ABSTRACT
ABSTRACT: Genomic instability contributes to cancer progression and is at least partly due to dysregulated homologous recombination (HR). Here, we show that an elevated level of ABL1 kinase overactivates the HR pathway and causes genomic instability in multiple myeloma (MM) cells. Inhibiting ABL1 with either short hairpin RNA or a pharmacological inhibitor (nilotinib) inhibits HR activity, reduces genomic instability, and slows MM cell growth. Moreover, inhibiting ABL1 reduces the HR activity and genomic instability caused by melphalan, a chemotherapeutic agent used in MM treatment, and increases melphalan's efficacy and cytotoxicity in vivo in a subcutaneous tumor model. In these tumors, nilotinib inhibits endogenous as well as melphalan-induced HR activity. These data demonstrate that inhibiting ABL1 using the clinically approved drug nilotinib reduces MM cell growth, reduces genomic instability in live cell fraction, increases the cytotoxicity of melphalan (and similar chemotherapeutic agents), and can potentially prevent or delay progression in patients with MM.
Subject(s)
Antineoplastic Agents , Multiple Myeloma , Humans , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Melphalan/pharmacology , Genomic Instability , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic useABSTRACT
PSMD4/Rpn10 is a subunit of the 19S proteasome unit that is involved with feeding target proteins into the catalytic machinery of the 26S proteasome. Because proteasome inhibition is a common therapeutic strategy in multiple myeloma (MM), we investigated Rpn10 and found that it is highly expressed in MM cells compared with normal plasma cells. Rpn10 levels inversely correlated with overall survival in patients with MM. Inducible knockout or knockdown of Rpn10 decreased MM cell viability both in vitro and in vivo by triggering the accumulation of polyubiquitinated proteins, cell cycle arrest, and apoptosis associated with the activation of caspases and unfolded protein response-related pathways. Proteomic analysis revealed that inhibiting Rpn10 increased autophagy, antigen presentation, and the activation of CD4+ T and natural killer cells. We developed an in vitro AlphaScreen binding assay for high-throughput screening and identified a novel Rpn10 inhibitor, SB699551 (SB). Treating MM cell lines, leukemic cell lines, and primary cells from patients with MM with SB decreased cell viability without affecting the viability of normal peripheral blood mononuclear cells. SB inhibited the proliferation of MM cells even in the presence of the tumor-promoting bone marrow milieu and overcame proteasome inhibitor (PI) resistance without blocking the 20S proteasome catalytic function or the 19S deubiquitinating activity. Rpn10 blockade by SB triggered MM cell death via similar pathways as the genetic strategy. In MM xenograft models, SB was well tolerated, inhibited tumor growth, and prolonged survival. Our data suggest that inhibiting Rpn10 will enhance cytotoxicity and overcome PI resistance in MM, providing the basis for further optimization studies of Rpn10 inhibitors for clinical application.
Subject(s)
Multiple Myeloma , Proteasome Endopeptidase Complex , Humans , Proteasome Endopeptidase Complex/metabolism , Ubiquitin/metabolism , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Proteomics , Leukocytes, Mononuclear/metabolism , Carrier Proteins/genetics , Proteins/metabolism , RNA-Binding ProteinsABSTRACT
Long noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.
Subject(s)
MicroRNAs , Multiple Myeloma , RNA, Long Noncoding , Humans , Animals , Mice , RNA, Long Noncoding/genetics , Multiple Myeloma/genetics , Chromatin , MicroRNAs/metabolism , Cell Proliferation , Gene Expression Regulation, NeoplasticABSTRACT
BACKGROUND & AIMS: The purpose of this study was to identify drivers of genomic evolution in esophageal adenocarcinoma (EAC) and other solid tumors. METHODS: An integrated genomics strategy was used to identify deoxyribonucleases correlating with genomic instability (as assessed from total copy number events in each patient) in 6 cancers. Apurinic/apyrimidinic nuclease 1 (APE1), identified as the top gene in functional screens, was either suppressed in cancer cell lines or overexpressed in normal esophageal cells and the impact on genome stability and growth was monitored in vitro and in vivo. The impact on DNA and chromosomal instability was monitored using multiple approaches, including investigation of micronuclei, acquisition of single nucleotide polymorphisms, whole genome sequencing, and/or multicolor fluorescence in situ hybridization. RESULTS: Expression of 4 deoxyribonucleases correlated with genomic instability in 6 human cancers. Functional screens of these genes identified APE1 as the top candidate for further evaluation. APE1 suppression in EAC, breast, lung, and prostate cancer cell lines caused cell cycle arrest; impaired growth and increased cytotoxicity of cisplatin in all cell lines and types and in a mouse model of EAC; and inhibition of homologous recombination and spontaneous and chemotherapy-induced genomic instability. APE1 overexpression in normal cells caused a massive chromosomal instability, leading to their oncogenic transformation. Evaluation of these cells by means of whole genome sequencing demonstrated the acquisition of changes throughout the genome and identified homologous recombination as the top mutational process. CONCLUSIONS: Elevated APE1 dysregulates homologous recombination and cell cycle, contributing to genomic instability, tumorigenesis, and chemoresistance, and its inhibitors have the potential to target these processes in EAC and possibly other cancers.
