Although osteolysis is a common complication in patients with multiple myeloma (MM), the biologic mechanisms involved in the pathogenesis of MM-induced bone disease are poorly understood. Two factors produced by stromal-osteoblastic cells seem critical to the regulation of bone resorption: osteoprotegerin (OPG) and its ligand (OPGL). OPGL stimulates osteoclast differentiation and activity, whereas OPG inhibits these processes.

The proteasome inhibitor bortezomib may increase osteoblast-related markers in multiple myeloma (MM) patients; however, its potential osteoblastic stimulatory effect is not known. In this study, we show that bortezomib significantly induced a stimulatory effect on osteoblast markers in human mesenchymal cells without affecting the number of osteoblast progenitors in bone marrow cultures or the viability of mature osteoblasts. Consistently we found that bortezomib significantly increased the transcription factor Runx2/Cbfa1 activity in human osteoblast progenitors and osteoblasts without affecting nuclear and cytoplasmatic active ␤-catenin levels. IntroductionMultiple myeloma (MM) is a plasma cell malignancy characterized by the high capacity to induce osteolytic bone lesions. 1 Hystomorphometric studies showed that osteoblast formation and function are profoundly impaired in MM patients and critical in the bone lesion development. [1][2][3] Several mechanisms are involved in MMinduced osteoblast suppression 1,4,7 including the production of Wnt inhibitors as DKK-1 or sFRP-2 5,6 or the impairment of osteoblast formation and differentiation through the block of the critical osteoblast transcription factor Runx2/Cbfa1. 7 In turn, osteoblastic cells also regulate myeloma cell growth 8,9 and the increase of bone formation in mice results in a reduction of tumoral burden. 10 Recent data suggest that ubiquitin-proteasome pathway, which is the major cellular degradative system and therapeutic target in myeloma cells, 11 also regulates osteoblast differentiation. [12][13][14] The ubiquitin-proteasome pathway can modulate the BMP-2 expression, 12 which can induce osteoblast differentiation through the Wnt signaling 13 and regulates the proteolytic degradation of the osteoblast transcription factor Runx2/ Cbfa1. 14 Recently, Garrett et al 12 demonstrated that proteasome inhibitors as PS1 and epoximicin stimulate bone formation in neonatal murine calvarial bones and in vivo in mice. 12 A strong correlation between the capacity of these compounds to inhibit proteasomal activity in osteoblasts and their bone-forming activity was also demonstrated. 12 Preliminary observations obtained in MM patients treated with the proteasome inhibitor bortezomib show an increase of serum bone-specific alkaline phosphatase and other osteoblast related markers suggesting a potential osteoblast stimulatory effect of this drug. [15][16][17][18] Currently it is not known whether the proteasome inhibitor bortezomib may have a direct effect on osteoblast differentiation and formation in vitro in human cultures and in vivo in MM patients. Patients, materials, and methods DrugsBortezomib was purchased from Janssen-Cilag (Milan, Italy). For in vitro studies, the drug was reconstituted in DMSO at a stock concentration of 50 mM, and this stock was diluted in medium just before use, so that the concentration of DMSO never exceeded 0.1%. The proteasome inhibitors MG-132 and MG-262 were purchased from BIOMOL International (Plymouth Meeting, PA; DBA srl, M...

Key Points• Myeloma cells produce ammonium in the presence of glutamine, showing high glutaminase and low glutamine synthetase expression.• Myeloma cells show high expression of glutamine transporters and inhibition of ASCT2 transporter hinders myeloma growth.The importance of glutamine (Gln) metabolism in multiple myeloma (MM) cells and its potential role as a therapeutic target are still unknown, although it has been reported that human myeloma cell lines (HMCLs) are highly sensitive to Gln depletion. In this study, we found that both HMCLs and primary bone marrow (BM) CD138 1 cells produced large amounts of ammonium in the presence of Gln. MM patients have lower BM plasma Gln with higher ammonium and glutamate than patients with indolent monoclonal gammopathies. Interestingly, HMCLs expressed glutaminase (GLS1) and were sensitive to its inhibition, whereas they exhibited negligible expression of glutamine synthetase (GS). High GLS1 and low GS expression were also observed in primary CD138 1 cells. Gln-free incubation or treatment with the glutaminolytic enzyme L-asparaginase depleted the cell contents of Gln, glutamate, and the anaplerotic substrate 2-oxoglutarate, inhibiting MM cell growth. Consistent with the dependence of MM cells on extracellular Gln, a gene expression profile analysis, on both proprietary and published datasets, showed an increased expression of the Gln transporters SNAT1, ASCT2, and LAT1 by CD138 1 cells across the progression of monoclonal gammopathies. Among these transporters, only ASCT2 inhibition in HMCLs caused a marked decrease in Gln uptake and a significant fall in cell growth. Consistently, stable ASCT2 downregulation by a lentiviral approach inhibited HMCL growth in vitro and in a murine model. In conclusion, MM cells strictly depend on extracellular Gln and show features of Gln addiction. Therefore, the inhibition of Gln uptake is a new attractive therapeutic strategy for MM. (Blood. 2016;128(5):667-679)

