scholarly journals Lyn Kinase Activity Is Required for Akt Mediated Erythroleukemia Cell Differentiation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 24-24
Author(s):  
Janice H. C. Plani-LAM ◽  
Mhairi Maxwell ◽  
Neli Slavova-Azmanova ◽  
Nicole Kucera ◽  
Alison Louw ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Epo Receptor ◽  
Lyn Kinase

Erythroleukemia (M6 subtype of Acute Myeloid Leukaemia) is uncommon but has a poor prognosis, with reports of successful differentiation therapy using erythropoietin (Epo). Signaling through the Epo-receptor, which involves JAK2 and Lyn tyrosine kinases, controls red blood cell progenitor development. We have highlighted the importance of Lyn for regulating downstream Akt, and feed-back inhibitory signaling of the Epo-receptor through analysis of Lyn-/-, Lynup/up (hyperactive Lyn) and Cbp-/- (Csk-binding protein, a negative regulator of Lyn) erythroid cells. However, the importance of maintaining Lyn activity as opposed to Lyn protein for erythroid cell development and signaling, has not been delineated. To address this, we generated LynKD/KD mice (expressing a kinase dead K275M mutant Lyn), and analysed their erythroid compartment and signaling in immortalized erythroid progenitors. We show that LynKD/KD mice display splenic extramedullary erythropoiesis and have evidence of elevate bone marrow erythropoiesis, similar to Lyn-/- mice but with a less severe phenotype. Immortalized erythroid progenitors from LynKD/KD mice show impaired Epo-induced differentiation and a greater dependence on Epo for viability, but unaltered proliferation, compared to wild-type cells. Epo-induced signaling of LynKD/KD cells showed enhanced pJAK2/pSTAT5, reduced pAkt/pGAB2, and substantially reduced ALAS-e levels, compared to wild-type cells. Importantly, elevating Akt signaling in LynKD/KD cells by addition of phosphatase inhibitors (okadaic acid or Calyculin A), or via expression of active Akt, restored their differentiation capacity (and ALAS-e levels) and reduced their dependence on Epo for viability. We have unveiled that Lyn kinase activity, and not just its expression, is required for correct signaling of Akt to GATA-1 to maintain ALAS-e expression in erythroid cells, enabling hemoglobin production and viability during differentiation. Disclosures No relevant conflicts of interest to declare.

GNF-2, An Allosteric BCR-ABL Inhibitor, Identifies a Novel Myristoylation-Mediated Mechanism Regulating the Ability of BCR-ABL to Activate HCK and IGF-1 Signaling.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 40-40 ◽  
Author(s):  
Ron Geyer ◽  
Teodora Zlateva ◽  
Asha Lakshmikuttyamma ◽  
David P. Sheridan ◽  
John F. DeCoteau
Keyword(s):  
Binding Sites ◽  
Cellular Localization ◽  
Kinase Activity ◽  
Conflicts Of Interest ◽  
Biological Effects ◽  
Binding Pocket ◽  
Negative Regulator ◽  
Wild Type ◽  
Abl Kinase ◽  
Competitive Inhibitors

