Myeloma Bone Research Articles

Gene signatures to therapeutics: Assessing the potential of ivermectin against t(4;14) multiple myeloma.

Multiple myeloma (MM) is a terminal differentiated B-cell tumor disease characterized by clonal proliferation of malignant plasma cells and excessive levels of monoclonal immunoglobulins in the bone marrow. The translocation, (t)(4;14), results in high-risk MM with limited treatment alternatives. Thus, there is an urgent need for identification and validation of potential treatments for this MM subtype. Microarray data and sequencing information from public databases could offer opportunities for the discovery of new diagnostic or therapeutic targets. To elucidate the molecular basis and search for potential effective drugs of t(4;14) MM subtype by employing a comprehensive approach. The transcriptional signature of t(4;14) MM was sourced from the Gene Expression Omnibus. Two datasets, GSE16558 and GSE116294, which included 17 and 15 t(4;14) MM bone marrow samples, and five and four normal bone marrow samples, respectively. After the differentially expressed genes were identified, the Cytohubba tool was used to screen for hub genes. Then, the hub genes were analyzed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. Using the STRING database and Cytoscape, protein-protein interaction networks and core targets were identified. Potential small-molecule drugs were identified and validated using the Connectivity Map database and molecular docking analysis, respectively. In this study, a total of 258 differentially expressed genes with enriched functions in cancer pathways, namely cytokine receptor interactions, nuclear factor (NF)-κB signaling pathway, lipid metabolism, atherosclerosis, and Hippo signaling pathway, were identified. Ten hub genes (cd45, vcam1, ccl3, cd56, app, cd48, btk, ccr2, cybb, and cxcl12) were identified. Nine drugs, including ivermectin, deforolimus, and isoliquiritigenin, were predicted by the Connectivity Map database to have potential therapeutic effects on t (4;14) MM. In molecular docking, ivermectin showed strong binding affinity to all 10 identified targets, especially cd45 and cybb. Ivermectin inhibited t(4;14) MM cell growth via the NF-κB pathway and induced MM cell apoptosis in vitro. Furthermore, ivermectin increased reactive oxygen species accumulation and altered the mitochondrial membrane potential in t(4;14) MM cells. Collectively, the findings offer valuable molecular insights for biomarker validation and potential drug development in t(4;14) MM diagnosis and treatment, with ivermectin emerging as a potential therapeutic alternative.

Aberrant metabolic processes promote the immunosuppressive microenvironment in multiple myeloma.

Multiple myeloma (MM) is still an incurable plasma cell malignancy. The efficacy of immunotherapy on MM remains unsatisfactory, and the underlying molecular mechanisms still are not fully understood. In this study, we delineated the dynamic features of immune cell in MM bone marrow (BM) along with elevated tumor cell infiltration by single-cell RNA sequencing (scRNA-seq), and investigated the underlying mechanisms on dysfunction of immune cells associated with myelomagenesis. We found that immune cells were activated in those patients with low infiltration of tumor cells, meanwhile suppressed with elevated infiltration of MM cells, which facilitated MM escaping from immune surveillance. Besides PD-1, abnormal expression of PIM kinases, KLRB1 and KLRC1 were involved in the defect of immune cells in MM patients. Importantly, we found aberrant metabolic processes were associated with the immunosuppressive microenvironment in MM patients. Disordered amino acid metabolism promoted the dysfunction of cytotoxicity CD8 T cells as well as lipid metabolism disorder was associated with the dysregulation of NK and DCs in MM. As metabolic checkpoints, PIM kinases would be potential effective strategies for MM immunotherapy. In summary, redressing the disordered metabolism should be the key points to get promising effects in immune-based therapies.

HNRNPA2B1-mediated m6A modification of TLR4 mRNA promotes progression of multiple myeloma

BackgroundMultiple myeloma (MM) is a malignancy of plasma cells that remains incurable. Toll-like receptor 4 (TLR4) acts as a stress-responsive signal, protecting mitochondria during proteasome inhibitor (PI) exposure, maintaining mitochondrial metabolism and increasing drug resistance in MM. However, the mechanism of TLR4 regulation remains elusive.AimsThe purpose of this study was to investigate the methylation pattern of multiple myeloma and its effect on the expression of HNRNPA2B1 and downstream targets.MethodsThe methylation level in MM and normal bone marrow specimens was detected using a colorimetric assay. HNRNPA2B1 gene knockdown was achieved in RPMI 8226 MM cells via adenovirus transfection. CCK8 and flow cytometric assays were used to detect proliferation and apoptosis, respectively. Transcriptome sequencing and m6A methylation MeRIP sequencing were applied, and differentially expressed genes (DEGs) were detected. Three independent NCBI GEO datasets were applied to examine the effects of HNRNPA2B1 and TLR4 expression on MM patient survival.ResultsHNRNPA2B1 promoted MM progression. Clinical data from database revealed that HNRNPA2B1 was adverse prognostic factor for survival among MM patients. Furthermore, transcriptome sequencing and methylation sequencing showed that HNRNPA2B1 recognized and was enriched at the m6A sites of TLR4 and TLR4 was down-regulated of both the m6A level and transcription level in HNRNPA2B1-knockdown MM cells. Moreover, TLR4 was an adverse survival prognostic factor based on database analysis.ConclusionOverall, our study implies that the RNA-binding protein HNRNPA2B1 increases cell proliferation and deregulates cell apoptosis in MM through TLR4 signaling. Our study suggests HNRNPA2B1 as a potential therapeutic target for MM.

Infiltration of the spinal cord and peripheral nerves in multiple myeloma.

