scholarly journals GATA2 Zinc Finger Mutations Influence Erythroid Differentiation and Chemotherapy Response

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1459-1459
Author(s):  
Georg Leubolt ◽  
Enric Redondo Monte ◽  
Anja Wilding ◽  
Paul Kerbs ◽  
Monica Cusan ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Research Funding ◽  
Blast Crisis ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Therapy Resistance ◽  
Chemotherapy Response ◽  
Dependent Manner ◽  
Monocytic Differentiation ◽  
Hematopoietic Stem

The transcription factor GATA2 plays an important role in cell lineage decisions during hematopoiesis. GATA2 Zinc-Finger (ZF) mutations are associated with distinct entities of myeloid malignancies. Alterations of the N-terminal ZF1 were identified in AML patients with biallelic CEBPA mutations, whereas the C-terminal ZF2 is typically affected by germline mutations predisposing to MDS and AML, or by somatic lesions in CML blast crisis. Nevertheless, the context-dependent mechanisms underlying GATA2 ZF mutations remain mostly unclear. Here, we set out to study the functional consequences of GATA2 ZF mutations. To test the effect on differentiation, we expressed GATA2 wild-type (WT) or GATA2 ZF mutants in human CD34+ hematopoietic stem and progenitor cells, stimulated with appropriate cytokines. Differentiation was evaluated by FACS-measurements of surface marker expression (erythroid markers: CD71, CD235a; granulocytic/monocytic markers: CD15, CD14). GATA2 WT caused a block of erythroid differentiation that is overcome by the ZF1 mutants (A318T and G320D), whereas the ZF2 mutant L359V may aggravate this block. For granulocytic/monocytic differentiation an overall block was observed for GATA2 WT and all the ZF mutants tested (Figure 1 A, B). Recently, we and others observed GATA2 mutation gain in AML relapse (Greif et al., 2018 Clin Cancer Res; Christopher et al., 2018, NEJM), pointing towards a potential role in therapy resistance. Therefore, we treated K562 cells stably expressing GATA2 WT or mutants with Daunorubicin (one of the two drugs commonly used in AML chemotherapy). Expression of GATA2 A318T in K562 cells correlated with higher sensitivity to Daunorubicin and lower expression levels of IDH2. (Figure 1 C, D). Interestingly, this particular ZF1 mutation was the only one that got lost at relapse in the study by Christopher and colleagues, consistent with therapy sensitivity, whereas most of the GATA2 mutations gained at relapse were localized in the ZF2 domain. In summary, GATA2 ZF mutations influence hematopoietic differentiation and chemotherapy response in a position-dependent manner. In the present study, we report distinct roles for individual GATA2 mutations depending on the affected ZF domain and altered amino acid positions. Understanding the oncogenic collaboration of GATA2 mutations with other driver genes in distinct patient subgroups is a challenge ahead. Figure.1 Disclosures Hiddemann: Roche: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria; Bayer: Research Funding; Vector Therapeutics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding.

VITAMIN E ACTIVATES EXPRESSION OF C/EBP ALPHA TRANSCRIPTION FACTOR AND G-CSF RECEPTOR IN LEUKEMIC K562 CELLS

2018 ◽  
Vol 40 (4) ◽  
pp. 328-331
Author(s):  
L P Shvachko ◽  
M P Zavelevich ◽  
D F Gluzman ◽  
I V Kravchuk ◽  
G D Telegeev
Keyword(s):  
Transcription Factor ◽  
Vitamin E ◽  
Blast Crisis ◽  
Antioxidant Properties ◽  
K562 Cells ◽  
Therapy Resistance ◽  
Myelogenous Leukemia ◽  
Effective Control ◽  
Granulocytic Differentiation ◽  
Hematopoietic Stem

Background: Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder associated with the activity of BCR-ABL fusion oncogene. Tyrosine kinase inhibitors are the current treatment of CML, but secondary mutations finally contribute to therapy resistance and blast crisis of the disease. The search for the novel compounds for the effective control of CML is now in the spotlight. The progression of CML to blast crisis is correlated with down-modulation of C/EBP alpha. Therefore, C/EBP alpha may be considered as a putative target in differentiation therapies in myeloid leukemias. The aim of the study was to assess the potential of vitamin E as the possible inducer of C/EBP alpha expression in BCR-ABL-positive CML K562 cells. Materials and Methods: RNA extracted from K562 cells cultured with valproic acid or vitamin E was converted to cDNA, RT-PCR reactions were carried out using HotStarTaq DNA polymerase with primers for C/EBP alpha and granulocyte colony-stimulating factor receptor (G-CSFR). Results: We have not found detectable expression of C/EBP alpha in K562 cells. Upon 48-h culture with vitamin E at a dose of 100 µM, K562 cells expressed both C/EBP alpha and GCSFR. Conclusion: Vitamin E restored the expression of C/EBP alpha mRNA in chronic myelogenous leukemia K562 cells. In this setting, G-CSFR expression in vitamin E treated K562 cells seems to suggest the activation to granulocytic differentiation. It should be further elucidated whether such effects of vitamin E on C/EBP alpha transcription factor are direct or mediated indirectly due to antioxidant properties of vitamin E.

