bluuebunny/crossref_metadata_2025 huggingface dataset:
14-3-3 Integrates Pro-Survival Signals in Hematopoietic Cells Transformed by Diverse Leukemogenic Tyrosine Kinases, and Represents a Common Target.
Authors: Dong Shaozhong , Kang Sumin , Gu Ting-lei , Kardar Sean , Lonial Sagar , Khoury H. Jean , Khuri Fadlo , Fu Haian , Chen Jing
Published: April 2020

Constitutively activated tyrosine kinases associated with recurrent chromosomal abnormalities play an essential role in the pathogenesis and disease progression of a variety of hematopoietic malignancies. Selective tyrosine kinase inhibitors such as imatinib are effective in treating some forms of leukemia such as t(9;22) CML associated with expression of BCR-ABL fusion tyrosine kinase. However, they are not curative and clinical resistance may develop, prompting the design of alternate and/or complementary therapeutic strategies. To better understand the signaling properties of constitutively activated tyrosine kinases associated with different hematopoietic malignancies, we examined whether BCR-ABL, FLT3-ITD, NPM-ALK, TEL-PDGFbetaR, TEL-FGFR3 and ZNF198-FGFR1 activate the same set of signaling pathways. We found that they all activated AKT and MAPK signaling pathways. Activated AKT resulted in phosphorylation of FOXO3a at Thr-32 but not BAD at Ser-136, whereas activated MAPK led to phosphorylation of BAD at Ser-112. These phosphorylated residues subsequently sequestered the pro-apoptotic FOXO3a and BAD to 14-3-3, suggesting that 14-3-3 integrates pro-survival signals from AKT and MAPK pathways. We utilized a peptide-based 14-3-3 competitive antagonist, R18 to disrupt 14-3-3/ligand association. Expression of R18 effectively induced apoptosis in hematopoietic Ba/F3 cells transformed by these tyrosine kinases with significantly enhanced sensitivity compared to the control Ba/F3 cells. Moreover, doxycycline-induced expression of R18 significantly attenuated the disease latency and penetrance in mice induced by intravenous injection of representative ZNF198-FGFR1-transformed Ba/F3 cells. Co-immunoprecipitation experiments indicate that induced R18 expression disrupted interaction between 14-3-3 and FOXO3a, but not 14-3-3/BAD association. R18 induced apoptosis by rescuing the nuclear localization of FOXO3a and up-regulating FOXO3a transcription targets Bim and p27 in cells expressing ZNF198-FGFR1. Furthermore, fluorescent confocal microscopy revealed that expression of R18 generally resumed FOXO3a nuclear localization in cells transformed by the spectrum of diverse leukemogenic tyrosine kinases. Together, these data support a model that 14-3-3 functions as a general integrator of pro-survival signals in hematopoietic transformation induced by diverse leukemogenic fusion/mutant tyrosine kinases. Disrupting 14-3-3/ligand association may be a common and effective therapeutic strategy for hematopoietic neoplasms associated with these tyrosine kinases.
Prevention of Human Interferon-alpha-Induced Thrombocytopenia by a Non-Peptidyl Human Thrombopoietin Receptor Activator, NIP-004.
Authors: Yamane Akiko , Nakamura Takanori , Ito Mamoru , Ohnishi Yasuyuki , Ikeda Yasuo , Miyakawa Yoshitaka
Published: April 2020

Human interferon-α (IFN) is useful for the treatment of chronic hepatitis C and neoplastic disorders; however, one major adverse effect is that thrombocytopenia often leads to a dose reduction or discontinuation of therapy. Since around 40% of patients with chronic hepatitis C will develop liver cancer, it is very important to prevent the transition from chronic hepatitis to liver cirrhosis and liver cancer with IFN treatment. However, some patients cannot receive IFN treatment because of thrombocytopenia resulting from liver cirrhosis and autoimmunity. This prompted us to evaluate the effects of a novel non-peptidyl human thrombopoietin (TPO) receptor (c-Mpl) activator, NIP-004, to prevent IFN-induced thrombocytopenia. We recently reported that NIP-004 at 30 μg/kg/day for 2 weeks increased human platelets 4-fold in immunodeficient NOD/Shi-scid, IL-2Rγnull (NOG) mice transplanted with 1 × 105 human cord blood-derived CD34+ cells (Blood, 2006). When PEG-hIFN-α2b (IFN-α2b) at 30 μg/kg was administered three times a week for 3 weeks to NOG mice transplanted with human CD34+ cells, the number of human platelets was significantly reduced by 40% compared to the control mice. Moreover, the number of human platelets was restored to the same level as the control mice with daily administration of 30 mg/kg NIP-004 for 2 weeks during the course of IFN treatment. These results indicated that NIP-004 is effective in preventing IFN-α2b-induced thrombocytopenia in vivo. Although IFN is believed to directly inhibit megakaryopoiesis, its mechanisms are not clearly understood. We analyzed the inhibitory effects of IFN-α2b on the colony formation of human megakaryocytes (CFU-MK) and the intracellular signaling induced by IFN and NIP-004. NIP-004 prevented the inhibition of CFU-MK formation by IFN-α2b in a dose dependent manner. IFN-α2b modestly inhibited the proliferation of human leukemia UT-7/EPO cells expressing human c-Mpl, and they were stimulated with TPO and NIP-004, respectively. We analyzed some form of cross-talk between TPO and IFN receptors by Western blot and quantitative RT-PCR analysis. NIP-004 induced the expression of SOCS-3 and CIS in UT-7/EPO-hMpl cells. We suggest that NIP-004 induces inhibitory signaling such as SOCS-3 and CIS and inhibits IFN-induced intracellular signaling. In conclusion, NIP-004 was shown to prevent IFN-α2b-induced thrombocytopenia in vivo and in vitro, suggesting the future clinical application of non-peptidyl c-Mpl activators for the treatment of patients with chronic hepatitis C by IFN.
Epo-Independent Proliferation and Differentiation of Genetically Modified Erythroid Progenitor Cells Using a Chemical Inducer of Dimerization.
Authors: Miller Chris P. , Zheng Songmao , Blau C. Anthony
Published: April 2020

