SDF-1 Stimulation Research Articles

Abstract 14306: Mural Cell SDF1 Signaling is Associated With the Pathogenesis of Pulmonary Arterial Hypertension

Introduction: Pericytes and pulmonary artery smooth muscle cells (PASMCs) are NG2+ mural cells that provide structural support to pulmonary vessels. In pulmonary arterial hypertension (PAH), both mural cell types contribute to PA muscularization, but whether distinct mechanisms that are responsible for their behavior is unclear. Methods: RNA-Seq was used to compare the gene profile of pericytes and PASMCs isolated from PAH and healthy lungs. NG2-CreER mice were used to generate tamoxifen-inducible NG2-selective reporter mice (NG2tdT fl/+ ) for cell lineage identification and for NG2-selective SDF1 knockout (SDF1 NG2-KO ). Results: Hierarchical clustering of RNA-seq data demonstrated that the genetic profile of PAH pericytes and PASMCs is significantly similar. Cellular lineage staining studies on NG2tdT mice in chronic hypoxia showed that tdT+ cells accumulate in muscularized arteries. RNAseq analysis performed on these hypoxic tdT+ cells demonstrated significant upregulation of SDF1, a chemokine involved in chemotaxis and angiogenesis. Compared to controls, SDF1 NG2-KO mice in chronic hypoxia had reduced RVSP, fulton index, less muscularization and lower abundance of NG2+ cells around microvessels (Fig. 1). In addition, SDF1 stimulation in human healthy pericytes induced greater contractility, impaired their capacity to establish endothelial-pericyte tube formation and increased expression of SMC markers SMA, Calponin and SM22. In contrast, SDF1 knockdown reduced PAH pericyte proliferation, contractility and improved endothelial cell tube formation on matrigel. Conclusions: SDF1 is upregulated in NG2+ mural cells. High expression of SDF1 could drive pericyte detachment and gain an SMC-like phenotype that contributes to abnormal muscularization and vascular obstruction. Targeting SDF1 could help prevent and reverse muscularization in PAH.

Mural Cell SDF1 Signaling Is Associated with the Pathogenesis of Pulmonary Arterial Hypertension.

Pulmonary artery smooth muscle cells (PASMCs) and pericytes are NG2+ mural cells that provide structural support to pulmonary arteries and capillaries. In pulmonary arterial hypertension (PAH), both mural cell types contribute to PA muscularization, but whether similar mechanisms are responsible for their behavior is unknown. RNA-seq was used to compare the gene profile of pericytes and PASMCs from PAH and healthy lungs. NG2-Cre-ER mice were used to generate NG2-selective reporter mice (NG2tdT) for cell lineage identification and tamoxifen-inducible mice for NG2-selective SDF1 knockout (SDF1NG2-KO). Hierarchical clustering of RNA-seq data demonstrated that the genetic profile of PAH pericytes and PASMCs is highly similar. Cellular lineage staining studies on NG2tdT mice in chronic hypoxia showed that, similar to PAH, tdT+ cells accumulate in muscularized microvessels and demonstrate significant upregulation of SDF1, a chemokine involved in chemotaxis and angiogenesis. Compared with control mice, SDF1NG2-KO mice in chronic hypoxia had reduced muscularization and lower abundance of NG2+ cells around microvessels. SDF1 stimulation in healthy pericytes induced greater contractility and impaired their capacity to establish endothelial-pericyte communications. In contrast, SDF1 knockdown reduced PAH pericyte contractility and improved their capacity to associate with vascular tubes in coculture. SDF1 is upregulated in NG2+ mural cells and is associated with PA muscularization. Targeting SDF1 could help prevent and/or reverse muscularization in PAH.

Prognostic significance of USP33 in advanced colorectal cancer patients: new insights into β-arrestin-dependent ERK signaling.

Patients with liver metastases of colorectal cancer (CRCLM) have a poorer prognosis compared to colorectal cancer (CRC) patients in local stage. Evaluating the recurrence and overall survival of advanced patients is critical in improving disease treatment and clinical outcome. Here we investigated the expression pattern of USP33, a deubiquitinating enzyme, in both primary CRC tissues and liver metastases tissues. Univariate and multivariate analyses identified that low expression of USP33 in CRCLM tissues indicated high recurrence risk and poor overall prognosis. Overexpression of USP33 can significantly inhibit cell proliferation, migration, and invasion. On the other hand, USP33 knock-down promoted cell proliferation and invasion under SDF-1 stimulation; whereas dynasore (an internalization inhibitor) pretreatment in USP33 silencing cells showed a distinct antipromoting effect, revealing the participation of CXCR4 internalization in regulating tumor progress. Further results verified that USP33 can deubiquitinate β-arrestin2, subsequently block the internalization of SDF-1-stimulated CXCR4, and disrupt β-arrestin-dependent ERK activation. The existence and functions of β-arrestin-dependent signaling have been previously determined in several Gs-coupled receptors, such as β2-adrenergic receptor and angiotensin receptor subtype 1a; however, little is known about this in Gi-coupled receptors. Our study not only established USP33 as a novel prognosis biomarker in advanced CRCLM patients, but also highlighted the significance of β-arrestin-dependent ERK signaling in cancer development.

Intranasal Oncolytic Virotherapy with CXCR4-Enhanced Stem Cells Extends Survival in Mouse Model of Glioma.

