Deregulated CXCR4-CXCL12 Signaling Impacts on the Pathogenesis of DLBCL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1519-1519
Author(s):  
Katrin Pansy ◽  
Julia Feichtinger ◽  
Barbara Ehall ◽  
Barbara Uhl ◽  
Hildegard T. Greinix ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Cell Lines ◽  
Cxcr4 Expression ◽  
Lymphoma Cell ◽  
Cxcl12 Expression ◽  
Bone Marrow Biopsies ◽  
Cxcr4 Antagonists ◽  
Apoptotic Effects

Introduction: The interaction of the chemokine receptor CXCR4 and its ligand CXCL12 appears to be implicated in many important biological processes such as proliferation, survival, migration, and/or invasion. Furthermore, it is important for normal leukocyte trafficking. Deregulation of this axis is frequently observed in several hematologic malignancies. In diffuse large B cell lymphomas (DLBCL), the CXCR4-CXCL12 axis is still largely unexplored and published data are contradictive. Hence, we comprehensively studied the CXCR4-CXCL12-axis in our DLBCL cohort as well as the effects of CXCR4 antagonists on lymphoma cell lines in vitro. Methods: We determined the CXCR4 and CXCL12 expression levels in NGCB- and GCB-DLBCL consisting of primary and transformed follicular lymphomas (n=77 in total), the corresponding bone marrow samples (n=63) and non-neoplastic germinal center-B cells (GC-B, n=5) serving as non-malignant control. To investigate the effects of CXCR4 antagonists in vitro and their function in regulation of important pathways (JNK, ERK1/2 and NF-κB) known to be involved in lymphomagenesis, we treated lymphoma cell lines with three different CXCR4 antagonists, AMD070, AMD3100 and WK1 (a novel nicotinic acid derivative of AMD070 - synthesized by us), followed by functional assays and gene expression profile. Results: CXCR4 was 140-fold higher expressed in DLBCL compared to non-neoplastic GC-B cells. Interestingly, higher CXCR4 expression correlated to a clinically advanced stage, to bone marrow infiltration and worse cancer-specific survival in DLBCL. Further expression analysis by using the corresponding bone marrow biopsies demonstrated that CXCL12 expression correlated to the lymphoma infiltration rate and that CXCR4 expression was reduced in remission under therapy. Moreover, two CXCR4 antagonists - AMD070 and especially WK1 - exerted pro-apoptotic effects on CXCR4 positive lymphoma cells in vitro and induced the expression of certain pro-apoptotic genes in CXCR4 positive cell lines. Remarkably, these effects were more pronounced for the WK1. Finally, WK1 treatment resulted in reduced expression of JNK-, ERK1/2- and NFκB/BCR-target genes. Conclusion: Our data demonstrate that the CXCR4-CXCL12 axis is involved in the pathogenesis of DLBCL. Since CXCR4 antagonists exert pro-apoptotic effects and impact lymphoma relevant pathways, they represent interesting molecules to develop novel therapeutic agents. Disclosures No relevant conflicts of interest to declare.

scholarly journals The CXCR4–CXCL12-Axis Is of Prognostic Relevance in DLBCL and Its Antagonists Exert Pro-Apoptotic Effects In Vitro

2019 ◽  
Vol 20 (19) ◽  
pp. 4740 ◽  
Author(s):  
Katrin Pansy ◽  
Julia Feichtinger ◽  
Barbara Ehall ◽  
Barbara Uhl ◽  
Miriam Sedej ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Bone Marrow Involvement ◽  
B Cell Lymphoma ◽  
Cxcr4 Expression ◽  
Lymphoma Cell ◽  
Bone Marrow Biopsies ◽  
Cxcr4 Antagonists ◽  
Apoptotic Effects

In tumor cells of more than 20 different cancer types, the CXCR4-CXCL12-axis is involved in multiple key processes including proliferation, survival, migration, invasion, and metastasis. Since data on this axis in diffuse large B cell lymphoma (DLBCL) are inconsistent and limited, we comprehensively studied the CXCR4-CXCL12-axis in our DLBCL cohort as well as the effects of CXCR4 antagonists on lymphoma cell lines in vitro. In DLBCL, we observed a 140-fold higher CXCR4 expression compared to non-neoplastic controls, which was associated with poor clinical outcome. In corresponding bone marrow biopsies, we observed a correlation of CXCL12 expression and lymphoma infiltration rate as well as a reduction of CXCR4 expression in remission of bone marrow involvement after treatment. Additionally, we investigated the effects of three CXCR4 antagonists in vitro. Therefore, we used AMD3100 (Plerixafor), AMD070 (Mavorixafor), and WKI, the niacin derivative of AMD070, which we synthesized. WK1 demonstrated stronger pro-apoptotic effects than AMD070 in vitro and induced expression of pro-apoptotic genes of the BCL2-family in CXCR4-positive lymphoma cell lines. Finally, WK1 treatment resulted in the reduced expression of JNK-, ERK1/2- and NF-κB/BCR-target genes. These data indicate that the CXCR4-CXCL12-axis impacts the pathogenesis of DLBCL and represents a potential therapeutic target in aggressive lymphomas.

