A Novel Role For Histone Deacetylase 11 (HDAC11) As a Regulator Of Neutrophil Function and Differentiation In Normal and Malignant Hematopoesis

Abstract Histone Deacetylase 11 (HDAC11) is the newest member of the HDAC family of enzymes, which we have previously reported to function as a negative regulator of IL-10 expression in macrophages and dendritic cells. Thus far, its role in other hematopoietic cells has not been completely elucidated. We hereby report for the first time a lineage-restricted over-expression of HDAC11 in neutrophils, committed neutrophil precursors and myeloid leukemias exhibiting neutrophilic differentiation demonstrating a novel physiological role of HDAC11 as a negative regulator of neutrophil cytokine production. Leukocyte subpopulations from murine bone marrow and spleen were flow-sorted and analyzed by qRT-PCR for HDAC11 mRNA, revealing a higher level of mRNA expression on neutrophils and promyelocytes, as compared to monocytes and lymphoid subsets. Similarly, sorted human peripheral blood leukocytes from normal donors, showed higher levels of HDAC11 mRNA in neutrophils, as compared to monocytes. To further investigate the transcriptional activity of HDAC11 in myeloid and lymphoid cells, we utilized a HDAC11 promoter-driven eGFP reporter mice, where eGFP expression indicates HDAC11 transcription (Heintz, N Nat. Rev. Neuroscience 2001). Using multiparametric flow cytometry with lineage-specific markers on this mouse model, we confirmed a marked over-expression of HDAC11 on neutrophils, compared to other subpopulations including monocytes, B-cell, T-cells, NK cells and plasma cells. Furthermore, analysis of bone marrow hematopoietic cells revealed a swift over-expression of HDAC11 at the promyelocyte stage of neutrophil differentiation, with low to undetectable expression in upstream uncommitted common myeloid progenitors and lineage-unrelated monocytic precursors. To study whether this lineage-specific overexpression applies to malignant processes, we studied human cell lines and found overt overexpression of HDAC11 in the acute promyelocytic leukemia cell line NB4, as compared to the myeloblastic cell line Kasumi and two monocyte/macrophage cell lines U937 and THP1. Moreover, in-vitro maturation of the differentiation-inducible myeloid cell line HL60 demonstrated a marked increase in HDAC11 mRNA, paralleling the acquisition of nuclear segmentation characteristic of neutrophil maturation. In order to investigate the physiologic role of HDAC11 overexpression on neutrophils, we utilized a model of germline-HDAC11KO mice. Surprisingly, highly purified neutrophils lacking HDAC11 showed an overt overproduction of TNF-alpha and IL-6 upon stimulation with LPS, as compared to their wild type counterparts. We hereby report a previously un-described lineage-specific over-expression of HDAC11 in neutrophils and its precursors, which actively functions as a physiological repressor of cytokine production and possibly involved in their regulation. Given the predominance of neutrophils which account for 70% of leukocytes in the peripheral blood, and their pivotal role in the first line of defense, results highlight a novel mechanism for HDAC11, as a key regulator and modulator of neutrophil cytokine production with potential implications for autoimmunity, inflammation, and infection. Disclosures: No relevant conflicts of interest to declare.

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BCL2A1 Is A Survival and Immortalization Factor for Primitive Myeloid Hematopoietic Cells.

Blood ◽

10.1182/blood.v110.11.3365.3365 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3365-3365

Author(s):