Subject(s)
Adenocarcinoma , Drug Resistance, Neoplasm , Male , Animals , Mice , Humans , Drug Resistance, Neoplasm/genetics , In Situ Hybridization, Fluorescence , Cell Line, Tumor , Adenocarcinoma/drug therapy , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Carcinogenesis/genetics , Cell Transformation, Neoplastic/genetics , Homologous Recombination , Cell Cycle , Genomic Instability , Genomics , Chromosomal Instability/genetics , Deoxyribonucleases/genetics , Evolution, MolecularABSTRACT
The accessibility of cell surface proteins makes them tractable for targeting by cancer immunotherapy, but identifying suitable targets remains challenging. Here we describe plasma membrane profiling of primary human myeloma cells to identify an unprecedented number of cell surface proteins of a primary cancer. We used a novel approach to prioritize immunotherapy targets and identified a cell surface protein not previously implicated in myeloma, semaphorin-4A (SEMA4A). Using knock-down by short-hairpin RNA and CRISPR/nuclease-dead Cas9 (dCas9), we show that expression of SEMA4A is essential for normal myeloma cell growth in vitro, indicating that myeloma cells cannot downregulate the protein to avoid detection. We further show that SEMA4A would not be identified as a myeloma therapeutic target by standard CRISPR/Cas9 knockout screens because of exon skipping. Finally, we potently and selectively targeted SEMA4A with a novel antibody-drug conjugate in vitro and in vivo.
Subject(s)
Multiple Myeloma , Semaphorins , Cell Membrane/metabolism , Humans , Immunologic Factors , Immunotherapy , Membrane Proteins , Multiple Myeloma/genetics , Multiple Myeloma/therapy , Proteomics , Semaphorins/genetics , Semaphorins/metabolismABSTRACT
Clonal evolution drives tumor progression, dissemination, and relapse in multiple myeloma (MM), with most patients dying of relapsed disease. This multistage process requires tumor cells to enter the circulation, extravasate, and colonize distant bone marrow (BM) sites. Here, we developed a fluorescent or DNA-barcode clone-tracking system on MM PrEDiCT (progression through evolution and dissemination of clonal tumor cells) xenograft mouse model to study clonal behavior within the BM microenvironment. We showed that only the few clones that successfully adapt to the BM microenvironment can enter the circulation and colonize distant BM sites. RNA sequencing of primary and distant-site MM tumor cells revealed a progression signature sequentially activated along human MM progression and significantly associated with overall survival when evaluated against patient data sets. A total of 28 genes were then computationally predicted to be master regulators (MRs) of MM progression. HMGA1 and PA2G4 were validated in vivo using CRISPR-Cas9 in the PrEDiCT model and were shown to be significantly depleted in distant BM sites, indicating their role in MM progression and dissemination. Loss of HMGA1 and PA2G4 also compromised the proliferation, migration, and adhesion abilities of MM cells in vitro. Overall, our model successfully recapitulates key characteristics of human MM disease progression and identified potential new therapeutic targets for MM.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Biomarkers, Tumor/metabolism , Disease Models, Animal , Gene Expression Regulation, Neoplastic , HMGA1a Protein/metabolism , Multiple Myeloma/pathology , Neoplasm Recurrence, Local/pathology , RNA-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Animals , Apoptosis , Biomarkers, Tumor/genetics , Bone Marrow/metabolism , Bone Marrow/pathology , CRISPR-Cas Systems , Cell Adhesion , Cell Movement , Cell Proliferation , Clonal Evolution , Disease Progression , Female , HMGA1a Protein/antagonists & inhibitors , HMGA1a Protein/genetics , Humans , Mice , Mice, SCID , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/metabolism , Prognosis , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/genetics , Survival Rate , Tumor Cells, CulturedABSTRACT