SummaryThe challenges to effective drug delivery to brain tumors are twofold: (1) there is a lack of non-invasive methods of local delivery and (2) the blood-brain barrier limits systemic delivery. Intranasal delivery of therapeutics to the brain overcomes both challenges. In mouse model of malignant glioma, we observed that a small fraction of intranasally delivered neural stem cells (NSCs) can migrate to the brain tumor site. Here, we demonstrate that hypoxic preconditioning or overexpression of CXCR4 significantly enhances the tumor-targeting ability of NSCs, but without altering their phenotype only in genetically modified NSCs. Modified NSCs deliver oncolytic virus to glioma more efficiently and extend survival of experimental animals in the context of radiotherapy. Our findings indicate that intranasal delivery of stem cell-based therapeutics could be optimized for future clinical applications, and allow for safe and repeated administration of biological therapies to brain tumors and other CNS disorders.

Crosstalk between SDF-1/CXCR4 and SDF-1/CXCR7 in cardiac stem cell migration.

Stromal cell-derived factor 1 (SDF-1) is a chemokine that can be expressed in injured cardiomyocytes after myocardial infarction (MI). By combining with its receptor CXCR4, SDF-1 induced stem and progenitor cells migration. CXCR7, a novel receptor for SDF-1, has been identified recently. We aimed to explore the roles of SDF-1/CXCR4 and SDF-1/CXCR7 pathway and their crosstalk in CSCs migration. In the present study, CXCR4 and CXCR7 expression were identified in CSCs. Transwell assay showed that SDF-1 caused CSCs migration in a dose- and time-dependent manner, which could be significantly suppressed by CXCR4 or CXCR7 siRNA. Phospho-ERK, phospho-Akt and Raf-1 significantly elevated in CSCs with SDF-1 stimulation. Knockdown of CXCR4 or CXCR7 significantly decreased phospho-ERK or phospho-Akt, respectively, and eventually resulted in the inhibition of CSCs migration. Moreover, western blot showed that MK2206 (Akt inhibitor) increased the expression of phospho-ERK and Raf-1, whereas PD98059 (ERK inhibitor) had no effect on phospho-Akt and Raf-1. GW5074 (Raf-1 inhibitor) upregulated the expression of phospho-ERK, but had no effect on phospho-Akt. The present study indicated that SDF-1/CXCR7/Akt and SDF-1/CXCR4/ERK pathway played important roles in CSCs migration. Akt phosphorylation inhibited Raf-1 activity, which in turn dephosphorylated ERK and negatively regulated CSCs migration.

Gab1 regulates SDF-1-induced progression via inhibition of apoptosis pathway induced by PI3K/AKT/Bcl-2/BAX pathway in human chondrosarcoma.

In recent decades, the stromal cell-derived factor-l (SDF-1) and Gab1 have been investigated to be involved in oncogenesis. However, it is scarcely reported that SDF-1-Gab1 pathway mediates proliferation and apoptosis in human chondrosarcoma (CS). In this study, we assessed the expression of Gab1 in 90 CS solid tumors by immunohistochemistry, immunoblotting, and qRT-PCR, and then, some in vitro assays were also applied to CS cells treated with SDF-1. We observed that the overexpression of Gab1 was positively correlated with lung metastasis and recurrence, and acts as an independent prognostic factor for CS patients. Gab1 expression was up-regulated in response to SDF-1 stimulation in CS cell line JJ012, SW1353, L3252. Overexpression of Gab1 increased Bcl-2/BAX ratio to promote cell growth via PI3K/AKT. On the other hand, silencing of Gab1 accelerated apoptosis and repressed the growth of CS cells, which further caused the inhibition of G1/S phase transition and decreased invasion capacity in CS cell lines. In vivo assay identified that the knockdown of Gab1 interfered with the tumor mass formation. In conclusion, our data identified overexpression of Gab1 in CS tissues, and Gab1 can be recommended as a novel biomarker for diagnosis and prognosis in patients with CS. Additionally, PI3K/AKT/Bcl-2/BAX axis was involved in Gab1-induced CS progression, indicating Gab1 might act as a new target for the treatment of CS patients.

Further evidence that paroxysmal nocturnal haemoglobinuria is a disorder of defective cell membrane lipid rafts.

The glycolipid glycosylphosphatidylinositol anchor (GPI-A) plays an important role in lipid raft formation, which is required for proper expression on the cell surface of two inhibitors of the complement cascade, CD55 and CD59. The absence of these markers from the surface of blood cells, including erythrocytes, makes the cells susceptible to complement lysis, as seen in patients suffering from paroxysmal nocturnal haemoglobinuria (PNH). However, the explanation for why PNH-affected hematopoietic stem/progenitor cells (HSPCs) expand over time in BM is still unclear. Here, we propose an explanation for this phenomenon and provide evidence that a defect in lipid raft formation in HSPCs leads to defective CXCR4- and VLA-4-mediated retention of these cells in BM. In support of this possibility, BM-isolated CD34+ cells from PNH patients show a defect in the incorporation of CXCR4 and VLA-4 into membrane lipid rafts, respond weakly to SDF-1 stimulation, and show defective adhesion to fibronectin. Similar data were obtained with the GPI-A− Jurkat cell line. Moreover, we also report that chimeric mice transplanted with CD55−/− CD59−/− BM cells but with proper GPI-A expression do not expand over time in transplanted hosts. On the basis of these findings, we propose that a defect in lipid raft formation in PNH-mutated HSPCs makes these cells more mobile, so that they expand and out-compete normal HSPCs from their BM niches over time.

