CXCL12-CXCR4 Interaction Research Articles

10000-SPE-1 STEM CELL-BASED GENE THERAPY FOR MALIGNANT GLIOMA USING GENOME-EDITED HUMAN INDUCED PLURIPOTENT STEM CELLS

Abstract Glioblastoma is the most aggressive primary brain tumor, and is characterized by diffuse infiltration into the normal brain parenchyma. New therapeutic approaches targeting invasive biological behaviour are warranted. In the present study, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited induced pluripotent stem cells (iPSCs) have high tumor-trophic migratory capacity and stable constitutive therapeutic transgene expression, which leads to strong anti-tumor effects against glioma stem cell (GSC) models. The present study provides answers to some important research questions associated with stem cell-based gene therapy. First, the tumor-trophic migratory capacities of human iPSC-derived NSCs (iPSC-NSCs), fetal NSCs, and mesenchymal stem cells (MSCs) were quantitatively evaluated by spatiotemporal methodologies. We demonstrated that iPSC-NSCs have a higher tumor-trophic migratory capacity than MSCs in the brain. Self-repulsive action and pathotropism were important for the migration of iPSC-NSCs: ephrin ligand/receptor mediated repulsion of iPSC-NSCs and CXCL12-CXCR4 interactions between GSCs and iPSC-NSCs. Second, a prodrug converting enzyme fusion gene was selected as a therapeutic gene in human iPSCs. In general, stable constitutive transgene expression by viral vectors was difficult in human iPSCs. Furthermore, viral vectors integrate randomly into the host genome, which raises concerns about transgene silencing, insertional mutagenesis, and oncogene activation. In the present study, several common insertion sites including GAPDH, ACTB, and AAVS1, were compared. The most appropriate gene locus that achieved stable constitutive transgene expression was determined via CRISPR/Cas9-mediated genome editing. Third, we revealed the novel mechanism of action using iPSC-NSCs expressing CD-UPRT, in which ferroptosis was associated with enhanced anti-tumor immune responses. We demonstrated that the established iPSC-NSCs had strong therapeutic efficacy in GSC animal models. Finally, predictive biomarkers for the efficacy of the present treatment strategy were established. We will conduct a clinical trial of this treatment strategy. This research concept can disseminate biological, medical and engineering advances.

Abstract 2262: Spatial architecture and cellular interactions of tumor immune microenvironment to discover biomarkers and predict immune checkpoint inhibitor response in gastric cancer

Abstract Background: While immune checkpoint inhibitors (ICI) extend survival for patients suffering from a variety of cancers, most gastric cancer (GC) patients do not respond to ICI. There is a critical need to identify novel biomarkers that guide the use of ICI or strategies to improve outcomes for GC patients. We aim to gain a deeper understanding of the GC tumor microenvironment (TME) to refine the use of ICI using spatial transcriptomes from ICI-treated gastric tumors. Method: We collected 12 pre-ICI treatment gastric tumor samples from five responders and seven nonresponders and performed spatial profiling to investigate spatial heterogeneity, functions, and phenotypes of tumor and neighboring stromal and immune cells to identify features associated with ICI response. We used pan-cytokeratin (pan-CK) to mark epithelial cells, CD45 for immune cells, and smooth muscle actin (SMA) for fibroblasts. We generated gene expression profiles of each cell type within regions of interest (ROI) with a NanoString GeoMx Cancer Transcriptome Atlas panel, comprised of 1,800 genes. A total of 58 ROIs were analyzed from responders and 62 ROIs from nonresponders. Results: We measured the spatial heterogeneity of each cell type in ROIs within and across tumors. We found the tumor, immune, and fibroblasts from nonresponders are transcriptionally more heterogeneous, compared to those from responders. This might indicate a higher degree of intratumor transcriptional heterogeneity is linked to ICI resistance. We found that nonresponse was associated with higher expression of VEGFA and VEGFB in tumor cells and higher ACTA2 in fibroblasts, not in other cell types. These findings are intriguing when considering our other finding that increased endothelial cell and fibroblast proportions in the TME are associated with ICI nonresponse. We also found that the TME of nonresponders had fewer M1 and more M2 macrophages, and there were more T cells that overexpressed gene programs consistent with dysfunction and exhaustion. These findings indicate that ICI-resistant TMEs are densely surrounded by immune-suppressive cellular components which might reduce ICI efficacy and result in ICI resistance. Finally, we performed cell-cell interaction and identified ligand-receptor interactions that are stronger in nonresponders, including CXCL12-CXCR4, CD74-MIF, and LGALS9-LRP1 ligand-receptor interactions. The predicted ligand-receptor interactions are present in cell-type-specific manners, indicating the functional role of each cell type in facilitating ICI response. Conclusion: We investigated how tumor and neighboring stromal and immune cells interact within GC TME to mediate ICI response, which demonstrate that spatial analysis could be used to identify novel biomarkers that predict response to ICI, and new therapies to enhance ICI efficacy in GC patients. Citation Format: Sunho Park, Changjing Hong, Jae-Ho Cheong, Sam C. Wang, Matthew R. Porembka, In-Ho Kim, Sung Hak Lee, Tae Hyun Hwang. Spatial architecture and cellular interactions of tumor immune microenvironment to discover biomarkers and predict immune checkpoint inhibitor response in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2262.

