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Multiple myeloma (MM) is a virtually incurable clonal plasma cell malignancy which mainly resides in the bone marrow. Like in other cancers, numerous epigenetic aberrations have been described in MM, resulting in deregulated gene expression, disease progression and drug resistance. Targeting epigenetic modifiers involved in this deregulated epigenetic landscape therefore represents an interesting therapeutic approach. G9a (EHMT2) and GLP (EHMT1) are 2 histone methyltransferases which catalyze mono- and dimethylation of histone 3 lysine 9 (H3K9). Importantly, G9a is overexpressed in several cancers, correlating with a poor prognosis. Currently, data about the expression and role of G9a/GLP in MM is lacking. The aim of this study is therefore to investigate the functional role of G9a and GLP in MM pathogenesis. Here we report that high expression levels of both G9a and GLP are associated with a worse disease outcome in the UAMS-TT2 newly diagnosed MM patients cohort (n=345, GSE4581). ...

BackgroundPD1/PDL1-directed therapies have been unsuccessful for multiple myeloma (MM), an incurable cancer of plasma cells in the bone marrow (BM). Therefore, other immune checkpoints such as extracellular adenosine and its immunosuppressive receptor should be considered. CD39 and CD73 convert extracellular ATP to adenosine, which inhibits T-cell effector functions via the adenosine receptor A2A (A2AR). We set out to investigate whether blocking the adenosine pathway could be a therapy for MM.MethodsExpression of CD39 and CD73 on BM cells from patients and T-cell proliferation were determined by flow cytometry and adenosine production by Liquid chromatograpy-mass spectrometry (HPCL/MS). ENTPD1 (CD39) mRNA expression was determined on myeloma cells from patients enrolled in the publicly available CoMMpass study. Transplantable 5T33MM myeloma cells were used to determine the effect of inhibiting CD39, CD73 and A2AR in mice in vivo.ResultsElevated level of adenosine was found in BM pl...

Thymosin beta 4 (Tβ4) is a small cytoplasmic and nuclear protein involved in different cellular processes including differentiation, migration and proliferation. It is overexpressed in several malignant cells, resulting in an increased angiogenic respons and metastatic potential of tumor cells. In the current study we wanted to evaluate the expression of Tβ4 in primary human multiple myeloma cells (MM) and murine 5TMM cells. Microarray analysis and qRT-PCR of 171 MM patients treated with high dose therapy and autologous stem cell transplantation, showed that MM cells display a decreased Tβ4 expression compared to plasma cells from healthy individuals. We investigated whether Tβ4 expression in MM cells influences the event free (EFS) and overall survival (OAS) of these patients. As Tβ4 was expressed in all MM cells, we compared survival of patients with Tβ4 expression in MM cells in the upper thirtile (Tβ4high MMC) against the lowest thirtile (Tβ4low MMC). Patients with Tβ4high MMC h...

Multiple myeloma (MM) is well-known for the development of drug resistance, leading to relapse. Therefore, finding novel treatment strategies remains necessary. By performing a lipidomics assay on MM patient plasma, we aimed to identify new targets. We observed a dysregulation in the sphingolipid metabolism, with the upregulation of several ceramides and downregulation of sphingomyelin. This imbalance suggests an increase in sphingomyelinase, the enzyme responsible for hydrolyzing sphingomyelin into ceramide. We confirmed the upregulation of acid sphingomyelinase (ASM) in primary MM cells. Furthermore, we observed an increase in ASM expression in MM cell lines treated with melphalan or bortezomib, as well as in their exosomes. Exosomes high in ASM content were able to transfer the drug-resistant phenotype to chemosensitive cells, hereby suggesting a tumor-protective role for ASM. Finally, inhibition of ASM by amitriptyline improved drug sensitivity in MM cell lines and primary MM ce...