Anti-CD38 monoclonal antibody (MoAb) treatments including daratumumab (DARA) are effective therapies for both newly diagnosed and relapsed multiple myeloma (MM). In this study, we examined the soluble factors that modulate CD38 expression and are associated with sensitivity to DARA-mediated antibody-dependent cellular cytotoxicity (ADCC) in the bone marrow (BM) microenvironment. Importantly, primary BM stromal cell (BMSC) culture supernatant (BMSC-sup) and interleukin-6 (IL-6) downregulated CD38 expression and reduced DARA-mediated ADCC. Both cytokine profiling of the BMSC-sup and genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) knockout screening in MM cell lines identified and validated the JAK-STAT3 signaling pathway mediating CD38 downregulation, whereas the JAK-STAT1 pathway mediated CD38 upregulation. STAT3 knockdown abrogated BMSC-sup- and IL-6-induced CD38 downregulation on MM cell lines. We also confirmed that STAT3 and CD38 is negatively correlated in primary MM cells. To assess potential clinical relevance, pharmacological inhibition of the JAK-STAT pathway on BMSC-sup-induced CD38 downregulation was further examined. JAK inhibitor ruxolitinib inhibited STAT3 phosphorylation in MM cell lines, upregulated CD38 expression in MM cell lines and primary patient MM cells, and augmented DARA-mediated ADCC against MM cell lines. Taken together, our results suggest that CD38 expression on MM cells in the BM microenvironment is regulated by both STAT1 (positively) and STAT3 (negatively), and that inhibition of the JAK-STAT3 pathway represents a novel therapeutic option to enhance CD38 expression and anti-CD38 MoAb-mediated MM cytotoxicity.
Subject(s)
ADP-ribosyl Cyclase 1/metabolism , Janus Kinases/metabolism , Membrane Glycoproteins/metabolism , Multiple Myeloma/metabolism , STAT Transcription Factors/metabolism , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Bone Marrow/metabolism , Bone Marrow/pathology , Humans , Janus Kinases/drug effects , Multiple Myeloma/pathology , Nitriles , Pyrazoles/pharmacology , Pyrimidines , STAT Transcription Factors/drug effects , Signal Transduction/drug effects , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunologyABSTRACT
High protein load is a feature of multiple myeloma (MM), making the disease exquisitely sensitive to proteasome inhibitor (PIs). Despite the success of PIs in improving patient outcome, the majority of patients develop resistance leading to progressive disease; thus, the need to investigate the mechanisms driving the drug sensitivity vs resistance. With the well-recognized chaperone function of 14-3-3 proteins, we evaluated their role in affecting proteasome activity and sensitivity to PIs by correlating expression of individual 14-3-3 gene and their sensitivity to PIs (bortezomib and carfilzomib) across a large panel of MM cell lines. We observed a significant positive correlation between 14-3-3ε expression and PI response in addition to a role for 14-3-3ε in promoting translation initiation and protein synthesis in MM cells through binding and inhibition of the TSC1/TSC2 complex, as well as directly interacting with and promoting phosphorylation of mTORC1. 14-3-3ε depletion caused up to a 50% reduction in protein synthesis, including a decrease in the intracellular abundance and secretion of the light chains in MM cells, whereas 14-3-3ε overexpression or addback in knockout cells resulted in a marked upregulation of protein synthesis and protein load. Importantly, the correlation among 14-3-3ε expression, PI sensitivity, and protein load was observed in primary MM cells from 2 independent data sets, and its lower expression was associated with poor outcome in patients with MM receiving a bortezomib-based therapy. Altogether, these observations suggest that 14-3-3ε is a predictor of clinical outcome and may serve as a potential target to modulate PI sensitivity in MM.