ET-1 STEM CELL-BASED GENE THERAPY FOR MALIGNANT GLIOMA USING GENOME-EDITED HUMAN INDUCED PLURIPOTENT STEM CELLS

Abstract Glioblastoma is the most aggressive primary brain tumor, and is characterized by diffuse infiltration into the normal brain parenchyma. New therapeutic approaches targeting invasive biological behaviour are warranted. In the present study, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited induced pluripotent stem cells (iPSCs) have high tumor-trophic migratory capacity and stable constitutive therapeutic transgene expression, which leads to strong anti-tumor effects against glioma stem cell (GSC) models. The present study provides answers to some important research questions associated with stem cell-based gene therapy. First, the tumor-trophic migratory capacities of human iPSC-derived NSCs (iPSC-NSCs), fetal NSCs, and mesenchymal stem cells (MSCs) were quantitatively evaluated by spatiotemporal methodologies. We demonstrated that iPSC-NSCs have a higher tumor-trophic migratory capacity than MSCs in the brain. Self-repulsive action and pathotropism were important for the migration of iPSC-NSCs: ephrin ligand/receptor mediated repulsion of iPSC-NSCs and CXCL12-CXCR4 interactions between GSCs and iPSC-NSCs. Second, a prodrug converting enzyme fusion gene was selected as a therapeutic gene in human iPSCs. In general, stable constitutive transgene expression by viral vectors was difficult in human iPSCs. Furthermore, viral vectors integrate randomly into the host genome, which raises concerns about transgene silencing, insertional mutagenesis, and oncogene activation. In the present study, several common insertion sites including GAPDH, ACTB, and AAVS1, were compared. The most appropriate gene locus that achieved stable constitutive transgene expression was determined via CRISPR/Cas9-mediated genome editing. Third, we revealed the novel mechanism of action using iPSC-NSCs expressing CD-UPRT, in which ferroptosis was associated with enhanced anti-tumor immune responses. We demonstrated that the established iPSC-NSCs had strong therapeutic efficacy in GSC animal models. Finally, predictive biomarkers for the efficacy of the present treatment strategy were established. We will conduct a clinical trial of this treatment strategy. This research concept can disseminate biological, medical and engineering advances.

Abstract 4: Discovery of novel and highly potent small molecule inhibitors of chemokine receptor CXCR4

Abstract Introduction: CXCR4 is a member of chemokine receptor and G protein coupled receptor (GPCR) families. Its interaction with the chemotactic ligand stromal cell-derived factor 1 (SDF-1 or CXCL12) plays important roles in physiological and pathological processes. Recent studies have showed that the blockade of CXCL12-CXCR4 interaction by small molecules can be used in several clinical applications, including sensitizing cancer cells to chemotherapy, mobilizing hematopoietic stem cells (HSCs) to the blood for HSC collection, and inhibiting the tumor metastasis. To date, AMD3100 (Plerixafor) is the only clinically approved drug used in combination with G-CSF for mobilizing HSCs. Here, we report the discovery and development of a new class of highly potent small molecule CXCR4 inhibitors which can strongly antagonize CXCL12-CXCR4 interaction and mobilizing HSCs. Methods: Based on the crystal structures of CXCR4 showing a major and minor subpockets, we applied a fragment-based combinatorial design (FBCD) strategy to design a new class of hybrid molecules to recognize these two subpockets. A series of designed compounds were chemically synthesized and tested in a panel of biological assays including competitive CXCR4 binding, chemotaxis, calcium mobilization and CXCR4 internalization to characterize their biological activities. Furthermore, site-directed mutagenesis coupled with molecular docking simulation was carried out to determine the binding modes of the most potent compounds. Results: More than 30 compounds were synthesized and characterized. Among them, compound HFX51116 showed the most potent CXCR4 binding activity (IC50 = 12.2 nM), which was more potent than AMD3100 tested in parallel (IC50 = 325.3 nM). Furthermore, HFX51116 displayed remarkable inhibition of calcium mobilization (IC50 = 1.478 nM) and chemotaxis activity (IC50 = 252.7 nM), both of which are better than those of AMD3100. In addition, mutational studies and molecular docking simulation suggested that HFX51116 recognizes the minor subpocket mainly and major subpocket partially. Conclusions: Our findings have demonstrated that HFX51116 is a promising novel lead compound for developing a new class of small molecule therapeutics targeting CXCR4. Citation Format: Xiong Fang, Qian Meng, Xiao Fang, Siyu Zhu, Yazi Huang, Jing An, Yan Xu, Ziwei Huang. Discovery of novel and highly potent small molecule inhibitors of chemokine receptor CXCR4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4.