scholarly journals The CXCR4-CXCL12-Axis is of Prognostic Relevance in DLBCL and Its Antagonists Exert Pro-Apoptotic Effects in Vitro

2019 ◽  
Author(s):  
Katrin Pansy ◽  
Julia Feichtinger ◽  
Barbara Ehall ◽  
Barbara Uhl ◽  
Miriam Sedej ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Target Genes ◽  
Bone Marrow Involvement ◽  
B Cell Lymphoma ◽  
Cxcr4 Expression ◽  
Lymphoma Cell ◽  
Bone Marrow Biopsies ◽  
Apoptotic Effects

In tumour cells of more than 20 different cancer types, the CXCR4-CXCL12-axis is involved in multiple key processes including proliferation, survival, migration, invasion, and metastasis. Since data on this axis in diffuse large B cell lymphoma (DLBCL) are inconsistent and limited, we comprehensively studied the CXCR4-CXCL12-axis in our DLBCL cohort as well as the effects of CXCR4 antagonists on lymphoma cell lines in vitro. In DLBCL, we observed a 140-fold higher CXCR4 expression compared to non-neoplastic controls. Interestingly, high expression of CXCR4 was associated with poor clinical outcome. Furthermore, in corresponding bone marrow biopsies, we observed a correlation of CXCL12 expression and lymphoma infiltration rate as well as a reduction of CXCR4 expression in remission of bone marrow involvement after treatment. Furthermore, the niacin derivate of the CXCR4 antagonist AMD070, which was synthesized by us, demonstrated stronger pro-apoptotic effects than AMD070 in vitro and induced expression of certain pro-apoptotic genes in CXCR4 positive lymphoma cell lines. Finally, WK1 treatment resulted in reduced expression of JNK-, ERK1/2- and NFκB/BCR-target genes. These data indicate that the CXCR4-CXCL12-axis impacts the pathogenesis of DLBCL and represents a potential therapeutic target in aggressive lymphomas.

CXCR7 Regulates SDF-1 Induced Adhesion and Homing in Multiple Myeloma.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1674-1674 ◽  
Author(s):  
Nicholas Burwick ◽  
Anne-Sophie Moreau ◽  
Xiaoying Jia ◽  
Xavier Leleu ◽  
Judith Runnels ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Adhesion ◽  
Cell Lines ◽  
Cxcr4 Expression ◽  
Rt Pcr ◽  
Growth And Survival ◽  
Tumor Types

