Multiple Myeloma Patient Plasma Research Articles

Differences in the proteome within extracellular vesicles between premalignant and malignant plasma cell disorders.

Precursor plasma cell disorders such as monoclonal gammopathy of undetermined significance (MGUS) always precede the development of active malignancies such as multiple myeloma (MM). There is a need for novel biomarkers to identify those patients with such precursor plasma cell disorders who rapidly progress to MM. Plasma-derived extracellular vesicles (EVs) may serve as a reservoir of potential biomarkers that can shed light on the pathogenesis and disease biology of MM. This study isolated small EVs (SEVs) and large EVs (LEVs) from the platelet-poor peripheral blood plasma of MGUS (n = 9) and MM (n = 12) patients using the size exclusion chromatography-based method and evaluated their proteome using a label-free proteomics workflow. In total, 2055 proteins were identified in SEVs, while 2794 proteins were identified in LEVs. The transferrin receptor (or CD71) protein was upregulated in both populations of EVs derived from MM patients compared to MGUS patients and was of prognostic significance. Similarly, three isoforms of serum amyloid A (SAA) protein, SAA1, SAA2, and SAA4, were also highly upregulated in SEVs within MM patients relative to MGUS patients. Finally, CD40 expression was also higher in the LEVs derived from MM patients than in MGUS patients. This study demonstrates the feasibility of successfully isolating both SEVs and LEVs from the peripheral blood of patients with plasma cell disorders and quantifying protein biomarkers within these EVs that could be of prognostic and diagnostic interest.

Measuring Minimal Residual Disease in Plasma of Multiple Myeloma Patients By Intact Mass Spectrometry

Multiple Myeloma (MM) is a hematological cancer that is currently treatable but incurable, and is characterized by the accumulation of malignant plasma cells in bone marrow. Clinical studies have shown that post-cancer treatment, the minimal residual disease (MRD) status is a prognostic factor indicative of the extent of disease remission. MRD assessment has been primarily assessed in bone marrow (BM) using flow cytometry or next generation sequencing. Not only these measurements may lead to false-negative results due to spatial heterogeneity in the BM, hemodilution and/or extramedullary disease as indicated by Derman et al. in Blood Cancer Journal ((2021), 11:19) but the procedure for BM collection is invasive and painful for the patient. These authors present a peripheral blood (PB)-based method that potentially can overcome the barriers in assessing for residual disease in MM patients. We hereby present a non-invasive blood-test for measurement of MRD using mass spectrometry (intact mass approach) for the detection of Ig product in MM patients. We will describe the assay development, analytical characterization of assay performance, and data from the analysis of plasma/serum samples from MM patients enrolled in a clinical trial. A very low volume of patient plasma/serum sample (10 μL) is required to perform this assay. The plasma/serum sample is diluted in purification buffer, applied to Melon gel resin and incubated for 5 min, followed by centrifugation and reduction with DTT. The supernatant is then analyzed by LC-MS on a Q-Exactive mass spectrometer. Deconvolution of the intact protein m/z spectra is performed to yield intact mass of the specific protein target species of interest. Overall, the method can process up to 96 samples in parallel within 3 hrs, making this procedure highly scalable for clinical applications. Data obtained from the assay characterization indicate that the assay is sensitive with an LOD <500 ng/mL and differentiates MM patient from a healthy donor as well allows for the monitoring of MM patient before and after treatment. In conclusion, although still an exploratory analysis, the blood-based mass spectrometry method can be used to screen for MRD in patients who have undetectable disease by flow cytometry or next generation sequencing.

Metabolomics-Driven Systematic Research Reveals Different Metabolomic and Lipidomic Profiling of Bone Marrow Plasma in Patients with Multiple Myeloma

Myeloma cells resident in bone marrow (BM), therefore the microenvironment plays critical roles in the invasion, proliferation and migration. Obesity increases the morbidity and drug resistance in patient of multiple myeloma (MM) through changing the bone marrow environment. Extracellular environments such as the BM plasma likely have unique lipidomic metabolite profiles that predicts the prognosis in patients with multiple myeloma. However, the study of multiple omics on metabolic changes in BM of MM patients are still very limited. The present study utilized an untargeted metabolite and targeted complex lipid profiling (Liquid chromatography-mass spectrometry, LC-MS) analysis to identify significant differences in the metabolite levels of BM plasma between newly diagnosed MM patients with different BMI (≤24 kg/m 2, n = 10; ≥25 kg/m 2, n = 10). This was followed by verification of some of the metabolite differences of interest by targeted quantification of the metabolites using isotopic internal standards in the exploratory cohort as well as an independent validation cohort. Significant differences were noted in the amino acid profiles, lipid metabolism and amino sugar metabolism, such as increased L-Glutamate, L-Proline, D-Proline, palmitoleic acid and D-Fructose, etc between MM patients with different BMI ( Figure A-C). Glycerol phosphate shuttle, glycerolipid metabolism and de novo triacylglycerol biosynthesis were the top 3 enriched signaling pathways in BM of obese MM patients ( Figure D). Targeted complex lipid profiling analysis further showed that triglyceride (TG)，diglyceride (DG) were increased notably in obese BM plasma of MM patients and correlated significantly with BMI ( Figure E-G). Glycerolipids, triradylglycerols, diradylglycerols and fatty acyls metabolism were enriched in obese group by GSEA analysis ( Figure H). Furthermore, KEGG enrichment analysis showed differentiate expressed lipid metabolites in BM plasma of obese MM patients comparing to that of lean group, including regulation of lipolysis in adipocytes, glycerolipid metabolism, fat digestion and absorption, cholesterol metabolism and metabolic pathways, etc ( Figure I ). In summary, metabolite and complex lipid profiling of the BM plasma identifies differences in levels of metabolites and lipids between MM patients with different BMI. This may provide insight into the explanation that obesity increases the susceptibility to MM and drug resistance. Acknowledgement This project is supported by the Sun Yat-sen University Hundred Talents Program (PT19200101), Natural Science Foundation of Guangdong Province (2022A1515010290).