scholarly journals Human leukemia mutations corrupt but do not abrogate GATA-2 function

2018 ◽  
Vol 115 (43) ◽  
pp. E10109-E10118 ◽  
Author(s):  
Koichi R. Katsumura ◽  
Charu Mehta ◽  
Kyle J. Hewitt ◽  
Alexandra A. Soukup ◽  
Isabela Fraga de Andrade ◽  
...  
Keyword(s):  
Dna Binding ◽  
Progenitor Cells ◽  
Zinc Finger ◽  
Erythroid Differentiation ◽  
Cell Types ◽  
Myeloid Differentiation ◽  
Human Leukemia ◽  
Hematopoietic Stem ◽  
Amino Terminal ◽  
Disease Mutations

By inducing the generation and function of hematopoietic stem and progenitor cells, the master regulator of hematopoiesis GATA-2 controls the production of all blood cell types. Heterozygous GATA2 mutations cause immunodeficiency, myelodysplastic syndrome, and acute myeloid leukemia. GATA2 disease mutations commonly disrupt amino acid residues that mediate DNA binding or cis-elements within a vital GATA2 intronic enhancer, suggesting a haploinsufficiency mechanism of pathogenesis. Mutations also occur in GATA2 coding regions distinct from the DNA-binding carboxyl-terminal zinc finger (C-finger), including the amino-terminal zinc finger (N-finger), and N-finger function is not established. Whether distinct mutations differentially impact GATA-2 mechanisms is unknown. Here, we demonstrate that N-finger mutations decreased GATA-2 chromatin occupancy and attenuated target gene regulation. We developed a genetic complementation assay to quantify GATA-2 function in myeloid progenitor cells from Gata2 −77 enhancer-mutant mice. GATA-2 complementation increased erythroid and myeloid differentiation. While GATA-2 disease mutants were not competent to induce erythroid differentiation of Lin−Kit+ myeloid progenitors, unexpectedly, they promoted myeloid differentiation and proliferation. As the myelopoiesis-promoting activity of GATA-2 mutants exceeded that of GATA-2, GATA2 disease mutations are not strictly inhibitory. Thus, we propose that the haploinsufficiency paradigm does not fully explain GATA-2–linked pathogenesis, and an amalgamation of qualitative and quantitative defects instigated by GATA2 mutations underlies the complex phenotypes of GATA-2–dependent pathologies.

AHSP Knockdown in Human Erythroleukemia Cell Line and Human Hematopoietic Stem Cells Results in Alpha Hemoglobin Chain Precipitation, Decreased Hemoglobin Levels and Increased Cell Death.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1778-1778
Author(s):  
Flavia O. Pinho ◽  
Dulcineia M. Albuquerque ◽  
Sara T.O Saad ◽  
Fernando F. Costa
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Hematopoietic Stem Cells ◽  
Cell Line ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Erythroleukemia Cell ◽  
Human Red Cells