Subject(s)
14-3-3 Proteins/metabolism , Bortezomib/pharmacology , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Multiple Myeloma , Neoplasm Proteins/metabolism , Oligopeptides/pharmacology , Proteasome Inhibitors/pharmacology , Female , Humans , Male , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Tumor Cells, CulturedABSTRACT
BACKGROUND: In multiple myeloma (MM), impact of specific chromosomal translocations involving IgH (14q21 locus, including t(4;14), t(11;14), and t(14;16)) has been explored extensively. However, over 15% MM patients harboring IgH translocation with undefined partners have long been ignored. METHODS: A prospective non-randomized cohort study with a total of 715 newly-diagnosed MM cases was conducted, 13.6% of whom were t(14;undefined) positive. The whole cohort was divided into four groups: no IgH split (47.7%); t(14;undefined) (13.6%); t(11;14) (17.6%); and t(4;14) or t(14;16) group (21.1%). RESULTS: Median OS for the four groups was 84.2, not reached (NR), 58.7, and 44.2 months, respectively, with P values for t(14;undefined) vs no IgH split, t(11;14), and t(4;14)/t(14;16) groups of 0.197, 0.022, and 0.001, respectively. In bortezomib-based group, the survival advantage gained by t(14;undefined) group was much more significant compared to t(11;14) and t(4;14)/t(14;16) groups. Importantly, t(14;undefined) turned out to be an independent predictive factor for longer OS of MM patients in multivariate analysis, especially in the context of bortezomib treatment. Similar results were also observed in the PUMCH external validation cohort. CONCLUSION: Collectively, our data confirmed and externally validated the favorable prognosis of the t(14;undefined) groups, especially in the era of novel agents.
Subject(s)
Immunoglobulin Heavy Chains/genetics , Multiple Myeloma/genetics , Multiple Myeloma/mortality , Translocation, Genetic , Aged , Aged, 80 and over , Biomarkers, Tumor , Chromosomes, Human, Pair 11 , Chromosomes, Human, Pair 14 , Chromosomes, Human, Pair 16 , Chromosomes, Human, Pair 4 , Female , Gene Frequency , Humans , In Situ Hybridization, Fluorescence , Kaplan-Meier Estimate , Male , Middle Aged , Multiple Myeloma/diagnosis , Multiple Myeloma/therapy , Prognosis , Proportional Hazards ModelsABSTRACT
Multiple myeloma (MM) has proven clinically susceptible to modulation of pathways of protein homeostasis. Blockade of proteasomal degradation of polyubiquitinated misfolded proteins by the proteasome inhibitor bortezomib (BTZ) achieves responses and prolongs survival in MM, but long-term treatment with BTZ leads to drug-resistant relapse in most patients. In a proof-of-concept study, we previously demonstrated that blocking aggresomal breakdown of polyubiquitinated misfolded proteins with the histone deacetylase 6 (HDAC6) inhibitor tubacin enhances BTZ-induced cytotoxicity in MM cells in vitro. However, these foundational studies were limited by the pharmacologic liabilities of tubacin as a chemical probe with only in vitro utility. Emerging from a focused library synthesis, a potent, selective, and bioavailable HDAC6 inhibitor, WT161, was created to study the mechanism of action of HDAC6 inhibition in MM alone and in combination with BTZ. WT161 in combination with BTZ triggers significant accumulation of polyubiquitinated proteins and cell stress, followed by caspase activation and apoptosis. More importantly, this combination treatment was effective in BTZ-resistant cells and in the presence of bone marrow stromal cells, which have been shown to mediate MM cell drug resistance. The activity of WT161 was confirmed in our human MM cell xenograft mouse model and established the framework for clinical trials of the combination treatment to improve patient outcomes in MM.
