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1
Raje, Manoj, and Karvita B. Ahluwalia. "Motility of leukemic lymphocytes." Proceedings, annual meeting, Electron Microscopy Society of America 48, no. 3 (August 12, 1990): 368–69. http://dx.doi.org/10.1017/s0424820100159382.
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In Acute Lymphocytic Leukemia motility of lymphocytes is associated with dissemination of malignancy and establishment of metastatic foci. Normal and leukemic lymphocytes in circulation reach solid tissues where due to in adequate perfusion some cells get trapped among tissue spaces. Although normal lymphocytes reenter into circulation leukemic lymphocytes are thought to remain entrapped owing to reduced mobility and form secondary metastasis. Cell surface, transmembrane interactions, cytoskeleton and level of cell differentiation are implicated in lymphocyte mobility. An attempt has been made to correlate ultrastructural information with quantitative data obtained by Laser Doppler Velocimetry (LDV). TEM of normal & leukemic lymphocytes revealed heterogeneity in cell populations ranging from well differentiated (Fig. 1) to poorly differentiated cells (Fig. 2). Unlike other cells, surface extensions in differentiated lymphocytes appear to originate by extrusion of large vesicles in to extra cellular space (Fig. 3). This results in persistent unevenness on lymphocyte surface which occurs due to a phenomenon different from that producing surface extensions in other cells.
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Liu, Hsiao-Chuan, Eun Ji Gang, Hye Na Kim, Yongsheng Ruan, Heather Ogana, Zesheng Wan, Halvard Bönig, K. Kirk Shung, and Yong-Mi Kim. "Characterizing the Motility of Chemotherapeutics-Treated Acute Lymphoblastic Leukemia Cells by Time-Lapse Imaging." Cells 9, no. 6 (June 16, 2020): 1470. http://dx.doi.org/10.3390/cells9061470.
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Drug resistance is an obstacle in the therapy of acute lymphoblastic leukemia (ALL). Whether the physical properties such as the motility of the cells contribute to the survival of ALL cells after drug treatment has recently been of increasing interest, as they could potentially allow the metastasis of solid tumor cells and the migration of leukemia cells. We hypothesized that chemotherapeutic treatment may alter these physical cellular properties. To investigate the motility of chemotherapeutics-treated B-cell ALL (B-ALL) cells, patient-derived B-ALL cells were treated with chemotherapy for 7 days and left for 12 h without chemotherapeutic treatment. Two parameters of motility were studied, velocity and migration distance, using a time-lapse imaging system. The study revealed that compared to non-chemotherapeutically treated B-ALL cells, B-ALL cells that survived chemotherapy treatment after 7 days showed reduced motility. We had previously shown that Tysabri and P5G10, antibodies against the adhesion molecules integrins α4 and α6, respectively, may overcome drug resistance mediated through leukemia cell adhesion to bone marrow stromal cells. Therefore, we tested the effect of integrin α4 or α6 blockade on the motility of chemotherapeutics-treated ALL cells. Only integrin α4 blockade decreased the motility and velocity of two chemotherapeutics-treated ALL cell lines. Interestingly, integrin α6 blockade did not affect the velocity of chemoresistant ALL cells. This study explores the physical properties of the movements of chemoresistant B-ALL cells and highlights a potential link to integrins. Further studies to investigate the underlying mechanism are warranted.
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Okabe-Kado, Junko, Takashi Kasukabe, Yoshio Honma, and Yasuhiko Kaneko. "Inverse Correlation of NM23 Expression with Lysophosphatidic Acid Receptor EDG2/lpa1 Expression of Human Leukemia Cells during Myeloid Differentiation Induced by All-Trans Retinoic Acid." Blood 112, no. 11 (November 16, 2008): 4490. http://dx.doi.org/10.1182/blood.v112.11.4490.4490.
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Abstract Overexpression of the nm23 gene is found in many hematological malignancies, and predicts poor treatment outcome. Because the amount of intracellular NM23-H1 protein is inversely correlated with differentiation and exogenous over expression of the nm23-H1 in myeloid leukemia cells reduced the sensitivity to induction of myeloid differentiation, nm23-overexpression is considered to function as a differentiation-suppressor. However, the molecular mechanism of this phenomenon is unknown. Recently, lysophosphatidic acid (LPA) receptor EDG2/lpa1 was identified as a gene down-regulated by nm23-H1 and associated with cell motility/metastasis suppressor functions in breast cancer cells. Although the clinical significance of nm23-H1 overexpression in leukemia is completely opposite to that in breast cancer, there might be a common molecular mechanism between the metastasis-suppressing activity and differentiation-inhibiting activity of nm23-H1 overexpression, because many functional characteristics of macrophages/neutrophils are similar to those of motile and metastatic tumor cells. Here, we examined the EDG2/lpa1 expression level and its association with NM23 expression level and myeloid differentiation of leukemia cells. EDG2 and NM23 proteins were expressed at different levels in all leukemia cells tested (HL-60, NB4, THP-1, U937, K562, HEL, BALM-1, BALM-3, MOLT4, Jurkat). During all-trans retinoic acid (ATRA)-induced myeloid differentiation of HL-60 cells, the NM23 expression decreased and EDG2 expression inversely increased. Similar effects of ATRA were observed in myeloid differentiation of NB4 and THP-1 cells. NM23 expression levels and EDG2 expression levels modulated by ATRA treatment were inversely correlated (Spearman’s correlation coefficient, rs = −0.7538, p = 0.0237). However, these correlations were not found in the erythroid differentiation of ATRA-treated HEL and K562 cells. Cell motility is required for myeloid differentiation; therefore, there might be a common mechanism, the inhibition of cell motility thorough EDG2 down-regulation by NM23-H1 overexpression, between differentiation-inhibiting activity in leukemia cells and motility-inhibiting activity in breast cancer cells. The discrepancy in the clinical significance of NM23-H1 overexpression between leukemia and breast cancer might be resolved.
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Resar, Linda, Joelle Hillion, Surajit Dhara, Takita Felder Sumter, Mita Mukherjee, Francescopaolo Di Cello, Amy Belton, et al. "The HMGA1a-STAT3 axis: an “Achilles Heel” for Hematopoietic Malignancies Overexpressing HMGA1a?" Blood 112, no. 11 (November 16, 2008): 3810. http://dx.doi.org/10.1182/blood.v112.11.3810.3810.
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Abstract Although the high mobility group A1 (HMGA1) oncogene is widely overexpressed in high-risk hematopoietic malignancies and other aggressive cancers, the molecular mechanisms underlying transformation by HMGA1 are only beginning to emerge. The HMGA1 gene encodes the HMGA1a and HMGA1b protein isoforms, which function in regulating gene expression. We showed that HMGA1 induces leukemic transformation in cultured human lymphoid cells. Inhibiting HMGA1 expression blocks the transformed phenotype in cultured human leukemia and lymphoma cells. We also engineered HMGA1a transgenic mice and all mice develop aggressive lymphoid malignancy which closely models human T-cell acute lymphoblastic leukemia. Because HMGA1 participates in transcriptional regulation, we hypothesize that it drives leukemic transformation by dysregulating specific molecular pathways. To discover genes targeted by HMGA1 in leukemic transformation, we performed gene expression profile analysis. The signaltransducer andactivator oftranscription 3 (STAT3) gene was identified as a critical downstream target of HMGA1. STAT3 mRNA and protein are up-regulated in leukemic cells overexpressing HMGA1a and activated STAT3 recapitulates the transforming activity of HMGA1a. HMGA1a binds directly to a conserved region of the STAT3 promoter in vivo and activates transcription of the STAT3 promoter in human leukemia cells. Blocking STAT3 function with a small molecule, platinum compound inhibitor (CPA-7) induces apoptosis in leukemic cells from HMGA1 transgenic mice, but not in control cells. In primary, human leukemia samples, there is a positive correlation between HMGA1a and STAT3 mRNA. Moreover, blocking STAT3 function with a dominant-negative construct in human leukemia or lymphoma cells leads to decreased cellular motility and colony formation. We also showed that treatment with a small molecule, oligonucleotide inhibitor decreases the leukemic burden in the HMGA1a transgenic mice. Our results demonstrate that the HMGA1a-STAT3 axis is a potential “Achilles heel” that could be exploited therapeutically in selected hematopoietic malignancies.
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Schwieger, Maike, Andrea Schüler, Martin Forster, Afra Engelmann, Michael A. Arnold, Ruud Delwel, Peter J. Valk, et al. "Homing and invasiveness of MLL/ENL leukemic cells is regulated by MEF2C." Blood 114, no. 12 (September 17, 2009): 2476–88. http://dx.doi.org/10.1182/blood-2008-05-158196.
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Abstract Acute myelogenous leukemia is driven by leukemic stem cells (LSCs) generated by mutations that confer (or maintain) self-renewal potential coupled to an aberrant differentiation program. Using retroviral mutagenesis, we identified genes that generate LSCs in collaboration with genetic disruption of the gene encoding interferon response factor 8 (Irf8), which induces a myeloproliferation in vivo. Among the targeted genes, we identified Mef2c, encoding a MCM1-agamous-deficiens-serum response factor transcription factor, and confirmed that overexpression induced a myelomonocytic leukemia in cooperation with Irf8 deficiency. Strikingly, several of the genes identified in our screen have been reported to be up-regulated in the mixed-lineage leukemia (MLL) subtype. High MEF2C expression levels were confirmed in acute myelogenous leukemia patient samples with MLL gene disruptions, prompting an investigation of the causal interplay. Using a conditional mouse strain, we demonstrated that Mef2c deficiency does not impair the establishment or maintenance of LSCs generated in vitro by MLL/ENL fusion proteins; however, its loss led to compromised homing and invasiveness of the tumor cells. Mef2c-dependent targets included several genes encoding matrix metalloproteinases and chemokine ligands and receptors, providing a mechanistic link to increased homing and motility. Thus, MEF2C up-regulation may be responsible for the aggressive nature of this leukemia subtype.
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Hogeman, P. H. G., A. J. P. Veerman, D. R. Huismans, C. H. van Zantwijk, and P. D. Bezemer. "Motility of Leukemic Cells in Collagen Gel Related to Immunological Phenotype in Childhood Acute Lymphoblastic Leukemia." Acta Haematologica 78, no. 4 (1987): 229–32. http://dx.doi.org/10.1159/000205883.
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Kornblau, Steven M., Andrew Pierce, Stefan Meyer, Farhad Ravandi, Gautam Borthakur, Kevin R. Coombes, Nianxiang Zhang, and Anthony Whetton. "Transglutaminase2 Expression in Acute Myeloid Leukemia: Association with Adhesion Molecule Expression and Leukemic Blast Motility." Blood 120, no. 21 (November 16, 2012): 1427. http://dx.doi.org/10.1182/blood.v120.21.1427.1427.
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Abstract Abstract 1427 Background: In prior proteomic analysis on Acute myeloid leukemia (AML) cell lines evaluating the effects of several leukemogenic oncogenes we observed that transglutaminase2 (TG2) was expressed at greater levels as a consequence of oncogenic transformation. TG2 is a multi-domain, multi-functional enzyme with diverse biological functions, including extracellular matrix formation, integrin-mediated signalling, and signal transduction. It's normal roles remain obscure, but it is linked to the pathogenesis of celiac sprue, neurodegenerative diseases, and some cancers. In malignancy it is reported to be an anti-apoptotic mediator of hypoxia inducible factor (HIF) conferring a growth advantage to tumor cells. Expression has been associated with resistance to chemotherapy and apoptosis. We therefore assess the expression of TG2 protein in primary AML patient samples. METHODS: We analyzed 511 AML samples from patients with newly diagnosed AML using a custom made reverse phase proteomic array. This array included 11 normal bone marrow derived CD34+ samples as controls and had 140 paired same day blood and marrow samples and 49 paired diagnosis and relapse samples. The array was probed with antibodies against 203 targets including TG2 (Abcam, ab2386, UK). Supercurve algorithms were used to generate a single value from the five serial dilutions. Loading control and topographical normalization procedures accounted for protein concentration and background staining variations. RESULTS: Expression of TG2 was statistically similar (p= 0.43) in paired blood and marrow samples and in protein prepared from fresh cells or from cryopreserved cells (p= 0.71). Expression was above, equal to or below that of normal CD34+ cells in 12%, 62%, and 27% of patients. Levels were significantly higher at relapse compared to diagnosis in the 49 paired samples (p = 0.003). Levels were higher in FAB M6 and M7 (P =<0.00001 and < 0.008) and lower in patients with inversion16. Higher TG2 expression was strongly inversely correlated with total WBC (r=.035, p < 0.0001) and the absolute blood blast count (r = −.30, p <0.0001). Patients with higher TG2 level had a shorter but not statistically significant overall survival in the entire cohort, and was not prognostic in subsets stratified based on cytogenetics or mutations (FLT3, NPM1, RAS). Likewise, patients with higher TG2 levels had shorter remission durations, but again this was not significant in the entire cohort or in subsets. Expression of TG2 was significantly correlated with 55 of 203 proteins. Notable among these were numerous integrin and adhesion proteins. Hierarchical clustering of these demonstrated that AML is characterized by two large cohorts, one in which TG2 is elevated and is positively correlated with CD49B, Integrinβ3, FAK, Fibronectin and IGFB2, a second in which TG2 is low and negatively correlated with high expression of Osteopontin, CD11 and, CD44 and a 3rd in which only Caveolin1 is expressed. A Cytoscape interaction plot based on online databases of known protein-protein interactions revealed that TG2 has known interactions with Fibronectin, which it binds and post-translationally modifies, and integrinβ3. In combination this suggests that there is canonical interaction between TG2 and integrin and adhesion proteins active in AML. TG2 expression also correlated positively with numerous anti-apotptosis proteins. CONCLUSION: TG2 is expressed in the majority of cases of AML at levels comparable to normal bone marrow CD34+ cells and levels became significantly higher at relapse suggesting that the protein expression signature associated with high TG2 levels may be selected for, or confer a subtle survival advantage to leukemic blasts. In support of this, while the level of TG2 was not statistically significantly prognostic for either overall survival or remission duration, patents with higher levels were somewhat more likely to relapse, and less likely to be alive beyond 3 years. TG2 has previously been linked to drug resistance in cancer and given the negative correlation between TG2 levels and peripheral blasts observed increased TG2 levels may lead to the protection of the leukemic stem cell due to increased adhesion/reduced motility. TG2 may therefore form part of a network of proteins that define poor outcome in AML patients and potentially offer a target to sensitize AML stem cells to drug treatment. Disclosures: Off Label Use: Clofarabine in AML.
