Expression and Functional Differences of CBFA2T (ETO, MTG) Gene Family Members in Hematopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4208-4208
Author(s):  
Fariborz Mortazavi ◽  
ShriHari Kadkol ◽  
Annette Bruno ◽  
Kristine Baraoidan ◽  
Steven Ackerman ◽  
...  
Keyword(s):  
Gene Family ◽  
Cell Line ◽  
Cell Lines ◽  
Family Members ◽  
Fold Increase ◽  
Erythroid Differentiation ◽  
Macrophage Differentiation ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract The CBFA2T (ETO, MTG) family has three similar family members - CBFA2T1-T3. CBFA2T1 (ETO, MTG8) and CBFA2T3 (ETO2, MTG16) are targeted by chromosomal translocations in acute myeloid leukemia. To better understand the usual hematopoietic function of this gene family, we examined the expression of CBFA2T RNA using RQ-PCR in cell-lines and human CD34+ hematopoietic cells during macrophage and erythroid differentiation. RQ-PCR on extracted RNA was performed with an icyclerQ instrument (Bio-Rad) using the Quantitect SYBR Green RT-PCR kit (Qiagen) and in vitro transcribed RNA to construct standard curves. CBFA2T3 was the most highly expressed family member in human CD34+ cells, the erythro-leukemia line K562, the myeloid line MPD, the T cell line Jurkatt and the B-cell line LCL-11. However, CBFA2T3 expression decreased by >50% during both macrophage and erythroid differentiation of human CD34+ cells. In contrast, CBFA2T1 expression was almost undetectable in human CD34+ cells and all cell lines except K562 but increased more than 20 fold during erythroid (but not macrophage) differentiation of human CD34+ cells. Extrinsic over-expression of CBFA2T1, but not CBFA2T2, significantly increased glycophorin-A and hemoglobin A expression in K562 cells, consistent with a regulatory role for CBFA2T1 in erythroid differentiation. CBFA2T2 (MTGR1) was moderately expressed in human CD34+ cells and all the cell lines and demonstrated a 2.5 fold increase in expression with macrophage differentiation but essentially no change with erythroid differentiation of human CD34+ cells. These findings suggest that despite their similarity, the CBFA2T family members have distinctive regulatory roles in hematopoietic differentiation.

C.E.R.A. Acts Differently at the Erythropoietin (EPO) Receptor Compared with Epoetin Beta: UT-7 and CD34+ Cell Stimulation Assays.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4206-4206 ◽  
Author(s):  
M. Jarsch ◽  
M. Brandt ◽  
M. Kubbies ◽  
A. Haselbeck
Keyword(s):  
Blood Cell ◽  
Cell Line ◽  
Glycophorin A ◽  
Epo Receptor ◽  
Epoetin Beta ◽  
Human Cd34 ◽  
Maximal Stimulation ◽  
Cd34 Cells ◽  
Stimulation Of

Abstract Continuous Erythropoietin Receptor Activator (C.E.R.A.), an innovative erythropoietic agent with unique receptor activity, is currently in development to provide correction of anemia and stable maintenance of hemoglobin (Hb) levels at extended administration intervals up to once monthly in patients with all stages of chronic kidney disease (CKD), and is also in development for the treatment of chemotherapy-induced anemia. In vitro studies show that C.E.R.A. has a 45-fold lower affinity for the EPO receptor than epoetin beta, due mainly to a reduced association rate. To further investigate the in vitro activity of C.E.R.A., two cell stimulation studies were undertaken. Study 1 evaluated an assay for the analysis of the molecular mechanism of C.E.R.A.- and epoetin beta-mediated cell activation. UT-7 cells were activated with C.E.R.A. or epoetin beta for 72 h or 96 h, followed by WST (tetrazolium salt) staining and spectrophotometric detection. UT-7 is a human myeloid leukemia cell line expressing the EPO receptor, and has growth dependency on EPO if no other growth factors are present. Results showed that the EC50 (concentration giving half maximal stimulation) value was approximately 10-fold higher for C.E.R.A. (range 300–400 pM) than for epoetin beta (30–60 pM). Maximal activation of UT-7 cells was achieved at C.E.R.A. 1000–2000 pM and epoetin beta 100–200 pM, but the maximal stimulation of cells was similar for both agents. Study 2 investigated the effects of C.E.R.A. and epoetin beta on stimulation of the proliferation and differentiation of human CD34+ cells. Human CD34+ stem cells from cord blood and bone marrow were cultivated with C.E.R.A. or epoetin beta for 8–14 days. After labeling, using fluorescence-tagged antibodies to proteins specific for erythroid cells (glycophorin A) and other blood cell types (CD13, CD14, CD16, CD41, CD42b, and CD61), cells were analyzed using three-color flow cytometry with a FACScan instrument (Becton Dickinson, CA). The maximal number of glycophorin A positive cells at plateau phase was used for EC50 calculation. Following stimulation of CD34+ cells, glycophorin A+ cells increased to a similar level with C.E.R.A. and epoetin beta. This stimulation was specific for erythroid precursors since the differentiation of white blood cells and megakaryocytes was not affected by C.E.R.A. or epoetin beta. Notably, mean EC50 values were 43.4-fold higher with C.E.R.A. (2.807 nM) than with epoetin beta (0.076 nM). In conclusion, C.E.R.A. and epoetin beta activate UT-7 cells and induce differentiation and expansion of CD34+ cells. These studies provide further evidence that C.E.R.A. has different EPO receptor binding properties compared with epoetin beta and demonstrate its specificity for the red blood cell line. Preclinical studies have shown that these properties translate into more continuous stimulation of erythropoiesis in vivo compared with epoetin beta and Phase III data indicate that C.E.R.A. achieved and maintained stable Hb levels in all patients with CKD.