Subject(s)
Antineoplastic Agents/therapeutic use , Bortezomib/therapeutic use , Histone Deacetylase Inhibitors/therapeutic use , Hydroxamic Acids/therapeutic use , Multiple Myeloma/drug therapy , Proteasome Inhibitors/therapeutic use , Terphenyl Compounds/therapeutic use , Anilides/pharmacology , Anilides/therapeutic use , Animals , Antineoplastic Agents/pharmacology , Bortezomib/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Drug Resistance, Neoplasm , Histone Deacetylase 6/antagonists & inhibitors , Histone Deacetylase 6/metabolism , Histone Deacetylase Inhibitors/pharmacology , Humans , Hydroxamic Acids/pharmacology , Male , Mice , Multiple Myeloma/metabolism , Proteasome Inhibitors/pharmacology , Terphenyl Compounds/pharmacology , Tubulin/metabolism , Tumor Cells, CulturedABSTRACT
Patient-derived multiple myeloma (MM) cells are difficult to establish in culture or propagate in vivo in murine model. Here, we describe a zebrafish xenograft model that permits rapid, reliable growth of human MM cells injected into the perivitelline space of albino zebrafish (Casper) embryos 48 hours postfertilization. MM1S and MM1R MM cell lines and primary CD138(+) MM cells were stained with CM-Dil red fluorescent dye and suspended in Matrigel prior to their injection. The cells grew at the site of injection and disseminated throughout the developing embryos and larvae. Tumor size was quantified by fluorescent microscopy, and cell fate was followed for 4 days. All of the cell line xenografts showed responses similar to those previously observed with in vitro assays. CD138(+) plasma cell xenografts derived from MM patients also grew and were inhibited by the same drugs patients had responded to clinically. Using this technique, we can assess drug sensitivity or resistance with a small number of MM cells in a short period. This raises the possibility that one might be able to assess drug sensitivity in real time with readily obtainable clinical samples.
Subject(s)
Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Zebrafish/metabolism , Animals , Cell Line, Tumor , Heterografts , Humans , Neoplasm TransplantationABSTRACT
Multiple myeloma (MM) cell lines and primary tumor cells are addicted to the MYC oncoprotein for survival. Little is known, however, about how MYC expression is upregulated in MM cells. The mucin 1 C-terminal subunit (MUC1-C) is an oncogenic transmembrane protein that is aberrantly expressed in MM cell lines and primary tumor samples. The present studies demonstrate that targeting MUC1-C with silencing by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 editing or with the GO-203 inhibitor is associated with downregulation of MYC messenger RNA and protein. The results show that MUC1-C occupies the MYC promoter and thereby activates the MYC gene by a ß-catenin/transcription factor 4 (TCF4)-mediated mechanism. In this way, MUC1-C (1) increases ß-catenin occupancy on the MYC promoter, (2) forms a complex with ß-catenin and TCF4, and, in turn, (3) drives MYC transcription. Analysis of MM cells using quantitative real-time reverse transcription polymerase chain reaction arrays further demonstrated that silencing MUC1-C is associated with downregulation of MYC target genes, including CCND2, hTERT, and GCLC Analysis of microarray data sets further demonstrated that MUC1 levels positively correlate with MYC expression in MM progression and in primary cells from over 800 MM patients. These findings collectively provide convincing evidence that MUC1-C drives MYC expression in MM.
Subject(s)
Down-Regulation , Gene Expression Regulation, Neoplastic , Mucin-1/biosynthesis , Multiple Myeloma/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Response Elements , Transcription, Genetic , CRISPR-Cas Systems , Cell Line, Tumor , Cyclin D2/biosynthesis , Cyclin D2/genetics , Glutamate-Cysteine Ligase/biosynthesis , Glutamate-Cysteine Ligase/genetics , Humans , Mucin-1/genetics , Multiple Myeloma/genetics , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Proteins c-myc/genetics , Telomerase/biosynthesis , Telomerase/genetics , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
The number and activity of osteoclasts (OCs) are strongly enhanced by myeloma cells, leading to significant bone lesions in patients with multiple myeloma (MM). Mechanisms remain elusive as to whether myeloma-supporting OCs also induce suppressive immune bone marrow (BM) microenvironment. Here, we first show that OCs significantly protect MM cells against T-cell-mediated cytotoxicity via direct inhibition of proliferating CD4(+) and CD8(+) T cells. The immune checkpoint molecules programmed death ligand 1 (PD-L1), Galectin-9, herpesvirus entry mediator (HVEM), and CD200, as well as T-cell metabolism regulators indoleamine 2, 3-dioxygenase (IDO), and CD38 are significantly upregulated during osteoclastogenesis. Importantly, the levels of these molecules, except CD38, are higher in OCs than in MM cells. Anti-PD-L1 monoclonal antibody (mAb) and IDO inhibitor partly overcome OC-inhibited T-cell responses against MM cells, confirming their roles in OC-suppressed MM cell lysis by cytotoxic T cells. In addition, Galectin-9 and a proliferation-induced ligand (APRIL), secreted by OCs, are significantly upregulated during osteoclastogenesis. Galectin-9 specifically induces apoptosis of T cells while sparing monocytes and MM cells. APRIL induces PD-L1 expression in MM cells, providing additional immune inhibition by OCs. Moreover, CD38 is significantly upregulated during osteoclastogenesis. When targeted by an anti-CD38 mAb, suppressive T-cell function by OCs is alleviated, associated with downregulation of HVEM and IDO. Taken together, these results define the expression of multiple immune proteins and cytokines in OCs essential for suppressive MM BM milieu. These results further support the combination of targeting these molecules to improve anti-MM immunity.