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Casalegno-Garduño, R., C. Meier, A. Schmitt, A. Spitschak, I. Hilgendorf, S. Rohde, C. Hirt, M. Freund, B. M. Pützer, and M. Schmitt. "Immune Responses to RHAMM in Patients with Acute Myeloid Leukemia after Chemotherapy and Allogeneic Stem Cell Transplantation." Clinical and Developmental Immunology 2012 (2012): 1–9. http://dx.doi.org/10.1155/2012/146463.
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Leukemic blasts overexpress immunogenic antigens, so-called leukemia-associated antigens like the receptor for hyaluronan acid-mediated motility (RHAMM). Persistent RHAMM expression and decreasing CD8+T-cell responses to RHAMM in the framework of allogeneic stem cell transplantation or chemotherapy alone might indicate the immune escape of leukemia cells. In the present study, we analyzed the expression of RHAMM in 48 patients suffering from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Furthermore, we correlated transcripts with the clinical course of the disease before and after treatment. Real-time quantitative reverse transcriptase polymerase chain reaction was performed from RNA of peripheral blood mononuclear cells. T cell responses against RHAMM were assessed by tetramer staining (flow cytometry) and enzyme-linked immunospot (ELISPOT) assays. Results were correlated with the clinical outcome of patients. The results of the present study showed that almost 60% of the patients were RHAMM positive; specific T-cells recognizing RHAMM could be detected, but they were nonfunctional in terms of interferon gamma or granzyme B release as demonstrated by ELISPOT assays. Immunotherapies like peptide vaccination or adoptive transfer of RHAMM-specific T cells might improve the immune response and the outcome of AML/MDS patients.
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Ikeyama, S., M. Koyama, M. Yamaoko, R. Sasada, and M. Miyake. "Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP-1/CD9) DNA." Journal of Experimental Medicine 177, no. 5 (May 1, 1993): 1231–37. http://dx.doi.org/10.1084/jem.177.5.1231.
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Previously we showed that motility-related protein (MRP-1) is an antigen recognized by monoclonal antibody (mAb) M31-15 inhibiting cell motility and that the sequence of MRP-1 coincides with that of CD9. In the present study, plasmid was constructed in which human MRP-1/CD9 cDNA is expressed under the control of the Abelson murine leukemia virus promoter sequence. The expression plasmid for MRP-1/CD9 was introduced into Chinese hamster ovary cells, human lung adenocarcinoma cell line MAC10 (MRP-1 positive), and human myeloma cell line ARH77 (MRP-1 negative). All of the MRP-1/CD9 (over)expressing clones obtained from these transfected cells showed suppressed cell motility (penetration and phagokinetic track assays) depending on the degree of expression of MRP-1/CD9. Overexpression of MRP-1/CD9 by MAC10 cells resulted in the suppression of cell motility (maximally 73%) associated with considerable inhibition of the cell growth (maximally 48%). However, the inhibition of the growth of MAC10 cells by mAb M31-15 was < 17% at an antibody concentration of 1-5 micrograms/ml, which inhibits cell motility by > 90%. These results suggest that MRP-1/CD9 directly regulates cell motility and may also affect cell growth. Effects on metastasis by the expression of MRP-1 CD9 were investigated with mouse melanoma BL6 cells-BALB/c nu/nu mouse system. Metastatic potential of all transformants expressing MRP-1/CD9 was lower than that of parent BL6 cells.
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Tavor, Sigal, Isabelle Petit, Svetlana Porozov, Polina Goichberg, Abraham Avigdor, Sari Sagiv, Arnon Nagler, Elizabeth Naparstek, and Tsvee Lapidot. "Motility, proliferation, and egress to the circulation of human AML cells are elastase dependent in NOD/SCID chimeric mice." Blood 106, no. 6 (September 15, 2005): 2120–27. http://dx.doi.org/10.1182/blood-2004-12-4969.
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Abstract The role of the proteolytic enzyme elastase in motility and proliferation of leukemic human acute myeloblastic leukemia (AML) cells is currently unknown. We report a correlation between abnormally high levels of elastase in the blood of AML patients and the number of leukemic blast cells in the circulation. In AML cells, we observed expression of cell-surface elastase, which was regulated by the chemokine stromal cell-derived factor-1 (SDF-1). In vitro inhibition of elastase prevented SDF-1-induced cell polarization, podia formation, and reduced migration of human AML cells as well as their adhesion. Elastase inhibition also significantly impaired in vivo homing of most human AML cells to the bone marrow (BM) of nonobese diabetic-severe combined immunodeficient (NOD/SCID)/beta-2 microglobulin knock-out (B2mnull) mice that underwent transplantation. Moreover, in vitro proliferation of AML cells was elastase dependent. In contrast, treatment with elastase inhibitor enhanced the proliferation rate of human cord blood CD34+ cells, including primitive CD34+/CD38- cells, and their in vivo homing. Finally, NOD/SCID mice previously engrafted with human AML cells and treated with elastase inhibitor had significantly reduced egress of leukemic cells into the circulation. Taken together, our data demonstrate that human AML cells constitutively secrete and express SDF-1-dependent cell-surface elastase, which regulates their migration and proliferation. (Blood. 2005;106:2120-2127)
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Riches, John C., Conor J. O’Donovan, Sarah J. Kingdon, Fabienne McClanahan, Andrew J. Clear, Donna S. Neuberg, Lillian Werner, et al. "Trisomy 12 chronic lymphocytic leukemia cells exhibit upregulation of integrin signaling that is modulated by NOTCH1 mutations." Blood 123, no. 26 (June 26, 2014): 4101–10. http://dx.doi.org/10.1182/blood-2014-01-552307.
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Key Points Trisomy 12 CLL cells exhibit upregulated integrin signaling and enhanced VLA-4-directed adhesion and motility. The increased expression of β2-integrins on trisomy 12 CLL cells is modulated by intercurrent NOTCH1 mutations.
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Dander, Erica, Federica Portale, Daniela Silvestri, Giulia Cricrì, Silvia Bresolin, Stefania Gaspari, Barbara Russo, et al. "Activin A, a Potential Key Factor of the Malignant Bone Marrow Niche, Enhances B-Cell Precursor-Acute Lymphoblastic Leukemic Cell Migratory and Invasive Properties." Blood 132, Supplement 1 (November 29, 2018): 1296. http://dx.doi.org/10.1182/blood-2018-99-116668.
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Abstract The bone marrow (BM) represents a peculiar microenvironment characterized by a high concentration of growth factors and cytokines necessary for hematopoiesis, that make it a sanctuary for leukemic cell homing, survival and proliferation. B-cell precursor-Acute Lymphoblastic Leukemia (BCP-ALL) reprogram the BM stroma to create a leukemia-supporting and chemoprotective niche. Strategies to modulate the microenvironment could offer new approaches for anti-leukemia therapies. We identified ActivinA, a TGF-β family member, with a well-described promoting role in several solid malignancies, as a new potentially targetable leukemia-favoring factor. ActivinA resulted overexpressed in the BM plasma of 108 BCP-ALL pediatric patients compared to 44 Healthy Donors (HDs), as evaluated by ELISA assay. Upon in vitro culture, primary BCP-ALL cells significantly increased ActivinA secretion by mesenchymal stromal cells (MSCs) derived from HD BM. Interestingly this effect was achieved both by cell-to-cell contact-mediated mechanisms (direct contact) and by soluble factor release (transwell-mediated co-culture). Interestingly, MSCs isolated from the BM of leukemic patients showed an intrinsic ability to secrete higher amounts of ActivinA (mean=983.6±362.9 pg/mL), compared to their normal counterpart (mean=218.5±31.99 pg/mL). In addition, we demonstrated that the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α are increased in the leukemic BM and that they were able to synergize with leukemic blasts in inducing ActivinA release by MSCs (>100 fold increase compared to basal production). Both type I and type II Activin receptors were found to be expressed by leukemic cells as demonstrated by flow cytometry analysis of ALK4, ACVR2A and ACVR2B receptors and western blot analysis of ALK2. Of note, ActivinA exposure could increase the mRNA expression levels of type I receptors ALK2 and ALK4, thus suggesting a possible self-reinforcing mechanism. Gene expression analysis of ActivinA-treated BCP-ALL cells showed that this protein was able to significantly influence motility-associated molecular pathways. Accordingly, time lapse microscopy analyses revealed that ActivinA significantly increased random motility of leukemic cells (p<0.0001). In addition, ActivinA was able to almost double the migration of BCP-ALL primary cells in response to CXCL12, as demonstrated by in vitro chemotaxis assays. The specificity of the observed effect was demonstrated by using the ALK4 specific inhibitor SB431542. CXCL12 reduction is one of the typical microenvironmental alterations occurring in the leukemic BM. In line with literature data, we showed 6-fold decrese of CXCL12 levels in the BM of BCP-ALL patients compared to HDs (p<0.0001). Dose-responses chemotaxis experiments revealed that ActivinA was able to sensitize leukemic cells to suboptimal CXCL12 concentrations. On the other site, ActivinA exerted an opposite effect on CD34+ cells isolated both from HD cord blood or BM. In detail, in HD-CD34+ cells ActivinA severely impaired CXCL12-induced migration (p<0.0001). This opposite effect could be explained by ActivinA ability to increase free cytosolic calcium only in leukemic cells, both basally and after addition of CXCL12 (flow cytometry analysis of Fluo-4 NW stained cells). Of note, calcium levels of HD-CD34+ cells resulted unaffected or even decreased (in 2 out of 3 experiments performed) by ActivinA treatment. Since calcium is critically involved in boosting cytoskeleton dynamics, we analysed, by flow cytometry, actin polymerization in phalloidin stained BCP-ALL cells. Interestingly, ActivinA treatment of BCP-ALL cells significantly increased the rate of conversion of globular into filamentous actin, which is a prerequisite of site-directed migration, as soon as CXCL12 was added. Moreover, ActivinA resulted a leukemia-promoting factor: protein treatment significantly increased the in vitro migration of BCP-ALL cells through Matrigel-coated transwells in response to CXCL12, thus stimulating leukemic cell invasiveness. Overall, ActivinA resulted a key factor conferring a migratory advantage to leukemic cells over healthy hematopoiesis within the leukemic niche. Indeed, our in vitro findings provide the biological rationale for designing novel therapeutical approaches targeting the leukemia-stroma interplay. Disclosures Locatelli: bluebird bio: Consultancy; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.
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Till, KJ, J. Burthem, A. Lopez, and JC Cawley. "Granulocyte-macrophage colony-stimulating factor receptor: stage- specific expression and function on late B cells." Blood 88, no. 2 (July 15, 1996): 479–86. http://dx.doi.org/10.1182/blood.v88.2.479.bloodjournal882479.
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Granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors (GMR) are expressed on myeloid cells throughout their maturational sequence. During myelopoiesis, GM-CSF induces the proliferation of precursors and has multiple effects on more mature cells; such effects include induction of maturation and priming for subsequent stimulation. GMR is expressed on a range of other cell types including acute leukemic blasts of myeloid and lymphoid lineage, but has been little studied on more mature lymphoid cells. Using sensitive triple-layer immunophenotypic techniques, we show here that both the alpha and beta c chains of the GMR are expressed on hairy cells (HCs) and myelomatous plasma cells (PCs), but not on chronic lymphocytic leukemia (CLL) or prolymphocytic leukemia (PLL) lymphocytes. The receptor was demonstrable on normal PCs in tonsil, but not on either activated or resting tonsillar B cells or on circulating normal B lymphocytes. The expression of the receptor is therefore stage specific, rather than a feature of activation. Perhaps, surprisingly, in view of its effects on myeloid cells, GM-CSF did not stimulate the proliferation or differentiation of HCs and did not protect them from apoptosis. However, the cytokine had a profound effect on the interaction of the HC with its environment. Thus, the cytokine caused a major cytoskeletal reorganization resulting in the inhibition of motility and loss of adhesion to cellular and matrix ligands. These studies indicate the importance of GM-CSF outside myelopoiesis and demonstrate a previously unrecognized stage specific role for the cytokine in B-cell biology. Taken together with our previous report that M-CSF enhances B-cell motility, the present findings indicate that myeloid growth factors act in concert to facilitate the controlled migration of certain B cells into and within tissues.
