A novel circRNA-miRNA-mRNA network revealed exosomal circ-ATP10A as a biomarker for multiple myeloma angiogenesis.

The importance of angiogenesis in multiple myeloma (MM) is unquestionable; however, to date, the success of antiangiogenic therapies has been fairly limited. Exosomal circular RNAs (circRNAs) have been proven to be pivotal players in angiogenesis in various cancers. Nevertheless, their role in MM remains unknown. Therefore, we aimed to identify differentially expressed circRNAs in peripheral blood exosomes from MM patients and explore their diagnostic and prognostic values. We screened 2,052 circRNAs with significant differential expression between MM patients and healthy controls via high-throughput sequencing. qRT-PCR confirmed that the expression of circ-ATP10A was significantly increased in MM patients. The bioinformatics analyses suggested that circ-ATP10A can act as a microRNA (miRNA) sponge and regulate the expression of downstream vascular endothelial growth factor-B (VEGFB), hypoxia-inducible factor-1alpha (HIF1A), platelet-derived growth factor subunit A (PDGFA), and fibroblast growth factor (FGF). The immunohistochemical results indicated that the circ-ATP10A level was positively correlated with the protein levels of VEGFB and marrow microvessel density (MVD) in MM patients, and the receiver operating characteristic (ROC) curve, area under the ROC curve (AUC) and Kaplan-Meier survival curve analyses confirmed it as a prognostic biomarker. Collectively, our study indicates that exosomal circ-ATP10A is a valuable prognostic biomarker in MM and may promote MM angiogenesis by targeting hsa-miR-6758-3p/hsa-miR-3977/hsa-miR-6804-3p/hsa-miR-1266-3p/hsa-miR-3620-3p and modulating their downstream mRNAs, such as VEGFB, HIF1A, PDGF, and FGF.

Tumor-associated macrophages modulate angiogenesis and tumor growth in a xenograft mouse model of multiple myeloma.

Tumor-associated macrophages (TAMs) are closely associated with poor multiple myeloma (MM) prognosis. Therefore, in-depth understanding of the mechanism by which TAM supports MM progression may lead to its effective treatment. We used the MM nude mouse subcutaneous xenograft model to evaluate the efficacy of the macrophage-depleting agent clodronate liposome (Clo) against MM and elucidate the mode of action of this therapy. At the same time, observe whether the elimination of TAM in vivo while silencing the expression of VEGFA has the same effect as in vitro experiments. We also used Clo to eliminate macrophages and reinjected M1 or M2 TAM through mouse tail veins to investigate the effects of various macrophage subtypes on MM xenograft tumor growth. We applied qRT-PCR, immunohistochemistry, and enzyme-linked immunosorbent assay to quantify VEGFA, CD31, and CD163 expression in tumor tissues and sera. Removal of TAMs from the tumor microenvironment impeded tumor growth. The combination of Clo plus VEGFA siRNA had a stronger inhibitory effect on tumor growth than Clo alone, and M2 and M1 macrophages promoted and inhibited tumor growth, respectively. Macrophage depletion combined with cytokine blocking is a promising MM treatment. Targeted M2 macrophage elimination together with cytokine block may be more effective at inhibiting MM growth than either treatment alone. The results of the present study lay an empirical foundation for the development of novel therapeutic strategies for MM.

JunB is a key regulator of multiple myeloma bone marrow angiogenesis.

Bone marrow (BM) angiogenesis significantly influences disease progression in multiple myeloma (MM) patients and correlates with adverse prognosis. The present study shows a statistically significant correlation of the AP-1 family member JunB with VEGF, VEGFB, and IGF1 expression levels in MM. In contrast to the angiogenic master regulator Hif-1α, JunB protein levels were independent of hypoxia. Results in tumor-cell models that allow the induction of JunB knockdown or JunB activation, respectively, corroborated the functional role of JunB in the production and secretion of these angiogenic factors (AFs). Consequently, conditioned media derived from MM cells after JunB knockdown or JunB activation either inhibited or stimulated in vitro angiogenesis. The impact of JunB on MM BM angiogenesis was finally confirmed in a dynamic 3D model of the BM microenvironment, a xenograft mouse model as well as in patient-derived BM sections. In summary, in continuation of our previous study (Fan et al., 2017), the present report reveals for the first time that JunB is not only a mediator of MM cell survival, proliferation, and drug resistance, but also a promoter of AF transcription and consequently of MM BM angiogenesis. Our results thereby underscore worldwide efforts to target AP-1 transcription factors such as JunB as a promising strategy in MM therapy.