Apoptosis plays a key role, not only in normal homeostasis but also in protection against genomic instability. Protection against apoptosis is a hallmark of cancer and is mainly regulated by the overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-Xl or Mcl-1. This results in increased survival of the tumor cells and resistance to therapy. This presentation will focus on MCL-1 (myeloid cell leukemia 1), its expression and its role as potential target in multiple myeloma (MM). MCL1 gene regions are one the most amplified gene regions in several human cancers and Mcl-1 activity is often associated with therapy resistance and relapse. Mcl-1 binds to and sequesters the pro-apoptotic BH3 proteins, thereby preventing apoptosis. Mcl-1 is overexpressed on MM cells from newly diagnosed patients compared to normal plasma cells and in MM cells at relapse. This overexpression is furthermore associated with a shorter survival of these patients. Increased Mcl-1 expression can result eithe...

Introduction Multiple myeloma (MM) is well-known for the development of drug resistance, leading to the need for multiple treatment lines at times of relapse or progression. Even then, most patients ultimately will succumb to this cancer. Therefore, there is a need for new therapeutic strategies to conquer this drug resistance. Lipidomics has recently gained more attention in the search for new cancer therapies. Lipids are mainly found in biological membranes and function as building blocks, but are also important metabolites that can influence energy, structure and signaling cascades. Lipid dysfunction has been correlated to other cancers, like prostate and breast cancer. In this study, we identified changes in lipid content in MM patients and further investigated this altered metabolism in vitro. Methods We performed a lipidomics assay to compare plasma from healthy volunteers to MM patients. For all in vitro experiments, we used four human MM cell lines (JJN3, OPM2, LP1, U266) an...

AXL belongs to the TAM (TYRO3, AXL, and MERTK) receptor family, a unique subfamily of the receptor tyrosine kinases. Their common ligand is growth arrest-specific protein 6 (GAS6). The GAS6/TAM signaling pathway regulates many important cell processes and plays an essential role in immunity, hemostasis, and erythropoiesis. In cancer, AXL overexpression and activation has been associated with cell proliferation, chemotherapy resistance, tumor angiogenesis, invasion, and metastasis; and has been correlated with a poor prognosis. In hematological malignancies, the expression and function of AXL is highly diverse, not only between the different tumor types but also in the surrounding tumor microenvironment. Most research and clinical evidence has been provided for AXL inhibitors in acute myeloid leukemia. However, recent studies also revealed an important role of AXL in lymphoid leukemia, lymphoma, and multiple myeloma. In this review, we summarize the basic functions of AXL in various ...

2966 The great challenges in multiple myeloma (MM) treatment are to overcome drug resistance and to prevent relapse. The bone marrow (BM) microenvironment plays a critical role in MM cell growth and survival. The Notch pathway was found to be activated in MM. In this study we aimed at investigating the role of Dll1/Notch interaction in MM clonogenic growth and in vivo engraftment, as well as the role of Notch pathway in BM-induced drug resistance. Dll1 is a Notch ligand expressed in BM stromal cells. Cocultures were performed using murine 5T33MM cells with Dll1 ligand or MS5.Dll1 (Dll1-overexpressing) stromal cells. Notch downstream target genes (Hes1, Hes5, Hey1, Hey2, and HeyL) were investigated for Notch pathway activation, showing that 5T33MM cells expressed Notch target genes and that Hes5 and HeyL were up-regulated both on mRNA (Real-Time PCR) and protein level (western blot) after Dll1/Notch interaction. This up-regulation could be reverted by blocking Notch signaling with DA...

Destructive bone lesions due to osteolytic bone disease are a major cause of morbidity and mortality in multiple myeloma patients, occurring in more than 80% of cases. Underlying osteolytic bone disease is an uncoupling of the bone remodeling process, with an increased activity of osteoclasts and a decreased activity of osteoblasts. Current strategies to treat osteolytic bone disease focus on anti-resorptive agents, which do not rebuild bone loss. Src kinase has been implicated in both osteoclast and osteoblast function. In this study, we assessed the effect of Src inhibition with AZD0530 (saracatinib, Astra Zeneca) on the development of multiple myeloma and its associated osteolytic bone disease. We first determined Src family kinase expression in the multiple myeloma microenvironment and found that patient-derived myeloma cells express Src at low levels but disease stage does not correlate with Src expression levels. In accordance with the literature, Src mRNA expression was found...