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Hofbauer, Sebastian W., Peter W. Krenn, Sylvia Ganghammer, Daniela Asslaber, Ulrike Pichler, Karin Oberascher, Reinhard Henschler, et al. "Tiam1/Rac1 signals contribute to the proliferation and chemoresistance, but not motility, of chronic lymphocytic leukemia cells." Blood 123, no. 14 (April 3, 2014): 2181–88. http://dx.doi.org/10.1182/blood-2013-08-523563.
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Key Points Motility of resting CLL cells requires chemokine-mediated RhoA activation but is independent of Tiam1/Rac signals. Tiam1/Rac signals are indispensible for CLL cell proliferation and chemoresistance.
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Nishio, Mitsufumi, Kohki Kumano, Katsuya Fujimoto, Tomoyuki Endo, Satoshi Yamamoto, Akio Shigematsu, Takeshi Kondo, et al. "DOCK180, a New Surrogate Marker to Distinguish CD34 Positive Acute Leukemia Cells from CD34 Positive Normal Hematopoietic Stem/Progenitor Cells." Blood 112, no. 11 (November 16, 2008): 1476. http://dx.doi.org/10.1182/blood.v112.11.1476.1476.
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Abstract DOCK180, which is an archetype of the CDM family protein, plays a pivotal role in the regulation of cell motility and phagocytosis through the activation of small GTPase Rac as a guanine nucleoside exchange factor (GEF). The expression of DOCK180 is ubiquitous except in hematopoietic cells, while the expression of another CDM family protein, DOCK2, is strictly limited in hematopoietic cells. However, DOCK180 was recently induced in two types of hematopoietic cells, namely, macrophages and dendritic cells, during their differentiation from CD14 positive monocytes (Fujimoto et al. ASH 2005, abstract #3077). Therefore, the expression of DOCK180 was analyzed in various sorts of normal hematopoietic cells, including CD34 positive hematopoietic stem/progenitor cells (HSC/P). Real time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine the relative levels of DOCK180 mRNA expression (defined in 293T cells as 1000). Consistent with the findings of a previous report, there was a negligible amount of DOCK180 mRNA and protein in the peripheral blood or bone marrow mononuclear cells from normal volunteers. On the other hand, purified CD34 positive cells from healthy human bone marrow (N=4) and G-CSF mobilized peripheral blood (N=4) expressed a high amount of DOCK180 mRNA (1302 ± 883 Units and 1455 ± 689 Units, respectively). Furthermore, the expression of DOCK180 protein was confirmed by Western blotting in these CD34 positive cells. To investigate the association of the DOCK180 expression and granulocytic differentiation, CD34 positive cells were cultured with stem cell factor, IL-3 and G-CSF in vitro to induce them to mature into a granulocytic lineage and thus lose their surface CD34 expression. Interestingly, there was a gradual reduction in the DOCK180 expression accompanied by a loss of the surface CD34 expression, thus suggesting that the DOCK180 expression may therefore be limited in CD34 positive, immature HSC/P. Since a large number of immature acute leukemia cells also express CD34 antigen on their surface, the expression of DOCK180 was investigated in CD34 positive leukemic cells from 25 acute leukemia patients. The subjects consisted of 16 patients with acute myeloid leukemia (AML: M0 2, M1 2, M2 4, M4 3, M7 1, MDS overt leukemia 2, secondary AML 2), 6 with acute lymphoblastic leukemia (ALL: Pro B 3, Ph1 positive 2, common 1), and 3 in blastic crisis of chronic myeloid leukemia (BC CML: 2 lymphoid BC and 1 myeloid BC). The leukemic cells were collected at the first onset of the disease from 19 patients and from 6 other patients at the time of relapse. While there was no difference in the expression of DOCK2 between leukemic and normal CD34 positive cells, the expression of DOCK180 in leukemic cells was significantly lower than that detected in normal CD34 positive cells (median 17 Units vs. 1096 Units, range 0–4746 Units and 279–2812 Units, respectively, p<0.01). In addition, the CD34 positive cells from four patients in remission after successful therapy expressed high levels of both DOCK180 mRNA and protein. Interestingly, three out of four patients whose leukemic cells had a compatible DOCK180 mRNA expression to that of normal CD34 positive cells showed a refractory response to either conventional induction therapy or bone marrow transplantation. Taken together, DOCK180 may therefore be useful as a new surrogate marker to distinguish most leukemic CD34 positive cells from those of normal HSC/P, thereby suggesting that DOCK180 may thus play a possible role as a marker for the detection of minimal residual disease. Moreover, further study to determine the significance of DOCK180 expression in the prognosis of acute leukemia is thus called for.
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Schulz, Angela, Claudia Dürr, Thorsten Zenz, Hartmut Döhner, Stephan Stilgenbauer, Peter Lichter, and Martina Seiffert. "Lenalidomide reduces survival of chronic lymphocytic leukemia cells in primary cocultures by altering the myeloid microenvironment." Blood 121, no. 13 (March 28, 2013): 2503–11. http://dx.doi.org/10.1182/blood-2012-08-447664.
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Key Points Lenalidomide treatment of primary CLL/nurse-like cell cocultures resulted in significantly decreased viability of CLL cells. Lenalidomide increased IL-10 levels, activation of STAT1, expression of ICAM-1, and migration-related genes, and reduced CLL cell motility.
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Till, Kathleen J., David G. Spiller, Robert J. Harris, Haijuan Chen, Mirko Zuzel, and John C. Cawley. "Chronic Lymphocytic Leukemia Cells Are Dependent on Autocrine VEGF and α4β1 Integrin for Chemokine-Induced Motility." Blood 104, no. 11 (November 16, 2004): 2808. http://dx.doi.org/10.1182/blood.v104.11.2808.2808.
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Abstract VEGF is a multifunctional cytokine involved in tumourigenesis. In chronic lymphocytic leukaemia (CLL), it is known that the malignant cells secrete VEGF and possess VEGF receptors. This suggests that an autocrine loop might be important in the pathogenesis of CLL. Here we show that, in patients with lymphadenopathy, autocrine VEGF and α4β1-integrin are involved in the chemokine-dependent motility of CLL cells on and through endothelium - processes important for the invasion of lymphoreticular tissues. In contrast, normal lymphocytes were not dependent on autocrine VEGF or α4β1 for either type of cell movement. Moreover, in contrast to normal B lymphocytes, CLL cells failed to cluster and activate αLβ2 in response to chemokines, unless VEGF receptor(s) and α4β1 were also engaged by their respective ligands. This is the first demonstration that autocrine VEGF is involved in CLL-cell motility and that the αLβ2 on the malignant cells is functionally altered in the disease. Taken together, the results suggest that VEGF and α4 blockade may be therapeutically useful in CLL patients with tissue disease.
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Turley, EA, AJ Belch, S. Poppema, and LM Pilarski. "Expression and function of a receptor for hyaluronan-mediated motility on normal and malignant B lymphocytes." Blood 81, no. 2 (January 15, 1993): 446–53. http://dx.doi.org/10.1182/blood.v81.2.446.446.
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Abstract Migration through extracellular matrix is fundamental to malignant invasion. A receptor for hyaluronan-mediated motility (RHAMM) has previously been shown to play a fundamental role in locomotion of ras- transformed cells as well as functioning in signal transduction. Expression of RHAMM was characterized on B lymphocytes from normal and malignant lymphoid tissues using multiparameter phenotypic immunofluorescence analysis as well as functional analysis of its role in locomotion of malignant hairy cell leukemia B cells. RHAMM is not detectable on most normal B cells located in blood, spleen, or lymph node, but it is detectable on bone marrow and thymic B cells. Among B- cell malignancies, it is expressed on most terminally differentiated B cells from multiple myeloma bone marrows, is present on a subset of non- Hodgkin's lymphomas, and is absent on B chronic lymphocytic leukemia. Activation of peripheral blood B cells by Staphylococcus A cowan (SAC), but not by pokeweed mitogen, induced transient expression of RHAMM at day 3 of culture, suggesting RHAMM may be used by antigen-activated normal B cells. For malignant cells, expression of RHAMM increased on long-term culture of bone marrow plasma cells from multiple myeloma patients, indicating prolonged expression in contrast to the transient expression on SAC-activated normal B cells. Intriguingly, RHAMM was expressed on hairy leukemia cells located in spleen but absent from those in peripheral blood of the same patient. RHAMM, as expressed on splenic hairy cells, was a 58-Kd molecule that binds hyaluronan, is encoded by a 5.2-kb messenger RNA, and participates in locomotion by these cells. Hairy cells locomoted in response to hyaluronan at 4 mu per minute. Monoclonal antibody to RHAMM inhibited this locomotion almost completely as detected using video time-lapse cinemicrography. These observations are consistent with a role for RHAMM in malignant invasion and metastatic growth.
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Turley, EA, AJ Belch, S. Poppema, and LM Pilarski. "Expression and function of a receptor for hyaluronan-mediated motility on normal and malignant B lymphocytes." Blood 81, no. 2 (January 15, 1993): 446–53. http://dx.doi.org/10.1182/blood.v81.2.446.bloodjournal812446.
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Migration through extracellular matrix is fundamental to malignant invasion. A receptor for hyaluronan-mediated motility (RHAMM) has previously been shown to play a fundamental role in locomotion of ras- transformed cells as well as functioning in signal transduction. Expression of RHAMM was characterized on B lymphocytes from normal and malignant lymphoid tissues using multiparameter phenotypic immunofluorescence analysis as well as functional analysis of its role in locomotion of malignant hairy cell leukemia B cells. RHAMM is not detectable on most normal B cells located in blood, spleen, or lymph node, but it is detectable on bone marrow and thymic B cells. Among B- cell malignancies, it is expressed on most terminally differentiated B cells from multiple myeloma bone marrows, is present on a subset of non- Hodgkin's lymphomas, and is absent on B chronic lymphocytic leukemia. Activation of peripheral blood B cells by Staphylococcus A cowan (SAC), but not by pokeweed mitogen, induced transient expression of RHAMM at day 3 of culture, suggesting RHAMM may be used by antigen-activated normal B cells. For malignant cells, expression of RHAMM increased on long-term culture of bone marrow plasma cells from multiple myeloma patients, indicating prolonged expression in contrast to the transient expression on SAC-activated normal B cells. Intriguingly, RHAMM was expressed on hairy leukemia cells located in spleen but absent from those in peripheral blood of the same patient. RHAMM, as expressed on splenic hairy cells, was a 58-Kd molecule that binds hyaluronan, is encoded by a 5.2-kb messenger RNA, and participates in locomotion by these cells. Hairy cells locomoted in response to hyaluronan at 4 mu per minute. Monoclonal antibody to RHAMM inhibited this locomotion almost completely as detected using video time-lapse cinemicrography. These observations are consistent with a role for RHAMM in malignant invasion and metastatic growth.
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Jamieson, Catriona, Sidd Jaiswal, David Traver, Jason Gotlib, Mark Chao, and Irving L. Weissman. "Increased Expression of CD47 Is a Constant Marker in Mouse and Human Myeloid Leukemias." Blood 106, no. 11 (November 16, 2005): 3260. http://dx.doi.org/10.1182/blood.v106.11.3260.3260.
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Abstract CD47, also known as integrin associated protein, is a ubiquitously expressed cell surface glycoprotein that interacts with a number of integrins, modulating leukocyte adhesion, migration, cell motility and platelet activation. CD47 is also the ligand for the macrophage inhibitory receptor signal regulatory protein α (SIRP α) and thus, impairs macrophage-mediated phagocytosis. Recent reports suggest that increased CD47 expression may play a role in the pathogenesis of lymphoproliferative disorders such as CLL (Mateo et al, Nat Med.1999; 5:1277) and multiple myeloma, and that a bivalent single-chain antibody fragment against human CD47 induces apoptosis of myeloma cells (Kikuchi et al, Leukemia Research2005; 29:445). However, little is known about the role of CD47 in the pathogenesis of myeloid leukemias or the stage of hematopoiesis at which CD47 is expressed. In order to identify the stage of hematopoiesis at which alterations in CD47 arise and its role in the pathogenesis of myeloid leukemias, we analyzed CD47 expression in hematopoietic stem cells (HSC), progenitors, and lineage-positive cells derived from three mouse transgenic models of myeloid leukemia including: 1) mice homozygous for overexpression of human bcl-2 in the myeloid lineage (hMRP8 bcl-2 x bcl-2); 2) mice deficient in Fas together with myeloid targeted overexpression of bcl-2 (Faslpr/lpr hMRP8 bcl-2); and 3) mice with both human bcl-2 and BCR-ABL targeted to the myeloid lineage (hMRP8-BCR-ABLxhMRP8-bcl-2). Quantitative RT-PCR analysis demonstrated a 3.01+/− 1.54 fold increase in CD47 transcripts in leukemic compared with control (bcl2+) bone marrow (normalized to b-actin) while FACS analysis revealed approximately a 10-fold increase in CD47 protein expression, as measured by mean fluorescence intensity (MFI), in leukemic GMP compared with wild type GMP. Moreover, transplantation experiments revealed that all mice with both primary (n=14 mice) and secondary (n=19 mice) leukemic transplantation potential had an expansion of granulocyte-macrophage progenitors (GMP) with high level CD47 expression. Human CD47 expression analysis was performed via FACS on human normal, pre-leukemic myeloproliferative disorder (MPD) or AML HSC, progenitors, and lineage positive cells derived from marrow or peripheral blood. MPD samples (n=63) included polycythemia vera (PV; n=15), post-polycythemic myeloid metaplasia/myelofibrosis (PPMM/MF; n=5), essential thrombocythemia (ET; n=8), atypical chronic myelogenous leukemia (aCML; n=2), CML (n=7), chronic eosinophilic leukemia (CEL; n=1), chronic myelomonocytic leukemia (CMML; n= 13) and acute myelogenous leukemia (AML; n=12). As noted with the transgenic leukemic mouse models, progression of human myeloproliferative disorders to AML (n=12) was associated with an expansion of the GMP pool (70.6% +/− S.D. 2.15) compared with normal bone marrow (14.7% +/− S.D. 2.3). Furthermore, FACS analysis revealed that CD47 expression first increased 1.7 fold in AML compared with normal HSC and then increased to 2.2 fold greater than normal with commitment of AML progenitors to the myeloid lineage. CD47 was over-expressed by AML primitive progenitors and their progeny but not by the majority of MPD (MFI 2.3+/−S.D. 0.43) compared with normal bone marrow (MFI 1.9 +/−S.D. 0.07). Thus, increased CD47 expression may serve as a useful diagnostic marker for progression to AML and may represent a novel therapeutic target.