Recombinant human erythropoietin (Epo) revolutionized the care of anemia in cancer. In light of emerging (albeit controversial) roles for Epo in supporting tumor growth, it may be useful to develop Epo-independent methods for regulating red blood cell production. Previous studies in this laboratory established that marrow transduced with conditional derivatives of fibroblast growth factor receptor-1 (F36VFGFR1) and the thrombopoietin receptor (F36VMpl) can support the chemical inducer of dimerization (CID)-dependent production of erythroid cells in transplanted mice. In the current study, we used human CD34+ cord blood (CB) cells to test whether CID-regulated red cell production might require collaboration between CID-initiated signals and those provided by Epo. CD34+ CB cells cultured in the absence of Epo did not proliferate and retained expression of the myeloid marker CD33. Epo (5U/mL) promoted proliferative expansion (66.2-fold in 12 days) and CD33 expression was lost as the cells differentiated to mature, glycophorin A+ erythroid cells (92.9 +/− 1.8%, n=3). Addition of a soluble human Epo receptor extracellular domain (shEpoR) at a concentration of 3ug/mL completely blocked Epo-dependent proliferation and similar to cells cultured without Epo, they retained expression of CD33. Addition of CID (100nM AP20187) to F36VMpl-transduced CD34+ CB cells in the absence of Epo promoted proliferative expansion (89.8-fold in 12 days) and differentiation as glycophorin A+ erythroid cells (77.2 +/− 4.8%, n=3). These CID responses were not significantly affected by the addition of shEpoR, as CID induced both mitogenic expansion (84.5 fold in 12 days) and differentiation as glycophorin A+ erythroid cells (74.2 +/− 6.7%, n=3) in the presence of concentrations of shEpoR that blocked Epo responses. These data suggest that F36VMpl does not require Epo signaling to support the proliferation and differentiation of human erythroid progenitor cells, and further define an Epo-independent method of erythroid cell production.
Leukemia Associated Mutants of SHP2 Protein Tyrosine Phosphatase Inhibit Nf1-Expression by Decreasing ICSBP and IRF2 Phosphorylation.
Authors: Huang Weiqi , Horvath Elizabeth , Eklund Elizabeth A.
Published: April 2020

Neurofibromin 1 (Nf1) is a ubiquitous protein with Ras-GAP activity. In myeloid cells, Nf1 down regulates Ras which has been activated by hematopoietic cytokines. Congenital absence of Nf1 is associated with the myeloproliferative/dysplastic disorder Juvenile Chronic Myelomonocytic Leukemia (JMMoL). JMMoL is characterized by hypersensitivity to cytokines such as GM-CSF, SCF and M-CSF. In previous studies, we found that cytokine stimulation of myeloid progenitor cells increases transcription of the NF1-gene, thereby increasing Nf1-expression. We identified a cis element in the NF1-promoter which is activated by the interferon consensus sequence binding protein (ICSBP). This is of interest because ICSBP-deficiency confers hypersensitivity to the same cytokines as does Nf1-deficiency. In the current studies, we find that interferon regulatory factor 2 (IRF2) cooperates with ICSBP to activate the NF1-cis element in differentiating myeloid cells. This interaction requires specific phosphorylation of conserved tyrosine residues in the IRF-domains of IRF2 and ICSBP. Phosphorylation of both proteins occurs in differentiating myeloid progenitor cells and myeloid cell lines. In myeloid progenitor cells, ICSBP and IRF2 are maintained in a non-phosphorylated state by the protein tyrosine phosphatases SHP1 and SHP2. During myeloid differentiation, activity of these PTPs decreases, permitting increased phosphorylation of ICSBP and IRF2. Leukemia associated, activating mutants of SHP2-PTP have been recently described in JMMoL, MDS and AML. Such mutations induce conformational changes in SHP2, resulting in constitutive activation. When introduced into myeloid progenitor cells, such SHP2-mutants induced a profile of cytokine hypersensitivity which is similar to those seen in Nf1-deficiency or ICSBP-deficiency. We find that expression of one of these leukemia associated SHP2 mutants blocks cytokine-induced phosphorylation of ICSBP and IRF2, impairing Nf1-expression in differentiating myeloid cells. This results in hypersensitivity and sustained proliferation in response to differentiating cytokines. Therefore, our studies mechanistically associate three molecular defects that have been associated with myeloproliferation and cytokine hypersensitivity; ICSBP-deficiency, Nf1-deficiency and expression of activated SHP2 mutants. Our results suggest that hematopoietic cytokine signaling induces tyrosine phosphorylation of IRF2 and ICSBP. These phospho-proteins activate transcription of the NF1-gene. Increased Nf1-expression down regulates cytokine-activated Ras. However, expression of constitutively active forms of SHP2-PTP blocks this process by impairing cytokine induced phosphorylation of IRF2 and ICSBP, decreasing Nf1-expression, and resulting in unopposed Ras activation. These results may be important to targeting this pathway in myeloid disorders characterized by cytokine hypersensitivity and myeloproliferation.
Essential Role of Kindlin 3 in Zebrafish Thrombopoiesis.
Authors: Mann James J. , Langer Nathaniel B. , Woo Andrew , Moran Tyler B. , Schindler Yocheved , Sullivan Matthew , Paw Barry H. , Cantor Alan B.
Published: April 2020

