Pharmacological Inhibition Of Histone Deacetylase (HDAC) 1, 2 Or 3 Have Distinct Effects On Cellular Viability, Erythroid Differentiation, and Fetal Globin (HbG) Induction

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 564-564
Author(s):  
Jeffrey R Shearstone ◽  
John H van Duzer ◽  
Simon S Jones ◽  
Matthew Jarpe
Keyword(s):  
Bone Marrow ◽  
Cell Viability ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Erythroid Differentiation ◽  
Employment Equity ◽  
Human Cd34 ◽  
Ic50 Values ◽  
Equity Ownership ◽  
Marrow Cells

Abstract Induction of HbG is an established therapeutic strategy for the treatment of sickle cell disease (SCD), and could also be effective in treating beta-thalassemia (bT). Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of HbG expression (Bradner JE, Proc Natl Acad Sci, 2010). Furthermore, we have previously shown that selective chemical inhibitors of HDAC1 and 2 elicit a dose and time dependent induction of HbG mRNA and fetal hemoglobin (HbF) protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). While a variety of selective HDAC inhibitors have been used successfully to induce HbF, further clinical development has been limited by variable efficacy and concerns over off-target side-effects observed in clinical trials, potentially due to inhibition of HDAC3. Additionally, it remains to be determined if HDAC1 or HDAC2 is the preferred therapeutic target. In this work we present data that investigates the effects of selective inhibitors of HDAC1, 2, or 3 on cytotoxicity, erythroid differentiation, and HbG induction in cultured human CD34+ bone marrow cells. Acetylon Pharmaceuticals has generated a library of structurally distinct compounds with a range of selectivity for each of HDAC1, 2, or 3 (Class I HDAC) as determined in a biochemical assay platform. From our initial chemical series, we identified ACY-822 as a Class I HDAC inhibitor with IC50 values of 5, 5, and 8 nM against HDAC1, 2, and 3, respectively. In contrast, ACY-1112 is 30-fold selective for HDAC1 and 2, with IC50 values of 38, 34, and 1010 nM against HDAC1, 2, and 3, respectively. Treatment of cells for 4 days with ACY-822 (1 μM) resulted in a 20-fold decrease in cell viability, while ACY-1112 (1 μM) treatment resulted in a minimal reduction in viability (1.2-fold) and a 2-fold increase in the percentage of HbG relative to other beta-like globin transcripts. This result suggests that pharmacological inhibition of HDAC3 is cytotoxic and is consistent with the therapeutic rationale for the design selective inhibitors of HDAC1 and 2. To investigate if HDAC1 or HDAC2 is the preferred therapeutic target, we utilized a second series of structurally distinct compounds. We identified ACY-957 as an HDAC1/2 selective compound biased towards HDAC1 with IC50 values of 4, 15, and 114 nM for HDAC1, 2, and 3, respectively. In contrast ACY-1071 showed balanced HDAC1 and 2 selectivity with IC50 values of 27, 24, and 247 nM for HDAC1, 2, and 3, respectively. Treatment of cells for 6 days with 1 μM of ACY-957 or ACY-1071 resulted in a 3-fold increase in the percentage of HbG relative to other beta-like globin transcripts. However, we found that ACY-957 treatment resulted in an approximately 3-fold decrease in cell viability after 6 days of treatment, while ACY-1071 treatment resulted in a minimal reduction (1.2-fold) in cell viability. Decreased cell viability observed with ACY-957 was associated with a reduction of cells positive for the erythroid differentiation markers CD71 and glycophorinA. This result is consistent with the Mx-Cre mouse model where HDAC1KO; HDAC2het had reduced numbers of erythrocytes, thrombocytes, and total bone marrow cells, while the HDAC1het; HDAC2KO was unaffected (Wilting RH, EMBO Journal, 2010). Our results suggest that compounds with a pharmacological profile of increased selectivity towards HDAC2 inhibition versus HDAC1 may be less cytotoxic and minimize effects on differentiation, while still inducing HbG in human CD34+ bone marrow cells. Disclosures: Shearstone: Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.

scholarly journals Pharmacological Inhibition of Histone Deacetylases 1 and 2 (HDAC1/2) Induces Fetal Hemoglobin (HbF) through Activation of Gata2

