Apoptosis Repressor with Caspase Recruitment Domain (ARC) Increases AML Cell Migration and Adhesion in Vitro and in Vivo by Regulating Leukemia-Stroma Interactions.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2626-2626
Author(s):  
Bing Z Carter ◽  
Po Yee Mak ◽  
Ye Chen ◽  
Rodrigo Jacamo ◽  
Vivian Ruvolo ◽  
...  
Keyword(s):  
Adhesion Molecules ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Caspase Recruitment Domain ◽  
Control Mscs ◽  
D Cells ◽  
And Migration

Abstract Abstract 2626 Hematopoietic cells express a wide range of adhesion molecules and bone marrow (BM) stroma cells produce their corresponding ligands. Through these ligand-receptor pairs, hematopoietic cells interact with their BM microenvironment. The same system is hijacked by AML and often adhesion molecules in leukemia cells and/or their ligands in stroma cells are upregulated, promote leukemia-stroma interactions, and protect leukemia cells from therapeutic agents. Understanding the underlying mechanisms is critical for therapeutic strategies aimed at disrupting leukemia-stroma interactions. For example, pharmacological blockage of the CXCR4-SDF1a axis has been shown to result in chemosensitization of AML cells in vitro, in vivo and in clinical trials (Zeng Z et al., Blood 2009; Uy GL et al., Blood 2012; Andreeff M et al., ASCO 2012). ARC (Apoptosis repressor with caspase recruitment domain) is an antiapoptotic protein. We recently determined ARC expression in samples from 511 newly diagnosed AML patients by reverse-phase protein array and reported that ARC is one of the strongest adverse prognostic markers in AML (Carter BZ et al., Blood 2011). In the same sample set, we also probed the expression of more than 200 additional proteins enabling us to correlate ARC expression with the expressions of other proteins. Surprisingly, ARC expression was correlated with multiple proteins involved in cell adhesion and migration. We generated stable ARC overexpressing (O/E) KG-1 cells, ARC knock down (K/D) OCI-AML3 and Molm13 cells, and ARC K/D BM derived mesenchymal stroma cells (MSCs) to investigate ARC's roles in leukemia-stroma interactions. Expression levels of FAK, integrinb3, fibronectin, and VLA4 were increased in ARC O/E and decreased in ARC K/D cells, compared to controls. CXCR4 mRNA and cell surface CXCR4 protein were higher in ARC O/E KG-1 (3.80- and 1.53-fold, P<0.01; respectively) and lower in ARC K/D cells (OCI-AML3: 0.43- and 0.70-fold, P=0.01, and Molm13: 0.46- and 0.74-fold, P=0.02, respectively), while levels of SDF1a mRNA as well as secreted SDF-1a protein determined by ELISA (P=0.013) were lower in ARC K/D as compared to control MSCs. Interestingly, MSCs co-cultured with AML cells significantly increased SDF1a secretion and this increase was greatly diminished when ARC was knocked down in MSCs. Migration of leukemic cells towards MSCs was significantly higher for ARC O/E and lower for ARC K/D AML cells (P<0.001) and migration of leukemic cells to ARC K/D MSCs was significantly diminished compared to control MSCs for both, OCI-AML3 (P<0.001) and primary AML cells (18.1±0.8% vs. 32.8±0.5%, n=4, P<0.001). In addition, adhesion to MSCs and to VCAM1 coated plates was increased for ARC O/E and decreased for ARC K/D AML cells (P<0.01). Furthermore, adhesion to ARC K/D MSCs was decreased compared to control MSCs for AML cells for both cell lines (KG-1, 43.6±1.2% vs. 58.2±1.7%; OCI-AML3, 34.8±1.0% vs. 48.7±0.7%; Molm13, 9.2±2.2% vs. 16.8±2.7%) and primary patient samples (52.1±1.6% vs. 66.3±6.4%, P=0.02 and 42.1±5.0% vs. 59.6±7.4%, P=0.03). The role of ARC in leukemia-stroma interactions was further assessed using a novel human extramedullary BM model in mice recently developed by our group (Chen Y et al., Blood 2012). Luciferase/GFP Molm13 cells were injected into Nod/ScidIL2Rg−/− mice after extramedullary human BM containing either ARC K/D or control MSCs was established in the flanks of mice. Significantly fewer AML cells engrafted in the human extramedullary BM developed from ARC K/D than from control MSCs as determined by immunohistochemistry staining for human CD45+ cells and quantitative image analysis (P<0.01). In conclusion, we here demonstrate that antiapoptotic ARC regulates migration and adhesion of leukemia cells to BM stroma in vitro and in vivo via activation of multiple mechanisms, not only in AML cells but also in MSCs, thus enhancing its anti-apoptotic activity. Given the fact that both intrinsic apoptosis resistance and extrinsic environmental factors contribute to drug resistance and relapse in AML, ARC may play a central role in AML and be an excellent therapeutic target: ARC inactivation may disrupt leukemia-stroma interactions and increase leukemia cell sensitivity to chemotherapy by increasing susceptibility to apoptosis. We speculate that ARC, a CARD containing protein, acts at least in part via activating NF-kB signaling, which is known to be regulated by CARD proteins. Disclosures: No relevant conflicts of interest to declare.

