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

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

Blood ◽

10.1182/blood-2008-08-173948 ◽

2009 ◽

Vol 113 (20) ◽

pp. 4894-4902 ◽

Cited By ~ 42

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.

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

Blood ◽

10.1182/blood.v116.21.128.128 ◽

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.

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IL-3 Inhibits Osteoblast Formation in Multiple Myeloma Via CD45+ Cells.

Blood ◽

10.1182/blood.v104.11.2347.2347 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2347-2347 ◽

Cited By ~ 1

Author(s):

Lori A. Ehrlich ◽

Masahiro Ito ◽

Sun Jin Choi ◽

G. D. Roodman

Keyword(s):

Cell Lines ◽

Cell Cultures ◽

Stromal Cells ◽

Stromal Cell ◽

Bone Destruction ◽

Osteoclast Formation ◽

Marrow Stromal Cells ◽

Inhibitory Effects ◽

Myeloma Cells ◽

Primary Mouse

Abstract Normally, osteoclast activation is coupled to an increase in osteoblast activity. In Multiple Myeloma (MM), bone remodeling is uncoupled, and bone destruction occurs both by markedly increased osteoclastic bone destruction and severely impaired osteoblastic bone formation. Although several reports have shown that conditioned media from MM cell lines suppress osteoblast (OBL) differentiation, the identity of the OBL inhibitor(s) is unknown. A recent report by Tian et al (NEJM 2003) has identified DKK1, an inhibitor of the WNT signaling pathway, as a putative OBL inhibitor in MM. However, it is likely that other inhibitors of OBL differentiation are present in the myeloma microenvironment, just as there are several potent stimulators of osteoclast formation produced or induced by myeloma cells. We recently reported that IL-3 levels in bone marrow plasma of patients with MM are increased compared to normal controls and that IL-3 in MM marrow plasma stimulates osteoclast formation. We also demonstrated that IL-3 is produced by primary myeloma cells and that it increased MM cell growth in vitro. However, the effects of IL-3 on OBL are unknown. Therefore, to determine if IL-3 could affect OBL growth and differentiation, we tested the effects of IL-3 on OBL differentiation in primary mouse marrow stromal cells. We found that murine marrow stromal cells and OBL-like cell lines expressed IL-3 receptor alpha (IL-3R) by RT-PCR. Staining for IL-3R in marrow stromal cell cultures after treatment with IL-3 confirmed that the IL-3R was present on thirty percent of the cells. Importantly, treatment of primary murine stromal cell cultures with IL-3 (0.01–10 ng/mL) inhibited basal and BMP-2 stimulated osteoblast formation in a dose dependent manner, without affecting cell growth. At 10 ng/mL IL-3 inhibited OBL differentiation by 80%. Time course studies demonstrated that IL-3 affected the later stages of osteoblast differentiation. Further, the inhibitory effects of IL-3 were not due to induction of TNFalpha, a known OBL inhibitor. TNFalpha levels were very low (0–20pg/ml) in the conditioned media of these cultures, and treating the cultures with anti-mouse TNFalpha did not block the IL-3 effect. IL-3 did not inhibit alkaline phosphatase activity in the osteoblast-like cell lines, MC3T3-E1 and C2C12 suggesting that IL-3 may act indirectly through another cell type in the mixed cell population in the primary mouse osteoblast culture system. Since IL-3 acts primarily on hematopoietic cells, we determined if IL-3 was acting indirectly by increasing CD45+ cells in the cultures. IL-3 increased the number of CD45+ hematopoietic cells in the primary culture from approximately 15% to 30%. Further, depletion of the CD45+ cells abolished the inhibitory effects of IL-3 but not TNFalpha on osteoblasts, and adding back CD45+ cells to the stromal cell cultures restores the inhibitory effects of IL-3. This data suggests that IL-3 is an important mediator of bone destruction in MM by both inducing osteoclast formation and indirectly inhibiting osteoblast formation.

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

Blood ◽

10.1182/blood.v108.11.513.513 ◽

2006 ◽

Vol 108 (11) ◽

pp. 513-513 ◽

Cited By ~ 1

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.

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

Blood ◽

10.1182/blood.v106.11.630.630 ◽

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.

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

Blood ◽

10.1182/blood.v114.22.2857.2857 ◽

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.

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Expression of XBP1s in bone marrow stromal cells is critical for myeloma cell growth and osteoclast formation

Blood ◽

10.1182/blood-2011-05-353300 ◽

2012 ◽

Vol 119 (18) ◽

pp. 4205-4214 ◽

Cited By ~ 47

Author(s):

Guoshuang Xu ◽

Kai Liu ◽

Judy Anderson ◽

Kenneth Patrene ◽

Suzanne Lentzsch ◽

...