Regulatory role of DEPTOR­mediated cellular autophagy and mitochondrial reactive oxygen species in angiogenesis in multiple myeloma.

DEPTOR, an inhibitor of mammalian target of rapamycin (mTOR), is essential for the survival of multiple myeloma (MM) cells. The expression level of DEPTOR is closely related to the prognosis of patients with MM treated with the antiangiogenic agent thalidomide; however, its role in the regulation of angiogenesis has not yet been elucidated. In the present study, the expression levels of DEPTOR and vascular endothelial growth factor (VEGF), and the microvessel density (MVD) of bone marrow (BM) from patients with MM assessed. DEPTORoverexpression plasmid or CRISPR­associated protein 9 (Cas9) and single guided RNAs (sgRNAs) were used to modulate DEPTOR expression. The DEPTOR­mediated angiogenic effects were assessed using a tube formation assay of human umbilical vein endothelial cells (HUVECs) cultured in the collected conditioned medium from MM cell lines with different expression levels of DEPTOR. It was found that the expression level of DEPTOR negatively correlated with the VEGF level and BM MVD in MM. Autophagic activity was regulated by DEPTOR expression, but was not related to thalidomide­binding protein CRBN, which is required for thalidomide to play an anti­tumor and antiangiogenic role in MM cells. The disruption of DEPTOR protein decreased cellular autophagy, increased VEGF expression in MM cells, and inhibited the tube formation of HUVECs, while a high expression of DEPTOR exerted the opposite effect. Moreover, targeting DEPTOR also resulted in the production of mitochondrial reactive oxygen species (mtROS), the phosphorylation of nuclear factor­κB (NF­κB) and an increase in interleukin 6 (IL­6) secretion. Of note, these effects are fully abrogated by treatment with autophagy activator (SMER28) or mitochondrial­specific antioxidant (Mito­TEMPO). Taken together, the present study demonstrates the role of DEPTOR in the regulation of autophagy/mtROS and subsequent angiogenesis. The results provide a novel mechanism for the further understanding of the therapeutic effects of thalidomide on MM.

Heparanase: A Dynamic Promoter of Myeloma Progression.

It has been speculated for many years that heparanase plays an important role in the progression of cancer due largely to the finding that its expression is weak or absent in normal tissues but generally as tumors become more aggressive heparanase expression increases. However, it is only in the last decade or so that we have begun to understand the molecular mechanism behind the sinister role that heparanase plays in cancer. In this review, we describe the many functions of heparanase in promoting the growth, angiogenesis and metastasis of multiple myeloma, a devastating cancer that localizes predominantly within the bone marrow and spreads throughout the skeletal system devouring bone and ultimately leading to death of almost all patients diagnosed with this disease. We also explore recent discoveries related to how heparanase primes exosome biogenesis and how heparanase enhances myeloma tumor chemoresistance. Discovery of these multiple tumor-promoting pathways that are driven by heparanase identified the enzyme as an ideal target for therapy, an approach recently tested in a Phase I trial in myeloma patients.

Diffuse Infiltration of Multiple Myeloma With Initial Manifestation of Cavernous Sinus Syndrome Unveiled by 18F-FDG PET/CT.

Intracranial infiltration is a rare complication of multiple myeloma (MM), which is usually seen in advanced stage. Here, we report a 47-year-old woman with only cavernous sinus syndrome as first manifestation. Brain MR revealed cavernous sinus masses, leptomeningeal nodules, and osseous erosion, which were considered metastases. F-FDG PET/CT, which was further performed to identify potential primary lesions, showed increased uptake not only in cavernous sinus and leptomeninges but also in diffuse bone lesions, hepatic nodules, lymph nodes, subcutaneous nodules, and bilateral ovarian masses. Multiple myeloma with intracranial involvement was confirmed by biopsy and cerebrospinal fluid examination.