Differentiation arrest and stromal cell-independent growth of murine erythroleukemia cells are associated with elevated expression of ets-related genes but not with mutation of p53

1993 ◽  
Vol 13 (9) ◽  
pp. 5582-5592
Author(s):  
R J Nibbs ◽  
K Itoh ◽  
W Ostertag ◽  
P R Harrison
Keyword(s):  
Stromal Cells ◽  
Stromal Cell ◽  
Gene Activation ◽  
Leukemic Cells ◽  
Term Survival ◽  
Elevated Expression ◽  
D Cells ◽  
Murine Erythroleukemia

The ELM erythroleukemia is novel in that long-term survival of leukemic cells in culture (ELM-D cells) is dependent on contact with a bone marrow-derived stromal feeder cell layer. However, a number of stroma-independent (ELM-I) mutants that vary in their ability to differentiate in vitro in response to erythropoietin and interleukin-3 have been derived. We have attempted to define the genetic changes responsible for these different phenotypes. At the p53 locus in the primary leukemic cells, one copy of the gene has been lost whereas the other contains an 18-bp depletion, implicating its mutation as an early step in the development of the leukemia. Changes in ets gene expression have also been found. The Fli-1 gene region is rearranged in the primary tumor because of the insertion of a retrovirus inserted upstream of one Fli-1 allele, but this does not result in Fli-1 gene activation in any of the ELM-D or ELM-I cell lines except one. It seems significant that this line is the only one to have lost the ability to differentiate in response to erythropoietin. In addition, up-regulation of erg is associated with stromal cell-independent growth, since all ELM-I mutants have moderate levels of erg mRNA, whereas only low or undetectable levels are found in primary leukemic cells in vivo or in ELM-D cells in vitro. This up-regulation of erg mRNA seems to be important for stromal cell-independent growth, since ELM-D cells show elevated expression of the erg gene after separation from stromal cells. This seems to be made permanent in ELM-I mutants, since they do not down-regulate erg mRNA when grown in contact with stromal cells. We therefore propose that ets family members regulate both the survival and differentiation of erythroid cells.

scholarly journals Oligonucleotide uptake in human hematopoietic cells is increased in leukemia and is related to cellular activation

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1788-1795 ◽  
Author(s):  
Q Zhao ◽  
X Song ◽  
T Waldschmidt ◽  
E Fisher ◽  
AM Krieg
Keyword(s):  
T Cells ◽  
Cell Activation ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Cellular Activation ◽  
Therapeutic Drugs ◽  
Normal Human ◽  
Novel Strategy

Abstract The use of antisense oligonucleotides as tools for modulating gene expression represents a novel strategy for designing drugs to treat a variety of diseases. Several factors, including cellular uptake and internalization of the oligonucleotides, are important parameters in determining the effectiveness of antisense agents such as therapeutic drugs. We have studied oligonucleotides uptake in normal and leukemic human hematopoietic cells, such as peripheral blood, bone marrow (BM), and HL-60 cell line; and have found that, in normal human blood and BM, myeloid cells and B cells preferably took up more oligonucleotides than T cells. There was no marked difference in oligonucleotide uptake between CD4+ helper T cells and CD8+ cytolytic T cells. Leukemic cells had greater oligonucleotide uptake than their normal counterparts. Furthermore, oligonucleotide uptake was closely related to cell activation status and can be modulated by growth factors or inhibitors. These studies provide a basis for using oligonucleotides as therapeutic drugs both in vitro and in vivo.