The bone microenvironment plays a critical role in supporting the growth and survival of myeloma cells and the development of osteolytic bone disease. Signalling through p38 α MAPK mediates synthesis of myeloma cell survival factors by stromal cells; whereas, inhibiting p38 α MAPK reduces myeloma cell proliferation and inhibits osteoclast formation in vitro. However, it is unclear whether p38 α MAPK inhibition will prevent the growth and survival of myeloma cells and the bone disease in vivo. The aim of this study was to determine whether SCIO-469, a selective p38 α MAPK inhibitor, would inhibit myeloma growth and prevent the development of bone disease in the 5TMM syngeneic models of myeloma. Treatment of 5TMM cells, in vitro, with SCIO-469 resulted in a clear inhibition of p38 phosphorylation, as assessed by Western blotting and an inhibition up to 35% of stromal cell induced 5T33MM proliferation. Injection of 5T2MM murine myeloma cells into C57Bl/KaLwRij mice resulted in the grow...

1840 One of the greatest challenges in multiple myeloma (MM) treatment is to overcome drug resistance. More and more evidence showed that not only the MM tumor cells should be targeted but also the bone marrow (BM) micro-environment. Interactions of MM cells with the BM micro-environment have a pivotal role in MM cell proliferation, survival, migration, angiogenesis as well as drug resistance. Many pathways are involved including the conserved Notch signaling pathway. The interaction of Notch receptors and ligands between adjacent cells induces proteolytic cleavage and release of the intracellular domain of the Notch receptor, also called Notch intracellular domains (NICD). NICD will then enter the nucleus and modify the expression of downstream target genes. Notch receptors are expressed by MM cells and Notch ligand Dll1 is present on bone marrow (BM) stromal cells. We investigated whether Notch activation in myeloma cells by the interaction with Dll1 on stromal cells contributes t...

In recent years, researchers have showed a growing interest in the mechanistic relationship between bone marrow adipose tissue and adjacent tumors. However, the impact of bone marrow adipocytes on development of hematological malignancies, particularly multiple myeloma is unknown. With aging, bone marrow changes occur and fatty deposits can occupy up to 70% of the BM cavity. Interactions of bone marrow adipose tissue with bone cells and other immune cells, possibly suggest indirect ways in which bone marrow adipocytes may affect MM disease progression. Leptin, an adipokine released by adipocytes and crucial in energy homeostasis, displays immune modulatory properties but its role in anti-tumor immunity remains unclear. In this study we aimed to investigate the intriguing relationship between leptin receptor activation and invariant natural killer T (iNKT) cell mediated anti-tumor immunity in multiple myeloma. The murine immunocompetent 5T33MM model, mimicking the human disease close...

S100A9 belongs to a family of low-molecular-weight calcium-binding proteins and is involved in many biological processes including inflammation, cell migration and angiogenesis. It has been reported that S100A9 knockdown reduced myeloid-derived suppressor cell (MDSC) accumulation and Multiple Myeloma (MM) cell growth in MM models. MDSC are a heterogeneous population of immature myeloid cells and contribute to MM disease by immunosuppression, induction of angiogenesis and secretion of cytokines and growth factors. S100A9 is therefore proposed as an attractive drug target and compounds inhibiting the interaction of S100A9 with its receptors RAGE and TLR4 have been developed. In this study we evaluated the therapeutic relevance of S100A9 inhibition in MM using the preclinical immunocompetent murine 5T33MM model and two different small molecule inhibitors for S100A9 interactions (Active Biotech AB, Sweden). The presence of S100A9 was investigated in different cell populations. S100A9 is...