The zinc finger transcription factor GATA-1 is required for proliferative inhibition and terminal maturation of megakaryocytes, and is mutated in Down Syndrome Transient Myeloproliferative Disorder (TMD) and Acute Megakaryoblastic Leukemia (DS-AMKL). Yet the molecular mechanisms that regulate GATA-1 activity in megakaryopoiesis remain incompletely understood. Many transcription factors, in addition to binding DNA, make important protein-protein interactions that modulate their activity. In order to further understand GATA-1’s function, and possibly identify new factors involved in megakaryopoiesis, we purified GATA-1 containing multiprotein complexes from the murine L8057 megakaryocytic cell line. We generated stable L8057 cell lines expressing metabolically biotinylated and FLAG epitope tagged GATA-1, and then performed a tandem anti-FLAG immunoaffinity and streptavidin affinity purification. Using mass spectrometry (LC/MS/MS), we identified the known GATA-1 associated proteins Friend of GATA-1 (FOG-1), SCL, Ldb1, Runx-1/Cbf-β. SP1 and all components of the NuRD complex (which binds FOG-1) as co-purifying proteins. In addition, we reproducibly obtained several novel proteins. We previously reported the identification of the kruppel-type zinc finger transcription factor zfp148 (also called ZBP-89), and showed that it plays an essential role in megakaryopoiesis and definitive erythropoiesis. Here we report the identification of Kindlin 3 (also called URP2 for UNC-112 related protein 2), a member of a family of PH and FERM domain containing proteins that are thought to play a role in integrin-mediated processes. Expression of Kindlin 3 is restricted to hematopoietic cells, principally megakaryocytes and lymphocytes. It is first expressed at ~E9.5 during murine embryogenesis, and is abundant in fetal liver megakaryocytes by day E14.5. In order to begin to assess the role of Kindlin 3 in megakaryopoiesis in vivo, we performed morpholino-mediated knockdown of Kindlin 3 expression in CD41-GFP transgenic zebrafish embryos. In contrast to control embryos, embryos injected with Kindlin 3 specific morpholinos exhibited nearly complete loss of GFP+ thrombocytes (equivalent to mammalian megakaryocyte/platelets). Erythroid development (equivalent to mammalian primitive erythropoiesis at this stage of development) was not significantly affected, similar to embryos injected with zfp148-specific morpholinos. Given the role of integrin outside-to-inside signaling in megakaryopoiesis, we propose that Kindlin 3 may play a role linking extracellular signals to megakaryocyte maturation and growth control via GATA-1 transcription complexes. Further analysis in murine systems is underway to test this hypothesis.
Acetylation of GATA-1 Is Required for Chromatin Occupancy.
Authors: Lamonica Janine M. , Vakoc Christopher R. , Blobel Gerd A.
Published: April 2020

All three hematopoietic GATA transcription factors GATA-1, GATA-2, and GATA-3 are acetylated, although the in vivo role of this modification remains unclear. It has been proposed that acetylation of GATA-1 increases its affinity for DNA in vitro, although this finding has not been observed by others. To study the role of GATA-1 acetylation, we examined the functions of an acetylation-defective mutant of GATA-1 in maturing erythroid cells. We found that removal of the acetylation sites in GATA-1 largely abrogates its biological activity but does not impair its nuclear localization, steady state protein levels, or its ability to bind naked GATA elements in vitro. However, chromatin immunoprecipitation (ChIP) experiments revealed that mutant GATA-1 was dramatically impaired in binding to its cellular target sites in vivo, including genes that are normally activated (α- and β-globin, EKLF, FOG-1, Band3, and AHSP) and repressed (GATA-2 and c-kit) by GATA-1. Together, these results suggest that acetylation is required for GATA-1 chromatin occupancy. These findings point to a novel function for transcription factor acetylation, perhaps by facilitating protein interactions required for stable association with chromatin templates in vivo. To identify proteins that interact with acetylated GATA-1, we performed peptide affinity chromatography using acetylated GATA-1 peptides. Using this technique coupled with mass spectrometry, several proteins that bind to GATA-1 peptides in an acetylation-dependent manner were identified. The identified proteins contain known acetyl-lysine binding modules (bromodomains) consistent with their binding properties. The in vivo role of these proteins with regard to GATA-1 function is being examined and will be discussed.
Hypomorphic Alleles of PU.1 Result in Impaired Myelopoiesis and Target Gene Expression.
Authors: Houston Isaac B. , Kamath Meghana B. , Schweitzer Brock L. , Chlon Timothy M. , Ondr Jennifer K. , DeKoter Rodney P.
Published: April 2020

