insight into new DPP4-mediated cell regulation. DPP4 posttranslationally modifies proteins and peptides with essential roles in hematopoietic cell regulation, stem cell transplantation, and malignancy. Targets include secreted signaling factors and may include membrane proteins and transcription factors critical for different hematopoietic functions. Knowing these targets and functions can provide insight into new regulatory roles for DPP4 that may be targeted to enhance transplantation, treat disease, and better understand different regulatory pathways of hematopoiesis.

Chromatin Remodeling Subunit Brm Regulates Hematopoietic Stem Cell Self-Renewal By Limiting Intracellular Valine Levels

Chromatin remodeling complexes facilitate gene expression and control cell fate decisions. The ATPase subunit of chromatin remodeling complex BRG1 is essential for stem cell function, but the role of its paralog Brm remains essentially unknown. To assess a role(s) for Brm in hematopoietic cell regulation in vivo, we studied hematopoietic stem (HSCs) and progenitor cells (HPCs) in bone marrow (BM) of Brm -/- vs. wildtype (WT) control mice. While BM from Brm -/- mice contain increased numbers of rigorously-defined phenotypic populations of long- and short-term repopulating HSCs and granulocyte macrophage progenitors (GMPs) and increased numbers and cycling status of functional HPC (assessed by CFU-GM, BFU-E, and CFU-GEMM colony assays), they were defective in self-renewal capacity upon in vivo serial transplantation using congenic mice (CD45.2+ donor cells, CD45.1+ competitor cells, and F1 (CD45.2+/CD45.1+) recipient mice). Increased numbers of HSCs from Brm-/- BM failed to show competitive advantage over wild type (WT) control BM cells in primary (1°) transplantation in lethally irradiated mice (based on month 4 donor cell chimerism and phenotypically defined HSC numbers). Moreover, 2° and 3° engraftment at 4 months post transplantation each, a measure of HSC self-renewal capacity, revealed much reduced engraftment of donor Brm -/- BM cell chimerism and HSC numbers compared to the extensive 2° and 3° engraftment of control WT BM. No significant differences in myeloid/lymphoid ratios were noted in 1° or 2° engrafted mice, suggesting no differentiation lineage bias of donor Brm -/- BM cells. This demonstrates a critical role for Brm in controlling in vivo self-renewal of mouse BM HSCs. Valine [(2S)-2 amino-3 methylbutanoic acid (C5H11N02)] is implicated in hematopoietic regulation, since depleting dietary valine permitted non-myeloablative mouse HSC transplantation (Taya et. al. Science 354:1152-1155, 2016). Metabolic analysis of lineage negative (lin-) cells demonstrated that valine, but not leucine, levels were very highly elevated in Brm -/- BM cells, thus linking intracellular valine levels with Brm expression. Exogenously added valine significantly increased basal oxygen consumption rates of both total WT BM and WT lin- cells, but not of total or lin-Brm -/- BM cells in vitro (via Seahorse machine analysis). To study effects of valine on HPCs, we assessed the addition of exogenously added valine on mouse BM and human cord blood (CB) cells cultured in the presence of cytokines with either non-dialyzed or dialyzed fetal bovine serum (FBS). Valine, but not leucine, dose-dependently enhanced HPC (CFU-GM, BFU-E, and CFU-GEMM) colony formation and secondary replating capacity of cytokine stimulated CFU-GM and CFU-GEMM derived colonies of normal mouse BM cells in vitro in presence of non-dialyzed FBS; additional enhanced valine effects were noted when dialyzed FBS (lacking valine and other amino acids) was used. Valine also enhanced mouse BM HPC survival in vitro in context of delayed addition of growth factors, and cytokine stimulated (SCF, FL, TPO) ex-vivo expansion of normal mouse BM HSCs and HPCs. Valine enhancement of the above noted functional mouse BM HPC assays in the presence of dialyzed FBS was also apparent with low density and CD34+ purified CB cells, demonstrating that valine effects are not species specific. Our results suggest that valine is an enhancing factor for HSC maintenance of self-renewal capacity and HPC proliferation, and that Brm gene expression limits intracellular valine levels, thereby controlling HSC self-renewal and HPC proliferation. This information is of potential use for future translation to modulate self-renewal of HSCs and survival and proliferation of HPCs for clinical advantage. Disclosures No relevant conflicts of interest to declare.