IntroductionAngiogenesis has a critical role in the pathophysiology and progression of multiple myeloma (MM) supporting the growth and survival of MM cells. [1][2][3][4][5] The angiogenic process in MM is sustained mainly by the overexpression of proangiogenic factors directly by MM cells including VEGF, 6 angiopoietin-1 (ANG-1), 7 osteopontin (OPN), . 9 Nevertheless, the molecular mechanisms underlying the regulation of angiogenesis in MM have not been completely elucidated.The new candidate tumor-suppressor gene inhibitor of growth family member 4 (ING4) has been recently implicated in solid tumors as a repressor of tumor growth and angiogenesis through the association with NF-B. ING4 is a nuclear factor expressed in all normal tissues and markedly reduced in glioblastoma cells and head and neck squamous cell carcinoma, with levels inversely correlated with tumor grade. 10,11 Inhibition of ING4 expression strongly promotes the growth of glioma cells in vivo, whereas its overexpression leads to growth inhibition through ING4's capability to interact with p65 subunit of NF-B. 10 Interestingly, it has been also shown that tumors lacking ING4 showed increased vascularization compared with ING4-expressing tumors. 12 Moreover ING4 down-regulated the angiogenic-related molecules including IL-8 and the hypoxia inducible factor-1␣ (HIF-1 ␣) activity in hypoxic condition through the involvement of HIF prolyl hydroxylase 2 (HPH-2) 10,13 In turn, the role of hypoxia has been recently highlighted in the promotion of angiogenesis. 14 The expression of ING4 by MM cells, as well as its potential role in MM-induced angiogenesis, has never been investigated. In this study, we evaluated the expression of ING4 in malignant MM cells and the potential relationship between ING4 and the production of proangiogenic molecules by MM cells in normoxic and hypoxic conditions, as well as its relationship with the "in vitro and in vivo" angiogenesis. Submitted February 15, 2007; accepted September 4, 2007. Prepublished online as Blood First Edition paper, September 11, 2007; DOI 10.1182 DOI 10. /blood-2007 The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on August 29, 2018. by guest www.bloodjournal.org From Patients, materials, and methods Cells and cell culture conditionsCell lines. Human myeloma cell lines (HMCLs) XG-6, XG-1, and JJN3 were obtained from Dr Bataille (Nantes, France). U266 was obtained from the American Type Culture Collection (Rockville, MD). OPM2 and RPMI-8226 were purchased from DSM (Braunschweig, Germany). ARP-1 and H929 were generously received from Dr Shaughnessy's laboratory (Little Rock, AR).Cell cultures. HMCLs were incubated in RPMI medium at 10% FCS (Invitrogen Life Technologies, Milan, Italy) and maintained with or without IL-6 (3 ng/mL; Endogen Woburn, MA). In a series of experiments, HMCLs were incubated with the HPH-2 in...

Hypoxia-inducible transcription factor-1 (HIF-1a) is overexpressed in multiple myeloma (MM) cells within the hypoxic microenvironment. Herein, we explored the effect of persistent HIF-1a inhibition by a lentivirus short hairpin RNA pool on MM cell growth either in vitro or in vivo and on the transcriptional and pro-angiogenic profiles of MM cells. HIF-1a suppression did not have a significant impact on MM cell proliferation and survival in vitro although, increased the antiproliferative effect of lenalidomide. On the other hand, we found that HIF-1a inhibition in MM cells downregulates the pro-angiogenic genes VEGF, IL8, IL10, CCL2, CCL5 and MMP9. Pro-osteoclastogenic cytokines were also inhibited, such as IL-7 and CCL3/MIP-1a. The effect of HIF-1a inhibition was assessed in vivo in nonobese diabetic/severe combined immunodeficiency mice both in a subcutaneous and an intratibial MM model. HIF-1a inhibition caused a dramatic reduction in the weight and volume of the tumor burden in both mouse models. Moreover, a significant reduction of the number of vessels and vascular endothelial growth factors (VEGFs) immunostaining was observed. Finally, in the intratibial experiments, HIF-1a inhibition significantly blocked bone destruction. Overall, our data indicate that HIF-1a suppression in MM cells significantly blocks MM-induced angiogenesis and reduces MM tumor burden and bone destruction in vivo, supporting HIF-1a as a potential therapeutic target in MM.