Glutamine Addiction of Multiple Myeloma Cells Reprograms Mesenchymal Stromal Cell Enzymes and Transporters Towards a Pro-Tumor Phenotype

Metabolic alterations in cancer are not only functional to ensure malignant cell growth but also shape a pro-tumor behavior of normal cell populations in the tumor microenvironment. Multiple myeloma (MM) is the only human cancer that is both glutamine-addicted and glutamine-auxotroph, a feature that renders MM growth completely dependent upon extracellular glutamine availability. Indeed, plasma cells from most MM patients do not express Glutamine Synthetase (GS), the only enzyme able to synthetize glutamine from glutamate and ammonium, while express high levels of Glutaminase (GLS), which catalyze the first step of glutaminolysis by deamidating glutamine into glutamate and ammonium. As a consequence, the bone marrow (BM) plasma of MM patients has low-glutamine/high glutamate levels compared to patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS), as firstly demonstrated by our group. This particular metabolic microenvironment induces GS expression in mesenchymal stromal cells (MSCs) impairing osteoblast (OB) differentiation of MSCs, thus favoring MM bone lesions typical of active MM. The impact of these metabolic alterations on other BM cell populations is much less defined and it has been investigated in this study. For instance, although MM BM displays an oversized adipocyte population, which sustain MM growth by providing free fatty acids, the effects of MM metabolism on MSCs and their adipocyte differentiation remain to be characterized. First we show that 13C 5-Gln isotopomer distribution revealed that almost 50% of total glutamate in human myeloma cell lines (HMLCs) directly derives from glutamine deamidation by GLS. Furthermore, in standard culture conditions, HMLCs secreted glutamate likely through the SLC7A11 transporter, whose activity was higher in HMLCs than in MSCs. On the other hand, HMCLs were not able to take up glutamate from the extracellular space, while human primary MSCs isolated from healthy donors displayed high activity of the sodium-dependent glutamate transporters of the EAAT family. Moreover, glutamate uptake was higher in undifferentiated MSCs than in MSC incubated for 14 days in osteogenic medium (10 -8 M dexamethasone and 50 µg/ml ascorbic acid). Consistently, a public transcriptional profile of MSCs and OBs obtained from bone biopsies of healthy donors (n=7) or MM patients (n=16) revealed that the expression of the inward Glu transporter EAAT3 is higher in MSCs compared to OBs. In glutamine-free conditions, MSCs produced and secreted glutamine, a process boosted by extracellular glutamate in a dose-dependent manner. Glutamine secretion was hindered by either l-methionine sulfoximine (MSO), an irreversible inhibitor of GS, or D-aspartate (D-Asp), a high-affinity inhibitor of EAAT3 that hinders glutamate uptake. In a co-culture system, HMCLs induced the expression of SNAT5 glutamine efflux transporter in MSCs. Consistently, upon glutamine withdrawal, primary undifferentiated MSCs from healthy donors sustained HMCLs growth, while this nutritional support was markedly impaired by either inhibition or silencing of GS and EAAT3, with a substantial decrease in MM cell viability. Lastly, primary human MSCs were incubated under adipogenic conditions (0.5 mM 3-Isobutyl-1-methylxanthine, 5 µM indomethacin, 50 µM dexamethasone and 10 mg/ml human insulin) in the presence or in the absence of glutamine to mimic, respectively, normal or MM BM microenvironment. Glutamine deprivation increased lipogenesis, assessed with Oil Red O staining, as well as the expression of the adipocyte markers PPARG, LEP, and ADIPOQ. These data point to a MM-driven metabolic pro-tumor BM niche in which MSCs feed glutamine-addicted MM cells, and MSC differentiation is skewed from osteogenesis to adipogenesis. Several steps of these deranged pathways are amenable to pharmacological inhibition, pointing to possible novel therapeutic approaches to counteract MM growth and its effects on the BM niche.

Proteomic and Metabolomic Analysis of Bone Marrow and Plasma from Patients with Extramedullary Multiple Myeloma Identifies Distinct Protein and Metabolite Signatures.

