ABSTRACT
BACKGROUND: The proteasome inhibitor bortezomib is one of the primary therapies used for the haematological malignancy multiple myeloma (MM). However, intrinsic or acquired resistance to bortezomib, via mechanisms that are not fully elucidated, is a barrier to successful treatment in many patients. Our previous studies have shown that elevated expression of the chemokine receptor CCR1 in MM plasma cells in newly diagnosed MM patients is associated with poor prognosis. Here, we hypothesised that the poor prognosis conferred by CCR1 expression is, in part, due to a CCR1-mediated decrease in MM plasma cell sensitivity to bortezomib. METHODS: In order to investigate the role of CCR1 in MM cells, CCR1 was knocked out in human myeloma cell lines OPM2 and U266 using CRISPR-Cas9. Additionally, CCR1 was overexpressed in the mouse MM cell line 5TGM1. The effect of bortezomib on CCR1 knockout or CCR1-overexpressing cells was then assessed by WST-1 assay, with or without CCL3 siRNA knockdown or addition of recombinant human CCL3. NSG mice were inoculated intratibially with OPM2-CCR1KO cells and were treated with 0.7â¯mg/kg bortezomib or vehicle twice per week for 3 weeks and GFP+ tumour cells in the bone marrow were quantitated by flow cytometry. The effect of CCR1 overexpression or knockout on unfolded protein response pathways was assessed using qPCR for ATF4, HSPA5, XBP1, ERN1 and CHOP and Western blot for IRE1α and p-Jnk. RESULTS: Using CCR1 overexpression or CRIPSR-Cas9-mediated CCR1 knockout in MM cell lines, we found that CCR1 expression significantly decreases sensitivity to bortezomib in vitro, independent of the CCR1 ligand CCL3. In addition, CCR1 knockout rendered the human MM cell line OPM2 more sensitive to bortezomib in an intratibial MM model in NSG mice in vivo. Moreover, CCR1 expression negatively regulated the expression of the unfolded protein response receptor IRE1 and downstream target gene XBP1, suggesting this pathway may be responsible for the decreased bortezomib sensitivity of CCR1-expressing cells. CONCLUSIONS: Taken together, these studies suggest that CCR1 expression may be associated with decreased response to bortezomib in MM cell lines.
Subject(s)
Multiple Myeloma , Humans , Animals , Mice , Bortezomib/pharmacology , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Cell Line, Tumor , Receptors, Chemokine , Endoribonucleases , Protein Serine-Threonine Kinases , Receptors, CCR1/genetics , Receptors, CCR1/metabolismABSTRACT
Expression of myeloperoxidase (MPO), a key inflammatory enzyme restricted to myeloid cells, is negatively associated with the development of solid tumours. Activated myeloid cell populations are increased in multiple myeloma (MM); however, the functional consequences of myeloid-derived MPO within the myeloma microenvironment are unknown. Here, the role of MPO in MM pathogenesis was investigated, and the capacity for pharmacological inhibition of MPO to impede MM progression was evaluated. In the 5TGM1-KaLwRij mouse model of myeloma, the early stages of tumour development were associated with an increase in CD11b+ myeloid cell populations and an increase in Mpo expression within the bone marrow (BM). Interestingly, MM tumour cell homing was increased towards sites of elevated myeloid cell numbers and MPO activity within the BM. Mechanistically, MPO induced the expression of key MM growth factors, resulting in tumour cell proliferation and suppressed cytotoxic T-cell activity. Notably, tumour growth studies in mice treated with a small-molecule irreversible inhibitor of MPO (4-ABAH) demonstrated a significant reduction in overall MM tumour burden. Taken together, our data demonstrate that MPO contributes to MM tumour growth, and that MPO-specific inhibitors may provide a new therapeutic strategy to limit MM disease progression.