BackgroundMultiple myeloma (MM) is a hematological malignancy, and intramedullary spinal cord metastasis is extremely rare.MethodsClinical and radiological data were collected from electronic medical records as well as a literature review of reported cases.ResultsWe report a rare case of IgA-LAM stage IIB MM with involvement of the spinal cord and peripheral nervous system. Laboratory studies showed elevated levels of serum β2-macroglobulin and cerebrospinal fluid protein. Electromyography revealed a demyelinating process with motor conduction blocks. On MRI, the lesions of MM bone marrow are characterized as a type of diffuse infiltration. MR neurography demonstrated an enhanced nodule in the thoracic segment with swelling of the cervicothoracic segments of the spinal cord. Moreover, swelling and hypertrophy of the entire nerve branchial, lumbosacral plexus, and cauda equina were detected, accompanied by myofascitis and denervated muscles. Ultimately, the condition of the patient deteriorated quickly and she died with a diagnosis of refractory MM.ConclusionMRI not only has the advantage of displaying the primary involved site of the bone marrow but also facilitates detecting extramedullary hematopoietic MM, such as infiltrating sites of the central and/or peripheral nervous system.

Abstract 1729: Single-cell transcriptome profiling of multiple myeloma bone marrow samples suggests that disease progression interplays with tumor and tumor microenvironment in The MMRF CoMMpass Study

Abstract Multiple Myeloma (MM) is a hematologic malignancy marked by uncontrolled clonal expansion of plasma cells. Previous research has examined single-cell transcriptome profiles of Monoclonal gammopathy of undetermined significance (MGUS) and MM tumor microenvironment (TME) and found that natural killer (NK) cell abundance is elevated in the early stages and correlated with altered chemokine receptor expression. This study suggested the critical role of immune cells on myeloma progression from asymptomatic MGUS to symptomatic MM. Up to date, however, there are no published studies comprehensively comparing tumor and immune populations differences between MM NON-progressors (NPs) and FAST-progressors (FPs) and investigating how clonal plasma cells affect disease progression in a large cohort. Therefore, understanding how tumor and immune cells influence disease progression within symptomatic MM is of great interest.Here, we subjected CD138-negative Bone Marrow Mononuclear cells (BMMC) samples from 418 MM patients to scRNA-seq. From MMRF CoMMpass study (NCT01454297), we also have whole exome sequencing (WES) and bulk RNA-seq from CD138-positive fraction of BMMC samples. Based on time to progressive disease (TTPD), we classified patients into 2 categories. Patients with TTPD less than 18 months were classified as FAST-progressors (FPs), whereas patients with TTPD more than 5 years were classified as NON-progressors (NPs). By analyzing patient genomic alterations and its association with progression, we found that there was a significant association of slow MM progression with t(11;14) (p = 0.048), consistent with previous study. In our preliminary analysis, we profiled 83 CD138-sorted MM bone marrow samples using scRNA-seq. Interestingly, we found plasma cells from samples with the same genetic alterations tend to cluster together, highlighting the important role of genetic drivers in transcriptome profiles of plasma cells. Moreover, integrated analysis of bone marrow samples from 83 MM patients and 4 healthy donors revealed an atypical naïve-B cell subset with enrichment of cells from fast-progressors and partial expression of MS4A1. Differentially expressed genes for this naïve-B cell subset includes KLF9, BCL2L11, JOSD1, and IRS2, etc. Overall, as part of MMRF immune profiling research, this study will help to interrogate how genetic alterations and disease progression interplay MM tumor and TME and provide a sufficiently broad and valuable dataset for systematically characterizing MM at single-cell resolution. Hopefully, this study could identify novel targets for MM immunotherapies, and ultimately identify patients with high risk of fast progression for early intervention in the clinic. Citation Format: Lijun Yao, Tianjiao Wang, Kazuhiro Sato, Reyka Jayasinghe, I-Ling Chiang, Darwin D'souza, William Pilcher, Edgar Gonzalez-Kozlova, Yered Pita-Juarez, Taxiarchis Kourelis, Deon Bryant Doxie, Beena Thomas, Brian Lee, Swati Sharma Bhasin, Upadhyaya Bhaskar, Mark Fiala, Julie Fortier, Travis Dawson, John Leech, Shaji Kumar, Hearn Cho, Seunghee Kim-Schulze, Bee Raj, Stephen Oh, the MMRF Immune Profiling Research Team, John Dipersio, Ravi Vij, Adeeb Rahman, Ionnis Vlachos, Shaadi Mehr, Mark Hamilton, Daniel Auclair, Surendra Dasari, David Avigan, Madhav Dhodapkar, Sacha Gnjatic, Manoj Bhasin, Li Ding. Single-cell transcriptome profiling of multiple myeloma bone marrow samples suggests that disease progression interplays with tumor and tumor microenvironment in The MMRF CoMMpass Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1729.

Abstract 6374: BMP6 overcomes multiple myeloma-induced bone disease by inducing the expression of small leucine-rich proteoglycans