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Ortlepp, Claudia, Christine Steudel, Sina Koch, Angela Jacobi, Satu Kyttaelae, Martin Ryser, Sebastian Brenner, Martin Bornhaeuser, Gerhard Ehninger, and Christian Thiede. "Expression and Functional Analysis of Autotaxin, a Relevant Motility and Survival Factor, in FLT3-ITD Positive Acute Myeloid Leukemia and Primary Hematopoietic Stem Cells." Blood 112, no. 11 (November 16, 2008): 1206. http://dx.doi.org/10.1182/blood.v112.11.1206.1206.
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Abstract FLT3-mutations are among the most common abnormalities in adult acute myeloid leukemia. These mutations induce constitutive activation of the receptor and downstream signaling pathways, including RAS/ERK, PI3K and STAT5-signalling. The most frequent aberration is an internal tandem duplication (ITD) of the juxtamembrane domain coding sequence, which is present in up to 27% of the patients with AML and has been associated with inferior prognosis. We have recently demonstrated by microarray analysis that leukemic samples of patients with FLT3-ITD mutations have significantly upregulated expression levels of Autotaxin (ATX). The ATX protein acts as a secreted lysophospholipase D (lysoPLD) through generating lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). LPA has several important functions in cell migration and proliferation and acts via G-protein coupled receptors. It has been shown that ATX represents an aberrantly expressed motility and growth factor in a variety of cancer cells. However data on ATX in myeloid leukemias and especially in AML are missing. To study more deeply the role of ATX in leukemogenesis, we screened a series of human leukemic model cell lines for ATX expression. Using retroviral transduction, two alternatively spliced transcripts of ATX were expressed in several human leukemic cell lines without detectable levels of endogenous ATX. To investigate the ATX function and expression in normal haematopoiesis we used CD34+ human progenitor cells. The expression of ATX transcripts was confirmed at both mRNA and protein levels by RT-PCR and Western blotting, respectively. Transwell migration assays in the presence of LPC or LPA were performed to study effects of ATX on cell motility. Proliferation and clonogenic potential were investigated using MTT and colony forming assays. Moreover, we examined the effect of the FLT3 inhibitor N-benzoylstaurosporine (PKC412) on ATX expression and ATX mediated migration in MV4-11 cells. High ATX expression was found primarily in malignant cells. Western blot analysis showed that detectable levels of ATX are secreted into medium within 2 hours. In normal cells, highest ATX mRNA expression was found in purified CD34 cells, but could also be found at lower levels in T-cells. Stable overexpression of FLT3-ITD in OCI-AML3 cells induced an increased ATX expression (up to 6 fold). Vice versa, inhibition of FLT3-ITD by sublethal doses of PKC412 in MV4-11 cells resulted in a significant reduction of ATX expression down to 10% of the initial expression level. Furthermore, PKC412 treatment resulted in a complete loss of LPC or LPA induced specific migratory capacity in MV4-11 cells. The transduction of ATX increased the colony-forming capacity by 75% and significantly increased the short term proliferation. LPA increased chemotaxis in human leukemic cell lines and human CD34+ progenitors in a dose dependent manner and induced significantly higher migratory rates by at least 50%. LPC induced chemotaxis by 80–200% only in cells with high expression of endogenous or exogenous ATX, demonstrating the autocrine activity of ATX. Ongoing studies on the mechanism of ATX expression showed that ionomycin, an activator of the Ca2+–calcineurin–NFAT signalling pathway, upregulated ATX mRNA in several leukemic cell lines as well as in human primary progenitors up to 5-fold, which could be completely blocked by cyclosporine A treatment, indicating an involvement of NFAT in the regulation of ATX. Our data suggest that the production of bioactive LPA through ATX is involved in controlling proliferation and migration of hematopoietic stem cells and its deregulation may contribute to the pathogenesis of AML, especially in patients with FLT3-ITD mutations.
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WANG, YINGCHAO, LINA MA, CHUNMEI WANG, GUANGYAO SHENG, LEI FENG, and CHUYUN YIN. "Autocrine motility factor receptor promotes the proliferation of human acute monocytic leukemia THP-1 cells." International Journal of Molecular Medicine 36, no. 3 (June 30, 2015): 627–32. http://dx.doi.org/10.3892/ijmm.2015.2267.
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Till, Kathleen J., David G. Spiller, Robert J. Harris, Haijuan Chen, Mirko Zuzel, and John C. Cawley. "CLL, but not normal, B cells are dependent on autocrine VEGF and α4β1 integrin for chemokine-induced motility on and through endothelium." Blood 105, no. 12 (June 15, 2005): 4813–19. http://dx.doi.org/10.1182/blood-2004-10-4054.
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Abstract Vascular endothelial cell growth factor (VEGF) is a multifunctional cytokine involved in tumor formation. In chronic lymphocytic leukemia (CLL), it is known that the malignant cells secrete VEGF and possess VEGF receptors. This suggests that an autocrine loop might be important in the pathogenesis of CLL. Here we show that, in patients with lymphadenopathy, autocrine VEGF and α4β1 integrin are involved in the chemokine-dependent motility of CLL cells on and through endothelium—processes important for the invasion of lymphoreticular tissues, a major determinant of disease outcome. In contrast, normal lymphocytes were not dependent on autocrine VEGF or α4β1 for either type of cell movement. Moreover, in contrast to normal B lymphocytes, CLL cells failed to cluster and activate αLβ2 in response to chemokines, unless VEGF receptor(s) and α4β1 were also engaged by their respective ligands. This is the first demonstration that autocrine VEGF is involved in CLL-cell motility, and that the αLβ2 on the malignant cells is functionally altered compared with that of normal B cells in not undergoing activation in response to chemokine alone. Given the importance of cell motility for tissue invasion, the present results provide a rationale for a trial of VEGF and α4 blockade in patients with CLL who have tissue disease. (Blood. 2005;105:4813-4819)
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Badger-Brown, Karla M., Monica L. Bailey, Josef Penninger, and Dwayne L. Barber. "Cbl-b Deficiency Enhances Motility and Impairs Leukemogenesis by Bcr-Abl." Blood 110, no. 11 (November 16, 2007): 1019. http://dx.doi.org/10.1182/blood.v110.11.1019.1019.
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Abstract One of the prominent tyrosine phosphorylated substrates of BCR-ABL identified in cells from chronic-phase Chronic Myeloid Leukemia (CML) patients is the Cbl ubiquitin ligase and adaptor protein. Bone marrow transplantation studies have revealed that Cbl is dispensable for CML development, as animals receiving Cbl-deficient bone marrow cells expressing BCR-ABL develop a myeloproliferative disease (MPD) with a similar latency and phenotype as wild type bone marrow recipients (Dinulescu et al. 22: 8852–60, 2003). Cbl belongs to a family of E3 ubiquitin ligases that also contains Cbl-b and Cbl-3. In contrast to the equivalent Cbl protein expression displayed in hematopoietic cell lines expressing BCR-ABL, addition of the oncogene leads to a decreased expression of the related family member, Cbl-b. The objective of this study was to determine whether Cbl-b is a critical signaling intermediate downstream of BCR-ABL. To investigate this objective, we performed retroviral transduction of wild type- and Cbl-b- deficient bone marrow with BCR-ABL and utilized these cells for bone marrow transplantation of lethally irradiated mice. Recipients of wild type donor cells transduced with the p210 isoform of BCR-ABL, succumb to a rapidly fatal CML-like MPD, while the lack of Cbl-b produces a significant delay in morbidity (average latency of 32 days as compared with 18 days for recipients of wild-type marrow). Expansion of differentiated neutrophils, as visualized by histopathological analysis, is evident in the peripheral blood, bone marrow and spleen of all transplanted animals. However, circulating white blood cells are augmented in number (average wbc count for Cbl-b(+/+), 150 × 106 cells / mL; Cbl-b(−/−), 83 × 106 cells / mL) and enhanced spleen size is observed in Cbl-b(+/+) mice (35 mg / g of body mass) as compared to Cbl-b(−/−) (19 mg/g) animals. Liver masses were comparable between the two transplants. In depth flow cytometric studies of splenic cells reveals the characteristic decrease in B and T cells and enhanced myeloid lineages. Notably, an increased number of granulocytes (Gr-1+/Mac-1+ cells) is observed in the Cbl-b-knockout animals, suggesting enhanced migration but decreased survival of leukemic cells in the Cbl-b−/− spleen. In support of this hypothesis, fibroblasts deficient in Cbl-b display enhanced chemotactic migration toward Fetal Calf Serum. As migration is a key determinant of cellular retention in the bone marrow, the loss of Cbl-b could ultimately be affecting cell localization. Therefore, our data supports a continued study of Cbl-b as a motility regulator in BCR-ABL-dependent CML progression.
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Spiegel, Asaf, Orit Kollet, Amnon Peled, Loya Abel, Arnon Nagler, Bella Bielorai, Gideon Rechavi, Josef Vormoor, and Tsvee Lapidot. "Unique SDF-1–induced activation of human precursor-B ALL cells as a result of altered CXCR4 expression and signaling." Blood 103, no. 8 (April 15, 2004): 2900–2907. http://dx.doi.org/10.1182/blood-2003-06-1891.
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Abstract The mechanisms governing migration and extramedullary dissemination of leukemic cells remain obscure. In this study the migration and in vivo homing to the bone marrow of nonobese diabetic severe combined immunodeficient (NOD/SCID) mice injected with human precursor-B acute lymphoblastic leukemia (ALL) cells in comparison to normal CD34+ progenitors (both cord blood and mobilized peripheral blood) was investigated. Although migration and homing of both cell populations was dependent on stromal cell-derived factor 1 (SDF-1)/CXCR4 interactions, major differences in receptor expression as well as the migratory capacity toward various concentrations of SDF-1 were found. Furthermore, unlike normal CD34+ progenitors, in vivo homing of the leukemic cells was superior when recipient NOD/SCID mice were not irradiated prior to transplantation. In addition, we report differences in the adhesion molecules activated following SDF-1 stimulation, documenting a major role for very late antigen 4 (VLA-4), but not VLA-5 and lymphocyte function-associated antigen-1 (LFA-1), in homing of precursor-B ALL cells. Interestingly, Toxin-B and pertussis toxin inhibited the homing of the leukemic cells but not that of normal CD34+ progenitors or normal CD10+/CD19+ precursor-B cells, revealing differences in CXCR4 signaling pathways that are based on changes that acquired by the leukemic cells. Altogether, our data provide new insights into different SDF-1–induced signaling, activation, and consequent motility between normal CD34+ and precursor-B ALL progenitors, which may lead to improved clinical protocols. (Blood. 2004;103: 2900-2907)
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Khan, Naveed I., Kenneth Francis Bradstock, and Linda J. Bendall. "The Wnt Pathway Modulates Expression of Growth and Survival Genes in Acute Lymphoblastic Leukemia Cells." Blood 108, no. 11 (November 16, 2006): 1850. http://dx.doi.org/10.1182/blood.v108.11.1850.1850.