Abstract Abstract 40 Pharmacologic inhibition of BCR-ABL, using clinically active ATP-competitive inhibitors imatinib, nilotinib, and dasatinib, has been used to investigate BCR-ABL kinase activated signaling pathways. However, these agents show cross reactivity with other kinases (e.g. Kit, PDGFR, SRC family members), and their multi-targeted nature complicates assigning biological effects to the inhibition of a specific kinase target. Allosteric kinase inhibitors modulate the catalytic activity of protein kinases by binding to a site distant from the active site and inducing a protein conformation that inhibits kinase activity. These agents show promise as clinical agents and may offer advantages over ATP-competitive inhibitors in studying the function of specific kinases because they exploit binding sites and regulatory mechanisms that are unique to a particular kinase. GNF-2, a mono-selective BCR-ABL inhibitor that targets wild-type BCR-ABL and many clinically relevant imatinib resistant mutants, was recently discovered and provided the first demonstration that c-ABL kinase activity could be modulated by an inhibitor that binds outside the ATP or substrate binding sites. GNF-2 binds to a myristoyl-binding pocket in the C-lobe of the c-ABL kinase domain but its mechanism of inhibiting specific BCR-ABL kinase targets remains unclear. We previously reported that BCR-ABL activates an autocrine IGF-1 pro-survival signaling pathway in CML blast crisis cells through HCK-mediated activation of STAT5b. As GNF-2 is known to inhibit STAT5b phosphorylation, and HCK myristolyation is known to regulate its cellular localization, we hypothesized that GNF-2 inhibits BCR-ABL activation of HCK by binding to the ABL myristoyl-binding pocket and blocking access to the HCK myristoyl moiety. In support of this hypothesis, we now show that GNF-2 inhibits HCK phosphorylation and IGF-1 activation, but not HCK binding to BCR-ABL. To confirm the importance of the HCK myristoyl moiety in HCK activation, we mutated the myristoyl attachment site at position 2 in HCK from glycine to alanine. The mutant G2A HCK still interacted with BCR-ABL in co-immunoprecipitation assays but showed significantly lower levels of phosphorylation compared to wild-type HCK. To confirm that the decrease in phosphorylation was not due to mislocalization of G2A HCK, we mutated the myristoylation binding pocket of BCR-ABL by changing glutamic acid at position 505 to lysine. Similar to G2A HCK, E505K BCR-ABL still interacted with HCK, but the phosphorylation levels of HCK were dramatically reduced. To confirm that the HCK myristoyl moiety directly interacted with the ABL myristoyl-binding pocket, we used fluorescent spectroscopy to measure the ability of a myristoylated peptide corresponding to the six N-terminal amino acids of HCK to displace GNF-2. The fluorescence of GNF-2 is enhanced when it associates with the myristoyl-binding pocket of ABL. Using this assay, we calculated the Kd of GNF-2 to be 180 nM. We then assayed the ability of myristolyated HCK peptide to displace GNF-2 from ABL. We calculated the IC50 of the myristolyated HCK peptide to be 25 μM when ABL was saturated with 300 nM GNF-2. Myristate showed an IC50 of 213 μM, which is ∼ 10-fold higher than the myristoylated peptide. No binding was detected between the non-myristoylated peptide and ABL. Together, our study highlights a novel acquired function resulting from the fusion of BCR to the N-terminus of ABL, which converts the myristoyl-binding pocket in ABL from a negative regulator of kinase activity to an HCK activation motif that activates downstream IGF-1 signaling. These results also reveal the mechanism of action of the mono-selective BCR-ABL inhibitor GNF-2 and highlight the ABL myristoyl-binding pocket as a therapeutic target for inhibiting BCR-ABL activity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Global Chromatin Occupancy and Epigenetic Signature Analysis Reveal New Insights into the Function of GATA1 N-Terminus in Erythropoiesis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 533-533
Author(s):  
Te Ling ◽  
Yehudit Birger ◽  
Monika Stankiewicz ◽  
Nissim Ben-Haim ◽  
Itamar Kanter ◽  
...  
Keyword(s):  
Research Funding ◽  
Regulatory Elements ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Cell Counts ◽  
Depth Analysis ◽  
N Terminus