Abstract Alpha Hemoglobin Stabilizing Protein (AHSP) binds alpha hemoglobin chain (αHb), avoiding its precipitation and its pro-oxidant activity. In the presence of beta hemoglobin chain (βHb), the αHb-AHSP complex is dismembered and βHb displaces AHSP to generate the quaternary structure of hemoglobin. These data have been obtained in vitro and in mouse cells, but strongly suggest the importance of AHSP for normal hemoglobin synthesis in humans. To the best of our knowledge, the relationship between hemoglobin formation and alterations in AHSP expression has not yet been described in human red cells. Hence, to investigate the consequences of a reduced AHSP synthesis in human red cells, we established the RNA interference-mediated knockdown of AHSP expression in human erythroleukemia cell line (K562 cells) and human hematopoietic stem cells (CD34+ cells) induced to erythroid differentiation, and analyzed the consequent cellular and molecular aspects of AHSP knockdown in these cells. shRNA expression vectors, aimed at the AHSP mRNA target sequence, were cloned and transfected into K562 and CD34+ cells using a non-liposomal lipid reagent. Following transfection, K562 cells that stably expressed AHSP-shRNA and CD34+ cells that transiently expressed AHSP-shRNA were selected. K562 and CD34+ cells were stimulated to erythroid differentiation by hemin and erythropoietin (EPO) respectively. The cells were examined in terms of gene expression using quantitative real-time PCR; production of reactive oxygen species (ROS), apoptosis and hemoglobin production through flow cytometry assays; and immunofluorescence assays for globin chains. AHSP-shRNA hemin-induced K562 cells and AHSP-shRNA EPO-induced CD34+ cells presented 71% and 75% decreases in AHSP expression levels, respectively. The RNAi-mediated knockdown of AHSP expression resulted in a considerable αHb precipitation, as well as in a significant decrease in fetal hemoglobin formation. In addition, AHSP-knockdown cells demonstrated an increased ROS production and increased rate of apoptosis. These findings strengthen the hypothesis that AHSP stabilizes the alpha hemoglobin chain, avoiding its precipitation and its ability to generate ROS which implicate in cell death. Moreover, data indicate that AHSP may be highly significant for human hemoglobin formation and suggest that AHSP is a key chaperone protein during human erythropoiesis.

CoCoA/CCAR1 Pair-Mediated Recruitment of Mediator Complex Indicates Novel Pathway for the Function of GATA1 in Erythroid Differentiation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1302-1302
Author(s):  
Chihiro Kaminaga ◽  
Shumpei Mizuta ◽  
Tomoya Minami ◽  
Kasumi Oda ◽  
Haruka Fujita ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Direct Interaction ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Luciferase Reporter ◽  
Zinc Finger Domain ◽  
C Terminus ◽  
N Terminus ◽  
Reporter Assays ◽  
Globin Promoter

Abstract Abstract 1302 The mammalian multi-protein complex Mediator, originally identified by ourselves as a nuclear receptor-specific coactivator complex, is a phylogenetically-conserved subcomplex of the RNA polymerase II holoenzyme and serves as an end-point integrator of diverse intracellular signals and transcriptional activators. The 220-kDa Mediator subunit MED1 is a specific coactivator not only for nuclear receptors but for GATA family activators, and serves as a GATA1-specific coactivator that is essential for optimal GATA1-mediated erythropoiesis. In this study, we show a novel nuclear signaling pathway for MED1 action in GATA1-induced transcriptional activation during erythroid differentiation. First, we identified the amino acid residues 681–715 of human MED1 (MED1(aa.681-715)) to be responsible for the direct interaction with GATA1. When MED1 in K562 human erythroleukemic cells was knocked down during hemin-induced erythroid differentiation, the erythroid differentiation was significantly attenuated as assessed by an erythroid differentiation score defined by the number of cells positive for benzidine staining, and the expressions of the GATA1-targeted and erythroid differentiation marker genes, β-globin, γ-globin, PBGD and ALAS-E, were prominently attenuated. However, overexpressions of the N-terminal MED1 truncations without and with nuclear receptor recognition motifs, MED1(aa.1–602) and MED1(aa.1–703), respectively, but neither of which could bind to GATA1 (above), prominently enhanced erythroid differentiation of K562 cells. Luciferase reporter assays by using the human γ-globin promoter and Med1−/− mouse embryonic fibroblasts (MEFs) showed that these N-terminal MED1 truncations rescued GATA1-mediated transactivation, indicating that MED1(a.a.1–602) served as the functional interaction surface for GATA1. Hence, a putative bypass for GATA1-MED1 pathway appears to exist, and is expected to interact with the N-terminus of MED1. As a candidate bypass system, we tested both the recently reported bypass molecule for a nuclear post-activator signaling, CCAR1, and its partner coactivator CoCoA. CCAR1 was reported by others to bypass the estrogen receptor-mediated transactivation by a simultaneous binding of CCAR1 with the estrogen receptor and the N-terminus of MED1. Functionally, serial luciferase reporter assays by using the γ-globin promoter and MEFs demonstrated cooperative transactivation by combinations of GATA1, CCAR1, CoCoA and/or the N-terminus of MED1, but the transactivation mediated by the N-terminus of MED1 was not as prominent as the one mediated by the full-length MED1. An overexperssion of CCAR1 or CoCoA in K562 cells prominently enhanced both the GATA1-mediated erythroid differentiation and the expressions of the GATA1-targeted genes. Next, the mechanisms underlying the CCAR1- and CoCoA-mediated GATA1 functions were analyzed by serial GST-pulldown and mammalian two-hybrid assays, and the following results were obtained. (i) The N-terminus of CCAR1 interacted with the C-terminus of CoCoA. (ii) The N-terminus of MED1 interacted with both the N- and C-termini of CCAR1. (iii) While the N-terminal zinc-finger domain of human GATA1 (GATA1(a.a.204–228)) is known to bind to the well-known GATA1 partner FOG1, intriguingly, the C-terminal zinc-finger domain of GATA1 (GATA1(a.a.258–272)) interacted with all three of the following cofactors; MED1 (MED1(aa.681–715)), CCAR1 (at the C-terminus) and CoCoA (at both the N- and C-termini). The affinity of CoCoA to bind to GATA1 appeared to be a little higher than the other. Thus, the GATA1(a.a.258-272) zinc finger appears to serve as a docking surface for multiple coactivating proteins, where both MED1 and CoCoA/CCAR1 pair can interact, probably in a competitive manner, or perhaps simultaneously. Here, both CoCoA/CCAR1 as a pair and CCAR1 by itself can serve as a bypass. Finally, ChIP assays of hemin-treated K562 cells showed that GATA1, CCAR1/CoCoA and MED1 were all recruited onto the γ-globin promoter during transactivation. Taken together, besides a direct interaction between GATA1 and MED1, the CoCoA/CCAR1 pair appears to relay the GATA1 signal to MED1. The multiple modes of mechanisms for transcription mediated by the GATA1-MED1 axis might contribute to a fine tuning of the GATA1 function, not only during erythropoiesis but also in other GATA1-mediated homeostasis events, within a living animal. Disclosures: No relevant conflicts of interest to declare.