Subject(s)
Multiple Myeloma , Peroxidase , Tumor Microenvironment , Animals , Mice , Bone Marrow/pathology , Disease Models, Animal , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Myeloid Cells/pathology , Peroxidase/metabolismABSTRACT
Multiple myeloma (MM) disease progression is dependent on the ability of MM plasma cells (PCs) to egress from the bone marrow (BM), enter the circulation and disseminate to distal BM sites. Expression of the chemokine CXCL12 by BM stromal cells is crucial for MM PC retention within the BM. However, the mechanisms which overcome CXCL12-mediated retention to enable dissemination are poorly understood. We have previously identified that treatment with the CCR1 ligand CCL3 inhibits the response to CXCL12 in MM cell lines, suggesting that CCL3/CCR1 signalling may enable egress of MM PC from the BM. Here, we demonstrated that CCR1 expression was an independent prognostic indicator in newly diagnosed MM patients. Furthermore, we showed that CCR1 is a crucial driver of dissemination in vivo, with CCR1 expression in the murine MM cell line 5TGM1 being associated with an increased incidence of bone and splenic disseminated tumours in C57BL/KaLwRij mice. Furthermore, we demonstrated that CCR1 knockout in the human myeloma cell line OPM2 resulted in a >95% reduction in circulating MM PC numbers and BM and splenic tumour dissemination following intratibial injection in NSG mice. Therapeutic targeting of CCR1 with the inhibitor CCX9588 significantly reduced OPM2 or RPMI-8226 dissemination in intratibial xenograft models. Collectively, our findings suggest a novel role for CCR1 as a critical driver of BM egress of MM PCs during tumour dissemination. Furthermore, these data suggest that CCR1 may represent a potential therapeutic target for the prevention of MM tumour dissemination.
Subject(s)
Multiple Myeloma , Animals , Cell Line, Tumor , Humans , Mice , Mice, Inbred C57BL , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Plasma Cells , Receptors, CCR1/geneticsABSTRACT
The protein SAMSN1 was recently identified as a putative tumor suppressor in multiple myeloma, with re-expression of Samsn1 in the 5TGM1/KaLwRij murine model of myeloma leading to a near complete abrogation of intramedullary tumor growth. Here, we sought to clarify the mechanism underlying this finding. Intratibial administration of 5TGM1 myeloma cells into KaLwRij mice revealed that Samsn1 had no effect on primary tumor growth, but that its expression significantly inhibited the metastasis of these primary tumors. Notably, neither in vitro nor in vivo migration was affected by Samsn1 expression. Both knocking-out SAMSN1 in the RPMI-8226 and JJN3 human myeloma cell lines, and retrovirally expressing SAMSN1 in the LP-1 and OPM2 human myeloma cell lines had no effect on either cell proliferation or migration in vitro. Altering SAMSN1 expression in these human myeloma cells did not affect the capacity of the cells to establish either primary or metastatic intramedullary tumors when administered intratibially into immune deficient NSG mice. Unexpectedly, the tumor suppressive and anti-metastatic activity of Samsn1 in 5TGM1 cells were not evidenced following cell administration either intratibially or intravenously to NSG mice. Crucially, the growth of Samsn1-expressing 5TGM1 cells was limited in C57BL/6/Samsn1-/- mice but not in C57BL/6 Samsn1+/+ mice. We conclude that the reported potent in vivo tumor suppressor activity of Samsn1 can be attributed, in large part, to graft-rejection from Samsn1-/- recipient mice. This has broad implications for the design and interpretation of experiments that utilize cancer cells and knockout mice that are mismatched for expression of specific proteins.