scholarly journals Curcumin and Its Analogue Induce Apoptosis in Leukemia Cells and Have Additive Effects with Bortezomib in Cellular and Xenograft Models

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
L. I. Nagy ◽  
L. Z. Fehér ◽  
G. J. Szebeni ◽  
M. Gyuris ◽  
P. Sipos ◽  
...  
Keyword(s):  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Great Promise ◽  
Human Leukemia ◽  
Human Leukemia Cell Line ◽  
Apoptotic Effects

Combination therapy of bortezomib with other chemotherapeutics is an emerging treatment strategy. Since both curcumin and bortezomib inhibit NF-κB, we tested the effects of their combination on leukemia cells. To improve potency, a novel Mannich-type curcumin derivative, C-150, was synthesized. Curcumin and its analogue showed potent antiproliferative and apoptotic effects on the human leukemia cell line, HL60, with different potency but similar additive properties with bortezomib. Additive antiproliferative effects were correlated well with LPS-induced NF-κB inhibition results. Gene expression data on cell cycle and apoptosis related genes, obtained by high-throughput QPCR, showed that curcumin and its analogue act through similar signaling pathways. In correlation with in vitro results similar additive effect could be obsereved in SCID mice inoculated systemically with HL60 cells. C-150 in a liposomal formulation given intravenously in combination with bortezomib was more efficient than either of the drugs alone. As our novel curcumin analogue exerted anticancer effects in leukemic cells at submicromolar concentration in vitro and at 3 mg/kg dose in vivo, which was potentiated by bortezomib, it holds a great promise as a future therapeutic agent in the treatment of leukemia alone or in combination.

ARC Is Regulated by MAPK and PI3K and Confers Drug Resistance and Survival Advantage to AML in Vitro and in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 893-893
Author(s):  
Po Yee Mak ◽  
Duncan H Mak ◽  
Yuexi Shi ◽  
Vivian Ruvolo ◽  
Rodrigo Jacamo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Apoptosis Resistance ◽  
Control Mscs ◽  
Extrinsic Apoptosis ◽  
Induced Apoptosis ◽  
And Control

Abstract Abstract 893 ARC (Apoptosis repressor with caspase recruitment domain) is a unique antiapoptotic protein that has been shown to suppress the activation of both intrinsic and extrinsic apoptosis. We previously reported that ARC is one of the most potent adverse prognostic factors in AML and that high ARC protein expression predicted shorter survival and poor clinical outcome in patients with AML (Carter BZ et al., Blood 2011). Here we report how ARC is regulated and its role in inhibition of AML apoptosis and in cell survival. We provide evidence that ARC expression is regulated by MAPK and PI3K signaling. Inhibition of MAPK and PI3K pathways decreased ARC mRNA and protein levels in AML cells. ARC expression in AML cells is upregulated in co-cultures with bone marrow-derived mesenchymal stromal cells (MSCs) and the upregulation is suppressed in the presence of MAPK or PI3K inhibitors. To investigate the role of ARC in apoptosis resistance in AML, we generated stable ARC overexpressing (O/E) KG-1 and stable ARC knock down (K/D) OCI-AML3 and Molm13 cells and treated them with Ara-C and agents selectively inducing intrinsic (ABT-737) or extrinsic (TRAIL) apoptosis. We found that ARC O/E cells are more resistant and ARC K/D cells more sensitive to Ara-C, ABT-737, and TRAIL-induced apoptosis: EC50s of Ara-C, ABT-737, or TRAIL treatment at 48 hours for ARC O/E KG-1 and control cells were 1.5 ± 0.1 μM vs. 83.5 ± 4.6 nM, 2.2 ± 0.2 μM vs. 60.2 ± 3.1 nM, or 0.97 ± 0.03 μg/mL vs. 0.17 ± 0.08 μg/mL, respectively and for ARC K/D OCI-AML3 and control cells were 0.33 ± 0.02 μM vs. 3.4 ± 0.2 μM, 0.24 ± 0.01 μM vs. 1.3 ± 0.1 μM, or 0.13 ± 0.09 μg/mL vs. 0.36 ± 0.03 μg/mL, respectively. Bone marrow microenvironment is known to play critical roles in AML disease progression and in protecting leukemia cells from various therapeutic agent-induced apoptosis. Leukemia cells were co-cultured with MSCs in vitro study to mimic the in vivo condition. ARC was found to be highly expressed in MSCs and stable ARC K/D MSCs were generated. AML cell lines and primary patient samples were co-cultured with ARC K/D or control MSCs and treated with Ara-C, ABT-737, or TRAIL. Interestingly, ARC K/D MSCs lost their protective activity for leukemia cells treated with these agents. EC50s for OCI-AML3 cells co-cultured with ARC K/D or control MSCs for 48 hours treated with Ara-C, ABT-737, or TRAIL were 1.0 ± 0.04 μM vs. 4.5 ± 0.2 μM, 0.15 ± 0.06 μM vs. 0.53 ± 0.02 μM, or 1.4 ± 0.8 μg/mL vs. 8.1 ± 0.3 μg/mL, respectively. In addition, ARC O/E KG-1 cells grew faster and ARC K/D OCI-AML3 and Molm13 cells and ARC K/D MSCs grew slower than their respective controls. We then injected KG-1 cells into mice and found that NOD-SCID mice harboring ARC O/E KG-1 had significantly shorter survival than mice injected with the vector control KG-1 (median 84 vs. 111 days) as shown in the figure. Collectively, results demonstrate that ARC plays critical roles in AML. ARC is regulated by MSCs through various signaling pathways in AML cells, protects leukemia cells from apoptosis induced by chemotherapy and by agents selectively inducing intrinsic and extrinsic apoptosis. ARC regulates leukemia cell growth in vitro and in vivo. The results suggest that ARC is a potential target for AML therapy. In addition, targeting ARC in MSCs suppresses microenvironmental protection of AML cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Leukocyte-endothelial adhesion molecules