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 335-335
Author(s):  
Jeffrey R Shearstone ◽  
Olga Golonzhka ◽  
Apurva Chonkar ◽  
Matthew Jarpe
Keyword(s):  
Bone Marrow Cells ◽  
Fetal Hemoglobin ◽  
Fold Increase ◽  
Erythroid Differentiation ◽  
Beta Globin ◽  
Employment Equity ◽  
Regulatory Regions ◽  
Genetic Ablation ◽  
Equity Ownership ◽  
Highly Correlated

Abstract Induction of HbF is an established therapeutic strategy for the treatment of sickle cell disease, and could also be effective in treating beta-thalassemia. Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of the fetal beta-like globin gene (HbG) transcript (Bradner JE, Proc Natl Acad Sci, 2010). We have previously shown that selective chemical inhibitors of HDAC1/2 elicit a dose and time dependent induction of HbG mRNA and HbF protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). In this work, we have utilized our proof of concept molecule ACY-957, a selective inhibitor of HDAC1/2, to discover a novel role for Gata2 in the activation of HbG. To identify genes affected by HDAC1/2 inhibition, CD34+ bone marrow cells undergoing erythroid differentiation were treated with ACY-957 or vehicle, followed by mRNA expression profiling. Among the genes differentially regulated by both pharmacological inhibition and genetic ablation of HDAC1/2 were Bcl11a and Sox6, known HbG repressors, and Gata2, a potential HbG activator. Quantitative real time PCR (QRT-PCR) time course experiments confirmed that ACY-957 treatment leads to a 2-fold and 10-fold decrease in Bcl11A and Sox6, respectively, and an 8-fold increase in Gata2 mRNA. Unlike Bcl11a and Sox6, Gata2 induction by ACY-957 was highly correlated with HbG induction, suggesting a possible role for this transcription factor in the direct activation of HbG. To investigate this possibility, lentiviral infection was utilized to overexpress full length Gata2 transcript in differentiating primary erythroblasts. After 5 days of differentiation, Gata2 overexpression resulted in a 2.5-fold increase in HbG mRNA, while the level of the major adult beta-like globin chain (HbB) mRNA was unaffected. HbG mRNA remained elevated by Gata2 overexpression at day 7 of differentiation, while HbB was reduced by 1.6-fold. Gata2 overexpression appeared to have minimal effect on cell differentiation, as determined by the cell surface markers CD71 and GlycophorinA, a finding consistent with observations in ACY-957 treated cells with elevated Gata2. Furthermore, lentiviral delivery of short hairpin RNA (shRNA) targeting Gata2, attenuated HbG induction by ACY-957. These data suggest that elevated levels of Gata2 resulting from HDAC1/2 inhibition is sufficient to induce HbG at early stages of erythroid cell differentiation. To understand how HDAC1/2 inhibition drives Gata2 activation, chromatin immunoprecipitation coupled with either next generation sequencing (ChIP-seq) or QRT-PCR was performed in ACY-957 and vehicle treated cells. HDAC1 and HDAC2 were present throughout the Gata2 gene body and promoter regions, and HDAC1/2 binding levels were highly correlated, suggesting co-occupancy of these enzymes at this locus. ACY-957 treatment led to elevated histone acetylation at previously described Gata2 gene regulatory regions (Bresnick et. al. 2010, J Biol Chem). Specifically, the -1.8 kb and -2.8 kb regulatory regions showed a 6-fold increase in histone H3K9 and H2BK5 acetylation, while the +9.5 kb and -3.9 kb regions showed a 3-fold increase. The Gata2 protein showed increased binding at these regulatory regions in response to ACY-957 treatment, with a maximum increase of 3-fold at the -1.8 kb region. This finding is consistent with the known positive autoregulation of the Gata2 gene. Taken together, these data suggest that selective inhibition of HDAC1/2 leads to elevated Gata2 through acetylation-induced activation of a positive autoregulatory loop. The tight temporal correlation between Gata2 and HbG activation following HDAC1/2 inhibition argues that Gata2 may affect the beta-globin locus directly. ChIP-seq data across the 70-kb beta-globin locus demonstrated that ACY-957 treatment altered Gata2 binding only at a single region, lying within the promoter for delta globin. This region is suspected in playing a role in switching from fetal to adult globin during development, as naturally occurring deletions of this region are associated with elevated fetal hemoglobin in adults (Sankaran et. al. 2011, NEJM). Whether the change in GATA2 binding to this region is responsible for the increased expression of HbG in cells treated with HDAC1/2-selective inhibitors is under investigation. Disclosures Shearstone: Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Golonzhka:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Chonkar:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.