Multiple myeloma (MM) is an incurable haematological malignancy of plasma cells in the bone marrow. In rare cases, an aggressive form of MM called extramedullary multiple myeloma (EMM) develops, where myeloma cells enter the bloodstream and colonise distal organs or soft tissues. This variant is associated with refractoriness to conventional therapies and a short overall survival. The molecular mechanisms associated with EMM are not yet fully understood. Here, we analysed the proteome of bone marrow mononuclear cells and blood plasma from eight patients (one serial sample) with EMM and eight patients without extramedullary spread. The patients with EMM had a significantly reduced overall survival with a median survival of 19 months. Label-free mass spectrometry revealed 225 proteins with a significant differential abundance between bone marrow mononuclear cells (BMNCs) isolated from patients with MM and EMM. This plasma proteomics analysis identified 22 proteins with a significant differential abundance. Three proteins, namely vascular cell adhesion molecule 1 (VCAM1), pigment epithelium derived factor (PEDF), and hepatocyte growth factor activator (HGFA), were verified as the promising markers of EMM, with the combined protein panel showing excellent accuracy in distinguishing EMM patients from MM patients. Metabolomic analysis revealed a distinct metabolite signature in EMM patient plasma compared to MM patient plasma. The results provide much needed insight into the phenotypic profile of EMM and in identifying promising plasma-derived markers of EMM that may inform novel drug development strategies.

Targeted Metabolomics Approach Identifies Alterations in the Plasma Metabolome of Multiple Myeloma Patients with and without Extramedullary Spread

Introduction: Metabolomics refers to the identification and quantitation of metabolites in cells, tissues and biofluids. The metabolome (complete set of metabolites in a biological sample) reflects the biochemical events occurring in an organism at a given time, thus providing a valuable source to analyse metabolic changes in a variety of diseases, including cancer. Metabolic profiling of blood cancers represents a useful tool for the detection of novel biomarkers and therapeutic targets. Multiple myeloma (MM) accounts for less than 2% of all new cancer cases in the United States. The rare and aggressive sub-entity of MM, extramedullary multiple myeloma (EMM), develops when malignant plasma cells escape the bone marrow microenvironment and colonize distal tissues or organs. The prognosis of EMM is poor, and the incidence of EMM increases at disease progression, occurring in up to 30% of relapsed MM patients. The pathogenic mechanisms of EMM are poorly understood with no targeted therapies currently available to strategize treatment regimens. Here, we use a targeted metabolomics approach to identify metabolic changes in the plasma of MM patients with and without extramedullary spread. Methods: Targeted metabolomic analysis of age and gender-matched medullary MM (n=8) and EMM (n=9) blood plasma samples was performed using the MxP® Quant 500 kit (Biocrates Life Sciences AG, Innsbruck, Austria) with a SCIEX QTRAP 6500plus mass spectrometer. The MxP® Quant 500 kit is capable of quantifying more than 600 metabolites from 26 compound classes. Quality control (QC) samples were employed to monitor the performance of the analysis with metabolite concentration in each sample normalised based on these QC samples. Isotopically labelled internal standards and seven-point calibration curves were used in the quantitation of amino acids and biogenic amines. Semi-quantitative analysis of other metabolites was performed using internal standards. Data quality was evaluated by checking the accuracy and reproducibility of QC samples. Metabolites were included only when the concentrations of the metabolites were above the limit of detection (LOD) in >75% of plasma samples. Data was imported into MetaboAnalyst 5.0 for further analysis. Feature filtering was performed based on relative standard deviation (RSD) and the resulting data was autoscaled. Metabolites of interest were identified based on p-value < 0.01 and fold-change > 1.5 between experimental groups. Supervised statistical approaches were used to further interrogate the data. Results: Using a targeted metabolomic technique, we compared the metabolic profile of MM and EMM patient plasma. Univariate analysis using a t-test identified 5 metabolites of interest; HexCer(d18:1/20:0), TG(16:0_34:2), TG(22:4_32:0), TG(18:2_32:0), and Taurine (Figure 1(A)). The supervised clustering technique orthogonal projection to latent structure discriminant analysis (OPLS-DA) was used to determine separation between the two groups (MM and EMM). OPLS-DA scores plot illustrated a distinct separation between MM patients with extramedullary spread (red dots) compared to those without extramedullary spread (green dots) (Figure 1(B)). A permutation test (n=1000) was performed to ensure there was no overfitting of the data. Permutation analysis results (Q2 = 0.478, p = 0.023; R2Y = 0.978, p = 0.033) demonstrated the model was of good predictive quality. Discriminatory variables responsible for the group separation were identified using the OPLS-DA variable importance in projection (VIP) score to identify metabolites with a score greater than 1.5. HexCer(d18:1/20:0), TG(16:0_34:2), TG(22:4_32:0), TG(18:2_32:0), and Taurine had VIP scores of 2.5, 2.3, 2.1, 2.2 and 2.4, respectively. The diagnostic potential of these metabolites as EMM biomarkers was evaluated by receiver operating characteristic (ROC) curve analysis. All 5 metabolites demonstrated high diagnostic potential with area under the curve (AUC) values greater than 0.84. Conclusion: Investigating disease-associated metabolomes presents an opportunity to identify dysregulated metabolic processes and novel biomarkers. This pilot targeted metabolomic analysis of EMM plasma samples reveals metabolites of interest for further analysis and contributes to our understanding of EMM pathophysiology. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Proteomic Characterisation of the Plasma Proteome in Extramedullary Multiple Myeloma Identifies Potential Prognostic Biomarkers