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2068-2101 ◽  
Author(s):  
TM Carlos ◽  
JM Harlan
Keyword(s):  
Adhesion Molecules ◽  
Sufficient Information ◽  
Leukocyte Integrins ◽  
Endothelial Adhesion Molecules ◽  
And Migration ◽  
Definition Of ◽  
The Individual ◽  
Exciting Area

In the 9 years since the last review on leukocyte and endothelial interactions was published in this journal many of the critical structures involved in leukocyte adherence to and migration across endothelium have been elucidated. With the advent of cell and molecular biology approaches, investigations have progressed from the early descriptions by intravital microscopy and histology, to functional and immunologic characterization of adhesion molecules, and now to the development of genetically deficient animals and the first phase I trial of “anti-adhesion” therapy in humans. The molecular cloning and definition of the adhesive functions of the leukocyte integrins, endothelial members of the Ig gene superfamily, and the selectins has already provided sufficient information to construct an operative paradigm of the molecular basis of leukocyte emigration. The regulation of these adhesion molecules by chemoattractants, cytokines, or chemokines, and the interrelationships of adhesion pathways need to be examined in vitro and, particularly, in vivo. Additional studies are required to dissect the contribution of the individual adhesion molecules to leukocyte emigration in various models of inflammation or immune reaction. Certainly, new adhesion structures will be identified, and the current paradigm of leukocyte emigration will be refined. The promise of new insights into the biology and pathology of the inflammatory and immune response, and the potential for new therapies for a wide variety of diseases assures that this will continue to be an exciting area of investigation.

scholarly journals Proliferation and differentiation of human myeloid leukemic cells in immunodeficient mice: electron microscopy and cytochemistry

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 1015-1022 ◽  
Author(s):  
EA Machado ◽  
DA Gerard ◽  
CB Lozzio ◽  
BB Lozzio ◽  
JR Mitchell ◽  
...  
Keyword(s):  
Nude Mice ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Immunodeficient Mice ◽  
Human Leukemia Cells

Abstract To study the influence of a biologic environment on cultured human leukemia cells, KG-1, KG-1a, and HL-60 cells were inoculated subcutaneously into newborn nude mice. The cells developed myelosarcomas at the site of inoculation and in lungs and kidneys. KG-1 and HL-60 myelosarcomas were successfully passaged through adult nude mice, whereas KG-1a tumors proliferated only after transplantation into newborn hosts. The human nature of the cells forming myelosarcomas in mice was assessed by chromosomal analyses and detection of cross- reactivity with an antibody to the human leukemia cell line K562. We undertook electron microscopic and cytochemical examinations of the cells proliferating in vitro and in the mice. The granules of KG-1 cells in vivo did not react for acid phosphatase, as observed in vitro, and the HL-60 cells proliferating in mice lost the perinuclear myeloperoxidase (MPO) demonstrated in cultured cells. Although the influence of an in vivo selection of cell subpopulations cannot be ruled out, the enzymatic changes are compatible with induced cell differentiation. Conclusive evidence of differentiation in vivo was observed in the KG-1a cell subline. The undifferentiated KG-1a blasts developed cytoplasmic granules and synthesized MPO during proliferation in vivo. These observations indicate that human leukemia cells from established cell lines proliferate in nude mice and may acquire new differentiated properties in response to the in vivo environment.