Abstract Multiple myeloma (MM), the second most common hematologic malignancy in adults, is characterized by clonal proliferation of malignant plasma cells and tumor-induced complications, including MM-induced bone disease (MBD). Up to 90% of MM patients develop MBD during the course of their disease, which is a major cause of morbidity and mortality. MM therapy focuses primarily on agents that seek to eradicate the tumor cells and others which inhibit bone resorption. In contrast, no drugs are currently available to promote bone formation in MM patients and reverse MBD. Towards addressing this unmet therapeutic need, we applied a modified version of our “humanized” bone marrow (BM)-like scaffold (huBMsc)-based xenograft model, where MM cells and bone marrow mesenchymal stromal cells (BMMSCs) are co-implanted. In this system, we investigated the impact of MM cells on BMMSCs to identify markers that can be targeted with the intent to reverse MBD. After confirming that MM cells indeed inhibit bone formation in vivo, we performed transcriptional analysis of ex vivo expanded “unexposed” control BMMSCs vs. “MM-exposed” BMMSCs and found 332 differentially expressed genes. Next, we compared these data with transcriptional profiles of BMMSCs after in vitro differentiation towards osteoblasts, as well as publicly available data of transcriptional profiles of BMMSCs obtained from MM, ALL, and AML patients. Combined, this analysis identified 8, MM-specific, differentially expressed genes, including the small leucine-rich proteoglycans (SLRPs), ASPN, OGN and OMD, that are 1) suppressed in the presence of MM (xenograft model and patient-derived BMMSCs); 2) induced during in vitro osteogenic differentiation; and 3) not dysregulated in BMMSCs in other hematological malignancies. As reported previously, we observed that bone morphogenetic protein (BMP)6 can upregulate the expression of these SLRPs in BMMSCs, while inducing osteogenic differentiation. Moreover, incubation of MM cells with BMP6 in either mono- or co-cultures with stromal cell lines, resulted in reduced MM cell viability. In addition, pretreatment of BMMSCs with BMP6 completely abolished their support of MM proliferation. Interestingly, BMP6, in contrast to BMP2 and BMP4, did not induce, but inhibited receptor activator of nuclear kappa-B ligand (RANKL)-dependent osteoclast generation. Collectively, our analyses identify BMP6 as a master regulator that promotes osteogenic differentiation, inhibits RANKL-dependent osteoclastogenesis and can directly induce MM cell death. Therefore, by reshaping the BM microenvironment to become less tumor-supportive, BMP6 represents an attractive candidate for MM treatment. Citation Format: Jiaxian Wang, Thomas Baardemans, Ricardo de Matos Simoes, Olga Dashevsky, Huipin Yuan, Joost D. de Bruijn, Anton C. Martens, Constantine S. Mitsiades, Richard W. Groen. BMP6 overcomes multiple myeloma-induced bone disease by inducing the expression of small leucine-rich proteoglycans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6374.

Down-regulation of circ_0058058 suppresses proliferation, angiogenesis and metastasis in multiple myeloma through miR-338-3p/ATG14 pathway

BackgroundMultiple myeloma (MM) is one of the most frequently diagnosed hematological malignancy. Dysregulation of circular RNAs (circRNAs) has important impacts on MM process. Herein, this work aimed to investigate the role and mechanism of circ_0058058 in MM progression.MethodsLevels of genes and proteins were detected by real-time reverse transcription PCR (RT-qPCR) and Western blot. CCK-8 assay, colony formation assay, EdU assay, flow cytometry, tube formation assay, transwell assay and Western blot were utilized to detect the proliferation, apoptosis, angiogenesis and metastasis of MM cells. The target relationship between miR-338-3p and circ_0058058 or ATG14 (autophagy related 14) was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. In vivo experiments were performed using Xenograft assay.ResultsCirc_0058058 was up-regulated in MM bone marrow aspirates and cells, knockdown of circ_0058058 reduced MM cell proliferation, angiogenesis and metastasis, but induced apoptosis in vitro. In a MM xenograft mouse model, circ_0058058 silencing reduced MM tumor growth and cell proliferation. Mechanistically, circ_0058058 acted as a sponge for miR-338-3p to up-regulate ATG14 expression, which was validated to be a target of miR-338-3p. Rescue assay showed that miR-338-3p inhibition reversed the antitumor effects of circ_0058058 knockdown on MM cell. Moreover, forced expression of miR-338-3p suppressed MM cell malignant phenotype, which was abolished by ATG14 up-regulation.ConclusionCirc_0058058 functions as a sponge for miR-338-3p to elevate ATG14 expression to promote MM cell proliferation, metastasis and angiogenesis, affording a potential therapeutic target for MM prevention.

Single-Cell Transcriptomic Analysis Reveals Loss of Activated Bone Marrow NK Cells in Multiple Myeloma Patients Which Associates with Disease Progression in Mice