The Hemoglobin-Haptoglobin Receptor (CD163) Is Expressed by a Subpopulation of Erythroid and Granulo-Macrophagic Progenitors and Can Be Stimulated in Vitro and in Vivo by Physiological and Pharmacological Ligands to Stimulate Hematopoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1221-1221
Author(s):  
Kathryn Matthews ◽  
Nicole Worsham ◽  
Neeta Rugg ◽  
Jose A. Cancelas ◽  
David Bell
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Hematopoietic Progenitor ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Marrow Cells

Abstract Abstract 1221 The receptor for the hemoglobin (Hb)-haptoglobin (Hp) complex, CD163, is expressed on the surface of a subpopulation of hematopoietic stem/progenitor cells (HPCs) (Matthews et al, 2006). The purpose of the studies presented here were two-fold – to demonstrate that the CD34+CD163+ double positive population could be isolated from normal adult bone marrow cells and these cells were functional as HPCs and, in addition, that these cells could be stimulated in vivo by ligands to CD163 to affect hematopoiesis. To investigate the clonogenic potential of CD34+/CD163+ HPCs, bone marrow CD34+ cells were examined for CD163 co-expression, sorted by fluorescence activated cell sorting (FACS) and plated into colony-forming assays (CFAs). 4.2% ± 1.4% (n=4) of CD34+ cells were found to co-express CD163 and this population consisted of two distinct sub-populations, CD34++ (hi)CD163+ and CD34+(lo)CD163+, each of which represented approximately half of the total CD34+CD163+ population. All three sorted populations (CD34+(all)CD163−, CD34++(hi) CD163+, CD34+(lo)CD163+) were plated into CFAs (n=4) and were assessed for erythroid and myeloid colony formation. The clonogenic efficiency of CD34++(hi)CD163+ had a 2.5-fold increase in the number CFU-E and CFU-GM when compared to both CD34+ (total) CD163− and CD34+(lo) CD163+ cells. In contrast, CD34+(hi an low)CD163+cells produced fewer BFU-E. To determine how the expression of CD163 expression on progenitor cells may play a role in hematopoiesis, we investigated the effects of the natural ligand to CD163 (Hb/Hp) as well as an agonistic antibody to CD163 (TBI 304) on HPCs in vivo. NOD-scid IL2R gammanull (NSG) mice (HuMurine Technologies) were engrafted with human CD34+cells and animals with < 30% human CD45+ cells in the peripheral blood were administered either 2 mg Hb/mouse, or 100 or 500 μg/mouse TBI 304 every 4 days. At study termination (day 14), bone marrow cells (BMC) were examined by flow cytometry and enriched for CD34+ cells for enumeration in CFAs. Hb administration resulted in an increase of human CD34+cells ranging from 4% to 7% of BMC and a corresponding 57% increase in colony-forming cells (CFC) when compared to control (PBS-administered) animals. In contrast, TBI 304 produced a dose dependent decrease in CD34+ and CFC, possibly reflecting a depletion of CD34+/CD163+ cells from overstimulation due to the longer circulating antibody. To investigate this, human CD34+ cell engrafted animals were given a single dose of 10 or 100 μg/mouse of TBI 304 and bone marrow cells were examined on day 7. TBI 304 provided a 3.5-fold increase in human CD34+ cells as well as a 1.8 to 6.7-fold increase in bone marrow erythroid lineage engraftment (huGlyA+, huCD36+ and huCD71+) and a 2-fold increase in erythroid and myeloid colony-forming cells. No overall toxicities were observed with the administration of TBI 304 or Hb. We have demonstrated that CD163 is expressed on a population of CD34+ hematopoietic progenitor cells, these cells have increased hematopoietic progenitor activity in vitro and that administration of physiological or pharmacological agonists of the CD163 receptor can measurably stimulate hematopoiesis in vivo. Disclosures: Matthews: Therapure Biopharma: Employment. Bell:Therapure Biopharma: Employment.