Multiple myeloma (MM) is a progressive monoclonal B cell malignancy, for which survival and progression largely relies on the crosstalk of tumor cells with the bone marrow (BM) microenvironment, inducing immune escape, angiogenesis, bone destruction and drug resistance. Despite great therapeutic advances, most of the MM patients still relapse and remain incurable. Over the past years, immunotherapy has emerged as a new field in cancer therapy. Here, the immune cells of the patients themselves are activated to target the tumor cells. In MM, several effector cells of the immune system are present in the BM microenvironment; unfortunately, they are mostly all functionally impaired. In this review, we focus on the role of innate-like T cells in MM, particularly CD1d- and MR1- restricted T cells such as respectively invariant natural killer T (iNKT) cells and mucosal associated invariant T (MAIT) cells. These cells have the capacity upon activation to rapidly release copious amounts of cytokines affecting a wide range of innate and adaptive immune responses, and could therefore play a key protective role in anti-tumor immunity. We describe recent observations with regard to functional exhaustion of iNKT and MAIT cells in MM pathology and discuss the potential application of checkpoint inhibition as an attractive target for prolonged activation of these immunomodulatory T cells in the treatment of MM.

Cancer is known for its cellular changes contributing to tumour growth and cell proliferation. As part of these changes, metabolic rearrangements are identified in several cancers, including multiple myeloma (MM), which is a condition whereby malignant plasma cells accumulate in the bone marrow (BM). These metabolic changes consist of generation, inhibition and accumulation of metabolites and metabolic shifts in MM cells. Changes in the BM micro-environment could be the reason for such adjustments. Enhancement of glycolysis and glutaminolysis is found in MM cells compared to healthy cells. Metabolites and enzymes can be upregulated or downregulated and play a crucial role in drug resistance. Therefore, this review will focus on changes in glucose and glutamine metabolism linked with the emergence of drug resistance. Moreover, metabolites do not only affect other metabolic components to benefit cancer development; they also interfere with transcription factors involved in proliferati...

RAS mutations occur frequently in multiple myeloma (MM), but apart from driving progression they can also stimulate antitumor effects by activating tumor suppressive RASSF proteins. While this family of death effector molecules are often silenced in cancers, functional data about RASSF proteins in MM are lacking. Here we report that RASSF4 is downregulated during MM progression and correlates with a poor prognosis. Promoter methylation analysis in human cell lines revealed an inverse correlation between RASSF4 mRNA levels and methylation status. Epigenetic modulating agents restored RASSF4 expression. Enforced expression of RASSF4 induced G2 phase cell cycle arrest and apoptosis in human cell lines, reduced primary MM cell viability, and blocked MM growth in vivo. Mechanistic investigations showed that RASSF4 linked RAS to several pro-death pathways including those regulated by the kinases MST1, JNK and p38. By activating MST1 and the JNK/c-Jun pathway, RASSF4 sensitized MM cells to...

Multiple myeloma (MM) bone disease is a major cause of morbidity and mortality in MM patients and persists even in patients in remission. This bone disease is caused by an uncoupling of bone remodeling, with increased osteoclast and decreased osteoblast activity and formation, culminating in lytic bone destruction. Bisphosphonates are the current standard of care but new therapies are needed. As the molecular mechanisms controlling MM bone disease are increasingly well understood, new therapeutic targets are extensively explored in the preclinical setting and initial clinical trials with novel compounds now show promising results. In this review, we will provide a comprehensive overview of the biology of MM bone disease, summarize its current clinical management and discuss preclinical and clinical data on next generation therapies.