IntroductionMultiple Myeloma (MM) disease progression and therapy response are the net result of tumor cell-intrinsic features and tumor cell-extrinsic cues from the bone marrow (BM) microenvironment. Natural killer (NK) cells are mediators of the cytotoxic immune response against MM and are important effector cells in antibody-based immune therapies, especially anti-CD38 monoclonal antibodies such as Daratumumab. Classically, NK cells are divided into a cytotoxic CD56 dim subset, important for antibody-dependent cellular cytotoxicity, and a cytokine-producing CD56 bright subset releasing inflammatory mediators such as IFNγ, TNFα and GM-CSF. However, accumulating evidence suggests greater heterogeneity in the NK cell compartment and modulation of these NK cell subsets could impact disease progression and response to NK cell-driven immunotherapies. Here, we combined the 5TGM1 murine model of MM with single-cell RNA sequencing of bone marrow (BM) NK cells of newly diagnosed MM patients to map NK cell heterogeneity and to investigate their role in MM progression.ResultsTo gain insight in NK cell heterogeneity in MM disease we performed single-cell RNA sequencing on immune cells of viably frozen BM aspirates from 19 newly diagnosed MM patients and 5 non-cancer control patients. NK cells were identified in silico by transcription of KLRF1, KLRD1, GNLY and NKG7 resulting in a single-cell transcriptomic dataset of 30,373 NK cells from MM patients and 8,865 NK cells from control patients. Conventional CD56 bright and CD56 dim NK-cells were identified by increased transcription of GZMK or GZMB, respectively. The GZMK +CD56 bright NK cells contained clusters of naïve and activated NK cells. The GZMB+CD56 dim NK cells consisted of 5 subclusters. To identify MM-induced alterations in NK cell subsets, we compared GZMK +CD56 bright vs GZMB+CD56 dim cluster composition and distribution between controls and MM patients. Control BM was dominated by GZMB-transcribing cytotoxic CD56 dim NK cells, resulting in a low ratio of cytokine-producing GZMK +CD56 bright vs cytotoxic GZMB+CD56 dim NK cells. In contrast, MM bone marrow was characterized by heterogeneity of this ratio with a subset of patients presenting with complete reversal of this ratio . In this subset of patients, the altered composition was due to a loss of cytotoxic GZMB +CD56 dim NK cells, and more specifically a loss of NK cells with a transcriptome suggesting recent activation.To better examine the significance of cytotoxic NK cells in MM disease course we utilized the well-established 5TGM1 mouse model. C57Bl/6 and KaLwRij mice both received 10 6 5TGM1-GFP cells intravenously. Three weeks after tumor injection all KaLwRij mice (18/18) developed MM, defined by >5% tumor cells in BM (“unrestrained tumor”) and serum M-protein >2mg/ml. Interestingly, while 39% (7/18) of C57Bl/6 mice had no tumor, 44% (8/18) had low but detectable levels of MM cells (0.1-5% of BM cells, “restrained tumor”) and 17% (3/18) presented with an unrestrained MM with BM tumor load similar to that seen in KaLwRij mice. With time the percentage of mice with unrestrained tumor increased (5/12, 42%) at the expense of restrained tumor (2/12, 16%). We hypothesized that C57Bl/6 mice with low tumor load could represent a model of immune-mediated tumor control. Detailed analysis of the NK cell compartment revealed an expansion of activated mature (CD69 + CD11b +CD27 +) NK cells in C57Bl/6 mice with restrained BM MM (p=0.0031). In contrast, high BM tumor burden in both genotypes was associated with a sharp decline in absolute numbers of activated NK cells.Conclusion:Through a combination of single-cell transcriptomic analyses of the BM immune microenvironment in MM patients and experimental mouse models we found a loss of activated NK cells in a subset of patients and mice. Our data suggests that loss of these activated NK cells is associated with MM progression in vivo. A subset of MM patients presented with a loss of activated cytotoxic GZMB +CD56 dim NK cells in the BM, suggestive of reduced cytotoxic anti-tumor responses. Meanwhile, in vivo, high disease burden only occurred in mice with an absence of activated NK cells. Current analyses are focused on differences in human disease progression and efficacy of Daratumumab-based therapies in patients with various NK cell phenotypes. DisclosuresBroijl: Janssen, Amgen, Sanofi, Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sonneveld: Janssen: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; Celgene/BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; SkylineDx: Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.

The Novel Role of Checkpoint Inhibitor PD-1H/VISTA in Osteoclast Activation and Multiple Myeloma Bone Disease

IntroductionMultiple myeloma (MM)-induced bone disease remains one of its most devastating complications, caused by increased bone resorption by overactivated osteoclasts coupled with impaired bone formation. MM cells produce osteoclast-activating factors that induce osteoclast activation and extensive bone resorption. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor that is highly secreted by MM cells (Fu J etc. JCI. 2016). We also identified that the checkpoint inhibitor, programmed death-1 homolog (PD-1H/VISTA), serves as the MMP-13 receptor in osteoclasts and mediates MMP-13-dependent osteoclastogenic function which is largely blocked in Pd-1h -/-osteoclasts (Fu J etc. ASH 2019, 2020). While the inhibitory role of PD-1H/VISTA in T-cells has recently been described (ElTanbouly MA etc. Science 2020), its cellular binding proteins remain unclear, and its role in osteoclast activation andMM bone disease have not been addressed.Methods and ResultsTo identify its interacting proteins, PD-1H-His 6 recombinant protein was expressed in mouse bone marrow mononuclear cells, the associated proteins pulled down by Ni-NTA agarose beads from cell lysates and identified by mass spectrometry. Functional annotation charting of the 75 proteins enriched in PD-1H pull-down samples (with signal ratio of PD-1H-His 6 pull-down vs control >2) indicated that almost 30% of the interacting targets were either cytoskeletal or cytoskeleton-associated proteins.Given that the F-actin cytoskeleton undergoes dynamic reorganization during osteoclast differentiation and plays critical roles in bone resorption, we further addressed the role of PD-1H in F-actin cytoskeleton regulation. Initially, osteoclasts form F-actin-rich adhesive structures, termed podosomes. At later stages, podosomes collectively rearrange into clusters and rings and finally into sealing belts to mediate osteoclast spreading, migration and bone resorption (Teitelbaum SL. Ann N Y Acad Sci. 2011). By confocal immunofluorescence microscopy, we found that PD-1H co-localized with F-actin podosome clusters, rings and sealing belts during osteoclast differentiation (Figure 1A). The functional role of PD-1H in F-actin cytoskeleton reorganization was addressed using Pd-1h -/- osteoclast wherein Pd-1h knockout lead to the disruption of podosome clusters at early stages relative to WT controls, while at later stages, Pd-1h -/-osteoclasts exhibited significantly fewer F-actin rings and belts (Figure 1B). Further, binding of MMP-13 to PD-1H increased the number of osteoclasts forming F-actin rings and belts, as well as the size of F-actin belts, which was blocked in Pd-1h -/- osteoclasts.To determine the role of PD-1H in the development of myeloma-induced lytic bone lesions, 5TGM1 myeloma cells were bilaterally intratibially injected into Pd-1h wtRag2 -/- or Pd-1h -/-Rag2 -/- mice (n=10) to induce lytic bone lesions. Three weeks following intratibial 5TGM1 injection, tibiae were harvested for micro-computed tomography. Subsequent quantitative histomorphology analyses of the trabecular and cortical bones confirmed that the knockout of Pd-1h reduced MM-induced bone destruction with significantly less decrease in trabecular bone volume (Tb. [BV/TV]), trabecular bone number (Tb. N.), trabecular bone thickness (Tb. Th.), as well as less increase in trabecular bone spacing (Tb. Sp.) and bone specific surface (Tb. [BS/BV]) compared to Pd-1h wtRag2 -/- mice. Similar effects were observed in cortical bone with less decrease in cortical bone thickness (CT. Th.), cortical bone area fraction (CT. [BA/TA]), and cortical tissue mineral density (CT. TMD) in 5TGM1 bearing Pd-1h -/-Rag2 -/- mice vs Pd-1h wtRag2 -/- mice (Table 1).ConclusionsTaken together, our study, for the first time, reveals the novel role of checkpoint inhibitor, PD-1H/VISTA, in osteoclasts and myeloma bone disease. PD-1H associates with cytoskeleton proteins and regulates the F-actin cytoskeleton reorganization which is critical for osteoclast bone resorption activity. Further, PD-1H mediates MMP-13-induced osteoclast fusion, F-actin belts formation, and osteoclast activation. Pd-1h -/-in recipient mice significantly impairs MM-induced bone loss, demonstrating that PD-1H/VISTA plays a critical role in MM bone disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Netrin-1 expression and targeting in multiple myeloma