Keyword(s):

Cell Growth ◽

Protein Expression ◽

Stromal Cells ◽

Bone Destruction ◽

Myeloma Cell ◽

Osteoclast Formation ◽

Healthy Human ◽

Unfolded Protein

Abstract BM stromal cells (BMSCs) are key players in the microenvironmental support of multiple myeloma (MM) cell growth and bone destruction. A spliced form of the X-box–binding protein-1 (XBP1s), a major proximal effector of unfolded protein response signaling, is highly expressed in MM cells and plays an indispensable role in MM pathogenesis. In the present study, we found that XBP1s is induced in the BMSCs of the MM microenvironment. XBP1s overexpression in healthy human BMSCs enhanced gene and/or protein expression of VCAM-1, IL-6, and receptor activator of NF-κB ligand (RANKL), enhancing BMSC support of MM cell growth and osteoclast formation in vitro and in vivo. Conversely, deficiency of XBP1 in healthy donor BMSCs displayed a range of effects on BMSCs that were opposite to those cells with overexpression of XBP1s. Knock-down of XBP1 in MM patient BMSCs greatly compromised their increased VCAM-1 protein expression and IL-6 and RANKL secretion in response to TNFα and reversed their enhanced support of MM-cell growth and osteoclast formation. Our results demonstrate that XBP1s is a pathogenic factor underlying BMSC support of MM cell growth and osteoclast formation and therefore represents a therapeutic target for MM bone disease.

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Interference with Bone Marrow Stroma Derived Factors CXCL12 and TGFBI Increases Apoptosis in ALL Cells and Enhances Antileukemia Effects of Dexamethasone, Methotrexate, Vincristine and 6-Mercaptopurine

Blood ◽

10.1182/blood.v126.23.4774.4774 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4774-4774

Author(s):

Sana Usmani ◽

Olena Tkachencko ◽

Leti Nunez ◽

Craig A. Mullen

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Stromal Cells ◽

Stromal Cell ◽

Low Dose ◽

Marrow Stromal Cells ◽

Bone Marrow Stroma ◽

Marrow Stroma ◽

The Impact

Abstract Background: Bone marrow stroma provides a favorable microenvironmental niche for ALL cell survival. We and others have demonstrated that bone marrow stromal cells contribute to prevention of apoptosis in ALL cells. Objective: Identify potentially "drug-able" molecules derived from marrow stromal cells that contribute to prevention of ALL cell apoptosis. Methods: We have developed an in vitro system to identify stromal gene products that deliver antiapoptotic signals to ALL cells. Primary human ALL cells are co-cultured with human bone marrow stromal cells. We manipulate stromal cells with siRNA directed against candidate stromal cell genes. Two days later the siRNA is washed out of culture and primary ALL cells are added to the stromal cells. Controls include irrelevant siRNA. Five days later we measure viability and apoptosis in ALL cells by flow cytometry. Results: (1) Knockdown of stroma cell CXCL12 or TGFBI reduces ALL survival. We performed global gene expression analysis upon human marrow stromal cells using RNASeq technology. Using bioinformatic approaches we are selecting some of the expressed stromal genes as candidates for the molecular mechanisms by which stromal cells prevent ALL apoptosis. We present preliminary results for two of our early candidates. (A) CXCL12 is a paracrine chemokine known to have activity in the marrow microenvironment upon hematopoietic cells and we hypothesized it may participate in the effect. Knockdown of CXCL12 with siRNA increased ALL cell death in the co-culture system. As measured by quantitative reverse transcriptase PCR stromal cell CXCL12 mRNA was reduced approximately 75% by siRNA treatment. Figure 1 displays representative results of the impact of CXCL12 knockdown in stromal cell on the survival of ALL cells in the coculture. The magnitude of effect was ~40% increase in ALL cell death. (B) TGFBI (transforming growth factor beta induced) is expressed by stromal cells. The gene is involved in cell-collagen interactions and we hypothesized it played a role. siRNA reduced stromal gene expression by about 90%. Figure 2 displays representative results in which ALL cell death increased by about 50%. (2) Validation of results using inhibitors to CXCL12. The gene knockdown experiments suggested a potential role for CXCL12 in prevention of ALL cell apoptosis. To further test this we tested the effect of plerixafor, a specific inhibitor of CXCL12/CXCR4 interactions, on survival of ALL. ALL cells express CXCR4. In a dose dependent manner (25 - 400 micromolar) we observed a 31-39% reduction in ALL survival in stromal co-cultures including plerixafor. Figure 3 depicts representative results with plerixafor 200 micromolar. We are evaluating small molecules to block TGFBI. (3) Potential augmentation of chemotherapy drug effects on ALL. We hypothesize that interference with stromal cell molecules that prevent apoptosis in ALL cells may increase the effectiveness of conventional antileukemia drugs. In our stromal cell/ALL coculture system we have identified the effective in vitro concentrations of the most commonly used ALL drugs. We measured the impact of combination of low dose plerixafor (LD10) and these individual drugs (used at approximately the LD50 concentrations). Figure 4 demonstrates increased antileukemia effects related to plerixafor for dexamethasone, vincristine, and 6-mercaptopurine. Results are plotted as a percentage of ALL cells surviving in the absence of any drugs. The low dose plerixafor alone control did not produce a statistically significant reduction in ALL survival. Conclusions: Marrow stromal cell-produced CXCL12 may contribute to prevention of apoptosis in human ALL cells. Pharmacological interference with its effect may enhance the effectiveness of some conventional chemotherapy drugs. Marrow stromal cell-produced TGFBI may also contribute to prevention of apoptosis in human ALL cells. Disclosures No relevant conflicts of interest to declare.