Abstract 4050: A central role for mTORC1 in CXCR4-mediated directional migration and metastasis

Abstract Tumor cells can co-opt the promigratory activity of chemokines and their cognate G protein-coupled receptors to disseminate to regional lymph nodes and distant organs. Indeed, the migration to SDF-1 of tumor cells expressing its receptor, CXCR4, is implicated in lymphatic and organ-specific metastasis in multiple human malignancies. However, the critical role of CXCR4 in bone marrow stem cell retention has hampered the development of CXCR4 inhibitors for metastasis prevention. Therefore the study of CXCR4 downstream signaling circuitry may provide new opportunities for the treatment of many human malignancies that depend on CXCR4 for their metastatic spread. The PI3K/mTOR pathway represents a major player in normal and aberrant cell growth. Consistently, mTOR inhibitors targeting its complex 1 (mTORC1) alone or mTORC1/mTORC2 have already shown promising therapeutic responses in many tumor types. Our data show that mTOR is activated upon SDF-1 stimulation of CXCR4 on epithelial-derived cancer cells, and that the disruption of mTORC1 or mTORC2 abrogates CXCR4-mediated directional migration. To study the CXCR4/mTOR axis in vivo we developed a simple and robust model for CXCR4-mediated spontaneous metastasis. Surprisingly, disruption of mTORC1 alone was sufficient to decrease tumor cell proliferation, angiogenesis, lymphangiogenesis, and CXCR4-mediated metastasis, whereas mTORC2 impairment had no effect on tumor dissemination or growth in vivo. Taken together, our data suggest that mTORC1 blockade could inhibit the migration of CXCR4 expressing cancer cells to their secondary sites, while disrupting the establishment of a permissive tumor microenvironment thereby halting the spread of highly aggressive tumors that require CXCR4 to metastasize. Citation Format: Patricia Dillenburg-Pilla, Vyomesh Patel, Constantinos M. Mikelis, Carlos Rodrigo Zarate-Blades, Panomwat Amornphimoltham, Zhiyong Wang, Daniel Martin, Kantima Leelahavanichkul, Colleen L. Doçi, Robert T. Dorsam, Andrius Masedunskas, Nijiro Nohata , Roberto Weigert, Alfredo A. Molinolo, J. Silvio Gutkind . A central role for mTORC1 in CXCR4-mediated directional migration and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4050. doi:10.1158/1538-7445.AM2014-4050

Glia Maturation Factor-Gamma Mediates Human Monocytes Migration Through Regulating β1/β2-Integrin Recycling

Recruitment of monocytes is essential for effective control and clearance of invading pathogens by migrating to the sites of infection, but recruited monocytes also contribute to the pathogenesis of chronic inflammatory and degenerative diseases, such as rheumatoid arthritis, multiple sclerosis, atherosclerosis and cancer. Thus, understanding the mechanisms controlling monocytes migration within different environments is of paramount importance. Although it is clear that adhesion signaling via integrin receptors and the surrounding ECM play a significant role in regulating migration of monocytes to site of inflammation, the underlying cellular and molecular mechanisms responsible for these process is still not fully characterized. Defining the molecular circuits through which integrins regulate monocytes motility is therefore important for gaining a better understanding of monocytes function. Glia maturation factor gamma (GMFG), a novel ADF/cofilin superfamily protein that is predominantly expressed in inflammatory cells, has been implicated in regulating actin reorganization. We have previously demonstrated that GMFG plays a role in regulating neutrophil chemotaxis and migration. We now examine whether GMFG has similar effects on monocytes and the cellular mechanism for these effects by using small-interfering RNA to knockdown GMFG in human primary monocytes. Knockdown of endogenous GMFG results in significantly reduced (220.6 ± 9.4 to 89.0 ± 3.2, p<0.003) chemotactic migration toward SDF-1 and enhanced adhesion on fibronectin, VCAM-1 and ICAM-1 compared with control siRNA transfected cells. Flow cytometry analysis shows that knockdown of GMFG enhances the expression of β1-, β2−integrin on the cell surface compared with control cells. Confocal microscopy analysis exhibited that GMFG is colocalized with internalized β1-, β2-integrin in early endosomes in primary monocytes. However, an internalization assay shows that β1- and β2-integrin were internalized with similar kinetics during the initial uptake time points both in control or GMFG knockdown primary monocytes. These data demonstrated that internalization of β1- and β2-integrin was not impaired in GMFG knockdown cells, suggesting that GMFG regulates β integrins recycling. Antibody-based recycling assays showed that GMFG knockdown cells resulted in enhanced plasma membrane exocytosis of β1- and β2-integrin compared with control cells. Western blot analysis revealed that SDF-1 stimulated the phosphoryalation of FAK (Tyr397) is moderately (∼ 40%) increased in GMFG knockdown monocytes compared with control cells. These results suggest that impaired monocytes migrated toward SDF-1 stimulation in GMFG depletion monocytes is due to enhanced adhesion, which is caused by accelerated recycling of β1- and β2-integrin to the surface. Taken together, these results indicate that GMFG is required to maintain the proper expression levels of β1- and β2-integrin on the plasma membrane and is fundamental for integrin-mediated monocytes motility. Disclosures:No relevant conflicts of interest to declare.