Dramatic Down-Regulation Of MNDA Expression In CP-CML Cells From The LSC Compartment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2717-2717
Author(s):  
Céline Bourgne ◽  
Alexandre Janel ◽  
Juliette Berger ◽  
Agnès Guerci ◽  
Caroline Jamot ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Differentiation ◽  
Cell Line ◽  
Cell Lines ◽  
Fold Increase ◽  
Positive Control ◽  
Polymorphonuclear Cells ◽  
Primary Cells ◽  
Down Regulation ◽  
Cd34 Cells

Abstract Introduction Chronic Phase - Chronic Myeloid Leukemia (CP-CML) is a myeloproliferative disorder characterized by malignant proliferation of the granulocytic lineage without the arrest of cell differentiation. Tyrosine Kinase Inhibitors (TKI) have revolutionized CML treatment but several studies showed that a combination of TKI and Interferon alpha (IFNα) provides better clinical response. Myeloid Nuclear Differentiation Antigen (MNDA), which belongs to the hematopoietic interferon-inducible nuclear proteins with the 200-amino-acid repeat (HIN200) gene family, encodes a protein expressed in myeloid cells but whose function remains poorly understood. Because of its high expression in polymorphonuclear cells, its involvement in cell differentiation and apoptosis, and its induction by IFNα, we evaluated MNDA expression in CML cells and its modulation after incubation with IFNα. Material and methods We tested MNDA expression in several cell lines (K562, KCL22, LAMA84, TF1 and U937 (positive control)), in polymorphonuclear cells from healthy donors (HD-PMN, n=13) and in primary cells from patients with CP-CML at diagnosis (CP-CML; n=17). The relative expression of the MNDA transcript was analyzed using the 2-ΔΔCt method and was normalized to the endogenous reference gene GAPDH. HD-PMN were used as calibrator. We developed a multiparametric flow cytometry assay (CD45-V500/CD14-APC-H7/CD15-PerCpCy5.5/CD34-PC7/CD38-V450/MNDA-FITC) to detect MNDA protein in the different cell subsets, particularly in CD34+cells. Results As previously described, MNDA was poorly expressed in the K562 cell line. Similarly, mRNA was detected at low levels in two other CML cell lines (KCL22, LAMA84) and in TF1 cells, but at a high level in the U937 cell line, used as a positive control. In each cell line, the transcript expression was correlated to the protein level, as evaluated by flow cytometry (MFI ratio: 2.04±0.21, 2.36±0.24, 1.59±0.14, 1.88±0.11 and 8.77±0.54 for K562, KCL22, LAMA84, TF1 and U937, respectively (n=3)). In CP-CML primary cells, MNDA expression was greatly diminished as compared with HD-PMN in both mRNA (0.20±0.08 (n=17) vs. 1.32±0.21 (n=10); p=1.52x10-6) and protein (MFI ratio: 6.9±0.98 vs. 16.31±1.25, p=0.001). After having verified that IFNα (2000 U/ml, 16 hours) induced MNDA expression in HD mononuclear cells but not in PMN, we observed that induction of MNDA was moderate in CML cell lines K562 and LAMA84 (2-fold increase, n=3) whereas the level of MNDA mRNA was significantly increased in TF1 cells (28-fold increase, n=4). Induction in primary CML cells was variable (3/5 patients). Aiming to evaluate the expression of MNDA in leukemic stem cells (LSC), we first analyzed MNDA expression in CD34+ and CD34+/CD38- cells from HD. We observed that MNDA is down-regulated in healthy CD34+ and CD34+/CD38- cells compared to mature cells (mRNA: about 4 logs, protein: 8-10 fold lower, n=4), but we always detected a significant signal in CD34+cells (MFI ratio: 2.76±0.46, n=3). However, MNDA was not expressed by CML cells from the LSC compartment (n=4). This inhibition does not seem to be antagonized by nilotinib or IFNα (n=2). Discussion/Conclusion MNDA expression appears to be clearly down-regulated in CP-CML cells and dramatically so in the LSC compartment. In some patients, we observed sustained sensitivity to IFNα, but only in the compartment of more mature cells. This suggests early deregulation of MNDA expression which seems to be only partially dependant on differentiation. The mechanisms involved in this down-regulation remain to be elucidated but could be independent to TK activity of BCR-ABL protein and resistant to IFNα in the LSC compartment. This marked deregulation of MNDA in the LSC compartment is an additional argument in favor of intrinsic changes specific to primitive cells. Disclosures: No relevant conflicts of interest to declare.