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Rationale: Twenty-eight percent of people with mild to moderate obstructive sleep apnea experience daytime sleepiness, which interferes with daily functioning. It remains unclear whether treatment with continuous positive airway pressure improves daytime function in these patients. Objectives: To evaluate the efficacy of continuous positive airway pressure treatment to improve functional status in sleepy patients with mild and moderate obstructive sleep apnea. Methods: Patients with self-reported daytime sleepiness (Epworth Sleepiness Scale score .10) and an apnea-hypopnea index with 3% desaturation and from 5 to 30 events per hour were randomized to 8 weeks of active or sham continuous positive airway pressure treatment. After the 8-week intervention, participants in the sham arm received 8 weeks of active continuous positive airway pressure treatment. Measurements and Main Results: The Total score on the Functional Outcomes of Sleep Questionnaire was the primary outcome measure.The adjusted mean change in the Total score after the first 8-week intervention was 0.89 for the active group (n ¼ 113) and 20.06 for the placebo group (n ¼ 110) (P ¼ 0.006). The group difference in mean change corresponded to an effect size of 0.41 (95% confidence interval, 0.14-0.67). The mean (SD) improvement in Functional Outcomes of Sleep Questionnaire Total score from the beginning to the end of the crossover phase (n ¼ 91) was 1.73 6 2.50 (t Author Contributions: T.E.W. was responsible for the study design, conduct of the study, data collection and interpretation, and writing of the manuscript. C.M. served as Project Manager, and was responsible for execution of study procedures, study quality control, and data collection. G.M. was the blinded biostatistician on the study, collaborated on the study, conducted the primary data analysis, provided interpretation of the results, and contributed to the writing of the manuscript. J.C. served as the unblinded biostatistician on the study and conducted required data analysis for the DSMB before unblinding. B.S. was responsible for study design and data collection and interpretation. J.R.L. contributed to study design and served for a period of time as Executive Secretary of the Data Safety Monitoring Board. K.A.F., C.F.P.G., D.A.S., H.G., D.M.R., J.A.W., and T.L.-C. contributed to study design, supervised data collection, interpreted the data, and edited and reviewed the manuscript. I.G. provided interpretation and quality control for the blood pressure obtained from the 48-hour ambulatory monitoring data. S.T.K. was responsible for study design, data collection, interpretation of the data, and writing of the manuscript.Correspondence and requests for reprints should be addressed to Terri E. Weaver, Ph.D., R.N., University of Illinois at Chicago College of Nursing, 845 South Damen Avenue (MC 802), Chicago, IL 60612. E-mail: teweaver@uic.edu This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org What This Study Adds to the Fi...

Osteopontin (OPN) is a multifunctional bone matrix glycoprotein that is involved in angiogenesis, cell survival and tumor progression. In this study we show that human myeloma cells directly produce OPN and express its major regulating gene Runx2/Cbfa1. The activity of Runx2/Cbfa1 protein in human myeloma cells has also been demonstrated. Moreover, using small interfering RNA (siRNA) to silent Runx2 in myeloma cells, we suppressed OPN mRNA and protein expression. OPN production in myeloma cells was stimulated by growth factors as IL-6 and IFG-1 and in turn OPN stimulated myeloma cell proliferation. In an 'in vitro' angiogenesis system we showed that OPN production by myeloma cells is critical for the proangiogenic effect of myeloma cells. The expression of OPN by purified bone marrow (BM) CD138 þ cells has also been investigated in 60 newly diagnosed multiple myeloma (MM) patients, finding that 40% of MM patients tested expressed OPN. Higher OPN levels have been detected in the BM plasma of MM patients positive for OPN as compared to controls. Moreover, significantly higher BM angiogenesis has been observed in MM patients positive for OPN as compared to those negative. Our data highlight that human myeloma cells with active Runx2/Cbfa1 protein directly produce OPN that is involved in the pathophysiology of MM-induced angiogenesis.