A Novel Role for Bst2 and E-Selectin in IFNg-Stimulated HSC Niche Relocalization

Prolonged exposure to proinflammatory conditions, such as during chronic infection, can lead to the development of bone marrow failure syndromes including aplastic anemia. Indeed, we have previously shown that chronic infection activates hematopoietic stem cells (HSCs), ultimately leading to bone marrow suppression due to an IFNgamma (IFNγ)-dependent depletion of HSCs. Defining the mechanisms behind inflammation mediated HSC loss may provide important insight into how inflammation affects hematopoiesis in many clinical conditions including infection, ageing, and myelodysplastic syndrome.HSC quiescence is maintained in large part through interactions with the surrounding cells of the bone marrow niche. In particular, HSCs closely colocalize with perivascular stromal cells that secrete high levels of the chemokine Cxcl12 and other hematopoietic maintenance factors such as angiopoietin, IL7, and stem cell factor. We hypothesized that IFNγ-induced activation may affect the positioning of HSCs relative to supporting cells within the bone marrow microenvironment. We used 3-D intravital imaging to assess the localization of HSCs relative to Cxcl12-abundant reticulocytes (CAR cells). When CMTMR-stained HSCs were transplanted into Cxcl12-GFP mice we found that IFNγ treatment led to a distancing of HSCs away from quiescence-enforcing CAR cells. In contrast, TNF-alpha treatment, which does not induce HSC proliferation at the concentration used, resulted in no such relocalization. Furthermore, no displacement occurred in response to IFNγ treatment when Ifngr1-deficient HSCs were transplanted, suggesting a cell-autonomous mechanism of relocalization.RNA expression analysis and chemotaxis assays showed that IFNγ-treated HSCs are not impaired in their ability to respond to Cxcl12 signaling, indicating that a change in Cxcl12-Cxcr4 interactions does not account for the movement of HSCs away from CAR cells. To further assess HSC-specific changes that may account for IFNγ-dependent relocalization, we performed gene expression analysis of HSCs from control or IFNγ-treated mice. We saw no change in the expression of common HSCs receptors that are thought to play a role in maintaining HSC quiescence, such as cKit, Cdh2, Mpl, Itgb1, Itbg2, Itga4, and Itga1. However, the surface protein, Bst2, also known as tetherin, was significantly upregulated in HSCs upon IFNγ stimulation. This was confirmed at both the RNA and protein level. A prior report identified Bst2 as a noncanonical E-selectin ligand. This finding is particularly interesting given that HSCs found in close proximity to E-selectin-expressing endothelial cells exist in an activated, proliferative state. Furthermore, the interaction between HSCs and E-selectin+ endothelial cells was previously noted to be independent of canonical E-selectin ligands such as Psgl1, CD44, or the Lewis family antigens. Thus we hypothesized that IFNγ-induced upregulation of surface Bst2 may facilitate proliferation by promoting HSC binding to E-selectin. Indeed, in vitro E-selectin binding assays showed that IFNγ treatment increased binding of progenitor cells to E-selectin, but not to the closely related adhesin, P-selectin.Inflammation plays an important role in the development of many cancers including acute myeloid leukemia (AML), leading us to investigate if Bst2 upregulation is an important factor in AML biology. Indeed, we found that increased levels of Bst2 are associated with poor survival in AML patients. Further, murine studies have shown that leukemic blasts have greatly increased E-selectin binding capacity following transformation. Using two independent AML cell lines, we found that IFNγ treatment upregulates Bst2 expression and increases E-selectin binding capacity. These findings suggest a mechanism by which inflammation could drive cell proliferation in AML.In summary, we have uncovered a potential new mechanism for IFNγ-induced HSC activation, whereby HSCs are relocalized in a Bst2-dependent manner from the quiescence-enforcing CAR niche to an active E-selectin-positive niche. Furthermore, we demonstrate that IFNγ-mediated Bst2 expression also occurs in AML, suggesting a mechanistic link between inflammation and AML progression. This study may open up new potential therapeutic avenues for the treatment of patients with chronic infection, inflammatory diseases, and cancer. DisclosuresNo relevant conflicts of interest to declare.

CXCL12-CXCR4 Axis Is Required for Contact-Mediated Human B Lymphoid and Plasmacytoid Dendritic Cell Differentiation but Not T Lymphoid Generation.

We investigated the involvement of CXCL12-CXCR4 interactions in human lymphohematopoiesis by coculture with telomerized human stromal cells. CXCR4 expression was low in CD34+CD38-CD45RA-CD10-CD7-CD19- immature hematopoietic stem/precursor cells (HSPCs) but higher in CD34+CD38-CD45RA+CD10+CD7+/-CD19- early lymphoid precursors and even higher in CD34+CD38+CD45RA+CD10+CD7-CD19+ pro-B cells. Inhibition of the effect of stromal cell-produced CXCL12 by an anti-CXCR4-blocking Ab suppressed the generation of CD45RA+CD10-CD7+CD19- early T lymphoid precursors (ETPs) and CD45RA+CD10+CD7-CD19+/- B lymphoid precursors on stromal cells, but it did not affect the generation of ETPs in conditioned medium of stromal cell cultures. Replating assays showed that contact with stromal cells was critical for HSPC-derived CD45RA+CD10+CD7-CD19- B lineage-biased precursors to differentiate into CD19+ pro-B cells, which was suppressed by the anti-CXCR4 Ab. Conversely, HSPC-derived ETPs possessed T and B lymphoid and monocytic differentiation potential; stromal cell contact was not required for their growth but rather promoted B lymphoid differentiation. The anti-CXCR4 Ab did not affect the growth of ETPs in conditioned medium, but it suppressed their B lymphoid differentiation on stromal cells. CD14-CD11c-HLA-DR+CD123highCD303+ plasmacytoid dendritic cells developed from HSPCs and ETPs exclusively in contact with stromal cells, which was suppressed by the anti-CXCR4 Ab. These data indicate that CXCL12 plays an essential role in stromal cell contact-mediated B lymphoid and plasmacytoid dendritic cell differentiation from immature hematopoietic and early T lymphoid precursors with a multilineage differentiation potential, but it does not participate in contact-independent generation of early T lymphoid precursors.