Abstract BACKGROUND: Multiple myeloma (MM) is a plasma cell malignancy that depends on interactions with the bone marrow (BM) microenvironment for growth and survival. In turn, adhesion of MM cells to the BM stroma provides a mechanism of resistance from standard chemotherapeutic agents. Recently, our lab has shown that by disrupting this adhesion using a selective CXCR4 inhibitor named AMD3100, MM cells are more sensitive to the proteasome inhibitor Bortezomib (Ghobrial lab, unpublished data). CXCR4 has been a particularly attractive target because its ligand SDF-1 is known to induce p42/44 MAPK, AKT, and the down-stream anti-apoptotic protein bad in MM cells, leading to increased MM growth and survival. Until recently, CXCR4 was thought to be a canonical receptor for the SDF-1 ligand. However, a second chemokine receptor for SDF-1 was subsequently discovered and named CXCR7. CXCR7 is a novel chemokine receptor that is important in cell adhesion, growth and survival in several tumor types. However, the role of CXCR7 in multiple myeloma (MM) has yet to be explored. Furthermore, the ability of SDF-1 ligand to regulate MM function via CXCR7 has not been studied. METHODS: The MM cell lines (U266, MM1.S, RPMI, OPM2, OPM1) were used. After informed consent was obtained, primary bone marrow samples from MM patients were collected. CD138 positive mononuclear cells were isolated by microbead selection. The expression of CXCR7 on MM cell lines and patient samples was confirmed using flow cytometry and RT-PCR analysis. For functional in vitro and ex-vivo assays, the CXCR7 selective antagonist 733 was used (ChemoCentryx Inc., Mountain View, CA). RESULTS: Here we show that CXCR7 was expressed on all tested MM cell lines and primary patient samples as demonstrated by flow cytometry and RT-PCR. Furthermore, CXCR7 was found to regulate SDF-1 induced MM cell adhesion, as demonstrated by in vitro assays using a small molecule compound specific for CXCR7 (733). The CXCR7 antagonist showed significant inhibition of adhesion of MM cell lines and patient samples to fibronectin, endothelial cells and stromal cells, with 50% reduction of adhesion at 5nM of the CXCR7 inhibitor, and with similar activity compared to 20uM of AMD3100 (CXCR4 inhibitor). However, unlike CXCR4, CXCR7 did not effect trans-well migration to SDF-1 chemokine. Interestingly, both receptors were found to be important for trans-endothelial migration of MM cells. Moreover, pre-treatment with 733 reduced homing of MM cells to the BM niche in vivo. Previous studies have failed to show signaling in response to CXCR7 in many tumor types. Here, we demonstrate that treatment with 733 inhibited SDF-1 induced pERK and pAKT, ribosomal pS6Kinase, pGSK3, pSTAT3, pFAK and pPAK signaling pathways, confirming a role for CXCR7 in facilitating SDF-1 signaling. This effect was further confirmed using immunofluorescence. To investigate whether CXCR7 and CXCR4 interact directly, we examined the effect of 733 and AMD3100 on CXCR4 expression and found that AMD3100 significantly inhibited CXCR4 expression, while 733 had no effect on CXCR4 expression, even in the presence of SDF-1. The CXCR7 inhibitor had no effect on the survival of MM cells using MTT and flow cytometry analysis, while high doses of 733 (1uM) had modest inhibition of proliferation. Interestingly, 733 prevented the growth advantage induced by 30nM SDF-1 at 24 hrs. CONCLUSION: Together, these results demonstrate the importance of CXCR7 in regulating MM adhesion and homing, and highlight the differential effects of CXCR4 and CXCR7 in regulating SDF-1 signaling in MM, thus providing a rationale for targeting the SDF-1/CXCR7 axis in MM.

The Novel CXCR4 Antagonist POL5551 Decreases Surface CXCR4 (s-CXCR4) Expression, Inhibits Chemotaxis, and Enhances Chemosensitivity in Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 780-780
Author(s):  
Edward Allan R. Sison ◽  
Daniel Magoon ◽  
Eric Chevalier ◽  
Klaus Dembowsky ◽  
Patrick Brown
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Cxcr4 Expression ◽  
Leukemia Cells ◽  
The Novel ◽  
Cxcr4 Antagonist ◽  
Induced Apoptosis

