Sharmin Khan

The proteasome inhibitor, bortezomib suppresses primary myeloma and stimulates bone formation in myelomatous and nonmyelomatous bones in vivo

Multiple myeloma (MM), a hematologic malignancy of terminally differentiated plasma cells is closely associated with induction of osteolytic bone disease, induced by stimulation of osteoclastogenesis and suppression of osteoblastogenesis. The ubiquitin‐proteasome pathway regulates differentiation of bone cells and MM cell growth. The proteasome inhibitor, bortezomib, is a clinical potent antimyeloma agent. The main goal of this study was to investigate the effect of bortezomib on myeloma‐induced bone resorption and tumor growth in SCID‐rab mice engrafted with MM cells from 16 patients. Antimyeloma response of bortezomib, which was evident in >50% of 16 experiments and resembled clinical response, was associated with significant increased bone mineral density (BMD) and osteoblast numbers, and reduced osteoclast numbers in myelomatous bones. This bone anabolic effect, which was also visualized on X‐ray radiographs and confirmed by static and dynamic histomorphometric analyses, was unique to bortezomib and was not observed in hosts responding to melphalan, a chemotherapeutic drug widely used to treat MM. Bortezomib also increased BMD and osteoblasts number and reduced osteoclasts number in nonmyelomatous implanted bones. In vitro bortezomib directly suppressed human osteoclast formation and promoted maturation of osteoblasts. We conclude that bortezomib promotes bone formation in myelomatous and nonmyelomatous bones by simultaneously inhibiting osteoclastogenesis and stimulating osteoblastogenesis. As clinical and experimental studies indicate that bone disease is both a consequence and necessity of MM progression our results suggest and that bortezomibs effects on bone remodeling contribute to the antimyeloma efficacy of this drug. Am. J. Hematol., 2009.

The ephrinB2/EphB4 axis is dysregulated in osteoprogenitors from myeloma patients and its activation affects myeloma bone disease and tumor growth

Myeloma bone disease is caused by uncoupling of osteoclastic bone resorption and osteoblastic bone formation. Bidirectional signaling between the cell-surface ligand ephrinB2 and its receptor, EphB4, is involved in the coupling of osteoblastogenesis and osteoclastogenesis and in angiogenesis. EphrinB2 and EphB4 expression in mesenchymal stem cells (MSCs) from myeloma patients and in bone cells in myelomatous bones was lower than in healthy counterparts. Wnt3a induced up-regulation of EphB4 in patient MSCs. Myeloma cells reduced expression of these genes in MSCs, whereas in vivo myeloma cell-conditioned media reduced EphB4 expression in bone. In osteoclast precursors, EphB4-Fc induced ephrinB2 phosphorylation with subsequent inhibition of NFATc1 and differentiation. In MSCs, EphB4-Fc did not induce ephrinB2 phosphorylation, whereas ephrinB2-Fc induced EphB4 phosphorylation and osteogenic differentiation. EphB4-Fc treatment of myelomatous SCID-hu mice inhibited myeloma growth, osteoclastosis, and angiogenesis and stimulated osteoblastogenesis and bone formation, whereas ephrinB2-Fc stimulated angiogenesis, osteoblastogenesis, and bone formation but had no effect on osteoclastogenesis and myeloma growth. These chimeric proteins had similar effects on normal bone. Myeloma cells expressed low to undetectable ephrinB2 and EphB4 and did not respond to the chimeric proteins. The ephrinB2/EphB4 axis is dysregulated in MM, and its activation by EphB4-Fc inhibits myeloma growth and bone disease.

Human placenta-derived adherent cells prevent bone loss, stimulate bone formation, and suppress growth of multiple myeloma in bone.