Design, synthesis, and biological characterization of novel PEG-linked dimeric modulators for CXCR4

CXCR4 dimerization has been widely demonstrated both biologically and structurally. This paper mainly focused on the development of structure-based dimeric ligands that target CXCL12–CXCR4 interaction and signaling. This study presents the design and synthesis of a series of [PEG]n linked dimeric ligands of CXCR4 based on the knowledge of the homodimeric crystal structure of CXCR4 and our well established platform of chemistry and bioassays for CXCR4. These new ligands include [PEG]n linked homodimeric or heterodimeric peptides consisting of either two DV3-derived moieties (where DV3 is an all-d-amino acid analog of N-terminal modules of 1–10 (V3) residues of vMIP-II) or hybrids of DV3 moieties and CXCL121–8. Among a total of 24 peptide ligands, four antagonists and three agonists showed good CXCR4 binding affinity, with IC50 values of <50nM and <800nM, respectively. Chemotaxis and calcium mobilization assays with SUP-T1 cells further identified two promising lead modulators of CXCR4: ligand 4, a [PEG3]2 linked homodimeric DV3, was an effective CXCR4 antagonist (IC50=22nM); and ligand 21, a [PEG3]2 linked heterodimeric DV3–CXCL121–8, was an effective CXCR4 agonist (IC50=407nM). These dimeric CXCR4 modulators represent new molecular probes and therapeutics that effectively modulate CXCL12–CXCR4 interaction and function.

Distinct Bone Marrow Blood Vessels Differentially Regulate Normal and Malignant Hematopoietic Stem and Progenitor Cells

Bone marrow (BM) endothelial cells (BMECs) form a network of blood vessels (BVs) that regulate both leukocyte trafficking and hematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance between these dual regulatory roles and if these events occur at the same vascular site. We define the BM architecture of functionally distinct BVs, their spatial localization and association with specific stromal precursors, which cooperatively regulate HSPC fate. BM stem and progenitor cell maintenance in a metabolically non-active state and leukocyte trafficking occur at separate sites and are differentially regulated by specific BVs with distinct permeability properties. BM arteries were found to be mostly encircled by aSMA+ pericytes whereas the ensuing small-diameter endosteal and trabecular arterioles were predominantly surrounded by stem cell-niche supporting stromal precursor cells. Live imaging and flow analysis revealed that endosteal arteriole BVs exhibited high flow rate, low permeability to external plasma from the peripheral blood, and high levels of adhesion- and tight-junction molecules. Primitive HSPCs located in peri-arteriole regions were found in a non-activated, low reactive oxygen species (ROS) state. Exposure of BM HSPCs to peripheral blood plasma, enhanced their metabolic activity, exhibited by enhanced intracellular ROS levels, and glucose uptake. The same was also evident for circulating HSPCs in the blood. Plasma-exposed HSPCs displayed enhanced motility alongside with reduced long-term repopulation potential. Live imaging showed that all immature and mature leukocyte bi-directional trafficking occurred exclusively at the more permeable sinusoids, located downstream to the endosteal arterioles. Of note, BM sinusoids contained a higher prevalence of ROShigh cells in their microenvironment, including HSPCs. Rapid AMD3100-induced HSPC mobilization preferentially affected sinusoidal but not arterial BVs permeability and CXCL12 chemokine release. Endothelial specific in vivo interference with CXCL12-CXCR4 interactions, via conditional CXCR4 genetic deletion, hampered BM barrier integrity resulting in enhanced HSPC egress. In line with these results we found that during conditions favoring BM stem and progenitor cells expansion, endothelial integrity was enhanced along with reduced HSPC bi-directional trafficking. Conversely, conditional endothelial specific induced genetic or pharmacologic disruption of barrier integrity augmented ROS levels in HSPCs, enhancing their bi-directional trafficking and differentiation while reducing their BM pool size and maintenance in a metabolically non-active state. Of note, humanized mice engrafted with pre-B ALL cells exhibited reduced BM barrier permeability most probably due to BM endothelium modification via FGF-2 secretion by the leukemic clone. Interestingly, human pre-B ALL cells displayed hypersensitivity to plasmatic exposure. We hypothesize that malignant cells modify BM endothelium to provide themselves with a supportive and protective microenvironment composed of undifferentiated BM stromal progenitors and tightly sealed endothelial barrier. In conclusion, our study identifies anatomically distinct BM BVs with different barrier functions serving as systemic leukocyte trafficking or HSPC BM maintenance sites with clinical therapeutic relevance. DisclosuresRafii:Angiocrine Bioscience: Consultancy, Equity Ownership.

Targeting the CXCR4-CXCL12 Pathway Using an Anti-CXCR4 IgG1 Antibody (PF-06747143) in Chronic Lymphocytic Leukemia