Jean-Yves Metais ◽

Ashley E. Dunfee ◽

Rodrigo T. Calado ◽

Cynthia E. Dunbar

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Gene Product ◽

Myeloid Leukemia ◽

Hematopoietic Cells ◽

Marker Genes ◽

Empty Vector ◽

Myeloid Progenitor ◽

The Impact

Abstract We recently reported development of an acute myeloid leukemia in a rhesus macaque transplanted with autologous CD34+ cells transduced with a murine stem cell virus-derived replication defective retrovirus vector expressing only marker genes under control of the strong MCSV LTR. This animal had an unusual clonal reconstitution pattern the first year following transplant, with a single transduced myeloid progenitor cell clone accounting for up to 80% of then normal myelopoiesis (Kelly, 2005). The same vector-containing clone then transformed to AML five years following transplantation, and each tumor cell was shown to contain two vector insertions, one localized 20 kb upstream the CDw92 gene on chromosome 9, and the second localized in the first intron of BCL2A1 on chromosome 15 (Seggewiss, 2006), a gene in the anti-apoptotic BCL2 family not previously linked to myeloid leukemia. BCL2A1 was highly expressed in the tumor cells. This tumor was the first hematopoietic malignancy reported in a recipient of primitive cells transduced with a replication-incompetent vector containing only marker genes, and suggested that BCL2A1 could have potent effects on myeloid cell behavior. To investigate the impact of the BCL2A1 gene product on hematopoietic cells, we cloned the murine and human HA-tagged BCL2A1 cDNAs into lentivirus vectors and transduced the murine BaF3 hematopoietic cell line as a model to study the impact of expression of these proteins on hematopoiesis. We confirmed overexpression of the proteins in the producer cell line as well as in transduced cells by western blot using an anti-HA monoclonal antibody. BaF3 cell proliferation and survival are dependant on IL-3, and under IL-3 replete conditions overexpression of murine or human BCL2A1 did alter proliferation compared with untransduced cells or cells transduced with an empty vector. Removal of IL-3 from the cell culture media leads to rapid apoptosis of BaF3 cells, with cell cycle arrest in the G1 and an apoptotic subpopulation appearing within 24 hours of IL-3 removal. 45% untransduced or empty vector cells were apoptotic, and this fraction decreased to 30% and 15% respectively for BaF3 cells expressing murine or human BCL2A1. These results were confirmed by direct analysis of apoptosis. Only BaF3 cells over-expressing human BCL2A1 were still alive and arrested in G1 after 3 days of culture without IL-3. The murine BCL2A1 had similar but less striking effects. Gene expression analyses on the BaF3 cell populations are ongoing, to identify potential downstream targets of the BCL2A1 protein. The BCL2A1 and empty vectors were also utilized in murine bone marrow cell immortalization assay, previously utilized to identify genes impacting on the survival and expansion of primary myeloid progenitor cells (Du, 2005). In an initial set of experiments, clonal clonal expansion was obtained with marrow cells expressing murine (4 clones) and human (5 clones) BCL2A1 but not for empty vector or untransduced murine marrow. Mice have also been transplanted with primary bone marrow cells transduced with the BCL2A1 and control vectors, and are being followed for in vivo expansion of transduced clones and development of leukemia. In conclusion, we have confirmed the role of BCL2A1 as an anti-apoptotic protein, now in myeloid hematopoietic cells, and will continue to investigate the role of this gene product in hematopoiesis and leukemogenesis.

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Unexpected Evidence That Inducible Nitric Oxide Synthase (iNOS) Is a Negative Regulator of Hematopoietic Stem/Progenitor Cell Mobilization

Blood ◽

10.1182/blood.v128.22.3372.3372 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3372-3372

Author(s):

Mateusz Adamiak ◽

Ahmed Abdelbaset-Ismail ◽

Marcin Wysoczynski ◽

Ahmed Abdel-Latif ◽

Joseph B Moore ◽

...

Keyword(s):

Nitric Oxide ◽

Cell Migration ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Hematopoietic Cell ◽

Negative Regulator ◽

Heme Oxygenase 1 ◽

Cell Trafficking ◽

Cell Mobilization

Abstract Background . Nitric oxide (NO) is gaseous free radical molecule involved in several biological processes related to inflammation, tissue damage, and infections. NO is synthesized by three isoforms of nitric oxide synthetase: two constitutively expressed isoforms, neuronal NOS (nNOS) and endothelial NOS (eNOS), and one isoform (iNOS) that is induced in response to inflammation. Interestingly, iNOS activity is enhanced during complement cascade (ComC) activation, which is a crucial element of innate immunity, and its cleavage fragments C3 and C5 (C3a and C5a, respectively) orchestrate the egress of HSPCs from BM into peripheral blood (PB). Mobilization studies in eNOS-/- mice revealed that the lack of constitutively expressed eNOS in endothelium reduces vascular endothelial growth factor (VEGF)-induced mobilization of endothelial progenitor cells (EPCs) and increases murine mortality after myelosuppression (Nature Med. 2003, 9:1370-6). However, no mobilization studies have been performed so far to assess the role of inducible hematopoietic cell-expressed iNOS in the mobilization of HSPCs. Because of this research void, we became interested in the potential role of iNOS in mobilization of HSPCs. This interest had two motivations. First, it was demonstrated in a recent paper that NO inhibits migration of granulocytes and monocytes, and it is known that these cells are crucial in facilitating egress of HSPCs across the bone marrow (BM)-peripheral blood (PB) barrier. Second, NO, which promotes inflammation, may have anti-inflammatory activity, and inflammation itself is one of the driving forces in HSPC mobilization. Aim of the study. To fill in this knowledge gap, we focused on the role of iNOS in the trafficking of hematopoietic cells as well as mobilization and homing of HSPCs. Materials and Methods. iNOS was upregulated or downregulated in established hematopoietic cell lines to study migration and adhesion of these cells. HSPC mobilization studies were performed in iNOS-/- mice, and, in parallel, homing and engraftment of iNOS-/- BM cells was evaluated in wild type animals. Since activation of the ComC is negatively regulated by heme oxygenase 1 (HO-1), and the ComC may downregulate HO-1, we tested the effect of HO-1 on iNOS-mediated cell trafficking. Results. Our results indicate that iNOS is a negative regulator of hematopoietic cell migration and prevents egress of HSPCs into PB during the mobilization process. At the molecular level, downregulation of iNOS resulted in downregulation of HO-1, and, vice versa, upregulation of iNOS enhanced HO-1 activity. Since, as we observed recently, HO-1 is a negative regulator of cell migration, the inhibitory effects of iNOS on the mobilization of HSPCs can be at least partially explained by its enhancing the HO-1 level in BM cells. Moreover, our studies, in which we generated hematopoietic chimeras, revealed that the iNOS effect depends on its expression in hematopoietic cells. Conclusions. We identified iNOS as a negative regulator of cell trafficking and mobilization of HSPCs. Further studies are needed to establish the mutual relationship between activation of iNOS and activation of HO-1 in hematopoietic cells, as both inducible enzymes are potent negative regulators of HSPC trafficking and together and regulate stem cell mobilization and homing. Therefore, inhibition of iNOS or HO-1 in HSPCs by employing small molecular inhibitors could find practical clinical application. Disclosures No relevant conflicts of interest to declare.