Abstract Mutations in GATA1 are seen in rare cases of dyserythropoietic anemia and in a subset of patients with Diamond Blackfan Anemia (DBA). Of note the truncation mutations in DBA, known as GATA1s, closely resemble those that are more commonly associated with acute megakaryoblastic leukemia in children with Down syndrome (DS). Studies with a mouse model of the Gata1s mutation revealed that replacement of the full-length protein by the shortened isoform led to a marked yet transient enhancement in megakaryopoiesis, similar in some respects to transient myeloproliferative disorder in DS. Furthermore, these mutant mice displayed impaired embryonic erythropoiesis but ostensibly no defects in adult hematopoiesis. In our efforts to better understand the connection between GATA1s and DBA, we comprehensively studied erythropoiesis in the Gata1s mouse strain. We observed a striking impairment in erythropoiesis in fetuses at E10.5 though E12.5, but saw improvement as the animals progressed through E14.5 and beyond. Defects included impaired terminal maturation and reduced numbers of erythroid progenitors, likely at the expense of expanded megakaryopoiesis. RNA-sequencing revealed that both erythroid genes and megakaryocytic genes were altered by the Gata1s mutation. Epiproteomic histone modification analysis further revealed there was an accumulation of H3K27 methylation in the R3 (CD71hiTer119hi) erythroid progenitor population, which suggests that GATA1 has a link to the epigenetic machinery that is altered in Gata1s mutant cells. Despite a global increase in H3K27me3, critical Gata2 regulatory elements in Gata1s mutant erythroid progenitors were marked by substantially less H3K27me3 than in wild-type littermates. Given that overexpression of GATA2 has been reported to impair erythropoiesis, we investigated whether reducing the GATA2 levels would ameliorate the phenotype. Indeed, we observed that haploinsufficiency for Gata2 rescued the erythroid defects of Gata1s fetuses. Next, to comprehensively study the effect of absence of the GATA1 N-terminus genome-wide, we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) with H3K27me3, GATA1 or GATA1s antibodies on wild-type versus Gata1s expressing fetal erythroid cells. Our data indicated that there is a substantial reduction in H3K27me3 along regulatory elements of the Runx1 gene at the late stage (R3) of fetal erythropoiesis in Gata1s mice. Along with an increase in Runx1 expression we observed strong downregulation of Klf1, a repressive target of RUNX1. Thus, failure of GATA1s to facilitate trimethylation of Runx1 and Gata2 regulatory elements appears to cause the defects in erythroid cell and megakaryocyte development. In parallel, we performed an in-depth analysis of the phenotype of adult Gata1s mice and discovered that they have reduced red cell counts, lower hemoglobin and hematocrit, increased extramedullary hematopoiesis and impaired stress erythropoiesis compared to control littermates. Although there were significantly more megakaryocyte erythrocyte progenitors (MEPs, Lin-c-Kit+Sca-1-CD34-FcgR-) in Gata1s mouse bone marrow, there were fewer pre-colony-forming unit erythroid cells (preCFU-E, Lin-c-Kit+Sca-1-CD41-FcgR-CD150hiCD105hi), likely at the expense of expanded megakaryocyte progenitors (MkP, Lin-c-Kit+Sca-1-CD41+CD150hi).Gata1s mice also developed an MDS-like disease with age. Together, our integrated genomic analysis of transcriptome, GATA1/GATA1s chromatin binding profile and chromatin signature reveal that, although Gata1s mice do not precisely model DBA, they provide novel insights into the role of the N-terminus of GATA1 in gene transcriptional regulation, lineage determination and red blood cell maturation. Disclosures Crispino: Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

scholarly journals Neocytolysis Is Mediated by down Regulation of Catalase By Mir-21 Resulting in defective Clearance of reticulocyte mitochondrial ROS

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1336-1336
Author(s):  
Song Jihyun ◽  
Yoon Donghoon ◽  
Robert D. Christensen ◽  
Perumal Thiagarajan ◽  
Josef T. Prchal
Keyword(s):  
Nadh Oxidase ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Erythroid Cell ◽  
Ros Scavenging ◽  
Erythroid Progenitors ◽  
Hypoxia Exposure ◽  
Oxygen Homeostasis ◽  
Mitochondrial Ros ◽  
Hypoxia Treatment