Introduction: Extramedullary multiple myeloma (EMM) is an aggressive manifestation of multiple myeloma (MM) reported to occur in approximately 7% of patients at diagnosis and up to 30% of patients at relapse. EMM is characterised by the spread of malignant plasma cells from the bone marrow microenvironment to distal tissues or organs. It is associated with an adverse prognosis, correlating with a significant reduction in overall survival. Currently there are no validated, established biomarkers to predict EMM. Furthermore, EMM is often treated similarly to high-risk MM with no targeted therapeutic strategies. In-depth proteomic studies on EMM are lacking and the underlying molecular mechanisms that facilitate extramedullary transition are yet to be fully defined. Novel biomarkers and therapeutic targets are urgently required. To enhance our understanding of EMM and to identify novel prognostic biomarkers, we performed a mass spectrometry-based proteomic study on plasma from MM patients with and without extramedullary spread. Methods: Label-free liquid chromatography mass spectrometric analysis of age and gender-matched medullary MM (n=8) and EMM (n=9) blood plasma samples was carried out using a Thermo Q-Exactive mass spectrometer (Thermo Fisher Scientific). Proteome Discoverer 2.2 using Sequest HT (Thermo Fisher Scientific) and a percolator were employed for the identification of peptides and proteins. Several parameters were defined for protein identification: MS/MS mass tolerance was set to 0.02 Da; peptide mass tolerance was set to 10ppm; methionine oxidation was set as a variable modification; carbamido-methylation was set as a fixed modification; and up to two missed cleavages were tolerated. Peptide probability was set to high confidence. Data was imported into Perseus (1.6.14.0) for further analysis. Proteins with less than 70% valid values were removed from the analysis. Proteins of interest were identified based on an FDR-adjusted p-value ≤0.1, fold change >1.5 between experimental groups. Six proteins were selected for further validation using DuoSet enzyme linked immunosorbent assay (ELISA) kits (R&D Systems). We performed receiver operating characteristic (ROC) and area under the curve (AUC) analyses to determine the diagnostic potential of the validated proteins. Results: The median age was 65. Survival analysis revealed a statistically significant change in overall survival (OS) between the two patient cohorts (Log-rank = 3.977, P = 0.046). The median OS of patients with EMM and those without extramedullary spread was 19 months and 83 months, respectively. Our quantitative MS-based proteomic analysis identified 21 proteins of differential abundance between EMM and MM patient plasma (False discovery rate (FDR)-adjusted p-value < 0.1, fold change > 1.5) (Fig. 1A). Antibody-based validation using ELISAs was performed on six proteins (vascular cell adhesion molecule 1 (VCAM1), hepatocyte growth factor activator (HGFA), pigment epithelial-derived factor (PEDF), alpha-2-macroglobulin (A2M), cholinesterase (BCHE), aminopeptidase N (CD13)). VCAM1, HGFA and PEDF were confirmed as being significantly altered between the two cohorts (FDR-adjusted p-value < 0.05). VCAM1, HGFA and PEDF were subject to ROC analyses, demonstrating high discriminatory power for EMM diagnosis (AUC = 0.96, AUC = 0.85, and AUC = 0.97, respectively). The diagnostic efficacy was further enhanced by combining these biomarkers using a logistic regression model (AUC = 1). Conclusion: Our mass spectrometry and antibody-based study identified proteins of differential abundance in the blood plasma of MM patients with and without extramedullary spread. VCAM1, PEDF and HGFA represent promising predictive biomarkers and warrant further investigation in a larger cohort of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification a Novel Molecular Mechanism Underlying the Anti-CD38-Based Treatment Resistance in Multiple Myeloma Patients

Introduction: Anti-CD38 antibody (Daratumumab (DARA)/Isatuximab (ISA))-mediated therapy is now used in combination therapies to treat both newly diagnosed and relapsed/refractory multiple myeloma (MM). Despite a high extent and frequency of response to DARA/ISA containing therapy, MM patient relapse is still common. Several resistance mechanisms to DARA/ISA have been reported including MM cell clonal selection towards reduced CD38 expression; loss of MM cell surface CD38 upon the formation of a CD38-DARA complex leading to immunosuppression; and immunomodulatory effects after the DARA/ISA treatment via the downregulation of intracellular signaling pathways in bone marrow stromal cells. Given the widespread use of anti-CD38-directed treatment in MM, the identification of anti-CD38 based treatment resistance is an urgent clinical need. Methods/results: To understand the underlying molecular mechanism of anti-CD38-based treatment resistance, we assessed post-transcriptional alterations on CD38 transcripts. We performed total and single-cell RNA sequencing of six samples from MM patients treated with DARA/ISA in combination with other MM drugs. Specifically, we examined mRNA splicing aberrations on the CD38 transcripts in 44 MM patient plasma cells (PCs). Four CD38 splice variants (CD38-Va, -Vb, -Vc, and Vd (CD38 SpVs)) were identified in CD138+PCs from DARA-resistant patient samples. These CD38-SpVs are the result of one or more cassette-exon splicing. Some of these splicing events cause in-frame shifts, which lead to the activation of a cryptic stop codon on the CD38 transcript and encode truncated proteins. The cryptic stop codon location at the specific proximity of the exon-intron junction, allowing these transcripts escaping degradation via non-sense mediated decay. In MM PCs, CD38 splice variant transcripts were overexpressed at different levels (2-4 fold) in various combinations with one or more novel splice variants and CD38 full-length (CD38-FL) transcripts. These splice variants were absent in MM PCs from BM aspirates from age-matched healthy donors. Next, we evaluated the expression of the CD38 SpVs and FL transcripts in PCs from MM patients at the time they were sensitive to DARA/ISA treatment and when resistance developed. We identified CD38 splice variant transcripts that were selectively expressed MM PCs at the time of resistance to DARA/ISA treatment. We also identified CD38 splice variant transcripts that are overexpressed in DARA/ISA treatment-sensitive patient samples. Our findings were further validated in DARA resistant patient sample by single-cell RNA-seq analysis, which showed overexpression of CD38Vb in >80% of PCs. We identified splice site mutations that contribute to the expression of CD38 SpVs. To evaluate whether CD38 aberrant splicing is associated with DARA resistance, we generated a KMS11 DARA-resistant cell line (KMS11 DARA-Res) and evaluated the expression of splice variants in these cells. This analysis demonstrated that KMS11DARA-Res over-express CD38 SpVs. To determine whether CD38 splice variants encode functional proteins, we cloned, sequenced, and stably expressed CD38 SpVs transcripts into H929 cells (H929-CD38 SpVs). CD38 SpVs expressions were evaluated by flow cytometry and live cell imaging. These analyses demonstrated that CD38 SpVs encode proteins. Importantly, CD38 splice variant protein folding analyses showed that splicing alterations abrogate DARA/ISA binding sites. Then, to evaluate the functional effects of aberrant splicing of CD38 in MM cells, we carried out DARA/ISA binding assays and evaluated cell killing with ADCC assays in H929- CD38SpV cells. Importantly, these analyses showed that both DARA/ISA binding and ADCC was lost in H929-CD38SpV cells. Conclusions: We identified a novel molecular mechanism that underlies the resistance to anti-CD38-based treatment, both DARA and ISA. By this mechanism, CD38 cognate epitopes are removed from the cell surface of MM cells without an entire disposal of the target protein. The existence of such splicing-based mechanisms of resistance indicates that other antibody-based therapeutics or future CARs should be developed against CD38 epitopes that are created as a result of splicing. This approach may allow for selective targeting of MM PCs while spearing other CD38 expressing normal cells, enhancing cytotoxicity and improving therapeutic index.

