The Serine/Threonine Kinase Pim-2 Is a Novel Anti-Apoptotic Mediator in Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 243-243
Author(s):  
Jin Asano ◽  
Masahiro Abe ◽  
Shiro Fujii ◽  
Osamu Tanaka ◽  
Ai Mihara ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Growth ◽  
Stromal Cells ◽  
Osteoblast Differentiation ◽  
Family Members ◽  
Bone Marrow Microenvironment ◽  
Serine Threonine Kinase ◽  
Growth And Survival ◽  
Threonine Kinase ◽  
Cell Growth And Survival

Abstract Myeloma (MM) cells stimulate bone resorption by enhancing osteoclast (OC) formation and suppress bone formation by inhibiting osteoblast differentiation, leading to destructive bone lesions. In these lesions, OCs and stromal cells with defective osteoblast differentiation create a microenvironment suitable for myeloma cell growth and survival (a MM niche) to protect MM cells from various apoptotic insults. IL-6 and the TNF family members BAFF and APRIL have been demonstrated to be among predominant anti-apoptotic cytokines for MM cells elaborated by the bone marrow microenvironment in MM. The serine/threonine kinase Pim-2 is a novel apoptotic inhibitor which is transcriptionally up-regulated to promote survival of hematopoietic cells in response to environmental growth factors and cytokines. Up-regulation of Pim-2 expression has also been observed in various malignancies including MM. However, the roles for Pim-2 in growth and survival of MM cells are largely unknown. In the present study we therefore investigated the regulatory mechanism for Pim-2 expression in MM cells and the impact of Pim-2 on MM cell growth and survival with special reference to the interaction between MM cells and bone marrow components. Pim-2 protein is constitutively overexpressed in the absence of IL-6 in IL-6-dependent INA-6 as well as IL-6-independent RPMI8226 and U266 MM cell lines. Addition of IL-6, BAFF and TNFalpha up-regulated Pim-2 protein expression in INA-6 and RPMI8226 cells. A JAK/STAT3 inhibitor, cucurbitacin I, suppresses Pim-2 expression induced by IL-6, indicating Pim-2 as a downstream target of a JAK/STAT3 pathway. Stromal cells and OCs are regarded as a predominant cell type in MM bone marrow microenvironment to produce IL-6 and the TNF family members BAFF and APRIL, respectively. Co-cultures with stromal cells as well as OCs enhanced Pim-2 expression in INA-6 cells, suggesting up-regulation of Pim-2 in MM cells by surrounding cells in the bone marrow. In order to clarify the roles for Pim-2 in growth and survival of MM cells we next looked at the effects of Pim-2 siRNA. Suppression of Pim-2 expression by Pim-2 siRNA partly reduced the proliferation of INA-6 cells stimulated by IL-6 as well as the co-cultures with stromal cells or OCs. Pim-2 silencing also enhanced the cytotoxic effects of dexamethason on MM cells. Interestingly, further addition of rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces cell death in concert with Pim-2 silencing in INA-6 cells, suggesting a cooperative roles for PI3K/Akt and Pim-2-mediated pathways in growth and survival of MM cells. Furthermore, Pim-2 silencing induced the cleavage of caspase9 but not caspase8; enforced expression of Pim-2 phosphorylated the BH3 only protein Bad; Pim-2 silencing suppressed phosphorylation of Bad by IL-6. Thus, Pim-2 appears to activate the intrinsic pathway of apoptotic machinery involving Bad phosphorylation. Taken together, our results suggest that Pim-2 is an important prosurvival mediator in MM cells, and that up-regulation of its expression in MM cells by bone marrow components may at least in part contribute to resistance to spontaneous and drug-induced apoptosis in MM cells. Therefore, Pim-2 may become a target for novel therapeutic strategies against MM.

scholarly journals Annexin II Interactions with the Annexin II Receptor Enhance Multiple Myeloma Cell Adhesion and Growth in the Bone Marrow Microenvironment,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3942-3942
Author(s):  
Sonia D'Souza ◽  
Noriyoshi Kurihara ◽  
Yusuke Shiozawa ◽  
Jeena Joseph ◽  
Russell Taichman ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Cell Lines ◽  
Stromal Cells ◽  
Critical Role ◽  
Myeloma Cell ◽  
Bone Marrow Microenvironment ◽  
Annexin Ii