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Abstract Wnt proteins are important bone marrow-derived growth factors known to support normal hematopoietic progenitor and stem cell development. Here we report that B cell progenitor acute lymphoblastic leukemia (pre-B ALL) cells express Wnt proteins, including Wnt-2b in 33%, Wnt-5a in 42%, Wnt-10b in 58% and Wnt-16b in 25% of cases. The Wnt receptors, Frizzled (Fz)-7 and -8 were also expressed in most cases while Fz-3, -4 and -9 were occasionally detected. Stimulation of pre-B ALL cells with Wnt-3a activated canonical Wnt signaling with increased expression and nuclear translocation of β-catenin. This resulted in a 1.7 to 5.3-fold increase in cell proliferation, which was associated with enhanced cell cycle entry. Wnt-3a also significantly increased the survival of pre-B ALL cells under conditions of serum deprivation. To determine the mechanisms involved we examined the effects of Wnt-3a on gene expression using the leukemic pre-B ALL cell line NALM6 and a cancer specific microarray (GEArray® OHS-802), which contains 440 known cancer genes. Expression of 83 genes (19%) could be detected on the array. Exposure to Wnt-3a for 24 hours resulted in increased (>1.5 fold) expression of 29 genes and reduced (<50% of control) expression of 3 genes. The most highly regulated genes in response to Wnt-3a were MYBL2, E2F1, CD10, VDAC1, CDC25B (upregulated) and TRAIL-R2 (downregulated). Using qRT-PCR, we confirmed regulation of these genes in NALM6 cells and/or in another leukemic cell line LK63. These genes play important roles in the control of cell cycle (MYBL2, E2F1 and CDC25B), apoptosis (VDAC1 and TRAIL-R2) and motility (CD10) in cancer cells. Our results suggest that Wnt signalling regulates cell growth and proliferation in leukemic cells by modulating the expression of a number of genes. To our knowledge this is the first study examining the gene expression profile following Wnt stimulation in leukemic cells and potentially identifies new therapeutic targets for treatment.
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Zal, Tomasz, Malgorzata Anna Zal, Mateusz Rytelewski, Kyung Hee Chang, Rodrigo Jacamo, William E. Fogler, John L. Magnani, and Michael Andreeff. "Dual CXCR4 and E-Selectin Inhibition (GMI-1359) Rapidly Increases AML Cellular Motility Prior to Intravasation and Vascular Niche Depletion Observed By Intravital Bone Marrow Two-Photon Microscopy." Blood 136, Supplement 1 (November 5, 2020): 14–15. http://dx.doi.org/10.1182/blood-2020-143420.
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BACKGROUND: Acute myelogenous leukemia (AML) cells that reside in bone marrow (BM) receive a great deal of protection from the cytotoxic effects of therapeutic agents. In contrast, the circulating leukemia cells are typically more chemosensitive compared to those embedded in BM niches. The BM homing of AML cells is mediated by multiple adhesive and chemokinetic interactions including, respectively, by sialylated glycoproteins on the cancer cells binding to E-selectin on the endothelium and by CXCR4-mediated sensing of SDF-1/CXCL12 gradients. Consequently, both E-selectin and CXCR4 inhibitors have been pursued as targets for add-on therapeutic strategies aimed to "mobilize" AML cells out of the BM. A next generation "mobilizing" agent GMI-1359 combines, in one molecular structure, the E-selectin and CXCR4 inhibitory moieties. In a previous study, GMI-1359 markedly reduced leukemia cell adhesion to endothelial cells, leukemia cellularity in BM (Zhang et al., 2016) and it enhanced the survival of cytarabine/daunorubicin therapy in a FLT3-mutated AML model (Zhang et al., 2015). APPROACH: Here, we used intravital 2-photon microscopy of calvarial BM to study the behavioral response of AML cells to the dual CXCR4 and E-selectin inhibition (GMI-1359) in vivo. We used a mTurquoise2 fluorescence-tagged transplantable mouse AML model (characterized by MLL, ENL-FLT3, ITD, p53-/-). To delineate the bone and vascular niches, the syngeneic immune-competent recipient mice harbored cell lineage fluorescence reporter genes such as Col2.3-GFP, hCD2-DsRed and CD11c-EYFP with the bone collagen and blood highlighted by, respectively, second harmonic generation (SHG) and fluorescent dextran. Intravenously infused AML cells (5x10E4) homed to BM where they gradually displaced most endogenous cells. In this experimental system, GMI-1359 or vehicle were infused into the tail vein while recording AML cell motilities in calvarial BM stroma in 3-D over 4 hours. RESULTS: In untreated or control vehicle treated mice, AML cells were slowly motile, migrating with the average velocity of ~2 µm/min. Cellular trajectories were random within the average 400 µm confinement radius corresponding to the size of BM cavities. Most AML cells localized in the vascular niche, defined as within a 50 µm distance to the nearest blood vessel. A minority of AML cells were also present in the proximity to osteoblasts and the bone (i.e. in the bone niche). In contrast, GMI-1359 infusion resulted in a 66% increase of the average speed of cellular motility of AML cells within 20 min and up to 100% increase within 3.5 hours. The motility pattern remained largely random within the bone cavity confinement volume and was concentrated in the vicinity of vasculature. Thereafter, we observed multiple instances of AML cell intravasation into the capillary lumens followed by several minute-long vessel wall attachment and sudden outflow. This cellular dynamics resulted in a substantial, yet structurally biased decrease of BM AML cellularity whereby the vascular niche was emptied preferentially whilst the bone niche remained populated by AML cells. In no case we observed a complete BM depletion of AML cells. Interestingly, as we reported before, a single, CXCR4-only inhibitor caused a protracted (24-48 h) depletion of AML cells in BM without significant cellular motility enhancement. CONCLUSIONS: Our observations reveal an unexpected mechanism of action by a dual E-selectin/CXCR4 inhibitor involving cellular migratory motility enhancement in BM stroma prior to AML cell intravasation. We envisage that, besides the designed blocking of E-selectin and CXCR4 molecules from binding their corresponding ligands, the dual moiety GMI-1359 agent has a capacity to generate motility-enhancing signals in AML cells that single inhibitors do not seem to trigger. Among several mechanisms possible, this action could involve E-selectin and CXCR4 crosslinking by a dual moiety molecular structure or binding avidity enhancement, requiring further investigation. In addition, our results highlight a likely mechanism of AML resistance to therapeutic "mobilization", one that involves cancer cell persistence in the bone/osteoblastic niches. Disclosures Zal: Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding. Fogler:GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Andreeff:Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Amgen: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees.
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Lautenschläger, Franziska, Stephan Paschke, Stefan Schinkinger, Arlette Bruel, Michael Beil, and Jochen Guck. "The regulatory role of cell mechanics for migration of differentiating myeloid cells." Proceedings of the National Academy of Sciences 106, no. 37 (August 26, 2009): 15696–701. http://dx.doi.org/10.1073/pnas.0811261106.
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Migration of cells is important for tissue maintenance, immune response, and often altered in disease. While biochemical aspects, including cell adhesion, have been studied in detail, much less is known about the role of the mechanical properties of cells. Previous measurement methods rely on contact with artificial surfaces, which can convolute the results. Here, we used a non-contact, microfluidic optical stretcher to study cell mechanics, isolated from other parameters, in the context of tissue infiltration by acute promyelocytic leukemia (APL) cells, which occurs during differentiation therapy with retinoic acid. Compliance measurements of APL cells reveal a significant softening during differentiation, with the mechanical properties of differentiated cells resembling those of normal neutrophils. To interfere with the migratory ability acquired with the softening, differentiated APL cells were exposed to paclitaxel, which stabilizes microtubules. This treatment does not alter compliance but reduces cell relaxation after cessation of mechanical stress six-fold, congruent with a significant reduction of motility. Our observations imply that the dynamical remodeling of cell shape required for tissue infiltration can be frustrated by stiffening the microtubular system. This link between the cytokeleton, cell mechanics, and motility suggests treatment options for pathologies relying on migration of cells, notably cancer metastasis.
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Spranger, Stefani, Irmela Jeremias, Susanne Wilde, Matthias Leisegang, Lilian Stärck, Barbara Mosetter, Wolfgang Uckert, Mirjam H. M. Heemskerk, Dolores J. Schendel, and Bernhard Frankenberger. "TCR-transgenic lymphocytes specific for HMMR/Rhamm limit tumor outgrowth in vivo." Blood 119, no. 15 (April 12, 2012): 3440–49. http://dx.doi.org/10.1182/blood-2011-06-357939.
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Abstract The hyaluronan-mediated motility receptor (HMMR/Rhamm) is overexpressed in numerous tumor types, including acute lymphoid leukemia and acute myeloid leukemia (AML). Several studies have reported the existence of T-cell responses directed against HMMR in AML patients that are linked to better clinical outcome. Therefore, we explored the use of HMMR-specific TCRs for transgenic expression in lymphocytes and their in vivo impact on HMMR+ solid tumors and disseminated leukemia. We obtained TCRs via an in vitro priming approach in combination with CD137-mediated enrichment. Recipient lymphocytes expressing transgenic TCR revealed the specific tumor recognition pattern seen with the original T cells. Adoptive transfer experiments using a humanized xenograft mouse model resulted in significantly retarded solid tumor outgrowth, which was enhanced using IL-15–conditioned, TCR-transgenic effector memory cells. These cells also showed an increased potency to retard the outgrowth of disseminated AML, and this was further improved using CD8-enriched effector memory cells. To define a safe clinical setting for HMMR-TCR gene therapy, we analyzed transgenic T-cell recognition of hematopoietic stem cells (HSCs) and found on-target killing of HLA-A2+ HSCs. Our findings clearly limit the use of HMMR-TCR therapy to MHC- mismatched HSC transplantation, in which HLA-A2 differences can be used to restrict recognition to patient HSCs and leukemia.
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Pillozzi, Serena, Maria Felice Brizzi, Pietro Antonio Bernabei, Benedetta Bartolozzi, Roberto Caporale, Venere Basile, Vieri Boddi, Luigi Pegoraro, Andrea Becchetti, and Annarosa Arcangeli. "VEGFR-1 (FLT-1), β1 integrin, and hERG K+ channel for a macromolecular signaling complex in acute myeloid leukemia: role in cell migration and clinical outcome." Blood 110, no. 4 (August 15, 2007): 1238–50. http://dx.doi.org/10.1182/blood-2006-02-003772.
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Abstract Leukemia cell motility and transendothelial migration into extramedullary sites are regulated by angiogenic factors and are considered unfavorable prognostic factors in acute leukemias. We have studied cross talk among (1) the vascular endothelial growth factor receptor-1, FLT-1; (2) the human eag-related gene 1 (hERG1) K+ channels; and (3) integrin receptors in acute myeloid leukemia (AML) cells. FLT-1, hERG1, and the β1 integrin were found to form a macromolecular signaling complex. The latter mostly recruited the hERG1B isoform of hERG1 channels, and its assembly was necessary for FLT-1 signaling activation and AML cell migration. Both effects were inhibited when hERG1 channels were specifically blocked. A FLT-1/hERG1/β1 complex was also observed in primary AML blasts, obtained from a population of human patients. The co-expression of FLT-1 and hERG1 conferred a pro-migratory phenotype to AML blasts. Such a phenotype was also observed in vivo. The hERG1-positive blasts were more efficient in invading the peripheral circulation and the extramedullary sites after engraftment into immunodeficient mice. Moreover, hERG1 expression in leukemia patients correlated with a higher probability of relapse and shorter survival periods. We conclude that in AML, hERG1 channels mediate the FLT-1–dependent cell migration and invasion, and hence confer a greater malignancy.
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Schmitt, Michael, Li Li, Mark Ringhoffer, Thomas Barth, Markus Wiesneth, Hartmut Dohner, and Jochen Greiner. "Characterization of T Cell Epitopes of the Receptor for Hyaluronic Acid Mediated Motility (RHAMM/CD168) in Acute Myeloid Leukemia." Blood 104, no. 11 (November 16, 2004): 2540. http://dx.doi.org/10.1182/blood.v104.11.2540.2540.
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Abstract To improve the clinical outcome of patients with acute myeloid leukemia (AML), immune therapies targeting leukemia associated antigens (LAAs) might be an approach complementary to chemotherapy and transplantation of hematopoetic stem cells. The receptor for hyaluronic acid mediated motility (RHAMM/CD168) has been defined as a LAA with specific expression. To define T cell epitopes of RHAMM/CD168 towards specific T cell immunotherapies, ten peptides were synthesized considering different computer algorithms and subjected to ELISPOT assays for interferon gamma and granzyme B, and to Cr-51 release assays. CD8+ T cells taken from the peripheral blood (PB) of 13 AML patients and presensitized with the RHAMM/CD168-derived peptides R3 (ILSLELMKL) or R5 (SLEENIVIL) did specifically recognize T2 cells pulsed with R3/R5. In contrast, CD8+ T cells isolated from the PB of 21 healthy volunteers were not able to lyse R3 or R5 pulsed T2 cells, even after presensitization. COS7 cells co-transfected with HLA-A*0201 and RHAMM/CD168 were lysed by R3 or R5 presensitized CD8+ T cells. Single HLA-A*0201 or RHAMM/CD168 transfected COS7 were not recognized. Cross-reactivity of the T cells was excluded by the use of unrelated peptides. K562 cells positive for RHAMM/CD168, but lacking HLA-class I molecules were not recognized indicating T cells and not NK cells as effector cells. The HLA class-I restricted lysis of COS-7 HLA-A*0201 and RHAMM/CD168 double- transfectants was confirmed by HLA class-I blocking antibody experiments. In an AML patient having received AML blast-derived dendritic cells, a higher frequency of RHAMM/CD168-peptide specific T cells was observed after four vaccinations when compared to his T cell status before vaccination. RHAMM/CD168 is also expressed in patients with other hematological malignancies which suggests a broad clinical applicability of its newly characterized T cell epitope peptides as a potential cancer vaccine.
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Matsumoto, Taichi, Shiro Jimi, Junji Suzumiya, Shuji Hara, and Kazuo Tamura. "RARα-Specific Retinoid Am-80 Prevents SDF-1-Mediated Chemotaxis in CXCR4 Expressing Tumor Cells." Blood 110, no. 11 (November 16, 2007): 4194. http://dx.doi.org/10.1182/blood.v110.11.4194.4194.