Spatial and Temporal Fluctuations In Marrow SDF-1 Following Radiation Injury Regulate Megakaryocyte-Vascular Niche Interactions and Circulating Platelet Levels

The development of megakaryocytes (MKs) in the bone marrow progresses spatially from the endosteal niche, which promotes MK progenitor proliferation, to the sinusoidal vascular niche, the site of terminal maturation and thrombopoiesis. The chemokine SDF-1 (CXCL12), signaling through receptor CXCR4, is produced by stromal cell populations throughout the marrow and is implicated in the maturational chemotaxis of MKs to the sinusoids. Understanding the regulation of MK localization has significance not only for optimal platelet production and the development of therapies for thrombocytopenia, but also in light of the recently proposed role for MKs in supporting hematopoietic stem cells (Heazlewood et al. 2013). In the injury setting of lethal total body irradiation (TBI), it was observed that radioresistant mature MKs relocate to the endosteal niche (Dominici et al. 2009, Olson et al. 2013). Complicating the study of marrow niches post-TBI is the vascular dilation that accompanies the drastic loss of marrow cells. Having confirmed that MKs relocate to the endosteum in our model of sublethal radiation-induced thrombocytopenia (4Gy TBI), we asked whether this localization is due to changes in the spatial distribution of the vasculature or to altered microenvironmental SDF-1.In agreement with other TBI models, we find a significant elevation in SDF-1 transcript levels in the marrow at days 1-3 following 4Gy TBI. Radioresistant MKs, which do not decrease in number until after 3 days, have significantly increased CXCR4 surface expression, a finding we also observe following SDF-1 stimulation of MKs both in vitro and in vivo. In situ hybridization was used to localize the spatial distribution of SDF-1 RNA in femoral marrow. At 2 days post-4Gy, a significant SDF-1 gradient develops with 30% higher SDF-1 message adjacent to the endosteum than in the central marrow. However, this gradient is dynamically eliminated 24 hours later at 3 days post-TBI. These shifts in SDF-1 expression are accompanied by parallel changes in the spatial distribution of MKs by immunohistochemistry. At 2 days post-TBI, there is over a 40% increase in MK in the endosteal niche. In contrast, MKs in the endosteal niche decrease by more than 15% at 3 days, coincident with a significant increase in the MKs associated with vascular endothelium. Thus, these data suggest that the spatial distribution of MKs is dependent upon the localization of SDF-1 in the rapidly fluctuating post-injury bone marrow.To determine if SDF-1 functionally contributes to MK niche changes, we stabilized endogenously-produced SDF-1 using Diprotin A, an inhibitor of SDF-1-inactivating protease DPP4. In uninjured marrow, Diprotin A treatment causes over a 30% rise in MK association with vasculature and a 20% increase in circulating platelets 24 hours later, with no change in MK number. Elevation of vascular SDF-1 by intravenous (IV) administration yields similar results. These data indicate that an endogenous SDF-1 gradient toward the vasculature contributes to homeostatic megakaryopoiesis and thrombopoiesis.At 2 days post-TBI, when endosteal SDF-1 message is increased, stabilization with Diprotin A results in a 40% decrease in MKs associated with vasculature and a small but significant decrease in platelets 24 hours later. Further supporting a role for altered SDF-1 gradients, elevating vascular levels with IV SDF-1 at 2 days causes the opposite effect of Diprotin A, with more MKs found in the vascular niche and a rise in peripheral platelet count. In contrast, at 3 days post-TBI, stabilization of endogenous SDF-1 with Diprotin A causes a further 25% increase in MKs in the vascular niche and a 10% rise in circulating platelets, consistent with the rapid loss of the endosteal SDF-1 gradient.Taken together, our data demonstrate that changes in microenvironmental SDF-1 regulate the spatial distribution of MKs in the post-TBI bone marrow. Importantly, the observed SDF-1 changes have functional consequences for platelet production, as the movement of MKs toward the endosteum decreases circulating platelets, while MK association with the vasculature increases circulating platelets. This knowledge will ultimately lead to improved therapeutic strategies to enhance platelet output in the setting of thrombocytopenia and highlights the need to carefully optimize the timing of therapeutic interventions. Disclosures:No relevant conflicts of interest to declare.

AB0164 Stromal cell-derived factor (SDF)-1 induces rankl in rheumaroid arthritis synovial fibroblasts and CD4+ T cells

BackgroundStromal cell-derived factor (SDF)-1 (CXCL12) is a chemokine promoting the migration of inflammatory cells into the inflamed synovium in rheumatoid arthritis (RA). The expression of SDF-1 is elevated in serum,...

Abstract 2108: Differential signaling and efficacy responses to SDF-1 and plerixafor in neuroblastoma cells.