Dimeric Naphthoquinones: Novel Anti-Leukemic Agents Modulating Cellular Redox Status

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1290-1290 ◽  
Author(s):  
Ashkan Emadi ◽  
Mariola Sadowska ◽  
Brandon Carter-Cooper ◽  
Omasiri Wonodi ◽  
Nidal Muvarak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Therapeutic Index ◽  
Fold Increase ◽  
Leukemia Cells ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Ic50 Values ◽  
Marrow Cells

Abstract Background The oxidative state is significantly perturbed in leukemia cells, compared to normal cells, due to generation of increased reactive oxygen species (ROS) and/or dysregulated antioxidant mechanisms. In a molecularly heterogeneous disease such as acute myeloid leukemia (AML), targeting the oxidative state, a fundamental physiological property, is an attractive strategy for developing novel anti-leukemic agents. We hypothesized that regardless of oncogenic mutations, therapeutic augmentation of ROS in AML cells with dysregulated antioxidants would kill leukemia cells and leukemia stem cells, while normal stem cells would remain relatively intact. Quinones can initiate and propagate free radical reactions. The electron-accepting nature of quinones could in principle be tuned to yield selective cytotoxicity for cells with a particular redox signature. Dimeric naphthoquinones (BiQs), with known ability to generate ROS in cancer cells, are a novel class of compounds with unique characteristics that make them excellent candidates to be tested against leukemia cells. The primary objective of this study was to determine the potency of BiQs in leukemia cells and to assess the therapeutic index of BiQs in AML cells, in relation to normal hematopoietic stem cells. The secondary objective was to determine whether BiQs induce apoptosis, increase ROS, target mitochondrial membrane potential, and/or affect antioxidant proteins. Methods We tested two BiQs (E6a and B1a) and one mononaphthoquinone in two AML cell lines, MOLM-14 (FLT3-ITD) and THP-1 (FLT3-WT), two normal karyotype primary AML samples (AML15, AML16), both with FLT3-WT, and fresh normal bone marrow cells. IC50 values were determined by exposing cell lines and bone marrow cells to BiQs for 72 h and 48 h, respectively. Each experiment was terminated with Alamar Blue (Sigma). Cell viability assays were carried out similarly except that the endpoint was trypan blue exclusion. For clonogenic assays, cells were plated in methylcellulose and exposed to BiQs either prior to plating or both prior to plating and during culture. Colonies were counted using an automated colony counter (Microbiology Intl.). Apoptosis was measured using the FITC Annexin V Kit (BD Pharmingen), and mitochondrial potential was assessed using the MitoRed Kit (Millipore); cells were analyzed by FACScan (BD Biosciences) and Flow Jo Software (Tree Flo). Standard Western blot techniques were used for measurement of caspase 3 and Nrf2 protein. Numbers are mean ± SD. Results The IC50 values (µM) of E6a for MOLM-14, THP1, AML15 and AML16 were 5.5 ± 0.8, 4.2 ± 1.9, 5.1, and 0.4, respectively. The IC50 values (µM) of B1a for MOLM-14, THP1, AML15 and AML16 were 6.2 ± 0.7, 5.6 ± 0.1, 5.4, and 3.0, respectively. The IC50 value of E6a for normal bone marrow cells was 14.5 µM. The mononaphthoquinone did not show anti-leukemic activity. Viability tests showed a 52 ± 8% and 33 ± 9% increase in MOLM-14 cell kill after exposure to 5 µM E6a and 5 µM B1a, compared to vehicle, versus 69 ± 12% and 59 ± 2% in THP-1. Treatment with E6a, compared to vehicle, resulted in a concentration-dependent decrease in colony formation in MOLM-14 (52 ± 17 [10 µM] vs 228 ± 170 [5 µM] vs 338 ± 72 colonies). Interestingly, when MOLM-14 cells were treated with E6a for 24 h then placed in methylcellulose in continuous presence of E6a for 9 days, colony formation was completely inhibited with 5 µM E6a compared to vehicle (2.5 ± 1 vs 294 ± 43 colonies). Treatment of AML16 with 5 µM E6a for 24 h resulted in a 1.5-fold increase in apoptosis, and a 1.6-fold increase in loss of mitochondrial membrane potential compared to vehicle. After 6 h exposure of MOLM-14 and AML16 to 5 µM E6a, cleavage of caspase 3 was detected. After 2 h exposure of AML16 to 5 µM E6a and 5 µM B1a, Nrf2 proteins was significantly increased in response to oxidative stress. Conclusions We have demonstrated that BiQs possess anti-leukemia activity with a reasonable therapeutic index in relation to normal bone marrow cells. Measurements of effects on intracellular ROS and on proteins involved in oxidative stress are ongoing, and drug transport by multidrug resistance-associated ATP-binding cassette proteins including P-glycoprotein will be studied. The more active BiQ will be studied further in an AML primagraft model. Disclosures: No relevant conflicts of interest to declare.