PU.1 is an Ets-family transcription factor that is critical for normal hematopoiesis. Knock-out studies of PU.1 demonstrate that it is required for the development of myeloid and lymphoid cells. PU.1 is expressed at graded levels in the immune system, with higher expression in myeloid lineage cells. Previous research has suggested that differences in PU.1 concentration are involved in hematopoietic cell fate decisions. However, the mechanism by which target genes in developing cells sense and respond to different PU.1 concentrations is unknown. To address this question, we inserted a beta-lactamase reporter gene and a neomycin resistance cassette into the first coding exon of PU.1. Mice homozygous for the targeted allele (PU.1BN), and an allele in which neomycin was removed (PU.1BLAC), had graded reductions in PU.1 expression and function. PU.1BN/BN mice display a substantial reduction in fetal and neonatal myelopoiesis and are macrophage deficient, resulting in a failure of vascular regression in the developing eye. A fraction of these mice survive until weaning and have an accumulation of c-Kit-expressing immature myeloid cells in the spleen and bone marrow. In addition, these mice acquire a myelomonocytic infiltration in the retina and hyaloid vessels of the eye. Mice homozygous for the second allele (PU.1BLAC/BLAC) survive less than one week and have a severe reduction in PU.1 activity and a block to myeloid development. Fetal livers from both types of mice lack any discernable mature myeloid cells and demonstrate a graded reduction in colony forming ability. To determine if these two alleles have an effect on gene expression, we established IL-7-dependent pro-B cell lines or IL-3-dependent cell lines from these mice as well as from wild type or PU.1 null controls. We found that PU.1-dependent target genes in these cell lines are expressed in a graded manner. In particular, expression of the gene encoding the low-affinity IgG Fc receptor (FcγRIIb) is directly responsive to PU.1 activity. In summary, these results demonstrate the critical importance of an appropriate threshold of PU.1 activity for normal hematopoiesis. The hypomorphic PU.1 alleles provide an excellent model for determining the mechanism of graded PU.1 levels in hematopoiesis.
STAT5 Is a Negative Regulator of Megakaryopoiesis.
Authors: Drayer Lyndsay , Olthof Sandra , Schuringa Jan Jacob , Vellenga Edo
Published: April 2020

The transcription factor STAT5 plays a critical role in self-renewal and lineage commitment of hematopoietic stem cels (HSCs). We have recently shown in CB CD34+ cells that persistent activation of STAT5 results in enhanced self-renewal and induces erythroid differentiation, while myelopoiesis was severely impaired. In this study we analyzed the function of STAT5 during megakaryocyte (MK) differentiation. Using RNA interference we downregulated STAT5 expression in peripheral blood CD34+ cells. Cells were transduced with a lentiviral construct encoding eGFP and a short-hairpin RNA for STAT5 or with a control vector expressing YFP. Transduction efficiencies were determined by flow cytometric detection of eGFP/YFP and ranged from 40%–80%. Decreased STAT5 expression was verified by Western blot and quantitative RT-PCR (65% lower expression in STAT5 RNAi cells versus YFP+ control cells, p=<0.01). To assess the effects of decreased STAT5 expression on the progenitor pool, the transduced populations were sorted and enumerated in colony assays. Downregulation of STAT5 significantly increased the number of MK progenitors (1.9-fold, p=0.02) and resulted in a decrease of erythroid progenitors (0.6-fold, p<0.01), but did not affect the number of granulocyte/monocyte progenitors. Prospective isolation of immature and commited progenitors are currently being performed to distinguish if certain progenitor subsets are re-directed to the MK lineage by low STAT5 expression. Next, we analyzed the role of STAT5 during MK differentiation in unilineage suspension cultures. Transduced cells were cultured in serum-free medium containing TPO and SCF and scored for cell counts and expression of MK markers weekly. The percentage of transduced cells and their number did not differ when STAT5 RNAi cells were compared to controls, indicating that downregulation of STAT5 provides no proliferative advantage or disadvantage. However, the expression of glycoproteins IIb/IIIa (CD41) and Ib (CD42) was upregulated, and an increase in large, polyploid cells was observed in STAT5 RNAi cultures (15.2% polyploid cells versus 4.9% in control cells at day 7, p<0.01). RT-PCR analysis of transcription factors predominantly expressed in MK and erythroid lineages revealed that Runx1 and Erg were increased in STAT5 RNAi cells, and the level of Scl was decreased compared to control cells. These observations correlate with the increased MK differentiation observed in STAT5 downregulated cells. Together, these data demonstrate that the expression level of STAT5 has important effects regarding the type of hematopoietic cell development, with high levels favouring erythroid development and low levels enhancing MK differentiation and maturation.
Regulation of RUNX1 Transcriptional Function by GATA-1 Kinase Recruitment: A Novel Pathway for Megakaryocytic Gene Regulation.
Authors: Elagib Kamaleldin E. , Mihaylov Ivailo S. , Delehanty Lorrie L. , Gonias Sara L. , Caronia Jill F. , Goldfarb Adam N.
Published: April 2020

RUNX1 and GATA-1 both play essential roles in the transcriptional programming of normal mammalian megakaryocytic development, deficiencies of either factor having similar phenotypic consequences. We have previously characterized physical and functional interactions between these two factors, and others have confirmed analogous cooperations in Drosophila and Danio homologs. We now present data on molecular mechanisms for the cooperation of these two factors in the transcriptional activation of the megakaryocytic aIIb integrin promoter. In these studies, GATA-2 also physically interacted with RUNX1 but failed to cooperate in transcriptional activation. In fact, increasing amounts of GATA-2 repressed the functional interplay between GATA-1 and RUNX1. Through generation of GATA-2/GATA-1 chimeras, we identified a conserved subdomain within the GATA-1 amino terminus that was both necessary and sufficient for transcriptional cooperation with RUNX1. Coexpression of wild type GATA-1 or of cooperating GATA-2/GATA-1 chimeras, but not of GATA-2 or of non-cooperating chimeras, induced a mobility shift in wild type RUNX1. Using immunoprecipitation followed by immunoblot with a panel of phosphospecific antibodies, we found GATA-1 to induce RUNX1 phosphorylation at recognition sites for cyclin-dependent kinases (cdks). Treatment of cells with roscovitine, a specific cdk inhibitor, blocked the transcriptional cooperation of GATA-1 with RUNX1 and eliminated the RUNX1 mobility shift caused by GATA-1 coexpression. Mutagenesis of RUNX1 identified a cluster of serine/threonine-proline (S/TP) sites collectively required for the transcriptional augmentation and mobility shift induced by GATA-1. In addition, intact DNA binding by RUNX1 was required for cooperation with GATA-1. These results provide a new paradigm for cooperation of interacting transcription factors, in which one partner recruits a kinase leading to phosphorylation and activation of the other partner. Furthermore, these results provide a biochemical basis for the previously inexplicable functional differences between GATA-1, which promotes megakaryocytic maturation, and GATA-2 which promotes proliferation without maturation.
Orientation-Dependent Regulation of RUNX1 and GATA-1 Transcriptional Function by the p300/CBP Coactivators.
Authors: Mihaylov Ivailo S. , Elagib Kamaleldin E. , Delehanty Lorrie L. , Gonias Sara L. , Caronia Jill F. , Goldfarb Adam N.
Published: April 2020