Extracellular Vesicles Isolated from Plasma of Multiple Myeloma Patients Treated with Daratumumab Express CD38, PD-L1, and the Complement Inhibitory Proteins CD55 and CD59.

Daratumumab (DARA) has improved the outcome of treatment of multiple myeloma (MM). DARA acts via complement-dependent and -independent mechanisms. Resistance to DARA may result from upregulation of the complement inhibitory proteins CD55 and CD59, downregulation of the DARA target CD38 on myeloma cells or altered expression of the checkpoint inhibitor ligand programmed death ligand-1 (PD-L1) or other mechanisms. In this study, EVs were isolated from peripheral blood (PB) and bone marrow (BM) from multiple myeloma (MM) patients treated with DARA and PB of healthy controls. EV size and number and the expression of CD38, CD55, CD59 and PD-L1 as well as the EV markers CD9, CD63, CD81, CD147 were determined by flow cytometry. Results reveal that all patient EV samples express CD38, PD-L1, CD55 and CD59. The level of CD55 and CD59 are elevated on MM PB EVs compared with healthy controls, and the level of PD-L1 on MM PB EVs is higher in patients responding to treatment with DARA. CD147, a marker of various aspects of malignant behaviour of cancer cells and a potential target for therapy, was significantly elevated on MM EVs compared with healthy controls. Furthermore, mass spectrometry data suggests that MM PB EVs bind DARA. This study reveals a MM PB and BM EV protein signature that may have diagnostic and prognostic value.

Paeoniflorin Ameliorates BiPN by Reducing IL6 Levels and Regulating PARKIN-Mediated Mitochondrial Autophagy.

BackgroundBortezomib-induced peripheral neuropathy (BiPN) is a common complication of multiple myeloma (MM) treatment that seriously affects the quality of life of patients. The purpose of the present study was to explore the therapeutic effect of paeoniflorin on BiPN and its possible mechanism.MethodsELISA was used to measure the level of interleukin-6 (IL6) in the plasma of MM patients, and bioinformatics analysis was used to predict the mechanism underlying the effect of paeoniflorin on peripheral neuropathy. Cell and animal models of BiPN were constructed to evaluate mitochondrial function by measuring cell viability and mitochondrial quality and labeling mitochondria with MitoTracker Green. Nerve injury in mice with BiPN was assessed by behavioral tests, evaluation of motor nerve conduction velocity, hematoxylin-eosin (HE) staining, electron microscopy and analysis of the levels of reactive oxygen species (ROS). Western blotting and immunohistochemistry (IHC) were used to assess the expression of autophagy-related proteins.ResultsIn MM patients, IL6 levels were positively correlated with the degree of PN. The results of bioinformatics analysis suggested that paeoniflorin ameliorated PN by altering inflammation levels and mitochondrial autophagy. Paeoniflorin increased PC12 cell viability and mitochondrial autophagy levels, alleviated mitochondrial damage, and reduced IL6 levels. In addition, paeoniflorin effectively improved the behavior of mice with BiPN, relieved sciatic nerve injury in mice, increased the expression of LC3II/I, beclin-1, and Parkin in sciatic nerve cells, and increased the expression of LC3B and Parkin in the nerve tissue.ConclusionThe present study confirmed that paeoniflorin significantly ameliorated peripheral neuropathy (PN) caused by bortezomib, possibly by reducing IL6 levels to regulate PARKIN-mediated mitochondrial autophagy and mitochondrial damage.

Proteomic analysis of the bone marrow microenvironment in extramedullary multiple myeloma patients.