Conditional deletion of Ahr alters gene expression profiles in hematopoietic stem cells.

The aryl hydrocarbon receptor (AHR) is a ligand activated bHLH transcription factor that belongs to the Per-Arnt-Sim (PAS) superfamily of proteins involved in mediating responses to cellular environment regulating normal physiological and developmental pathways. The AHR binds a broad range of naturally derived and synthetic compounds, and plays a major role in mediating effects of certain environmental chemicals. Although our understanding of the physiological roles of the AHR in the immune system is evolving, there is little known about its role in hematopoiesis and hematopoietic diseases. Prior studies demonstrated that AHR null (AHR-KO) mice have impaired hematopoietic stem cell (HSC) function; they develop myeloproliferative changes in peripheral blood cells, and alterations in hematopoietic stem and progenitor cell populations in the bone marrow. We hypothesized mice lacking AHR expression only within hematopoietic cells (AHRVav1 mice) would develop similar changes. However, we did not observe a complete phenocopy of AHR-KO and AHRVav1 animals at 2 or 18 months of age. To illuminate the signaling mechanisms underlying the alterations in hematopoiesis observed in these mice, we sorted a population of cells highly enriched for HSC function (LSK cells: CD34-CD48-CD150+) and performed microarray analyses. Ingenuity Pathway and Gene Set Enrichment Analyses revealed that that loss of AHR within HSCs alters several gene and signaling networks important for HSC function. Differences in gene expression networks among HSCs from AHR-KO and AHRVav1 mice suggest that AHR in bone marrow stromal cells also contributes to HSC function. In addition, numerous studies have suggested a role for AHR in both regulation of hematopoietic cells, and in the development of blood diseases. More work is needed to define what these signals are, and how they act upon HSCs.

The Importance of the Number of Transplanted Cells with Dipeptidyl Peptidase-4 Expression on the Hematopoietic Recovery and Lymphocyte Reconstitution in Patients with Multiple Myeloma after Autologous Hematopoietic Stem-Cell Transplantation

IntroductionAutologous hematopoietic stem cell transplantation (AHSCT) remains the treatment of choice in multiple myeloma (MM) patients (pts). In earlier research it has been suggested that the expression of dipeptidyl peptidase-4 (DPP4, CD26) influences both the homing and lymphocyte reconstitution after AHSCT in pts with lymphoproliferative neoplasms. The aim of the study is to investigate the effect of transplanted CD26 positive cells of the hematopoietic recovery and lymphocyte reconstitution in MM pts after AHSCT.Patients and methodsForty eight pts with MM with median age 56 (range 21-76) were undergoing AHSCT in our center in years 2011-2013. Conditioning regimen was Melphalan 200. Number of all CD26+ cells, CD26+ lymphocytes, CD26+ monocytes and CD26+ and CD34+ cells were measured in harvested material. Number of lymphocyte's subpopulations (all lymphocytes CD3+, helpers CD3+CD4+, suppressors CD3+CD8+, natural killer (NK) CD3-CD16+CD56+, cytotoxic NK CD3+CD16+CD56+, lymphocytes B CD3-CD19+) were measured in peripheral blood during regeneration period after AHSCT. In both flow cytometry was used. The hematopoietic regeneration was measured as following: the day of white blood cells' regeneration when WBC count reached >1,0x109/L, the day of granulocytes' regeneration when ANC >0,5x109/L and the day of platelets' regeneration when PLT >20x109/L.ResultsAll pts successfully engrafted. The results of AHSCT are shown in table nr 1.Table 1The number of transplanted cells and regeneration during the procedure AHSCT in pts with MM.ParameterMedianRangeMeanStandard deviationNumber of transplanted WBCx108/kg b.w.4,260,73-18,85,434,36Number of transplanted CD34+cells x106/kg b.w.3,362,2-8,23,521,28Number of transplanted CD26+ lymphocytes [109/L]46,59-14853,630,8Number of transplanted CD26+ monocytes [109/L]3,650-828,0313,05Number of all transplanted CD26+ cells [109/L]50,429,6-21362,523,2RegenerationWBC >1x109/L (day)1310-20132,64ANC >0.5x109/L (day)139-2013,32,16PLT >20x109/L (day)1411-2014,12,18As regards regeneration of hematopoietic cells after AHSCT it was shown that a higher number of transplanted CD26+ monocytes improves the reconstitution of suppressor (p=0,019) and NK lymphocytes (p=0,0237). A higher number of all transplanted CD26+ lymphocytes has a positive impact of the reconstitution of suppressor lymphocytes (p=0,0054), whereas a higher number of all transplanted the CD26+ cells improves the regeneration of cytotoxic NK (p=0,0126) and helper lymphocytes (p=0,0261). There were no confirmed adverse effects of the number of CD26+ cells on the hematopoietic regeneration0 and lymphocytes B reconstitution after AHSCT.DiscussionOur research shows that the number of transplanted CD26-positive cells may improve immune reconstitution after AHSCT in patients with multiple myeloma, which was not clearly demonstrated before. As is well known faster lymphocyte reconstitution after AHSCT is associated with improved patient survival. Therefore, the greater the number of transplanted autologous CD26-positive cells may be associated with improved survival, which, however, needs further investigation. DisclosuresNo relevant conflicts of interest to declare.