The transcription factors RUNX1 and GATA-1, as well as the coactivators p300/CBP, have been implicated in the regulation of primary megakaryopoiesis through studies of knockout mice. In particular, p300/CBP has previously been shown to serve as a coactivator for both RUNX1 and GATA-1 in the transactivation of hematopoietic target genes. Coactivator orientation within transactivating complexes is generally not considered to influence the degree of transcriptional activity, reflecting an inherent flexibility in the spatial requirements for coactivator function. Experiments to further explore this issue showed coexpression of p300 to enhance cooperative transcriptional activation by wild type RUNX1 and GATA-1. Enforced recruitment of p300/CBP to GATA-1 through fusion of GATA-1 with the p300/CBP docking module of adenoviral E1A, E1A(1–89), enhanced GATA-1 activity alone, regardless of whether the fusion was to the amino or carboxy terminal of GATA-1. However, enforced recruitment of p300/CBP to the amino terminus of GATA-1 completely eliminated cooperation with RUNX1, and enforced recruitment of p300/CBP to the carboxy terminus of GATA-1 diminished cooperation with RUNX1. Both GATA-1 fusions retained physical interaction with RUNX1. Similarly, fusion of E1A(1–89) directly to the amino terminus of RUNX1 completely eliminated its transcriptional activity, while fusion to the carboxy terminus diminished RUNX1 transcriptional activity. For both the GATA-1 and the RUNX1 fusions, these repressive effects were attributable to the ectopic recruitment of p300/CBP because a mutation within E1A(1–89) known to specifically diminish p300/CBP recruitment, R2G, rescued the transcriptional activities. Addressing the mechanism of repression by ectopic p300/CBP, we found that E1A(1–89)-GATA-1 caused diminished serine phosphorylation within RUNX1, an effect opposite to that of wild type GATA-1 which enhanced RUNX1 phosphorylation. Similarly, E1A(1–89)-RUNX1 showed complete loss of phosphorylation on cdk target sites, as compared with wild type RUNX1. RUNX1-E1A(1–89) showed diminished phosphorylation on cdk target sites, as compared with wild type RUNX1. From these results, we conclude that p300/CBP may function as a coactivator for the RUNX1-GATA-1 complex when recruited to endogenous, wild type domains. By contrast, ectopic recruitment of p300/CBP to RUNX1, particularly to the amino terminus, targets RUNX1 for repression through inhibition or reversal of phosphorylation. Our results thus offer a novel paradigm in which the function of p300/CBP, coactivator versus repressor, may be determined by its mode of recruitment to certain transcriptional complexes. Notably, some transcription factors, such as GATA-1, have relaxed requirements for the topology of coactivator recruitment, whereas other transcription factors, such as RUNX1, have stringent requirements in this regard.
MicroRNAs 17-5p/20a/106a Function as a Master Gene Complex Controlling Monocytopoiesis through AML1 Targeting.
Authors: Fontana Laura , Pelosi Elvira , Greco Paolo , Testa Ugo , Liuzzi Francesca , Croce Carlo Maria , Brunetti Ercole , Peschle Cesare
Published: April 2020

MicroRNAs (miRs) are a novel regulatory class of non-coding, single-stranded RNAs of ~ 22 nucleotides, identified in plants and animals. MiRs repress protein expression at post-transcriptional level, mostly through base pairing to the 3′ untranslated region (UTR) of the target mRNA, thus leading to its degradation and/or reduced translation. MiRs have been shown to control basic biological functions, such as cell proliferation and differentiation. However, little is known on their role in hematopoiesis. Furthermore, in diverse types of cancer miRs may function as “oncomirs”.

We investigated the role of miR-17-5p, -20a and -106a, located in the miR-17-5p-92 and miR-106a-92 clusters respectively, in monocytic-macrophage (Mo) differentiation-maturation. In unilineage Mo culture generated by cord blood (CB) hematopoietic progenitor cells (HPC), these miRs are gradually and markedly downmodulated, whereas AML1 is upmodulated at protein but not at mRNA level. The acute myeloid leukaemia-1 protein (AML1, also known as Runt-related transcription factor 1, Runx1) is the DNA-binding subunit of the hematopoietic transcription factor CBF, which plays a pivotal role in Mo differentiation-maturation. Luciferase assay shows that miR-17/20/106 bind the AML1 mRNA 3′UTR, implying that during monocytopoiesis their decline unblocks AML1 translation. Mo culture transfection with miR-17/20/106 stimulates blast cell proliferation and inhibits differentiation-maturation: this is coupled with AML1 translation blockade, leading in turn to inhibition of M-CSF receptor (M-CSFR) expression. Equivalent results were observed in Mo clonogenic culture of HPCs. Knockdown of endogenous miR-17/20/106 by anti-miR oligonucleotide transfection causes opposite effects. Altoghether, our studies indicate that miR-17/20/106 downmodulation during monocytopoiesis “unblocks” the translation of AML1, which in turn transactivates the M-CSFR, thereby allowing Mo differentiation and maturation. We hence propose that these miRs function as a master gene complex underlying the monocytopoiesis gene program, at least in part through the regulation of two pivotal proteins, AML1 and M-CSFR.

