ABSTRACT
OBJECTIVES: Two promising epigenetic therapeutic targets have emerged for the treatment of hematologic malignancies, BET and CBP/EP300 proteins. Several studies have shown that targeting these individual classes of proteins has anti-tumor activity in multiple myeloma (MM), as well as other cancers. Here, we present the first data exploring the anti-tumor activity of two novel dual inhibitors, NEO2734 and NEO1132, of both BET and CBP/EP300 proteins in MM. METHODS: Sixteen MM cell lines (MMCLs) were treated with the dual inhibitors NEO2734 and NEO1132, the single BET inhibitors JQ1, OTX015, IBET-762, and IBET-151, and a single CBP/EP300 inhibitor CPI-637. RESULTS: The dual inhibitor NEO2734 showed strong anti-tumor activity and was consistently highly active against all MMCLs, being as potent as JQ1 and more so than other single inhibitors. NEO2734 and NEO11132 induced a significant G1 cell cycle arrest and decreased c-MYC and IRF4 protein levels in MMCLs compared to the other single inhibitors. Sensitivity to the dual inhibitors was not dependent on a specific MM molecular subgroup but correlated with c-MYC protein expression levels. CONCLUSIONS: The dual inhibition of BET and CBP/EP300 has potential therapeutic benefits for patients with MM.
Subject(s)
Antineoplastic Agents/pharmacology , Benzimidazoles/pharmacology , E1A-Associated p300 Protein/antagonists & inhibitors , Proteins/antagonists & inhibitors , Pyridones/pharmacology , Antineoplastic Agents/therapeutic use , Benzimidazoles/therapeutic use , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Humans , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/metabolism , Multiple Myeloma , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Pyridones/therapeutic useABSTRACT
MYC is a widely acting transcription factor and its deregulation is a crucial event in many human cancers. MYC is important biologically and clinically in multiple myeloma, but the mechanisms underlying its dysregulation are poorly understood. We show that MYC rearrangements are present in 36.0% of newly diagnosed myeloma patients, as detected in the largest set of next generation sequencing data to date (n=1,267). Rearrangements were complex and associated with increased expression of MYC and PVT1, but not other genes at 8q24. The highest effect on gene expression was detected in cases where the MYC locus is juxtaposed next to super-enhancers associated with genes such as IGH, IGK, IGL, TXNDC5/BMP6, FAM46C and FOXO3 We identified three hotspots of recombination at 8q24, one of which is enriched for IGH-MYC translocations. Breakpoint analysis indicates primary myeloma rearrangements involving the IGH locus occur through non-homologous end joining, whereas secondary MYC rearrangements occur through microhomology-mediated end joining. This mechanism is different to lymphomas, where non-homologous end joining generates MYC rearrangements. Rearrangements resulted in overexpression of key genes and chromatin immunoprecipitation-sequencing identified that HK2, a member of the glucose metabolism pathway, is directly over-expressed through binding of MYC at its promoter.
Subject(s)
Genes, myc , Multiple Myeloma , RNA, Long Noncoding/genetics , Genes, Immunoglobulin Heavy Chain , Humans , In Situ Hybridization, Fluorescence , Multiple Myeloma/genetics , Protein Disulfide-Isomerases , Translocation, GeneticABSTRACT
The small GTPase RhoA plays a crucial role in the regulation of neuronal signalling to generate behaviour. In the developing nervous system RhoA is known to regulate the actin cytoskeleton, however the effectors of RhoA-signalling in adult neurons remain largely unidentified. We have previously shown that activation of the RhoA ortholog (RHO-1) in C. elegans cholinergic motor neurons triggers hyperactivity of these neurons and loopy locomotion with exaggerated body bends. This is achieved in part through increased diacylglycerol (DAG) levels and the recruitment of the synaptic vesicle protein UNC-13 to synaptic release sites, however other pathways remain to be identified. Dopamine, which is negatively regulated by the dopamine re-uptake transporter (DAT), has a central role in modulating locomotion in both humans and C. elegans. In this study we identify a new pathway in which RHO-1 regulates locomotory behaviour by repressing dopamine signalling, via DAT-1, linking these two pathways together. We observed an upregulation of dat-1 expression when RHO-1 is activated and show that loss of DAT-1 inhibits the loopy locomotion phenotype caused by RHO-1 activation. Reducing dopamine signalling in dat-1 mutants through mutations in genes involved in dopamine synthesis or in the dopamine receptor DOP-1 restores the ability of RHO-1 to trigger loopy locomotion in dat-1 mutants. Taken together, we show that negative regulation of dopamine signalling via DAT-1 is necessary for the neuronal RHO-1 pathway to regulate locomotion.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Cholinergic Neurons/metabolism , Dopamine/metabolism , Locomotion , Motor Neurons/metabolism , Signal Transduction , rho GTP-Binding Proteins/metabolism , Acetylcholine/metabolism , Animals , Caenorhabditis elegans Proteins/genetics , Mutation/genetics , Neuropeptides/metabolism , Phenotype , Transcription, Genetic , Up-Regulation/geneticsABSTRACT
