p62 as a Therapeutic Target for Myeloma Cell Growth and Osteoclast Formation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2857-2857
Author(s):  
Fumito Ishizuka ◽  
Jolene Windle ◽  
David Roodman ◽  
Noriyoshi Kurihara
Keyword(s):  
Cell Growth ◽  
P38 Mapk ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Critical Role ◽  
Mapk Signaling ◽  
Marrow Stromal Cells ◽  
Fusion Peptides ◽  
Binding Domains ◽  
Cell Support

Abstract Abstract 2857 Poster Board II-833 We reported that sequestosome 1 (p62) plays a critical role in the formation of signaling complexes that result in NF-kB, p38 MAPK, and PI3K activation in the marrow microenvironment of patients with multiple myeloma (MM), and that p62 is a potential therapeutic target for MM. In contrast to treating patients with inhibitors of each of the multiple signaling pathways activated in marrow stromal cells by MM cells (e.g. NF-kB or p38 MAPK), blocking the function of p62 should inhibit the activation of the multiple pathways mediated by p62 and have a broader effect on the bone marrow microenvironment. The goal of this study was to identify the domains of p62 responsible for increased MM cell growth and osteoclast (OCL) formation mediated by NF-kB and p38 MAPK signaling in marrow stromal cells when they interact with myeloma cells, and develop inhibitory peptides as potential therapeutic agents that interfere with p62's role in these signaling complexes. To pursue this objective, we transfected p62−/− stromal cells with p62 deletion constructs and assessed their effects on NF-kB and p38 MAPK signaling induced by MM cells or TNF-a. p62−/− stromal cells support of MM growth or OCL formation was significantly decreased compared to WT stromal cells. We made a series of 5' deletion constructs of p62 that lacked specific p62 domains: ΔPB1 (Δ1) lacks homodimerization domain and binding to PKCz, ΔPB1, ZZ (Δ2) lacks PB1 and RIP1 binding domains, and ΔPB1, ZZ, TBS, (Δ3) the PB1, RIP1, p38 and TRAF6 binding domains have been deleted. These constructs were tested for their capacity to restore p62 function in p62−/−stromal cells and support MM cell growth and OCL formation. GFP-labeled MM1.S myeloma cells were cocultured with p62−/− and WT marrow stromal cells transduced with the different p62 deletion constructs. Transduction of p62−/− stromal cells with the full-length p62 construct restored the capacity of p62−/− stromal cells to enhance the growth of MM cells to levels induced by WT stromal cells. Transduction of p62−/− stromal cells with the Δ1 construct also restored stromal cell support of MM growth. Therefore, the PB1 domain is not important for this function. Transduction of p62−/− stromal cells with the Δ2 construct, resulted in an inability of the stromal cells to support MM cell growth. Additional loss of the p38 and TRAF6 binding domains did not further impair p62−/− stromal cells support of MM cell growth. These results suggest that the RIP1 binding domain plays a critical role in supporting the growth of MM cells by marrow stromal cells. We then examined the capacity of p62−/− stromal cells transduced with various p62 deletion constructs to support OCL formation. Normal CFU-GM, a source of OCL precursors, were cocultured with p62−/− stromal cells transfected with the different p62 cDNA deletion constructs. The Δ1 construct completely rescued p62−/− support of OCL formation. However, Δ2 construct transduced p62−/− stromal cells only partly restored stromal cell support of OCL formation. Transduction of the Δ3 construct did not restore the capacity of the p62−/− stromal cells to support OCL formation. Similarly, transduction of the Δ2 and Δ3 construction decreased WT stromal cell support of MM cell growth. We then tested the feasibility of using transduction domain (PTD) fusion peptides as a potential means of delivering dominant negative p62 constructs into stromal cells in vitro and in vivo to block MM cell growth and VCAM-1 expression induced by marrow stromal cells. PTD binding domain fusion peptides containing NEMO binding protein that blocks NF-kB activity was used as a model system to determine the feasibility of transducing marrow stromal cells with p62 constructs. Addition of PTD-NEMO fusion peptides to stromal cells significantly inhibited WT stromal cell to enhance MM cell growth and VCAM-1 expression on stromal cells, which is the capacity of dependent, in part, on NF-kB signaling. These results demonstrate that the ZZ, p38 MAPK and TRAF-6 domains of p62 together are required for stromal cell support of MM cell growth and OCL formation and suggest that PTD constructs containing dominant negatives for p62 may be a feasible method for blocking p62 function in the MM marrow microenvironment. Disclosures: Roodman: Novartis: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy; Acceleron: Consultancy.