Subject(s)
B7-H1 Antigen/immunology , Monocytes/immunology , Multiple Myeloma/immunology , Osteoclasts/immunology , T-Lymphocytes, Cytotoxic/immunology , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Proliferation , Cells, Cultured , Cytokines/metabolism , Humans , Immunosuppression Therapy , Monocytes/cytology , Monocytes/metabolism , Multiple Myeloma/metabolism , Multiple Myeloma/therapy , Osteoclasts/cytology , Osteoclasts/metabolismABSTRACT
Here we show that overexpression or activation of B-cell maturation antigen (BCMA) by its ligand, a proliferation-inducing ligand (APRIL), promotes human multiple myeloma (MM) progression in vivo. BCMA downregulation strongly decreases viability and MM colony formation; conversely, BCMA overexpression augments MM cell growth and survival via induction of protein kinase B (AKT), MAPK, and nuclear factor (NF)-κB signaling cascades. Importantly, BCMA promotes in vivo growth of xenografted MM cells harboring p53 mutation in mice. BCMA-overexpressing tumors exhibit significantly increased CD31/microvessel density and vascular endothelial growth factor compared with paired control tumors. These tumors also express increased transcripts crucial for osteoclast activation, adhesion, and angiogenesis/metastasis, as well as genes mediating immune inhibition including programmed death ligand 1, transforming growth factor ß, and interleukin 10. These target genes are consistently induced by paracrine APRIL binding to BCMA on MM cells, which is blocked by an antagonistic anti-APRIL monoclonal antibody hAPRIL01A (01A). 01A is cytotoxic against MM cells even in the presence of protective bone marrow (BM) myeloid cells including osteoclasts, macrophages, and plasmacytoid dendritic cells. 01A further decreases APRIL-induced adhesion and migration of MM cells via blockade of canonical and noncanonical NF-κB pathways. Moreover, 01A prevents in vivo MM cell growth within implanted human bone chips in SCID mice. Finally, the effect of 01A on MM cell viability is enhanced by lenalidomide and bortezomib. Taken together, these data delineate new molecular mechanisms of in vivo MM growth and immunosuppression critically dependent on BCMA and APRIL in the BM microenvironment, further supporting targeting this prominent pathway in MM.
Subject(s)
B-Cell Maturation Antigen/physiology , Bone Marrow/physiology , Cell Proliferation/genetics , Cellular Microenvironment , Immune Tolerance/genetics , Multiple Myeloma/pathology , Tumor Necrosis Factor Ligand Superfamily Member 13/physiology , Animals , B-Cell Maturation Antigen/genetics , Bone Marrow/pathology , Cell Line, Tumor , Cellular Microenvironment/genetics , Gene Expression Regulation, Neoplastic , Heterografts , Humans , Mice , Mice, SCID , Multiple Myeloma/genetics , Osteoclasts/pathology , Osteoclasts/physiology , Tumor Necrosis Factor Ligand Superfamily Member 13/geneticsABSTRACT
Multiple myeloma (MM) is characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DNA damage response. Here, we show that the NAD(+)-dependent deacetylase SIRT6 is highly expressed in MM cells, as an adaptive response to genomic stability, and that high SIRT6 levels are associated with adverse prognosis. Mechanistically, SIRT6 interacts with the transcription factor ELK1 and with the ERK signaling-related gene. By binding to their promoters and deacetylating H3K9 at these sites, SIRT6 downregulates the expression of mitogen-activated protein kinase (MAPK) pathway genes, MAPK signaling, and proliferation. In addition, inactivation of ERK2/p90RSK signaling triggered by high SIRT6 levels increases DNA repair via Chk1 and confers resistance to DNA damage. Using genetic and biochemical studies in vitro and in human MM xenograft models, we show that SIRT6 depletion both enhances proliferation and confers sensitization to DNA-damaging agents. Our findings therefore provide insights into the functional interplay between SIRT6 and DNA repair mechanisms, with implications for both tumorigenesis and the treatment of MM.