ABSTRACT
In most instances, multiple myeloma (MM) plasma cells (PCs) are reliant on factors made by cells of the bone marrow (BM) stroma for their survival and growth. To date, the nature and cellular composition of the BM tumor microenvironment and the critical factors which drive tumor progression remain imprecisely defined. Our studies show that Gremlin1 (Grem1), a highly conserved protein, which is abundantly secreted by a subset of BM mesenchymal stromal cells, plays a critical role in MM disease development. Analysis of human and mouse BM stromal samples by quantitative PCR showed that GREM1/Grem1 expression was significantly higher in the MM tumor-bearing cohorts compared to healthy controls (p < 0.05, Mann-Whitney test). Additionally, BM-stromal cells cultured with 5TGM1 MM PC line expressed significantly higher levels of Grem1, compared to stromal cells alone (p < 0.01, t-test), suggesting that MM PCs promote increased Grem1 expression in stromal cells. Furthermore, the proliferation of 5TGM1 MM PCs was found to be significantly increased when co-cultured with Grem1-overexpressing stromal cells (p < 0.01, t-test). To examine the role of Grem1 in MM disease in vivo, we utilized the 5TGM1/KaLwRij mouse model of MM. Our studies showed that, compared to immunoglobulin G (IgG) control antibody-treated mice, mice treated with an anti-Grem1 neutralizing antibody had a decrease in MM tumor burden of up to 81.2% (p < 0.05, two-way ANOVA). The studies presented here demonstrate, for the first time, a novel positive feedback loop between MM PCs and BM stroma, and that inhibiting this vicious cycle with a neutralizing antibody can dramatically reduce tumor burden in a preclinical mouse model of MM.
ABSTRACT
N-cadherin is a homophilic cell-cell adhesion molecule that plays a critical role in maintaining vascular stability and modulating endothelial barrier permeability. Pre-clinical studies have shown that the N-cadherin antagonist peptide, ADH-1, increases the permeability of tumor-associated vasculature thereby increasing anti-cancer drug delivery to tumors and enhancing tumor response. Small molecule library screens have identified a novel compound, LCRF-0006, that is a mimetic of the classical cadherin His-Ala-Val sequence-containing region of ADH-1. Here, we evaluated the vascular permeability-enhancing and anti-cancer properties of LCRF-0006 using in vitro vascular disruption and cell apoptosis assays, and a well-established pre-clinical model (C57BL/KaLwRij/5TGM1) of the hematological cancer multiple myeloma (MM). We found that LCRF-0006 disrupted endothelial cell junctions in a rapid, transient and reversible manner, and increased vascular permeability in vitro and at sites of MM tumor in vivo. Notably, LCRF-0006 synergistically increased the in vivo anti-MM tumor response to low-dose bortezomib, a frontline anti-MM agent, leading to regression of disease in 100% of mice. Moreover, LCRF-0006 and bortezomib synergistically induced 5TGM1 MM tumor cell apoptosis in vitro. Our findings demonstrate the potential clinical utility of LCRF-0006 to significantly increase bortezomib effectiveness and enhance the depth of tumor response in patients with MM.
ABSTRACT
Approximately 15% of patients with multiple myeloma (MM) harbour the t(4;14) chromosomal translocation, leading to the overexpression of the histone methyltransferase NSD2. Patients with this translocation display increased tumour dissemination, accelerated disease progression and rapid relapse. Using publicly available gene expression profile data from NSD2high (n = 135) and NSD2low (n = 878) MM patients, we identified 39 epithelial-mesenchymal transition (EMT)-associated genes which are overexpressed in NSD2high MM plasma cells. In addition, our analyses identified Twist-1 as a key transcription factor upregulated in NSD2high MM patients and t(4;14)-positive cell lines. Overexpression and knockdown studies confirmed that Twist-1 is involved in driving the expression of EMT-associated genes in the human MM cell line KMS11 and promoted the migration of myeloma cell lines in vitro. Notably, Twist-1 overexpression in the mouse MM cell line 5TGM1 significantly increased tumour dissemination in an intratibial tumour model. These findings demonstrate that Twist-1, downstream of NSD2, contributes to the induction of an EMT-like signature in t(4;14)-positive MM and enhances the dissemination of MM plasma cells in vivo, which may, in part, explain the aggressive disease features associated with t(4;14)-positive MM.