The ZZ Domain of Sequestosome-1/p62 Plays An Important Role In Stromal Cell Support of Myeloma Cell Growth and Osteoclast Formation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 128-128
Author(s):  
Jumpei Teramachi ◽  
Jolene J. Windle ◽  
David Roodman ◽  
Noriyoshi Kurihara
Keyword(s):  
Cell Growth ◽  
Cell Line ◽  
P38 Mapk ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Bone Destruction ◽  
Mapk Signaling ◽  
Sequestosome 1 ◽  
Stromal Cell Line ◽  
Cell Support

Abstract Abstract 128 The marrow microenvironment provides a critical supportive role in myeloma (MM) and enhances both tumor growth and bone destruction through activation of multiple signaling pathways in stromal cells. We reported that sequestosome 1 (p62) plays a key role in the formation of signaling complexes that result in NF-kB, p38 MAPK, and PI3K activation in the marrow microenvironment of patients with MM. These results suggest that p62 is a potential therapeutic target for blocking the supportive effects of the marrow microenvironment in MM. The goal of this study was to identify the domains of p62 responsible for increased MM cell growth and osteoclast (OCL) formation mediated by NF-kB and p38 MAPK signaling, as a means to develop inhibitory peptides/molecules as potential therapeutic agents for MM. To pursue this objective, we generated deletion constructs of p62 that lacked specific p62 domains: ΔSH2, ΔPB1, ΔZZ, Δp38, ΔTBS and ΔUBA domains. We then transfected these constructs into a p62-knockout (KO) stromal cell-line we established from p62-KO mice and examined their RANKL, IL-6, and VCAM-1 expression induced by TNF-a. GFP-labeled MM1.S myeloma cells or normal CFU-GM, a source of OCL precursors, were then co-cultured with the p62-KO cells transduced with the different p62 deletion constructs and compared to wild type (WT) stromal cells. IL-6 production and VCAM-1 expression induced by TNF-a was 50% lower in non-transduced p62-KO stromal cells compared to WT stromal cells. Further, in contrast to WT cells, RANKL was not induced by TNF-a in the p62-KO stromal cell-line, and OCL formation in co-cultures of p62-KO stromal cells with CFU-GM was very low. Transduction of p62-KO stromal cells with the ΔSH2, ΔPB1, Δp38 and ΔUBA constructs restored stromal cell support of MM growth, VCAM-1 and IL-6 production. However, p62-KO stromal cells transduced with the ΔZZ construct did not increase MM cell growth, or increase IL-6 and VCAM-1 expression, or fully restore the capacity of the p62-KO stromal cells to support OCL formation. These results demonstrate that the ZZ domain of p62 is required for stromal cell support of MM cell growth, increased IL-6 and VCAM-1 expression, and OCL formation. These results suggest that dominant negative constructs or small molecules that target the ZZ domain of p62 should block p62 function and inhibit support of MM cells and OCL formation by the marrow microenvironment. Disclosures: Roodman: Amgen, Celgene, Acceleron & Millennium: Consultancy.