Reacting to Inflammatory Signals

Specialized cellular niches in the bone marrow (BM) modulate critical functions of the hematopoietic stem and progenitor cells, such as self-renewal, cell-fate decisions and the balance between proliferation and differentiation. Constituents of the BM niche, such as osteoblasts, endothelial cells and macrophages, respond to inflammation with secretion of pro-inflammatory cytokines, and serve as effectors of the inflammatory circuitry; thus their excessive activation can potentially impact on the regulation of hematopoietic cells, and on the initiation and progression of myeloid malignancies. Our current understanding of the mechanisms for Bcr/Ab-negative myeloproliferative neoplasia (MPN) involves recognition of driver mutations (targeting i.e. Tet2, Jak2, Mpl, or Asxl) and of an inflammatory microenvironment. However, despite accumulating evidences on the role of inflammation and of the microenvironment in the maintenance and progression of myeloid malignancies, it is still unclear how the inflamed BM niche contributes to disease establishment and progression. Similarly, the causes leading to an inflammatory microenvironment in MPN are not fully understood. We use genetically-controlled animal model of BM inflammation to study the contribution of the inflammatory microenvironment to MPN. We found that loss of Notch signaling in the BM microenvironment results in an inflammatory state of the BM niche promoting myeloproliferation and marrow fibrosis. Using this model, we discovered that loss of Notch/RBPJ signaling leads to transcriptional upregulation of the pro-inflammatory microRNA miR-155, especially in BM endothelial and mesenchymal cells, resulting in miR-155-dependent inhibition of the NF-κB inhibitor κB-Ras1. Decreased levels of kB-Ras1 resulted in the heightened and persistent activation of NF-κB and in the increased production of NF-kB dependent pro-inflammatory cytokines, leading to uncontrolled myeloproliferation. Deletion of miR-155 in the stroma of RBPJ-/- mice prevented the development of the myeloproliferative disease, and patients affected by MPN exhibit elevated expression of miR155 in their BM. Therefore, we hypothesize that the Notch/miR155/NF-kB axis regulates the level of the inflammatory tonus in the BM niche, and that persistent deregulation of this pathway may contribute to the establishment and progression of MPN. In this presentation, we will discuss our findings and ongoing studies in the context of other recent insights on the crosstalk between tumor cells and the BM niche, and their implications for disease initiation, progression and therapeutic approaches. Disclosures No relevant conflicts of interest to declare.