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Abstract Chemokine (C-X-C motif) receptor 4 (CXCR4) plays an important role on hematogenous metastasis of tumor cells to the organs comprising constitutively stromal cell-derived facter-1 (SDF-1). Retinoids have been utilized as a drug for treatment of acute promyelocytic leukemia (APL), in which tumor cells give rise to differentiation and cell death by activation of retinoic acid receptors (RARs). However, the effects of retinoid on chemokine-mediated cell motility are totally unknown. In this study, we examined the effects of retinoids, including all-trans retinoic acid (ATRA) and RARα-specific retinoid Am-80, on CXCR4-mediated chemotactic activity using five myeloid and lymphoid leukemia cells (HL-60, K562, MOLT-3, Jurkat, and U266B1). When cells were incubated with ATRA or Am-80 at doses up to 10 mM for 3 days, comparable effects were found; cytotoxic effect occurred only in HL-60, and growth inhibition was albeit detected in all of other cells. The extents of cell surface CXCR4 in all of the cells detected by flow cytometric analysis tended to be correlated with SDF-1-induced migration activity. Cell surface CXCR4 was slightly decreased by ATRA treatment, while it was significantly decreased by Am-80 treatment, and the alterations were in time- and dose-dependent fashions. In terms of CXCR4 expression, Jurkat was the most sensitive cells. Cell migration in response to SDF-1, was monitored by a modified Boyden chamber method. SDF-1 induced significant chemotaxis in HL-60, MOLT-3 and Jurkat cells, but did not in K562 and U266B1 cells. After treatment with the retinoids, HL-60 was only a cell type increasing random cell motility, however in retinoids-pretreated MOLT-3 and Jurkat cells, no such effect was found, and therefore suggesting that up-regulation of the motility in HL-60 is maybe due to a response by dyeing cells. However, in a case of pretreatment with not ATRA but Am-80, SDF-1-induced chemotaxis of MOLT-3 and Jurkat cells was dose-dependently repressed in accordance with decrease in cell surface CXCR4 expression. This was also confirmed by a time-laps horizontal migration system, indicated that pseudopodia formation accompanied with F-actin rearrangement by SDF-1 was strongly inhibited by Am-80-pretreatment. These results indicate that RARα-specific retinoid Am-80 may induce not only growth inhibition, but also prevention of SDF-1/CXCR4-mediated hematogenous metastasis in CXCR4 expressing tumor cells.
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Taraboletti, Giulia, Patrizia Borsotti, Renato G. S. Chirivi, Veronica Vergani, Anna Falanga, Tiziano Barbui, Raffaella Giavazzi, and Alessandro Rambaldi. "Effect of alltrans-retinoic acid (ATRA) on the adhesive and motility properties of acute promyelocytic leukemia cells." International Journal of Cancer 70, no. 1 (January 6, 1997): 72–77. http://dx.doi.org/10.1002/(sici)1097-0215(19970106)70:1<72::aid-ijc11>3.0.co;2-f.
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You, Zhi-Mei, Liang Zhao, Jing Xia, Qiang Wei, Yu-Min Liu, Xiao-Yan Liu, Di-Long Chen, and Jing Li. "Down-regulation of Phosphoglucose Isomerase/Autocrine Motility Factor Enhances Gensenoside Rh2 Pharmacological Action on Leukemia KG1α Cells." Asian Pacific Journal of Cancer Prevention 15, no. 3 (February 1, 2014): 1099–104. http://dx.doi.org/10.7314/apjcp.2014.15.3.1099.
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Bendall, Linda, Rana Baraz, Julius Juarez, Sylvie Shen, and Ken Bradstock. "Defective P38 MAPK Signalling Impairs Chemotactic but Not Proliferative Responses to SDF-1 in Acute Lymphoblastic Leukemia." Blood 104, no. 11 (November 16, 2004): 997. http://dx.doi.org/10.1182/blood.v104.11.997.997.
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Abstract The chemokine SDF-1 regulates leukemic cell motility and proliferation but the importance of these functions in the growth and dissemination of leukemia is unclear. We examined SDF-1-mediated responses of cells from 27 cases of acute lymphoblastic leukemia (ALL). Although cells from the majority of cases showed chemotactic and proliferative responses to SDF-1, a subset of cases (18%) did not undergo chemotaxis in response to SDF-1 (unresponsive cases). These unresponsive cases also failed to increase b1 integrin-mediated adhesion to fibronectin or adhesion to bone marrow fibroblast layers. However the unresponsive cases could still elicit a calcium flux in response to SDF-1 and three of the four cases internalised the receptor, CXCR4, following exposure to SDF-1. In contrast, the CXCR4 antagonist, TC14012, inhibited proliferation of both responsive and unresponsive cases in stromal-dependent cultures, demonstrating that the unresponsive cases were still able to undergo proliferative responses to SDF-1. Examination of the signalling pathways activated by SDF-1 in responsive cells revealed increased phosphorylation of AKT, ERK and p38 MAPK 2 to 5 minutes following stimulation. However, cells from the unresponsive cases failed to phosphorylate p38 MAPK kinase when stimulated with SDF-1, while phosphorylation of AKT and ERK were comparable with that observed in responsive ALL cases. Inhibition of p38 MAPK by SB203500 completely inhibited the chemotaxis of responsive ALL cells to SDF-1 gradients suggesting that signalling through p38 MAPK is essential for ALL cell chemotaxis. Therefore it is likely that the absence of p38 MAPK phosphorylation in unresponsive cases underlies their lack of chemotaxis to this chemokine. The ability of the unresponsive cases to undergo SDF-1 driven proliferation in the absence of p38 MAPK phosphorylation suggests that, despite being absolutely required for chemotactic responses, induction of phosphorylation of p38 MAPK is not required for proliferative responses. No correlation was observed between CXCR4 expression and chemotactic function, in vitro migration into bone marrow stromal layers, and engraftment of leukemic cells in NOD/SCID mice. This study suggests that signalling through p38 MAPK is required for ALL cell chemotaxis, but not for proliferation, and that chemotactic responses to SDF-1 are not essential for leukemic cell engraftment.
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Satake, Noriko, Astra Chang, Bridget McLaughlin, Sara Bauman, James Chan, Susmita Sarangi, Ping Zhou, et al. "Evaluation of CD9 as a Candidate Leukemia Stem Cell Marker In Childhood Precursor B Cell Acute Lymphoblastic Leukemia." Blood 116, no. 21 (November 19, 2010): 3241. http://dx.doi.org/10.1182/blood.v116.21.3241.3241.
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Abstract Abstract 3241 Leukemia cells are believed to arise from leukemia stem cells (LSC). It is also known that LSC are responsible for relapse in certain types of leukemia, such as acute myeloid leukemia (AML). However, the existence and role of LSC in acute lymphoblastic leukemia (ALL) is unclear. CD9 was reported to be a marker for LSC in B-ALL using cell lines (Nishida H. et al., 2009). CD9 is a tetraspanin and is believed to be involved in cell adhesion, motility, and signaling events. It is also involved in metastasis; however, the mechanisms are unknown. Since childhood ALL is a heterogeneous group of diseases and cell lines can be different from primary leukemia cells, we tested the role of CD9 as a candidate LSC marker using primary precursor B (preB) ALL cells from pediatric patients. Two methods, Raman spectroscopy and serial transplantation of sorted leukemia cells in NOD/SCID/IL2R g null (NSG) mice, were used to confirm LSC. Raman spectroscopy is a laser-based technique for the single cell analysis of intrinsic molecular vibrations reflecting cellular biochemical information. It can provide a quantitative assessment of the levels of DNA, RNA, proteins, lipids, and carbohydrates in the cell, as well as molecular-level conformational changes. Previous studies by our group showed that unique Raman fingerprints were identified in normal blood cells, ALL cells, and stem cells, including hematopoietic stem cells and embryonic stem cells. Four preB ALL samples were stained for CD9 and sorted by flow cytometry. ALL samples were obtained from patients at diagnosis or freshly harvested from NSG mice engrafted with primary leukemia samples. All samples showed heterogeneous expression of CD9. CD9 high-positive cells and negative cells were flow sorted. Raman spectra of freshly sorted CD9 high-positive and negative cells were obtained. 10 to 20 cells were analyzed in each sample. CD34 positive cells, which were isolated from normal donors, were also analyzed by Raman spectroscopy as a control. No unique Raman fingerprints were identified to separate CD9 high-positive cells from negative cells using Principal Component Analysis (PCA). Furthermore, CD9 high-positive and negative cells from three preB ALL samples were transplanted into NSG mice via intra-bone marrow injection. Equal cell numbers (5×105 to 1.5×106 cells) were used for positive and negative samples in each injection. The majority of the mice from both groups (transplanted with CD9 high-positive or negative cells) developed leukemia 3 to 4 months after injection. Leukemia phenotype was confirmed to be the same as the original leukemia. In conclusion, although CD9 was shown to be a marker for LSC in B-ALL cell lines, it does not appear to be an LSC marker in primary preB ALL. Since childhood preB ALL is a heterogeneous group of diseases, larger cohorts are necessary to confirm our findings. Raman spectroscopy may be a useful screening tool for analysis of cellular intrinsic markers. Disclosures: No relevant conflicts of interest to declare.
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Fu, Jen-Fen, and Lee-Yung Shih. "Activated K-RAS Down-Regulated Small GTPases and Cell Motility of Myeloid Sarcoma-Inducing Leukemia Cells Through Erk Signaling Cascade." Blood 118, no. 21 (November 18, 2011): 2459. http://dx.doi.org/10.1182/blood.v118.21.2459.2459.
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Abstract Abstract 2459 The molecular mechanism of myeloid sarcoma (MS) formation in the patients with acute myeloid leukemia (AML) is not clear. By analyzing the genetic mutations, we found that AML patients with combined MLL/AF10 and activated N-/K-RAS mutation had a high occurrence of association with MS. We had previously performed a retroviral transduction/transplantation assay and demonstrated that the activated KRAS mutation could not immortalize murine bone marrow (BM) cells, whereas MLL/AF10 alone induced myeloproliferative disease-like myeloid leukemia (MPD) in the transplanted mice. Combination ofMLL/AF10 and activated K-RAS mutation induced MPD in a shorter latency, in addition, multiple MS were detected in the abdominal-, subcutaneous-, or intramuscular fat of the transplanted mice. To understand the role of activated KRAS mutation in cooperation with MLL/AF10 in the MS formation, we performed an in vivo imaging analysis to compare cell migration between cells carrying single and combined mutations. Our results showed that the cells carrying combined mutations were slowly migrating to the omental and gonadal adipose tissues of the transplanted mice after intra-peritoneal injection. On the contrary, the cells carrying only MLL/AF10 were rapidly condensed and entered into blood stream. An in vitro transwell migration assay confirmed that the cells carrying combined mutations exhibited a reduced cell motility in response to SDF-1 treatment. Analysis of the Cxcr4 gene expression did not show significant difference between cells carrying single and combined mutations, suggesting that the slow migration was not attributed to lacking receptor for SDF-1. Further determination of the cell migration-related small GTPases revealed that the amount of total and GTP–bound Rac, Cdc42 and Rho was reduced in the cells carrying combined mutations. Moreover, treatment of SDF-1 further down-regulated protein expression and activity of these small GTPases. To understand which signaling pathway(s) linked the KRAS mutation to the down-regulated small GTPases, we analyzed the amount of total and phosphorylated signaling molecules and found that Erk, but not Jnk1 and p38 Mapk, had a similar protein expression and activation pattern as those of the GTPases, suggesting that the reduction in cell motility of the MS-inducing leukemia cells might be mediated via KRAS-Mek-Erk axis. Disclosures: No relevant conflicts of interest to declare.
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Palmi, Chiara, Grazia Fazio, Ilaria Brunati, Valeria Cazzaniga, Valentina André, Silvano Sozzani, Antonello Villa, et al. "TEL-AML1 Affects the Regulation of Cytoskeleton and Causes Alteration In Cellular Adhesive and Migratory Properties In An In Vitro Model of Pre-Leukemia." Blood 116, no. 21 (November 19, 2010): 3624. http://dx.doi.org/10.1182/blood.v116.21.3624.3624.