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Transendothelial Migration of CD34+ and Mature Hematopoietic Cells: An In Vitro Study Using a Human Bone Marrow Endothelial Cell Line

Blood ◽

10.1182/blood.v89.1.72.72_72_80 ◽

1997 ◽

Vol 89 (1) ◽

pp. 72-80 ◽

Cited By ~ 2

Author(s):

Robert Möhle ◽

Malcolm A.S. Moore ◽

Ralph L. Nachman ◽

Shahin Rafii

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Progenitor Cells ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Transendothelial Migration ◽

Plating Efficiency ◽

Cd34 Cells ◽

Upper Chamber

To study the role of bone marrow endothelial cells (BMEC) in the regulation of hematopoietic cell trafficking, we have designed an in vitro model of transendothelial migration of hematopoietic progenitor cells and their progeny. For these studies, we have taken advantage of a human BMEC-derived cell line (BMEC-1), which proliferates independent of growth factors, is contact inhibited, and expresses adhesion molecules similar to BMEC in vivo. BMEC-1 monolayers were grown to confluency on 3 μm microporous membrane inserts and placed in 6-well tissue culture plates. Granulocyte-colony stimulating factor (G-CSF )–mobilized peripheral blood CD34+ cells were added to the BMEC-1 monolayer in the upper chamber of the 6-well plate. After 24 hours of coincubation, the majority of CD34+ cells remained nonadherent in the upper chamber, while 1.6 ± 0.3% of the progenitor cells had transmigrated. Transmigrated CD34 cells expressed a higher level of CD38 compared with nonmigrating CD34+ cells and may therefore represent predominantly committed progenitor cells. Accordingly, the total plating efficiency of the transmigrated CD34+ cells for lineage-committed progenitors was higher (14.0 ± 0.1 v 7.8% ± 1.5%). In particular, the plating efficiency of transmigrated cells for erythroid progenitors was 27-fold greater compared with nonmigrating cells (8.0% ± 0.8% v 0.3% ± 0.1%) and 5.5-fold compared with unprocessed CD34+ cells (2.2% ± 0.4%). While no difference in the expression of the β1-integrin very late activation antigen (VLA)-4 and β2-integrin lymphocyte function-associated antigen (LFA)-1 was found, L-selectin expression on transmigrated CD34+ cells was lost, suggesting that shedding had occurred during migration. The number of transmigrated cells was reduced by blocking antibodies to LFA-1, while L-selectin and VLA-4 antibodies had no inhibitory effect. Continuous coculture of the remaining CD34+ cells in the upper chamber of the transwell inserts resulted in proliferation and differentiation into myeloid and megakaryocytic cells. While the majority of cells in the upper chamber comprised proliferating myeloid precursors such as promyelocytes and myelocytes, only mature monocytes and granulocytes were detected in the lower chamber. In conclusion, BMEC-1 cells support transmigration of hematopoietic progenitors and mature hematopoietic cells. Therefore, this model may be used to study mechanisms involved in mobilization and homing of CD34+ cells during peripheral blood progenitor cell transplantation and trafficking of mature hematopoietic cells.

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Role of Focal Adhesion Kinase in Myelotoxicity, Inflammatory Stress Response and Myeloid Cell Functions.

Blood ◽

10.1182/blood.v114.22.3615.3615 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3615-3615

Author(s):

Sasidhar Vemula ◽

Baskar Ramdas ◽

Philip Hanneman ◽

Hillary Beggs ◽

Reuben Kapur

Keyword(s):

Bone Marrow ◽

Stress Response ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Myeloid Cells ◽