Hypoxia-Inducible Factor 1-Mediated Human GATA1 Induction Promotes Erythroid Differentiation Under Hypoxic Conditions

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4794-4794
Author(s):  
Jun-Wu Zhang ◽  
Feng-Lin Zhang ◽  
Guo-Min Shen ◽  
Xiao-Ling Liu ◽  
Fang Wang
Keyword(s):  
Rna Interference ◽  
Conflicts Of Interest ◽  
Hypoxia Inducible Factor ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Flanking Sequence ◽  
Differentiation Markers ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Hypoxic Conditions

Abstract Abstract 4794 The stimulation of red blood cell (RBC) production is one of the systemic adaptions to hypoxia. Hypoxia-inducible factor (HIF) promotes erythropoiesis through coordinated cell type-specific hypoxia responses. Hematopoietic transcription factor GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34+ hematopoietic stem/progenitor cells (HPCs) derived from human cord blood. Enforced HIF1Á expression increased GATA1 expression, while HIF1Á knock-down by RNA interference decreased GATA1 expression in K562 cells. We searched the human GATA1 gene sequence on NCBI and identified a putative HRE in the 3'-flanking sequence of the gene. The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1Á or GATA1. Flow cytometry analysis also indicated that hypoxia or desferrioxamine or CoCl2 induced expression of erythroid surface marker CD71 and CD235a, while expression repression of HIF1Á or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 upregulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions. Disclosures: No relevant conflicts of interest to declare.

Identification of the Epigenetic Reader BRD4 As a Novel Potential Target in Ph+ CML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1571-1571
Author(s):  
Barbara Peter ◽  
Gregor Eisenwort ◽  
Gabriele Stefanzl ◽  
Daniela Berger ◽  
Wolfgang R Sperr ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Drug Target ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Potential Drug ◽  
Ku812 Cells