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Abstract Abstract 3624 Introduction: The t(12;21) chromosome translocation generating TEL-AML1 chimeric fusion gene is a frequent initiating event in childhood leukaemia. Its impact is to generate a clone of covert, clinically silent pre-leukemic B cell progenitors. The leukemia arises only following second, post-natal hit/genetic events occurring years later. Moreover, relapse of leukemia is frequently arising from the pre-leukemic clone. Aim of our study is to investigate how TEL-AML1 expression can sustain this covert condition for many years. In a recent paper we described that the fusion gene rendered the B precursors resistant to the inhibitory activity of TGFbeta. Here we want to inquire into other factors that can explain the positive selection of the pre-leukemic clones over the normal counterpart. In particular, given the importance of the interaction with the microenvironment for survival signals for normal and leukemic stem cells, we question if the fusion gene causes changes in cellular adhesive and migratory properties. Methods: the study was performed by using two different models: i) a TEL-AML1 inducible expression system on the murine pro-B Ba/F3 cell line and ii) murine primary B lymphocytes (pre-BI cells) isolated from fetal liver, stably transduced with the pMIGR1-TEL-AML1-IRES-GFP construct. Gene expression assays were performed by using TaqMan (Applied Biosystems) and PCR Array technologies (SABioscences). Results: The expression of TEL-AML1 in Ba/F3 cell line causes over-expression of genes regulators of the cytoskeleton, specifically involved in cellular movement and in the regulation of actin dynamics. This gene expression alteration results in changes in the cellular morphology and phenotype: the cells acquire long extensions and several molecules involved in cell adhesion and migration are disregulated. Moreover, the TEL-AML1 positive cells present an increased ability to adhere to the ICAM1 substrate, but they also show a significant defect in the chemotactic response to CXCL12 in transwell migration assays in vitro, although the expression and the recycling of CXCR4 receptor are unaffected. This inability is not due to defects to migrate in general, as spontaneous motility is enhanced, but it is associated with a defect in CXCR4 signaling. In particular, CXCL12 calcium flux and ERK phosphorylation were inhibited. Those results have been confirmed in murine PreBI primary cells. Conclusions: in our murine models, TEL-AML1 affects the cytoscheleton regulation and causes alteration in cellular adhesive and migratory properties. We are now investigating how these alterations can give advantages to the pre-leukemic cells in the pathogenesis of TEL-AML1–expressing leukemia. Disclosures: No relevant conflicts of interest to declare.
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Giannopoulos, Krzysztof, Iwona Hus, Li Li, Agnieszka Bojarska-Junak, Jochen Greiner, Jacek Rolinski, Anna Dmoszynska, Hartmut Doehner, and Michael Schmitt. "The Receptor for Hyaluronic Acid Mediated Motility (RHAMM/CD168) Is a Potential Target for Immunotherapy of Patients with B-Cell Chronic Lymphocytic Leukemia." Blood 106, no. 11 (November 16, 2005): 53. http://dx.doi.org/10.1182/blood.v106.11.53.53.
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Abstract Definition of appropriate target antigens might open new avenues to antigen targeted immunotherapies for patients with B-cell chronic lymphocytic leukemia (B-CLL). We screened the mRNA expression of tumor associated antigens (TAAs), from the literature (fibromodulin, survivin, OFA-iLRP, BAGE, G250, MAGE1, PRAME, proteinase, Syntaxin, hTERT, WT-1), and TAAs defined earlier by serological analysis of cDNA expression libraries from leukemic cells (PINCH, HSJ2, MAZ, MPP11, RHAMM/CD168, NY-Ren60). Peripheral blood mononuclear cells from 43 B-CLL patients and 20 healthy volunteers (HVs) were examined by conventional and quantitative RT-PCR. mRNA of RHAMM/CD168, fibromodulin, syntaxin and NY-Ren60 was expressed in 55–90%, mRNA of HSJ2, MAZ and OFA-iLRP in 90–100% of the patients. No expression of WT-1, h-TERT, BAGE, G250, MAGE1 and survivin was observed. Low (2–20%) expression frequencies of MPP11, PINCH, PRAME and proteinase were detected. RHAMM/CD168, fibromodulin, PRAME and MPP11 showed expression in B-CLL patients, but not in HVs. Because of the exquisite tissue expression of RHAMM/CD168 and its high expression frequency in B-CLL patients even in early stages of disease, mixed lymphocyte peptide culture (MLPC), enzyme-linked immunosorbent spot (ELISPOT) and flow cytometry were performed for antigen specific T cells. In MLPC, RHAMM specific responses by CD8+HLA-A2/R3tetramer+CCR7-CD45RAhigh effector T cells were detected. Moreover, we observed an enhanced RHAMM/CD168 specific CD8+ T cell response after vaccination in one from four HLA-A2 positive B-CLL patients vaccinated with tumor cell lysate pulsed dendritic cells. Therefore, RHAMM/CD168 is an interesting candidate antigen for future immunotherapies in both ZAP-70 (+) and ZAP-70 (−) B-CLL patients.
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Burthem, J., PK Baker, JA Hunt, and JC Cawley. "The function of c-fms in hairy-cell leukemia: macrophage colony- stimulating factor stimulates hairy-cell movement." Blood 83, no. 5 (March 1, 1994): 1381–89. http://dx.doi.org/10.1182/blood.v83.5.1381.1381.
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Abstract Hairy cells (HCs) and some activated B cells express high levels of macrophage colony-stimulating factor (M-CSF) (CSF-1) receptor, but the functional effects of the cytokine on B cells have not been previously identified. Using video microscopy, image analysis, and migration assays, M-CSF was shown to induce chemokinetic and chemotactic movement of HCs. This movement response involved transition to a highly mobile, rounded cell form and was accompanied by distinctive changes in F-actin polymerization and distribution. Furthermore, the M-CSF-induced motility was substantially modified by the adhesive protein used as a substratum and involved qualitative changes in the function of the alpha v beta 3 integrin of HCs. It is suggested that the findings are relevant to the pathophysiology of hairy-cell leukemia (HCL) in particular, and to the biology of B-cell migration in general.
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Burthem, J., PK Baker, JA Hunt, and JC Cawley. "The function of c-fms in hairy-cell leukemia: macrophage colony- stimulating factor stimulates hairy-cell movement." Blood 83, no. 5 (March 1, 1994): 1381–89. http://dx.doi.org/10.1182/blood.v83.5.1381.bloodjournal8351381.
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Hairy cells (HCs) and some activated B cells express high levels of macrophage colony-stimulating factor (M-CSF) (CSF-1) receptor, but the functional effects of the cytokine on B cells have not been previously identified. Using video microscopy, image analysis, and migration assays, M-CSF was shown to induce chemokinetic and chemotactic movement of HCs. This movement response involved transition to a highly mobile, rounded cell form and was accompanied by distinctive changes in F-actin polymerization and distribution. Furthermore, the M-CSF-induced motility was substantially modified by the adhesive protein used as a substratum and involved qualitative changes in the function of the alpha v beta 3 integrin of HCs. It is suggested that the findings are relevant to the pathophysiology of hairy-cell leukemia (HCL) in particular, and to the biology of B-cell migration in general.
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Ramsay, Alan G., Rachel Evans, Lena Svensson, Shahryar Kiaii, Nancy Hogg, and John G. Gribben. "Defective LFA-1 Mediated T Cell Motility In Chronic Lymphocytic Leukemia Is Mediated by Defects In the Rho GTPase Signaling Pathway." Blood 116, no. 21 (November 19, 2010): 914. http://dx.doi.org/10.1182/blood.v116.21.914.914.
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Abstract Abstract 914 T lymphocytes have an essential role in adaptive immunity and rely on tightly regulated signaling through integrin lymphocyte function-associated antigen (LFA)-1 to migrate into lymph nodes and interact with antigen-presenting cells. Malignant cells modify their immune microenvironment to prevent effective host anti-tumor responses, promote tumor progression, and suppress the therapeutic benefit of immunotherapy treatments. Here we assessed LFA-1-mediated cell migration of highly purified T cells from treatment naïve chronic lymphocytic leukemia (CLL) patients compared to age-matched healthy donor T cells using CXCL12 stimulation and immobilized ICAM-1, the principal integrin ligand. Video microscopy with motility tracking analysis identified that both CD4 and CD8 T cells from CLL patients (n=14) exhibited significantly reduced migration rates (P < .01) compared to healthy donor T cells (5.5 ± 0.3 (SEM) μm/min and 4.4 ± 0.2 μm/min compared to 8.2 ± 0.3 μm/min and 7.5 ± 0.3 μm/min respectively). We further identified that direct CLL cell contact, and not soluble factors alone, induced similar T cell motility dysfunction in previously healthy CD3 T cells. Primary co-culture of healthy donor T cells with CLL cells caused a significant decrease in the speed of migration on ICAM-1 compared to coculture with control healthy B cells (6.2 ± 0.3 μm/min versus 9.5 ± 0.6 μm/min) (n=9) (P < .05). Next we sought to repair this T cell defect in CLL using a clinically relevant agent. We identify that treatment of CLL patient T cells (n=9) with lenalidomide restores rapid LFA-1 mediated migration on ICAM-1. Ex vivo treatment of CLL T cells with lenalidomide (1μM for 24 hours) significantly increased the speed of T cell migration compared to untreated patient T cells (7 ± 0.4 μm/min versus 2.5 ± 0.7 μm/min) (P < .05) and the rescued T cell migratory function of lenalidomide exposed patient T cells was comparable to healthy donor T cells treated with or without drug. Interference reflection microscopy (IRM) examining the contact zone between migrating T cells and ICAM-1 identified a significant CLL patient T cell adhesion defect (P < .05) with reduced spreading area and strength of adhesive contacts (pixel density) compared to healthy donor T cells. IRM was further utilized with pharmacological inhibitors to demonstrate that exposure to lenalidomide rescued CLL T cell adhesion by acting on the Rho family GTPases that are dysregulated in cancer patient T cells. Lenalidomide significantly increased (P < .05) levels of active RhoA in CLL patient T cells compared to untreated cells. In addition, untreated CLL patient T cells adhering to ICAM-1 exhibited significantly reduced expression levels of phosphorylated myosin light chain (MLC) compared to healthy donor T cells (P < .05) and this defect was repaired following lenalidomide treatment. MLC is normally phosphorylated by MLC kinase at the T cell leading edge and by the RhoA target, ROCK at the trailing edge, and is an important downstream signaling molecule during LFA-1-mediated T cell motility. Further expression analysis identified that lenalidomide significantly increased (P < .01) ICAM-1-engaged high-affinity LFA-1 in CLL patient T cells to levels comparable to healthy donor T cells. Overall, our results show that T cells in CLL patients have dysfunctional tumor-induced cytoskeletal signaling via the Rho GTPase signaling pathway, and this is reversed by lenalidomide, rescuing dynamic LFA-1 mediated outside-in signalling and migration. Lenalidomide's immunomodulatory activity was highly cancer T cell specific: rescuing defective LFA-1 migration and signaling in CLL T cells, but with no detectable effects on healthy donor T cells. These findings provide important mechanistic insight into the action of lenalidomide, and highlight the potential clinical utility of immunomodulatory drugs to rescue normal immune function in cancer. Disclosures: Gribben: Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.
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Sun, Hongbo, Zhifu Zhang, Wei Luo, Junmin Liu, Ye Lou, and Shengmei Xia. "MiR-7 Functions as a Tumor Suppressor by Targeting the Oncogenes TAL1 in T-Cell Acute Lymphoblastic Leukemia." Technology in Cancer Research & Treatment 19 (January 1, 2020): 153303382093413. http://dx.doi.org/10.1177/1533033820934130.
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Background: T-cell acute lymphoblastic leukemia is a hematologic malignancy characterized by T-cell proliferation, and in many cases, the ectopic expression of the oncogenic transcription factor T-cell acute lymphocytic leukemia protein 1 (TAL1). MicroRNA-7 has been shown to play a critical role in proliferation, migration, and treatment sensitivity in a diverse array of cancers. In this study, we sought to establish a novel link between microRNA-7 and T-cell acute lymphoblastic leukemia oncogenesis. Material and Method: To do so, we characterized gene expression of microRNA-7 as well as TAL1 in both T-cell acute lymphoblastic leukemia patient-derived tissue and cell lines, as well as performing functional luciferase assays to assess microRNA-7 binding to the TAL1 3′-untranslated region. We also performed growth, apoptosis, and migration experiments using 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide, Annexin V, and transwell assays in the context of microRNA-7 overexpression. Results: We found that microRNA-7 expression is attenuated and inversely correlated with TAL1 expression in TAL1 + T-cell acute lymphoblastic leukemia cells. Additionally, microRNA-7 directly targets and suppresses TAL1 levels. Finally, microRNA-7 overexpression reduces growth, motility, and migration while inducing apoptosis in T-cell acute lymphoblastic leukemia cells, phenotypes that can be rescued by concomitant overexpression of TAL1. Conclusions: These results indicate that microRNA-7 functions as a potent tumor suppressor by inhibiting the oncogene TAL1 and suggest microRNA-7 could function as a prognostic biomarker and possible therapeutic in the clinical management of T-cell acute lymphoblastic leukemia.
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Michailov, Yulia, Eitan Lunenfeld, Joseph Kapilushnik, Shevach Friedler, Eckart Meese, and Mahmoud Huleihel. "Acute Myeloid Leukemia Affects Mouse Sperm Parameters, Spontaneous Acrosome Reaction, and Fertility Capacity." International Journal of Molecular Sciences 20, no. 1 (January 8, 2019): 219. http://dx.doi.org/10.3390/ijms20010219.
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Leukemia is one of the most common cancers in patients of reproductive age. It is well known that chemotherapy, used as anti-cancer therapy, adversely affects male fertility. Moreover, the negative effect of leukemia on sperm quality, even before chemotherapy treatment, has been reported. However, the mechanisms behind this disease’s effect on sperm quality remains unknown. In this study, we examine the direct effect of leukemia and chemotherapy alone and in combination on sperm parameters and male fertility. For this, we developed an acute myeloid leukemia (AML) mouse model (mice were treated with AML cells C1498 and developed leukemia); these mice then received cytarabine chemotherapy. Our findings reveal a significant reduction in sperm concentration and motility and a significant increase in abnormal morphology and spontaneous acrosome reaction of the sperm following AML and chemotherapy treatment, alone and in combination. We also found a reduction in male fertility and the number of delivered offspring. Our results support previous findings that AML impairs sperm parameters and show for the first time that AML increases spontaneous acrosome reaction and decreases male fertility capacity and number of offspring.