Multiple myeloma (MM) is a heterogeneous hematological malignancy characterized by the uncontrolled clonal proliferation of bone marrow (BM) plasma cells. The poor prognosis of patients is associated with the presence of extramedullary disease (EMD). Previously, different mechanisms involved in the colonization of BM niches by MM cells and their escape during EMD have been described. Thus, we aimed to investigate the expression of selected cytokines in the BM plasma of MM patients as well as EMD patients to reveal novel molecules involved in EMD pathogenesis. Expression of 120 different cytokines was measured in BM plasma of 13 MM and 11 EMD patients using Proteome Profiler Antibody Arrays. The correlation between statistically significant cytokines and clinicopathological parameters of patients was determined using the Spearman correlation analysis. Finally, protein-protein interactions were analyzed, and GO and KEGG pathways enrichment analysis was performed. In total, 27 cytokines were found to be differently expressed between MM and EMD patients. After the Benjamini-Hochberg correction for multiple testing, the statistical significance of two cytokines downregulated in EMD (EGF, BDNF) and six cytokines upregulated in EMD (NAP-2, ADIPOQ, CRP, MIG, BAFF, and THBS1) was maintained. Correlation analysis proved a significant association between the expression of these molecules and selected clinical-pathological features of MM/EMD patients. Protein association network analysis revealed important protein-protein interactions between THBS1/EGF, MIG/NAP-2, THBS1/NAP-2, EGF/NAP-2, and ADIPOQ/CRP. Finally, identified cytokines were proved to be significantly involved in focal adhesion, PI3K/AKT, and MAPK signaling pathways, and regulation of cell development, localization, proliferation, migration, differentiation, immune system processes, and stress response. Obtained results confirm the key function of the BM microenvironment in the pathogenesis of MM and indicate the essential role of numerous cytokines in disease progression and EMD development. However, the exact mechanisms need to be further clarified.

The Potential Role of Proinflammatory Cytokines and Complement Components in the Development of Drug-Induced Neuropathy in Patients with Multiple Myeloma.

The launch of novel chemotherapeutic agents—in particular, proteasome inhibitors and immunomodulatory drugs—dramatically changed multiple myeloma (MM) therapy, improving the response rate and prolonging progression-free survival. However, none of the anti-MM drugs are deprived of side effects. Peripheral neuropathy (PN) seems to be one of the most pressing problems. Despite extensive research in this area, the pathogenesis of drug-induced peripheral neuropathy (DiPN) has not yet been fully elucidated. In the present study, we aimed to assess the potential relationship between proinflammatory factors and the development of PN in MM patients with particular emphasis on the application of VTD (bortezomib, thalidomide, dexamethasone) regimen. Our analysis identified increased concentrations of CCL2, IL-1β, and IFN-γ in plasma of MM patients during treatment, both with and without symptoms of PN, compared with untreated neuropathy-free MM patients. At the same time, the plasma concentration of IL-1β in patients with neuropathy was significantly increased compared with patients without PN before and during treatment. Moreover, the results were enhanced at the transcript level by performing global mRNA expression analysis using microarray technology. The most significant changes were observed in the expression of genes responsible for regulating immunological and apoptotic processes. An in-depth understanding of the mechanisms responsible for the development of DiPN might in the future reduce the incidence of PN and accelerate diagnosis, allowing the choice of neuropathy-free treatment strategies for MM.

A Molecular Signature of Circulating MicroRNA Can Predict Osteolytic Bone Disease in Multiple Myeloma.

Simple SummaryMultiple myeloma bone disease (MMBD) is one of the most important complications of multiple myeloma with a great impact on quality of life. Recent advances in the field of imaging techniques provided clinicians with a variety of imaging modalities with high sensitivity for the diagnosis of MMBD. However, no circulating biomarkers are available to support the diagnosis of MMBD in cases where the results are inconclusive. The aim of our study was to investigate the clinical utility of 19 miRNAs implicated in osteoporosis in MMBD. Our results suggest that the levels of circulating let-7b-5p, miR-143-3p, miR-17-5p, miR-335-5p, and miR-214-3p (standalone or combined in multi-miRNA models) can effectively predict the presence of MMBD in newly diagnosed MM patients.Background: Multiple myeloma bone disease (MMBD) constitutes a common and severe complication of multiple myeloma (MM), impacting the quality of life and survival. We evaluated the clinical value of a panel of 19 miRNAs associated with osteoporosis in MMBD. Methods: miRNAs were isolated from the plasma of 62 newly diagnosed MM patients with or without MMBD. First-strand cDNA was synthesized, and relative quantification was performed using qPCR. Lastly, we carried out extensive biostatistical analysis. Results: Circulating levels of let-7b-5p, miR-143-3p, miR-17-5p, miR-214-3p, and miR-335-5p were significantly higher in the blood plasma of MM patients with MMBD compared to those without. Receiver operating characteristic curve and logistic regression analyses showed that these miRNAs could accurately predict MMBD. Furthermore, a standalone multi-miRNA–based logistic regression model exhibited the best predictive potential regarding MMBD. Two of those miRNAs also have a prognostic role in MM since survival analysis indicated that lower circulating levels of both let-7b-5p and miR-335-5p were associated with significantly worse progression-free survival, independently of the established prognostic factors. Conclusions: Our study proposes a miRNA signature to facilitate MMBD diagnosis, especially in ambiguous cases. Moreover, we provide evidence of the prognostic role of let-7b-5p and miR-335-5p as non-invasive prognostic biomarkers in MM.