Abstract Chronic myelogenous leukemia (CML) is a bone marrow-derived hematopoietic neoplasm in which BCR/ABL1 acts as a major driver of proliferation, differentiation and survival of leukemic cells. In a majority of all patients with CML, leukemic cells can be kept under control by BCR/ABL1 tyrosine kinase inhibitors (TKI), including imatinib, nilotinib, dasatinib, bosutinib, and ponatinib. Nevertheless, resistance or intolerance against one or more of these TKI may occur. Therefore, current research is focusing on novel potential drug targets in CML. A promising class of targets may be epigenetic regulators of cell growth, such as members of the bromodomain and extra-terminal domain (BET) family. The epigenetic reader and BET family member BRD4 has recently been identified as a novel potential drug target in acute myeloid leukemia (AML). However, so far, little is known about the expression and function of BRD4 in CML cells. The aims of the present study were to determine the expression of BRD4 and its downstream target MYC in CML cells and to explore whether BRD4 can serve as a novel drug target in this disease. As determined by qPCR, primary CML cells (chronic phase patients, n=7) as well as the CML cell lines KU812 and K562 expressed BRD4 mRNA. In addition, both CML cell lines stained positive for BRD4 in our immunocytochemistry staining experiments. In one patient with accelerated phase CML, putative leukemic (CD34+/CD38-) stem cells were sorted to near homogeneity and found to express BRD4 mRNA by qPCR. In order to examine the functional role of BRD4 in CML cells, a BRD4-specific shRNA was applied. In these experiments, the shRNA-induced knockdown of BRD4 in KU812 cells and K562 resulted in reduced growth compared to a control shRNA. Furthermore, the BRD4-targeting drug JQ1 was found to inhibit 3H-thymidine uptake and thus proliferation in KU812 cells in a dose-dependent manner (IC50: 0.25-0.75 µM). In addition, we were able to show that JQ1 inhibits growth of primary CML cells with variable IC50 values (0.1-5 µM). However, no substantial growth-inhibitory effects of JQ1 were seen in K562 cells (IC50: >5 µM). As determined by Annexin V/PI staining, JQ1 induced apoptosis in KU812 cells whereas no apoptosis-inducing effect of JQ1 was observed in K562 cells. Nevertheless, we were able to show that both CML cell lines as well as primary CML cells express MYC mRNA, and treatment of KU812 cells or K562 cells with JQ1 resulted in a decreased expression of MYC mRNA and MYC protein. Next, we analyzed whether MYC expression in CML cells can be blocked by BCR/ABL1 TKI. We found that imatinib, nilotinib, dasatinib, and ponatinib decrease MYC mRNA- and MYC protein expression in KU812 and K562 cells. Finally, we found that JQ1 cooperates with imatinib, nilotinib, ponatinib and dasatinib in inhibiting the proliferation of KU812 and K562 cells. Together, our data show that BRD4 serves as a potential new target in CML cells, and that the BRD4 blocker JQ1 cooperates with BCR/ABL1 TKI in inducing growth-inhibition. Whether BRD4 inhibition is a pharmacologically meaningful approach in patients with TKI-resistant CML remains to be determined in clinical trials. Disclosures Sperr: Ariad: Consultancy; Celgene: Consultancy. Zuber:Mirimus Inc.: Consultancy, Other: Stock holder; Boehringer Ingelheim: Research Funding. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria.

Anti-Leukemic Effects of Venetoclax on Philadelphia Chromosome Positive Leukemia Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5428-5428 ◽  
Author(s):  
Seiichi Okabe ◽  
Tetsuzo Tauchi ◽  
Yuko Tanaka ◽  
Kazuma Ohyashiki
Keyword(s):  
Research Funding ◽  
Combined Treatment ◽  
Philadelphia Chromosome ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Apoptotic Cells ◽  
Dependent Manner ◽  
T315i Mutation ◽  
Mutant Cells ◽  
Dose Dependent