Subject(s)
DNA Damage , Multiple Myeloma/enzymology , Multiple Myeloma/pathology , Sirtuins/metabolism , Acetylation , Cell Line, Tumor , Cell Proliferation , DNA Repair , Doxorubicin/pharmacology , Histones/metabolism , Humans , Lysine/metabolism , MAP Kinase Signaling System , Models, Biological , Mutagens/toxicity , Prognosis , ets-Domain Protein Elk-1/metabolismABSTRACT
Lenalidomide (LEN) acts directly on multiple myeloma (MM) cells by inducing cereblon-mediated degradation of interferon regulatory factor 4, Ikaros (IKZF)1 and IKZF3, transcription factors that are essential for MM cell survival. The mucin 1 (MUC1) C-terminal transmembrane subunit (MUC1-C) oncoprotein is aberrantly expressed by MM cells and protects against reactive oxygen species (ROS)-mediated MM cell death. The present studies demonstrate that targeting MUC1-C with GO-203, a cell-penetrating peptide inhibitor of MUC1-C homodimerization, is more than additive with LEN in downregulating the WNT/ß-catenin pathway, suppressing MYC, and inducing late apoptosis/necrosis. We show that the GO-203/LEN combination acts by synergistically increasing ROS and, in turn, suppressing ß-catenin. LEN resistance has been linked to activation of the WNT/ß-cateninâCD44 pathway. In this regard, our results further demonstrate that targeting MUC1-C is effective against LEN-resistant MM cells. Moreover, GO-203 resensitized LEN-resistant MM cells to LEN treatment in association with suppression of ß-catenin and CD44. Targeting MUC1-C also resulted in downregulation of CD44 on the surface of primary MM cells. These findings, and the demonstration that expression of MUC1 and CD44 significantly correlate in microarrays from primary MM cells, provide support for combining GO-203 with LEN in the treatment of MM and in LEN-resistance.
Subject(s)
Mucin-1/drug effects , Multiple Myeloma/pathology , Thalidomide/analogs & derivatives , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Computational Biology/methods , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic/drug effects , Humans , Hyaluronan Receptors/metabolism , Lenalidomide , Molecular Targeted Therapy/methods , Mucin-1/metabolism , Multiple Myeloma/metabolism , Oxidation-Reduction/drug effects , Peptides/administration & dosage , Peptides/pharmacology , Reactive Oxygen Species/metabolism , Thalidomide/administration & dosage , Thalidomide/pharmacology , Tumor Cells, Cultured/drug effectsABSTRACT
CXCR4(WHIM) somatic mutations are distinctive to Waldenström Macroglobulinaemia (WM), and impact disease presentation and treatment outcome. The clonal architecture of CXCR4(WHIM) mutations remains to be delineated. We developed highly sensitive allele-specific polymerase chain reaction (AS-PCR) assays for detecting the most common CXCR4(WHIM) mutations (CXCR4(S338X C>A and C>G) ) in WM. The AS-PCR assays detected CXCR4(S338X) mutations in WM and IgM monoclonal gammopathy of unknown significance (MGUS) patients not revealed by Sanger sequencing. By combined AS-PCR and Sanger sequencing, CXCR4(WHIM) mutations were identified in 44/102 (43%), 21/62 (34%), 2/12 (17%) and 1/20 (5%) untreated WM, previously treated WM, IgM MGUS and marginal zone lymphoma patients, respectively, but no chronic lymphocytic leukaemia, multiple myeloma, non-IgM MGUS patients or healthy donors. Cancer cell fraction analysis in WM and IgM MGUS patients showed CXCR4(S338X) mutations were primarily subclonal, with highly variable clonal distribution (median 35·1%, range 1·2-97·5%). Combined AS-PCR and Sanger sequencing revealed multiple CXCR4(WHIM) mutations in many individual WM patients, including homozygous and compound heterozygous mutations validated by deep RNA sequencing. The findings show that CXCR4(WHIM) mutations are more common in WM than previously revealed, and are primarily subclonal, supporting their acquisition after MYD88(L265P) in WM oncogenesis. The presence of multiple CXCR4(WHIM) mutations within individual WM patients may be indicative of targeted CXCR4 genomic instability.