Soluble CX3CL1/Fractalkine Is a New Player in the Pro-Inflammatory Microenvironment of Multiple Myeloma Patients Related to Bone Marrow Plasma Cell Infiltration and Neo-Vascularization

Multiple myeloma (MM) is characterized by the tight dependence of plasma cells (PCs) to the bone marrow (BM) microenvironment that activates the production of several pro-inflammatory and growth factors involved in MM cell survival, bone remodeling alterations and increased angiogenesis. CX3CL1/Fractalkine is a unique chemokine synthetized as a membrane-bound protein and released as soluble protein after proteolytic cleavage by two metalloproteases as ADAM10 and ADAM17. CX3CL1 interacts with its single receptor CX3CR1 being involved in several physiological and pathophysiological processes including inflammation and angiogenesis. The role CX3CL1/CX3CR1 axis in MM is still unexplored and unknown.In this study, firstly we analyzed BM plasma levels of CX3CL1 in MM patients as compared to indolent monoclonal gammopathies and healthy controls. BM plasma was obtained from 38 MM patients (median age 70 years; 53% female and 47% male; ISS I= 32%, II= 34%, III=34%) 25 of them with newly diagnosed and 13 relapsed MM, and from sex-aged matched 14 Smoldering MM (SMM) patients and 11 monoclonal gammopathy of undetermined significance (MGUS) patients. 10 healthy donors (HD) (median age 70 years; 20% female and 80% male) were used as controls. We found that plasma levels were significantly increased in the BM plasma from MM patients compared to HD (median levels 0.88 ng/ml vs 0.56 ng/ml, p=0.004). Furthermore, increased CX3CL1 levels were observed in patients with active MM as compared to patients with asymptomatic disease, as MGUS and SMM (median levels MM vs MGUS: 0.88 ng/ml vs 0.67 ng/ml, p=0.0176; median levels MM vs SMM: 0.88 ng/ml vs 0.65ng/ml, p= 0.0329). Moreover, CX3CL1 levels increased in the advanced stages of disease (median levels ISS III vs ISS I: 1.18 ng/ml vs 0.72 ng/ml, p=0.0115; median levels ISS III vs ISS II: 1.18 ng/ml vs 0.74 ng/ml, p=0.0157). Accordingly, BM soluble CX3CL1 levels were positively correlated with the percentage of BM plasma cells in the cohort of MM patients (p=0.002). Subsequently, the possible link between soluble CX3CL1/fractalkine and BM angiogenesis has been investigated. CD34 expression was evaluated by immunohistochemistry on bone biopsies obtained from 28 MM patients, showed that BM plasma levels of CX3CL1 significantly correlated with both the number of CD34 positive vessels (p= 0.002) and the micro-vessels density (p=0.002). Thereafter, we performed experiments on chorioallantoic membranes to demonstrate the role of CX3CL1 in MM-induced angiogenesis. The incubation of BM plasma of MM patients for 12 days resulted in a significant angiogenic response in the form of numerous allantoic neovessels that were significantly inhibited by blocking anti-CX3CL1 antibody. Finally, to clarify the source of CX3CL1 into the BM of MM patients, we explored CX3CL1 expression either by CD138+ cells on a dataset of 133 MM patients at diagnosis (GSE16122) and 23 human myeloma cell lines (HMCLs) (GSE6205), or on a proprietary dataset of primary mesenchymal stromal cells (MSCs) and osteoblasts (OBs) of 16 MM and 7 HD. Isolated BM endothelial cells from 4 MM patients and controls were also analysed. Purified CD138+ cells, HMCLs and BM endothelial cells were negative for CX3CL1 mRNA expression whereas MSCs and OBs were positive for CX3CL1 even if any statistically significant difference was not observed across the different groups. On the other hand, we found that CD138+ cells and HMCLs expressed both ADAM10 and ADAM17, leading to the hypothesis that the expression of these metalloproteinases by MM cells is accountable for the increased levels of CX3CL1 into the BM microenvironment and consequently increase of the neo-vascularization.In conclusion, our data indicate that CX3CL1, present at high level into the BM of MM patients, is involved in MM-induced BM neo-vascularization and potentially in MM progression. DisclosuresNo relevant conflicts of interest to declare.

The Effect of miR-361-3p Targeting TRAF6 on Apoptosis of Multiple Myeloma Cells.

microRNA-361-3p (miR-361-3p) is involved in the carcinogenesis of oral cancer and pancreatic catheter adenocarcinoma, and has anti-carcinogenic effects on non-small cell lung cancer (NSCLC). However, its effect on multiple myeloma (MM) is less reported. Here, we found that upregulating the expression of miR-361-3p inhibited MM cell viability and promoted MM apoptosis. We measured expressions of tumor necrosis factor receptor-associated factor 6 (TRAF6) and miR-361-3p in MM cells and detected the viability, colony formation rate, and apoptosis of MM cells. In addition, we measured expressions of apoptosis-related genes Bcl-2, Bax, and Cleaved caspase-3 (C caspase-3). The binding site between miR-361-3p and TRAF6 was predicted by TargetScan. Our results showed that miR-361-3p was low expressed in the plasma of MM patients and cell lines, while its overexpression inhibited viability and colony formation of MM cells and increased the cell apoptosis. Furthermore, TRAF6, which was predicted to be a target gene of miR-361-3p, was highexpressed in the plasma of patients and cell lines with MM. Rescue experiments demonstrated that the effect of TRAF6 on MM cells was opposite to that of miR-361-3p. Upregulation of miR-361-3p induced apoptosis and inhibited the proliferation of MM cells through targeting TRAF6, suggesting that miR-361-3p might be a potential target for MM therapy.