Long-Term Culture of Human CD34+ Progenitors With FLT3-Ligand, Thrombopoietin, and Stem Cell Factor Induces Extensive Amplification of a CD34−CD14− and a CD34−CD14+ Dendritic Cell Precursor

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2244-2252 ◽  
Author(s):  
Jean-François Arrighi ◽  
Conrad Hauser ◽  
Bernard Chapuis ◽  
Rudolf H. Zubler ◽  
Vincent Kindler
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor ◽  
Fold Increase ◽  
Cell Number ◽  
Interleukin 4 ◽  
Flt3 Ligand ◽  
Gm Csf ◽  
Human Cd34 ◽  
Cd34 Cells

Current in vitro culture systems allow the generation of human dendritic cells (DCs), but the output of mature cells remains modest. This contrasts with the extensive amplification of hematopoietic progenitors achieved when culturing CD34+ cells with FLT3-ligand and thrombopoietin. To test whether such cultures contained DC precursors, CD34+ cord blood cells were incubated with the above cytokines, inducing on the mean a 250-fold and a 16,600-fold increase in total cell number after 4 and 8 weeks, respectively. The addition of stem cell factor induced a further fivefold increase in proliferation. The majority of the cells produced were CD34−CD1a− CD14+(p14+) and CD34−CD1a−CD14−(p14−) and did not display the morphology, surface markers, or allostimulatory capacity of DC. When cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), both subsets differentiated without further proliferation into immature (CD1a+, CD14−, CD83−) macropinocytic DC. Mature (CD1a+, CD14−, CD83+) DCs with high allostimulatory activity were generated if such cultures were supplemented with tumor necrosis factor- (TNF). In addition, p14− cells generated CD14+ cells with GM-CSF and TNF, which in turn, differentiated into DC when exposed to GM-CSF and IL-4. Similar results were obtained with frozen DC precursors and also when using pooled human serum AB+ instead of bovine serum, emphasizing that this system using CD34+ cells may improve future prospects for immunotherapy.

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.