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Chen, Jinfei, Anita Schmitt, Baoan Chen, Markus Rojewski, Jochen Greiner, Philippe Guillaume, Hartmut Dohner, Donald Bunjes, and Michael Schmitt. "Imatinib Inhibits Both CD4+ T Regulatory Cells and CD8+ T Lymphocytes Specifically Directed Against the Leukemia-Associated Antigen RHAMM/CD168." Blood 108, no. 11 (November 1, 2006): 2201. http://dx.doi.org/10.1182/blood.v108.11.2201.2201.
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Abstract Imatinib mesylate (IM, STI571, Gleevec®) is highly effective in the treatment of patients with chronic myelogenous leukemia (CML). In patients with residual disease or relapse after allogeneic stem cell transplantation (allo-SCT), the optimal strategy is a matter of debate: donor lymphocyte infusions (DLIs) alone, IM alone or a combination of both have been administered to CML patients in this clinical situation. As an impairment of anti-viral CD8+ T lymphocyte function by IM has been described, we questioned whether IM also affects specific anti-leukemic CD8+ T lymphocytes. Moreover, we asked to which extent CD4+CD25hi T regulatory cells might be affected by IM thus changing the balance between cytotoxic and tolerogenic T cells. After mixed lymphocyte peptide culture (MLPC), we assessed CD8+ T cells from healthy donors and patients with CML after allo-SCT by tetramer staining/multi-color flow cytometry and enzyme linked immunosorbent spot (ELISPOT) assays, as well as CD4+CD25hicells after 3 days of culture with anti-CD3 and anti-CD28. The release of interferon gamma and granzyme B by CD8+ T lymphocytes specific for R3 (ILSLELMKL), a T cell epitope peptide derived from the leukemia-associated antigen designated RHAMM/CD168 (receptor for hyaluronic acid mediated motility) was inhibited by IM in a clearly dose-dependent fashion in a range from 1 to 25 μM. These T cells were further characterized as CD8+ HLA-A2/R3_tetramer+ WT1_tetra-CD45RA+CD27-CD28-CCR7- effector T cells with the ability to lyse R3 peptide labeled T2 cells and CD34+ CML progenitor cells. The inhibition of CD8+ T lymphocytes was a function of the time of exposure to IM and reversible after removal of IM from the MLPC. On the other hand, the proliferation and function of CD4+CD25hiFoxP3+GITR+TGFß1+ CD69+CD152+ T regulatory cells were also significantly inhibited by IM in a dose-related fashion. These findings suggest that the clinical impact IM must not necessarily result in a reduced efficacy of the graft-versus-leukemia effect observed after allo-SCT and/or DLIs but might depend rather on the ratio of CD8+ cytotoxic T cells and CD4+CD25hi T regulatory cells.
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Karajannis, Matthias A., Loïc Vincent, Fan Zhang, Sergey V. Shmelkov, Joseph Cheung, Peter Bohlen, Zhenping Zhu, Paul Kussie, Haijun Sun, and Shahin Rafii. "Fibroblast Growth Factor Receptor-1 Expression and Signaling in Acute Myeloid Leukemia." Blood 104, no. 11 (November 16, 2004): 4477. http://dx.doi.org/10.1182/blood.v104.11.4477.4477.
Full textAbstract:
Abstract Fibroblast growth factor receptors (FGFRs) are tyrosine kinase receptors affecting cell proliferation, motility and survival. Fibroblast growth factor 2 (FGF2, basic FGF) represents the prototype FGFR ligand. Expression of FGFRs has been demonstrated in a subset of acute myeloid leukemias (AMLs) and FGF2 is overexpressed in the bone marrow of AML patients. The role of FGF/FGFR signaling in AML however is controversial, and downstream signaling cascades activated by FGFR1 are not known. Therefore, we hypothesized that subsets of primary acute leukemic cells may express functional FGFR1 mediating survival and proliferation of leukemic cells. We examined the role of the FGF/FGFR axis in primary AML blasts isolated from a 74 year old male (designated R81). Using RT-PCR we showed that FRFR1 was expressed in leukemic cells. To assess whether FGF2 stimulates FGFR1 signaling, R81 blasts were incubated in serum free medium with and without 20 ng/ml of FGF2 and/or a with 10 μg/ml of neutralizing monoclonal antibody to FGFR1. Viable cells were counted at 48 and 96 hours with trypan blue exclusion (see figure). The results show that FGF2 stimulates growth and/or survival of R81 AML blasts and this effect is abrogated by FGFR1 antibodies. Specifically, FGF2 induced a two-fold increase in the proliferation of leukemic blasts after 48 hours. To identify downstream signaling pathways involved in FGFR1 signaling, proliferation and survival of FGF2-treated R81 cells were examined in the presence or absence of selective PI3 kinase and MAP kinase inhibitors, LY-294002 and PD098059, respectively. The results indicated that FGF2 signaling exerts its proliferative and pro-survival effects primarily through PI3 kinase activation. In summary, we show that FGF/FGFR1 signaling plays a role in a subset of AMLs and may be blocked by a FGFR1 specific antibody. Currently, we are further elucidating the mechanisms involved in FGFR signaling and its effects on cell proliferation, migration, and apoptosis. In addition, animal experiments are underway to assess a possible therapeutic role of anti-FGFR antibodies for the treatment of FGFR-positive AML in murine xenotransplant models. Figure Figure
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Hoellenriegel, Julia, Dirk Zboralski, Christian Maasch, Nathalie Y. Rosin, William G. Wierda, Michael J. Keating, Anna Kruschinski, and Jan A. Burger. "The Spiegelmer NOX-A12, a novel CXCL12 inhibitor, interferes with chronic lymphocytic leukemia cell motility and causes chemosensitization." Blood 123, no. 7 (February 13, 2014): 1032–39. http://dx.doi.org/10.1182/blood-2013-03-493924.
Full textAbstract:
Key Points NOX-A12, a structured mirror-image RNA oligonucleotide that neutralizes CXCL12, interferes with CLL migration and drug resistance. NOX-A12 inhibits chemotaxis and sensitizes CLL cells toward cytotoxic drugs, providing a rationale for NOX-A12 combination therapy.
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Ramsay, Alan G., Lena Svensson, Nancy Hogg, and John G. Gribben. "Defective T Cell Migration in Chronic Lymphocytic Leukemia Is Repaired by Lenalidomide." Blood 112, no. 11 (November 16, 2008): 3117. http://dx.doi.org/10.1182/blood.v112.11.3117.3117.
Full textAbstract:
Abstract We have previously demonstrated that multiple gene expression abnormalities are induced in T cells from chronic lymphocytic leukemia (CLL) patients including defects within the actin cytoskeleton signaling pathways that control immune recognition and motility (Gullu et al. JCI, 2005). T cell immune surveillance requires rapid migratory responses and LFA-1 (CD11a/CD18; αLβ2) is a promigratory receptor that engages the cytoskeleton to control migration. We hypothesized that CLL T cells may exhibit dysfunctional migration in response to ICAM-1, the principal ligand for LFA-1. Using time lapse microscopy, we observed significantly reduced chemokine SDF-1 (CXCL12) induced migration on ICAM-1 of CLL CD4 and CD8 T cells compared to age-matched healthy donor T cells. Healthy T cells tracked for 45 min displayed a random course of migration with an average speed of ~ 8 μm/min, whereas CLL T cells were slower ~ 5 μm/min (n=14, ~ 30% reduction, p<0.01). We further postulated that direct contact of CLL tumor cells with healthy T cells would induce this migratory defect. Healthy CD4 or CD8 T cells were cocultured with either allogeneic CLL B cells or allogeneic healthy B cells and subsequently used in migration assays. Co-culture with CLL cells resulted in significantly reduced T cell migration compared with co-culture with healthy B cells (~ 44% reduction in migration, n=6, p<0.01). Evidence that direct contact was required to induce this migratory defect was shown when no effect was observed when cell-cell adhesion was prevented by pretreatment of CLL cells with anti-ICAM-1 blocking antibody prior to primary co-culture with healthy T cells. This cancer-induced migratory defect was repaired when CLL T cells were pretreated with the immunomodulatory drug Lenalidomide (1μM for 1hr). Treatment with this agent enhanced the migratory potential of CLL T cells to a speed comparable to untreated and treated healthy T cells. The finding that lenalidomide can restore rapid migration in patient T cells provides evidence that this agent may increase immune surveillance in CLL patients.
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Paggetti, Jerome, Franziska Haderk, Martina Seiffert, Bassam Janji, Yeoun Jin Kim, Ute Diestler, Wim Ammerlaan, et al. "Chronic Lymphocytic Leukemia-Exosomes Switch Endothelial and Mesenchymal Stromal Cells into Cancer-Associated Fibroblasts to Sustain Leukemic Cell Survival." Blood 124, no. 21 (December 6, 2014): 2927. http://dx.doi.org/10.1182/blood.v124.21.2927.2927.
Full textAbstract:
Abstract Chronic lymphocytic leukemia (CLL), the most common hematologic malignancy in Western countries, is mostly affecting the elderly over 65 year-old. CLL is characterized by the accumulation of mature but non-functional B lymphocytes of clonal origin in the blood and the primary lymphoid organs. CLL was previously considered as a relatively static disease resulting from the accumulation of apoptosis-resistant but quiescent B lymphocytes. However, recent studies using heavy water labeling indicated that CLL is in fact a very dynamic disease with alternation of proliferation phases and peripheral circulation. A focus on the trafficking of CLL cells in vivo has shown that leukemic cells circulate between the blood and the lymphoid organs but have a preference for the bone marrow. Recent next-generation sequencing of CLL cells indicated the presence of different genetic subclones. This intraclonal heterogeneity observed in CLL subpopulations may be in part determined by the interactions that leukemic cells entertain with their microenvironment when B cells migrate into the lymph nodes and the bone marrow. Indeed, tumor-stroma interactions are not only providing signals necessary for leukemic cells survival but may also influence the clonal architecture and evolution. One of these interactions involves CLL-derived exosomes. Here, we show that CLL-exosomes efficiently transfer nucleic acids, including functional microRNAs, and proteins, including MHC-Class II molecules and B-cell specific proteins, to bone marrow mesenchymal stem cells and endothelial cells. CLL-exosomes also activate signaling pathways, including PI3K and NF-κB pathways, in these stromal cells. As a consequence, gene expression is strongly modified indicating a switch towards a cancer-associated fibroblast phenotype. Functionally, exosome-stimulated stromal cells show a striking actin cytoskeleton remodeling characterized by the formation of stress fibers, and enhanced proliferation, motility and angiogenic properties. We also identified several proteins synthesized and secreted by stromal cells that promote leukemic cell adhesion and survival ex vivo. To confirm the involvement of CLL-exosomes in CLL pathology in vivo, MEC-1-eGFP cells were subcutaneously injected into immunocompromised NSG mice together with CLL-exosomes. We observed a significant increase in tumor size and a reduction in survival of exosome-treated animals. Flow cytometry analysis of selected organs indicated an enrichment in leukemic cells in the kidney, providing a potential explanation to the renal failures observed in CLL patients. In conclusion, the communication between CLL cells and stromal cells may be a critical factor influencing CLL progression by promoting leukemic cell survival. This study demonstrates the crucial role of exosomes as mediators of the communication between leukemic cells and their microenvironment. Exosomes could thus represent a suitable target for therapeutic intervention in CLL. Disclosures No relevant conflicts of interest to declare.
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Jolly, Puneet S., Meryem Bektas, Kenneth R. Watterson, Heidi Sankala, Shawn G. Payne, Sheldon Milstien, and Sarah Spiegel. "Expression of SphK1 impairs degranulation and motility of RBL-2H3 mast cells by desensitizing S1P receptors." Blood 105, no. 12 (June 15, 2005): 4736–42. http://dx.doi.org/10.1182/blood-2004-12-4686.
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Abstract Mast cells play a central role in inflammatory and immediate-type allergic reactions by secreting a variety of biologically active substances, including sphingosine-1 phosphate (S1P). Sphingosine kinase 1 (SphK1) and formation of S1P, which leads to transactivation of S1P receptors and their downstream signaling pathways, regulates mast-cell functions initiated by cross-linking of the high-affinity immunoglobulin E (IgE) receptor FcϵRI. Surprisingly, overexpression of SphK1 in rat basophilic leukemia (RBL)-2H3 mast cells impaired degranulation as well as migration toward antigen. These effects were reversed by serum withdrawal, yet the increased formation and secretion of S1P were the same as in the presence of serum. Nonetheless, serum increased localization of SphK1 at the plasma membrane. This restricted formation of S1P induced internalization and desensitization of S1P receptors on the surface of mast cells as determined by confocal immunofluorescence microscopy, aberrant S1P receptor signaling, and lack of S1P receptor coupling to G proteins. Serum starvation, which significantly reduced membrane-associated SphK1 activity, restored S1P receptor functions. Our results have important implications for mast-cell migration and degranulation as well as for the biologic functions of the S1P receptors on cells that are circulating in the bloodstream. (Blood. 2005;105:4736-4742)