Abstract Abstract 780 Background: The interaction between the cell surface receptor CXCR4 and the chemokine SDF-1 (CXCL12) is critical in signaling between leukemic blasts and the bone marrow microenvironment. We previously demonstrated that CXCR4 is an important mediator of chemotherapy resistance, as chemotherapy-induced upregulation of s-CXCR4 in acute myeloid leukemia (AML) cell lines and primary samples led to increased SDF-1-mediated chemotaxis and increased protection by normal human bone marrow stroma from chemotherapy-induced apoptosis. We also showed that stromal protection and chemotherapy resistance could be reversed by treatment with the FDA-approved CXCR4 inhibitor plerixafor, both in vitro in stromal co-cultures of pre-B cell ALL cell lines and in vivo in xenografts of primary samples of infant MLL-rearranged ALL. Therefore, disruption of the CXCR4/SDF-1 axis is a rational means to target extrinsic survival mechanisms in acute leukemia. The novel Protein Epitope Mimetic (PEM) POL5551 is a selective and potent antagonist of CXCR4. Treatment with POL5551 inhibits vascular accumulation of CXCR4+ smooth muscle cells but its effects on ALL have not been reported. We hypothesized that treatment of ALL cell lines with POL5551 would 1) decrease s-CXCR4 expression, 2) inhibit SDF-1-mediated chemotaxis, and 3) reverse stromal-mediated protection from chemotherapy-induced apoptosis. Methods/Results: Pre-B cell ALL (697, HB11;19, NALM-6, SEMK2) and T cell ALL cell lines (CCRF-CEM-1301, Jurkat, Molt-4) were treated with dose ranges of POL5551. Cells were harvested at multiple time points over 72 hours and s-CXCR4 was measured by FACS. S-CXCR4 was potently and markedly reduced in all cell lines, with IC50 levels of <5 nM at 1 hour and IC50 levels of <20 nM at 48 hours. In comparison, 3- to 30-fold higher doses of plerixafor were needed to achieve similar levels of reduction. Simultaneous measurement of cell proliferation using the WST-1 proliferation assay demonstrated that treatment with POL5551 neither increased nor decreased leukemia cell proliferation in a significant manner. To ascertain the functionality of s-CXCR4 inhibition, we performed chemotaxis assays. Leukemia cells were treated with 10 nM POL5551 or vehicle control and placed into hanging cell culture inserts. Migration through a permeable membrane toward an SDF-1 gradient was then measured after 24 hours. Compared to control-treated cells, POL5551-treated cells had significantly decreased SDF-1-induced chemotaxis (average 38% reduction in chemotaxis in pre-B cell lines, p<0.001; average 41% reduction in T cell lines, p=0.05). We also performed co-culture experiments with normal human bone marrow stroma in the presence and absence of POL5551 to further demonstrate the functional effects of s-CXCR4 inhibition. Specifically, we cultured leukemia cells off stroma (O), on stroma (S), or pretreated with POL5551 for 30 minutes prior to plating on stroma (P+S). Cells from each culture condition were then treated with dose ranges of chemotherapy. Following treatment, we measured apoptosis by staining with Annexin V/7-AAD. IC10 through IC90 values were obtained using Calcusyn. To quantify stromal protection, we calculated a Protective Index (PI), defined as the S IC values divided by the O IC values. Thus, PI >1 signified stromal protection, while PI ≤1 signified no stromal protection. To quantify the ability of POL5551 to reverse stromal protection, we calculated a Reversal Index (RI), defined as the P+S IC values divided by the O IC values. Therefore, PI > RI indicated a decrease in stromal protection, while RI ≤1 indicated a reversal of stromal protection. Overall, stroma protected leukemia cells from chemotherapy-induced apoptosis. Importantly, treatment with POL5551 abrogated stromal-mediated protection and restored chemosensitivity (eg, PI 1.182 vs. RI 0.956 for NALM-6 treated with daunorubicin +/− 20 nM POL5551, p<1×10e-9). Conclusions: The novel CXCR4 antagonist POL5551 is a potent inhibitor of CXCR4 in pre-B and T ALL cell lines with activity at nanomolar concentrations in decreasing s-CXCR4 expression, inhibiting SDF-1-induced chemotaxis, and reversing stromal-mediated protection from chemotherapy in vitro. Therefore, if our findings are confirmed in primary samples and in vivo, interruption of leukemia-microenvironment signaling with POL5551 may prove to be an effective strategy in the treatment of pre-B and T cell ALL. Disclosures: Chevalier: Polyphor Ltd: Employment. Dembowsky:Polyphor Ltd: Employment.

scholarly journals Epstein-Barr virus transformation of human pre-B cells.

1983 ◽  
Vol 158 (2) ◽  
pp. 616-622 ◽  
Author(s):  
M Hansson ◽  
K Falk ◽  
I Ernberg
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Bone Marrow Cells ◽  
Fetal Bone ◽  
Barr Virus ◽  
Epstein Barr

In vitro infection of human B lymphocytes with Epstein-Barr virus (EBV) results in establishment of B lymphoblastoid cell lines that reflect normal B cell phenotypes. In this study we have investigated whether immature B cells from fetal bone marrow and liver can serve as targets for EBV. The fetal bone marrow cells were readily transformed by EBV. Among the resulting cell lines, five were surface Ig (sIg)-negative. Three B cell-associated antigens defined by monoclonal antibodies were expressed to the same extent on the fetal cell lines, whether they belonged to the sIg- or sIg+ group. The various differentiation stages that these cell lines may represent are discussed.

CD31/PECAM-1 Is Overexpressed On Mantle-Cell Lymphoma (MCL) Primary Cells and Positively Regulates Engraftment and Growth of MCL Cell Lines in Immunodeficient Mice.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2681-2681
Author(s):  
Jan Molinsky ◽  
Magdalena Klanova ◽  
Bokang Maswabi ◽  
Josef Karban ◽  
Martin Spacek ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Cell Lymphoma ◽  
Immunodeficient Mice ◽  
Mantle Cell ◽  
Original Cell ◽  
Cd31 Expression