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells in the bone marrow (BM). Recently, several studies have highlighted the role of pathogens in either promoting or dampening malignancies of unrelated origin. Trypanosoma brucei is an extracellular protozoan parasite which causes sleeping sickness. Our group has previously demonstrated that trypanosome infection affects effector plasma B cells. Therefore, we hypothesized that T. brucei infection could have an impact on MM development. Using the immunocompetent 5T33MM model, we demonstrated a significant reduction in BM-plasmacytosis and M-protein levels in mice infected with T. brucei, resulting in an increased survival of these mice. Blocking IFNγ could only partially abrogate these effects, suggesting that other mechanisms are involved in the destruction of malignant plasma cells. We found that T. brucei induces intrinsic apoptosis of 5T33MM cells in vivo, and that this was associated with reduced e...

Overcoming drug resistance is one of the greatest challenges in the treatment of multiple myeloma (MM). The interaction of myeloma cells with the bone marrow (BM) microenvironment is a major factor contributing to drug resistance. Tumour-associated macrophages (TAMs) with different polarization states constitute an important component of this microenvironment. Previous studies have revealed a role of TAMs in MM cell survival and drug resistance; however, the impact of macrophage polarization (anti-tumoural 'M1' versus pro-tumoural 'M2'-like phenotype) in this process has not yet been described. Here, the presence of TAMs was confirmed in BM sections from MM patients, both at diagnosis and relapse, with two M2 markers, CD163 and CD206. By following different TAM subpopulations during disease progression in the syngeneic murine 5T33MM model, we demonstrated a decrease in the number of inflammatory monocytes and an increase in the number of M2-oriented TAMs in BM. Co-culture experiments demonstrated that macrophages provide a survival benefit to myeloma cells that is maintained after treatment with several classes of anti-myeloma agent (melphalan and bortezomib); the greatest effect was observed with M2-polarized macrophages. The pro-survival effect was associated with activation of the STAT3 pathway in 5T33MM cells, less cleavage of caspase-3, and thus less apoptosis. AZD1480, an ATP-competitive JAK2 inhibitor, abrogated the observed TAM-mediated MM cell survival, and partially inhibited resistance to bortezomib. Despite having only a small quantitative impact on myeloid cells in vivo, AZD1480 treatment alone and in combination with bortezomib significantly reduced tumour load. In conclusion, M2 TAMs are present in the MM microenvironment, and contribute to MM cell survival and protection from drug-induced apoptosis. As a result of TAM-induced activation of the STAT3 pathway, 5T33MM cells are sensitized to AZD1480 treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The bone marrow (BM) represents a complex microenvironment containing stromal cells, immune cells, osteoclasts, osteoblasts, and hematopoietic cells, which are crucial for the immune response, bone formation, and hematopoiesis. Apart from soluble factors and direct cell-cell contact, extracellular vesicles (EVs), including exosomes, were recently identified as a third mediator for cell communication. Solid evidence has already demonstrated the involvement of various BM-derived cells and soluble factors in the regulation of multiple biological processes whereas the EV-mediated message delivery system from the BM has just been explored in recent decades. These EVs not only perform physiological functions but can also play a role in cancer development, including in Multiple Myeloma (MM) which is a plasma cell malignancy predominantly localized in the BM. This review will therefore focus on the multiple functions of EVs derived from BM cells, the manipulation of the BM by cancer-derived...

Multiple myeloma (MM) pathogenesis and progression largely rely on the cells and extracellular factors in the bone marrow (BM) microenvironment. Compelling studies have identified tumour exosomes as key regulators in the maintenance and education of the BM microenvironment by targeting stromal cells, immune cells, and vascular cells. However, the role of MM exosomes in the modification of the BM microenvironment and MM progression remains unclear. Here, we explored the functions of MM exosomes in angiogenesis and immunosuppression in vitro and in vivo. Murine MM exosomes carrying multiple angiogenesis-related proteins enhanced angiogenesis and directly promoted endothelial cell growth. Several pathways such as signal transducer and activator of transcription 3 (STAT3), c-Jun N-terminal kinase, and p53 were modulated by the exosomes in endothelial and BM stromal cells. These exosomes promoted the growth of myeloid-derived suppressor cells (MDSCs) in naive mice through activation of t...