Abstract Oxygen homeostasis is tightly controlled by the number of red blood cells (RBCs). Hypoxia increases RBCs by enhanced erythropoiesis mediated by hypoxia-inducible factors (HIFs). Upon return to normoxia, the increase in RBCs is overcorrected by preferential destruction of young RBCs, a process termed neocytolysis. Neocytolysis was first described in astronauts and later in people descending from high altitude. The molecular mechanism of neocytolysis is obscure. We hypothesized that neocytolysis occurs because of rapid transient changes of HIF levels, resulting in increased reactive oxygen species (ROS) from mitochondria in reticulocytes and defective antioxidant protection of young RBCs generated in hypoxia. We developed a neocytolysis model by exposing mice to 12% oxygen (equivalent to 4500m altitude) for 10 days. This model recapitulates the RBC changes observed in humans exposed to hypoxia for ~30 days. Upon return to normoxia, ROS were markedly increased in reticulocytes and mature RBCs, but not in neutrophils, B- or T-cells, or monocytes. Reduction of ROS by antioxidant (N-acetyl-L cysteine) treatment attenuated hemolysis and decreased hematocrit. To test whether HIFs (transcription factors regulating hypoxic response) contribute to neocytolysis, we repeated these experiments using Chuvash mice (which bear a VHLR200Wmutation, resulting in constitutively high HIF). These mice also showed attenuated hemolysis and decreased hematocrit; in addition, their reticulocyte half-life was higher (36.6 vs.17.8 hours in wild type). Similar findings were also observed with treatment of mice with dimethyloxallyl glycine (DMOG), an inhibitor of prolyl hydroxylase (another negative regulator of HIF). These experiments indicate the essential role of HIF pathways in neocytolysis. Mitochondria are a major source of ROS in cells. During terminal differentiation of RBCs, mitochondria are removed from reticulocytes by mitophagy. After hypoxia treatment, mitochondria mass increased in reticulocytes concomitant with the reduction of HIF-regulated Bnip3L (a mediator of mitophagy) transcripts. These increased ROS were of mitochondrial origin, as detected by Mito-Sox staining. To pursue the mechanism behind the preferential destruction of young RBCs, we investigated antioxidant enzymes. After hypoxia treatment, catalase decreased by 30%, but not glutathione peroxidase, superoxide dismutase or NADH oxidase. The decreased catalase in RBCs produced during hypoxia was unexpected, as it was shown previously that catalase is regulated by HIF2 (as we also show, to a lower degree by HIF1 in our Hif1a-/-embryo) suggesting alternate negative regulator(s) of catalase in hypoxia. Several hypoxia-regulated microRNAs (miRs) are reported to control oxidative stress; we found that miR-451, miR-205 and miR-21 were expressed in erythroid progenitors and reticulocytes and induced after 10 day-hypoxia exposure. To verify whether these miRs regulate catalase expression, we overexpressed and downregulated these miRs in K562 and HEL erythroid cell lines, and found that only miR-21 regulated catalase. Further, we found increased miR-21 after 10 day-hypoxia exposure, with a concomitant decrease of catalase transcripts and activity resulting in impaired ROS scavenging. We conclude that neocytolysis is mediated by excessive generation of ROS from increased mitochondrial mass due to reduced Bnip3L in reticulocytes upon return to normoxia. The reticulocyte ROS then interact with hypoxia-produced young RBCs having miR-21-downregulated catalase, resulting in their preferential destruction. We show that increased mitochondrial ROS and miR-21-downregulated catalase provide the molecular basis of neocytolysis. Disclosures No relevant conflicts of interest to declare.

Lyn kinase plays important roles in erythroid expansion, maturation and erythropoietin receptor signalling by regulating inhibitory signalling pathways that control survival

2014 ◽  
Vol 459 (3) ◽  
pp. 455-466 ◽  
Author(s):  
Neli S. Slavova-Azmanova ◽  
Nicole Kucera ◽  
Alison Louw ◽  
Jiulia Satiaputra ◽  
Adley Handoko ◽  
...  
Keyword(s):  
Cell Survival ◽  
Cell Development ◽  
Erythropoietin Receptor ◽  
Erythroid Cell ◽  
Signalling Pathways ◽  
Erythroid Cells ◽  
Src Family Kinase ◽  
Receptor Signalling ◽  
Lyn Kinase ◽  
Control Survival

In erythroid cells both positive viability signals and feedback inhibitory signalling require the Src family kinase Lyn, influencing cell survival and their ability to differentiate. This illustrates that Lyn is critical for normal erythropoiesis and erythroid cell development.

scholarly journals Roles of focal adhesion kinase (FAK) in megakaryopoiesis and platelet function: studies using a megakaryocyte lineage–specific FAK knockout