Knockdown of HCK Reduces Cell Death and Erythroid Differentiation in Human CD34+ Hematopoietic Progenitor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2860-2860
Author(s):  
Fernanda Marconi Roversi ◽  
Fernando Vieira Pericole ◽  
Adriana da Silva Santos Duarte ◽  
Karla Priscila Ferro ◽  
Flávia Adolfo Corrocher ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Specific Inhibitor ◽  
Erythroid Differentiation ◽  
Fold Change ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Myelodysplastic syndromes (MDS) are clonal disorders characterized by ineffective hematopoiesis and increased risk of transformation to acute myeloid leukemia (AML). The identification of genes and cellular pathways active in leukemia cells but not in normal hematopoietic stem/progenitor cells (HSC) may help to understand the key steps in the MDS and AML pathogenesis and lead to new approaches to further enhance the treatment of both diseases, considered incurable with non-transplantation therapy. Src kinase family (SFK) is a central mediator in multiple oncogenic signaling pathways and some SFK members (Hck, Lyn, Fgr, Fyn) had previously been described as overexpressed or activated in leukemic cells. However, to this moment, the role of hematopoietic cell kinase (HCK), the unique SFK member restricted expressed in hematopoietic cells, had not been characterized in MDS and AML pathogenesis as well as in HSC. In order to better understand the HCK importance in hematopoiesis, we used lentiviral shRNA vectors to knockdown the HCK expression in primary human CD34+ HSC. The HCK levels were reduced in approximately 70-80% (shHCK) compared to the control lentiviral shRNA (shControl-GFP). To promote erythroid differentiation, human CD34+ transduced cells were grown in methylcellulose for 7 days and in liquid media for another 6 days. During this experiment, shHCK cells showed decreased cell viability (fold change compared to shControl-GFP = 0.55, P<.0001, n=3) combined with an increase in CD71+ expression (fold change compared to shControl-GFP = 3, P<.01, n=3), indicating a delay in erythroid differentiation. As expected, shControl-GFP cells showed a decreased GATA1 expression during erythroid differentiation. Meanwhile, shHCK cells did not modulate GATA1 expression. Interestingly, without any stimulus, HCK knockdown in CD34+ cells significantly decreased apoptosis (AnnexinV+ cells) compared to shControl-GFP (fold change = 0.52, P<.01, n=4). Attempts have been made to overexpress HCK in CD34+ HSC, however more than 80% cells were apoptotic and further assays were not possible. Thus, in HSC, HCK participates of erythroid differentiation and apoptosis signaling. According to the HCK importance on HSC and that SFK inhibitors are undergoing early phase clinical testing, a specific inhibitory activity compound for HCK, named iHCK-37, had been developed by Dr Maurizzio Botta. We tested this compound on primary normal human CD34+ cells originated from healthy donors bone marrow samples and also from cord blood units. The iHCK-37 treatment did not change proliferation, survival and death of these normal CD34+ cells. Conversely, MDS and AML CD34+ cells treated with the same drug exhibited a dose-dependent growth inhibition. Likewise, following iHCK-37 treatment of MDS and AML total bone marrow mononuclear cells, the BFU-E and CFUs colony numbers were significantly decreased compared to untreated cells (vehicle). We also observed a potent in vitro antiproliferative activity of iHCK-37 against a panel of leukemia cell lines, with uM IC50 values in AML (5.0 - 5.8uM) and chronic myeloid leukemia (9.1 - 19.2uM). In addition, the combinatory in vitro treatment of iHCK-37 and 5-Azacitidine (Aza) also demonstrated additive effects relative to either drug alone. Interestingly, iHCK-37 or iHCK-37 plus Aza treatments of dysplastic and leukemia cells enhanced apoptosis and resulted in increased BAX and reduced BCL-XL protein levels. This result could be clinically relevant for MDS, as Aza is the only treatment available for higher-risk MDS, but with low response rates and frequent induced resistance and refractoriness over time. In summary, we herein have shown that HCK mRNA knockdown of normal CD34+ cells resulted in growth inhibition, decreased cell death and reduced erythroid differentiation, suggesting that HCK is essential for normal hematopoiesis. We presume that the deregulation of HCK pathway in leukemic cells might be crucial for MDS and AML pathogenesis. On the other hand, the inhibition of HCK protein activity with a specific inhibitor was able to restore the apoptotic pathways of leukemic cells, acting on cancer cells without alter any signaling of normal cells. Moreover, the specific inhibitor may have antineoplastic effect that can even be additive to current available drugs. Our study adds new insights to the role of HCK in MDS and AML as well as into potential new anticancer treatment strategies. Disclosures No relevant conflicts of interest to declare.

Long-Term Culture of Human CD34+ Progenitors With FLT3-Ligand, Thrombopoietin, and Stem Cell Factor Induces Extensive Amplification of a CD34−CD14− and a CD34−CD14+ Dendritic Cell Precursor