Abstract Abstract 2681 Mantle-cell lymphoma (MCL) is a lymphoma subtype with poor prognosis. Recently, new drugs targeting at least partially the angiogenesis cascade have been successfully tested in the therapy of relapsed MCL patients, including temsirolimus, enzastaurin and lenalidomide. To reveal molecular mechanisms that regulate MCL-induced angiogenesis (and that might represent potential new druggable targets), we established and analyzed two mouse models of human MCL. Immunodeficient mice were subcutaneously (s.c.) xenografted with MCL cell lines JEKO-1 and HBL-2, and when tumor diameters reached 3cm in any size, the mice were sacrificed, and the excized tumors subjected to immunohistochemical (IHC) analysis. Alternatively, ex vivo obtained MCL cells were magnetically sorted using CD45 microbeads, and subjected to gene expression and flow cytometry analyses compared to the in vitro growing controls. IHC analysis proved that the tumors were neovascularized. Gene expression profiling by TaqMan Human Angiogenesis Array revealed that the most upregulated gene in both JEKO-1 and HBL-2 in vivo vs. in vitro growing cells was platelet/endothelial cell adhesion molecule CD31/PECAM-1 (fold change 148.9 ± 19.4, and 127.6 ± 10.5, respectively). Recently, Boyd et al. reported upregulation of CD31 in three of five primary MCL samples compared to normal B cells by Western blotting. By flow cytometry we assessed surface expression of CD31 on primary cells obtained from peripheral blood and bone marrow of 25 MCL patients before therapy. The percentage of CD31 positive cells was significantly higher in the subset of CD19+CD5+ peripheral blood MCL cells compared to CD19+CD5+ healthy donor B-cells (78.6± 4.4 vs. 14.7±2.6, p< 0.001), as well as in the subset of CD19+CD5+ bone marrow MCL cells compared to CD19+CD5+ B-cells obtained from the bone marrow of patients with various lymphoma subtypes with no detectable bone marrow involvement (81.9±3.9 vs. 20.5±4.8, p< 0.0001). In addition to surface CD31 we asked if patients with MCL have increased levels of soluble form of CD31 (sCD31) compared to healthy volunteers. We measured concentrations of sCD31 in plasma samples obtained from 17 MCL patients before therapy by ELISA. Despite the fact that MCL patients demonstrated higher variability in sCD31 concentrations (range 10.7–135.6 ng/ml) compared to healthy volunteers (range 43.3– 92.0 ng/ml), the medians were not statistically different (56.4 vs. 53.3 ng/ml). We also measured concentrations of sCD31 in plasma of two patients with MCL before treatment and after three cycles of chemotherapy, and found that the post-chemotherapy levels of sCD31 were lower in both patients (91.8 vs. 60.7 ng/ml; 56.4 vs. 30.4 ng/ml). To investigate the role of CD31 in the biology of MCL we derived JEKO-1 and HBL-2 clones with stable downregulation of CD31 by siRNA approach. In addition, two JEKO-1 subclones with upregulated CD31 were established by limiting dilution from the original cell line (=controls). The limiting dilution approach was not feasible in HBL-2 cells, which completely lack CD31 expression. While HBL-2 clones with downregulated CD31 engrafted constantly, JEKO-1 clones with downregulated CD31 engrafted only in 2 out of 8 mice. Growth of the tumors derived from both HBL-2 and JEKO-1 clones with downregulated CD31 was significantly slower compared to that of control tumors (HBL-2: 2.1±0.4 g versus 4.1±0.2 g, p=0.002; JEKO-1: 0.5±0.1 g versus 2.2±0.2 g, p=0.0019). Xenotransplantation of JEKO-1 clones with upregulated CD31 resulted in accelerated tumor growth compared to controls (3.4±0.2 g versus 2.2±0.2 g, p=0.0027). Importantly, the in vitro proliferation rate between the clones with changed CD31 expression and the original cell lines were not statistically different suggesting that the different growth pattern of tumors was a consequence of altered interaction between the tumor cells and the murine microenvironment. In summary, CD31/PECAM-1 antigen is overexpressed on primary CD19+CD5+ MCL cells obtained from the peripheral blood and bone marrow of MCL patients before therapy compared to control CD19+CD5+ B-cells. The upregulated CD31/PECAM-1 appears to play important role in MCL biology, and might represent potential druggable target. Financial Support: IGA-MZ NT13201-4/2012, GAUK 259211/110709, GAUK 446211, UNCE 204021, PRVOUK P24/LF1/3, PRVOUK 1–5101–280002 PVK, SVV-2012–254260507 Disclosures: No relevant conflicts of interest to declare.