Angiopoietin-1 supports induction of hematopoietic activity in human CD34− bone marrow cells

2007 ◽  
Vol 35 (12) ◽  
pp. 1872-1883 ◽  
Author(s):  
Yoshihiko Nakamura ◽  
Takashi Yahata ◽  
Yukari Muguruma ◽  
Tomoko Uno ◽  
Tadayuki Sato ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Human Cd34 ◽  
Hematopoietic Activity ◽  
Angiopoietin 1 ◽  
Marrow Cells

RAP-536 Promotes Terminal Erythroid Differentiation and Reduces Anemia in a Murine Model of Myelodysplastic Syndromes

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3796-3796 ◽  
Author(s):  
Rajasekhar NVS Suragani ◽  
Robert Li ◽  
Dianne Sako ◽  
Asya Grinberg ◽  
R. Scott Pearsall ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Erythroid Differentiation ◽  
Severe Anemia ◽  
Ineffective Erythropoiesis ◽  
Employment Equity ◽  
Erythroid Hyperplasia ◽  
Erythroid Precursors ◽  
Equity Ownership ◽  
Terminal Erythroid Differentiation

Abstract Abstract 3796 Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell disorders characterized by peripheral blood cytopenias such as anemia, neutropenia or thrombocytopenia. Ineffective erythropoiesis due to increased proliferation and abortive maturation of precursors leads to severe anemia, the most common cytopenia observed in MDS syndromes. Despite elevated erythropoietin (EPO) and erythroid hyperplasia, MDS patients are often given recombinant EPO therapy to stimulate erythropoiesis. However, only a small proportion of patients respond to EPO therapy. Frequent blood transfusions as supportive care result in iron overloading and recently iron overloading is also linked to enhanced progression to AML. Therefore, alternative therapies are necessary to treat anemia in MDS patients. Signaling by members of the TGFβ superfamily are known regulators of erythropoiesis. We developed ACE-536, a ligand trap consisting of a modified activin receptor Type IIB extracellular domain linked to a human Fc domain. In vitro assays revealed that ACE-536 inhibits smad 2/3 ligands of the signaling pathway but not smad 1/5/8 ligands. Dose dependent studies using ACE-536 in mice, rats and monkeys revealed that ACE-536 treatment resulted in increased red blood parameters but did not affect other cell types. These data suggests that ACE-536 inhibits smad 2/3 phosphorylation modulating the expression of downstream genes involved in erythroid development pathway. BFU-E and CFU-E colony formation assays from bone marrow and spleen in mice following ACE-536 treatment revealed that ACE-536 did not affect the proliferation stages of erythropoiesis. In mice, terminal erythroid differentiation analysis by flow cytometry at 72hrs following RAP-536 (10mg/kg) treatment demonstrated decreased basophilic and increased ortho- and poly-chromatophilic erythroblasts and reticulocytes compared to VEH treatment. Cell cycle analysis of bone marrow and splenic erythroblasts counterstained with BrdU and 7-AAD after RAP-536 (10mg/kg, for 24 hours) or VEH treatment to EPO pre-treated (1500 units/kg, for 40 hours) mice (N=5/group) revealed that EPO+RAP-536 treatment resulted in significant decrease in S-phase and increase in G1/G2-phases of cell cycle compared to EPO+VEH treatment. In addition, EPO+RAP-536 treatment resulted in a greater increase in RBC parameters than either of the treatments alone. Together, these results demonstrate that ACE-536 increases red blood cell formation by promoting maturation of late stage erythroblasts. We then investigated the effect of ACE-536 on anemia in NUP98-HOXD13 (NHD13) transgenic murine model of MDS. NHD13 mice develop anemia, neutropenia and lymphopenia, with normal or hyper cellular bone marrow. A Majority of the mice die by 14 months due to severe pancytopenia or progression to acute myeloid leukemia. In this study, mice were divided into three groups based on age. Early (∼4 months old), mid (∼8 months old) and late stage (∼10 months) groups were randomized and dosed with either RAP-536 at 10 mg/kg or VEH twice per week for 6–8 weeks. NHD13 mice in each group had severe anemia characterized by reduced RBC, Hemoglobin and HCT and compared to wild-type littermates prior to treatment. Treatment of RAP-536 for 6–8 weeks significantly increased RBC parameters and reversed anemia at all stages. Peripheral blood smear analysis revealed no indication of increased leukemic progression due to RAP-536 treatment. Cell differential and flow cytometric evaluation of erythroid precursors from bone marrow demonstrated decreased erythroid precursors and hyperplasia after RAP-536 treatment compared to vehicle treated control. Our data demonstrate that RAP-536 can increase hematology parameters by enhancing maturation of terminally differentiated red blood cells. We have shown RAP-536 corrects ineffective erythropoiesis, decreases erythroid hyperplasia and normalizes myeloid: erythroid ratios without enhanced progression to AML in a murine MDS model. Therefore ACE-536 may represent a novel treatment for anemia associated with MDS, particularly in patients that are refractory to EPO therapy. ACE-536 has completed Phase I clinical trials in healthy human volunteers and Phase II study in MDS patients is planned. Disclosures: Suragani: Acceleron Pharma Inc: Employment, Equity Ownership. Li:Acceleron Pharma Inc: Employment, Equity Ownership. Sako:Acceleron Pharma Inc: Employment, Equity Ownership. Grinberg:Acceleron Pharma Inc: Employment, Equity Ownership. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership. Kumar:Acceleron Pharma Inc: Employment, Equity Ownership.