Inflammatory Stress

Abstract Abstract 3615 Poster Board III-551 Focal adhesion kinase (FAK) is a key signaling molecule in focal adhesion signaling and a potential integrator of integrin and growth factor receptor mediated signals. FAK has been implicated in various cellular functions such as growth, survival, migration, adhesion and cytoskeletal reorganization in fibroblasts but its role in hematopoietic stem and progenitors is unknown. To demonstrate the role of FAK in normal and stress-induced hematopoiesis, we generated FAK deficient mice by using a Cre/loxP method from here on termed FAKflox/flox (WT) mice. FAK deletion was induced by injecting poly (I)-poly(C) to FAK flox/flox mice containing the Mx.Cre transgene for one month (FAK-/-). PCR and western blot analysis revealed that after one month of poly (I)-poly(C) induction, hematopoietic cells failed to express detectable levels of FAK in bone marrow (BM), spleen and thymus. To determine the effect of FAK deletion on the development of hematopoietic cells a thorough analysis of the hematopoietic compartment in FAK-/- mice was performed. Total and differential cell counts of peripheral blood revealed significantly high red blood cell distribution width {RDW (%)} and mean cell volume (MCV) in FAK-/- mice compared to WT (n=13, WT; 18.6, 47.2 vs. FAK-/-; 20.06, 48.7, *p<0.05), respectively. In addition, differential basophil counts were significantly less in FAK-/- mice compared to WT (n=13, WT; 0.68 vs. FAK-/-; 0.3 *p<0.04) but all leukocyte populations were present at normal frequencies. Furthermore, platelet counts were significantly higher in FAK-/- peripheral blood compared to WT controls (n=13, WT; 759 vs FAK-/-; 978, *p<0.01). Under basal steady-state conditions, granulopoiesis appeared to be significantly altered in FAK deficient bone marrow (BM), as frequency of granulocytes, but not of other myeloid cells was reduced (n=10, WT; 44.14% vs. FAK-/-; 34.4%, *p<0.0001). Interestingly the frequency of Lin-, c-Kit+, Sca-1+ was also impaired in FAK deficient BM compared to controls (n=9, *p<0.05). FAK deficient BM progenitors displayed significantly lower frequency of colony-forming units compared to WT controls in response to various cytokine combinations (n=6, *p<0.01), which was associated with higher apoptosis in vitro (n=9, *p<0.006). Under conditions of stress, recovery of BM myeloid compartment and Lin−,c-Kit+, Sca-1+ cells following 5-Fluorouracil myeloablation was much slower in FAK-/- mice compared to WT controls (n=3, *p<0.05). Furthermore, the response of myeloid cells to acute inflammatory stress inflicted by intraperitoneal injection of thioglycollate was impaired in FAK-/- mice compared to WT mice (Macrophages: WT; 7.47 × 106 vs. FAK−/−; 3.1 × 106, n=8, *p <0.01. Neutrophils: WT; 5.47 × 106 vs. FAK−/−; 2.1 × 106, n=3, *p <0.05). These results led us to more closely examine the myeloid compartment in these mice. In vitro, FAK-/- macrophage progenitors show reduced growth in response to M-CSF stimulation (n=4, *p <0.01). In addition, deficiency of FAK in macrophages resulted in significant reduction in haptotactic migration in response to M-CSF on extracellular matrix proteins such as fibronectin, laminin and collagen (n=4, *p <0.01). Consistently, a significant reduction in the migration of FAK-/- macrophages was also observed in a wound healing assay which was associated with reduced activation of Rho GTPases including Rac. The reduction in migration of FAK-/- macrophages was associated with a significant decrease in adhesion on fibronectin, laminin and collagen. The impaired migration and adhesion of FAK-/- macrophages was observed in spite of comparable levels of F4/80 as well as integrin (α4β1 & α5β1) expression. Consistent with enhanced neutrophil apoptosis and reduced frequency under basal conditions, FAK deficient BM derived neutrophil progenitors (BMNs) show reduced growth and cycling in response to G-CSF stimulation (n=4, *p <0.01). Deletion of FAK in BMNs led to increased apoptosis upon cytokine withdrawal, which was associated with reduced activation of AKT and increased caspase-3 cleavage compared to controls. Taken together, our findings indicate that FAK plays a vital role in modulating physiological stress response to myeloablation, inflammation as well as in regulating several functions in macrophages and neutrophils. Disclosures: No relevant conflicts of interest to declare.

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Transendothelial Migration of CD34+ and Mature Hematopoietic Cells: An In Vitro Study Using a Human Bone Marrow Endothelial Cell Line

Blood ◽

10.1182/blood.v89.1.72 ◽

1997 ◽

Vol 89 (1) ◽

pp. 72-80 ◽

Cited By ~ 96

Author(s):

Robert Möhle ◽

Malcolm A.S. Moore ◽

Ralph L. Nachman ◽

Shahin Rafii

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Progenitor Cells ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Transendothelial Migration ◽