New Insights in Anti-Angiogenesis in Multiple Myeloma.

Angiogenesis is a constant hallmark of multiple myeloma (MM) progression and involves direct production of angiogenic cytokines by plasma cells and their induction within the bone marrow microenvironment. This article summarizes the more recent literature data concerning the employment of anti-angiogenic therapeutic agents actually used in preclinical models and clinical settings for the treatment of multiple myeloma.

Endothelial progenitor cells in multiple myeloma neovascularization: a brick to the wall.

Multiple myeloma (MM) is characterized by the clonal expansion of plasma cells in the bone marrow that leads to events such as bone destruction, anaemia and renal failure. Despite the several therapeutic options available, there is still no effective cure, and the standard survival is up to 4 years. The evolution from the asymptomatic stage of monoclonal gammopathy of undetermined significance to MM and the progression of the disease itself are related to cellular and molecular alterations in the bone marrow microenvironment, including the development of the vasculature. Post-natal vasculogenesis is characterized by the recruitment to the tumour vasculature of bone marrow progenitors, known as endothelial progenitor cells (EPCs), which incorporate newly forming blood vessels and differentiate into endothelial cells. Several processes related to EPCs, such as recruitment, mobilization, adhesion and differentiation, are tightly controlled by cells and molecules in the bone marrow microenvironment. In this review, the bone marrow microenvironment and the mechanisms associated to the development of the neovasculature promoted by EPCs are discussed in detail in both a non-pathological scenario and in MM. The latest developments in therapy targeting the vasculature and EPCs in MM are also highlighted. The identification and characterization of the pathways relevant to the complex setting of MM are of utter importance to identify not only biomarkers for an early diagnosis and disease progression monitoring, but also to reveal intervention targets for more effective therapy directed to cancer cells and the endothelial mediators relevant to neovasculature development.

A Mini-Review on Thalidomide: Chemistry, Mechanisms of Action, Therapeutic Potential and Anti-Angiogenic Properties in Multiple Myeloma.

Thalidomide is a drug with interesting therapeutic properties but also with severe side effects which require a careful and monitored use. Potential immunomodulatory, antiinflammatory, anti-angiogenic and sedative properties make thalidomide a good candidate for the treatment of several diseases such as multiple myeloma. Through an increase in the degradation of TNFα-mRNA, thalidomide reduces the production of TNFα by monocytes and macrophages stimulated by lipopolysaccharide or by T lymphocytes induced by mitogenic stimuli. The decreased level of TNFα alters the mechanisms of intracellular transduction by preventing the activation of NF-kB and by decreasing the synthesis of proteins, in particular IL-6, involved in cell proliferation, inflammation, angiogenesis and protection from apoptosis. Furthermore, thalidomide affects VEGF levels by down-regulating its expression. Nowadays, new safer and less toxic drugs, analogs of thalidomide, are emerging as beneficial for a more targeted treatment of multiple myeloma and several other diseases such as Crohn';s disease, rheumatoid arthritis, sarcoidosis, erythema nodosum leprosum, graft-versus-host disease.

Nanoparticle delivery systems, general approaches, and their implementation in multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy that remains incurable, with relapse rates >90%. The main limiting factor for the effective use of chemotherapies in MM is the serious side effects caused by these drugs. The emphasis in cancer treatment has shifted from cytotoxic, non-specific chemotherapies to molecularly targeted and rationally designed therapies showing greater efficacy and fewer side effects. Traditional chemotherapy has shown several disadvantages such as lack of targeting capabilities, systemic toxicity, and side effects; low therapeutic index, as well as most anticancer drugs, has poor water solubility. Nanoparticle delivery systems (NPs) are capable of targeting large doses of chemotherapies into the target area while sparing healthy tissues, overcoming the limitations of traditional chemotherapy. Here, we review the current state of the art in nanoparticle-based strategies designed to treat MM. Many nanoparticle delivery systems have been studied for myeloma using non-targeted NPs (liposomes, polymeric NPs, and inorganic NPs), triggered NPs, as well as targeted NPs (VLA-4, ABC drug transporters, bone microenvironment targeting). The results in preclinical and clinical studies are promising; however, there remains much to be learned in the emerging field of nanomedicine in myeloma.