Subject(s)
Biomarkers, Tumor/metabolism , Epithelial-Mesenchymal Transition , Histone-Lysine N-Methyltransferase/metabolism , Multiple Myeloma/pathology , Nuclear Proteins/metabolism , Repressor Proteins/metabolism , Translocation, Genetic , Twist-Related Protein 1/metabolism , Animals , Apoptosis , Biomarkers, Tumor/genetics , Cell Proliferation , Chromosomes, Human, Pair 14/genetics , Chromosomes, Human, Pair 4/genetics , Gene Expression Regulation, Neoplastic , Histone-Lysine N-Methyltransferase/genetics , Humans , Mice , Mice, Inbred C57BL , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Nuclear Proteins/genetics , Prognosis , Repressor Proteins/genetics , Transcriptional Activation , Transcriptome , Tumor Cells, Cultured , Twist-Related Protein 1/genetics , Xenograft Model Antitumor AssaysABSTRACT
Multiple myeloma, a plasma cell malignancy, is a genetically heterogeneous disease and the genetic factors that contribute to its development and progression remain to be fully elucidated. The tumour suppressor gene GLIPR1 has previously been shown to be deleted in approximately 10% of myeloma patients, to inhibit the development of plasma cell tumours in ageing mice and to have reduced expression levels in the plasma cells of patients with light-chain amyloidosis, a myeloma-related malignancy. Therefore, we hypothesised that GLIPR1 may have tumour suppressor activity in multiple myeloma. In this study, we demonstrate that plasma cell expression of GLIPR1 is reduced in the majority of myeloma patients and Glipr1 expression is lost in the 5TGM1 murine myeloma cell line. However, overexpression of GLIPR1 in a human myeloma cell line did not affect cell proliferation in vitro. Similarly, re-expression of Glipr1 in 5TGM1 cells did not significantly reduce their in vitro proliferation or in vivo growth in C57BL/KaLwRij mice. In addition, using CRISPR-Cas9 genome editing, we generated C57BL/Glipr1-/- mice and showed that loss of Glipr1 in vivo did not affect normal haematopoiesis or the development of monoclonal plasma cell expansions in these mice up to one year of age. Taken together, our results suggest that GLIPR1 is unlikely to be a potent tumour suppressor in multiple myeloma. However, it remains possible that the down-regulation of GLIPR1 may cooperate with other genetic lesions to promote the development of myeloma.
Subject(s)
Down-Regulation , Intercellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Multiple Myeloma/pathology , Neoplasm Proteins/genetics , Nerve Tissue Proteins/genetics , Animals , Cell Line, Tumor , Cell Proliferation , Gene Editing , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , Multiple Myeloma/genetics , Neoplasm TransplantationABSTRACT
Multiple myeloma is a fatal plasma cell malignancy that is reliant on the bone marrow microenvironment. The bone marrow is comprised of numerous cells of mesenchymal and hemopoietic origin. Of these, macrophages have been implicated to play a role in myeloma disease progression, angiogenesis, and drug resistance; however, the role of macrophages in myeloma disease establishment remains unknown. In this study, the antimyeloma efficacy of clodronate-liposome treatment, which globally and transiently depletes macrophages, was evaluated in the well-established C57BL/KaLwRijHsd murine model of myeloma. Our studies show, for the first time, that clodronate-liposome pretreatment abrogates myeloma tumor development in vivo. Clodronate-liposome administration resulted in depletion of CD169+ bone marrow-resident macrophages. Flow cytometric analysis revealed that clodronate-liposome pretreatment impaired myeloma plasma cell homing and retention within the bone marrow 24â¯hours postmyeloma plasma cell inoculation. This was attributed in part to decreased levels of macrophage-derived insulin-like growth factor 1. Moreover, a single dose of clodronate-liposome led to a significant reduction in myeloma tumor burden in KaLwRij mice with established disease. Collectively, these findings support a role for CD169-expressing bone marrow-resident macrophages in myeloma disease establishment and progression and demonstrate the potential of targeting macrophages as a therapy for myeloma patients.