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2244-2252 ◽  
Author(s):  
Jean-François Arrighi ◽  
Conrad Hauser ◽  
Bernard Chapuis ◽  
Rudolf H. Zubler ◽  
Vincent Kindler
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor ◽  
Fold Increase ◽  
Cell Number ◽  
Interleukin 4 ◽  
Flt3 Ligand ◽  
Gm Csf ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Current in vitro culture systems allow the generation of human dendritic cells (DCs), but the output of mature cells remains modest. This contrasts with the extensive amplification of hematopoietic progenitors achieved when culturing CD34+ cells with FLT3-ligand and thrombopoietin. To test whether such cultures contained DC precursors, CD34+ cord blood cells were incubated with the above cytokines, inducing on the mean a 250-fold and a 16,600-fold increase in total cell number after 4 and 8 weeks, respectively. The addition of stem cell factor induced a further fivefold increase in proliferation. The majority of the cells produced were CD34−CD1a− CD14+(p14+) and CD34−CD1a−CD14−(p14−) and did not display the morphology, surface markers, or allostimulatory capacity of DC. When cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), both subsets differentiated without further proliferation into immature (CD1a+, CD14−, CD83−) macropinocytic DC. Mature (CD1a+, CD14−, CD83+) DCs with high allostimulatory activity were generated if such cultures were supplemented with tumor necrosis factor- (TNF). In addition, p14− cells generated CD14+ cells with GM-CSF and TNF, which in turn, differentiated into DC when exposed to GM-CSF and IL-4. Similar results were obtained with frozen DC precursors and also when using pooled human serum AB+ instead of bovine serum, emphasizing that this system using CD34+ cells may improve future prospects for immunotherapy.

scholarly journals CARM1 Inhibition: Evaluation of Response and Efficacy in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2719-2719
Author(s):  
Adnan K Mookhtiar ◽  
Sarah Greenblatt ◽  
Na Man ◽  
Daniel Karl ◽  
Vasileios Stathias ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Rna Sequencing ◽  
Cell Lines ◽  
Small Molecule ◽  
Colony Formation ◽  
Myeloid Differentiation ◽  
Cd34 Cells ◽  
Over Time

Abstract Small molecule protein arginine methyltransferase inhibitors (PRMTi) are being actively pursued for the treatment of a variety of cancers; however, the mechanisms of response to PRMTi remain poorly understood. CARM1, also known as PRMT4, is significantly overexpressed in AML, as well as many solid tumors, and regulates myeloid differentiation. We have shown the dependency of AML cells, but not normal blood cells, on CARM1 activity, based on CARM1 knockout, CARM1 knockdown, and chemical inhibition (Greenblatt et al. Cancer Cell 2018). These experiments showed that CARM1 regulates essential processes in leukemia cells, and is critical for leukemic transformation. Although several small molecule inhibitors of CARM1 have been reported recently, many display a lack of selectivity for CARM1 or fail to produce a biological response. The recent discovery of potent and selective CARM1 inhibitors (Drew et al., 2017), has made it possible to investigate the implications of pharmacological inhibition of CARM1 in vitro and in vivo. In vitro, a selective CARM1 inhibitor, EPZ025654, reduced the methylation of a CARM1 substrate, BAF155, in a time and concentration-dependent manner, while the specific histone targets of CARM1 remained unchanged. Translocation (8;21) AML samples in the Eastern Cooperative Oncology Group cohort, have significantly higher CARM1 expression compared to normal CD34+ controls. This led us to hypothesize that CARM1 is a direct target of the AML1-ETO fusion protein. Therefore, we assessed whether EPZ025654 could inhibit AML1-ETO driven gene expression. AML1-ETO specific target genes showed significant changes in expression following EPZ025654 treatment. AML1-ETO positive patient samples also displayed decreased colony formation in methylcellulose and increased myeloid differentiation in response to CARM1 inhibition. We next evaluated EZM2302, a compound structurally related to EPZ025654, that is highly orally bioavailable and is well tolerated in mice (Drew et al., 2017). We generated AE9a-GFP primary transplantation mice and treated them with 100 mg/kg of EZM2302 or vehicle twice-daily (BID). The inhibitor treated mice showed significantly improved survival as well as fewer GFP+ cells in the peripheral blood over time. GFP+ AE9a bone marrow cells also showed decreased colony formation in vitro and induced macrophage differentiation in methylcellulose. GFP+ cells were isolated by FACS and submitted for RNA-sequencing. Flow cytometry analysis post-treatment revealed a significant downregulation of c-Kit and increased differentiation of hematopoietic stem and progenitor cells. Resistance to epigenetic targeted therapeutics has been observed, often through the induction of kinase signaling pathways. Therefore, we explored synergistic combinations with CARM1 inhibition using RNA-sequencing and proteomics analysis in leukemia cell lines. We used L1000 profiling (Subramanian et al., 2017) to simultaneously profile the transcriptional response of 18 AML cell line and CD34+ cells after 6 days of treatment. The AML1-ETO positive cell lines exhibited an IC50 in the 0.4-3 μM range, while CD34+ cells and several AML cell lines appeared to be resistant to CARM1 inhibition. While gene expression changes resulting from alterations in RNA stability were observed, the most significant differences between sensitive and resistant cell lines were genes associated with the regulation of cell cycle progression. Gene expression changes were evaluated over time in an AML1-ETO positive cell line, SKNO-1. SKNO-1 cell lines showed an upregulation of a gene expression signature associated with PI3K/AKT/mTOR signaling, with the most significant gene expression changes occurring 7-14 days post treatment. We simultaneously profiled these cells using multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in response to CARM1 inhibition over time. A comparison of this response to chemical perturbation signatures in the L1000 database, identified several chemical inhibitors of the PI3K/AKT/mTOR axis that could reverse the gene expression changes induced by CARM1 inhibition. This finding elucidated a response mechanism for CARM inhibition and a synergistic therapeutic strategy that has the potential to improve CARM1 directed therapy. Disclosures No relevant conflicts of interest to declare.