Abstract Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adults in the Western world. This leukemia is not curable and resistance to therapy is promoted by factors present in the tumor microenvironment including the chemokine CXCL12 (SDF-1), which interacts with its receptor CXCR4 and is thought to promote cancer cell survival. Here we explored the therapeutic potential of blocking CXCL12-CXCR4 interactions using PF-06747143, a humanized IgG1 antibody specific for CXCR4, which is expressed at high levels by CLL cells. Using primary leukemia cells from CLL patients, we found that PF-06747143 inhibited CXCL12-induced cell migration and blocked cytoskeletal changes via F-actin polymerization similar to AMD-3100 (Mozobil, a small molecule inhibitor of CXCR4). In addition, PF-06747143 induced apoptosis on CLL cells cultured alone or in the presence of human bone marrow-derived stromal cells (stroma-NK-tert). The pro-apoptotic activity of PF-06747143 was independent of high-risk prognostic factors including IGHV mutation status, ZAP-70 expression or TP53 mutation / 17p-deletion. Interestingly, AMD-3100, which binds and inhibits signaling through CXCR4, did not induce cell death in CLL or any of the cell lines tested. PF-06747143 did not induce apoptosis on normal B and T cells, and the ability of this anti-CXCR4 antibody to induce cell death on CLL cells appeared to be dependent on the crosslinking of CXCR4. This was supported by the fact that a Fab only fragment derived from PF-06747143 did not induce apoptosis despite of its high binding affinity for CXCR4. We observed that PF-06747143 induced complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) in CLL cells. However, this antibody did not induce caspase activation but rather its cell death activity appeared to be dependent on the production of reactive oxygen species (ROS) in leukemia cells. This effect was similar to that observed with other ROS dependent antibodies such as obinutuzumab (Gazyva). ROS induction was observed with PF-06747143, but not its Fab derived fragment and preceded apoptosis suggesting that this is critical component of its mechanism of action. We evaluated synergism of PF-06747143 with other CLL therapeutic agents and observed that this antibody synergized with fludarabine, bendamustine, ibrutinib and rituximab in the majority of CLL patient samples tested. In summary, our studies showed that PF-06747143, a CXCR4 IgG1 antibody is a potent inhibitor of the CXCR4-CXCL12 pathway and induces cell death primarily in CLL cells but not in normal lymphocytes. The cytotoxic effect of PF-06747143 was similar in CLL cells cultured alone or with stromal cells, suggesting that this antibody has the potential to overcome the protective effect of the tumor microenvironment. We also showed that PF-06747143 induced programmed cell death on CLL cells was dependent on ROS production and that this antibody synergized with agents currently used for the treatment of CLL patients. Overall, these findings highlight the biological relevance of the CXCR4-CXCL12 pathway in CLL, and provide rationale for clinical evaluation of PF-06747143 in CLL and other cancers. Disclosures Choi: Gilead: Consultancy, Other: Advisory Board, Speakers Bureau; AbbVie: Consultancy, Other: Advisory Board, Research Funding. Kipps:Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor.

CXCL12-induced VLA-4 activation is impaired in trisomy 12 chronic lymphocytic leukemia cells: a role for CCL21.

Homing to distinct lymphoid organs enables chronic lymphocytic leukemia (CLL) cells to receive pro-survival and proliferative signals. Cytogenetic aberrations can significantly affect CLL cell compartmentalization. Trisomy 12 (tri12) defines a CLL subgroup with specific clinical features and increased levels of the negative prognostic marker CD49d, the α4-subunit of the integrin VLA-4, which is a key regulator of CLL cell homing to bone marrow (BM). Chemokine-induced inside-out VLA-4 activation, particularly via the CXCL12-CXCR4 axis, increases the arrest of various cell types on VCAM-1 presenting endothelium. Here, we demonstrate that high CD49d expression in tri12 CLL is accompanied by decreased CXCR4 expression. Dissecting functional consequences of these alterations, we observed that tri12 CLL cell homing to murine BM is not affected by CXCR4-CXCL12 blockage using AMD3100 or olaptesed pegol/NOX-A12. In line, CCL21-CCR7 rather than CXCL12-CXCR4 interactions triggered VLA-4-mediated arrests of tri12 CLL cells to VCAM-1 under blood flow conditions. Concordantly, in real-time kinetic analyses we found CCL21 but not CXCL12 being capable to induce inside-out VLA-4 conformational changes in this CLL subgroup. Our results provide novel insights into the peculiar clinico-biological behaviour of tri12 CLL and emphasize its specific chemokine and integrin utilization during pathophysiologically and therapeutically relevant interactions with the microenvironment.

Th2 lymphocytes migrating to the bone marrow under high-altitude hypoxia promote erythropoiesis via activin A and interleukin-9

The mechanism of accelerated erythropoiesis under the hypoxic conditions of high altitude (HA) remains largely obscure. Here, we investigated the potential role of bone marrow (BM) T cells in the increased production of erythrocytes at HA. We found that mice exposed to a simulated altitude of 6,000 m for 1-3 weeks exhibited a significant expansion of BM CD4+ cells, mainly caused by increasing T helper 2 (Th2) cells. Using a coculture model of BM T cells and hematopoietic stem/progenitor cells, we observed that BM CD4+ cells from hypoxic mice induced erythroid output more easily, in agreement with the erythroid-enhancing effect observed for Th2-condition-cultured BM CD4+ cells. It was further demonstrated that elevated secretion of activin A and interleukin-9 by BM Th2 cells of hypoxic mice promoted erythroid differentiation of hematopoietic stem/progenitor cells and the growth of erythroblasts, respectively. Our study also provided evidence that the CXCL12-CXCR4 interaction played an important role in Th2 cell trafficking to the BM under HA conditions. These results collectively suggest that Th2 cells migrating to the BM during HA exposure have a regulatory role in erythropoiesis, which provides new insight into the mechanism of high altitude polycythemia.