Abstract Purpose: We have previously demonstrated that CXCR4 is widely but heterogeneously expressed in neuroblastoma cells and that the selective CXCR4 drug Plerixafor has preclinical efficacy for reducing growth of ectopic neuroblastomas in mice. Despite this reports exist which show Plerixafor can exhibit positive effects on growth in other cancers. Knowledge of the biochemical effect of CXCR4 occupancy by SDF-1 or Plerixafor in neuroblastoma cells and how this might vary based on CXCR4 isotype is largely unknown. The purpose of this study is to evaluate the efficacy response of neuroblastoma cells to SDF-1 or Plerixafor and determine if any differences exist and is so are these differences influenced by CXCR4 isotype. Additionally, we seek to identify signaling pathways activated in neuroblastoma cells by SDF-1 and Plerixafor. Experimental Design: Patient-derived neuroblastoma cell lines that differ by level and nature of CXCR4 isoform expressed were evaluated for their response to SDF-1 or Plerixafor. Serum-starved cells were treated with either agent in microplates and calcium mobilization or cell growth was measured using FluoForte and MTT assay kits respectively. For measurement of Akt activation total protein was extracted from stimulated cells, followed by western blot detection of Phosphorylated Akt using phospho-specific antibody and enhanced chemiluminescence (ECL). Results: We observed dose-dependent increases in calcium-mobilization in response to SDF-1 and Plerixafor treatment in all cell lines. Effects on cell growth were heterogeneous and variable in response to SDF-1 stimulation. High-CXCR4 expressing cell line SK-N-SH showed a dose-dependent increase in growth subsequent to SDF-1stimulation, while the low-CXCR4 expressing cell lines SH-SY5Y and SK-N-AS showed stimulation and inhibition at different doses. Plerixafor treatment of SK-N-SH cells resulted in a dose-dependent inhibition of cell growth while the low-CXCR4 expressing lines showed differential efficacy responses, with lower doses stimulating growth and higher doses inhibiting growth. Both SDF-1 and Plerixafor stimulation resulted in increased phosphorylation of Akt. Conclusion: In neuroblastoma cells signaling through CXCR4 occurs in response to Plerixafor as well as SDF-1; signaling by these two agents involves utilization of similar downstream signals including activation of the PI3K/Akt pathway. Neuroblastoma cells show differential efficacy responses to both SDF-1 and Plerixafor with respect to cell growth and this response appears to be dependent on CXCR4 expression type. In addition to its inhibitory effect Plerixafor is partially agonistic as well, underscoring the fact that further understanding of the mechanisms associated with differential efficacy response to Plerixafor is critical to developing effective treatment strategies for neuroblastoma based on this modality. Citation Format: Waleed A. Osman, Jacqueline N. Shanks, Charise M. White, Alex J. Carlisle. Differential signaling and efficacy responses to SDF-1 and plerixafor in neuroblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2108. doi:10.1158/1538-7445.AM2013-2108

The Tandem PH Domain-Containing Protein 2 (TAPP2) Regulates Chemokine-Induced Cytoskeletal Reorganization and Malignant B Cell Migration

The intracellular signaling processes controlling malignant B cell migration and tissue localization remain largely undefined. Tandem PH domain-containing proteins TAPP1 and TAPP2 are adaptor proteins that specifically bind to phosphatidylinositol-3,4-bisphosphate, or PI(3,4)P2, a product of phosphoinositide 3-kinases (PI3K). While PI3K enzymes have a number of functions in cell biology, including cell migration, the functions of PI(3,4)P2 and its binding proteins are not well understood. Previously we found that TAPP2 is highly expressed in primary leukemic B cells that have strong migratory capacity. Here we find that SDF-1-dependent migration of human malignant B cells requires both PI3K signaling and TAPP2. Migration in a transwell assay is significantly impaired by pan-PI3K and isoform-selective PI3K inhibitors, or by TAPP2 shRNA knockdown (KD). Strikingly, TAPP2 KD in combination with PI3K inhibitor treatment nearly abolished the migration response, suggesting that TAPP2 may contribute some functions independent of the PI3K pathway. In microfluidic chamber cell tracking assays, TAPP2 KD cells show reduction in percentage of migrating cells, migration velocity and directionality. TAPP2 KD led to alterations in chemokine-induced rearrangement of the actin cytoskeleton and failure to form polarized morphology. TAPP2 co-localized with the stable F-actin-binding protein utrophin, with both molecules reciprocally localizing against F-actin accumulated at the leading edge upon SDF-1 stimulation. In TAPP2 KD cells, Rac was over-activated and localized to multiple membrane protrusions, suggesting that TAPP2 may act in concert with utrophin and stable F-actin to spatially restrict Rac activation and reduce formation of multiple membrane protrusions. TAPP2 function in cell migration is also apparent in the more complex context of B cell migration into stromal cell layers – a process that is only partially dependent on PI3K and SDF-1. In summary, this study identified TAPP2 as a novel regulator of malignant B cell migration and a potential therapeutic intervention target.

Whole Genome Sequencing Identifies Recurring Somatic Mutations in the C-Terminal Domain of CXCR4, Including a Gain of Function Mutation in Waldenstrom's Macroglobinemia.