Abstract Abstract 3942 Background: Multiple myeloma (MM) is an incurable B-cell malignancy that develops in the bone marrow. The marrow microenvironment plays a critical role in supporting homing, lodging, and growth of MM cells by activating signaling pathways in both MM and bone marrow stromal cells (BMSC). We previously showed that annexin II (AXII) is involved in prostate cancer cell lodgment to the bone marrow via the annexin II receptor (AXIIR) expressed on prostate cancer cells. We hypothesized that MM cells use a similar mechanism to lodge and grow in the bone marrow. In support of this hypothesis, we found that MM cell lines and primary MM cells from 8 MM patients express the AXIIR protein, and that MM cells adhered significantly better to BMSC from AXII+/+ mice than from AXII−/− mice. Further, knockdown of AXIIR by siRNA in MM1.S and ANBL-6 MM cells decreased AXII binding and decreased adherence of MM cells to human stromal cells and BMSC from AXII+/+ mice. Furthermore, addition of an anti-AXII antibody to MM1.S cells, did not effect MM cell growth demonstrating that AXII expressed by MM cells does not support MM cell growth. Importantly, soluble AXII was released by osteoclasts into their conditioned media which stimulated the growth of MM cells via ERK1/2 and AKT phosphorylation. In the further study, we further characterized the role of AXIIR in MM-BMSC interactions. Methods: AXIIR expression in MM cells was determined by RT-PCR, Western blotting, and immunocytochemistry. Adhesion and growth assays were performed between MM cells and BMSC or AXII to determine the contribution of the AXII/AXIIR axis in supporting adhesion and growth of MM cells. In addition, MM cells or CD138+ cells from MM patients were treated with AXII to determine AXII-dependent MM cell growth. Further, adhesion and growth assays were performed on MM cells expressing either siAXIIR or shAXIIR. Phosphorylation assays were performed to determine the pathways stimulated by AXII in MM cells. Since OCL secrete large amount of AXII, MM cell growth assays were performed with OCL-CM from AXII+/+ and AXII−/− mice in the presence of an AXII antibody. Results: We now report that in addition to MM1.S and ANBL-6 cells, other MM cell lines, including U266, H929, and OPM2 also express AXIIR, and that AXII stimulated the growth of RPMI8226, ANBL-6 and U266 in addition to MM1.S cells. Finally, an AXIIR antibody prevented adhesion of MM1.S cells to AXII, and that AXII upregulated the adhesion molecule, RhoA in MM cells. Additionally, AXII did not stimulate the proliferation of MM1.SshAXIIR cells compared to MM1.SshControl or untreated MM cells, demonstrating that AXII specifically acts through its receptor, AXIIR on MM cells to promote proliferation. More importantly, AXII stimulated the growth of CD138+ cells obtained from MM patients. Conclusions: Based on our results, we conclude that the interaction between AXII and AXIIR in the bone marrow microenvironment supports adhesion via RhoA and growth of MM cells by stimulating the Erk1/2 and Akt pathways, AXII produced by MM cells does not act in an autocrine manner on MM cell growth. Thus, AXII and AXIIR are key players in MM and targeting the AXII/AXIIR axis may be a novel therapeutic approach for MM. Disclosures: Roodman: Amgen: Consultancy; Millennium: Consultancy.

Human Monoclonal Antibody Targeting IL-17A (AIN457) Down-Regulates MM Cell-Growth and Survival and Inhibits Osteoclast Development In Vitro and In Vivo: A Potential Novel Therapeutic Application In Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 456-456
Author(s):  
Rao H Prabhala ◽  
Mariateresa Fulciniti ◽  
Dheeraj Pelluru ◽  
Puru Nanjappa ◽  
Christine Pai ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Growth ◽  
Stromal Cells ◽  
Human Monoclonal Antibody ◽  
Critical Role ◽  
Osteoclast Differentiation ◽  
Growth And Survival ◽  
Cell Growth And Survival