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Imatinib Mesylate (STI571) Considerably Affects Normal Human Bone Marrow Stromal Cell Growth in Vitro.

Blood ◽

10.1182/blood.v104.11.2330.2330 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2330-2330 ◽

Cited By ~ 1

Author(s):

Daniela Melzer ◽

Ulf Neumann ◽

Wolfram Ebell ◽

Karl Seeger ◽

Peter Neuhaus ◽

...

Keyword(s):

Bone Marrow ◽

Side Effects ◽

Cell Growth ◽

Imatinib Mesylate ◽

Stromal Cells ◽

Mononuclear Cells ◽

Marrow Stromal Cells ◽

Normal Human ◽

Dose Dependent

Abstract Imatinib mesylate (STI571) is a selective Bcr-Abl tyrosine kinase inhibitor used for the treatment of Philapdelphia chromosome positive leukemias and other malignancies. Typical non-hematological side effects of imatinib are mostly moderate whereas hematological side effects include all three lineages and are dose-dependent (e.g. 35% grade 3/4 neutropenia at 800 mg daily). Recently, a significant dose-dependent inhibitory effect of imatinib on normal hematopoietic progenitor cells has been reported. However, since alterations of the hematopoietic system might also result (at least in part) from a possible drug-induced damage of the hematopoietic environment, we studied the effects of imatinib on normal human marrow microenvironmental cells. Marrow stromal progenitor cell growth was considerably inhibited by imatinib in a dose-dependent fashion: CFU-F were reduced to 75 ± 35.5%, 52 ± 29.2%, 32 ± 25.7%, 33.7 ± 23.4%, and 20.6 ± 20.6% of control at 0.04, 0.16, 0.62, 2.5, and 10.0 μM imatinib, respectively (IC50: 0.44 μM). Marrow stromal cells (MSC, culture-derived from normal bone marrow mononuclear cells) were also affected dose-dependently: After two weeks in culture, MSC were reduced to 20.0 ± 2.2% (0.62 μM), 14.6 ± 1.2% (1.25 μM), 10.1 ± 0.3% (2.5 μM), 5.8 ± 0.5% (5 μM), and 2.8 ± 0.3% (10 μM) of controls. After 4 weeks, corresponding data were 3.1 ± 0.5%, 2.2 ± 0.4%, 1.8 ± 0.3%, 1.1 ± 0.2%, and 0.5 ± 0.1%. Furthermore, following a 7-day culture period in imatinib-containing medium (0.62 – 10 μM) and subsequent washing and resuspension, the growth kinetics of imatinib-treated MSC were delayed for up to 2 weeks after imatinib withdrawal. Moreover, the total cellular expansion of imatinib-pretreated MSC after 4 weeks of culture was lower when compared with controls. To assess whether imatinib would impair functional MSC capacities, long-term culture initiating cell assays were set up using imatinib-treated MSC (0 μM, 1.25 μM, 5 μM) as feeder cells and normal CD34pos-PBPC for inoculation. After 5 weeks, numbers of clonogenic progenitors assayed per well were not different (control: 20.0 ± 7.1, imatinib 1.25 μM: 16.2 ± 6.5, imatinib 5 μM: 21.3 ± 6). Experiments addressing a possible role of PDGF- and SCF-receptor signaling revealed that the growth inhibitory effects of imatinib (10 μM) clearly exceeded those of receptor blocking antibodies, thereby indicating that imatinib acts - at least in part - independently of PDGFR- and c-kit-signaling. Cell cycle analysis showed that the fraction of imatinib-treated MSC (0.62 - 10 μM) in S- and G2/M-phase was slightly lower albeit not significantly different when compared with controls. Furthermore, no differences with regard to the fraction of apoptotic cells were observed. Nevertheless, MSC cultures in maximum growth phase showed a markedly higher susceptibility to imatinib compared to those in the steady-state. Taken together, imatinib severely affected marrow stromal cells in-vitro. This effect was at least partly independent of PDGFR- and c-kit signaling and appeared to be related to the proliferative status of the cells. This information might be relevant for the administration of imatinib in situations with increased MSC turnover, such as regeneration after intensive chemo/radiotherapy.