Subject(s)
Bone Density Conservation Agents/administration & dosage , Clodronic Acid/administration & dosage , Disease Susceptibility , Liposomes , Macrophages/immunology , Macrophages/metabolism , Multiple Myeloma/etiology , Multiple Myeloma/metabolism , Animals , Biomarkers , Cell Count , Cell Line, Tumor , Cell Movement , Disease Models, Animal , Immunophenotyping , Mice , Multiple Myeloma/pathology , Osteoblasts , Tumor MicroenvironmentABSTRACT
The era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of individual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of AXL and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells.
Subject(s)
Multiple Myeloma/genetics , Multiple Myeloma/pathology , Neoplasm Recurrence, Local/genetics , Neoplastic Stem Cells/pathology , Stem Cell Niche/genetics , Animals , Humans , Mice , Neoplasm Recurrence, Local/pathology , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Transcriptome , Axl Receptor Tyrosine KinaseABSTRACT
Multiple myeloma (MM) is a largely incurable haematological malignancy defined by the clonal proliferation of malignant plasma cells (PCs) within the bone marrow. Clonal heterogeneity has recently been established as a feature in MM, however, the subclonal evolution associated with disease progression has not been described. Here, we performed whole-exome sequencing of serial samples from 10 patients, providing new insights into the progression from monoclonal gammopathy of undetermined significance (MGUS) and smouldering MM (SMM), to symptomatic MM. We confirm that intraclonal genetic heterogeneity is a common feature at diagnosis and that the driving events involved in disease progression are more subtle than previously reported. We reveal that MM evolution is mainly characterised by the phenomenon of clonal stability, where the transformed subclonal PC populations identified at MM are already present in the asymptomatic MGUS/SMM stages. Our findings highlight the possibility that PC extrinsic factors may play a role in subclonal evolution and MGUS/SMM to MM progression.
Subject(s)
Biomarkers, Tumor/genetics , Clonal Evolution , Monoclonal Gammopathy of Undetermined Significance/pathology , Multiple Myeloma/pathology , Cells, Cultured , Cohort Studies , Disease Progression , Follow-Up Studies , Humans , Longitudinal Studies , Monoclonal Gammopathy of Undetermined Significance/genetics , Multiple Myeloma/genetics , Prognosis , Exome SequencingABSTRACT
Multiple myeloma (MM) is a hematological malignancy resulting from the uncontrolled proliferation of antibody-producing plasma cells in the bone marrow. At diagnosis, independent plasma cell tumors are found throughout the skeleton. The recirculation of mutant plasma cells from the initial lesion and their recolonization of distant marrow sites are thought to occur by a process similar to solid tumor metastasis. However, the efficiency of this bone marrow homing process and the proportion of disseminated cells that actively divide and contribute to new tumor growth in MM are both unknown. We used the C57BL/KaLwRij mouse model of myeloma, lentiviral-mediated DNA barcoding of 5TGM1 myeloma cells, and next-generation sequencing to investigate the relative efficiency of plasma cell migration to, and growth within, the bone marrow. This approach revealed three major findings: firstly, establishment of metastasis within the bone marrow was extremely inefficient, with approximately 0.01% of circulating myeloma cells becoming resident long term in the bone marrow of each long bone; secondly, the individual cells of each metastasis exhibited marked differences in their proliferative fates, with the majority of final tumor burden within a bone being attributable to the progeny of between 1 and 8 cells; and, thirdly, the proliferative fate of individual clonal plasma cells differed at each bone marrow site in which the cells "landed." These findings suggest that individual myeloma plasma cells are subjected to vastly different selection pressures within the bone marrow microenvironment, highlighting the importance of niche-driven factors, which determine the disease course and outcome.