Suppression of Antioxidant Responses in Dyskeratosis Congenita Cells

Dyskeratosis Congenita (DC) is a bone marrow failure disorder characterized by a triad of leukoplakia, skin dyspigmentation and nail dystrophy. Pathologies found in these patients arise due to mutations found within a number of genes (DKC1, TERT, TERC, TINF2, TCAB1, CTC1, NOP10, C16orf57, NHP2 and PARN) that limit telomere maintenance/elongation, resulting in severely shortened telomeres. Previous studies in our lab have demonstrated impaired proliferation, limited lifespan and aberrant DNA damage response pathways in DC cells. These studies have also uncovered a significant reactive oxygen species (ROS) increase within every cell type investigated thus far. This ROS increase correlates with telomere dysfunction and the subsequent activation of the p53 DNA damage response pathway, which can be rescued by exogenous TERT or p53-shRNA expression. We have acquired skin punch biopsies from two patients with DC carrying either a TERT or DKC1 mutation. Here, we have investigated a potential candidate pathway largely characterized as a key antioxidant regulator in hematopoietic cells, NRF2 (NFE2L2). NRF2 is a redox-sensitive basic leucine zipper transcription factor that, together with its heterologous partners (small MAF proteins, cJun, ATF, etc), binds to antioxidant response elements (AREs) within gene promoters in a pro-oxidant environment. We compared the RNA expression via QRTPCR of NRF2 in control and DC skin fibroblasts and found a significant reduction in DC cells (TERT mutation: 1.5 fold; DKC1 mutation: 2.6 fold). Protein levels of NRF2 were also decreased in DC fibroblasts compared to controls. TXN is a gene whose expression is increased by NRF2 in a pro-oxidant environment. TXN expression was also significantly reduced (TERT mutation: 2.1 fold; DKC1 mutation: 2.2 fold). To test whether NRF2 suppression in DC cells is due to telomere dysfunction, we exogenously expressed TERT via retrovirus in DC and control fibroblasts. TERT expression led to dramatic increases in NRF2 (TERT mutation: 3.4 fold, DKC1 mutation: 3.7 fold) and TXN (TERT mutation: 3.7 fold, DKC1 mutation: 1.6 fold). In contrast, TERT expression in control cells increased NRF2 only 1.3 fold while TXN decreased 1.4 fold. Finally, we wanted to compare the expression of NRF2/TXN in low and elevated oxidative environments (4% vs 21% O2). Control cells increased the TXN expression in 21% O2 (NRF2: no change, TXN: 2.8 fold) while DC cells suppressed NRF2 (TERT mutation: no change, DKC1 mutation: 3 fold decrease) and TXN expression (TERT mutation: 1.4 fold decrease, DKC mutation: 2.3 fold decrease). Functional studies have found DC cells grown in low oxygen increase their proliferative capacity perhaps due to, in part, the NRF2 pathway. Together, these data support a hypothesis whereby shortened/dysfunctional telomeres suppress NRF2 activity and an antioxidant response to a pro-oxidant environment. Based upon previous research, this pathway is likely dependent on the activation of p53 as an intermediary between dysfunctional telomere signaling and the subsequent suppression of NRF2 activity. An abrogated antioxidant response in shortened telomere cells may promote entry into senescence and pathologies related to aging. Systemic pharmacological intervention that reduces ROS could reverse this process and form the basis to alleviate DC and related symptomology associated with this multi-organ disorder. DisclosuresNo relevant conflicts of interest to declare.

Advances in functional regulation of hematopoietic stem cells through soluble regulators

Hematopoietic microenvironment induces hematopoietic stem cell (HSC) to locate within hematopoietic microenvironment, balance self-renewal and differentiation state of HSC, and maintain a relatively stable state of HSC via soluble factors such as cytokines, chemokines and adhesion molecules. Soluble factors can keep HSC adhered to the hematopoietic microenvironment, which plays a vital role in the proliferation and differentiation of HSC. This article reviews literatures on the function of soluble regulatory factors which take part in the regulation of HSC. Key words: Stem cell niche; Hematopoietic stem cells; Cytokines; Signal transduction

Optimization of Cytostatic Leukemia Therapy in an Advection–Reaction–Diffusion Model

In this work, we study an advection---reaction---diffusion system involving normal and cancer hematopoietic cells (HC) and modeling the development of leukemia in the bone marrow. The effects of a targeted cytostatic therapy, which inhibits the regeneration of cancer HC, are studied through an optimal control system. We prove the existence of a positive bounded solution and the existence of an optimal controls by semigroup techniques. Conditions of optimality are in terms of the dual system. The effects of targeted cytostatic therapy, upon the dynamics of normal and cancer HC, are discussed through numerical simulations.

DNMT3A Arg882 mutation drives chronic myelomonocytic leukemia through disturbing gene expression/DNA methylation in hematopoietic cells

The gene encoding DNA methyltransferase 3A (DNMT3A) is mutated in ∼20% of acute myeloid leukemia cases, with Arg882 (R882) as the hotspot. Here, we addressed the transformation ability of the DNMT3A-Arg882His (R882H) mutant by using a retroviral transduction and bone marrow transplantation (BMT) approach and found that the mutant gene can induce aberrant proliferation of hematopoietic stem/progenitor cells. At 12 mo post-BMT, all mice developed chronic myelomonocytic leukemia with thrombocytosis. RNA microarray analysis revealed abnormal expressions of some hematopoiesis-related genes, and the DNA methylation assay identified corresponding changes in methylation patterns in gene body regions. Moreover, DNMT3A-R882H increased the CDK1 protein level and enhanced cell-cycle activity, thereby contributing to leukemogenesis.