Plating Efficiency ◽

Cd34 Cells ◽

Upper Chamber

Abstract To study the role of bone marrow endothelial cells (BMEC) in the regulation of hematopoietic cell trafficking, we have designed an in vitro model of transendothelial migration of hematopoietic progenitor cells and their progeny. For these studies, we have taken advantage of a human BMEC-derived cell line (BMEC-1), which proliferates independent of growth factors, is contact inhibited, and expresses adhesion molecules similar to BMEC in vivo. BMEC-1 monolayers were grown to confluency on 3 μm microporous membrane inserts and placed in 6-well tissue culture plates. Granulocyte-colony stimulating factor (G-CSF )–mobilized peripheral blood CD34+ cells were added to the BMEC-1 monolayer in the upper chamber of the 6-well plate. After 24 hours of coincubation, the majority of CD34+ cells remained nonadherent in the upper chamber, while 1.6 ± 0.3% of the progenitor cells had transmigrated. Transmigrated CD34 cells expressed a higher level of CD38 compared with nonmigrating CD34+ cells and may therefore represent predominantly committed progenitor cells. Accordingly, the total plating efficiency of the transmigrated CD34+ cells for lineage-committed progenitors was higher (14.0 ± 0.1 v 7.8% ± 1.5%). In particular, the plating efficiency of transmigrated cells for erythroid progenitors was 27-fold greater compared with nonmigrating cells (8.0% ± 0.8% v 0.3% ± 0.1%) and 5.5-fold compared with unprocessed CD34+ cells (2.2% ± 0.4%). While no difference in the expression of the β1-integrin very late activation antigen (VLA)-4 and β2-integrin lymphocyte function-associated antigen (LFA)-1 was found, L-selectin expression on transmigrated CD34+ cells was lost, suggesting that shedding had occurred during migration. The number of transmigrated cells was reduced by blocking antibodies to LFA-1, while L-selectin and VLA-4 antibodies had no inhibitory effect. Continuous coculture of the remaining CD34+ cells in the upper chamber of the transwell inserts resulted in proliferation and differentiation into myeloid and megakaryocytic cells. While the majority of cells in the upper chamber comprised proliferating myeloid precursors such as promyelocytes and myelocytes, only mature monocytes and granulocytes were detected in the lower chamber. In conclusion, BMEC-1 cells support transmigration of hematopoietic progenitors and mature hematopoietic cells. Therefore, this model may be used to study mechanisms involved in mobilization and homing of CD34+ cells during peripheral blood progenitor cell transplantation and trafficking of mature hematopoietic cells.

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Human Drug Efflux Transporter ABCC5 Confers Acquired Resistance to Pemetrexed in Breast Cancer

10.21203/rs.3.rs-105352/v2 ◽

2021 ◽

Author(s):

Jihui Chen ◽

Zhipeng Wang ◽

Shouhong Gao ◽

Kejin Wu ◽

Fang Bai ◽

...

Keyword(s):

Breast Cancer ◽

Cell Line ◽

Cell Lines ◽

Abc Transporters ◽

Control Group ◽

Apoptosis Pathway ◽

Over Expression ◽

Mcf 7

Abstract AimPemetrexed, a new generation antifolate drug, is approved for the treatment for locally advanced or metastatic breast cancer, but factors affecting the efficacy and resistance of it have yet to be fully explicit. ATP-binding cassette (ABC) transporters have been reported as prognostic and adverse effects predictors of many xenobiotics. This study was designed to explore whether ABC transporters affect pemetrexed resistance and may contribute to treatment regimen optimization for breast cancer.MethodsFirstly, the expression of ABC transporters family members was measured in cell lines, thereafter examined the potential role of ABC transporter in conferring resistance to pemetrexed in primary cancer cell lines isolated from 34 breast cancer patients, and then the role of ABCC5 in mediating transport of pemetrexed and apoptosis pathway in MCF-7 cell line was assessed. Finally, the functions of ABCC5 on therapeutic effect of pemetrexed was evaluated in breast cancer bearing mice.ResultsThe expressions of ABCC2, ABCC4, ABCC5 and ABCG2 were significantly increased in pan-resistance cell line, and the ABCC5, the most obvious one, was 5.21 times higher than that of the control group. The expression of ABCC5 was inversely correlated with sensitivity (IC50) of pemetrexed (r = 0.741; p<0.001) in breast cancer cells from 34 patients. Furthermore, we found that the expression of ABCC5 influenced the efflux and cytotoxicity of pemetrexed in MCF-7 cell line, and the IC50 were 0.06 μg/ml and 0.20 μg/ml in ABCC5 knock-down and over-expression cells, respectively. In in vivo study, we found ABCC5 affected the sensitivity of pemetrexed in breast cancer bearing mice, and the tumor volume was much larger in ABCC5 over-expression group than that in control group (2.7 folds vs 1.3 folds).ConclusionsOur results indicated ABCC5 expression was associated with pemetrexed resistance in vitro and in vivo, and may be a biomarker for regimen optimization of pemetrexed in breast cancer treatment.