Effects of Elevated BCR/ABL Expression Level on Cellular Transformation and Sensitivity to Imatinib in Primitive Human Hematopoietic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1998-1998
Author(s):  
Hardik Modi ◽  
Su Chu ◽  
Tinisha McDonald ◽  
Stephen Forman ◽  
Ravi Bhatia
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitor ◽  
Cell Transformation ◽  
Hematopoietic Cells ◽  
Fold Increase ◽  
Cellular Transformation ◽  
Pcr Analysis ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Elevated Expression

Abstract Increased levels of BCR/ABL (BA) expression in CML hematopoietic cells have been associated with disease progression and resistance to the tyrosine kinase inhibitor imatinib mesylate (IM). Although cell lines with varying levels of BA expression have been studied, the role of elevated BA expression in cell transformation and drug resistance has not been directly evaluated in the primitive human hematopoietic cells in which the disease arises. Here we have used a human transduction model of CML to determine the effects of varying BA expression levels on cellular transformation (proliferation, apoptosis and differentiation) as well as responsiveness to IM. Cord blood (CB) CD34+ cells were transduced with MSCV vectors expressing BA and GFP, or control vectors expressing GFP alone followed by CD34+GFP+ cells selection by flow cytometry sorting. For BA expressing cells, two separate populations were selected based on low or high GFP expression (BAlo and BAhi). Quantitative RT-PCR analysis confirmed increased expression of BA in high GFP expressing cells (5.9±1.5 fold increase in BA:B2M levels in BAhi compared with the BAlo cells, n=3). The proliferation rate of BAhi cells, measured by fold expansion after 3 days of growth factor (GF) culture, was 2.0±0.2 fold higher than BAlo cells, and 6.7±0.1 fold higher than control (n=3). Upon GF deprivation, BAhi cells demonstrated increased resistance to apoptosis (24.4±11.4%, n=3) compared with BAlo (42±12%, n=3) or control cells (45±12%, n=3). BA transduced CD34+ cells generated higher numbers of glycophorin A+ cells than control (35.6%) following GF culture for 7 days. This effect was enhanced in BAhi (91.5%) compared with BAlo cells (77.1%). This was accompanied by an increase in CD33+ myeloid cells and a decrease in CD11b+ cells (36.3, 60.1 and 65.2% CD33+ cells and 6.0, 1.8 and 0.5% CD11b+ cells for control, BAlo and BAhi cells respectively). In addition the frequency of CD41a+ megakaryocytic cells was higher in BAhi (7.0%) relative to BAlo (3.1%) and control cells (1.0%). Next, we asked whether elevated expression of BA resulted in altered sensitivity to IM (0.025μM to 1μM) in an MTS assay. We observed that BAhi cells were more sensitive (83% inhibition at 1μM) to the IM compared with BAlo cells (14% inhibition at 1μM). We also investigated whether elevated levels of expression of two BA kinase mutants, M351T and E255K, were associated with altered IM sensitivity in CD34+ cells. The M351T mutation leads to intermediate level of IM resistance in cell lines. As was observed for wild type BA, CD34+ cells expressing higher levels of M351T demonstrated increased sensitivity to IM. On the other hand, cells expressing high and low levels of the E255K mutant, which is associated with high levels of IM resistance, demonstrated similar levels of IM sensitivity. In conclusion, increased levels of BA expression in human CD34+ cells results in enhanced proliferation; increased resistance to apoptosis following GF withdrawal, and altered differentiation with increased expression of erythroid, megakaryocytic and early myeloid markers and reduced expression of mature myeloid markers. Interestingly, expression of high levels of BA was associated with enhanced rather than reduced sensitivity to IM. Taken together these observations suggest that the effects of varying levels of BA expression on imatinib sensitivity in primitive human hematopoietic cells are determined primarily by increased proliferation rather than reduced apoptosis resulting from enhanced BA expression.