Abstract Introduction: Chronic myeloid leukemia (CML) is characterized by the t(9:22) translocation known as the Philadelphia chromosome (Ph). Although ABL tyrosine kinase inhibitors (ABL TKI) such as imatinib, dasatinib and nilotinib have improved CML treatment, such therapies cannot cure patients with Philadelphia chromosome (Ph)-positive leukemia because of leukemia stem cells. Moreover, some patients develop BCR-ABL point mutations including T315I and become resistant to ABL TKI therapy. These leukemia stem cells are contained within a niche in the bone marrow and are often impervious to current treatments. Therefore, new approach against BCR-ABL mutant cells and LSCs may improve the outcome of Ph-positive leukemia patients. B cell lymphoma 2 (BCL-2) protein families are key regulator of apoptosis and highly promising targets for the development of anti-cancer treatment. Venetoclax, also known as ABT-199 is a selective, orally bioavailable BCL-2 inhibitor. Venetoclax is investigated in a pivotal phase 3 clinical trial against hematological malignancies such as chronic lymphocytic leukemia (CLL) and approved for the treatment of patients with CLL. Materials and methods: In this study, we investigated whether venetoclax could suppress Ph-positive leukemia cells including T315I mutation and primary samples. Results: BCL-2 expression was found in Ph-positive leukemia cells including primary samples, however, BCL-2 expression was reduced in K562 cells. We found 72 h venetoclax treatment inhibited the growth of Ba/F3 BCR-ABL and KCL-22 cells in a dose dependent manner. However, venetoclax activity was reduced in K562 cells. We examined the intracellular signaling after treatment of venetoclax. Phosphorylation of BCR-ABL and Crk-L was not reduced. However, activity of caspase 3, poly (ADP-ribose) polymerase (PARP) was increased. We next investigated the efficacy between ABL TKI and venetoclax by using these cell line. Combined treatment of Ba/F3 BCR-ABL cells with imatinib and venetoclax caused significantly more cytotoxicity than each drug alone. Apoptotic cells were also increased. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved caspase 3 and PARP activity was increased after imatinib and venetoclax treatment. We investigated the venetoclax activity against T315I positive cells. Venetoclax potently induced cell growth inhibition of Ba/F3 T315I mutant cells in a dose dependent manner. Combined treatment of Ba/F3 T315I mutant cells with ponatinib and venetoclax caused significantly more cytotoxicity than each drug alone. Apoptotic cells were also increased. Phosphorylation of BCR-ABL, Crk-L was reduced and cleaved PARP was increased after ponatinib and venetoclax treatment. To assess the activity of ponatinib and venetoclax, we examined tumor formation in mice model. We injected subcutaneously 1×107 Ba/F3 T315I mutant cells in nude mice. A dose of 20 mg/kg/day p.o of ponatinib and 50 mg/kg/day p.o of venetoclax inhibited tumor growth and reduced tumor volume compared with control mice. In the immunohistochemical analysis, we found that tumors in mice treated with ponatinib and venetoclax exhibited an increase in apoptotic cells. We also found that co-treatment with ponatinib and venetoclax increased mouse survival. The treatments were well tolerated with no animal health concerns observed. We also found that the treatment of venetoclax exhibits cell growth inhibition against CD34 positive CML samples. Conclusion: The results of our study indicate that the BCL-2 inhibitor venetoclax may be a powerful strategy against ABL TKI resistant cells including T315I mutation and enhance cytotoxic effects of ABL TKI against those Ph-positive leukemia cells. Disclosures Tauchi: Pfizer Inc.: Research Funding. Ohyashiki:Novartis International AG,: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding.

Total Saponins ofPanax GinsengInduces K562 Cell Differentiation by Promoting Internalization of the Erythropoietin Receptor

2009 ◽  
Vol 37 (04) ◽  
pp. 747-757 ◽  
Author(s):  
Guowei Zuo ◽  
Tao Guan ◽  
Dilong Chen ◽  
Chunli Li ◽  
Rong Jiang ◽  
...  
Keyword(s):  
Leukemia Cell ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Erythropoietin Receptor ◽  
Dependent Manner ◽  
Therapeutic Benefits ◽  
Effective Components ◽  
Wide Range ◽  
Electron Microscopes ◽  
K562 Cell Differentiation

Ginseng is a commonly used herbal medicine with a wide range of therapeutic benefits. Total saponins of Panax ginseng (TSPG) is one of the main effective components of ginseng. Our previous studies have shown that TSPG could promote the production of normal blood cells and inhibition of the leukemia cell proliferation. However, whether ginseng can induce the differentiation of leukemia cells is still unclear. This study was to examine the effect of TSPG or the combination of erythropoietin (EPO) and TSPG on the erythroid differentiation of K562 cells, and their corresponding mechanisms regarding erythropoietin receptor (EPOR) expression. Under light and electron microscopes, the TSPG- or TSPG + EPO-treated K562 cells showed a tendency to undergo erythroid differentiation; early and intermediate erythroblast-like cells were observed. Hemoglobin and HIR2 expressions were significantly increased. As determined by Western blotting analysis, the EPOR protein level in the K562 cytoplasmic membrane was significantly decreased after TSPG treatment, while its cytoplasm level increased in a dose-dependent manner. However, the total cellular EPOR level was unchanged. These results indicate that TSPG-induced erythroid differentiation of K562 cells may be accompanied by the internalization of EPOR. Thus, our study suggests that treatment with a combination of TSPG and EPO may induce erythroid differentiation of K562 cells at least in part through induction of EPOR internalization.

scholarly journals Implication of Hypoxia-Inducible Factor-1 (HIF-1) As a New Therapeutic Target in JAK2V617F Positive Myeloproliferative Neoplasms (MPN)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4318-4318 ◽  
Author(s):  
Julian Baumeister ◽  
Nicolas Chatain ◽  
Annika Hubrich ◽  
Caroline Küstermann ◽  
Stephanie Sontag ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Line ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Research Funding ◽  
Myeloproliferative Neoplasms ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Protein Levels