This study also sheds light on other relevant aspects:the regulatory action of miR-17/20/106 action on AML1 mRNA is restricted to the Mo lineage, implying a role for lineage-specific factor(s) to modulate the interaction of these miRs with their mRNA target;the subtle complexity of mechanisms controlling the transcription/processing of the miR-17-5p-92 cluster, implied by the differential expression patterns of the miRs comprised in the 17-5p-92 polycistron in Mo culture;the leukemogenesis mediated by AML1 fusion proteins.
EVI1 Interacts with and Represses RUNX1: A Novel Mechanism of Deregulated Hematopoiesis in EVI1-Positive Hematopoietic Cells.
Authors: Senyuk Vitalyi , Sinha Kislay K. , Yanamandra Sastry , Nucifora Giuseppina
Published: April 2020

Myelodysplastic syndrome (MDS) comprises a group of disorders with overlapping features. The majority of recurring chromosomal abnormalities associated with MDS are large deletions. There are two genes, RUNX1 and EVI1, whose deregulation is also known to be associated with MDS. RUNX1 is a master regulator of hematopoiesis that specifically binds to the DNA and regulates the expression of hematopoietic genes. Lately it has become clear that RUNX1 is frequently mutated in patients with MDS. Surprisingly, the mutations are limited to one allele, suggesting that a partial loss of function is sufficient to contribute to the disease. The second gene, EVI1, encodes a large nuclear protein characterized by two domains of zinc fingers. While the precise role of EVI1 is not known, this protein is not expressed in normal bone marrow and is inappropriately activated in very aggressive MDS by chromosomal translocations. Because alterations of both genes are not usually observed together, we have recently investigated whether there could be a potential link between the pathways regulated by RUNX1 and EVI1. Here, we report that in vivo and in vitro EVI1 physically interacts with RUNX1. We mapped the interaction domains to the eighth zinc finger motif of EVI1 and to the DNA-binding Runt domain of RUNX1. We found that this interaction alters the ability of RUNX1 to bind to its DNA consensus and represses the ability of RUNX1 to activate a reporter gene, suggesting that the interaction between EVI1 and RUNX1 could impair the hematopoietic programs regulated by RUNX1. These data suggest that inappropriate expression of EVI1 could contribute to hematopoietic transformation in part by a new mechanism that involves EVI1 association with key hematopoietic regulators leading to their functional impairment. Our results also indicate that the interaction region between RUNX1 and EVI1 could represent a novel target for therapeutic drugs for EVI1-associated leukemia.
Thalidomide Induces gamma-Globin Gene Expression in Adult Primary Erythroid Cells through Increased Intracellular Reactive Oxygen Species Mediated Activation of p38 MAPK Signal Pathway.
Authors: Aerbajinai Wulin , Zhu Jianqiong , Gao Peter , Chin Kyung , Rodgers Griffin P.
Published: April 2020

Thalidomide has been shown to be effective in some patients with myelodysplastic syndromes (MDS), including increases in both the total hemoglobin and in the proportion of the fetal hemoglobin in some patients in clinical trials. Also, it has been demonstrated that thalidomide increases the intracellular reactive oxygen species (ROS) in embryoid bodies. The mechanisms of thalidomide’s therapeutic effect are still being defined. We hypothesize that thalidomide induce the gamma-globin gene expression in adult erythropoiesis, and that this induction may be mediated by increased ROS formation. To investigate this hypothesis, we assessed the effect of increasing dosages of thalidomide (0.01uM to 100uM) on cell growth, globin gene expression and ROS generation using cultured primary human CD34+ progenitor cells. The effects of varying concentrations of thalidomide on the cultured CD34+ cells, demonstrate a significant increase in cell number at maximum thalidomide concentration of 100uM. Real time quantitative PCR analysis of gamma- and beta-globin gene expression demonstrated that thalidomide significantly induces gamma-globin gene expression in a dose-dependent manner. The averaged gamma/gamma+beta percentage ratio was 12.89% ± 0.11% in cultures treatment with the highest concentration of 100uM thalidomide compared with 3.21% ± 0.07% in Epo alone (P<0.01). Interestingly, we found that intracellular ROS level was significantly increased by treated with 100uM thalidomide for 48 hours, from day 3 to day 5. We can not rule-out an effect of thalidomide on ROS generation beyond day 6, as concomitant hemoglobin formation at this time also induce the H2DCF-DA dye generate the fluroscence. We also found by Western blot analysis that thalidomide activated the p38 MAPK signaling pathway in a time- and dose-dependent manner. Thalidomide also increased the histone H3 phospho-acetylation and histone H4 acetylation. In contrast, treatment with the anti-oxidant N-acetylcycteine (NAC) inhibited the thalidomide induced p38 activity and histone H3, H4 acetylation. These data indicate that thalidomide induced the gamma-globin gene increase via activate p38 MAPK signaling pathway as well as histone modification associated with the generation of ROS.
Computational Modeling of Nitric Oxide Bioavailability in Hemolytic Anemias.
Authors: Jeffers Anne , Gladwin Mark , Kim-Shapiro Daniel
Published: April 2020

Intravascular hemoglobin (Hb) scavenges nitric oxide (NO), which decreases the availability of endothelial-derived NO, thereby reducing smooth muscle relaxation. Under normal conditions, there are various mechanisms that limit consumption of NO by Hb in vivo includinga physical barrier to NO diffusion across the red blood cell (RBC) membrane,an unstirred layer creating a concentration gradient in NO outside the RBC, andand a cell-free zone between the endothelium where NO is made and the location of the RBCs.