Subject(s)
Bone Marrow/pathology , Clonal Evolution/genetics , DNA Barcoding, Taxonomic , Multiple Myeloma/genetics , Plasma Cells/metabolism , Plasma Cells/pathology , Tumor Microenvironment , Animals , Cell Line, Tumor , Cell Proliferation , Gene Library , High-Throughput Nucleotide Sequencing , Humans , Mice , Neoplasm MetastasisABSTRACT
Disease progression and relapse in multiple myeloma is dependent on the ability of the multiple myeloma plasma cells (PC) to reenter the circulation and disseminate throughout the bone marrow. Increased bone marrow hypoxia is associated with increased recirculation of multiple myeloma PCs. Accordingly, we hypothesized that during chronic hypoxia, activation of HIF-2α may overcome the bone marrow retention signal provided by stromal-derived CXCL12, thereby enabling dissemination of multiple myeloma PCs. Here we demonstrate that HIF-2α upregulates multiple myeloma PC CXCL12 expression, decreasing migration toward CXCL12 and reducing adhesion to mesenchymal stromal cells in vitro We also found that HIF-2α strongly induced expression of the chemokine receptor CCR1 in multiple myeloma PCs. CCR1 activation potently induces multiple myeloma PC migration toward CCL3 while abrogating the multiple myeloma PC migratory response to CXCL12. In addition, increased CCR1 expression by multiple myeloma PCs conferred poor prognosis in newly diagnosed multiple myeloma patients and was associated with an increase in circulating multiple myeloma PCs in these patients. Taken together, our results suggest a role for hypoxia-mediated CCR1 upregulation in driving the egress of multiple myeloma PCs from the bone marrow. Targeting CCR1 may represent a novel strategy to prevent dissemination and overt relapse in multiple myeloma. Cancer Res; 77(20); 5452-63. ©2017 AACR.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Chemokine CXCL12/metabolism , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Plasma Cells/pathology , Receptors, CCR1/metabolism , Receptors, CXCR4/metabolism , Adult , Aged , Aged, 80 and over , Disease Progression , Female , Gene Expression Profiling , Humans , Male , Middle Aged , Plasma Cells/metabolism , Tumor Cells, CulturedABSTRACT
The proteasome inhibitor bortezomib has proven to be invaluable in the treatment of myeloma. By exploiting the inherent high immunoglobulin protein production of malignant plasma cells, bortezomib induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), resulting in myeloma cell death. In most cases, however, the disease remains incurable highlighting the need for new therapeutic targets. Sphingosine kinase 2 (SK2) has been proposed as one such therapeutic target for myeloma. Our observations that bortezomib and SK2 inhibitors independently elicited induction of ER stress and the UPR prompted us to examine potential synergy between these agents in myeloma. Targeting SK2 synergistically contributed to ER stress and UPR activation induced by bortezomib, as evidenced by activation of the IRE1 pathway and stress kinases JNK and p38MAPK, thereby resulting in potent synergistic myeloma apoptosis in vitro. The combination of bortezomib and SK2 inhibition also exhibited strong in vivo synergy and favourable effects on bone disease. Therefore, our studies suggest that perturbations of sphingolipid signalling can synergistically enhance the effects seen with proteasome inhibition, highlighting the potential for the combination of these two modes of increasing ER stress to be formally evaluated in clinical trials for the treatment of myeloma patients.