Abstract Abstract 456 We have previously demonstrated that IL-17 producing TH17cells, a new subset of T helper cells, are significantly elevated in peripheral blood and bone marrow (BM) from patients with multiple myeloma (MM) and IL-17 produced by these cells promotes MM cell growth and survival, suppresses immune responses and induces osteoclast differentiation both in vitro and in vivo. Based on these observations we have investigated the effects of human anti-IL-17A monoclonal antibody (mAb), AIN-457, in MM. We observed that whereas IL-17A induced proliferation of MM cells (+30.7+2.7%) compared to control; anti-IL-17A mAb AIN-457 significantly inhibited MM cell growth both in presence and absence of BM stromal cells, as measured by thymidine incorporation (−18.7+1.5% and −22.7+2.6% respectively). We have further confirmed these inhibitory effects of anti-IL-17A antibody using MM cell colony forming assay with MethoCult agar plates. While presence of IL-17A increased the colony number from 80 in control plates to 188, presence of AIN-457 reduced the colonies to <40 per unit area (p < 0.01). Evaluating the mechanism of action, IL-17A induced IL-6 production (+289.6+38%; p<0.01); while AIN457 significantly down-regulated IL-6 production (−25+7%; p<0.05) in MM-BMSC co-culture. We also observed that AIN-457 significantly reduced adhesion of MM cells to stromal cells (27%, p=0.011). AIN457 significantly inhibited IL-6 production in human fetal bone chips in the presence of MM cells within 24 hours of ex-vivo culture (control − 487+39 pg/ml; IL-17 990+27 pg/ml; p<0.01 and AIN-457 − 326+7 pg/ml; p<0.01). Since IL-17A plays a critical role in bone damage, we further evaluated the effect of this mAb on the generation of osteoclasts. When normal BM cells were cultured for three weeks in osteoclast supporting medium, presence of AIN-457 significantly inhibited TRAP+ multinucleated osteoclast cell numbers by>60%. We next evaluated the efficacy of AIN-457 in vivo in the murine models of human myeloma; in the subcutaneous MM xenograft model, we observed significant reduction in tumor volumes by pre-treatment with AIN457 compared to control (142+77 mm versus 355+56 mm, p=0.019) while IL-17A significantly increased MM cell growth (727+135 mm, p=0.01). More importantly in the SCIDhu model of human myeloma where MM cells grow within the human microenvironment in the mice, administration of AIN-457 weekly for 4 weeks after the first detection of tumor in mice led to a significant inhibition of tumor growth as measured by human sIL-6 receptor compared to control mice (5.9±2.2 ng/ml versus 23.2±6.3 ng/ml; n=7; P <0.01). These pre-clinical in vitro and in vivo observations confirm the role of IL-17A produced by TH17 cells in MM and provide the rationale for clinical evaluation of AIN 457 for both anti-myeloma effects as well as to improve bone disease in myeloma. Disclosures: No relevant conflicts of interest to declare.

Stromal Interaction of Lymphoma Cells Regulates Survival and Chemoresistance

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5195-5195
Author(s):  
Gyeongsin Park ◽  
Byunghoo Song ◽  
Yuji Lee ◽  
Ahwon Lee ◽  
Yang-Guk Chung ◽  
...  
Keyword(s):  
Cell Growth ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Lymphoma Cell ◽  
Low Grade ◽  
High Grade ◽  
Lymphoma Cells ◽  
Growth And Survival ◽  
Stromal Microenvironment ◽  
Cell Growth And Survival

Abstract Abstract 5195 High-grade lymphomas are aggressive but largely curable, whereas low-grade lymphomas are indolent, but frequently recur to be incurable, paradoxically. Mesenchymal stromal cells (MSCs) have been known to participate for reconstituting microenvironment. Studies show that signalings between normal lymphoid cells and stromal cells were frequently altered in high grade lymphomas, but relatively conserved in low grade lymphomas. However, which cell and mechanism is responsible for lymphoma recurrence remains unclear. Here we hypothesized that the interaction with stroma may play a role for lymphoma cell growth and survival. For this, we investigated the effect of MSCs on lymphoma cell growth and chemo-resistance by using a coculture system with MSCs (derived from tonsil), as a stromal microenvironment for lymphoma cells. First, coculture of lymphoma cell line (Pfeiffer) and primary lymphoma cells with/without MSCs for 3 days showed that the MSC-cocultured cells grew more rapidly (1.4 times and 1.8 times for Pfeiffer and primary cells, respectively) than MSC-free lymphoma cells. To further investigate the underlying mechanism of promoting growth, lymphoma cells were cocultured with MSCs in the presence or absence of transwell filter. At day 4, the proliferation of lymphoma cells in the absence of transwell was 3.5 times higher than in the presence of transwell. Interestingly, the lymphoma cells cocultured with MSCs showed increased expression of CXCR4 compared with lymphoma cells cultured alone. When the cyto-protective effect of MSCs was examined by doxorubicin treatment (1ug/ml) in the presence or absence of MSCs, 91% of MSC-cocultured cells survived, whereas only 28% of stroma-free cultured cells survived. These results demonstrate that the direct contact interaction with stroma might be important for lymphoma cell growth and survival. In conclusion, our data suggest that the interaction with stromal microenvironment is an important factor for survival of lymphoma cells which cells might be responsible for chemoresistance, and raise the possibility that the stromal interaction could be a potential target for lymphoma treatment. Disclosures: No relevant conflicts of interest to declare. This research was supported by a grant (10172KFDA993) from Korea Food & Drug Administration in 2011. Disclosures: No relevant conflicts of interest to declare.