Deleted in colorectal cancer (DCC) and uncoordinated-5 (UNC5) receptors, play a key role in tumor progression of several solid tumors by inducing apoptosis when unbound to their ligand netrin-1. Netrin 1 is currently being evaluated as a therapeutic target. These receptors, known as dependence receptors, and their ligands, have not yet been extensively explored in hematological malignancies. Here, we performed a screening of various human myeloma cell lines and bone marrow samples from multiple myeloma patients for netrin-1 and its receptors to determine the expression of netrin 1 and its receptors in multiple myeloma as well as to assess the potential anti-myeloma activity of a novel anti-netrin-1 treatment (NP137). Our results showed heterogeneous expression of netrin-1 and its receptors DCC and UNC5H2(B) in six human myeloma lines. Additionally, immunohistochemistry and flow cytometry showed expression of these molecules in a majority of myeloma patient samples. In vitro NP137 did not induce apoptosis of myeloma cell lines yet enhanced the cytotoxicity of bortezomib and dexamethasone. In vivo, NP137 treatment of SCID mice with established RPMI8226 myeloma tumors led to a reduction of tumor size compared to controls. Ex vivo, NP137 lowered the plasma cells percentage in bone marrow aspirates in a fraction of the patient samples analyzed. These results suggest that netrin signaling could constitute a novel therapeutic target in multiple myeloma.

Investigating the Interplay between Myeloma Cells and Bone Marrow Stromal Cells in the Development of Drug Resistance: Dissecting the Role of Epigenetic Modifications.

Simple SummaryDespite advances made in the last two decades, multiple myeloma (MM) is still an incurable disease. The genetic complexity of MM and the presence of intra-clonal heterogeneity are major contributors to disease relapse and the development of treatment resistance. Additionally, the bone marrow microenvironment is known to play a pivotal role in MM disease progression. Together with genetic modifications, epigenetic changes have been shown to influence MM development and progression. However, epigenetic treatments for MM are still lacking. This is mainly due to the high rate of adverse events of epigenetic drugs in clinical practice. In this review, we will focus on the role of epigenetic modifications in MM disease progression and the development of drug resistance, as well as their role in shaping the interplay between bone marrow stromal cells and MM cells. The current and future treatment strategies involving epigenetic drugs will also be addressed.Multiple Myeloma (MM) is a malignancy of plasma cells infiltrating the bone marrow (BM). Many studies have demonstrated the crucial involvement of bone marrow stromal cells in MM progression and drug resistance. Together with the BM microenvironment (BMME), epigenetics also plays a crucial role in MM development. A variety of epigenetic regulators, including histone acetyltransferases (HATs), histone methyltransferases (HMTs) and lysine demethylases (KDMs), are altered in MM, contributing to the disease progression and prognosis. In addition to histone modifications, DNA methylation also plays a crucial role. Among others, aberrant epigenetics involves processes associated with the BMME, like bone homeostasis, ECM remodeling or the development of treatment resistance. In this review, we will highlight the importance of the interplay of MM cells with the BMME in the development of treatment resistance. Additionally, we will focus on the epigenetic aberrations in MM and their role in disease evolution, interaction with the BMME, disease progression and development of drug resistance. We will also briefly touch on the epigenetic treatments currently available or currently under investigation to overcome BMME-driven treatment resistance.

V-Domain Ig Suppressor of T Cell Activation (VISTA) Expression Is an Independent Prognostic Factor in Multiple Myeloma.