Subject(s)
Antineoplastic Agents/pharmacology , Bortezomib/pharmacology , Endoplasmic Reticulum Stress/drug effects , Multiple Myeloma/metabolism , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cell Death/drug effects , Cell Line, Tumor , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Synergism , Humans , Models, Biological , Molecular Targeted Therapy , Multiple Myeloma/drug therapy , Multiple Myeloma/mortality , Multiple Myeloma/pathology , Proteasome Inhibitors/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Multiple Myeloma (MM) is a haematological malignancy characterised by the clonal expansion of plasma cells (PCs) within the bone marrow. Despite advances in therapy, MM remains a largely incurable disease with a median survival of 6 years. In almost all cases, the development of MM is preceded by the benign PC condition Monoclonal Gammopathy of Undetermined Significance (MGUS). Recent studies show that the transformation of MGUS to MM is associated with complex genetic changes. Understanding how these changes contribute to evolution will present targets for clinical intervention. We discuss three models of MM evolution; the linear, the expansionist and the intraclonal heterogeneity models. Of particular interest is the intraclonal heterogeneity model. Here, distinct populations of MM PCs carry differing combinations of genetic mutations. Acquisition of additional mutations can contribute to subclonal lineages where "driver" mutations may influence selective pressure and dominance, and "passenger" mutations are neutral in their effects. Furthermore, studies show that clinical intervention introduces additional selective pressure on tumour cells and can influence subclone survival, leading to therapy resistance. This review discusses how Next Generation Sequencing approaches are revealing critical insights into the genetics of MM development, disease progression and treatment. MM disease progression will illuminate possible mechanisms underlying the tumour.
Subject(s)
Genomics/methods , Multiple Myeloma/genetics , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Disease Progression , Enzyme Inhibitors/therapeutic use , Epigenesis, Genetic/genetics , Forecasting , Genomics/trends , Humans , Immunologic Factors/therapeutic use , Multiple Myeloma/drug therapy , Mutation/geneticsABSTRACT
BACKGROUND: Multiple myeloma (MM) is an incurable haematological malignancy characterised by the clonal proliferation of malignant plasma cells within the bone marrow. We have previously identified pituitary tumour transforming gene 1 (Pttg1) as a gene that is significantly upregulated in the haematopoietic compartment of the myeloma-susceptible C57BL/KaLwRij mouse strain, when compared with the myeloma-resistant C57BL/6 mouse. Over-expression of PTTG1 has previously been associated with malignant progression and an enhanced proliferative capacity in solid tumours. METHODS: In this study, we investigated PTTG1 gene and protein expression in MM plasma cells from newly diagnosed MM patients. Gene expression profiling was used to identify gene signatures associated with high PTTG1 expression in MM patients. Additionally, we investigated the effect of short hairpin ribonucleic acid (shRNA)-mediated PTTG1 knockdown on the proliferation of the murine myeloma plasma cell line 5TGM1 in vitro and in vivo. RESULTS: PTTG1 was found to be over-expressed in 36-70 % of MM patients, relative to normal controls, with high PTTG1 expression being associated with poor patient outcomes (hazard ratio 2.49; 95 % CI 1.28 to 4.86; p = 0.0075; log-rank test). In addition, patients with high PTTG1 expression exhibited increased expression of cell proliferation-associated genes including CCNB1, CCNB2, CDK1, AURKA, BIRC5 and DEPDC1. Knockdown of Pttg1 in 5TGM1 cells decreased cellular proliferation, without affecting cell cycle distribution or viability, and decreased expression of Ccnb1, Birc5 and Depdc1 in vitro. Notably, Pttg1 knockdown significantly reduced MM tumour development in vivo, with an 83.2 % reduction in tumour burden at 4 weeks (p < 0.0001, two-way ANOVA). CONCLUSIONS: This study supports a role for increased PTTG1 expression in augmenting tumour development in a subset of MM patients.