Gliomas are a highly heterogeneous group of brain tumours that are refractory to treatment, highly invasive and pro-angiogenic. Glioblastoma patients have an average survival time of less than 15 months. Understanding the molecular basis of different grades of glioma, from well differentiated, low-grade tumours to high-grade tumours, is a key step in defining new therapeutic targets. Here we use a data-driven approach to learn the structure of gene regulatory networks from observational data and use the resulting models to formulate hypothesis on the molecular determinants of glioma stage. Remarkably, integration of available knowledge with functional genomics datasets representing clinical and pre-clinical studies reveals important properties within the regulatory circuits controlling low and high-grade glioma. Our analyses first show that low and high-grade gliomas are characterised by a switch in activity of two subsets of Rho GTPases. The first one is involved in maintaining normal glial cell function, while the second is linked to the establishment of multiple hallmarks of cancer. Next, the development and application of a novel data integration methodology reveals novel functions of RND3 in controlling glioma cell migration, invasion, proliferation, angiogenesis and clinical outcome.
Subject(s)
Brain Neoplasms/genetics , Gene Regulatory Networks/genetics , Glioma/genetics , Neoplasm Invasiveness/genetics , rho GTP-Binding Proteins/genetics , Apoptosis/genetics , Brain Neoplasms/pathology , Cell Cycle/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , DNA Copy Number Variations , Gene Expression Regulation, Neoplastic/genetics , Glioma/pathology , HEK293 Cells , Humans , RNA Interference , RNA, Small InterferingABSTRACT
The human epidermis is a self-renewing, stratified epithelial tissue that provides the protective function of the skin. The principal cell type within the epidermis is the keratinocyte, and normal function of the epidermis requires that keratinocyte proliferation, differentiation and cell death be carefully controlled. There is clear evidence that signalling through adhesion receptors such as integrins and cadherins plays a key role in regulating epidermal function. Previous work has shown that Rho family GTPases regulate cadherin- and integrin-based adhesion structures and hence epidermal function. In this study, we show that a member of this family, Rnd3, regulates desmosomal cell-cell adhesion in that loss of Rnd3 expression leads to an increase in desmosomes at sites of cell-cell adhesion and altered colony morphology. Loss of Rnd3 expression is also associated with resistance to cisplatin-mediated apoptosis in keratinocytes and this resistance is mediated through the desmosomal protein plakoglobin. We propose a novel plakoglobin-dependent role for Rnd3 in the regulation of keratinocyte cell death.
Subject(s)
Apoptosis , Desmoplakins/metabolism , Keratinocytes/cytology , rho GTP-Binding Proteins/metabolism , Adherens Junctions/genetics , Adherens Junctions/metabolism , Amides , Cell Communication , Cell Line , Cell Survival , Cisplatin/adverse effects , Desmoplakins/genetics , Desmosomes/genetics , Desmosomes/metabolism , Gene Expression Regulation , Humans , Keratinocytes/metabolism , Pyridines , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Time Factors , Transfection , gamma Catenin , rho GTP-Binding Proteins/genetics , rho-Associated Kinases/antagonists & inhibitors , rho-Associated Kinases/metabolismABSTRACT
BACKGROUND: ROCK1 and ROCK2 are serine/threonine kinases that function downstream of the small GTP-binding protein RhoA. Rho signalling via ROCK regulates a number of cellular functions including organisation of the actin cytoskeleton, cell adhesion and cell migration. METHODOLOGY/PRINCIPAL FINDINGS: In this study we use RNAi to specifically knockdown ROCK1 and ROCK2 and analyse their role in assembly of adhesion complexes in human epidermal keratinocytes. We observe that loss of ROCK1 inhibits signalling via focal adhesion kinase resulting in a failure of immature adhesion complexes to form mature stable focal adhesions. In contrast, loss of ROCK2 expression results in a significant reduction in adhesion complex turnover leading to formation of large, stable focal adhesions. Interestingly, loss of either ROCK1 or ROCK2 expression significantly impairs cell migration indicating both ROCK isoforms are required for normal keratinocyte migration. CONCLUSIONS: ROCK1 and ROCK2 have distinct and separate roles in adhesion complex assembly and turnover in human epidermal keratinocytes.