Conjugated Linoleic Acid Targets β2 Integrin Expression To Suppress Monocyte Adhesion

Chronic recruitment of monocytes and their subsequent migration through the activated endothelium contribute to atherosclerotic plaque development. Integrin-mediated leukocyte adhesion is central to this process. Conjugated linoleic acid (CLA) has the unique property of inducing regression of pre-established murine atherosclerosis via modulation of monocyte/macrophage function. Understanding the mechanisms through which CLA mediates its atheroprotective effect may help to identify novel pathways that limit or reverse atherosclerosis. In this study, we identified a novel mechanism through which CLA alters monocyte function. We show that CLA inhibits human peripheral blood monocyte cell adhesion to activated endothelial cells via loss of CD18 expression, the β2 chain of LFA-1 and Mac-1 integrins. In addition, using a static-adhesion assay, we provide evidence that CLA prevents monocytes from binding to ICAM-1 and subsequently reduces the capacity of these cells to polarize. CXCL12-CXCR4 interactions induce a conformational change in β2 integrins, facilitating leukocyte adhesion. In this study, we demonstrate that CLA inhibits CXCR4 expression, resulting in a failure of monocytes to directionally migrate toward CXCL12. Finally, using intravital microscopy, we show that, during CLA-induced regression of pre-established atherosclerosis in ApoE(-/-) mice, there is reduced leukocyte adhesion and decreased CD18 expression on Gr1(+)/CD115(+) proinflammatory monocytes. In summary, the data presented describe a novel functional role for CLA in the regulation of monocyte adhesion, polarization, and migration.

BMS-936564 (MDX1338): A Fully Human Anti-CXCR4 Antibody Induces Apoptosis in an in Vitro Model of Stromal – Leukemia Cell Interaction for Chronic Lymphocytic Leukemia.

AbstractAbstract 2887Chronic lymphocytic leukemia (CLL) remains incurable despite advances in the biology and treatment of this disease. Current data support the notion that resistance to therapy is promoted by a “protective” tumor microenvironment in which non-leukemia cells produce factors that enhance the resistance of CLL cells to spontaneous or drug-induced apoptosis. One such factor is the chemokine CXCL12, which interacts with its receptor CXCR4 on CLL cells to promote cancer cell survival.To examine the therapeutic potential of blocking CXCL12-CXCR4 interactions, we studied the effect of BMS-936564, a fully human IgG4 anti-CXCR4 antibody, using an in vitro co-culture model of human bone marrow derived stomal-NKter cells – leukemia cell interaction. Such stromal-NKter cells secrete CXCL12 and enhance the resistance of CLL cells to apoptosis in vitro.We observed that primary CLL cells co-cultured with stromal-NKter cells had significantly greater viability than CLL cells cultured alone (20–60% above baseline at 48 hours). Moreover, CLL cells co-cultured with stromal cells had enhanced resistance to drug-induced apoptosis. We found that BMS-936564 antibody at concentrations of 2–200nM could enhance the rate of apoptosis of CLL cells cultured alone or in the presence of stromal cells. CLL cells that expressed unmutated IgVH genes or ZAP-70 appeared equally susceptible to treatment with BMS-936564 as did CLL cells that lack these adverse prognostic markers, as did CLL cells that harbored deletions in 17p13.2 and that were resistant to chemotherapeutic agents, such a fludarabine monophosphate. BMS-936564 antibody inhibited CXCL12 mediated F-Actin polymerization in CLL cells at lower concentrations (20–200nM) compared to AMD-3100 (Mozobil), a small molecule CXCR4 inhibitor (50–150μM). In addition, AMD-3100 did not induce apoptosis in CLL cells (10–300μM).In summary, we observed that the anti-CXCR4 antibody BMS-936564 inhibited CXCL12 mediated activation of the CXCR4 receptor in CLL cells and induced apoptosis in leukemia cells. The pro-apoptotic activity of BMS-936564 was observed in cells cultured alone or together with stromal cells suggesting that this antibody had direct cytotoxic effect on leukemia cells and that it can overcome the protective tumor microenvironment. More over, the activity of BMS-936564 was independent of the presence of poor prognostic factors such as del(17p) suggesting that its mechanism of action is P53 independent. These findings show evidence that the CXCR4-CXCL12 pathway is a valid therapeutic target in CLL and provide additional biological rationale for ongoing clinical trials in CLL and other hematological malignancies using BMS-936564. Disclosures:Kuhne:Bristol-Myers Squibb: Employment. Sabbatini:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Shelat:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Kipps:Abbott: Consultancy, Research Funding.

AMD3100 sensitizes acute lymphoblastic leukemia cells to chemotherapy in vivo

AMD3100 sensitizes acute lymphoblastic leukemia cells to chemotherapy in vivo

The expression of CXCR4, CXCL12 and CXCR7 in malignant pleural mesothelioma

The chemokine CXCL12 and its receptors, CXCR4 and CXCR7, are involved in tumour progression, metastasis, and survival. We investigated the expression of CXCR4, CXCL12, and CXCR7 in malignant pleural mesothelioma to determine if they are possible biomarkers and potential therapeutic targets. Forty-one mesothelioma tumour tissues, ten normal human pleural tissues, and two mesothelioma cell lines were stained with anti-CXCR4, anti-CXCL12, anti-CXCR7, and anti-p-Akt antibodies. RT-PCR was performed to determine the expression of CXCR4, CXCL12, and CXCR7 in six human mesothelioma cell lines (H28, 211H, H2052, ms-1, H290, and H513) and one human normal mesothelial cell line, LP9. These seven cell lines were also stained with anti-CXCR7. We found that CXCR4 and CXCL12 were expressed in 97.6% and 78.0% mesothelioma tissue samples, concurrently with strong expression of p-Akt (R(2) = 0.739 and 0.620, respectively). In addition, CXCR7 expression was weaker than CXCR4 expression in mesothelioma tissues. Furthermore, RT-PCR showed that CXCR4 and CXCL12 were overexpressed in 5/6 mesothelioma cell lines (211H, H2052, ms-1, H290, and H513), whereas CXCR7 was overexpressed in only 2/6 (H513 and H2052). Moreover, we found that the CXCR4 antagonist AMD3100 inhibited the growth of all five mesothelioma cell lines that overexpress CXCR4 and CXCL12. Our results suggest that the Akt-mTOR pathway is involved during the interruption of the CXCL12/CXCR4 axis in these five mesothelioma cell lines. In conclusion, CXCR4 and CXCL12 are highly expressed in most mesothelioma cell lines and tumour tissues, suggesting that CXCR4 and CXCL12 may be used as biomarkers for patients with mesothelioma. The CXCL12-CXCR4 interaction may be a potential therapeutic target for mesothelioma.