Subject(s)
Cell Proliferation/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Multiple Myeloma/genetics , Securin/genetics , Animals , Blotting, Western , Cell Cycle/genetics , Cell Line, Tumor , Female , Male , Mice, Inbred C57BL , Microscopy, Confocal , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Oligonucleotide Array Sequence Analysis , Plasma Cells/metabolism , Plasma Cells/pathology , Prognosis , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Securin/metabolism , Survival Analysis , Tumor Burden/geneticsABSTRACT
BACKGROUND: Increased expression of the tetraspanin TSPAN7 has been observed in a number of cancers; however, it is unclear how TSPAN7 plays a role in cancer progression. METHODS: We investigated the expression of TSPAN7 in the haematological malignancy multiple myleoma (MM) and assessed the consequences of TSPAN7 expression in the adhesion, migration and growth of MM plasma cells (PC) in vitro and in bone marrow (BM) homing and tumour growth in vivo. Finally, we characterised the association of TSPAN7 with cell surface partner molecules in vitro. RESULTS: TSPAN7 was found to be highly expressed at the RNA and protein level in CD138(+) MM PC from approximately 50% of MM patients. TSPAN7 overexpression in the murine myeloma cell line 5TGM1 significantly reduced tumour burden in 5TGM1/KaLwRij mice 4 weeks after intravenous adminstration of 5TGM1 cells. While TSPAN7 overexpression did not affect cell proliferation in vitro, TSPAN7 increased 5TGM1 cell adhesion to BM stromal cells and transendothelial migration. In addition, TSPAN7 was found to associate with the molecular chaperone calnexin on the cell surface. CONCLUSION: These results suggest that elevated TSPAN7 may be associated with better outcomes for up to 50% of MM patients.
Subject(s)
Multiple Myeloma/metabolism , Nerve Tissue Proteins/genetics , Tetraspanins/genetics , Animals , Calnexin/genetics , Calnexin/metabolism , Cell Adhesion , Cell Line, Tumor , Cell Movement , Cell Proliferation , Coculture Techniques , Gene Expression , Humans , Mice, Inbred C57BL , Multiple Myeloma/mortality , Multiple Myeloma/pathology , Neoplasm Transplantation , Nerve Tissue Proteins/metabolism , Proportional Hazards Models , Tetraspanins/metabolism , Up-RegulationABSTRACT
Multiple myeloma (MM), a hematological malignancy characterized by the clonal growth of malignant plasma cells (PCs) in the bone marrow, is preceded by the benign asymptomatic condition, monoclonal gammopathy of undetermined significance (MGUS). Several genetic abnormalities have been identified as critical for the development of MM; however, a number of these abnormalities are also found in patients with MGUS, indicating that there are other, as yet unidentified, factors that contribute to the onset of MM disease. In this study, we identify a Samsn1 gene deletion in the 5TGM1/C57BL/KaLwRij murine model of myeloma. In addition, SAMSN1 expression is reduced in the malignant CD138+ PCs of patients with MM and this reduced expression correlates to total PC burden. We identify promoter methylation as a potential mechanism through which SAMSN1 expression is modulated in human myeloma cell lines. Notably, re-expression of Samsn1 in the 5TGM1 murine PC line resulted in complete inhibition of MM disease development in vivo and decreased proliferation in stromal cell-PC co-cultures in vitro. This is the first study to identify deletion of a key gene in the C57BL/KaLwRij mice that also displays reduced gene expression in patients with MM and is therefore likely to play an integral role in MM disease development.
Subject(s)
Adaptor Proteins, Vesicular Transport/genetics , Genes, Tumor Suppressor , Multiple Myeloma/genetics , Alternative Splicing , Animals , Cell Line, Tumor , Chromosome Mapping , DNA Methylation , DNA Mutational Analysis , Disease Models, Animal , Disease Progression , Female , Gene Deletion , Gene Expression , Gene Expression Profiling , Gene Order , Genetic Loci , Humans , Male , Mice , Monoclonal Gammopathy of Undetermined Significance/diagnosis , Monoclonal Gammopathy of Undetermined Significance/genetics , Monoclonal Gammopathy of Undetermined Significance/metabolism , Multiple Myeloma/diagnosis , Multiple Myeloma/metabolism , Promoter Regions, GeneticABSTRACT
Leukemia, with its origin in a specific genetic abnormality, will only arise if the cell properly folds and processes the oncogenic protein encoded by the mutant gene. In this issue of Blood, Tsukahara and Maru describe a set of proteins that control the processing of the nascent BCR-ABL oncoprotein, providing new avenues for potential therapeutic intervention in chronic myelogenous leukemia (CML).