Inhibition of CML Stem Cells with an Alkaloid That Reduces β-Catenin

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1882-1882
Author(s):  
Shawnya J. Michaels ◽  
Ngoc DeSouza ◽  
Yi Shan ◽  
Shaoguang Li ◽  
Saira Bates
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Marrow Transplantation ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Solvent Control ◽  
Equity Ownership ◽  
Marrow Cells

Abstract Although the majority of CML patients initially respond positively to BCR-ABL tyrosine kinase inhibitors (TKIs), they fail to eradicate the leukemia stem cells (LSCs) from which the disease arises. Only a minority of patients is able to discontinue TKI therapy, presumably due to the survival of LSCs. Therefore, the development of new therapeutics which ablate CML stem cells through a non-TKI, BCR-ABL independent pathway is needed. The Wnt/β-catenin pathway has been identified as an LSC survival pathway which provides proliferative signals and controls the stability of BCR-ABL1 through the increased expression of β-catenin. While previous research has demonstrated that Wnt/β-catenin is necessary for the survival and self-renewal of all CML cells and LSCs, it is not essential for maintenance of normal hematopoietic stem cells (HSCs). Tetrandrine (ES-3000, TET) is a natural product alkaloid used clinically in China as an analgesic and an anti-inflammatory. Its known mechanism of action is the inhibition of voltage-gated calcium channels and calcium activated big potassium (BK) channels which are commonly overexpressed in malignancies. However, TET has recently been demonstrated to inhibit the Wnt/β-catenin pathway resulting in a reduced expression of β-catenin, putatively through the inhibition of CaMKII-γ activation. This study investigated the efficacy of TET in models of CML stem cells. To demonstrate that TET can reduce β-catenin in leukemic cells, an in vitro assay with leukemic K562 cells was performed. Cells were exposed to TET for 24 hours at concentrations between 0-40 μM. Cell lysates were assayed by Western blot for β-catenin and actin. The results demonstrated that TET reduces β-catenin expression in a dose dependent manner. The effectiveness of TET was tested on CML stem cells using an in vivo mouse CML model. After priming donor C57BL/6 (B6) mice with intravenous injections of 5-fluorouracil for four days, bone marrow cells were harvested from femurs and tibia, then transfected twice with retrovirus containing MSCV-BCR-ABL-IRES-GFP. Recipient mice were lethally irradiated by two doses of 550 cGy before bone marrow transplantation by intravenous injection with 5x105 cells/mouse. Blood from recipient mice was tested for disease induction one week after transduction by FACS analysis for GFP. All mice tested positive. Treatments started on day 8 after bone marrow transplantation. Mice were randomized into four groups and treated orally with vehicle [3x/day, 2x], imatinib [100 mg/kg, 2x/day], TET [150 mg/kg, 1x/day] or imatinib + TET [3x/day: 2x with imatinib, 1x with TET]. The results of this study demonstrated that TET given orally once a day is superior to imatinib given twice a day in inhibiting the development of both circulating leukemic cells and leukemic stem cells while the combination of TET with imatinib further improves efficacy (See Figure). To determine whether TET has efficacy on human CML stem-like cells, a colony forming cell (CFC) assay with bone marrow cells from a de-novo CML patient was performed. The bone marrow cells were treated with 10 µM (IC50) TET for 14 days. After treatment, primary and secondary colonies were grown and analyzed by qPCR to determine BCR-ABL or ABL only cells. Replating efficiency of TET treated cells was 54% compared to 67% of solvent controls. In primary colonies, 95% of colonies from solvent control cultures were BCR-ABL positive compared to 70% of colonies treated with ES-3000. In secondary colonies (representing stem-ness), the TET treatment group was negative for BCR-ABL colonies while 79% of solvent control colonies still tested positive for BCR-ABL, indicating efficacy of TET in CML stem-like cells (See Table). We conclude that TET reduces leukemic stem cells in both a murine model of CML and a CFC assay which demonstrates its potential for development as an adjuvant therapy for CML patients demonstrating a lack of optimal response to TKIs, alone. Figure Figure. Table Table. Disclosures Michaels: Escend Pharmaceuticals, Inc.: Equity Ownership. Bates:Escend Pharmaceuticals, Inc.: Equity Ownership.

scholarly journals NR2F6 (EAR-2) Is a Novel Leukemia Oncogene Whose Cellular Function Is to Regulate Terminal Differentiation of Erythrocytes at the Proerythroblast Stage

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1337-1337
Author(s):  
Christine Victoria Ichim ◽  
Dzana Dervovic ◽  
David Koos ◽  
Marciano D. Reis ◽  
Alden Chesney ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Bone Marrow Cells ◽  
Erythroid Differentiation ◽  
Erythroid Progenitor ◽  
Over Expression ◽  
Marrow Cells