Abstract Myeloproliferative neoplasms (MPN) are a heterogeneous group of malignancies including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The JAK2V617F mutation can be found in 90% of PV and approximately 50% of ET and PMF patients. Hypoxia-inducible factors (HIFs) are master transcriptional regulators of the response to decreases in cellular oxygen levels. Unveiling the function of deregulated HIF-1 signaling in normal and malignant hematopoiesis was the aim of several recent publications, highlighting the importance of HIF-1 for the maintenance of leukemic stem cells (LSCs) in acute and chronic myeloid leukemia (AML/CML). In a JAK2V617F knock-in mouse model and in patients, JAK2V617F was shown to induce the accumulation of reactive oxygen species (ROS) in the hematopoietic stem cell compartment, leading to a stabilization of HIF-1α protein. Further, aberrant STAT5 and PI3K/AKT/mTOR signaling induced HIF-1α expression on the transcriptional and translational level. Ruxolitinib treatment inhibited growth and reduced the expression of HIF-1α and its target gene VEGF in the JAK2V617F human erythroleukemia cell line HEL. In several leukemic cell lines constitutive expression of HIF-1α was reported, even under normoxic conditions. However, it still remains unknown whether HIF-1α plays a role in JAK2V617F positive MPN. In this study, we investigated the role HIF-1α signaling in JAK2V617F positive MPN in vitro. We retrovirally transduced the murine bone marrow cell line 32D with JAK2V617F or JAK2WT. Western blot analysis revealed significant increases in HIF-1α protein levels in JAK2V617F positive cells compared to JAK2WT controls after cultivation in normoxic conditions and this effect was abrogated by treatment with the JAK1/JAK2 inhibitor ruxolitinib. Inhibition of HIF-1, binding to hypoxia response elements (HRE), by low doses of echinomycin (1 nM), significantly impaired proliferation and survival. Using an Annexin-V/7-AAD flow cytometry assay apoptosis was found to be selectively induced in JAK2V617F positive, but not JAK2WT cells after echinomycin treatment. Additionally, BrdU/7-AAD cell cycle analysis revealed that only JAK2V617F positive cells were significantly arrested in G0/1 phase. These findings were consistent with shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F transduced 32D cells in presence but not the absence of HIF-2 antagonist 2. Inhibition of HIF-2 was necessary due to a compensatory increase of HIF-2α protein levels, shown by Western Blot analysis, counteracting HIF-1α-KD mediated effects. We isolated PBMCs and BMMNCs from JAK2V617F positive patients or healthy controls using Ficoll density gradient centrifugation. Echinomycin significantly abrogated the colony formation ability alone and in combination with ruxolitinib. In vitro treatment with echinomycin significantly decreased cell number and viability of 8 JAK2V617F positive BMMNC samples (4 PV, 3 PMF, 1 preMF; p[1nM]=0.0169, p[5nM]=0.0009) and 7 PBMC samples (6 PV, 1 PMF; p[1nM]=0.0156, p[5nM]=0.0156) in a dose-dependent manner. In contrast, PBMCs from 6 healthy donors were unaffected by the treatment. The same effect was observed in heterozygous and homozygous iPS cell-derived progenitors from JAK2V617F positive PV patients, whereas JAK2WT cells were unaffected by the treatment. Collectively, our data indicate that targeting HIF-1 might represent a novel therapeutic approach in classical Philadelphia-chromosome-negative MPN. Disclosures Brümmendorf: Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy.

scholarly journals AVID200, a Potent Trap for TGF-β Ligands Inhibits TGF-β1 Signaling in Human Myelofibrosis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1791-1791 ◽  
Author(s):  
Lilian Varricchio ◽  
John Mascarenhas ◽  
Anna Rita Migliaccio ◽  
Maureen O'Connor-McCourt ◽  
Gilles Tremblay ◽  
...  
Keyword(s):  
Stem Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Type I Collagen ◽  
Normal Donor ◽  
Type I ◽  
Cell Transplant ◽  
Dependent Manner ◽  
Advisory Committees ◽  
Hematopoietic Stem