Human placenta has emerged as a valuable source of transplantable cells of mesenchymal and hematopoietic origin for multiple cytotherapeutic purposes, including enhanced engraftment of hematopoietic stem cells, modulation of inflammation, bone repair, and cancer. Placenta‐derived adherent cells (PDACs) are mesenchymal‐like stem cells isolated from postpartum human placenta. Multiple myeloma is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. We evaluated the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDACs in the severe combined immunodeficiency (SCID)–rab model of medullary myeloma‐associated bone loss. Intrabone injection of PDACs into nonmyelomatous and myelomatous implanted bone in SCID‐rab mice promoted bone formation by stimulating endogenous osteoblastogenesis, and most PDACs disappeared from bone within 4 weeks. PDACs inhibitory effects on myeloma bone disease and tumor growth were dose‐dependent and comparable with those of fetal human mesenchymal stem cells (MSCs). Intrabone, but not subcutaneous, engraftment of PDACs inhibited bone disease and tumor growth in SCID‐rab mice. Intratumor injection of PDACs had no effect on subcutaneous growth of myeloma cells. A small number of intravenously injected PDACs trafficked into myelomatous bone. Myeloma cell growth rate in vitro was lower in coculture with PDACs than with MSCs from human fetal bone or myeloma patients. PDACs also promoted apoptosis in osteoclast precursors and inhibited their differentiation. This study suggests that altering the bone marrow microenvironment with PDAC cytotherapy attenuates growth of myeloma and that PDAC cytotherapy is a promising therapeutic approach for myeloma osteolysis. STEM CELLS 2011;29:263–273

Role of Bruton's tyrosine kinase in myeloma cell migration and induction of bone disease

Myeloma cells typically grow in bone, recruit osteoclast precursors and induce their differentiation and activity in areas adjacent to tumor foci. Brutons tyrosine kinase (BTK), of the TEC family, is expressed in hematopoietic cells and is particularly involved in B‐lymphocyte function and osteoclastogenesis. We demonstrated BTK expression in clinical myeloma plasma cells, interleukin (IL)−6– or stroma–dependent cell lines and osteoclasts. SDF‐1 induced BTK activation in myeloma cells and BTK inhibition by small hairpin RNA or the small molecule inhibitor, LFM‐A13, reduced their migration toward stromal cell‐derived factor‐1 (SDF‐1). Pretreatment with LFM‐A13 also reduced in vivo homing of myeloma cells to bone using bioluminescence imaging in the SCID‐rab model. Enforced expression of BTK in myeloma cell line enhanced cell migration toward SDF‐1 but had no effect on short‐term growth. BTK expression was correlated with cell‐surface CXCR4 expression in myeloma cells (n = 33, r = 0.81, P < 0.0001), and BTK gene and protein expression was more profound in cell‐surface CXCR4‐expressing myeloma cells. BTK was not upregulated by IL‐6 while its inhibition had no effect on IL‐6 signaling in myeloma cells. Human osteoclast precursors also expressed BTK and cell‐surface CXCR4 and migrated toward SDF‐1. LFM‐A13 suppressed migration and differentiation of osteoclast precursors as well as bone‐resorbing activity of mature osteoclasts. In primary myeloma‐bearing SCID‐rab mice, LFM‐A13 inhibited osteoclast activity, prevented myeloma‐induced bone resorption and moderately suppressed myeloma growth. These data demonstrate BTK and cell‐surface CXCR4 association in myeloma cells and that BTK plays a role in myeloma cell homing to bone and myeloma‐induced bone disease. Am. J. Hematol. 88:463–471, 2013.

Consequences of Daily Administered Parathyroid Hormone on Myeloma Growth, Bone Disease, and Molecular Profiling of Whole Myelomatous Bone

Background Induction of osteolytic bone lesions in multiple myeloma is caused by an uncoupling of osteoclastic bone resorption and osteoblastic bone formation. Current management of myeloma bone disease is limited to the use of antiresorptive agents such as bisphosphonates. Methodology/Principal Findings We tested the effects of daily administered parathyroid hormone (PTH) on bone disease and myeloma growth, and we investigated molecular mechanisms by analyzing gene expression profiles of unique myeloma cell lines and primary myeloma cells engrafted in SCID-rab and SCID-hu mouse models. PTH resulted in increased bone mineral density of myelomatous bones and reduced tumor burden, which reflected the dependence of primary myeloma cells on the bone marrow microenvironment. Treatment with PTH also increased bone mineral density of uninvolved murine bones in myelomatous hosts and bone mineral density of implanted human bones in nonmyelomatous hosts. In myelomatous bone, PTH markedly increased the number of osteoblasts and bone-formation parameters, and the number of osteoclasts was unaffected or moderately reduced. Pretreatment with PTH before injecting myeloma cells increased bone mineral density of the implanted bone and delayed tumor progression. Human global gene expression profiling of myelomatous bones from SCID-hu mice treated with PTH or saline revealed activation of multiple distinct pathways involved in bone formation and coupling; involvement of Wnt signaling was prominent. Treatment with PTH also downregulated markers typically expressed by osteoclasts and myeloma cells, and altered expression of genes that control oxidative stress and inflammation. PTH receptors were not expressed by myeloma cells, and PTH had no effect on myeloma cell growth in vitro. Conclusions/Significance We conclude that PTH-induced bone formation in myelomatous bones is mediated by activation of multiple signaling pathways involved in osteoblastogenesis and attenuated bone resorption and myeloma growth; mechanisms involve increased osteoblast production of anti-myeloma factors and minimized myeloma induction of inflammatory conditions.

