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)

No abstract

Summary Amplification of 1q21 occurs in approximately 30% of de novo and 70% of relapsed multiple myeloma (MM) and is correlated with disease progression and drug resistance. Here, we provide evidence that the 1q21 amplification-driven overexpression of ILF2 in MM promotes tolerance of genomic instability and drives resistance to DNA-damaging agents. Mechanistically, elevated ILF2 expression exerts resistance to genotoxic agents by modulating YB-1 nuclear localization and interaction with the splicing factor U2AF65, which promotes mRNA processing and the stabilization of transcripts involved in homologous recombination in response to DNA damage. The intimate link between 1q21-amplified ILF2 and the regulation of RNA splicing of DNA repair genes may be exploited to optimize the use of DNA-damaging agents in patients with high-risk MM.

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.

The involvement of osteocytes in multiple myeloma (MM)-induced osteoclast (OCL) formation and bone lesions is still unknown. Osteocytes regulate bone remodelling at least partially, as a result of their cell death triggering OCL recruitment. In this study, we found that the number of viable osteocytes was significantly smaller in MM patients than in healthy controls, and negatively correlated with the number of OCLs. Moreover, the MM patients with bone lesions had a significantly smaller number of viable osteocytes than those without, partly because of increased apoptosis. These findings were further confirmed by ultrastructural in vitro analyses of human preosteocyte cells cocultured with MM cells, which showed that MM cells increased preosteocyte death and apoptosis. A micro-array analysis showed that MM cells affect the transcriptional profiles of preosteocytes by upregulating the production of osteoclastogenic cytokines such as interleukin (IL)-11, and increasing their pro-osteoclastogenic properties. Finally, the osteocyte expression of IL-11 was higher in the MM patients with than in those without bone lesions. Our data suggest that MM patients are characterized by a reduced number of viable osteocytes related to the presence of bone lesions, and that this is involved in MM-induced OCL formation.Leukemia ( Keywords: multiple myeloma; osteocytes; bone disease; osteoclasts INTRODUCTIONThe presence of osteolytic bone lesions or osteoporosis is the hallmark of multiple myeloma (MM), and leads to bone fragility and fractures in MM patients. The bone lesions are characterized by severely unbalanced and uncoupled bone remodelling in the area of plasma cell infiltration due to increased osteoclast (OCL) formation and activity, and osteoblast (OB) suppression. 1 --3 Local factors produced by MM cells and the bone marrow microenvironment are involved in the MM-induced increase in osteoclastogenesis, and include an imbalance in the receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG) ratio that favours RANKL, 2,4 and the MM cell production of osteoclastogenic factors such as Chemokine (C --C motif) ligand 3 (CCL3)/macrophage inflammatory proteins (MIP)-1a. 5 --7 OB exhaustion is mainly due to a reduction in OB differentiation and formation, from mesenchymal stem cells and OB progenitors, that is induced by MM cells as a result of cell-to-cell contacts and the release of soluble factors. 3,8 --11 Osteocytes are differentiated cells of osteogenic lineage 12 located in the lacuno-canalicular system of the bone (see the review by Bonewald). 13 There is increasing evidence that they regulate physiological local bone remodelling, 13 --16 partly as a result of the cell death and apoptosis that triggers OCL formation and bone resorption, 15 --19 and partly by secreting sclerostin, a molecule that is specifically produced by osteocytes that inhibits bone formation. 20,21 Interestingly, an apoptosis-related reduction in osteocyte viability has recently been demonstrated in

SUMMARY Myelodysplastic syndrome (MDS) risk correlates with advancing age, therapy-induced DNA damage, and/or shorter telomeres but whether telomere erosion directly induces MDS is unknown. Here, we provide the genetic evidence that telomere dysfunction-induced DNA damage drives classical MDS phenotypes and alters common myeloid progenitor (CMP) differentiation by repressing the expression of mRNA splicing/processing genes, including srsf2. RNA-Seq analyses of telomere dysfunctional CMP identified aberrantly spliced transcripts linked to pathways relevant to MDS pathogenesis such as genome stability, DNA repair, chromatin remodeling and histone modification, which are also enriched in mouse CMP haploinsufficient for srsf2 and in CD34+ CMML patient cells harboring srsf2 mutation. Together, our studies establish an intimate link across telomere biology, aberrant RNA splicing and myeloid progenitor differentiation.

Purpose: Multiple myeloma (MM) derives from plasmablast/plasma cells that accumulate in the bone marrow. Different microenvironmental factors may promote metastatic dissemination especially to the skeleton, causing bone destruction. The balance between osteoclast and osteoblast activity represents a critical issue in bone remodeling. Thus, we investigated whether interluekin-27 (IL-27) may function as an antitumor agent by acting directly on MM cells and/or on osteoclasts/osteoblasts.Experimental Design: The IL-27 direct antitumor activity on MM cells was investigated in terms of angiogenesis, proliferation, apoptosis, and chemotaxis. The IL-27 activity on osteoclast/osteoblast differentiation and function was also tested. In vivo studies were done using severe combined immunodeficient/nonobese diabetic mice injected with MM cell lines. Tumors from IL-27-and PBS-treated mice were analyzed by immunohistochemistry and PCR array.Results: We showed that IL-27 (a) strongly inhibited tumor growth of primary MM cells and MM cell lines through inhibition of angiogenesis, (b) inhibited osteoclast differentiation and activity and induced osteoblast proliferation, and (c) damped in vivo tumorigenicity of human MM cell lines through inhibition of angiogenesis.Conclusions: These findings show that IL-27 may represent a novel therapeutic agent capable of inhibiting directly MM cell growth as well as osteoclast differentiation and activity. Clin Cancer Res; 16(16); 4188-97. ©2010 AACR.

The bone marrow microenvironment in multiple myeloma is characterized by an increased microvessel density. The production of pro-angiogenic molecules is increased and the production of angiogenic inhibitors is suppressed, leading to an "angiogenic switch". Here we present an overview of the role of angiogenesis in multiple myeloma, the pro-angiogenic factors produced by myeloma cells and the microenvironment, and the mechanisms involved in the myeloma-induced angiogenic switch. Current data suggest that the increased bone marrow angiogenesis in multiple myeloma is due to the aberrant expression of angiogenic factors by myeloma cells, the subsequent increase in pro-angiogenic activity of normal plasma cells as a result of myeloma cell angiogenic activity, and the increased number of plasma cells overall. Hypoxia also contributes to the angiogenic properties of the myeloma marrow microenvironment. The transcription factor hypoxia-inducible factor-1α is overexpressed by myeloma cells and affects their transcriptional and angiogenic profiles. In addition, potential roles of the tumor suppressor gene inhibitor of growth family member 4 and homeobox B7 have also been recently highlighted as repressors of angiogenesis and pro-angiogenic related genes, respectively. This complex pathogenetic model of myeloma-induced angiogenesis suggests that several proangiogenic molecules and related genes in myeloma cells and the microenvironment are potential therapeutic targets.