Targeting p62ZIP in Marrow Stromal Cells Is Highly Effective at Inhibiting Myeloma Cell Growth and Osteoclast Formation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 630-630
Author(s):  
Noriyoshi Kurihara ◽  
Tadashi Honjo ◽  
Jolene J. Windle ◽  
J. Shin ◽  
G. D. Roodman
Keyword(s):  
Cell Growth ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Critical Role ◽  
Bone Destruction ◽  
Myeloma Cell ◽  
Marrow Stromal Cells ◽  
Wild Type ◽  
Myeloma Cells ◽  
Tnf Α

Abstract Marrow stromal cells play a critical role in osteolytic bone destruction in multiple myeloma, and promote tumor growth. In particular, adhesive interactions between myeloma cells and marrow stromal cells increase RANK ligand (RANKL), a potent inducer of osteoclast formation, IL-6 and TNF-α production by marrow stromal cells. IL-6 enhances the growth and prevents apoptosis of myeloma cells, and TNF increases production of RANKL and IL-6. Recently, a new member of NF-κB signaling pathway, p62ZIP, has been identified. p62ZIP plays a critical role in NF-κB activation induced by TNF-α and RANKL and is involved in multiple signaling pathways that result in enhanced IL-6 production, tumor cell survival and bone destruction. It is our hypothesis that inhibiting p62ZIP expression will profoundly diminish myeloma growth by blocking the effects of IL-6 produced in the tumor-bone microenvironment in response to TNF-α. Therefore, we used p62ZIP−/− mice to determine the effects of deleting p62ZIP in stromal cells on the growth of myeloma cells. Marrow cells from p62ZIP −/− or wild type mice were used to establish long-term Dexter-type marrow cultures to isolate marrow stromal cells. Marrow stromal cells from p62ZIP −/− or wild type mice were cocultured for 48 h with a GFP-labeled human MM.1S myeloma cell line (MM.1S) and murine and human RANKL, IL-6 and TNF-α levels were determined in conditioned media from these cocultures using commercial ELISA assays. Cocultures of MM.1S with wild type marrow stromal cells resulted in much greater upregulation of murine IL-6 than p62−/− stromal cell coculture (IL-6 in p62−/− stromal cell cultures; 114+70 vs. WT 1900+9 pg/ml). In addition, deleting p62ZIP in stromal cells markedly decreased the growth of tumor cells. Coculture with wild type stromal cells induce 1.4-fold greater increase in MM.1S cell growth at 72 h compared to p62−/− stromal cells. Further, addition of neutralizing antibodies to TNF-α and IL-6 to the cocultures of MM.1S cells with WT stromal cells similarly affected the growth of the MM.1S. Since TNF-alpha can increase the expression of adhesion molecules on stromal cells and tumor cells, we measured expression levels of ICAM-1 and VCAM-1 by Western blot. VCAM-1 and ICAM-1 levels on p62ZIP−/− bone marrow stromal cells were not changed compared to WT stromal cells. We then determined the capacity of p62 −/− cells to support OCL formation by normal spleen CFU-blast. OCL formation was decreased about 50 % in cocultures containing p62−/− stromal cells treated with PTH-rp, IL-6 and TNF-α compare with WT stromal cell culture. These results show that p62 plays an important role in myeloma cell growth and OCL formation induced by cytokines that are upregulated in the marrow microenvironment in patients with myeloma.

Chronic Lymphocytic Leukemia B Cells Express Functional CXCR4 Chemokine Receptors That Mediate Spontaneous Migration Beneath Bone Marrow Stromal Cells

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3658-3667 ◽  
Author(s):  
Jan A. Burger ◽  
Meike Burger ◽  
Thomas J. Kipps
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Pertussis Toxin ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Lymphocytic Leukemia ◽  
Marrow Stromal Cells