An Lysophosphatidic Acid Receptors 1 and 3 Axis Governs Cellular Senescence of Mesenchymal Stromal Cells and Promotes Growth and Vascularization of Multiple Myeloma.

Mesenchymal stromal cells (MSCs) are multipotent progenitor cells and there is much interest in how MSCs contribute to the regulation of the tumor microenvironment. Whether MSCs exert a supportive or suppressive effect on tumor progression is still controversial, but is likely dependent on a variety of factors that are tumor-type dependent. Multiple myeloma (MM) is characterized by growth of malignant plasma cells in the bone marrow. It has been shown that the progression of MM is governed by MSCs, which act as a stroma of the myeloma cells. Although stroma is created via mutual communication between myeloma cells and MSCs, the mechanism is poorly understood. Here we explored the role of lysophosphatidic acid (LPA) signaling in cellular events where MSCs were converted into either MM-supportive or MM-suppressive stroma. We found that myeloma cells stimulate MSCs to produce autotaxin, an indispensable enzyme for the biosynthesis of LPA, and LPA receptor 1 (LPA1) and 3 (LPA3) transduce opposite signals to MSCs to determine the fate of MSCs. LPA3-silenced MSCs (siLPA3-MSCs) exhibited cellular senescence-related phenotypes in vitro, and significantly promoted progression of MM and tumor-related angiogenesis in vivo. In contrast, siLPA1-MSCs showed resistance to cellular senescence in vitro, and efficiently delayed progression of MM and tumor-related angiogenesis in vivo. Consistently, anti-MM effects obtained by LPA1-silencing in MSCs were completely reproduced by systemic administration of Ki6425, an LPA1 antagonist. Collectively, our results indicate that LPA signaling determines the fate of MSCs and has potential as a therapeutic target in MM. Stem Cells 2017;35:739-753.

Antiproliferative and Antiangiogenic Effects of Punica granatum Juice (PGJ) in Multiple Myeloma (MM).

Multiple myeloma (MM) is a clonal B-cell malignancy characterized by an accumulation of clonal plasma cells (PC) in the bone marrow (BM) leading to bone destruction and BM failure. Despite recent advances in pharmacological therapy, MM remains a largely incurable pathology. Therefore, novel effective and less toxic agents are urgently necessary. In the last few years, pomegranate has been studied for its potential therapeutic properties including treatment and prevention of cancer. Pomegranate juice (PGJ) contains a number of potential active compounds including organic acids, vitamins, sugars, and phenolic components that are all responsible of the pro-apoptotic effects observed in tumor cell line. The aim of present investigation is to assess the antiproliferative and antiangiogenic potential of the PGJ in human multiple myeloma cell lines. Our data demonstrate the anti-proliferative potential of PGJ in MM cells; its ability to induce G0/G1 cell cycle block and its anti-angiogenic effects. Interestingly, sequential combination of bortezomib/PGJ improved the cytotoxic effect of the proteosome inhibitor. We investigated the effect of PGJ on angiogenesis and cell migration/invasion. Interestingly, we observed an inhibitory effect on the tube formation, microvessel outgrowth aorting ring and decreased cell migration and invasion as showed by wound-healing and transwell assays, respectively. Analysis of angiogenic genes expression in endothelial cells confirmed the anti-angiogenic properties of pomegranate. Therefore, PGJ administration could represent a good tool in order to identify novel therapeutic strategies for MM treatment, exploiting its anti-proliferative and anti-angiogenic effects. Finally, the present research supports the evidence that PGJ could play a key role of a future therapeutic approach for treatment of MM in order to optimize the pharmacological effect of bortezomib, especially as adjuvant after treatment.

Galectin-1 suppression delineates a new strategy to inhibit myeloma-induced angiogenesis and tumoral growth in vivo.