Deregulated Expression of a Novel Oncogene, Ahi-1, in Murine Hematopoietic Cells Confers a Proliferative Advantage In Vitro and Leukemogenic Activity In Vivo and Enhances the Effects of BCR-ABL.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 388-388
Author(s):  
Leon Zhou ◽  
Ashley Ringrose ◽  
Ann E.-J. Lin ◽  
Xiaoyan Jiang
Keyword(s):  
Growth Factor ◽  
Protein Expression ◽  
Western Blot ◽  
Proliferative Activity ◽  
Integration Site ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Biological Behavior

Abstract Ahi-1 (Abelson helper integration site-1) is a novel gene that we recently identified based on its common activation in v-abl or myc-induced murine leukemias and lymphomas. It encodes a unique protein with known signaling features, including SH3 and WD40-repeat domains, but its function is largely unknown. We have recently demonstrated that Ahi-1/AHI-1 transcript levels are normally down-regulated during both early murine and human hematopoietic cell differentiation and are highly increased in human leukemic cells, particularly in highly enriched populations of primitive BCR-ABL+ leukemic stem cells (lin−CD34+CD38−) in patients with chronic myeloid leukemia (CML). To investigate the potential cooperative activity of Ahi-1 in BCR-ABL-mediated signal transduction and leukemogenesis, we transduced IL-3-dependent BaF3 cells with MSCV-Ahi-1-IRES-YFP and/or MSCV-BCR-ABL-IRES-GFP retroviruses and compared the biological behavior of these cells in vitro and in vivo. All Ahi-1-transduced clonal cell lines showed increased proliferative activity and reduced apoptosis in the absence of IL-3, compared to parental BaF3 cells or control GFP-transduced cells. Interestingly, overexpression of both Ahi-1 and BCR-ABL caused more enhanced perturbations when compared to cells transduced with either Ahi-1 or BCR-ABL alone. Strikingly, intravenous injection of NOD/SCID-β2microglobulin−/−mice with BaF3 cells induced to overexpress Ahi-1 alone induces a lethal leukemia within 70 days. These leukemogenic activities were further increased by introduction of co-transduced Ahi-1 and BCR-ABL cells, producing a shorter latency of 30 days. A disease latency of 40 days was revealed by introduction of BCR-ABL-transduced cells alone. Western blot analysis showed that both protein expression and the tyrosine kinase activity of p210BCR-ABL were highly increased in cells co-transduced with Ahi-1 and BCR-ABL compared to BaF3 cells transduced with BCR-ABL alone. Similarly, we also observed higher levels of Ahi-1 protein expression in the same dually transduced cells (Ahi-1+BCR-ABL+) than in those transduced with Ahi-1 alone. We further demonstrated a similarly perturbed proliferative activity, growth factor independence and colony-forming cell (CFC) output in long-term culture initiated cell (LTC-IC) assays of 5-fluorouracil (5-FU)-treated primitive murine BM cells (Lin−Sca1+ cells) transduced with Ahi-1 and BCR-ABL, either alone or in combination. To investigate directly the cooperating oncogenic role of AHI-1 in BCR-ABL-mediated malignant transformation of CML cells, knockdown of AHI-1 expression in K562 cells, a cell line that was derived from a patient with CML and that is characterized by highly increased expression of AHI-1, was performed using retroviral-mediated RNA interference. Retroviral-mediated suppression specifically inhibited endogenous AHI-1 expression in transduced cells by 70% as evaluated by Q-RT-PCR and Western blot analyses. It further caused a significant reduction in their growth factor independence in semi-solid cultures (up to 5-fold) and in single cell cultures (2-fold) by comparison to cells transduced with a control vector. Taken together, these findings provide strong evidence of the transforming potential of Ahi-1/AHI-1 in primitive hematopoietic cells. This effect is additive with those of BCR-ABL, suggesting that AHI-1 and BCR-ABL can play a cooperate role in the development of BCR-ABL-associated diseases like CML.