Glucose-regulated protein (GRP) 78 is overexpressed in multiple myeloma (MM) and both, its surface expression as well as its biological significance as key sensor of the unfolded protein response make GRP78 an ideal candidate for immunotherapeutic intervention. The monoclonal antibody PAT-SM6 targets surface (s) GRP78 and leads to disease stabilization when used as single-agent in a clinical trial. In this paper, we evaluated expression of GRP78 in relapsed-refractory disease and explored PAT-SM6 therapy in combination regimens. GRP78 expression was immunohistochemically analyzed during disease progression and development of drug resistance throughout different stages of MM. Activity of PAT-SM6 was evaluated in combination with anti-MM agents lenalidomide, bortezomib and dexamethasone in vitro. Finally, we report on a MM patient with relapsed and refractory disease treated with PAT-SM6 in combination with bortezomib and lenalidomide. Although sGRP78 expression was present at all sta...

Thymosin beta 4 (Tβ4) is a small cytoplasmic and nuclear protein involved in different cellular processes including differentiation, migration and proliferation. It is overexpressed in several malignant cells, resulting in an increased angiogenic respons and metastatic potential of tumor cells. In the current study we wanted to evaluate the expression of Tβ4 in primary human multiple myeloma cells (MM) and murine 5TMM cells. Microarray analysis and qRT-PCR of 171 MM patients treated with high dose therapy and autologous stem cell transplantation, showed that MM cells display a decreased Tβ4 expression compared to plasma cells from healthy individuals. We investigated whether Tβ4 expression in MM cells influences the event free (EFS) and overall survival (OAS) of these patients. As Tβ4 was expressed in all MM cells, we compared survival of patients with Tβ4 expression in MM cells in the upper thirtile (Tβ4high MMC) against the lowest thirtile (Tβ4low MMC). Patients with Tβ4high MMC had an increased EFS (P< 0.05) and also their OAS tended to be longer. A similar observation was made in the 5TMM model where qRT-PCR and ELISA revealed a decreased expression of Tβ4 in BM samples from 5T33MM and 5T2MM diseased mice compared to control mice. To study the functionality of Tβ4, we overexpressed Tβ4 in 5T33MMvitro (5T33MMvt) cells by lentiviral transduction. These 5T33MMvt-Tβ4+ cells demonstrated a significantly decreased proliferative capacitity and an increased sensitivity to different anti-myeloma agents (bortezomib, melphalan, dexamethasone) compared to control 5T33MMvt cells. Furthermore, injection of 5T33MMvt-Tβ4+ cells in C57BlKaLwRij mice resulted in a significant decreased tumor formation (paraprotein concentration was 1.92 g/dl versus 3.74 g/dl for untransduced cells, p<0.05) and a prolonged survival compared to mice injected with untransduced 5T33MMvt cells (respectively 65.9 days versus 88.9 days, p< 0.05). In conclusion, we found a decreased Tβ4 expression in human and murine MM cells compared to normal plasma cells. In our cohort of MM patients treated with high-dose chemotherapy, low expression of TB4 identifies a group of patients with adverse prognosis while in the murine 5TMM33vt model, overexpression of Tβ4 resulted in an inhibitory effect on tumor formation suggesting a role of Tβ4 as a tumor suppressor gene in MM.

Cancer progression is in part determined by interactions between cancer cells and stromal cells in the tumor microenvironment (TME). The identification of cytotoxic tumor-infiltrating lymphocytes has instigated research into immune stimulating cancer therapies. Although a promising direction, immunosuppressive mechanisms exerted at the TME hamper its success. Myeloid-derived suppressor cells (MDSCs) have come to the forefront as stromal cells that orchestrate the immunosuppressive TME. Consequently, this heterogeneous cell population has been the object of investigation. Studies revealed that the transcription factor signal transducer and activator of transcription 3 (STAT3) largely dictates the recruitment, activation and function of MDSCs in the TME. Therefore, this review will focus on the role of this key transcription factor during the MDSC's life cycle and on the therapeutic opportunities it offers.