However, in hemolytic conditions, these mechanisms can be compromised, leading to reduced levels of NO reaching the smooth muscle cells to cause vasodilation. Although it is generally accepted that cell-free Hb scavenges NO more effectively than Hb encapsulated in the red blood cell (RBC), some do not believe that the low concentrations of cell-free Hb that are present in hemolytic conditions, such as sickle cell disease, can impact NO bioavailability in the presence of the large amounts of RBC encapsulated Hb (about 10 mM in heme) present in vivo. We have performed computational modeling looking at the effects of cell-free Hb on NO bioavailability within blood vessels. We have found that concentrations of cell-free Hb as low as one micromolar significantly reduces the availability of NO. Thus, 0.01% hemolysis has a significant affect on steady state NO levels. Extravasation of cell-free Hb into the interstitial space of as low as one micromolar further reduces the bioavailability of NO. At low hematocrit values, cell-free Hb scavenging of NO was found to be more efficient than at high hematocrit values. We have also found that the effect of RBC membrane permeability diminishes at cell-free Hb concentrations of only four micromolar and above. These theoretical results support experimental evidence indicating that cell-free Hb contributes to the pathology of hemolytic diseases. Supported by NIH grant RG0489.
Additive Effect of Butyrate and Hemin on Fetal Hemoglobin Induction in Erythroid Cells from Sickle Cell Disease and Beta-Thalassemia.
Authors: Fathallah Hassana , Taher Ali , Bazarbachi Ali , Atweh George F.
Published: April 2020

High levels of fetal hemoglobin (HbF) are known to ameliorate the pathophysiology of β-globin disorders. The objective of this study is twofold: the first is to evaluate the efficacy of hemin as an inducer of HbF in erythroid cells from patients with sickle cell disease (SCD) and β-thalassemia (β-thal); the second is to determine if the combination of butyrate and hemin can induce higher levels of expression of HbF than either agent alone. BFU-E derived cells from the peripheral blood of two patients with homozygous SCD, three patients with β-thal, one patient with sickle β-thalassemia (S/β-thal) and one normal individual (AA) were cultured in the absence (control) or presence of butyrate (B), hemin (H) or butyrate and hemin (B+H). As expected, the levels of γ-globin mRNA [expressed as % γ/(β+γ)] increased upon butyrate exposure in progenitor-derived erythroid cells from SS and S/β-thal patients, and to a lesser extent in patients with β-thal (P = 0.01). In contrast, butyrate did not increase γ-globin expression in BFU-E derived colonies from the AA individual. Moreover, hemin exposure increased the γ/(β+γ) ratio in all subjects (P = 0.02). These findings confirm that hemin can be an effective HbF inducing agent in SCD and β-thal. Although the mechanism of induction of HbF by hemin is not known, unlike butyrate, hemin is clearly not a direct inhibitor of histone deacetylases and is likely to induce HbF by a different mechanism of action. Thus, we investigated the effect of the combination of hemin and butyrate on γ-globin gene expression. Interestingly, the combination of butyrate and hemin resulted in additive increases in the γ/(β+γ) ratios in all patients compared to butyrate alone (P = 0.03) or hemin alone (P = 0.01) (Table I). Just as importantly, exposure to both drugs resulted in a decrease in the α/(β+γ) mRNA imbalance in β-thal, which is the predominant pathophysiological feature of this disorder. In conclusion, combination therapy consisting of butyrate and hemin, which are two agents with different mechanisms of action and different toxicity profiles, may provide a more effective way of inducing HbF in patients with SCD and β-thal.

Table I mRNA SCD β-Thal S/β-Thal AA n 2 3 1 1 %γ/(β+γ) Control 36 42 26 7.1 B 45 50 41 6.9 H 55 55 52 15 B+H 60 61 59 13 α/(β+γ) Control 3.1 8.9 1.8 1.9 B 2.0 7.7 2.9 1.7 H 3.0 7.5 1.7 1.0 B+H 2.9 6.4 2.2 1.3
Effects of Sickle Cell Disease on Hematopoietic Stem Cell Function and the Bone Marrow Microenvironment.
Authors: Javazon Elisabeth H. , Kean Leslie S. , Perry Jennifer , Butler Jessica , Archer David R.
Published: April 2020

Gene therapy and stem cell transplantation are attractive potential therapies for sickle cell disease (SCD). Previous studies have shown that the sickle environment is highly enriched for reactive oxygen species (ROS), but have not addressed whether or not the increased ROS may alter the bone marrow (BM) microenvironment or affect stem cell function. Using the Berkeley sickle mouse model, we examined the effects of sickle cell disease on hematopoietic stem cell function and the bone marrow microenvironment. We transplanted C57BL/6 (control) BM into C57BL/6 and homozygous sickle mice. Recipients received 2 × 106 BM cells and a conditioning regimen consisting of busulfan, anti-asialo GM1, and co-stimulation blockade (anti-CD40L and CTLA4-Ig). Following transplantation, sickle mice demonstrated increased donor cell engraftment in the peripheral blood compared to normal mice (58.3% vs. 33.1%, respectively). Similarly, BMT in a fully allogeneic system also resulted in enhanced engraftment in sickle recipients. Next we analyzed whether or not engraftment defects exist within the BM stem cell population of sickle mice. In vitro colony forming assays showed a significant decrease in progenitor colony formation in sickle compared to control BM. By flow cytometry, we determined that there was a significant decrease in the KSL (c-Kit+, Sca-1+, Lineage−) progenitor population within the BM of sickle mice. Cell cycle analysis of the KSL population demonstrated that significantly fewer sickle KSL cells were in G0 phase compared to control, suggesting that there are fewer quiescent stem cells in the BM of sickle mice. To assess the potential role of ROS and glutathione depletion in sickle mice, we tested the engraftment efficiency of KSL cells from untreated and n-acetyl-cysteine (NAC) treated control, hemizygous sickle (hemi), and sickle mice in a competitive repopulation experiment. Peripheral chimerism showed an engraftment defect from both hemizygous and homozygous sickle mice such that control KSL cells engrafted > hemi > sickle at a ratio of 1 : 0.4 : 0.25. Treatment with NAC for four months prior to transplantation partially restored KSL engraftment (control : hemi : sickle; 1 : 0.97 : 0.56 ). We have demonstrated that congenic and allogeneic BMT into sickle mice result in increased donor cell engraftment in the sickle recipients. Both the decreased number of KSL cells and the decreased percentage of quiescent KSL cells in the sickle mice indicate that more stem cells in the transgenic sickle mouse model are mobilized from the BM environment. The engraftment defect of sickle KSL cells that was partially ameliorated by NAC treatment suggests that an altered redox environment in sickle mice may contribute to the engraftment deficiencies that we observed.
Endothelial Cell-Derived Microparticules (EMPs) Can Induce In Vitro Maturation of Both Plasmocytoid and Myeloid Dendritic Cells.
Authors: Garnache-Ottou Francine , Angelot Fanny , Biichle Sabeha , PLumas Joel , George Francoise Dignat , Saas Philippe , Tiberghien Pierre , Seilles Estelle
Published: April 2020