Cyclin C Regulates the Quiescence of Human CD34+CD38- Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1273-1273
Author(s):  
Yasuhiko Miyata ◽  
Yan Liu ◽  
Vladimir Jankovic ◽  
Goro Sashida ◽  
Silvia Menendez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fold Increase ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cyclin C

Abstract The relative quiescence of adult hematopoietic stem cells (HSCs) at steady state represents an important regulatory mechanism for maintaining their self-renewal and engraftment capacity, as well as their resistance to cytotoxic insults. However, the specific mechanisms regulating the intermittent entry of HSCs into the cell cycle are not well characterized. Here we provide the evidence that cyclin C (CCNC) specifically promotes the G0/G1 transition of human CD34+CD38- HSCs, and thus can significantly affect the loss of HSC self-renewal capacity in in vitro culture. Based on the recently hypothesized specific function of CCNC in G0 exit of human fibroblasts, we have analyzed the effects of CCNC loss on the behavior of human cord blood HSCs. We achieved a highly efficient knockdown of CCNC expression (>90%) using lentiviral shRNA (shCCNC) transduction of freshly isolated human cord blood CD34+ cells, allowing the in vitro assessment of early cell cycle regulation in HSCs. First, we observed a 3-fold increase in the G0 fraction of shCCNC transduced CD34+ cells compared to the empty vector control, based on the Pyronin Y and Hoechst 33342 staining 72h after infection. The depletion of CCNC did not prevent cell cycle progression beyond the G1 entry, as we observed no significant changes in the G1/S/G2-M distribution, indicating that critical CCNC activities may be restricted to the G0 checkpoint. Consistent with the reported enrichment of functional HSCs in the G0 fraction, CCNC knockdown (CCNC KD) cells showed increased activity in all surrogate in vitro assays of stem cell-ness tested: a ∼3 fold increase in CD34+ population after long term culture, a ∼2.5 fold increase in long-term culture initiating cells (LTC-ICs) and a ∼3.5 fold increase in cobblestone area forming cells (CAFCs). In contrast, CFU assays using freshly sorted shCCNC cells (and cells obtained after one-week culture in cytokines) showed only a minimal decrease in total colony number, with no difference in colony composition or morphology, indicating no significant effect on hematopoietic progenitor cell differentiation. However, we did observe a prominent effect on secondary CFUs after 2 and 3 weeks in liquid culture (i.e. using the delta assay), namely a 2-fold and 30-fold increase in shCCNC over control culture respectively, again indicating a specific function of CCNC on the more primitive cells. Consistently, CCNC KD robustly enhanced CD34 expression and secondary CFU maintenance in sorted CD34+CD38- cells (HSCs); both markers of hematopoietic cell immaturity were rapidly lost in CD34+CD38+ cells (i.e. the committed progenitor cells) with no detectable effect of shCCNC transduction. Finally, we have found that these effects of CCNC depletion are likely the result of its initial loss of function, as transient CCNC KD, using siRNA transfection of CD34+ cells, produced similar biological effects as the constitutive lentiviral shCCNC expression. Collectively, these data indicate a cell context-dependent effect of CCNC KD on the initial rate of cell cycle entry by quiescent HSCs and suggest that this approach could be used to preserve their functional capacity in culture, potentially enhancing the ex vivo expansion of HSCs, as well as their use in gene therapy protocols. Transplantation of transduced CD34+ cells into sublethally irradiated immunodeficient mice is now under way to establish the potentially beneficial effects of CCNC KD on the engraftment and repopulating capability of cultured HSCs.