Simple SummaryMultiple myeloma (MM) is characterized by loss of anti-tumor T-cell immunity. The precise mechanisms by which malignant plasma cells escape T-cell immunity are unknown, although upregulation of checkpoint molecules is seen in progressive disease. The aim of our study was to investigate mechanisms of escape from T-cell immunity. We observed that the expression of V-domain Ig suppressor of T cell activation (VISTA) in the tumor microenvironment is an independent prognostic factor for survival in MM and its major cellular source is tumor infiltrating CD11B+ cells. The combination of high VISTA expression in the tumor combined with low infiltration of CD8+ cells compared to the surrounding stromal tissue is significantly associated with poor survival. These finding have identified VISTA as an interesting target for inhibition to circumvent escape of T-cell immunity.Multiple myeloma (MM) is characterized by loss of anti-tumor T cell immunity. Despite moderate success of treatment with anti-PD1 antibodies, effective treatment is still challenged by poor T cell-mediated control of MM. To better enable identification of shortcomings in T-cell immunity that relate to overall survival (OS), we interrogated transcriptomic data of bone marrow samples from eight clinical trials (n = 1654) and one trial-independent patient cohort (n = 718) for multivariate analysis. Gene expression of V-domain Ig suppressor of T cell activation (VISTA) was observed to correlate to OS [hazard ratio (HR): 0.72; 95% CI: 0.61–0.83; p = 0.005]. Upon imaging the immune contexture of MM bone marrow tissues (n = 22) via multiplex in situ stainings, we demonstrated that VISTA was expressed predominantly by CD11b+ myeloid cells. The combination of abundance of VISTA+, CD11b+ cells in the tumor but not stromal tissue together with low presence of CD8+ T cells in the same tissue compartment, termed a high VISTA-associated T cell exclusion score, was significantly associated with short OS [HR: 16.6; 95% CI: 4.54–62.50; p < 0.0001]. Taken together, the prognostic value of a combined score of VISTA+, CD11b+ and CD8+ cells in the tumor compartment could potentially be utilized to guide stratification of MM patients for immune therapies.

Elevation of miR-302b prevents multiple myeloma cell growth and bone destruction by blocking DKK1 secretion

BackgroundMyeloma bone disease (MBD) is a severe complication of multiple myeloma (MM) mainly due to an imbalance between enhanced osteoclast activity and reduced osteoblast function. Previous studies have demonstrated that miRNAs play a vital role in the osteogenic differentiation of mesenchymal stromal cells (MSCs) in MM. However, the value of miR‑302b in MBD remains to be further elucidated. The aim of this study is to explore the role of miR‑302b in the regulation of MBD osteogenic differentiation and evaluate the potential of a new therapeutic strategy for the clinical treatment of MBD.MethodOur previous research demonstrated that MiR-302b belongs to the miR-302 cluster and is able to inhibit tumor growth and osteolysis in an orthotopic osteosarcoma xenograft tumor mouse model. In this study, we first transfected miR-302b mimics, miR-302b inhibitor, and miR-302b NC into MM1.S and RPMI8226 MM cells to detect the correlation between miR-302b expression in the pathological specimens and the clinicopathological features by qPCR, the target correlation between miR-302b and DKK1 by immunohistochemistry, qPCR and Western blot, and the correlation between miR-302b and the Wnt/β-catenin signaling pathway by Western blot. The effect of miR-302b on osteoblastogenesis was also studied in a subperiosteal tumorigenesis model of NOD/SCID nude mice.ResultsWe found that increased miR-302b suppressed cell proliferation and induced cell apoptosis in RPMI 8226 and MM1.S cells. TargetScan online bioinformatic analysis predicted that miR-302b is able to bind to 3′UTR of DKK1 mRNA. Target binding of miR-302b to DKK1 was demonstrated by dual-luciferase reporter assay, qPCR, Western blot and immunohistochemistry, indicating that miR-302b is able to degrade DKK1 in RPMI 8226 and MM1.S cells. The model of co-culturing MM cells with preosteoblast MC3T3-E1 cells showed that miR-302b inhibits MM-induced suppression of osteoblast differentiation. Western blotting showed that miR-302b promotes the Wnt/β-catenin signaling pathway in MM cells. Micro-CT and immunohistochemistry results showed that miR-302b suppresses myeloma bone destruction in vivo.ConclusionmiR-302b is able to target DKK1 and promote the Wnt/β-catenin signaling pathway in MM.

Hypoxia-induced CREB cooperates MMSET to modify chromatin and promote DKK1 expression in multiple myeloma

Myeloma cells produce excessive levels of dickkopf-1 (DKK1), which mediates the inhibition of Wnt signaling in osteoblasts, leading to multiple myeloma (MM) bone disease. Nevertheless, the precise mechanisms underlying DKK1 overexpression in myeloma remain incompletely understood. Herein, we provide evidence that hypoxia promotes DKK1 expression in myeloma cells. Under hypoxic conditions, p38 kinase phosphorylated cAMP-responsive element-binding protein (CREB) and drove its nuclear import to activate DKK1 transcription. In addition, high levels of DKK1 were associated with the presence of focal bone lesions in patients with t(4;14) MM, overexpressing the histone methyltransferase MMSET, which was identified as a downstream target gene of hypoxia-inducible factor (HIF)-1α. Furthermore, we found that CREB could recruit MMSET, leading to the stabilization of HIF-1α protein and the increased dimethylation of histone H3 at lysine 36 on the DKK1 promoter. Knockdown of CREB in myeloma cells alleviated the suppression of osteoblastogenesis by myeloma-secreted DKK1 in vitro. Combined treatment with a CREB inhibitor and the hypoxia-activated prodrug TH-302 (evofosfamide) significantly reduced MM-induced bone destruction in vivo. Taken together, our findings reveal that hypoxia and a cytogenetic abnormality regulate DKK1 expression in myeloma cells, and provide an additional rationale for the development of therapeutic strategies that interrupt DKK1 to cure MM.

