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1.
bioRxiv ; 2023 May 30.
Article in English | MEDLINE | ID: mdl-37398413

ABSTRACT

Mutation in nucleophosmin (NPM1) causes relocalization of this normally nucleolar protein to the cytoplasm ( NPM1c+ ). Despite NPM1 mutation being the most common driver mutation in cytogenetically normal adult acute myeloid leukemia (AML), the mechanisms of NPM1c+-induced leukemogenesis remain unclear. Caspase-2 is a pro-apoptotic protein activated by NPM1 in the nucleolus. Here, we show that caspase-2 is also activated by NPM1c+ in the cytoplasm, and DNA damage-induced apoptosis is caspase-2-dependent in NPM1c+ AML but not in NPM1wt cells. Strikingly, in NPM1c+ cells, loss of caspase-2 results in profound cell cycle arrest, differentiation, and down-regulation of stem cell pathways that regulate pluripotency including impairment in the AKT/mTORC1 and Wnt signaling pathways. In contrast, there were minimal differences in proliferation, differentiation, or the transcriptional profile of NPM1wt cells with and without caspase-2. Together, these results show that caspase-2 is essential for proliferation and self-renewal of AML cells that have mutated NPM1. This study demonstrates that caspase-2 is a major effector of NPM1c+ function and may even be a druggable target to treat NPM1c+ AML and prevent relapse.

2.
Sci Rep ; 8(1): 9711, 2018 06 26.
Article in English | MEDLINE | ID: mdl-29946150

ABSTRACT

Evasion of the potent tumour suppressor activity of p53 is one of the hurdles that must be overcome for cancer cells to escape normal regulation of cellular proliferation and survival. In addition to frequent loss of function mutations, p53 wild-type activity can also be suppressed post-translationally through several mechanisms, including the activity of PRMT5. Here we describe broad anti-proliferative activity of potent, selective, reversible inhibitors of protein arginine methyltransferase 5 (PRMT5) including GSK3326595 in human cancer cell lines representing both hematologic and solid malignancies. Interestingly, PRMT5 inhibition activates the p53 pathway via the induction of alternative splicing of MDM4. The MDM4 isoform switch and subsequent p53 activation are critical determinants of the response to PRMT5 inhibition suggesting that the integrity of the p53-MDM4 regulatory axis defines a subset of patients that could benefit from treatment with GSK3326595.


Subject(s)
Nuclear Proteins/metabolism , Protein-Arginine N-Methyltransferases/metabolism , Proto-Oncogene Proteins/metabolism , RNA Splicing/genetics , Tumor Suppressor Protein p53/metabolism , Alternative Splicing/genetics , Antineoplastic Agents , Arginine/analogs & derivatives , Arginine/metabolism , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Cycle Proteins , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Enzyme Inhibitors/pharmacology , Humans , Nuclear Proteins/genetics , Protein Isoforms/genetics , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Tumor Suppressor Protein p53/genetics , snRNP Core Proteins/metabolism
3.
4.
Blood ; 123(5): 697-705, 2014 Jan 30.
Article in English | MEDLINE | ID: mdl-24335499

ABSTRACT

The bromodomain and extraterminal (BET) protein BRD2-4 inhibitors hold therapeutic promise in preclinical models of hematologic malignancies. However, translation of these data to molecules suitable for clinical development has yet to be accomplished. Herein we expand the mechanistic understanding of BET inhibitors in multiple myeloma by using the chemical probe molecule I-BET151. I-BET151 induces apoptosis and exerts strong antiproliferative effect in vitro and in vivo. This is associated with contrasting effects on oncogenic MYC and HEXIM1, an inhibitor of the transcriptional activator P-TEFb. I-BET151 causes transcriptional repression of MYC and MYC-dependent programs by abrogating recruitment to the chromatin of the P-TEFb component CDK9 in a BRD2-4-dependent manner. In contrast, transcriptional upregulation of HEXIM1 is BRD2-4 independent. Finally, preclinical studies show that I-BET762 has a favorable pharmacologic profile as an oral agent and that it inhibits myeloma cell proliferation, resulting in survival advantage in a systemic myeloma xenograft model. These data provide a strong rationale for extending the clinical testing of the novel antimyeloma agent I-BET762 and reveal insights into biologic pathways required for myeloma cell proliferation.