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Intact Rac Signaling Is Important for Leukemia Cell Survival

Blood ◽

10.1182/blood.v116.21.2885.2885 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2885-2885

Author(s):

Anja Troeger ◽

Pascal-David Johann ◽

Mumine Senturk ◽

Michael D. Milsom ◽

David A. Williams

Keyword(s):

Western Blot ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Hematopoietic Cells ◽

Leukemia Cell ◽

Jurkat Cells ◽

Knock Down ◽

Induced Apoptosis

Abstract Abstract 2885 Rho GTPases, Ras-related small G proteins, regulate multiple cell processes in hematopoietic cells. There is growing evidence that acute myeloid leukemia (AML) blasts and particularly MLL-rearranged AML blasts, rely on Rac activity (Mulloy JC et al, Blood, 2010). However, little is known about the role of these GTPases in acute lymphoblastic leukemia (ALL) and particularly precursor B cell ALL. To investigate the role of Rac and potential compensation by other GTPases in ALL, we first assessed the protein expression and activation of Rac in a number of B-ALL cell lines (SEM; RS4,11; REH; Nalm 6; Raji), compared with a T-ALL cell line (Jurkat) and several AML cell lines (ML2; MV4,11). Of these cell lines SEM; RS4,11; ML2 and MV4,11 are characterized by MLL-fusion genes. Jurkat and MLL-rearranged AML cell lines show higher expression of Rac proteins compared to B cell leukemia lines (Table 1). Overall, B-ALL cell lines exhibit highly variable levels of Rac expression and activity with no obvious correlation to the presence of MLL-fusion proteins. We then investigated proliferation and apoptosis in cell lines treated with the small molecule inhibitor NSC23766 (NSC), which blocks interaction of a subset of guanine exchange factors (GEFs) with Rac and thus inhibits its activation. Treatment with NSC led to ∼2-fold increase in cells arrested at G0/G1 and induced apoptosis in a dose-dependent fashion at NSC concentrations previously demonstrated to be non-toxic in normal hematopoietic cells (Muller LUW et al., Leukemia, 2008) (Table 2). The lymphoid cell lines Jurkat, Raji and SEM appeared less responsive to NSC with no increased apoptosis at 40μM NSC. There was no correlation between NSC response and baseline expression or activation status of Rac. However, cell lines resistant to NSC exhibited a paradoxical and transient early increase in Rac activation, suggesting the existence of compensatory activation mechanisms. To determine if the relative resistance observed in some cell lines was related to dependence on GEFs not targeted by NSC and to validate that the inhibitory effect of NSC was specifically due to Rac inhibition in sensitive cells, shRNAs were utilized to knock-down different members of the Rac subfamily. Effective shRNA-mediated knockdown was validated by western blot. Knockdown of Rac1 or Rac2 consistently induced apoptosis compared to non-targeting vector controls in NSC sensitive cell lines ML2 and Nalm6, with ML2 cells appearing slightly more sensitive to knock-down of Rac2 (Table 3). Knock-down of either Rac1 or Rac2 had little effect upon Jurkat cells which are resistant to NSC treatment. These data suggest that Jurkat cells are not dependent upon Rac signaling for survival; however we cannot discount the possibility that some compensation may occur between Rac1 and Rac2. These experiments demonstrate the importance of intact Rac signaling pathways for the survival of the majority of leukemia cell lines tested and demonstrate that dependence on Rac signaling is not restricted to leukemias characterized by MLL-rearrangements. Our observations also suggest that activation of different Rac isoforms may influence sensitivity towards pharmacological Rac inhibition. Table 1: Baseline Expression of Rac assessed by Western blot Cell line Jurkat ML-2 MV-4,11 RS-4,11 SEM Nalm 6 REH Raji Rac/b-actin expression* 1.6 2.5 1.7 0.5 0.7 0.8 1.0 1.0 (*arbitrary units, italics indicate cell lines carrying MLL-rearrangements) Table 2: % AnnexinV+ cells after treatment of the different cell lines with increasing doses the Rac-specific inhibitor NSC Cell line Jurkat ML-2 MV-4,11 RS-4,11 SEM Nalm 6 REH Raji control 6%+1.4 6%+1.3 9%+0.3 12%+3.6 9%+1.9 7%+1.5 9%+2 13%+2.3 20uM NSC 6%+1.4 9%+1.3 15%+0.3** 21%+8.5 8%+1.5 6%+1.9 25%+6.4 16%+3 40uM NSC 7%+1.8 24%+9.1 60%+4** 52%+11* 10%+1.3 10%+3.4 39%+11 16%+1.9 80uM NSC 15%+3.5* 73%+14.7** 97%+0.4** 80%+4** 17%+1.2* 46%+10.5** 62%+12.3* 22%+4 (Mean±SEM; n=5; * p<0.05; ** p<=0.01 versus control, bolded columns indicate increased NSC sensitivity) Table 3: % AnnexinV+ cells 7 days after lentiviral transduction of the different cell lines with Rac1 and Rac2-specific shRNA Cell line Jurkat ML-2 Nalm 6 non targeting control 4.3%+0.3 14.2%+8 11.4%+2.2 Rac1 shRNA* 8.0%+3.5 26.3%+7.9 36.8%+8.5 non targeting control 9.6%+4.2 8.1%+4.0 16.2%+3.1 Rac2 shRNA* 18.7%+4.5 35.5%+12.9 43.7%+7.1 (Mean±SEM; n=6; * second set of Rac1 and Rac2 shRNAs gave comparable results) Disclosures: No relevant conflicts of interest to declare.