Checkpoint Inhibitor PD-1H/VISTA Mediates MMP-13 Induced Osteoclast Activation and Multiple Myeloma Bone Disease

Introduction Multiple myeloma (MM) bone disease remains one of the most devastating complications of this incurable cancer, causing bone fractures, pain, mobility issues and neurological deficits. MM cells produce osteoclast-activating factors that induce osteoclast activation, thereby leading to excessive bone resorption and lytic bone lesions1. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor that is highly secreted by MM cells. MMP-13 induces osteoclast fusion and bone-resorption via a mechanism independent of its proteolytic activity2. We recently reported that MMP-13 binds to checkpoint inhibitor programmed death-1 homolog (PD-1H/VISTA), a surface receptor that is expressed in osteoclasts at high levels3. Binding of MMP-13 to PD-1H/VISTA induces osteoclast fusion and bone resorption activity whereas knockdown or knockout of PD-1H/VISTA largely block MMP-13 mediated effects on osteoclasts3. However, the function of PD-1H inMM bone disease in vitro or in vivo has not been previously defined. Methods and Results To confirm the role of PD-1H in MMP-13 induced bone disease in MM, we first conducted MM-osteoclast trans-well co-culture assay using murine MM cell line, 5TGM1 cells, and bone marrow mononuclear cells from Pd-1h-/- or wild type (WT) mice. 5TGM1 control cells or MMP-13 knockdown 5TGM1 cells were seeded in the upper wells of the transwell plates; while WT or Pd-1h-/- bone marrow mononuclear cells were seeded in the lower wells and cultured for osteoclast differentiation assessed by TRAP staining. Results show that 5TGM1 induced differentiation of WT osteoclasts with significantly increased osteoclast size and nuclei number/osteoclast. Consistent with our previous results2, MMP-13 knockdown blocked the 5TGM1 MM cells-induced activation of WT osteoclasts. In contrast, neither 5TGM1 MM cells nor MMP-13 knockdown cells had significant effects on Pd-1h-/- osteoclasts. Hence, knockout of Pd-1h abrogated MMP-13 mediated MM induction of osteoclasts, indicating that MMP-13/PD-1H interactions are critically involved in MM-induced osteoclast activation. The in vivo role of PD-1H in MM bone disease was investigated using the intratibial 5TGM1 Rag2-/- MM bone disease mice model2. For this purpose, Pd-1h-/-Rag2-/- mice were generated by crossbreeding C57BL/6 Pd-1h-/- with C57BL/6 Rag2-/- mice. 3x105 firefly luciferase expressing 5TGM1 cells (5TGM1-luc) were intratibially injected into age and sex-paired Rag2-/- or Pd-1h-/-Rag2-/- mice (N=5). Tumor progression was monitored by weekly bioluminescence imaging (BLI). 3 weeks after tumor inoculation, tibiae were harvested for quantitative micro-CT, followed by histological analysis. Histological staining showed that intratibial injection of 5TGM1-luc MM cells induced extensive lytic lesions and trabecular bone loss in Rag2-/- mice. In contrast, in Pd-1h-/-Rag2-/- mice,the bone structure was maintained with markedly less bone loss. Morphological analyses of trabecular bone across proximal tibiae further indicated that in Rag2-/- mice, 5TGM1 induced significant changes in bone microarchitecture, with decreased bone volume fraction (bone volume/tissue volume), connective density, trabecular bone numbers, and trabecular bone thickness, as well as increased trabecular bone spacing (Table 1). In contrast, in Pd-1h-/-Rag2-/- mice, 5TGM1 failed to induce significant loss of trabecular bone, confirming the critical role of PD-1H in MM induced bone disease in vivo. Conclusions Taken together, our study, for the first time, reveal that checkpoint inhibitor PD-1H/VISTA is the critical receptor for MMP-13 in osteoclasts, thereby mediating MMP-13-induced osteoclast fusion, activation and bone resorption. MM-induced trabecular bone loss was significantly lower in Pd-1h-/-mice, demonstrating that PD-1H/VISTA plays a critical role in MMP-13-induced MM bone disease. Given the checkpoint role of PD-1H/VISTA in cancer immunosuppression, we further posit that targeting the interaction of MMP-13 and PD-1H may represent a novel therapeutic strategy to treat MM bone disease and modulate the MM immune environment.

Preventative metformin treatment increases myeloma tumour burden and bone disease in vivo

Preventative metformin treatment increases myeloma tumour burden and bone disease in vivo

A Physiologically-Relevant Three-Dimensional In Vitro Model of Human Myeloma Bone Disease

Abstract Multiple myeloma (MM) is a hematologic cancer caused by mature B cell neoplasm that infiltrates the skeleton at several sites. The disease is characterized by uninhibited transformed plasma cell proliferation that disrupts skeletal homeostasis leading to increased bone resorption. Osteolytic lesions (OL) left in the bone, remain long after the treatment of the cancer and indicate disease progression to myeloma bone disease (MBD). Current combinatorial MM therapies inhibit malignant plasma cell proliferation, slow the progression towards MBD, and increase the mean five-year survival rate, but do little to improve osteoblastic function and restore skeletal homeostasis. Several novel MBD treatments have been developed to heal OLs but testing their efficacy against MBD is challenging. Therefore, we created a in vitro three-dimensional microphysiological human MM bone model to facilitate the identification of improved combinatorial treatments that suppress plasma cell proliferation while healing osteolytic lesions. Bone Marrow Stromal Cell derived osteoblasts and bone marrow macrophage derived osteoclasts were capable of bone formation and resorption in a homeostatic coculture as measured by gene expression profiling, alkaline phosphatase deposition, and alizarin red staining. The introduction of human plasmacytoma cell lines induced lesions in the mini-bones decreasing the cumulative hydroxyapatite (HA) content while increasing resorption markers and recapitulating physiological conditions of MBD. In the future, we hope to use this model of human MBD to evaluate the effectiveness of new combinatorial therapies that promise to restore skeletal homeostasis.