Design of Druglike Small Molecules with LYN-Specific Binding

Glioblastoma multiforme (GBM), a very aggressive brain tumor, has a median survival of only 14 months. LYN, an important kinase involved in regulation of hematopoietic cells, is significantly overexpressed in GBM. It is believed that LYN promotes migration of cancer cells, thus advancing the malignancy. This research addresses computational design of small druglike molecules that could potentially inhibit LYN and thus stave off the cancer advancement. LYN has a very similar binding site to the polo-box domain (PBD) in Polo-like kinase 1 (Plk1). Plk1 is a main regulator of mitosis. Considering the key cellular roles of both LYN and Plk1, it is important to design inhibitors that will specifically bind to LYN. In this work, physical and chemical properties of the binding sites of LYN and Plk1 were investigated and compared. Pertinent atomic distances within the LYN binding site were found to be smaller than those within the PBD of Plk1. The two sites also differed in their flexibilities. By utilizing the differences, novel molecules were designed that could potentially bind LYN with higher affinities than they could Plk1. Previously designed molecules that bonded both LYN and Plk1 were used as initial templates to design more specific inhibitors. Potential toxicities and drug-likeness of the molecules were evaluated. Molecules with no implied toxicities and optimal druglike properties were used for docking studies. Molecules that made the most stable docking configurations with LYN and with no other kinases were identified as LYN-specific. Binding energies of the stable complexes that these molecules formed with LYN were calculated. Possible utilization of the designed molecules against tumors with overexpressed LYN is discussed.

N-wasp is essential for the negative regulation of B cell receptor signaling.

Author SummaryMechanisms to shut down B-cell activation are necessary to ensure termination of an immune response when an infection has been cleared. When this negative regulation goes wrong, it can also lead to autoimmunity. To understand how this inhibitory process is regulated, here we utilized knockout mice containing B cells that are deficient for proteins potentially involved in their negative regulation. We focus on Wiskott–Aldrich syndrome protein (WASP), a key cytoskeletal regulator of hematopoietic cells, and neural WASP (N-WASP), which shares 50% homology with WASP and is ubiquitously expressed. Our study shows that mouse B cells that lack N-WASP protein are activated to a greater level and for longer periods than B cells that express this protein. Furthermore, in mice where B cells do not make N-WASP, the numbers of self-reactive B cells are elevated. We went on to identify molecules that promote or inhibit N-WASP activation and to examine the cellular mechanisms by which N-WASP inhibits B-cell activation. Based on these findings we propose that N-WASP is a critical inhibitor of B-cell activation and serves to suppress self-reactive B cells.

A Specific PTPRC/CD45 Phosphorylation Event Governed by Stem Cell Chemokine CXCL12 Regulates Primitive Hematopoietic Cell Motility

CXCL12 governs cellular motility, a process deregulated by hematopoietic stem cell oncogenes such as p210-BCR-ABL. A phosphoproteomics approach to the analysis of a hematopoietic progenitor cell line treated with CXCL12 and the Rac 1 and 2 inhibitor NSC23766 has been employed to objectively discover novel mechanisms for regulation of stem cells in normal and malignant hematopoiesis. The proteomic data sets identified new aspects of CXCL12-mediated signaling and novel features of stem cell regulation. We also identified a novel phosphorylation event in hematopoietic progenitor cells that correlated with motile response and governed by the chemotactic factor CXCL12. The novel phosphorylation site on PTPRC/CD45; a protein tyrosine phosphatase, was validated by raising an antibody to the site and also using a mass spectrometry absolute quantification strategy. Site directed mutagenesis and inhibitor studies demonstrated that this single phosphorylation site governs hematopoietic progenitor cell and lymphoid cell motility, lies downstream from Rac proteins and potentiates Src signaling. We have also demonstrated that PTPRC/CD45 is down-regulated in leukemogenic tyrosine kinase expressing cells. The use of discovery proteomics has enabled further understanding of the regulation of PTPRC/CD45 and its important role in cellular motility in progenitor cells.