Abstract The leukemia stem cell model suggests that elucidation of the genes that regulate growth ability within the leukemia cell hierarchy will have important clinical relevance. We showed that the expression of NR2F6 (EAR-2), is greater in clonogenic leukemia single cells than in leukemia cells that do not divide, and that this gene is over-expressed in patients with acute myeloid leukemia and myelodysplastic syndrome. In vivo, overexpression of EAR-2 using a retroviral vector in a chimeric mouse model leads to a condition that resembles myelodysplastic syndrome with hypercellular bone marrow, increased blasts, abnormal localization of immature progenitors, morphological dysplasia of the erythroid lineage and a competitive advantage over wild-type cells, that eventually leads to AML in a subset of the mice, or after secondary-transplantation. Interestingly, animals transplanted with bone marrow that over-expresses EAR-2 develop leukemia that is preceded by expansion of the stem cell compartment in the transplanted mice—suggesting that EAR-2 is an important regulator of hematopoietic stem cell differentiation. Here we report that over-expression of EAR-2 also has a profound effect on the differentiation of erythroid progenitor cells both in vitro and in vivo. Studies of the roles of EAR-2 in normal primary bone marrow cells in vitro showed that overexpression of EAR-2 profoundly impaired differentiation along the erythroid lineage. EAR-2 over-expressing bone marrow cells formed 40% fewer BFU-E colonies, but had greatly extended replating capacity in colony assays. While knockdown of EAR-2 increased the number of cells produced per BFU-E colony 300%. Normal mice transplanted with grafts of purified bone marrow cells that over-expressed EAR-2 developed a rapidly fatal leukemia characterized by pancytopenia, enlargement of the spleen, and infiltration of blasts into the spleen, liver and peripheral blood. Sick animals had profound reduction of peripheral blood cell counts, particularly anemia with a 55% reduction in hemoglobin levels. Anemia was evident even on gross inspection of the blood and the liver in EAR-2 overexpressing animals. Analysis of the leukemic cells revealed an erythroblastic morphology, with the immunophenotype lineageneg, CD71high, TER119med. Hence, we wondered weather EAR-2 caused leukemia by arresting erythroid progenitor cell differentiation. Examination of the bone marrow of pre-leukemic animals showed a four-fold increase in cells with a pro-erythroblastic immunophenotype (CD71highTER119med , region I), and a four-fold decrease in orthochromatophilic erythroblasts (CD71lowTER119high , region IV). We observed no change in the numbers of basophilic erythroblasts (CD71highTER119high , region II) or late basophilic and polychromatophilic erythroblasts (CD71medTER119high, region III). These data suggests that over-expression of EAR-2 blocks erythroid cell differentiation at the pro-erythroblastic stage. Since EAR-2 over-expressing recipients died within 4 week, we wanted to definitively test whether animals had compromised