Blood ◽  
2008 ◽  
Vol 111 (2) ◽  
pp. 596-604 ◽  
Author(s):  
Ian S. Hitchcock ◽  
Norma E. Fox ◽  
Nicolas Prévost ◽  
Katherine Sear ◽  
Sanford J. Shattil ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Platelet Function ◽  
Therapeutic Benefit ◽  
Negative Regulator ◽  
Null Mouse ◽  
Wild Type ◽  
Platelet Recovery ◽  
Lyn Kinase

Focal adhesion kinase (FAK) plays a key role in mediating signaling downstream of integrins and growth factor receptors. In this study, we determined the roles of FAK in vivo by generating a megakaryocyte lineage–specific FAK-null mouse (Pf4-Cre/FAK-floxed). Megakaryocyte and platelet FAK expression was ablated in Pf4-Cre/FAK-floxed mice without affecting expression of the FAK homologue PYK2, although PYK2 phosphorylation was increased in FAK−/− megakaryocytes in response to fibrinogen. Megakaryopoiesis is greatly enhanced in Pf4-Cre/FAK-floxed mice, with significant increases in megakaryocytic progenitors (CFU-MK), mature megakaryocytes, megakaryocyte ploidy, and moderate increases in resting platelet number and platelet recovery following a thrombocytopenic stress. Thrombopoietin (Tpo)–mediated activation of Lyn kinase, a negative regulator of megakaryopoiesis, is severely attenuated in FAK-null megakaryocytes compared with wild-type controls. In contrast, Tpo-mediated activation of positive megakaryopoiesis regulators such as ERK1/2 and AKT is increased in FAK-null megakaryocytes, providing a plausible explanation for the observed increases in megakaryopoiesis in these mice. In Pf4-Cre/FAK-floxed mice, rebleeding times are significantly increased, and FAK-null platelets exhibit diminished spreading on immobilized fibrinogen. These studies establish clear roles for FAK in megakaryocyte growth and platelet function, setting the stage for manipulation of this component of the Tpo signaling apparatus for therapeutic benefit.

STAT5 Requires the N-Domain for Suppression of miR15/16, Induction of Bcl-2, and Survival Signaling in Myeloproliferative Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 211-211
Author(s):  
Geqiang Li ◽  
Kristy L. Miskimen ◽  
Zhengqi Wang ◽  
Xiu Yan Xie ◽  
Jennifer Brenzovich ◽  
...  
Keyword(s):  
Mast Cells ◽  
Tyrosine Kinases ◽  
Conflicts Of Interest ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Myeloproliferative Disease ◽  
Wild Type ◽  
Virtual Absence ◽  
Protein Levels ◽  
Survival Signaling