The Dual E-Selectin/CXCR4 Inhibitor, GMI-1359, Enhances Efficacy of Anti-Leukemia Chemotherapy in FLT3-ITD Mutated Acute Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3790-3790 ◽  
Author(s):  
Weiguo Zhang ◽  
Nalini Patel ◽  
William E. Fogler ◽  
John L. Magnani ◽  
Michael Andreeff
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemic Cell ◽  
Cxcr4 Expression ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Acute Myeloid ◽  
Cells Cultured

Abstract Aberrant activation of the FMS-like tyrosine kinase-3 (FLT3) is driven by internal tandem duplication (ITD) mutations in the FLT3 gene, which are commonly observed in patients with acute myeloid leukemia (AML). Hence, FLT3 represents an attractive therapeutic target in AML (Weisberg et al., 2002). Indeed, several small molecule FLT3 inhibitors including sorafenib have showed encouraging efficacy in reducing leukemia blasts in the peripheral blood in FLT3 mutated AML patients. However, these agents have little effect on leukemic stem cells in the bone marrow (BM) microenvironment (Borthakur et al., 2011; Fathi and Chabner, 2011; Zhang et al., 2008). The BM microenvironment is enriched with cytokines and adhesion molecules, such as CXCR4 and E-selectin, which are believed to provide AML cells protection against chemotherapeutic agents (Horacek et al., 2013; Peled and Tavor, 2013). In fact, treatment with sorafenib markedly upregulated CXCR4 levels in FLT3 -mutated cells. In addition, leukemia cells can activate endothelial cells (EC) that induce adhesion of a sub-set of the leukemia cells through E-selectin. The adherent AML cells are sequestered in a nonproliferative state that further protects them from chemotherapy (Pezeshkian et al., 2013). Therefore, blocking CXCR4 and E-selectin in parallel could theoretically eliminate the protection provided by the interaction of leukemic cells with their BM microenvironment and enhance effectiveness of chemotherapy in FLT3-mutant AML patients. In the present study, we evaluated the effectiveness of a dual CXCR4 and E-selectin antagonist, GMI-1359 (GlycoMimetics, Inc., Rockville, MD), in targeting FLT3-ITD-mutant AML in vitro and in vivo. High levels of CXCR4 expression were observed in several human and murine AML cell lines, which was further increased in hypoxic (i.e., 1% oxygen) conditions that mimic the BM microenvironment. These FLT3 -ITD leukemic cell lines also expressed hypoxia-responsive, functional E-selectin ligands identified by reactivity with an antibody (HECA452) that binds the same carbohydrate epitope required for binding to E-selectin. One such E-selectin ligand CD44 increased in FLT3 -ITD cells cultured in hypoxia compared to those cultured in normoxia (i.e. 21% oxygen). In addition, hypoxia also enhanced CXCR4 expression on mesenchymal stem cells (MSC) and EC such as HUVEC. In hypoxic co-cultures of the FLT3 -ITD-mutant leukemia cells MV4-11 or MOLM14 with MSCs and ECs (i.e., HUVEC or TeloHAEC), the presence of the dual E-selectin/CXCR4 inhibitor GMI-1359 effectively reduced leukemic cell adhesion by ~ 50% to the MSC/EC feeder layer compared to the PBS-treated control (p<0.05), even in the presence of TNFa, which induces E-selectin expression in EC. However, an E-selectin specific inhibitor only reduced adhesion of MV4-11 and MOLM14 by ~ 20%. GMI-1359 markedly abrogated the protection provided by the BM microenvironment (i.e., hypoxia and/or MSC and EC) of Baf3-FLT3 -ITD leukemic cells treated with the FLT3 inhibitor sorafenib. Apoptosis was induced in 36.6%, 35.6% and 48.9% of leukemic cells cultured with sorafenib alone, sorafenib and an E-selectin inhibitor or sorafenib and GMI-1359, respectively. The significance of these in vitro findings were studied in vivo. Female SCID beige mice were injected iv with MV4-11 and followed for survival. Beginning 14 days post tumor injection, cohorts of mice (n=10/group) were treated with saline, GMI-1359 (40 mg/kg), standard chemotherapy cytarabine plus daunorubicin, or a combination of GMI-1359 and chemotherapy. Combined treatment of mice with GMI-1359 (40 mg/kg) and chemotherapy demonstrated a profound survival benefit compared to controls or chemotherapy alone at day 135 after leukemia cell injection (i.e., 67% vs. 11% or 30%, p=0.0011 and 0.0406, respectively). Single agent treatment with GMI-1359 was statistically indistinguishable from saline alone or chemotherapy alone. In a separate cohort of MV4.11-engrafted mice, the single administration of GMI-1359 increased circulating WBC and leukemic MV4-11cells, which persisted for at least 8 hrs. This effect was consistent with GMI-1359 disrupting the protective effects of the tumor microenvironment and mobilizing MV4-11 cells from the BM niche.. These findings provide the pre-clinical basis for the evaluation of GMI-1359 in patients with FLT3 -mutant AML. Figure 1. Figure 1. Disclosures Zhang: Karyopharm: Research Funding. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Differentiation arrest and stromal cell-independent growth of murine erythroleukemia cells are associated with elevated expression of ets-related genes but not with mutation of p53.