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Hemonectin mediates adhesion of engrafted murine progenitors to a clonal bone marrow stromal cell line from Sl/Sld mice

Blood ◽

10.1182/blood.v77.8.1691.1691 ◽

1991 ◽

Vol 77 (8) ◽

pp. 1691-1698 ◽

Cited By ~ 14

Author(s):

P Anklesaria ◽

JS Greenberger ◽

TJ Fitzgerald ◽

B Sullenbarger ◽

M Wicha ◽

...

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Cell Lines ◽

Stromal Cells ◽

Stromal Cell ◽

Cumulative Number ◽

Specific Staining ◽

Marrow Stromal Cell

Abstract Mutant Sl/Sld mice exhibit decreased marrow hematopoiesis. The defect is known to reside in the marrow microenvironment of these animals, which is reproduced in vitro by primary marrow explants as well as by cloned marrow stromal cell lines. Bone marrow progenitor cells are incapable of adhering to primary Sl/Sld stromal cells or cloned stromal cell lines derived from them to form cobblestone-islands and proliferate. The role of hemonectin, a marrow-specific adhesion protein in the defective hematopoiesis of the Sl/Sld mice, was studied. Indirect immunoperoxidase staining of marrow in situ from Sl/Sld mice showed little specific staining while specific staining was seen in a pericellular distribution in marrow from +/+ mice. Hemonectin expression in several cloned stromal cell lines from Sl/Sld mice was compared by immunoblotting with that in cloned stromal cell lines from normal +/+ littermates. Cell line Sld3, which has the least hematopoiesis supportive capacity in vitro, showed no detectable hemonectin by immunoblotting, while Sld1 and Sld2 showed detectable but greatly reduced amounts compared with normal +/+ 2.4, GBI/6, and D2XRII. Confluent cultures incubated with purified hemonectin and engrafted with enriched progenitors showed a significant increase in the cumulative number of cobbleston-islands and day 14 spleen colony- forming units (CFU-s) forming progenitors (39.15 +/- 3.6/dish; 16.3 +/- 3.1/dish, respectively), compared with untreated Sld3 cultures (cobblestone-islands 8.1 +/- 3.6/dish; CFU-s forming progenitors 8.8 +/- 0.05/dish). Hemonectin-mediated progenitor cell binding to the Sld3 stromal cells was specifically inhibited by antihemonectin but not by preimmune serum. These data support the role of hemonectin in early progenitor-stromal cell interactions.

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CLL Xenograft Models to Address the Impact of Single Gene Loss (ATM) on Proliferation and Cytotoxic Response

Blood ◽

10.1182/blood.v116.21.2439.2439 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2439-2439

Author(s):

Ceri E Oldreive ◽

Victoria J Weston ◽

Paul AH Moss ◽

Guy Pratt ◽

Pamela R. Kearns ◽

...

Keyword(s):