Abstract Abstract 211 Phosphorylated signal transducer and activator of transcription 5 (STAT5) can inform diagnosis and clinical outcome in myeloid leukemia. STAT5 may be an important therapeutic target for hematologic disease characterized by constitutively active tyrosine kinases. The most direct evidence that STAT5 is crucial for oncogenic transformation has come from mouse models, which showed that STAT5-deficient hematopoietic cells are resistant to transformation by oncogenic tyrosine kinases, such as TEL-JAK2, TEL-PDGF, or BCR-ABL. The oncogenic role of STAT5 has been recognized and likely extends to other activated kinases such as JAK2, MPL, and FLT3. Although up-regulation of genes such as bcl-2, bcl-XL, mcl-1, D-type cyclins, and myc by activated oncogenic tyrosine kinases has been demonstrated, the specific mechanisms how STAT5 can critically control pre-leukemic expansion in the myeloid lineage have not been defined. We have shown previously that lethal myeloproliferative disease (MPD) in mice mediated by persistently activated STAT5 (STAT5aS711F) requires the N-domain but the mechanism was not defined. We now demonstrate by retrovirally complementing STAT5abnull/null primary mast cells that STAT5a lacking the N-domain (STAT5aΔN) ineffectively protected against cytokine withdrawal-induced cell death relative to wild-type STAT5a. To study the mechanisms for this survival defect, bcl-2 and bcl-XL protein levels were analyzed by Western blot and shown to be greatly reduced. Whether bcl-2 is a direct target gene of STAT5 in native chromatin as validated by chromatin immunoprecipitation (ChIP) has not been previously shown. We identified 7 conserved STAT5 binding sites in the bcl-2 gene and tested these by ChIP. Only one site located within intron 2 was bound by STAT5a and STAT5aΔN in mast cells cultured in IL-3 alone, where bcl-2 mRNA levels were low. STAT5a add-back induced bcl-2 mRNA (10-fold) compared with STAT5aΔN (2- to 3-fold). Interestingly, when STAT5 was absent the mRNA levels of bcl-2 were not reduced when mast cells were grown in the presence of both IL-3 and SCF, despite the virtual absence of bcl-2 protein. MicroRNAs (miRs) are small, noncoding, single-stranded RNAs of ∼22 nucleotides that negatively regulate gene expression at the post-transcriptional level primarily through targeting the 3'-UTR of target mRNAs. There are no current reports of STAT5 mediated miR expression. We found that wild-type STAT5a and STAT5aS711F suppressed accumulation of miR15b/16 in primary mast cells and transduced BaF3 cells. Importantly, we show binding of STAT5 to a conserved STAT5 binding site in the promoter of miR15b/16 by ChIP. We propose that reciprocal induction of bcl-2 mRNA and suppression of miR15b/16 maintained bcl-2 protein levels. We also observed binding of STAT5aΔNS711F at both sites by ChIP, indicating that co-activator and co-repressor interactions with the N-domain determine regulation of bcl-2. To test whether these observations of bcl-2 regulation were physiologically relevant in the setting of activated STAT5 in myeloproliferative disease, retroviral complementation of STAT5abnull/null fetal liver cells was performed and followed by transplantation into lethally-irradiated recipients. Persistently active STAT5aS711F lacking the N-domain (STAT5aΔNS711F) was insufficient to protect c-Kit+Lin−Sca-1+ (KLS) cells from apoptosis as determined by Annexin V/DAPI staining. These cells were unable to induce bcl-2 expression determined by intracellular flow cytometry 15 days following injection of donor cells. In contrast, STAT5aS711F caused robust KLS cell expansion, induction of bcl-2, and 47-fold expansion of peripheral Gr-1+Mac-1+ cells. In the absence of the STAT5 N-domain a mild splenomegaly was observed with 50% of mice surviving greater than 90 days, instead of a rapidly lethal monocytic disease observed when full-length STAT5aS711F was present, with death of all mice by 35 days. Importantly, the modest 6-fold increase in peripheral Gr-1+Mac-1+ counts and better survival conferred by STAT5aΔNS711F could be reversed to 41-fold above control levels by adding back only bcl-2 through H2k/bcl-2 transgenic expression. Overall, these studies define N-domain dependent survival signaling as an Achilles' heel of persistent STAT5 activation and highlight the potential therapeutic importance of targeting STAT5 N-domain mediated regulation of bcl-2 family members. Disclosures: No relevant conflicts of interest to declare.

Klf1 Regulatory Networks in Primary Erythroid Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1462-1462
Author(s):  
Michael Tallack ◽  
Thomas Whitington ◽  
Brooke Gardiner ◽  
Eleanor Wainwright ◽  
Janelle Keys ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Es Cells ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Gene Promoters ◽  
Erythroid Cells ◽  
Red Cell Membrane ◽  
Sequencing Platform