Abstract 2715 Introduction:Waldenstrom's Macroglobulinemia (WM) is an indolent non-Hodgkin's lymphoma characterized by the accumulation of IgM secreting lymphoplasmacytic cells (LPC) in the bone marrow. Using paired normal/WM lymphoplasmacytic cell paired tissues and whole genome sequencing (WGS), we identified somatic mutations in the CXC chemokine receptor 4 (CXCR4) gene which were present in 16/55 (29%) WM patients. CXCR4 is a G-protein-coupled receptor, together with its ligand, the stromal cell- derived factor-1(CXCL12/SDF-1), play an important role in leukocyte and lymphocyte hematopoiesis and trafficking. Upon SDF-1 stimulation, CXCR4 is phosphorylated and interacts with b-arrestins, which then trigger extracellular signal-regulated kinase (ERK) MAPKs and chemotaxis. CXCR4 signaling is then terminated through receptor internalization which is mediated via phosphorylation of its C-terminal tail. Methods:Sanger sequencing was used to validate WGS results. To clarify the functional significance of one of the most common somatic mutation identified (C1013G), cloning by PCR was undertaken from CD19+ bone marrow cells from a WM patient with the C1013G CXCR4 (C1013G-CXCR4) mutation. Wild type (WT) and C1013G-CXCR4 cDNAs were subcloned into plenti-IRES-GFP vector, and transduced using an optimized lentiviral based strategy for WM cells into BCWM.1 WM cells. Five days after transduction, GFP positive cells were sorted and used for functional studies. Surface expression of CXCR4 was determined by FACS using PE-conjugated anti-CXCR4 monoclonal antibody 12G5. CXCR4 internalization was studied by comparing CXCR4 surface expression before and after SDF-1 stimulation. Chemotaxis studies were performed using a transwell assay. The expression of phosphorylated ERK1/2 and total ERK2 was determined by western blot. Results:Validated somatic mutations in CXCR4 were all present in the C-terminal domain and included premature stop codons (C1013A; C1013G), and frameshift mutations. Importantly, the identified mutations are similar to those reported in the germline of patients with WHIM (Warts, Hypogammaglobulinemia, Infections and Myelokathexis) Syndrome, a dominant autosomal genetic disorder caused by tonic CXCR4 activation by impairment of CXCR4 internalization, and stimulation of G-protein-dependent responses, and chemotaxis. Consistent with such a role, SDF-1a stimulation showed decreased internalization of CXCR4 in C1013G-CXCR4 versus WT CXCR4 transduced BCWM.1 WM cells. SDF-1a stimulated ERK1/2 phosphorylation was also more robust C1013G-CXCR4 versus WT CXCR4 expressing cells. Lastly, C1013G-CXCR4 transduced cells displayed stronger migratory response toward SDF-1a versus WT CXCR4 expressing BCWM.1 cells. Conclusions:C-terminal domain somatic mutations are common in WM and overlap with germline mutation identified in WHIM syndrome. Moreover, the most common of these mutations (C1013G) confers gain of function including decreased CXCR4 internalization, more robust ERK ½ phosphorylation, and chemotaxis. The findings provide new insights into the pathogenesis of WM, and a framework for the study of CXCR4 inhibitors for WM therapy. Disclosures:Treon:Onyx: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees.

The Hsp90 Inhibitor 17-DMAG Increases SOCS3 and Regulates Cytokine Production, Migration and Cell Death in Chronic Lymphocytic Leukemia

Abstract 1362Epigenetic or transcriptional silencing of important tumor suppressors has been described to contribute to cell survival and tumorigenesis in chronic lymphocytic leukemia (CLL). We investigated gene silencing in CLL using gene expression microarray analysis, and found that over 2000 genes are repressed more than 2-fold in CLL B cells compared to normal B cells, including genes involved in cell signaling and survival (Jun/Fos signaling, ATF family, cyclin dependent kinases and suppressors of cytokine signaling). In particular, the suppressor of cytokine signaling protein 3 (SOCS3) was decreased 60-fold in CLL B cells compared to peripheral blood B cells from normal donors. Despite this profound effect, few therapeutic approaches have focused on reversing this gene silencing in CLL. NF-κB has been shown to transcriptionally silence gene expression in several types of cancer, and our group has previously identified a similar role for this transcription factor in CLL. In addition, we have previously shown that the Hsp90 inhibitor 17-DMAG targets NF-κB signaling to induce apoptosis in CLL through transcriptional down-regulation of oncogenes such as MCL1 and BCL2. However, our microarray analysis revealed that treatment with 17-DMAG also leads to the re-expression of nearly 200 genes silenced in CLL compared to normal B cells. One of the genes significantly silenced in CLL and re-expressed by 17-DMAG is SOCS3. This increase in SOCS3 was evident as early as 8 hours following treatment with 17-DMAG, and peaking at 16–24 hours just prior to 17-DMAG induced cell death (up-regulated 5.6 fold at 8 hours, 59.8 fold at 16 hours, and 25.7 fold at 24 hours). The magnitude of induction in CLL cells was significantly greater than SOCS3 induction in peripheral blood B cells from normal donors, which correlates with a decreased apoptotic response of normal B cells to 17-DMAG (25.7 fold up-regulated in CLL versus 3.5 fold in normal B cells). While little is known about its regulation or functional impact in CLL, SOCS3 has been shown to be methylated in solid tumors as well as myeloid leukemia, leading to aberrant cytokine production and cell survival. While it is known that promoter hypermethylation and subsequent gene silencing contributes to CLL disease progression, we found that there was no significant methylation of the SOCS3 promoter in CLL compared to normal B lymphocytes, indicating an alternative mechanism of SOCS3 silencing in CLL. In order to further characterize the downstream effect of SOCS3 regulation, we investigated the pathways known to be regulated by this protein, specifically IL-6 and CXCR4 signaling. We found that 17-DMAG prevents phosphorylation of STAT3 induced by IL-6 stimulation, which leads to decreased production of pro-survival cytokines including negative feedback by decreasing IL-6 itself. While STAT3 is a known Hsp90 client protein, the effect on phosphorylation of STAT3 was evident before a decrease in the total protein was observed, indicating a distinct effect on the signaling pathway independent of Hsp90’s role as a protein chaperone. SOCS3 has also been shown to prevent phosphorylation of focal adhesion kinase (FAK) and therefore block both integrin and CXCR4 signaling pathways. We found that 17-DMAG prevents constitutive phosphorylation of FAK in primary CLL cells, and subsequently reduces AKT phosphorylation following recombinant SDF-1 stimulation. In order to determine if 17-DMAG inhibits migration of CLL cells towards recombinant SDF-1 as well as the direct signaling through the CXCR4 receptor, we performed transwell migration assays and found that 17-DMAG significantly inhibits migration towards both recombinant SDF-1 and CXCL13 (migration towards CXCL12, 12.4% with Vehicle vs. 8.9% with 17-DMAG, p=0.0061, towards CXCL13, 12.4% with Vehicle vs. 6.1% with 17-DMAG, p<0.0001). Similar results were obtained by over-expression of SOCS3 in a CLL B cell line, suggesting that 17-DMAG inhibits migration through an increase in SOCS3. Based on these results, we suggest that 17-DMAG reverses gene silencing in CLL, and through re-expression SOCS3 inhibits the migration and signaling associated with SDF-1/CXCR4, an important factor in the tumor microenvironment that contributes to CLL cell survival. Therefore Hsp90 inhibitors represent a novel approach to target transcriptional silencing in CLL and other B cell lymphoproliferative disorders. Disclosures:No relevant conflicts of interest to declare.