In Vitro and Ex Vivo Studies On Cell Lines and Primary Human Leukemia Cells of the Effects of APR-246 Alone and in Combination with Conventional Chemotherapeutic Drugs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2751-2751
Author(s):  
Kerstin Jonsson-Videsater ◽  
Dina Ali ◽  
Hareth Nahi ◽  
Sofia Bengtzen ◽  
Stefan Deneberg ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Ex Vivo ◽  
Fold Increase ◽  
Global Gene Expression ◽  
Chemotherapeutic Drugs ◽  
Cytotoxic Effects ◽  
Global Gene Expression Analysis ◽  
Active Caspase

Abstract Abstract 2751 Poster Board II-727 Introduction: Mutations of TP53 are associated with an extremely poor prognosis in hematopoietic malignancies and is found in 10 to 15% of patients with AML. APR-246 is a methylated form of the small molecule PRIMA-1. It primarily targets mutated p53 protein, but has cytotoxic and apoptosis inducing effects on primary leukemia cells from AML and CLL patients irrespectively of p53 status even though cells with mutated p53 have been shown to be more sensitive. In May 2009, a first-in-man trial has been initiated evaluating the effect of APR-246 in hematologic malignancies. Patients and Methods: Leukemic blast cells from 32 untreated patients with AML were isolated and analyzed. Sixteeen selected patients had normal karyotype and 16 selected patients had complex karyotype. All patient DNA were sequenced at exon 5-8 of TP53 gene. Cells were exposed to APR-246 (2.5μM and 5μM), Ara-C (0.5μM), daunorubicin (0,05μM) and fludarabin (1μM) alone and in combinations. Different timing schedules of the exposure were also used. Cell viability was assayed by bioluminescence measuring ATP. Expression of p53, Bax, Bcl-2 and active caspase –3 and induction of reactive oxygen species (DCF-fluorescence) was analyzed by flow cytometry. We also analyzed two leukemia cell lines, KBM3 and NB4, with and without p53mutation respectively, in order to study the combination effect with chemotherapeutic drugs and the role of preincubation with either of the drug. The Combination Index (CI) was calculated according to the additive model where a CI of less than 0.8 indicates synergy (Möllgård et al CCP, 2008). We also performed global gene expression analysis with Affymetrix platform 1.0 after 18h of APR-246 treatment at 15μM and 25μM in vitro. Results: APR-246 induced dose dependent cytotoxic effects in primary AML samples with a IC50 value of 5.0 μM after 4 days of incubation. A statistically significant increase in the expression of active caspase-3 after 48h treatment with APR-246 (p<0.001) and a tendency to p53 up-regulation (p=0.056) could be detected in patient cells with both normal and complex karyotype. Patients that up-regulated p53 after APR-246 exposure were more sensitive to APR-246 (p<0.05) and had a significantly lower level of Bax (p<0.05) before APR-246 treatment. The seven patients with TP53 mutation had significant lower sensitivity to daunorubicin and fludarabin (p<0.01) in vitro but not to APR-246, indicating a possible treatment alternative in multidrug resistant cells. In primary AML cells, the combination of APR-246 and daunorubicin was the most effective drug combination and gave strong synergy at simultaneous incubation. The TP53 mutated KBM3 cell line was significantly more sensitive to APR-246 (p<0.01) than wt TP53 NB4 cell line with IC50 of 15μM and 45μM respectively. Simultaneous exposure of APR-246 and conventional chemothperapeutic drugs showed synergy in drug combinations tested in both the mutated and the wild type cell line. Pre-incubation with APR-246 gave significantly stronger synergism with all tested drugs combined with 15μM APR-246 (CI for DNR 0.76, for Ara-C 0.65 and for fludarabin 0.60). Pre-incubation with conventional chemotherapeutic drugs showed significantly less synergy. Flow cytometry demonstrated a 2-fold increase in reactive oxygene species (ROS) after 24h and a 15-fold increase after 48h of APR-246 treatment in the TP53 mutated cell line. Global gene expression analysis showed that 50 % of the genes that were upregulated more than a 2.0 fold by APR-246 were associated to the redox status of the cells. Genes that were significantly upregulated were SLC7A11 and oxygenase 1, indicating that APR-246 may exert some of its cytotxic effects by incuding oxidative stress. Conclusions: We conclude that APR-246 exhibits a concentration and time dependent cytotoxic effects in wt and p53 mutated cell lines and primary AML cells ex vivo. APR-246 shows strong synergism together with conventional chemotherapeutic drugs, especially with pre-incubation with APR-246. The anti-leukemic effects are associated with an increase in ROS. A first-in-man trial with APR-246 in haematological malignancies has been initiated and an update of the trial will be given at the meeting. Disclosures: Paul: Aprea AB: Consultancy, Research Funding. Lehmann:Aprea AB: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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