Abstract Abstract 1462 Poster Board I-485 Klf1/Eklf regulates a diverse suite of genes to direct erythroid cell differentiation from bi-potent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which Klf1 works, we performed Klf1 ChIP-seq using the SOLiD deep sequencing platform. We mapped more than 10 million unique 35mer tags and found ∼1500 sites in the genome of primary fetal liver erythroid cells are occupied by endogenous Klf1. Many reside within well characterised erythroid gene promoters (e.g. b-globin) or enhancers (e.g. E2f2 intron 1), but some are >100kb from any known gene. We tested a number of Klf1 bound promoter and intragenic sites for activity in erythroid cell lines and zebrafish. Our data suggests Klf1 directly regulates most aspects of terminal erythroid differentiation including synthesis of the hemoglobin tetramer, construction of a deformable red cell membrane and cytoskeleton, bimodal regulation of proliferation, and co-ordination of anti-apoptosis and enucleation pathways. Additionally, we suggest new mechanisms for Klf1 co-operation with other transcription factors such as those of the gata, ets and myb families based on over-representation and spatial constraints of their binding motifs in the vicinity of Klf1-bound promoters and enhancers. Finally, we have identified a group of ∼100 Klf1-occupied sites in fetal liver which overlap with Klf4-occupied sites in ES cells defined by Klf4 ChIP-seq. These sites are associated with genes controlling the cell cycle and proliferation and are Klf4-dependent in skin, gut and ES cells, suggesting a global paradigm for Klfs as regulators of differentiation in many, if not all, cell types. Disclosures No relevant conflicts of interest to declare.

Kinase Activity of RTKs Is Dispensable for Factor Independent Growth and Transformation of a Myeloid Cell Line 32D in the Presence of Cbl Mutants.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3970-3970
Author(s):  
◽  
Srinivasa Rao Bandi ◽  
Marion Rensinghoff ◽  
Rebekka Grundler ◽  
Lara Tickenbrock ◽  
...  
Keyword(s):  
Cell Line ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Conflicts Of Interest ◽  
Myeloid Cell ◽  
Dominant Negative ◽  
Class Iii ◽  
Primary Resistance

Abstract Abstract 3970 Poster Board III-906 Purpose The Cbl proto-oncogene products have emerged as important components of the signal transduction cascades downstream of both non-receptor and receptor tyrosine kinases (RTKs). By regulation of receptor trafficking and degradation, they have been shown to tightly regulate the intensity and amplitude of RTK activation. c-Kit belongs to the family of the class-III RTKs and plays an important role in the pathogenesis of acute myeloid leukemia (AML). So far, very little is known about the role of c-Cbl mutants in the role of c-Kit signaling. Results We analyzed the interaction of c-Cbl with c-Kit and the functional relevance of this interaction in the IL-3-dependent murine myeloid progenitor cell line 32Dcl3. We recently identified the first c-Cbl mutation in human disease in an AML patient, named Cbl-R420Q. Co-expression of two different dominant negative mutants of c-Cbl (Cbl-R420Q or Cbl-70Z) with Kit induced cytokine-independent proliferation, survival and clonogenic growth. Importantly, transformation was observed also with kinase-dead forms of Kit and Flt3 in the presence of Cbl-70Z, but not in the absence of Kit or Flt3, suggesting a mechanism dependent on RTKs, but independent of their kinase activity. Instead, transformation appeared to depend on Src family kinases (SFKs), as c-Cbl co-immunoprecipitated with SFKs and SFK inhibition abolished transformation. Conclusion Our results indicate that c-Cbl has an important role in c-Kit signal mitigation. They demonstrate that disturbed mechanisms of c-Kit internalization have important implications for its transforming potential, possibly in the development of AML. Furthermore, these findings may explain primary resistance to tyrosine kinase inhibitors targeted at RTKs. Disclosures: No relevant conflicts of interest to declare.

Studies on Heme Oxygenase 1 During Erythroid Differentiation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4254-4254
Author(s):  
Daniel Garcia Santos ◽  
Jesse Eisenberg ◽  
Matthias Schranzhofer ◽  
Prem Ponka
Keyword(s):  
Aminolevulinic Acid ◽  
Conflicts Of Interest ◽  
Tissue Injury ◽  
Erythroid Differentiation ◽  
The Body ◽  
Cellular Damage ◽  
Erythroid Cell ◽  
Heme Oxygenase 1 ◽  
Erythroid Cells ◽  
Murine Erythroleukemia