Expression of Bcl-2 Pro-Survival Family Proteins Predicts Pharmacological Responses to ABT-199, a Novel and Selective Bcl-2 Antagonist, in Multiple Myeloma Models

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5028-5028 ◽  
Author(s):  
Deepak Sampath ◽  
Elizabeth Punnoose ◽  
Erwin R. Boghaert ◽  
Lisa Belmont ◽  
Jun Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Cell Lines ◽  
Single Agent ◽  
Employment Equity ◽  
Bone Marrow Biopsies ◽  
Xenograft Models ◽  
Equity Ownership

Abstract Abstract 5028 Multiple myeloma (MM) is a hematological malignancy of the bone marrow caused by the dysregulated proliferation of monoclonal antibody producing plasma cells. A hallmark feature of cancer is the ability to evade cell death signals induced by stress response cues. The Bcl-2 family of proteins regulates the intrinsic apoptosis pathways and consists of pro-apoptotic (Bax, Bak, Bad, Bim, Noxa, Puma) and pro-survival (Bcl-2, Bcl-xL, Mcl-1); the balance of which dictates the life or death status of MM tumor cells. Thus, there is a strong rationale to target members of the Bcl-2 proteins for the treatment of MM. ABT-199 is a potent BH3-only mimetic that selectively antagonizes Bcl-2 and is currently in phase I clinical trials for the treatment of hematological malignancies. Therefore, we evaluated the efficacy of ABT-199 as a single agent and in combination with standard of care drugs such as Velcade (bortezomib) in preclinical models of MM. A panel of 21 human MM cell lines was evaluated in vitro for to sensitivity to ABT-199. ABT-199 potently inhibited cell viability in a sub-set of MM cell lines (7/21) with EC50 values less than 1 μM. Expression of Bcl-2, Bcl-xL, Mcl-1, Bim and other Bcl-2 family proteins were evaluated by protein and mRNA. Cell line modeling identified thresholds for expression of Bcl-2, Bcl-xL and Mcl-1 that best predicted sensitivity and resistance to ABT-199 and the dual Bcl-2/Bcl-xL antagonist, navitoclax. Consistent with the target inhibition profile of these drugs, we found that MM lines that were Bcl-2high/Bcl-xLlow/Mcl-1low are the most sensitive to ABT-199 treatment. Whereas cell lines that are Bcl-xLhigh remain sensitive to navitoclax but not ABT-199. MM cell lines that are Mcl-1high are less sensitive to both ABT-199 and navitoclax, suggesting that Mcl-1 is a resistance factor to both drugs. Utilizing a novel Mesoscale Discovery based immunoassay we determined that levels of Bcl-2/Bim complexes also correlated with sensitivity of ABT-199 in the MM cell lines tested. In addition, the t(11;14) status in these cell lines associated with sensitivity to ABT-199. The clinical relevance of the Bcl-2 pro-survival expression pattern in MM cell lines, was determined by a collection of bone marrow biopsies and aspirates (n=27) from MM patients by immunohistochemistry for prevalence of Bcl-2 and Bcl-xL. Similar to our in vitro observations, the majority (75%) of the MM bone marrow biopsies and aspirates had high Bcl-2 levels whereas 50% had high Bcl-xL expression. Therefore, a subset of patient samples (33%) were identified with a favorable biomarker profile (Bcl-2high/Bcl-xLlow) that may predict ABT-199 single agent activity. ABT-199 synergized with bortezomib in decreasing cell viability in the majority of MM cell lines tested in vitro based on the Bliss model of independence analyses (Bliss score range = 10 to 40). However the window of combination activity was reduced due to high degree of sensitivity to bortezomib alone. Therefore, the combination efficacy of ABT-199 and bortezomib was further evaluated in vivo in MM xenograft models that expressed high levels of Bcl-2 protein (OPM-2, KMS-11, RPMI-8226, H929 and MM. 1s). Bortezomib treatment alone at a maximum tolerated dose resulted in tumor regressions or stasis in all xenograft models tested. ABT-199 at a maximum tolerated dose was moderately efficacious (defined by tumor growth delay) as a single agent in xenograft models that expressed high protein levels of Bcl-2 but relatively lower levels of Bcl-xL. However, the combination of ABT-199 with bortezomib significantly increased the overall response rate and durability of anti-tumor activity when compared to bortezomib, resulting in increased cell death in vivo. Treatment with bortezomib increased levels of the pro-apoptotic BH3-only protein, Noxa, in MM xenograft models that expressed high levels of Mcl-1. Given that the induction of Noxa by bortezomib results in neutralization of Mcl-1 pro-survival activity in MM models [Gomez-Bougie et al; Cancer Res. 67:5418–24 (2007)], greater efficacy may be achieved when Bcl-2 is antagonized by ABT-199 thereby inhibiting pro-survival activity occurring through either Bcl-2 or Mcl-1 and increasing cell death. Thus, our preclinical data support the clinical evaluation of ABT-199 in combination with bortezomib in MM patients in which relative expression of the Bcl-2 pro-survival proteins may serve as predictive biomarkers of drug activity. Disclosures: Sampath: Genentech: Employment, Equity Ownership. Punnoose:Genentech: Employment, Equity Ownership. Boghaert:Abbott Pharmaceuticals: Employment, Equity Ownership. Belmont:Genentech: Employment, Equity Ownership. Chen:Abbott Pharmaceuticals: Employment, Equity Ownership. Peale:Genentech: Employment, Equity Ownership. Tan:Genentech: Employment, Equity Ownership. Darbonne:Genentech: Employment, Equity Ownership. Yue:Genentech: Employment, Equity Ownership. Oeh:Genentech: Employment, Equity Ownership. Lee:Genentech: Employment, Equity Ownership. Fairbrother:Genentech: Employment, Equity Ownership. Souers:Abbott Pharmaceuticals: Employment, Equity Ownership. Elmore:Abbott Pharmaceuticals: Employment, Equity Ownership. Leverson:Abbott Pharmaceuticals: Employment, Equity Ownership.