Subject(s)
Mutation , Receptors, CXCR4/genetics , Waldenstrom Macroglobulinemia/genetics , Adult , Aged , Aged, 80 and over , Amino Acid Substitution , Female , Genomic Instability , Humans , Immunoglobulin M/blood , Lymphoma, B-Cell, Marginal Zone/genetics , Male , Middle Aged , Monoclonal Gammopathy of Undetermined Significance/genetics , Myeloid Differentiation Factor 88/genetics , Polymerase Chain Reaction/methods , Sequence Analysis, DNA/methodsABSTRACT
Proteasome inhibitors have demonstrated that targeting protein degradation is effective therapy in multiple myeloma (MM). Here we show that deubiquitylating enzymes (DUBs) USP14 and UCHL5 are more highly expressed in MM cells than in normal plasma cells. USP14 and UCHL5 short interfering RNA knockdown decreases MM cell viability. A novel 19S regulatory particle inhibitor b-AP15 selectively blocks deubiquitylating activity of USP14 and UCHL5 without inhibiting proteasome activity. b-AP15 decreases viability in MM cell lines and patient MM cells, inhibits proliferation of MM cells even in the presence of bone marrow stroma cells, and overcomes bortezomib resistance. Anti-MM activity of b-AP15 is associated with growth arrest via downregulation of CDC25C, CDC2, and cyclin B1 as well as induction of caspase-dependent apoptosis and activation of unfolded protein response. In vivo studies using distinct human MM xenograft models show that b-AP15 is well tolerated, inhibits tumor growth, and prolongs survival. Combining b-AP15 with suberoylanilide hydroxamic acid, lenalidomide, or dexamethasone induces synergistic anti-MM activity. Our preclinical data showing efficacy of b-AP15 in MM disease models validates targeting DUBs in the ubiquitin proteasomal cascade to overcome proteasome inhibitor resistance and provides the framework for clinical evaluation of USP14/UCHL5 inhibitors to improve patient outcome in MM.
Subject(s)
Antineoplastic Agents/pharmacology , Boronic Acids/pharmacology , Multiple Myeloma/drug therapy , Piperidones/pharmacology , Protease Inhibitors/pharmacology , Pyrazines/pharmacology , Ubiquitin Thiolesterase/antagonists & inhibitors , Animals , Apoptosis/drug effects , Bortezomib , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Endoplasmic Reticulum Stress/drug effects , Female , Gene Expression Regulation, Neoplastic , Humans , Mice , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Ubiquitin Thiolesterase/genetics , Up-RegulationABSTRACT
Clonal proliferation of plasma cells within the bone marrow (BM) affects local cells, such as mesenchymal stromal cells (MSCs), leading to osteolysis and fatality in multiple myeloma (MM). Consequently, there is an urgent need to find better mechanisms of inhibiting myeloma growth and osteolytic lesion development. To meet this need and accelerate clinical translation, better models of myeloma within the BM are required. Herein we have developed a clinically relevant, three-dimensional (3D) myeloma BM coculture model that mimics bone cell/cancer cell interactions within the bone microenvironment. The coculture model and clinical samples were used to investigate myeloma growth, osteogenesis inhibition, and myeloma-induced abnormalities in MM-MSCs. This platform demonstrated myeloma support of capillary-like assembly of endothelial cells and cell adhesion-mediated drug resistance (CAM-DR). Also, distinct normal donor (ND)- and MM-MSC miRNA (miR) signatures were identified and used to uncover osteogenic miRs of interest for osteoblast differentiation. More broadly, our 3D platform provides a simple, clinically relevant tool to model cancer growth within the bone-useful for investigating skeletal cancer biology, screening compounds, and exploring osteogenesis. Our identification and efficacy validation of novel bone anabolic miRs in MM opens more opportunities for novel approaches to cancer therapy via stromal miR modulation.