Chemokines play a central role for lymphocyte trafficking and homing. The mechanisms that direct the tissue localization of B cells from patients with chronic lymphocytic leukemia (B-CLL) are unknown. We found that CLL B cells express functional CXCR4 receptors for the chemokine stromal cell-derived factor-1 (SDF-1), as demonstrated by receptor endocytosis, calcium mobilization, and actin polymerization assays. Moreover, CLL B cells displayed chemotaxis to this chemokine that could be inhibited by monoclonal antibodies (MoAbs) against CXCR4, pertussis toxin, or Wortmannin, a phosphatidylinositol 3-kinase inhibitor. That this chemotaxis may be involved in the homing of CLL cells is argued by studies in which CLL B cells were cocultured with a murine marrow stromal cell line that secretes SDF-1. Within 2 hours, CLL B cells spontaneously migrated beneath such stromal cells in vitro (pseudoemperipolesis). This migration could be inhibited by pretreatment of CLL B cells with anti-CXCR4 MoAbs, SDF-1, or pertussis-toxin. Furthermore, we noted strong downmodulation of CXCR4 on CLL B cells that migrated into the stromal cell layer. These findings demonstrate that the chemokine receptor CXCR4 on CLL B cells plays a critical role for heterotypic adherence to marrow stromal cells and provide a new mechanism to account for the marrow infiltration by neoplastic B cells.

Chronic Lymphocytic Leukemia B Cells Express Functional CXCR4 Chemokine Receptors That Mediate Spontaneous Migration Beneath Bone Marrow Stromal Cells

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3658-3667 ◽  
Author(s):  
Jan A. Burger ◽  
Meike Burger ◽  
Thomas J. Kipps
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Pertussis Toxin ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Lymphocytic Leukemia ◽  
Marrow Stromal Cells

Abstract Chemokines play a central role for lymphocyte trafficking and homing. The mechanisms that direct the tissue localization of B cells from patients with chronic lymphocytic leukemia (B-CLL) are unknown. We found that CLL B cells express functional CXCR4 receptors for the chemokine stromal cell-derived factor-1 (SDF-1), as demonstrated by receptor endocytosis, calcium mobilization, and actin polymerization assays. Moreover, CLL B cells displayed chemotaxis to this chemokine that could be inhibited by monoclonal antibodies (MoAbs) against CXCR4, pertussis toxin, or Wortmannin, a phosphatidylinositol 3-kinase inhibitor. That this chemotaxis may be involved in the homing of CLL cells is argued by studies in which CLL B cells were cocultured with a murine marrow stromal cell line that secretes SDF-1. Within 2 hours, CLL B cells spontaneously migrated beneath such stromal cells in vitro (pseudoemperipolesis). This migration could be inhibited by pretreatment of CLL B cells with anti-CXCR4 MoAbs, SDF-1, or pertussis-toxin. Furthermore, we noted strong downmodulation of CXCR4 on CLL B cells that migrated into the stromal cell layer. These findings demonstrate that the chemokine receptor CXCR4 on CLL B cells plays a critical role for heterotypic adherence to marrow stromal cells and provide a new mechanism to account for the marrow infiltration by neoplastic B cells.

scholarly journals Increased signaling through p62 in the marrow microenvironment increases myeloma cell growth and osteoclast formation

Blood ◽  
2009 ◽  
Vol 113 (20) ◽  
pp. 4894-4902 ◽  
Author(s):  
Yuko Hiruma ◽  
Tadashi Honjo ◽  
Diane F. Jelinek ◽  
Jolene J. Windle ◽  
Jaekyoon Shin ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Growth ◽  
Signaling Pathways ◽  
P38 Mapk ◽  
Stromal Cells ◽  
Bone Destruction ◽  
Osteoclast Formation ◽  
Marrow Stromal Cells ◽  
Sequestosome 1 ◽  
Multiple Signaling

Adhesive interactions between multiple myeloma (MM) cells and marrow stromal cells activate multiple signaling pathways including nuclear factor κB (NF-κB), p38 mitogen-activated protein kinase (MAPK), and Jun N-terminal kinase (JNK) in stromal cells, which promote tumor growth and bone destruction. Sequestosome-1 (p62), an adapter protein that has no intrinsic enzymatic activity, serves as a platform to facilitate formation of signaling complexes for these pathways. Therefore, we determined if targeting only p62 would inhibit multiple signaling pathways activated in the MM microenvironment and thereby decrease MM cell growth and osteoclast formation. Signaling through NF-κB and p38 MAPK was increased in primary stromal cells from MM patients. Increased interleukin-6 (IL-6) production by MM stromal cells was p38 MAPK-dependent while increased vascular cell adhesion molecule-1 (VCAM-1) expression was NF-κB–dependent. Knocking-down p62 in patient-derived stromal cells significantly decreased protein kinase Cζ (PKCζ), VCAM-1, and IL-6 levels as well as decreased stromal cell support of MM cell growth. Similarly, marrow stromal cells from p62−/− mice produced much lower levels of IL-6, tumor necrosis factor-α (TNF-α), and receptor activator of NF-κB ligand (RANKL) and supported MM cell growth and osteoclast formation to a much lower extent than normal cells. Thus, p62 is an attractive therapeutic target for MM.