HGAL-a Germinal Center Specific Protein, Enhances B-Cell Receptor Signaling by Activation of Syk, Leading to Follicular Lymphoproliferation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 584-584
Author(s):  
Xiaoyu Jiang ◽  
Isabel Romero-Camarero ◽  
Xiaoqing Lu ◽  
Carolina Vicente-Dueñas ◽  
Ines Gonzalez-Herrero ◽  
...  
Keyword(s):  
B Cells ◽  
Transgenic Mice ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Lines ◽  
Germinal Center ◽  
Kinase Activity ◽  
Lymphoma Cell ◽  
Syk Kinase

Abstract Abstract 584 The Human Germinal center Associated Lymphoma (HGAL) gene is exclusively expressed in germinal center (GC) B-lymphocytes and GC-derived lymphomas. In patients with diffuse large B-cell lymphomas (DLBCL), HGAL expression identifies a subgroup of patients with biologically distinct tumors associated with improved survival. Our previous in vitro studies demonstrated that HGAL decreases spontaneous and chemoattractant-induced cell motility by activating the RhoA signaling pathway and by directly interacting and augmenting F-actin and myosin II binding. However, the major function of HGAL in GC lymphocytes remains largely unknown. Based on our previous observation of tyrosine phosphorylation of a modified ITAM motif in the HGAL by Lyn, we hypothesized that HGAL may be involved in B-cell receptor (BCR) signaling. Indeed, following BCR stimulation of two GCB-like lymphoma cell lines (Raji and VAL), we observed marked reduction of Syk, Btk and PLCγ phosphorylation upon knockdown of endogenous HGAL by specific but not control siRNAs. Concordantly, HGAL knockdown in BCR-stimulated Raji cells reduced Ca2+ mobilization and decreased NFAT transcriptional activity as analyzed by a luciferase reporter assay. HGAL expression in the BCR-stimulated HBL1 lymphoma cell line (lacking endogenous HGAL protein) resulted in increased Syk, Btk and PLCγ phosphorylation. Syk plays a major role in coupling BCR activation to downstream effectors. Endogenous HGAL was detected in immunoprecipitates of endogenous Syk and vice versa. Nanoscope microscopy studies confirmed co-localization of HGAL and Syk proteins in cell membranes, which was enhanced following BCR stimulation. In BCR-stimulated cells, Syk kinase activity was markedly increased following addition of HGAL protein as measured by an in vitro Syk kinase activity assay. To comprehensively examine HGAL effects on immune system and BCR signaling, we generated a transgenic mouse model in which HGAL is expressed under the control of the mouse Ly-6E.1 promoter in Sca1+ hematopoietic stem cells and progenitors of C57BL/6 × CBA mice. The Sca1-HGAL transgenic mice showed normal embryonic and post natal development, and at 8 weeks of age demonstrated normal lymphoid development without any significant changes in the major hematopoietic compartments (bone marrow (BM), spleen, thymus and peripheral lymph nodes) and in peripheral blood. They also exhibited normal GC development in response to a T-cell dependent antigen immunization. In contrast, at 12 months of age the Sca1-HGAL mice developed a decrease in BM immature B-cells at the expense of recirculating B-cells (B220+IgDhi) compared to the age-matched normal littermates, suggesting a defect in B-cell lymphopoiesis. All the Sca1-HGAL transgenic mice became ill from approximately 12 months of age and all died between 12 to 22 months of age with statistically shorter survival as compared to the wild type controls. Analysis of these animals showed massive splenomegaly with marked white pulp hyperplasia and presence of multiple, frequently contiguous nodules predominantly composed of polyclonal follicular (B220+CD21intCD23hi) B lymphocytes. Extra-lymphatic infiltration by similar B lymphocytes was observed in the liver, lungs and kidneys of Sca1-HGAL mice with advanced disease. IgG isotype titers in these animals tended to be higher than in the wild-type controls, reaching a statistically significant difference for the IgG1 isotype. Follicular hyperplasia in the Sca1-HGAL transgenic mice is likely attributable to increased RhoA activation and enhanced BCR signalling manifested by increased Syk phosphorylation, Ca2+ mobilization and in vitro B cell proliferation following BCR stimulation, in agreement with similar data observed in human DLBCL cell lines expressing HGAL. Gene expression profiling of lymphoid tissues confirmed significantly enhanced BCR signalling and RhoA pathway activation in Sca1-HGAL transgenic mice, corresponding to similar pathway activation in human lymphoma cell lines over-expressing HGAL. Overall, our findings demonstrate that HGAL, specifically expressed in GC B cells, enhances responsiveness to antigens by stimulating Syk kinase activity that without appropriate regulation may lead to lymphoproliferation. Further studies are needed to examine the role of HGAL in the pathogenesis of GC-derived lymphomas. Disclosures: No relevant conflicts of interest to declare.