Bone Marrow ◽

Tissue Distribution ◽

Cell Line ◽

Cell Lines ◽

Peripheral Blood ◽

Gene Loss ◽

Single Gene ◽

Isogenic Cell Line ◽

Xenograft Models ◽

The Impact

Abstract Abstract 2439 Chronic lymphocytic leukaemia (CLL) is a malignancy characterised by gradual accumulation of mature B cells in peripheral lymphoid organs. This accumulation is a consequence of both prolonged survival but also active proliferation restricted to a small population of tumour cells. Access to the proliferating tumour population in CLL patients is limited and there is a need for establishment of human CLL xenograft models that can recapitulate human disease. Several xenograft models of CLL have been already established, both with primary tumours and CLL cell lines, and they exhibit either a limited length of engraftment or tissue distribution that only partially recapitulates CLL disease. Furthermore, there is an absence of CLL xenograft models that address the impact of a single gene loss that confers a poor clinical outcome. The purpose of this study was to a) establish a range of CLL xenografts that exhibit different kinetics and distribution of CLL engraftment b) develop individual xenograft models with isogenic CLL cell lines, with and without the principal DNA damage response gene ATM whose loss confers chemoresistance, c) obtain simultaneous engraftment of differentially labelled ATM null and ATM wild type isogenic CLL cells and d) demonstrate that such CLL xenograft models can be used to measure the cytotoxic impact of new treatments. Initially, we addressed the kinetics of engraftment and tissue distribution of the following CLL cell lines: MEC-1 (Stacchini A at al. Leuk Res. 1999; 23:127–36), HG3 (Klein and Rosen, in prep), CII (Najfeld V et al.Int J Cancer. 1980;26:543–549) and PGA (Lewin N et al. Int J Cancer. 1988;41:892–895) administered intravenously into NOD/SCID mice. Engraftment and tissue distribution varied considerably between the different cell lines, with CII recapitulating CLL most closely. Detectable levels of peripheral blood engraftment of all cell lines occurred within 2–4 weeks. Mice engrafted with HG3 cells rapidly succumbed to CLL engraftment, surviving for a maximum of 6 weeks, whilst engraftment of other CLL cell lines was more prolonged (PGA up to 13 weeks; CII up to 16 weeks; MEC-1 up to 21 weeks). MEC-1 and CII cells were found to reside mainly in haemopoietic organs such as spleen, bone marrow and blood of injected animals, whereas HG3 and PGA showed a wider distribution that included kidneys, liver, uterus, gastrointestinal tract, bladder and neck. We subsequently established a subcutaneous model of the CLL cell line CII that both recapitulated CLL disease by dissemination into lymphoid organs but also allowed direct measurement of tumour size. Solid subcutaneous tumours were evident within 2 weeks and engraftment in the peripheral blood, spleen and bone marrow was demonstratable within 5 days of subcutaneous cell injection. In order to recapitulate the effect of ATM loss on engraftment and proliferation we simultaneously injected isogenic cell line pairs (MEC-1, HG3, CII), with and without stable ATM knock down. Isogenic cell line pairs were differentially labelled with either SNARF (ATM wild-type) or CFSE (ATM null), mixed in equal numbers (3×106) and intravenously injected into multiple NOD/SCID mice. The relative proportion of each cell type in the peripheral blood was assessed weekly by FACS analysis and using an IVIS whole body imager. We observed a predominance of ATM null CLL cells (MEC-1, HG3, CII) in the peripheral blood and spleen within three weeks of engraftment, whereas equivalent levels of ATM null and control isogenic cells were found in the bone marrow. Finally, to determine whether the CLL xenografts could serve as a valid models for testing new CLL treatments, the effect of a PARP inhibitor (Olaparib) on tumour load and animal survival was examined in a MEC-1 xenograft with stable ATM knockdown. Daily oral administration of olaparib (100mg/kg/mouse) to mice engrafted with ATM null MEC-1 cells compared to the vehicle-treated animals resulted in a reduction in the tumour load both in the bone marrow and to a lesser extent the spleen with concurrent lengthening of overall survival. We conclude that xenograft models with isogenic CLL cell lines provide a robust system where the impact of a single gene loss on engraftment, proliferation and treatment response can be addressed. Disclosures: No relevant conflicts of interest to declare.

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Hemonectin mediates adhesion of engrafted murine progenitors to a clonal bone marrow stromal cell line from Sl/Sld mice

Blood ◽

10.1182/blood.v77.8.1691.bloodjournal7781691 ◽

1991 ◽

Vol 77 (8) ◽

pp. 1691-1698

Author(s):

P Anklesaria ◽

JS Greenberger ◽

TJ Fitzgerald ◽

B Sullenbarger ◽

M Wicha ◽

...

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Cell Lines ◽

Stromal Cells ◽

Stromal Cell ◽

Cumulative Number ◽

Specific Staining ◽

Marrow Stromal Cell

Mutant Sl/Sld mice exhibit decreased marrow hematopoiesis. The defect is known to reside in the marrow microenvironment of these animals, which is reproduced in vitro by primary marrow explants as well as by cloned marrow stromal cell lines. Bone marrow progenitor cells are incapable of adhering to primary Sl/Sld stromal cells or cloned stromal cell lines derived from them to form cobblestone-islands and proliferate. The role of hemonectin, a marrow-specific adhesion protein in the defective hematopoiesis of the Sl/Sld mice, was studied. Indirect immunoperoxidase staining of marrow in situ from Sl/Sld mice showed little specific staining while specific staining was seen in a pericellular distribution in marrow from +/+ mice. Hemonectin expression in several cloned stromal cell lines from Sl/Sld mice was compared by immunoblotting with that in cloned stromal cell lines from normal +/+ littermates. Cell line Sld3, which has the least hematopoiesis supportive capacity in vitro, showed no detectable hemonectin by immunoblotting, while Sld1 and Sld2 showed detectable but greatly reduced amounts compared with normal +/+ 2.4, GBI/6, and D2XRII. Confluent cultures incubated with purified hemonectin and engrafted with enriched progenitors showed a significant increase in the cumulative number of cobbleston-islands and day 14 spleen colony- forming units (CFU-s) forming progenitors (39.15 +/- 3.6/dish; 16.3 +/- 3.1/dish, respectively), compared with untreated Sld3 cultures (cobblestone-islands 8.1 +/- 3.6/dish; CFU-s forming progenitors 8.8 +/- 0.05/dish). Hemonectin-mediated progenitor cell binding to the Sld3 stromal cells was specifically inhibited by antihemonectin but not by preimmune serum. These data support the role of hemonectin in early progenitor-stromal cell interactions.

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