Synergetic topography and chemistry cues guiding osteogenic differentiation in bone marrow stromal cells through ERK1/2 and p38 MAPK signaling pathway

2018 ◽  
Vol 6 (2) ◽  
pp. 418-430 ◽  
Author(s):  
Xinran Zhang ◽  
Haotian Li ◽  
Chucheng Lin ◽  
Congqin Ning ◽  
Kaili Lin
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
P38 Mapk ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Mapk Signaling ◽  
Marrow Stromal Cells ◽  
Ion Doping ◽  
Mapk Signaling Pathways

Ordered micro-patterned topography and Sr ion doping in HAp synergistically enhance osteogenesis through ERK1/2 and p38 MAPK signaling pathways.

Targeting p62 in Marrow Stromal Cells Is Effective at Inhibiting Myeloma Cell Growth.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 513-513 ◽  
Author(s):  
Noriyoshi Kurihara ◽  
Y. Hiruma ◽  
J. Windle ◽  
C.S. Hong ◽  
J. Shin ◽  
...  
Keyword(s):  
Cell Growth ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Bone Destruction ◽  
Myeloma Cell ◽  
Marrow Stromal Cells ◽  
Bone Microenvironment ◽  
Wild Type ◽  
Myeloma Cells ◽  
Tnf Α

Abstract Release of growth factors from bone by osteoclastic bone destruction and by tumor-stromal cell interactions play critical roles in promoting myeloma cell growth. In particular, expression of the cytokines RANK ligand (RANKL), a potent inducer of osteoclast (OCL) formation, IL-6, tumor necrosis factor-α (TNF-α) and MCP-1 are upregulated in the bone microenvironment in response to myeloma (MM) cells as well as by adhesive interactions between myeloma cells and marrow stromal cells through VCAM-1 on stromal cells. Therefore, agents that can both inhibit OCL formation and block the effects of myeloma stromal cell interactions should have a major impact on both bone destruction and tumor growth. p62 plays a critical role in NF-κB activation induced by TNF-α, RANKL and IL-1 and is involved in multiple signaling pathways that result in enhanced tumor cell survival and bone destruction. It is our hypothesis that inhibiting p62 expression will profoundly diminish osteolytic bone destruction and myeloma growth in patients, by blocking production of RANKL, MCP-1, TNF-α and IL-6 in the tumor-bone microenvironment, and the upregulation of VCAM-1 on stromal cells. Therefore, we used p62 −/− mice to determine the effects of deleting p62 in stromal cells on the growth of myeloma cells. Marrow cells from p62 −/− or wild type mice were used to establish long-term Dexter-type marrow cultures to isolate marrow stromal cells. IL-6 and TNF-α production by p62 −/− stromal cells was decreased compared to WT stromal cells. To determine the effects of the lack of p62 on MM cell growth, GFP-labeled MM.1S cells were co-cultured with p62 −/− stromal cells. Growth of MM.1S cells was decreased by 70% in cocultures of p62 −/− mice, and IL-6 and TNF-α levels were not increased in cocultures of tumor cells with p62 −/− stromal cells. Next, we measured the relative expression levels of VCAM-1 on marrow stromal cells by Western blot in cocultures of human myeloma cells with marrow stromal cells. Stromal cells from the p62 −/− or wild type were cultured with and without MM.1S cells for 3 days in separate experiments. The levels of VCAM-1 in p62 −/− stromal cells were lower than p62 +/− stromal cells. In addition, VCAM-1 levels on p62 −/− bone marrow stromal cells were decreased compared to p62 +/− stromal cells when cocultured with MM cells. The addition of 25 ng/ml mouse TNF-α to p62 −/− stromal cells cocultured with MM.1S cells resulted in enhanced MM.1S growth and VCAM-1 production to similar levels as seen with p62 +/− stromal cells cocultured with MM.1S cells. We then determined the capacity of p62 −/− stromal cells to increase MCP-1 production, a chemoattract for myeloma cells, when they were cocultured with human myeloma cells for 48 hours. The conditioned media were collected after 48 hours of culture. Wild type stromal cells produced increased levels of MCP-1 when cocultured with MM.1S cells. MCP-1 levels in p62 −/− stromal cell conditioned media were decreased compared to wild type stromal cell cultures, regardless of whether MM.1S cells were present in the culture (MCP-1 in p62 +/− stromal cell culture, 980± 70pg/ml vs. p62 −/− 380± 10 pg/ml). These results show that p62 plays an important role in myeloma cell growth through regulation of production of cytokines that are upregulated in the marrow microenvironment in response to myeloma, and suggest that p62 is a novel target for treating myeloma.