Therapeutic effects of intrabone and systemic mesenchymal stem cell cytotherapy on myeloma bone disease and tumor growth

The cytotherapeutic potential of mesenchymal stem cells (MSCs) has been evaluated in various disorders including those involving inflammation, autoimmunity, bone regeneration, and cancer. Multiple myeloma (MM) is a systemic malignancy associated with induction of osteolytic lesions that often are not repaired even after prolonged remission. The aims of this study were to evaluate the effects of intrabone and systemic injections of MSCs on MM bone disease, tumor growth, and tumor regrowth in the severe combined immunodeficiency (SCID)‐rab model and to shed light on the exact localization of systemically injected MSCs. Intrabone injection of MSCs, but not hematopoietic stem cells, into myelomatous bones prevented MM‐induced bone disease, promoted bone formation, and inhibited MM growth. After remission was induced with melphalan treatment, intrabone‐injected MSCs promoted bone formation and delayed myeloma cell regrowth in bone. Most intrabone or systemically injected MSCs were undetected 2 to 4 weeks after injection. The bone‐building effects of MSCs were mediated through activation of endogenous osteoblasts and suppression of osteoclast activity. Although a single intravenous injection of MSCs had no effect on MM, sequential weekly intravenous injections of MSCs prevented MM‐induced bone disease but had no effect on tumor burden. MSCs expressed high levels of anti‐inflammatory (eg, HMOX1) and bone‐remodeling (eg, Decorin, CYR61) mediators. In vitro, MSCs promoted osteoblast maturation and suppressed osteoclast formation, and these effects were partially prevented by blocking decorin. A subset of intravenously or intracardially injected MSCs trafficked to myelomatous bone in SCID‐rab mice. Although the majority of intravenously injected MSCs were trapped in lungs, intracardially injected MSCs were mainly localized in draining mesenteric lymph nodes. This study shows that exogenous MSCs act as bystander cells to inhibit MM‐induced bone disease and tumor growth and that systemically injected MSCs are attracted to bone by myeloma cells or conditions induced by MM and inhibit bone disease.

Inhibitor of DASH proteases affects expression of adhesion molecules in osteoclasts and reduces myeloma growth and bone disease

Dipeptidyl peptidase (DPP) IV activity and/or structure homologues (DASH) are serine proteases implicated in tumourigenesis. We previously found that a DASH protease, fibroblast activation protein (FAP), was involved in osteoclast‐induced myeloma growth. Here we further demonstrated expression of various adhesion molecules in osteoclasts cultured alone or cocultured with myeloma cells, and tested the effects of DASH inhibitor, PT‐100, on myeloma cell growth, bone disease, osteoclast differentiation and activity, and expression of adhesion molecules in osteoclasts. PT‐100 had no direct effects on viability of myeloma cells or mature osteoclasts, but significantly reduced survival of myeloma cells cocultured with osteoclasts. Real‐time PCR array for 85 adhesion molecules revealed upregulation of 17 genes in osteoclasts after coculture with myeloma cells. Treatment of myeloma/osteoclast cocultures with PT‐100 significantly downregulated 18 of 85 tested genes in osteoclasts, some of which are known to play roles in tumourigenesis and osteoclastogenesis. PT‐100 also inhibited osteoclast differentiation and subsequent pit formation. Resorption activity of mature osteoclasts and differentiation of osteoblasts were not affected by PT‐100. In primary myelomatous severe combined immunodeficient (SCID)‐hu mice PT‐100 reduced osteoclast activity, bone resorption and tumour burden. These data demonstrated that DASH proteases are involved in myeloma bone disease and tumour growth.

Fenretinide inhibits myeloma cell growth, osteoclastogenesis and osteoclast viability.