The Src Kinases Play a Crucial Role in the Growth of Hematopoietic Cells Transformed with Constitutively Activated Stat5 Mutants.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5041-5041
Author(s):  
Remy Nyga ◽  
Fabrice Gouilleux ◽  
Flora Cartier ◽  
Christian Pecquet ◽  
Aline Regnier ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Growth ◽  
Kinase Inhibitor ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Src Kinase ◽  
Hematopoietic Cell ◽  
Src Kinases ◽  
Growth And Survival ◽  
Cell Growth And Survival

Abstract Abstract 5041 Stat5A and Stat5B transcription factors (TF) play an important role in the control of hematopoietic cell proliferation and survival and are constitutively activated in number of hematological malignancies and solid tumours. Studies conducted with constitutively active (ca) Stat5 mutants (Stat51*6 and cS5F) have shown that deregulated Stat5 activity promotes leukemogenesis. In order to evaluate the role of Src kinases in the transforming properties of Stat5 TF, caStat5 expressing Ba/F3 cells and primary murine bone marrow cells were treated with the PP1 Src kinase inhibitor and with its inactive analogue PP3 as a control. We found that PP1 but not PP3 strongly inhibited Stat5A1*6- and Stat5B1*6- expressing Ba/F3 cell growth and survival while no changes were observed in IL-3 stimulated-parental Ba/F3 cells when treated with PP1. Similarly, we were able to demonstrate specific requirement of Src kinases in cS5F-induced primary bone marrow cell growth. We then examined the contribution of Src kinases to the phosphorylation of various signaling molecules involved in cell growth and survival like Stat5, PI3-K/Akt, Ras/Mapk and NF-kB. The treatment of caStat5 (Stat51*6 or cS5F)-expressing Ba/F3 cells with PP1 resulted in a strong decrease of Erk1/2 phosphorylation, but not of Stat5, Akt and IKK species. In addition, caStat5-expressing Ba/F3 cells were found to express a constitutive molecular complex comprising Stat5, Shc, Grb2, Sos, Erk, Gab2, p85, Lyn and Hck. Finally we found that the Lyn Src kinase was overexpressed in the caStat5-expressing cells. Our data suggest a direct implication of Src kinases in the proliferation of caStat5- Ba/F3 transformed cells through the Shc/Erk1/2 signaling pathway and that Src family members are required for caStat5 (Stat51*6 and cS5F)-induced hematopoietic cell growth. Disclosures No relevant conflicts of interest to declare.

PD-1 Is Expressed on B-Cells in Waldenstrom Macroglobulinemia and Promotes Malignant Cell Viability and Proliferation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3015-3015
Author(s):  
Stephen M. Ansell
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
Cell Growth ◽  
Cell Lines ◽  
Stromal Cells ◽  
Malignant Cell ◽  
Growth And Survival ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia

The tumor microenvironment plays an important role in regulating malignant cell growth and mechanisms to enhance anti-tumor immune function have been shown to improve patient outcome. Interactions between programmed death 1 (PD-1) and its ligands (PD-L1 and PD-L2) have been shown to be an important checkpoint in immune regulation. While it is well known that PD-1 is expressed on normal T cells and signaling through PD-1 inhibits T cell function, PD-1 is also expressed on a subset of B-cells but little is known about PD-1 signaling in B-cells. The goal of this study was to determine if the PD-1 is expressed on malignant B cells in Waldenstrom macroglobulinemia (WM) and whether this pathway plays a role in the survival and growth of malignant B cells in this B cell lymphoma. Using flow cytometry, we found that the cell lines MWCL-1, BCWM.1 and RPCI, all derived from patients with Waldenstrom macroglobulinemia, expressed PD-1 to varying degrees on their cell surface. PD-1 expression in the cell lines was further confirmed by RT PCR analysis. Using flow cytometry and immunohistochemistry to examine bone marrow specimens from WM patients, we further confirmed PD-1 expression on CD19+ CD138+ malignant B-cells. Furthermore, intense staining for the ligands PD-L1 and PD-L2 was found by in bone marrows of WM patients when compared to normal bone marrow specimens. When WM cell lines are co-cultured with stromal cells engineered to express PD-L1 or PD-L2, there was a consistent increase in cell viability compared to controls. When malignant B cells from WM patients were co-cultured with stromal cells expressing the ligands, viability was unchanged but there was an increase in cell proliferation, most noticeably when cocultured with cells expressing PD-L2. To determine potential mechanisms that account for upregulation of PD-1 on malignant B-cells, we tested whether cytokines that promote WM cell growth and survival, including IL-6, IL-21 and BAFF, increased PD-1 expression. We found that WM cell lines and patient derived CD19+CD138+WM B-cells (n=4) treated with IL-21 demonstrated an increase in PD-1 expression compared to untreated controls. We conclude that PD-1 is expressed on malignant B-cells in WM and that signaling through PD-1 may promote WM cell growth and survival. Blocking PD-1/PD ligand interactions may therefore be a potential therapeutic strategy in patients with Waldenstrom macroglobulinemia. Disclosures No relevant conflicts of interest to declare.

Non-overlapping Promoter and Super-enhancer Driven Processes Support Myeloma Cell Growth and Survival via Distinct Regulatory Axes

2017 ◽  
Vol 17 (1) ◽  
pp. e1
Author(s):  
Mariateresa Fulciniti ◽  
Charles Lin ◽  
Mehmet Samur ◽  
Rick Young ◽  
Kenneth C. Anderson ◽  
...  
Keyword(s):  
Cell Growth ◽  
Myeloma Cell ◽  
Growth And Survival ◽  
Super Enhancer ◽  
Cell Growth And Survival

scholarly journals Activation of 4-1BB signaling in bone marrow stromal cells triggers bone loss via the p-38 MAPK-DKK1 axis in aged mice

2021 ◽  
Author(s):  
Daqian Wan ◽  
Songtao Ai ◽  
Huoniu Ouyang ◽  
Liming Cheng
Keyword(s):  
Bone Marrow ◽  
Bone Loss ◽  
Osteogenic Differentiation ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Bone Marrow Microenvironment ◽  
Marrow Stromal Cells ◽  
Senile Osteoporosis ◽  
Aged Mice ◽  
Old Mice

AbstractSenile osteoporosis can cause bone fragility and increased fracture risks and has been one of the most prevalent and severe diseases affecting the elderly population. Bone formation depends on the proper osteogenic differentiation of bone marrow stromal cells (BMSCs) in the bone marrow microenvironment, which is generated by the functional relationship among different cell types in the bone marrow. With aging, bone marrow provides signals that repress osteogenesis. Finding the signals that oppose BMSC osteogenic differentiation from the bone marrow microenvironment and identifying the abnormal changes in BMSCs with aging are key to elucidating the mechanisms of senile osteoporosis. In a pilot experiment, we found that 4-1BBL and 4-1BB were more abundant in bone marrow from aged (18-month-old) mice than young (6-month-old) mice. Meanwhile, significant bone loss was observed in aged mice compared with young mice. However, very little data have been generated regarding whether high-level 4-1BB/4-1BBL in bone marrow was associated with bone loss in aged mice. In the current study, we found upregulation of 4-1BB in the BMSCs of aged mice, which resulted in the attenuation of the osteogenic differentiation potential of BMSCs from aged mice via the p38 MAPK-Dkk1 pathway. More importantly, bone loss of aged mice could be rescued through the blockade of 4-1BB signaling in vivo. Our study will benefit not only our understanding of the pathogenesis of age-related trabecular bone loss but also the search for new targets to treat senile osteoporosis.

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