Abstract Myelofibrosis (MF) is caused by driver mutations which upregulate JAK/STAT signaling. The only curative treatment for MF is hematopoietic stem cell transplant. Ruxolitinib alleviates many of the symptoms in MF but does not significantly alter survival. There is, therefore, an urgent need for additional rational therapies for MF. Bone marrow fibrosis and collagen deposition are hallmarks of MF which have been attributed to megakaryocyte (MK) derived TGFβ, which also plays a role in myelo-proliferation. There are three isoforms of TGFβ (TGFβ1, β2, and β3). AVID200, which was constructed by fusing TGFβR ectodomains to IgG Fc regions, is a potent TGFβ trap with pM potency against two of the three TGFβ ligands, TGFβ1 and β3 (IC50 values of ~ 3 pM ). AVID200's IC50 for TGFβ2 is ~4,000-fold higher indicating that it has minimal activity against TGFβ2, which is desirable since TGFβ2 is a positive regulator of hematopoiesis. We explored the therapeutic potential of AVID200 by culturing MF or normal donor (ND) mononuclear cells (MNCs) first in the presence of stem cell factor and thrombopoietin (TPO) and then TPO alone in order to generate MK-enriched populations. Although the percentage of mature MKs from ND and MF MNCs was similar, the absolute number of CD41+/CD42+ MKs generated from MF MNCs was two-fold greater than ND MNCs. To determine the levels of TGFβ secreted by the MKs we screened MF and ND MNC conditioned media (CM). We observed significantly higher levels of TGFβ1 but not TGFβ2 and TGFβ3 in MF MK CM. The effects of AVID200 on MKs were then evaluated by measuring the levels of phosphorylated SMAD2. Treatment with 0.001 - 0.1 nM AVID200 decreased phosphorylation of SMAD2, suggesting that AVID200 blocks autocrine MK TGFβ signaling. The increased levels of TGFβ in MF patients promote the proliferation and deposition of collagen by mesenchymal stem cells (MSCs). Cellular proliferation of MSCs was evaluated following treatment with either recombinant TGFβ1 or ND/MF CM in the presence or absence of AVID200. In the absence of AVID200, both recombinant TGFβ1 and MK-derived CM increased the proliferation of MSCs by 1.4- and 1.6-fold respectively, which returned to basal levels with the addition of increasing concentrations of AVID200. These data indicate that AVID200 directly blocks the effect of TGFβ1 on MSCs. MF stroma is characterized by an increase in Type I collagen. We therefore examined if treatment with AVID200 interferes with the ability of TGFβ1 to induce collagen expression by MSCs. MSCs were cultured in presence of recombinant TGFβ1 alone or in combination with varying concentrations of AVID200 for 72 hours. Recombinant TGFβ1 alone induced an increase in COL1A1 mRNA expression as compared to untreated controls (p<0.01). Addition of AVID200 eliminated the TGFβ-mediated increase in COL1A1 expression in a dose dependent manner. ND and MF MK-derived CM also increased COL1A1 expression by MSCs as compared to un-treated controls (p<0.01) and that effect was eliminated by AVID200 treatment (p<0.01). We next demonstrated that TGFβ1 activated pSMAD2 in MSCs without affecting total SMAD2/3 expression and that SMAD2 phosphorylation was reduced by adding AVID200. Furthermore, AVID200 treatment decreased pSTAT3 which is associated with the ability of TGFβ to induce fibrosis. We next investigated the effect of AVID200 on MF hematopoiesis. Briefly, MNCs (which produce TGFβ) from two JAK2V617F+ MF patients were incubated with or without 50 nM of AVID200 and plated in semi-solid media. Treatment with AVID200 did not affect the overall number of colonies generated, but reduced the numbers of JAKV617F+ colonies while increasing the numbers of WT colonies: for PT1, there were 32% JAKV617F+ CFUs in untreated cultures (11 JAKV617F+/34 total colonies) versus 16% JAKV617F+ CFUs (7 JAKV617F+/42 total CFUs) in AVID200 treated cultures; for PT2 there were 100% JAKV617F+ CFUs in untreated cultures (37 JAKV617F+/37 total CFUs) versus 94% JAKV617F+ CFUs (49 JAK2V617F+/52 total CFUs) in AVID200 treated cultures. The in vivo effects of AVID200 on the development of MF in GATA1 low mice will be presented at the meeting. These data indicate that AVID200 selectively suppresses TGFβ1 signaling associated with the proliferation of MSCs and type I collagen synthesis, and depletes MF MNCs of JAK2V617F+progenitor cells. We conclude that AVID200 is a promising agent for treating MF patients which will be evaluated in a phase 1 clinical trial. Disclosures Mascarenhas: Novartis: Research Funding; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding; Janssen: Research Funding; Promedior: Research Funding; Merck: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding. Iancu-Rubin:Incyte: Research Funding; Merck: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding. Hoffman:Incyte: Research Funding; Summer Road: Research Funding; Merus: Research Funding; Janssen: Research Funding; Formation Biologics: Research Funding.

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