C3a and C5a facilitates the metastasis of myeloma cells by activating Nrf2.

Multiple myeloma (MM) is still an incurable hematological malignancy, with even poorer prognosis in MM patients with distant invasion. The present study was designed to explore the effects of C3a and C5a on the migration, invasion, and adhesion of MM tumor cells and to investigate the underlying mechanisms. As a result, the levels of C3a and C5a in plasma of MM patients were significantly higher than those of healthy donors. Consistently, the expression of C3a and C5a receptors on myeloma cells of MM patients was also significantly higher than that on sorted plasma cells of normal donors. C3a and C5a have been confirmed to increase the migration, invasion and adhesion of MM cell lines by activating the MEK/ERK pathway and increasing the nuclear transfer of Nrf2 in vitro. Moreover, the MM cell line U266 with Nrf2 downregulation was incubated with C3a and C5a, followed by injection into the tail vein of NOD-SCID mice. We found that Nrf2 downregulation attenuated the migration of anaphylatoxin C3a and C5a to MM tumor cells in bone marrow, liver and lung in vivo. In conclusion, our results indicate that activation of the complement cascade in MM patients may contribute to the migration, invasion and adhesion of MM cells, and this type of tumor cells dissemination in MM is, at least partially, regulated by Nrf2. Thereby, complement suppression or Nrf2 downregulation might offer a novel therapeutic opportunity for MM.

Long non-coding RNA TUG1 knockdown hinders the tumorigenesis of multiple myeloma by regulating the microRNA-34a-5p/NOTCH1 signaling pathway.

Multiple myeloma (MM) is a serious health issue in hematological malignancies. Long non-coding RNA taurine-upregulated gene 1 (TUG1) has been reported to be highly expressed in the plasma of MM patients. However, the functions of TUG1 in MM tumorigenesis along with related molecular basis are still undefined. In this study, increased TUG1 and decreased microRNA-34a-5p (miR-34a-5p) levels in MM tissues and cells were measured by the real-time quantitative polymerase reaction assay. The expression of relative proteins was determined by the Western blot assay. TUG1 knockdown suppressed cell viability, induced cell cycle arrest and cell apoptosis in MM cells, as shown by Cell Counting Kit-8 and flow cytometry assays. Bioinformatics analysis, luciferase reporter assay, and RNA pull-down assay indicated that miR-34a-5p was a target of TUG1 and directly bound to notch receptor 1 (NOTCH1), and TUG1 regulated the NOTCH1 expression by targeting miR-34a-5p. The functions of miR-34a-5p were abrogated by TUG1 upregulation. Moreover, TUG1 loss impeded MM xenograft tumor growth in vivo by upregulating miR-34a-5p and downregulating NOTCH1. Furthermore, TUG1 depletion inhibited the expression of Hes-1, Survivin, and Bcl-2 protein in MM cells and xenograft tumors. TUG1 knockdown inhibited MM tumorigenesis by regulating the miR-34a-5p/NOTCH1 signaling pathway in vitro and in vivo, deepening our understanding of the TUG1 function in MM.

Conversion of ATP to adenosine by CD39 and CD73 in multiple myeloma can be successfully targeted together with adenosine receptor A2A blockade

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...

A T-cell–redirecting bispecific G-protein–coupled receptor class 5 member D x CD3 antibody to treat multiple myeloma

AbstractT-cell–mediated approaches have shown promise in myeloma treatment. However, there are currently a limited number of specific myeloma antigens that can be targeted, and multiple myeloma (MM) remains an incurable disease. G-protein–coupled receptor class 5 member D (GPRC5D) is expressed in MM and smoldering MM patient plasma cells. Here, we demonstrate that GPRC5D protein is present on the surface of MM cells and describe JNJ-64407564, a GPRC5DxCD3 bispecific antibody that recruits CD3+ T cells to GPRC5D+ MM cells and induces killing of GPRC5D+ cells. In vitro, JNJ-64407564 induced specific cytotoxicity of GPRC5D+ cells with concomitant T-cell activation and also killed plasma cells in MM patient samples ex vivo. JNJ-64407564 can recruit T cells and induce tumor regression in GPRC5D+ MM murine models, which coincide with T-cell infiltration at the tumor site. This antibody is also able to induce cytotoxicity of patient primary MM cells from bone marrow, which is the natural site of this disease. GPRC5D is a promising surface antigen for MM immunotherapy, and JNJ-64407564 is currently being evaluated in a phase 1 clinical trial in patients with relapsed or refractory MM (NCT03399799).

The Transfer of Sphingomyelinase Contributes to Drug Resistance in Multiple Myeloma.

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 cells. In summary, this study is the first to analyze differences in plasma lipid composition of MM patients and match the observed differences to an upregulation of ASM. Moreover, we demonstrate that amitriptyline is able to inhibit ASM and increase sensitivity to anti-myeloma drugs. This study, therefore, provides a rational to include ASM-targeting-drugs in combination strategies in myeloma patients.