Multiple functions of CXCL12 in a syngeneic model of breast cancer

BackgroundA growing body of work implicates chemokines, in particular CXCL12 and its receptors, in the progression and site-specific metastasis of various cancers, including breast cancer. Various agents have been used to block the CXCL12-CXCR4 interaction as a means of inhibiting cancer metastasis. However, as a potent chemotactic factor for leukocytes, CXCL12 also has the potential to enhance anti-cancer immunity. To further elucidate its role in breast cancer progression, CXCL12 and its antagonist CXCL12(P2G) were overexpressed in the syngeneic 4T1.2 mouse model of breast carcinoma.ResultsWhile expression of CXCL12(P2G) significantly inhibited metastasis, expression of wild-type CXCL12 potently inhibited both metastasis and primary tumor growth. The effects of wild-type CXCL12 were attributed to an immune response characterized by the induction of CD8+ T cell activity, enhanced cell-mediated cytotoxicity, increased numbers of CD11c+ cells in the tumor-draining lymph nodes and reduced accumulation of myeloid-derived suppressor cells in the spleen.ConclusionsThis study highlights the need to consider carefully therapeutic strategies that block CXCL12 signaling. Therapies that boost CXCL12 levels at the primary tumor site may prove more effective in the treatment of metastatic breast cancer.

Alternative implication of CXCR4 in JAK2/STAT3 activation in small cell lung cancer

Small cell lung cancer (SCLC) is an aggressive, rapidly metastasising tumour. Previously, we demonstrated the influence of CXCL12–CXCR4 interaction on processes involved in metastasis and chemoresistance in SCLC. We show here that STAT3 is expressed in both primary SCLC tumour tissues and SCLC cell lines. We investigated the function of STAT3 upon CXCL12 stimulation in SCLC cell lines. Small cell lung cancer cell lines present constitutive phosphorylation of STAT3, and in the reference cell lines NCI-H69 and NCI-H82 constitutive phosphorylation was further increased by CXCL12 stimulation. Further investigating this signalling cascade, we showed that it involves interactions between CXCR4 and JAK2 in both cell lines. However CXCL12-induced adhesion to VCAM-1 could be completely inhibited by the JAK2 inhibitor AG490 only in NCI-H82. Furthermore, CXCR4 antagonist but not AG490 inhibited cell adhesion whereas both antagonisms were shown to inhibit growth of the cells in soft agar, indicating the central involvement of this signalling in anchorage-independent growth of SCLC cells. Most interestingly, while using primary tumour material, we observed that in contrast to non-small-cell lung cancer samples from primary tumour tissues, all analysed samples from SCLC were strongly positive for tyrosine-phosphorylated STAT3. Taken together, these data indicate that STAT3 is constitutively phosphorylated in SCLC and is important in SCLC growth and spreading thus presenting an interesting target for therapy.

Enhanced Functional Response to CXCL12/SDF-1 Through Retroviral Overexpression of CXCR4 on M07e Cells: Implications for Hematopoietic Stem Cell Transplantation

The chemokine CXCL12 (stromal cell derived factor-1/SDF-1) stimulates hematopoietic stem and progenitor cells (HSCs/HPCs) through the corresponding chemokine receptor CXCR4. CXCL12 is thought to be important for both proper HSC homing, retention, and engraftment into the bone marrow (BM) and mobilization out of the BM. Previous studies suggest that breaking the CXCL12-CXCR4 interaction mobilizes HPCs, blocking CXCR4 inhibits HSC homing, and overexpression increases HSC/HPC repopulation. The efficiency of mobilization and engraftment therefore appears to be dependent on the response of HSCs/HPCs to CXCL12, which is in turn dependent upon levels of CXCR4 expressed on HSCs/HPCs. However, expression of CXCR4 on the surface of HSCs/HPCs appears to be variable. To study the function of CXCR4 on HSCs/HPCs, we used the MSCV-based bicistronic (EGFP) retroviral vector MIEG3 to overexpress CXCR4 on M07e cells, an established model of human HPC. CXCR4 overexpression resulted in significant increases in CXCL12-induced chemotaxis and cell survival. Most importantly, cells overexpressing CXCR4 responded to CXCL12 at levels typically too low induce a response. These data suggest that an increased transplant efficiency resulting from CXCR4 overexpression is likely a function of increased HSC/HPC homing and increased HSC/HPC survival in the recipient's BM. These experiments also validate the ability of the MIEG3-CXCR4 retroviral construct to overexpress CXCR4 efficiently and the use of MIEG3-CXCR4 M07e cells for further study. Finally, this information may have future potential therapeutic implications for improvements in transplant efficiency.