radioprotection. We showed that decreasing the size of the bone marrow graft, reduced survival of the EAR-2 over-expressing cohort by a week, but had no effect on control animals proving that EAR-2 over-expression has a profound effect on erythropoietic reconstitution in vivo. Mechanistically, we show that DNA binding is necessary for EAR-2 function, and that EAR-2 functions in an HDAC-dependent manner, regulating expression of several genes. Pre-leukemic pro-erythroblastic cells (CD71highTER119med) that over-expressed EAR-2 had lower expression of genes involved in erythroid differentiation such as GATA1, EBF1, inhibitor of NFKB (NFKBia), ETV6, CEBP/a, LMO2, and Nfe2, and increased expression of GATA2, GLI1, ID1 and PU.1 than GFP control pro-erythroblasts. These data establish that EAR-2 is a novel oncogene whose cellular function is to regulate terminal differentiation of erythroid cells at the proerythroblastic (CD71highTER119med) stage by deregulating gene expression necessary for erythroid differentiation. Disclosures Ichim: Entest BioMedical: Employment, Equity Ownership, Patents & Royalties, Research Funding. Koos:Entest BioMedical: Employment, Equity Ownership, Patents & Royalties, Research Funding.

scholarly journals Selective Expression of the Receptor Tyrosine Kinase, HTK, on Human Erythroid Progenitor Cells

Blood ◽  
1997 ◽  
Vol 89 (8) ◽  
pp. 2757-2765 ◽  
Author(s):  
Tomohisa Inada ◽  
Atsushi Iwama ◽  
Seiji Sakano ◽  
Mitsuharu Ohno ◽  
Ken-ichi Sawada ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Bone Marrow Cells ◽  
Regulatory System ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Marrow Cells

Abstract HTK is a receptor tyrosine kinase of the Eph family. To characterize the involvement of HTK in hematopoiesis, we generated monoclonal antibodies against HTK and investigated its expression on human bone marrow cells. About 5% of the bone marrow cells were HTK+, which were also c-Kit+, CD34low, and glycophorin A−/low. Assays of progenitors showed that HTK+c-Kit+ cells consisted exclusively of erythroid progenitors, whereas HTK−c-Kit+ cells contained progenitors of granulocytes and macrophages as well as those of erythroid cells. Most of the HTK+ erythroid progenitors were stem cell factor-dependent for proliferation, indicating that they represent mainly erythroid burst-forming units (BFU-E). During the erythroid differentiation of cultured peripheral CD34+ cells, HTK expression was upregulated on immature erythroid cells that corresponded to BFU-E and erythroid colony-forming units and downregulated on erythroblasts with high levels of glycophorin expression. These findings suggest that HTK is selectively expressed on the restricted stage of erythroid progenitors, particularly BFU-E, and that HTK is the first marker antigen that allows the purification of erythroid progenitors. Furthermore, HTKL, the ligand for HTK, was expressed in the bone marrow stromal cells. Our findings provide a novel regulatory system of erythropoiesis mediated by the HTKL-HTK signaling pathway.

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