Galectin-1 (Gal-1) is involved in tumoral angiogenesis, hypoxia and metastases. Actually the Gal-1 expression profile in multiple myeloma (MM) patients and its pathophysiological role in MM-induced angiogenesis and tumoral growth are unknown. In this study, we found that Gal-1 expression by MM cells was upregulated in hypoxic conditions and that stable knockdown of hypoxia inducible factor-1α significantly downregulated its expression. Therefore, we performed Gal-1 inhibition using lentivirus transfection of shRNA anti-Gal-1 in human myeloma cell lines (HMCLs), and showed that its suppression modified transcriptional profiles in both hypoxic and normoxic conditions. Interestingly, Gal-1 inhibition in MM cells downregulated proangiogenic genes, including MMP9 and CCL2, and upregulated the antiangiogenic ones SEMA3A and CXCL10. Consistently, Gal-1 suppression in MM cells significantly decreased their proangiogenic properties in vitro. This was confirmed in vivo, in two different mouse models injected with HMCLs transfected with anti-Gal-1 shRNA or the control vector. Gal-1 suppression in both models significantly reduced tumor burden and microvascular density as compared with the control mice. Moreover, Gal-1 suppression induced smaller lytic lesions on X-ray in the intratibial model. Overall, our data indicate that Gal-1 is a new potential therapeutic target in MM blocking angiogenesis.

Multiple myeloma exosomes establish a favourable bone marrow microenvironment with enhanced angiogenesis and immunosuppression.

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 the STAT3 pathway and changed their subsets to similar phenotypes to those seen in MM-bearing mice. Moreover, MM exosomes up-regulated inducible nitric oxide synthase and enhanced the immunosuppressive capacity of BM MDSCs in vivo. Our data show that MM exosomes modulate the BM microenvironment through enhancement of angiogenesis and immunosuppression, which will further facilitate MM progression. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Increased microcirculation detected by dynamic contrast-enhanced magnetic resonance imaging is of prognostic significance in asymptomatic myeloma.

This prospective study aimed to investigate the prognostic significance of dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) as a non-invasive imaging technique delivering the quantitative parameters amplitude A (reflecting blood volume) and exchange rate constant kep (reflecting vascular permeability) in patients with asymptomatic monoclonal plasma cell diseases. We analysed DCE-MRI parameters in 33 healthy controls and 148 patients with monoclonal gammopathy of undetermined significance (MGUS) or smouldering multiple myeloma (SMM) according to the 2003 IMWG guidelines. All individuals underwent standardized DCE-MRI of the lumbar spine. Regions of interest were drawn manually on T1-weighted images encompassing the bone marrow of each of the 5 lumbar vertebrae sparing the vertebral vessel. Prognostic significance for median of amplitude A (univariate: P < 0·001, hazard ratio (HR) 2·42, multivariate P = 0·02, HR 2·7) and exchange rate constant kep (univariate P = 0·03, HR 1·92, multivariate P = 0·46, HR 1·5) for time to progression of 79 patients with SMM was found. Patients with amplitude A above the optimal cut-off point of 0·89 arbitrary units had a 2-year progression rate into symptomatic disease of 80%. In conclusion, DCE-MRI parameters are of prognostic significance for time to progression in patients with SMM but not in individuals with MGUS.

Role of erythropoietin in the angiogenic activity of bone marrow endothelial cells of MGUS and multiple myeloma patients.

Increasing evidences suggest several biological roles for erythropoietin and its receptor (Epo and EpoR), unrelated to erythropoiesis, including angiogenesis. Here, we detected the expression of EpoR in bone marrow-derived endothelial cells from monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients (MGECs and MMECs, respectively) and assessed whether Epo plays a role in MGECs- and MMECs-mediated angiogenesis. We show that EpoR is expressed by both MGECs and MMECs even though at a higher level in the first ones. Both EC types respond to rHuEpo in terms of cell proliferation, whereas other responses, including activation of JAK2/STAT5 and PI3K/Akt pathways, cell migration and capillarogenesis are enhanced by Epo in MGECs, but not in MMECs. In addition, the conditioned media of both Epo-treated cells induce a strong angiogenic response in vivo in the chorioallantoic membrane assay, comparable to that of vascular endothelial growth factor (VEGF). Overall, these data highlight the effect of Epo on MGECs- and MMECs-mediated angiogenesis: MGECs are more responsive to Epo treatment than MMECs, probably because over-angiogenic phenotype of MMECs is already activated by their autocrine/paracrine loops occurring in the "angiogenic switch" from MGUS.