Mutual communication between multiple myeloma (MM) cells and mesenchymal stromal cells (MSC) plays a pivotal role in supporting MM progression. In MM, MSC exhibit a different genomic profile and dysregulated cytokine secretion compared to normal MSC, however the mechanisms involved in these changes are not fully understood. Here, we examined the miRNA changes in human MSC after culture with conditioned medium of MM cells and found 19 dysregulated miRNAs, including upregulated miR-146a. Moreover, exosomes derived from MM cells contained miR-146a and could be transferred into MSC. After overexpressing miR-146a in MSC, secretion of several cytokines and chemokines including CXCL1, IL6, IL-8, IP-10, MCP-1, and CCL-5 was elevated, resulting in the enhancement of MM cell viability and migration. DAPT, an inhibitor of the endogenous Notch pathway, was able to abrogate the miR-146a-induced increase of cytokines in MSC, suggesting the involvement of the Notch pathway. Taken together, our results demonstrate a positive feedback loop between MM cells and MSC: MM cells promote the increase of miR146a in MSC which leads to more cytokine secretion, which in turn favors MM cell growth and migration.

Multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of plasma cells in the bone marrow (BM). The success of the proteasome inhibitor bortezomib in the treatment of MM highlights the importance of the ubiquitin proteasome system (UPS) in this particular cancer. Despite the prolonged survival of MM patients, a significant amount of patients relapse or become resistant to therapy. This underlines the importance of the development and investigation of novel targets to improve MM therapy. The UPS plays an important role in different cellular processes by targeted destruction of proteins. The ubiquitination process consists of enzymes that transfer ubiquitin to proteins targeting them for proteasomal degradation. An emerging and promising approach is to target more disease specific components of the UPS to reduce side effects and overcome resistance. In this review, we will focus on different components of the UPS such as the ubiquitin activating enzyme E...

The anaphase promoting complex/cyclosome (APC/C) is an ubiquitin ligase involved in cell cycle. During the metaphase-anaphase transition the APC/C is activated by Cdc20. The aim of this study is to elucidate the importance and therapeutic potential of APC/C and its co-activator Cdc20 in multiple myeloma (MM). Gene expression analysis revealed that Cdc20 was expressed at higher levels in gene expression-based high-risk MM patients. Moreover, high Cdc20 expression correlated with poor prognosis. Treatment of human myeloma cell lines with proTAME, an APC/C inhibitor, resulted in an accumulation of APC/CCdc20 substrate cyclin B1 and an accumulation of cells in metaphase. Moreover we observed a significant dose-dependent decrease in viability and increase in apoptosis in MM cells upon proTAME treatment. The induction of apoptosis was accompanied with caspase 3, 8, 9 and PARP cleavage. A similar metaphase arrest and induction of apoptosis were obtained with specific knockdown of Cdc20. In...

Exosomes, extracellular nanovesicles secreted by various cell types, modulate the bone marrow (BM) microenvironment by regulating angiogenesis, cytokine release, immune response, inflammation, and metastasis. Interactions between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells play crucial roles in MM development. We previously reported that BMSC-derived exosomes directly promote MM cell growth, whereas the other possible mechanisms for supporting MM progression by these exosomes are still not clear. Here, we investigated the effect of BMSC-derived exosomes on the MM BM cells with specific emphasis on myeloid-derived suppressor cells (MDSCs). BMSC-derived exosomes were able to be taken up by MM MDSCs and induced their expansion in vitro. Moreover, these exosomes directly induced the survival of MDSCs through activating STAT3 and STAT1 pathways and increasing the anti-apoptotic proteins Bcl-xL and Mcl-1. Inhibition of these pathways blocked the enhancement of MDSC s...