CXCL12–CXCR4 interactions modulate prostate cancer cell migration, metalloproteinase expression and invasion

The mechanisms responsible for prostate cancer metastasis are incompletely understood at both the cellular and molecular levels. In this regard, chemokines are a family of small, cytokine-like proteins that induce motility of neoplastic cells, leukocytes and cancer cells. The current study evaluates the molecular mechanisms of CXCL12 and CXCR4 in prostate cancer cell migration and invasion. We report that functional CXCR4 is significantly expressed by prostate cancer cell lines, LNCaP and PC3, when compared with normal prostatic epithelial cells (PrEC). As measured using motility and invasion chamber assays, prostate cancer cells migrated and invaded through extracellular matrix components in response to CXCL12, at rates that corresponded to CXCR4 expression. Anti-CXCR4 antibodies (Abs) significantly impaired the migration and invasive potential of PC3 and LNCaP cells. CXCL12 induction also enhanced collagenase-1 (metalloproteinase-1 (MMP-1)) expression by LNCaP and PC3 cells. Collagenase-3 (MMP-13) was expressed by prostate cancer cells, but it was not expressed by PrEC cells or modulated by CXCL12. CXCL12 increased MMP-2 expression by LNCaP and PC3; however, MMP-9 expression was elevated only in PC3 cells after CXCL12–CXCR4 ligation. PC3 cells also expressed high levels of stromelysin-1 (MMP-3) after CXCL12 stimulation. CXCL12 also significantly increased stromelysin-2 (MMP-10) expression by LNCaP cells. Stromelysin-3 (MMP-11) was expressed by LNCaP cells, but not by PC3 or PrEC cells and CXCL12 induced PC3 MMP-11 expression. Membrane type-1 MMP (MMP-14) was not expressed by PrEC or LNCaP cells, but CXCL12 significantly enhanced MMP-14 expression by PC3 cells. These studies reveal important cellular and molecular mechanisms of CXCR4/CXCL12-mediated prostate cancer cell migration and invasion.

Enhanced Functional Response to CXCL12/SDF-1 through Retroviral Overexpression of CXCR4: Implications for Hematopoietic Stem & Progenitor Cell Homing and Engraftment.

The chemokine CXCL12 (stromal cell-derived factor 1/SDF-1) induces the migration of hematopoietic stem and progenitor cells (HSC/HPC) through the corresponding chemokine receptor, CXCR4. CXCL12 is thought to be important for proper homing and engraftment of HSC/HPC to the bone marrow (BM) and mobilization of HSC/HPC out of the BM. Previous studies have suggested that breaking the CXCL12-CXCR4 interaction mobilizes both human and mouse HPC (Liles WC, et al. Blood. 2003 102(8): 2728-30.) (Broxmeyer HE, et al. ASH 44th Annual Meeting. 2002 #2397). Other studies suggest that blocking CXCR4 inhibits homing (Peled A, et al. Science. 1999 283(5403): 845-8) and CXCR4 overexpression by lentiviral vector increases repopulation (Kahn J, et al. Blood. 2004 103(8): 2942-9) of CD34+ cells to the BM of NOD/SCID recipient mice. Expression of CXCR4 on the surface of HSC/HPC appears to be variable, depending highly on the cytokine and growth factor composition of the environment. This may partially explain the reduction in homing efficiency observed during the expansion of cord blood (CB). The efficiency of engraftment therefore appears to be dependent on the response of HSC/HPC to CXCL12 which is in turn dependent upon levels of CXCR4 expressed on HSC/HPC. In order to study the functional cellular response of HSC/HPC independent of variable levels of CXCR4 expression on the surface of cells we utilized the MSCV-based bicistronic (EGFP) retroviral vector, MIEG3, to overexpress human CXCR4 in M07e cells. The human megakaryocytic leukemia cell line, M07e, (a CD34+, c-Kit+, growth-factor-dependent human cell line) has been established as a model for human HPC. Cells infected with the MIEG3 empty vector or the MIEG3-CXCR4 construct were sorted based on their expression of GFP and CXCR4 expression was measured by flow cytometric analysis. MIEG3-CXCR4 M07e cells (GFP+) and MIEG3-CXCR4 bright M07e cells (GFP+++) express significantly higher levels of CXCR4 than M07e cells, MIEG3 M07e cells (GFP+), or MIEG3 bright M07e cells (GFP+++). Migratory response of cells to CXCL12 was assessed by chemotaxis assay. Increased CXCL12 induced chemotaxis was observed in MIEG3-CXCR4 M07e cells (p<0.05) and MIEG3-CXCR4 bright M07e cells (p<0.01). Overexpression of CXCR4 also resulted in a significant increase in CXCL12 induced cell survival during growth factor withdrawal, most markedly at low doses of CXCL12 (10 and 1.0ng/ml) (p<0.05). Thus, retroviral overexpression of CXCR4 enhances both the migratory response and survival of the human HPC line, M07e, to CXCL12. Most importantly cells respond to levels of CXCL12 that are normally too low. This data validates the ability of the MIEG3-CXCR4 construct to efficiently overexpress CXCR4 and validates the future use of MIEG3-CXCR4 M07e cells for further study of CXCR4 in HPC. This is important given the previously established ability of MIEG3 to infect CD34+ CB cells (Tao W et al. Gene Ther. 2004 11(1): 61-9). This information also suggests that previously reported increases in engraftment resulting from CXCR4 overexpression is a function of both increased HSC/HPC settlement and increased HSC/HPC survival in the transplant recipient's BM. This information may have potential therapeutic application for improvements in overall transplant efficiency.