Subject(s)
Antineoplastic Agents/therapeutic use , Benzodiazepines/therapeutic use , Heterocyclic Compounds, 4 or More Rings/therapeutic use , Multiple Myeloma/drug therapy , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Benzodiazepines/pharmacology , Cell Cycle Checkpoints/drug effects , Down-Regulation/drug effects , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Mice , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Proto-Oncogene Proteins c-myc/genetics , RNA-Binding Proteins/genetics , Transcription Factors , Transcriptional Activation/drug effects , Tumor Cells, Cultured
5.
Oncotarget ; 4(12): 2419-29, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24293458

ABSTRACT

BET (bromodomain and extra-terminal) proteins regulate gene expression through their ability to bind to acetylated chromatin and subsequently activate RNA PolII-driven transcriptional elongation. Small molecule BET inhibitors prevent binding of BET proteins to acetylated histones and inhibit transcriptional activation of BET target genes. BET inhibitors attenuate cell growth and survival in several hematologic cancer models, partially through the down-regulation of the critical oncogene, MYC. We hypothesized that BET inhibitors will regulate MYC expression in solid tumors that frequently over-express MYC. Here we describe the effects of the highly specific BET inhibitor, I-BET762, on MYC expression in prostate cancer models. I-BET762 potently reduced MYC expression in prostate cancer cell lines and a patient-derived tumor model with subsequent inhibition of cell growth and reduction of tumor burden in vivo. Our data suggests that I-BET762 effects are partially driven by MYC down-regulation and underlines the critical importance of additional mechanisms of I-BET762 induced phenotypes.


Subject(s)
Benzodiazepines/pharmacology , Nuclear Proteins/antagonists & inhibitors , Prostatic Neoplasms, Castration-Resistant/drug therapy , Protein Serine-Threonine Kinases/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cell Growth Processes/physiology , Cell Line, Tumor , Down-Regulation , Gene Expression Profiling , Humans , Male , Mice , Mice, SCID , Prostatic Neoplasms, Castration-Resistant/enzymology , Prostatic Neoplasms, Castration-Resistant/pathology , Xenograft Model Antitumor Assays
6.
PLoS One ; 8(8): e72967, 2013.
Article in English | MEDLINE | ID: mdl-24009722

ABSTRACT

BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.


Subject(s)
Benzodiazepines/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Gene Silencing , Neuroblastoma/genetics , Neuroblastoma/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Oncogene Proteins/genetics , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , RNA-Binding Proteins/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/toxicity , Apoptosis/drug effects , Apoptosis/genetics , Benzodiazepines/chemistry , Benzodiazepines/toxicity , Cell Cycle Proteins , Cell Proliferation/drug effects , Cluster Analysis , Disease Models, Animal , Female , Gene Expression Profiling , Gene Regulatory Networks , Humans , Kinetics , Mice , Models, Molecular , Molecular Conformation , N-Myc Proto-Oncogene Protein , Neuroblastoma/drug therapy , Neuroblastoma/pathology , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Protein Binding , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Signal Transduction/drug effects , Transcription Factors/chemistry , Transcription Factors/metabolism , Tumor Burden/drug effects , Tumor Burden/genetics , Xenograft Model Antitumor Assays
7.
J Biomol Screen ; 17(10): 1279-92, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22904200

ABSTRACT

Histone methyltransferases (HMT) catalyze the methylation of histone tail lysines, resulting in changes in gene transcription. Misregulation of these enzymes has been associated with various forms of cancer, making this target class a potential new area for the development of novel chemotherapeutics. EZH2 is the catalytic component of the polycomb group repressive complex (PRC2), which selectively methylates histone H3 lysine 27 (H3K27). EZH2 is overexpressed in prostate, breast, bladder, brain, and other tumor types and is recognized as a molecular marker for cancer progression and aggressiveness. Several new reagents and assays were developed to aid in the identification of EZH2 inhibitors, and these were used to execute two high-throughput screening campaigns. Activity assays using either an H3K27 peptide or nucleosomes as substrates for methylation are described. The strategy to screen EZH2 with either a surrogate peptide or a natural substrate led to the identification of the same tractable series. Compounds from this series are reversible, are [(3)H]-S-adenosyl-L-methionine competitive, and display biochemical inhibition of H3K27 methylation.


Subject(s)
High-Throughput Screening Assays/methods , Nucleosomes/metabolism , Peptides/metabolism , Polycomb Repressive Complex 2/metabolism , Drug Screening Assays, Antitumor/methods , Enhancer of Zeste Homolog 2 Protein , Humans , Indicators and Reagents , Kinetics , Peptides/antagonists & inhibitors , Polycomb Repressive Complex 2/antagonists & inhibitors , Polycomb Repressive Complex 2/chemistry , Reproducibility of Results
8.
Epigenetics ; 7(4): 340-3, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22419068

ABSTRACT

Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease.


Subject(s)
Gene Expression Regulation, Neoplastic , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Lysine/metabolism , Animals , Blotting, Western , Chromatin/genetics , Chromatin/metabolism , Enzyme Activation , Fibroblasts/metabolism , Fibroblasts/pathology , Genetic Complementation Test , HeLa Cells , Histone-Lysine N-Methyltransferase/genetics , Histones/genetics , Humans , Methylation , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutagenesis, Site-Directed , Peptide Library , Phenotype , Plasmids/genetics , Plasmids/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity
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