Abstract Abstract 4254 Heme is indispensable for the function of all aerobic cells as a prosthetic group of innumerable proteins. However, “free heme” (uncommitted) can initiate the formation of free radicals and cause lipid peroxidation, which can lead to cellular damage and tissue injury. Therefore, the rate of heme biosynthesis and catabolism must be well balanced by tight control mechanisms. The highest amounts of organismal heme (75-80%) are present in circulating red blood cells (RBC), whose precursors synthesize heme with rates that are at least one order of magnitude higher (on the per cell basis) than those in the liver – the second most active heme producer in the body. The degradation of heme is exclusively carried out by heme oxygenases 1 and 2 (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using both a murine erythroleukemia cell line (MEL) and primary erythroid cells isolated from mouse fetal livers, we have demonstrated that during erythroid differentiation HO1 is up-regulated at both mRNA and protein levels. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase. These data suggest that in developing RBC, in addition to the continuous assembly of heme with globin chains, there is an increase in levels of uncommitted heme, which upregulates HO1 expression. Additionally, we have shown that down-regulation of HO1 via siRNA increased hemoglobinization in differentiating MEL cells. In contrast, induction of HO1 expression by NaAsO2 reduced the hemoglobinization of MEL cells. This effect could be reversed to control levels by the addition of HO1 inhibitor tin-protophorphyrin (SnPP). These results show that in differentiating erythroid cells the balance between levels of heme and HO1 have to be tightly regulated to maintain hemoglobinization at appropriate levels. Our results lead us to propose that disturbances in HO1 expression could play a role in some pathophysiological conditions such as thalassemias. Disclosures: No relevant conflicts of interest to declare.

Heme Oxygenase 1 Plays An Unexpected Role During Erythroid Differentiation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 344-344
Author(s):  
Daniel Garcia Santos ◽  
Matthias Schranzhofer ◽  
José Artur Bogo Chies ◽  
Prem Ponka
Keyword(s):  
Red Blood Cells ◽  
Heme Oxygenase ◽  
Blood Cells ◽  
Erythroid Differentiation ◽  
Heme Biosynthesis ◽  
Erythroid Cell ◽  
Heme Oxygenase 1 ◽  
Erythroid Cells ◽  
Wild Type ◽  
Protein Levels

Abstract Abstract 344 Red blood cells (RBC) are produced at a rate of 2.3 × 106 cells per second by a dynamic and exquisitely regulated process known as erythropoiesis. During this development, RBC precursors synthesize the highest amounts of total organismal heme (75–80%), which is a complex of iron with protoporphyrin IX. Heme is essential for the function of all aerobic cells, but if left unbound to protein, it can promote free radical formation and peroxidation reactions leading to cell damage and tissue injury. Therefore, in order to prevent the accumulation of ‘free' heme, it is imperative that cells maintain a balance of heme biosynthesis and catabolism. Physiologically, the only enzyme capable of degrading heme are heme oxyganase 1 & 2 (HO). Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Heme oxygenase, in particular its heme-inducible isoform HO1, has been extensively studied in hepatocytes and many other non-erythroid cells. In contrast, virtually nothing is known about the expression of HO1 in developing RBC. Likewise, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using primary erythroid cells isolated from mouse fetal livers (FL), we have shown that HO1 mRNA and protein are expressed in undifferenetiated FL cells and that its levels, somewhat surprisingly, increase during erythropoietin-induced erythroid differentiation. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase, the second enzyme in the heme biosynthesis pathway. Moreover, we have found that down-regulation of HO1 via siRNA increases globin protein levels in DMSO-induced murine erythroleukemic (MEL) cells. Similarly, compared to wild type mice, FL cells isolated from HO1 knockout mice (FL/HO1−/−) exhibited increased globin and transferrin receptor levels and a decrease in ferritin levels when induced for differentiation with erythropoietin. Following induction, compared to wild type cells, FL/HO1−/− cells showed increased iron uptake and its incorporation into heme. We therefore conclude that the normal hemoglobinization rate appears to require HO1. On the other hand, MEL cells engineered to overexpress HO1 displayed reduced globin mRNA and protein levels when induced to differentiate. This finding suggests that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias. Disclosures: No relevant conflicts of interest to declare.

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