ABSTRACT The bone marrow (BM) microenvironment plays a critical role in the multiple myeloma (MM) cell growth and survival. As a highly conserved cell signaling system, the Notch pathway is considered to regulate cell-fate determination, stem cell self-renewal, proliferation, and apoptosis. Notch receptors and ligands are expressed both in MM cells and the BM microenvironment. In this review, we mainly discuss the canonical Notch signaling pathway including background and components of the signaling, its activation, the downstream targets, its regulation, and cross talk with other pathways. We further focus on the role of Notch signaling in multiple myeloma cell growth, angiogenesis, differentiation, drug resistance, bone disease, metastasis, and stem cell biology. A better understanding of Notch signaling in myeloma may provide new strategies to improve current treatment and overall survival.

Liver sinusoidal endothelial cells (LSECs) react to different anti-actin agents by increasing their number of fenestrae. A new structure related to fenestrae formation could be observed when LSECs were treated with misakinolide. In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics. High-resolution microscopy, including immunocytochemistry and a combination of fluorescence- and scanning electron microscopy was applied. Halichondramide and dihydrohalichondramide disrupt microfilaments within 10 minutes and double the number of fenestrae in 30 minutes. Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter. Correlative microscopy showed the absence of actin filaments (F-actin) in sieve plates and fenestrae-forming centers. Comparable experiments on umbilical vein endothelial cells and bone marrow sinusoidal endothelia...

Multiple myeloma (MM) is ultimately fatal in part because no effective cell cycle-based therapy has been available to control tumor cell proliferation. Loss of cell cycle control in MM cells stems from coordinated deregulation of Cdk4-cyclin D1 or Cdk6 (Cdk4)-cyclin D2. PD 0332991 is the first orally bioactive small molecule that potently and specifically inhibits Cdk4 and Cdk6. It represents a promising cell cycle-based therapy for myeloma owing to its ability to potently inhibit Cdk4/6 and induce G1 cell cycle arrest in primary human myeloma cells in BM stromal cell co-cultures and to control tumor progression in a xenograft model. However, the efficacy of PD 0332991 in the presence of an intact immune system is unknown. To optimize therapeutic targeting of Cdk4/6 with PD 0332991, we investigated the effectiveness of PD 0332991 in inhibiting Cdk4/6 and controlling myeloma tumor progression in the immunocompetent, bone migrating 5T33MM model. By quantitative real-time PCR analysis, we found that these myeloma cells express a normal plasma cell transcription program such as upregulated Blimp-1 and loss of Bcl-6 expression. However, they proliferate aggressively due to Cdk4 overexpression and impaired p27Kip1 expression, thereby mimicking relapse disease in human myeloma. PD 0332991 potently inhibits Cdk4/6 phosphorylation of Rb in primary 5T33MM cells and induces G1 cell cycle arrest, both in vivo and ex vivo. Accordingly, treatment with PD 0332991 significantly prolongs the survival of tumor-induced 5T33MM mice; a mean of 35 days in the PD 0332991-treated group (N=9) versus 25 days in the vehicle-treated group (N=9, p< 0.003). These findings demonstrate for the first time that PD 0332991 targets Cdk4/6 and controls tumor expansion in the presence of an intact immune system. To further optimize Cdk4/6 targeting in MM, we combined PD 0332991 with bortezomib, a cytotoxic drug widely used in MM treatment. Pretreatment of 5T33MMvt cells with PD 0332991 for 48 hours markedly augmented bortezomib killing, to the same level of twice the dose of bortezomib when it was used alone. These studies of PD 0332991 in the immunocompetent 5T33MM model demonstrate that, as a consequence of Cdk4/6 inhibition and induction of G1 arrest, PD 0332991 sensitizes MM cells to killing by a cytotoxic agent. Targeting Cdk4/6 in combination therapy, therefore, represents a novel and promising strategy for myeloma treatment.