Targeting vasculogenesis to prevent progression in multiple myeloma.

The role of endothelial progenitor cell (EPC)-mediated vasculogenesis in hematological malignancies is not well explored. Here, we showed that EPCs are mobilized from the bone marrow (BM) to the peripheral blood at early stages of multiple myeloma (MM); and recruited to MM cell-colonized BM niches. Using EPC-defective ID1+/- ID3-/- mice, we found that MM tumor progression is dependent on EPC trafficking. By performing RNA-sequencing studies, we confirmed that endothelial cells can enhance proliferation and favor cell-cycle progression only in MM clones that are smoldering-like and have dependency on endothelial cells for tumor growth. We further confirmed that angiogenic dependency occurs early and not late during tumor progression in MM. By using a VEGFR2 antibody with anti-vasculogenic activity, we demonstrated that early targeting of EPCs delays tumor progression, while using the same agent at late stages of tumor progression is ineffective. Thus, although there is significant angiogenesis in myeloma, the dependency of the tumor cells on EPCs and vasculogenesis may actually precede this step. Manipulating vasculogenesis at an early stage of disease may be examined in clinical trials in patients with smoldering MM, and other hematological malignancies with precursor conditions.

Wogonin inhibits multiple myeloma-stimulated angiogenesis via c-Myc/VHL/HIF-1α signaling axis.

Angiogenesis is associated with the progression of multiple myeloma (MM). Wogonin is an active mono-flavonoid with remarkable antitumor activity. However, its impact on MM-stimulated angiogenesis remains largely unknown. Here, we demonstrated that wogonin decreased expression and secretion of pro-angiogenic factors in MM cells via c-Myc/HIF-1α signaling axis, reducing MM-stimulated angiogenesis and MM cell proliferation in vivo. Overexpression of c-Myc in MM cells disrupted the balance between VHL SUMOylation and ubiquitination, and thus inhibited proteasome-mediated HIF-1α degradation. Impaired function of VHL ubiquitination complex in c-Myc-overexpressing cells was fully reversed by wogonin treatment via increasing HIF-1α-VHL interaction and promoting HIF-1α degradation. Collectively, our in vitro and in vivo studies reveal for the first time that wogonin represses MM-stimulated angiogenesis and tumor progression via c-Myc/VHL/HIF-1α signaling axis.

Interrelationship between angiogenesis, inflammation and oxidative stress in Indian patients with multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a B-cell malignancy characterized by the accumulation of clonal population of plasma cells in the bone marrow (BM). A variety of angiogenic factors, proteases, reactive oxygen species and inflammatory cytokines induce the formation of an extensive and suitable BM microenvironment. Previous studies have established the importance of angiogenic factors, inflammatory molecules and oxidative stress in MM but their interplay and effect on each other are not being taken together. METHODS: Circulatory levels of VEGF, angiopoietin-2 (Ang-2), IL-6 and TNF-α along with the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were investigated in 112 subjects including 62 MM patients and 50 healthy controls. Inter-stage analysis was done to evaluate the association of these molecules with the severity of disease. Pearson correlation was determined to find interrelationship, if any, between these molecules. RESULTS: We have observed elevated levels of VEGF, Ang-2, IL-6, TNF-α and decreased activity of SOD, GPx in MM patients in comparison to controls. All these molecules also showed a trend with the severity of disease. We have found strong association between these factors upon their correlation and regression analysis. CONCLUSION: This study is a step toward understanding the indepth contribution of angiogenesis, inflammation and oxidative stress together in making BM microenvironment suitable for growth, survival and proliferation of malignant plasma cells in MM.