Increased Signaling through p62 in the Marrow Microenvironment Increases Myeloma Cell Growth and Osteoclast Formation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 642-642 ◽  
Author(s):  
Yuko Hiruma ◽  
Noriyoshi Kurihara ◽  
Diane F. Jelinek ◽  
David Roodman
Keyword(s):  
Cell Growth ◽  
Tumor Growth ◽  
Signaling Pathways ◽  
P38 Mapk ◽  
Stromal Cells ◽  
Therapeutic Target ◽  
Bone Destruction ◽  
Marrow Stromal Cells ◽  
Tnf Α ◽  
Attractive Therapeutic Target

Abstract Marrow stromal cells in the bone microenvironment of multiple myeloma (MM) patients play a critical role in promoting both tumor growth and bone destruction. Binding of MM cells to marrow stromal cells, through VCAM-1 on stromal cells and α4β1 integrin on MM cells, results in increased production of TNF-α, receptor activator of NF-KB ligand (RANKL) and IL-6 by marrow stromal cells. These factors in turn increase osteoclast (OCL) formation and the growth of MM cells. Adhesive interaction between MM cells and marrow stromal cells also decrease the sensitivity of MM cells to chemotherapeutic agents. Many of the downstream effects of these adhesive interactions are mediated through the NF-KB, p38 MAPK and JNK signaling pathways. The adapter protein sequestosome-1 (p62) sits at the crossroads of these signaling pathways and mediates the effects of cytokines and other factors that activate NF-KB, p38 MAPK and JNK. However, the role that increased signaling through p62 plays in MM tumor growth and bone destruction is unknown. It is our hypothesis that increased signaling through p62 in marrow stromal cells is necessary for their capacity to increase the growth of MM cells and OCL formation in MM, and thus p62 may be an attractive therapeutic target for MM. To test these hypotheses, we established long-term Dexter-type marrow cultures to isolate marrow stromal cells from MM patients and normals and measured signaling through p62 and PKCζ activation in MM marrow. We found significantly elevated levels of phosho-PKCζ, total PKCζ and VCAM-1 in MM stromal cells. The enhanced IL-6 production resulted from increased p38 MAPK activity and the increased VCAM-1 expression was NF-KB dependent. We then examined the effects of blocking p62 activity in primary patients and normal stromal cells with p62 siRNA (10μg). We confirmed that p62 expression was decreased by at least 90% in both these stromal cells by Western blot analysis. Stromal cells expressing p62siRNA or control siRNA were cultured with or without MM1.S cells for 3 days in separate experiments. Knocking-down p62 in MM derived marrow stromal cells significantly decreased the levels of PKCζ, VCAM-1 and IL-6 in marrow stromal cells and markedly decreased stromal cell support of MM cell growth and OCL formation. Similarly, marrow stromal cells from p62−/− mice produced much lower levels of IL-6, TNF-α, VCAM-1 and RANKL and minimally supported MM cell growth and OCL formation compared to normal cells. Thus, increased signaling through p62 in marrow stromal cells in patients with MM plays an important role in the increased tumor growth and OCL formation in MM, and support p62 as an attractive therapeutic target for MM.