Fenretinide (4HPR), a nontoxic analog of ATRA, has been investigated in various malignancies but not in multiple myeloma (MM), a plasma cell malignancy associated with induction of osteolytic bone disease. Here we show that 4HPR induces apoptosis through increased level of ROS and activation of caspase-8, 9 and 3, and inhibits growth of several MM cell lines in a dose-dependent manner. Serum or co-culture with the supportive osteoclasts partially protects MM cells from 4HPR-induced growth inhibition. Sphingosine-1 phosphate (S1P) significantly protects MM cells from 4HPR-induced apoptosis suggesting that as in other malignancies, this drug up-regulates ceramide in MM cells. 4HPR has no toxic effects on non-malignant cells such as blood mononucleated cells, mesenchymal stem cells and osteoblasts, but markedly reduces viability of endothelial cells and mature osteoclasts and inhibits differentiation of osteoclasts and MM-induced tube formation. 4HPR is a potential anti-MM agent, affecting MM cells and MM-induced bone disease and angiogenesis.

Role of Bruton's tyrosine kinase (BTK) in growth and metastasis of INA6 myeloma cells

Brutons tyrosine kinase (BTK) and the chemokine receptor CXCR4 are linked in various hematologic malignancies. The aim of the study was to understand the role of BTK in myeloma cell growth and metastasis using the stably BTK knockdown luciferase-expressing INA6 myeloma line. BTK knockdown had reduced adhesion to stroma and migration of myeloma cells toward stromal cell-derived factor-1. BTK knockdown had no effect on short-term in vitro growth of myeloma cells, although clonogenicity was inhibited and myeloma cell growth was promoted in coculture with osteoclasts. In severe combined immunodeficient-rab mice with contralaterally implanted pieces of bones, BTK knockdown in myeloma cells promoted their proliferation and growth in the primary bone but suppressed metastasis to the contralateral bone. BTK knockdown myeloma cells had altered the expression of genes associated with adhesion and proliferation and increased mammalian target of rapamycin signaling. In 176 paired clinical samples, BTK and CXCR4 expression was lower in myeloma cells purified from a focal lesion than from a random site. BTK expression in random-site samples was correlated with proportions of myeloma cells expressing cell surface CXCR4. Our findings highlight intratumoral heterogeneity of myeloma cells in the bone marrow microenvironment and suggest that BTK is involved in determining proliferative, quiescent or metastatic phenotypes of myeloma cells.

Abstract 1648: Primary myeloma plasma cells are capable of growth in adult, normal whole human bone marrow environment .

Multiple myeloma (MM) is a neoplasm of low proliferative plasma cells that are highly dependent on bone marrow (BM) microenvironment for survival and growth. Primary human MM cells do not survive in conditions designed for cell lines and cell lines do not represent the primary cell heterogeneity and are microenvironment independent. Current in vitro and in vivo systems for primary human MM are limited to co-culture with specific BM cell type or growth in immunodeficient animal model. We and others have demonstrated the ability of the recognizable mature, C138+ or CD45-/CD38+ MM cells to produce MM in those experimental systems. The aim of the study was to establish and test the survival and growth of primary MM plasma cells in long term culture with a complete adult normal BM. This system is different from the autologous BM culture that is already affected by the disease. Whole BM cells from healthy donors were cultured in αMEM medium supplemented with 10% FBS and 10% serum from MM patients. Following 7-9 days the cultures were composed of adherent and nonadherent cellular compartments. The nonadherent compartment contained typical BM hematopoietic cells such monocytes, B and T lymphocytes and NK cells as assessed by flow cytometry, while the adherent compartment contained cells that morphologically resemble macrophages, osteoclasts, megakaryocytes and fibroblast-like cells. At this culture stage, CD138-selected MM cells from 13 patients were added to the BM cultures and survival and growth of MM cells were determined after 7 days by assessing proportion of CD45 low/intermediate /CD38 high MM plasma cells among total number of nonadherent cells. Low proportions of normal plasma cells (0.1-1.7%) were also detected in the cultures throughout the duration of experiments. In additional study, we used luciferase-expressing IL6-dependent INA6 MM cells and stroma-dependent MM line to assess MM growth within the whole BM cultures using bioluminescence analysis. Cell viability was constantly high (>90%) in the primary MM cell-normal BM cultures. Subsets of primary MM plasma cells were survived and detected in all culture cases while in 9 of 13 experiments, number of primary MM plasma cells was heterogeneously increased by 63±19% (p Citation Format: Rakesh Bam, Sathisha Upparahalli Venkateshaiah, Xin Li, Sharmin Khan, Wen Ling, Bart Barlogie, Joshua Epstein, Shmuel Yaccoby. Primary myeloma plasma cells are capable of growth in adult, normal whole human bone marrow environment . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1648. doi:10.1158/1538-7445.AM2013-1648