Checkpoint Inhibitor PD-1H/VISTA Functions As MMP-13 Receptor on Osteoclasts and Mediates MMP-13 Induced Osteoclast Activation in Multiple Myeloma

Introduction Multiple myeloma (MM) bone disease is characterized by the development of osteolytic bone lesions due to the over-activation of osteoclast and inhibition of osteoblast cells. MM cells secret pro-osteoclastogenic factors which lead to osteoclast (OCL) activation. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor which is highly secreted by MM cells. MMP-13 induces osteoclast fusion and bone-resorption by triggering the ERK1/2-dependent up-regulation of the cell fusogen, DC-STAMP. This process operates independently of the MMP-13 proteolytic activity. The fact that MMP-13 regulates OCL signaling via a proteolytic independent mechanism suggests that MMP-13 functions as a paracrine message protein mediating the crosstalk between MM cells and OCL. However, the mechanism is yet to be identified. Methods and Results To screen for MMP-13 cellular binding proteins/receptors, we performed a MMP-13 pull-down assay wherein recombinant MMP-13-His6 was incubated with mouse mononuclear bone marrow cells (BMCs) lysates and Ni-NTA magnetic beads were used to pull-down MMP-13- associated proteins. Following mass spectral analysis, programmed death-1 homolog (PD-1H) was identified as a major MMP-13-binding protein. PD-1H, also known as V-domain Ig suppressor of T cell activation (VISTA), is a critical negative checkpoint regulator expressed on myeloid cells and involved in immune responses. Binding of MMP-13 and PD-1H was confirmed by co-expressing both proteins in HEK293 cells and subsequent co-immunoprecipitation assay. Further, following PD-1H expression in HEK293 cells, an MMP-13-GFP fusion fluorescence protein docked to the cell surface where mutagenesis studies demonstrated that the PD-1H extracellular domain (ECD) mediates the specific binding interaction. Western blot and immunohistochemistry revealed that PD-1H was highly expressed in mononuclear BMCs, pre-OCL and mature OCL. ShRNA-mediated knockdown of PD-1H in mouse mononuclear BMCs blocked the ability of MMP-13 to induce osteoclast fusion and activation. These results were further confirmed by using BMCs from Pd-1h-/- mice and WT littermates for in vitro osteoclast differentiation. While MMP-13 induced WT OCL fusion and activation, these effects were completely blocked in Pd-1h-/-OCLs in tandem with a loss in MMP-13 induced ERK1/2 phosphorylation, NFATc1 and DC-STAMP upregulation. Conclusions Taken together, our study, for the first time, revealed that checkpoint inhibitor PD-1H is highly expressed in pre- and mature osteoclast. More importantly, we identified PD-1H as the critical receptor for MMP-13 in OCL, thereby mediating MMP-13-induced OCL fusion, activation and bone resorption. Hence, MMP-13 as a novel PD-1H ligand might not only induce bone disease, but also play a potential role in the regulation of T-cell activity in MM. As such, targeting MMP-13 and PD-1H interactions may represent a novel therapeutic strategy to treat MM bone disease and modulate the MM immune environment. Disclosures Lentzsch: Abbvie: Consultancy; Clinical Care Options: Speakers Bureau; Sanofi: Consultancy, Research Funding; Multiple Myeloma Research Foundation: Honoraria; International Myeloma Foundation: Honoraria; Karyopharm: Research Funding; Columbia University: Patents &amp; Royalties: 11-1F4mAb as anti-amyloid strategy; Bayer: Consultancy; Janssen: Consultancy; Takeda: Consultancy; BMS: Consultancy; Proclara: Consultancy; Caelum Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Supervised and home-based physical exercise in patients newly diagnosed with multiple myeloma\u2014a randomized controlled feasibility study

BackgroundThe study evaluated the feasibility and safety of the exercise intervention and physical test procedures of our ongoing randomized controlled trial, examining the effect of physical exercise in newly diagnosed patients with multiple myeloma.MethodsPatients are randomized 1:1 to a control group (usual care) or an intervention group (usual care and exercise) by block randomization with stratification of planned treatment, WHO performance status, and study site. The exercise intervention consists of eight supervised exercise sessions combined with home-based exercise over a 10-week period. Bone disease is systematically evaluated to determine limitations regarding physical testing and/or exercise. Feasibility outcome measures were study eligibility, acceptance, and attrition, and furthermore attendance, adherence, tolerability, and safety to the exercise intervention. Additionally, test completion, pain, and adverse events during the physical test procedures were evaluated. Outcome assessors were blinded to allocation.ResultsOf 49 patients screened, 30 were included. The median age was 69 years, range 38–90, 77% were males, and 67% had bone disease. Study eligibility was 82%, acceptance 75%, and attrition 20%. Attendance at supervised exercise sessions was 92%, and adherence to supervised exercise sessions and home-based exercise sessions was 99% and 89%, respectively. No serious adverse events attributed to exercise or physical tests were reported. All patients completed the physical tests, except for two patients, where physical test procedures were modified due to bone disease.DiscussionThe exercise intervention and physical test procedures were feasible and safe in patients with multiple myeloma, even in older patients with multiple myeloma and in patients with myeloma bone disease.Trial registrationClinicalTrials.gov. ID NCT02439112. Registered on May 7, 2015.

Whole-Body Low-Dose Computed Tomography for Multiple Myeloma (MM) Bone Disease Detection in a Single Center in Colombia

Whole-Body Low-Dose Computed Tomography for Multiple Myeloma (MM) Bone Disease Detection in a Single Center in Colombia