The implication of plasmocytoid dendritic cells (pDCs) in transplantation is more and more reported, especially because of their tolerogenic properties supported by the expansion of regulatory T cells. Endothelial microparticles (EMPs) are plasma membrane-derived vesicules shed from endothelial stimulation involved in physiological and pathological processes, including intercellular communication, hemostasis and immunity. However, the interaction between DC and EMP are not well-known, especially in the context of alloreactivity. Therefore, the aims of the current study were to explore the DC/EMP interactions, more specifically the capacity of EMPs to modulate the maturation of DC. Firstly, we validated an original co-culture model involving a tumoral pDC lineage (GEN 2.2) and EMPs generated in a standard fashion from an endothelial cell lineage (HMEC U608), stimulated or not with TNF-α . EMPs was analyzed by FACS (FITC labelled annexin V) and supernatant resulting from the last wash of EMPs was used as negative control. By incubating increasing amounts of EMPs we showed that EMPs can lead to GEN pDC maturation in a dose- effect manner as evidenced by a significative increase in the HLA-DR and CD83 expression, compared to the negative controls or, of importance, compared to the maturation with CPG-A (ligand of TLR9 mimicking bacterial DNA), CD40L or R848 (ligand of TLR7 mimicking viral RNA). To confirm and expand these findings, we then cocultured with EMPs (and similar controls)

circulating pDC obtained by an anti-BDCA-4 MoAb-mediated positive selection of healthy donors PBMC , and myeloid DC (mDC) obtained from peripheral blood monocytes cultured in the presence of GM-CSF and IL-4.

Maturation of both normal circulating pDC and mDC occurred in the presence of EMPs and once again, in a dose-dependant fashion. The cytokine secretion pattern of the EMPs- matured mDC and pDC, determined by the Luminex technology, was as expected for such cell populations. The EMPs matured pDC secreted IL-1, IL-6, IFN-α , IL-12 and IL-10 but not TGF-β . In contrast, the EMPs matured mDC secreted mainly proinflammatory cytokines : IL-1, IL-6 and IL-8 but not IL-10. Overall, our studies demonstrate that EMPs can induce the maturation of both pDC and mDC. Such endothelial microparticules, generated by inflammatory and hemostatic disorders, could play a role in post-transplant immune responses. Determining the factors resulting in EMPs-induced pDC vs mDC maturation may open the possibility for EMPs-related immuno-intervention.
ISG12 Is a Critical Modulator of Innate Immune Responses in Animal Models of Septis.
Authors: Schabbauer Gernot , Papac-Milicevic Nikolina , Uhrin Pavel , Binder Bernd
Published: April 2020

Sepsis is still a major burden for the society with a high incidence of morbidity and mortality each year. Molecular mechanisms underlying the systemic inflammatory response syndrome (SIRS) associated with sepsis are still ill defined and most therapies developed to target the acute inflammatory component of the disease are insufficent.

Recently the role of nuclear receptors (NRs) in transcriptional regulation of inflammatory processes became a major topic of interest. Nuclear receptors, such as the peroxisome proliferators-activated receptors (PPARs), have been found to exert anti-inflammatory properties by interfering with the NFkB pathway.

We are interested in the nuclear envelope protein, interferon stimulated gene 12 (ISG12), which directly interacts with NRs. ISG12 is a co-factor stimulating nuclear export of NRs, thereby reducing the anti-inflammatory potential of NRs such as PPARg or NR4A1. To examine the role of ISG12 in acute inflammation we generated ISG12 deficient mice.

We can demonstrate by reverse genetics in ISG12 deficient mice that lack of ISG12 is beneficial in experimental sepsis and endotoxemia. Furthermore we can show that several acute inflammatory parameters, such as systemic IL6 cytokine levels, are downregulated in septic ISG12-/- animals. Consistently, similar results could be obtained in in vitro experiments in peritoneal macrophages derived from ISG12 deficient mice.

In contrast, mice deficient for the nuclear receptor NR4A1 exhibited an exacerbated innate immune response and showed a significantly higher mortality after lethal septic challenge. This dramatic phenotype could be restored in ISG12/NR4A1 double deficient mice.

We conclude from our data in vitro and in vivo that ISG12 is a novel modulator of innate immune responses regulating anti-inflammatory nuclear receptors such as NR4A1.