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p62 Signaling Is Increased in Multiple Myeloma Microenvironment.

Blood ◽

10.1182/blood.v110.11.669.669 ◽

2007 ◽

Vol 110 (11) ◽

pp. 669-669

Author(s):

Yuko Hiruma ◽

Noriyoshi Kurihara ◽

David Roodman

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Western Blot ◽

Stromal Cells ◽

Bone Destruction ◽

Marrow Stromal Cells ◽

Myeloma Patient ◽

Bone Microenvironment ◽

Tnf Α ◽

Human Stromal Cells

Abstract The bone microenvironment plays a critical role in promoting both tumor growth and bone destruction in myeloma (MM). Marrow stromal cells produce factors, which stimulate both the growth of MM cells and osteoclastic bone destruction and are key regulators of these processes. Marrow stromal cells produce these factors in increased amounts when they bind MM cells through adhesive interactions mediated via VCAM-1 on stromal cells and β1 integrins on the surface of MM cells. We have examined the role of sequestosome-1 (p62), a recently described member of the NF-κB signaling pathway, in this process, since it sits at the crossroads of multiple signaling pathways potentially involved in both osteoclastogenesis and MM cell growth. In previous studies, we found that stromal cells lacking p62 minimally supported the growth of MM cells or osteoclast (OCL) formation compared to wild-type p62 containing stromal cells. We have further shown that stromal cells lacking p62 produce much lower levels of TNF-α, interleukin (IL)-6, MCP-1 and RANK ligand (RANKL), factors which increase OCL formation, and express lower levels of VCAM-1. However, the mechanisms responsible for this decreased cytokine production and VCAM-1 expression in multiple myeloma patient stromal cells lacking p62 are unknown. Further, it is unknown that targeting PKCζ, a downstream enzyme activity activated by p62, will have similar effects on myeloma patient stromal cells. Our hypotheses are that inhibiting PKCζ expression will block the effects of cytokines produced in the MM-bone microenvironment in response to TNF-α, decrease VCAM-1 expression in stromal cells, and markedly diminish osteolytic bone destruction and MM growth in MM patients. To test these hypotheses, we established long-term Dexter-type marrow cultures to isolate marrow stromal cells from MM patient and normals and screened for p62 and PKCζ/λ activation in MM marrow. We measured levels of PKCζ and p62 by Western blot analysis in marrow stromal cells from 13 patients and 11 healthy controls. We found significantly elevated levels of phosho-PKCζ/λ and total PKCζ in MM patients although the levels varied greatly among the patients. We next measured the relative expression levels of VCAM-1 on marrow stromal cells by Western blot in these marrow stromal cells. Since inhibiting p62 expression should 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, we assessed the effects of blocking p62 activity in human stromal cells. p62 siRNA (40 nM) was transduced into human stromal cells using a commercial transfection substrate. We confirmed that p62 expression was decreased by at least 50% in these stromal cells by Western blot analysis using an anti-p62 antibody. Stromal cells from the p62siRNA and control siRNA treatment and non-treatment stromal cells were cultured with or without MM1s cells for 3 days in separate experiments. The levels of VCAM-1 in p62siRNA transducer stromal cells were lower than control siRNA and untreated stromal cells. In addition, IL-6 production by MM1.s was increased when cocultured with control stromal cells but not with p62siRNA treated bone marrow stromal cells. These results show that p62 plays an important role in myeloma cell growth induced by cytokines that are upregulated in the marrow microenvironment and myeloma cells in patients with myeloma. Therefore, p62 is an attractive therapeutic target for MM growth and bone destruction.

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