1993 ◽  
Vol 13 (9) ◽  
pp. 5582-5592 ◽  
Author(s):  
R J Nibbs ◽  
K Itoh ◽  
W Ostertag ◽  
P R Harrison
Keyword(s):  
Stromal Cells ◽  
Stromal Cell ◽  
Gene Activation ◽  
Leukemic Cells ◽  
Term Survival ◽  
Elevated Expression ◽  
D Cells ◽  
Murine Erythroleukemia

The ELM erythroleukemia is novel in that long-term survival of leukemic cells in culture (ELM-D cells) is dependent on contact with a bone marrow-derived stromal feeder cell layer. However, a number of stroma-independent (ELM-I) mutants that vary in their ability to differentiate in vitro in response to erythropoietin and interleukin-3 have been derived. We have attempted to define the genetic changes responsible for these different phenotypes. At the p53 locus in the primary leukemic cells, one copy of the gene has been lost whereas the other contains an 18-bp depletion, implicating its mutation as an early step in the development of the leukemia. Changes in ets gene expression have also been found. The Fli-1 gene region is rearranged in the primary tumor because of the insertion of a retrovirus inserted upstream of one Fli-1 allele, but this does not result in Fli-1 gene activation in any of the ELM-D or ELM-I cell lines except one. It seems significant that this line is the only one to have lost the ability to differentiate in response to erythropoietin. In addition, up-regulation of erg is associated with stromal cell-independent growth, since all ELM-I mutants have moderate levels of erg mRNA, whereas only low or undetectable levels are found in primary leukemic cells in vivo or in ELM-D cells in vitro. This up-regulation of erg mRNA seems to be important for stromal cell-independent growth, since ELM-D cells show elevated expression of the erg gene after separation from stromal cells. This seems to be made permanent in ELM-I mutants, since they do not down-regulate erg mRNA when grown in contact with stromal cells. We therefore propose that ets family members regulate both the survival and differentiation of erythroid cells.

scholarly journals Leukocyte-endothelial adhesion molecules

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2068-2101 ◽  
Author(s):  
TM Carlos ◽  
JM Harlan
Keyword(s):  
Adhesion Molecules ◽  
Sufficient Information ◽  
Leukocyte Integrins ◽  
Endothelial Adhesion Molecules ◽  
And Migration ◽  
Definition Of ◽  
The Individual ◽  
Exciting Area

Abstract In the 9 years since the last review on leukocyte and endothelial interactions was published in this journal many of the critical structures involved in leukocyte adherence to and migration across endothelium have been elucidated. With the advent of cell and molecular biology approaches, investigations have progressed from the early descriptions by intravital microscopy and histology, to functional and immunologic characterization of adhesion molecules, and now to the development of genetically deficient animals and the first phase I trial of “anti-adhesion” therapy in humans. The molecular cloning and definition of the adhesive functions of the leukocyte integrins, endothelial members of the Ig gene superfamily, and the selectins has already provided sufficient information to construct an operative paradigm of the molecular basis of leukocyte emigration. The regulation of these adhesion molecules by chemoattractants, cytokines, or chemokines, and the interrelationships of adhesion pathways need to be examined in vitro and, particularly, in vivo. Additional studies are required to dissect the contribution of the individual adhesion molecules to leukocyte emigration in various models of inflammation or immune reaction. Certainly, new adhesion structures will be identified, and the current paradigm of leukocyte emigration will be refined. The promise of new insights into the biology and pathology of the inflammatory and immune response, and the potential for new therapies for a wide variety of diseases assures that this will continue to be an exciting area of investigation.

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