Eltrombopag Decreases Proliferation of Human Leukemia and Lymphoma Cell Lines In Vitro.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4089-4089
Author(s):  
Connie L. Erickson-Miller ◽  
Parrish L. Payne ◽  
Sherri Moore ◽  
Sherryann Wert ◽  
Richard D. May
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Cell Lines ◽  
Thymidine Incorporation ◽  
Lymphoma Cell ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Platelet Counts ◽  
Rpmi 8226

Abstract Eltrombopag is a novel, oral platelet growth factor that interacts with the thrombopoietin receptor on bone marrow progenitors to stimulate megakaryocyte and platelet production. Clinical trials have demonstrated that eltrombopag increases platelet counts in healthy volunteers and in patients with idiopathic thrombocytopenic purpura (ITP) and hepatitis C. Because thrombopoietin (TPO) receptors are expressed on lymphoma and leukemia cell lines and recombinant human TPO (rhTPO) has been shown to stimulate their proliferation in vitro, experiments were conducted to evaluate the effects of eltrombopag on the proliferation of leukemia and lymphoma cells in vitro. Five leukemia and lymphoma cell lines (ie, CCRF-CEM, K562, MOLT-4, RPMI-8226 and SR) representing lymphoblastic T cell leukemia, chronic myelogenous leukemia, acute lymphoblastic T cell leukemia, plasmacytoma and immunoblastic large cell lymphoma, were exposed to eltrombopag (0.1–40 ug/mL), rhTPO (100 ng/mL) or both for 3 days. Proliferation was measured by 18-hour incubation with tritiated thymidine incorporation. As expected, rhTPO alone led to a small, statistically significant increase in the proliferation of CCRF-CEM and RPMI-8226 cells, while it had no effect on the other cell lines. However, the addition of eltrombopag to rhTPO negated the increased proliferation seen with rhTPO alone. Even more remarkable, treatment with eltrombopag alone inhibited the proliferation of all five leukemia and lymphoma cell lines with an IC50 range of 0.56 to 5.9 ug/mL and 100% inhibition (IC100) of thymidine incorporation at 10 ug/mL (Table). Previous studies have shown that eltrombopag induces proliferation and differentiation of bone marrow progenitor cells in vitro and increases platelet counts in vivo. While eltrombopag and rhTPO interact with the TPO receptor to stimulate the production of platelets, there are differences in the site of receptor interaction and their signaling pathways. The findings of the current study suggest that eltrombopag may inhibit the proliferation of leukemia and lymphoma cell lines unlike the effect that has been demonstrated with rhTPO. Interestingly, eltrombopag may also serve to mitigate rhTPO-mediated proliferation of malignant hematologic cell lines. These findings merit further evaluation of the effects of eltrombopag on leukemia, lymphoma, and solid tumor cell proliferation. Cell line IC50 (ug/mL) IC100 (ug/mL) CCRF-CEM 0.74 10 K562 1.80 10 MOLT-4 0.56 4 RPMI-8226 5.90 10 SR 0.77 4

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