Heparin Disrupts the CXCR4/SDF-1 Axis and Impairs the Functional Capacity of Bone Marrow–Derived Mononuclear Cells Used for Cardiovascular Repair

Cell therapy is a promising option for the treatment of acute or chronic myocardial ischemia. The intracoronary infusion of cells imposes the potential risk of cell clotting, which may be prevented by the addition of anticoagulants. However, a comprehensive analysis of the effects of anticoagulants on the function of the cells is missing. Here, we investigated the effects of heparin and the thrombin inhibitor bivalirudin on bone marrow-derived mononuclear cell (BMC) functional activity and homing capacity. Heparin, but not bivalirudin profoundly and dose-dependently inhibited basal and stromal cell-derived factor 1 (SDF-1)-induced BMC migration. Incubation of BMCs with 20 U/mL heparin for 30 minutes abrogated SDF-1-induced BMC invasion (16±8% of control; P<0.01), whereas no effects on apoptosis or colony formation were observed (80±33% and 100±44% of control, respectively). Pretreatment of BMCs with heparin significantly reduced the homing of the injected cells in a mouse ear-wound model (69±10% of control; P<0.05). In contrast, bivalirudin did not inhibit in vivo homing of BMCs. Mechanistically, heparin binds to both, the chemoattractant SDF-1 and its receptor, chemokine receptor 4 (CXCR4), blocking CXCR4 internalization as well as SDF-1/CXCR4 signaling after SDF-1 stimulation. Heparin blocks SDF-1/CXCR4 signaling by binding to the ligand as well as the receptor, thereby interfering with migration and homing of BMCs. In contrast, the thrombin inhibitor bivalirudin did not interfere with BMC homing or SDF-1/CXCR4 signaling. These findings suggest that bivalirudin but not heparin might be recommended as an anticoagulant for intracoronary infusion of BMCs for cell therapy after cardiac ischemia.

Stromal cell-derived factor-1 overexpression induces gastric dysplasia through expansion of stromal myofibroblasts and epithelial progenitors

ObjectiveStromal cell-derived factor-1 (SDF-1/CXCL12), the main ligand for CXCR4, is overexpressed in human cancer. This study addressed the precise contribution of SDF-1 to gastric carcinogenesis.DesignSDF-1 transgenic mice were created and...

COMP-Ang1 stimulates HIF-1α–mediated SDF-1 overexpression and recovers ischemic injury through BM-derived progenitor cell recruitment

AbstractRecruitment and adhesion of bone marrow (BM)–derived circulating progenitor cells to ischemic tissue are important for vasculogenesis and tissue repair. Recently, we found cartilage oligomeric matrix protein (COMP)–Ang1 is a useful cell-priming agent to improve the therapeutic efficacy of progenitor cells. However, the effect and the underlying mechanisms of COMP-Ang1 on recruitment of BM-derived progenitor cells (BMPCs) to foci of vascular injury have not been well defined. Here, we found that COMP-Ang1 is a critical stimulator of stromal cell–derived factor 1 (SDF-1), the principal regulator of BM-cell trafficking. Furthermore, SDF-1 stimulation by COMP-Ang1 was blocked by small-interfering RNA (siRNA) against hypoxia-inducible factor-1α (HIF-1α). COMP-Ang1 increased the synthesis of HIF-1α by activating mammalian target of rapamycin (mTOR) in hypoxic endothelium. The intermediate mechanism transmitting the COMP-Ang1 signal to the downstream mTOR/HIF-1α/SDF-1 pathway was the enhanced binding of the Tie2 receptor with integrin-linked kinase (ILK), an upstream activator of mTOR. In the mouse ischemic model, local injection of COMP-Ang1 stimulated the incorporation of BMPCs into ischemic limb, thereby enhancing neovasculogenesis and limb salvage. Collectively, our findings identify the COMP-Ang1/HIF-1α/SDF-1 pathway as a novel inducer of BMPC recruitment and neovasculogenesis in ischemic disease.