Blocking the ZZ Domain of Sequestosome 1/p62 Suppress the Enhancement of Myeloma Cell Growth and Osteoclast Formation by Marrow Stromal Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 888-888 ◽  
Author(s):  
Jumpei Teramachi ◽  
Kyaw Ze Yar Myint ◽  
Rentian Feng ◽  
Xiangqun Xie ◽  
Jolene J. Windle ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Mononuclear Cells ◽  
Apoptotic Cell ◽  
Bone Destruction ◽  
Osteoclast Formation ◽  
Marrow Stromal Cells ◽  
Sequestosome 1

Abstract Abstract 888 The marrow microenvironment enhances both tumor growth and bone destruction in multiple myeloma (MM) through MM cell-induced activation of multiple signaling pathways in bone marrow stromal cells (BMSC) by TNFα. We reported that sequestosome-1 (p62) acts as a signaling hub for NF-kB, MAPK and PI3K activation in BMSC of MM patients and enhances MM growth and osteoclast (OCL) formation. p62 is composed of 5 domains that are involved in protein–protein interactions required for formation of these signaling complexes, but which domain of p62 mediates increase MM growth and OCL formation is unclear. Therefore, deletion constructs of p62 that lacked each of the 5 domains (PB1, ZZ, p38, TBS or UBA) were transfected into a p62−/− stromal cell line. We found that the ZZ domains mediated BMSC enhancement of MM cell growth, IL-6 production, VCAM-1 expression and OCL formation. Using virtual modeling of the ZZ domain, we identified 6 candidate p62-ZZ inhibitory molecules and tested them for their capacity to block enhanced MM cell growth, OCL formation, IL-6 production, and VCAM-1 expression on BMSC induced by TNFα. When MM1.S, RPMI8266, ANBL6 MM cell-lines or CD138+ primary MM cells were cultured in the absence of BMSC with p62-ZZ inhibitor #3 (10mM), this inhibitor directly induced cell death. The p62-ZZ inhibitor-induced cell death was characterized by an increase in reactive oxygen species (ROS) production by inhibiting NF-kB activation, and apoptotic cell death by triggering the activation of caspases 3, 7, and 9. This inhibitor had an IC50 of 4.6mM for MM1.S survival. In contrast, CFU-Blast formation by human CD34+ cells was not inhibited by p62-ZZ inhibitor #3 (10mM). p62-ZZ inhibitor #3 (10mM) treatment of human OCL precursors derived from CFU-GM, inhibited OCL formation by blocking precursor proliferation. To examine the specificity of the p62-ZZ inhibitor #3, we tested its effects on OCL formation by CD11b+ mononuclear cells from wild type (WT) and p62−/− mice cultured with TNFα for 7 days. p62-ZZ inhibitor #3 blocked WT OCL formation but did not block p62−/− OCL formation. The p62-ZZ inhibitor also blocked VCAM-1 expression and IL-6 production by normal and MM patient stromal cells induced by TNFα compared to vehicle. Importantly, the inhibitor (10mM) did not block stromal cell proliferation. Further, the inhibitor blocked TNFα induced PKCζ phosphorylation in stromal cells and MM1.S when the cells were pretreated with the p62-ZZ inhibitor for 3 hours. These results demonstrate that p62-ZZ inhibitor #3 specifically blocks both stromal cells independent and dependent MM cell growth and OCL formation but does not affect hematopoietic or stromal cell growth. These results support p62 as a potential novel therapeutic target for MM. Disclosures: Roodman: Amgen: Consultancy; Millennium: Consultancy.

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