Genetically modified adipose tissue−derived mesenchymal stem cells overexpressing CXCR4 display increased motility, invasiveness, and homing to bone marrow of NOD/SCID mice

This study evaluates usefulness of CXCR4 overexpression via retroviral transduction in adipose tissue-derived mesenchymal stem cells (AT-MSCs) as a strategy to increase their migration and engraftment ability. AT-MSCs were isolated from lipoaspirates from human healthy donors with liberase 3. Cells were transduced with retroviral vector carrying either CXCR4 or green fluorescent protein (GFP) complementary DNA, and neo-resistant colonies were selected and used in experiments. Chemotaxis, invasion through Matrigel, motor activity, gene expression, osteodifferentiation potential, and engraftment into bone marrow of nonobese diabetic/severe combined immunodeficient mice were analyzed for CXCR4-overexpressing cells and GFP-control cells. Approximately 90% of retrovirus-transduced AT-MSCs expressed CXCR4 or GFP and maintained their ability to differentiate into osteocytes. CXCR4-transduced AT-MSCs displayed enhanced migration and higher invasiveness toward SDF-1 gradient. The upregulation of CXCR4 led to phosphorylation of mitogen-activated protein and AKT kinases and an increase in metalloproteinase expression after SDF-1 stimulation. The transplantation of CXCR4-transduced AT-MSCs into nonobese diabetic/severe combined immunodeficient mice led to increased engraftment into bone marrow in comparison to GFP-transduced AT-MSCs. Adipose tissue is one of the alternative sources of MSCs to bone marrow. We showed that AT-MSCs overexpressing CXCR4 preserve their ability for osteodifferentiation. Enhanced migration and engraftment of the transduced AT-MSCs into bone marrow indicate the usefulness of this strategy in overcoming low engraftment of MSCs in clinical approaches of cellular therapies for bone disorders and can represent a powerful tool in regenerative medicine and gene therapies. Thus, these cells may be used as an alternative to bone marrow-derived MSCs.

Bone marrow-derived mesenchymal stem cells from early diffuse systemic sclerosis stimulate in vitro angiogenesis and foster revascularization of ischemic limbs

Systemic sclerosis (SSc) is a chronic connective tissue disorder characterized by widespread microangiopathy, lack of angiogenesis, and fibrosis. Bone marrow-derived mesenchymal stem cells (MSCs) produce and release high amounts of proangiogenic and antiapoptotic factors that inhibit apoptosis of endothelial cells under hypoxic conditions and promote the formation of capillary-like structures in vitro. In the present study, we characterized MSCs from SSc patients for the expression of factors implicated in MSC recruitment at sites of injury, angiogenesis, and fibrosis. We also analyzed whether SSc-MSCs could modulate dermal microvascular endothelial cell (MVEC) angiogenesis in vitro. MSCs were obtained from 5 patients with early diffuse SSc and 5 healthy donors. MSCs were expanded in culture and their phenotype was investigated by flow cytometry, CFU-F assay, and analysis of osteogenic/adipogenic differentiation. MSCs were stimulated with VEGF, TGFβ, or SDF-1. Transcript and protein levels of SDF-1 and its receptor CXCR4, VEGF, TGFβ1 and receptors TβRI/TβRII were evaluated by real-time RT-PCR, Western blotting, and confocal microscopy. VEGF, SDF-1, and TGFβ1 secretion in culture supernatant was measured by ELISA. MVEC capillary morphogenesis was performed on Matrigel with the addition of MSC-conditioned medium. SSc- and healthy-MSCs showed no significant differences in morphology, proliferation rate, CFU-F colony formation or osteogenic/adipogenic differentiation. In SSc-MSCs, the basal expression of proangiogenic SDF-1/CXCR4 and VEGF was significantly increased compared to healthy-MSCs. SSc-MSCs constitutively released higher levels of SDF-1 and VEGF. SDF-1/CXCR4 were upregulated after VEGF stimulation, and CXCR4 redistributed from cytoplasm to cell surface. VEGF was increased by SDF-1 challenge. VEGF, TGFβ and SDF-1 stimulation upregulated TGFβ1, TβRI, and TβRII in SSc-MSCs. SSc-MSC-conditioned medium showed a greater proangiogenic effect on MVECs than healthy-MSCs. Experiments with blocking antibodies demonstrated that MSC-derived cytokines were responsible for this potent proangiogenic effect. One patient who developed gangrene of the upper and lower limbs was treated with 3 intravenous infusions of expanded autologous MSCs. Areas of necrotic skin were reduced after the first MSC infusion. After the third infusion, angiography showed revascularization of the patient’s extremities. Skin section analysis revealed cell clusters with tube-like structures, and angiogenic factors were strongly expressed. SSc-MSCs constitutively overexpress and release proangiogenic factors, and potentiate dermal MVEC angiogenesis in vitro. In SSc patients with severe peripheral ischemia, intravenous infusion of expanded autologous MSCs may foster the recovery of the vascular network.