ABSTRACT
Pharmacological induction of fetal hemoglobin (HbF) expression is an effective therapeutic strategy for the management of beta-hemoglobinopathies such as sickle cell disease. DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-aza) and 5-aza-2'-deoxycytidine (decitabine) have been shown to induce fetal hemoglobin expression in both preclinical models and clinical studies, but are not currently approved for the management of hemoglobinopathies. We report here the discovery of a novel class of orally bioavailable DNMT1-selective inhibitors as exemplified by GSK3482364. This molecule potently inhibits the methyltransferase activity of DNMT1, but not DNMT family members DNMT3A or DNMT3B. In contrast with cytidine analog DNMT inhibitors, the DNMT1 inhibitory mechanism of GSK3482364 does not require DNA incorporation and is reversible. In cultured human erythroid progenitor cells (EPCs), GSK3482364 decreased overall DNA methylation resulting in de-repression of the gamma globin genes HBG1 and HBG2 and increased HbF expression. In a transgenic mouse model of sickle cell disease, orally administered GSK3482364 caused significant increases in both HbF levels and in the percentage HbF-expressing erythrocytes, with good overall tolerability. We conclude that in these preclinical models, selective, reversible inhibition of DNMT1 is sufficient for the induction of HbF, and is well-tolerated. We anticipate that GSK3482364 will be a useful tool molecule for the further study of selective DNMT1 inhibition both in vitro and in vivo.
Subject(s)
Anemia, Sickle Cell , Fetal Hemoglobin , Anemia, Sickle Cell/drug therapy , Anemia, Sickle Cell/genetics , Animals , Azacitidine/pharmacology , DNA Methylation , Fetal Hemoglobin/genetics , Mice , gamma-Globins/geneticsABSTRACT
Lysine specific demethylase 1 (LSD1) is a histone modifying enzyme that suppresses gene expression through demethylation of lysine 4 on histone H3. The anti-tumor activity of GSK2879552 and GSK-LSD1, potent, selective irreversible inactivators of LSD1, has previously been described. Inhibition of LSD1 results in a cytostatic growth inhibitory effect in a range of acute myeloid leukemia cell lines. To enhance the therapeutic potential of LSD1 inhibition in this disease setting, a combination of LSD1 inhibition and all-trans retinoic acid was explored. All-trans retinoic acid is currently approved for use in acute promyelocytic leukemia in which it promotes differentiation of abnormal blast cells into normal white blood cells. Combined treatment with all-trans retinoic acid and GSK2879552 results in synergistic effects on cell proliferation, markers of differentiation, and, most importantly, cytotoxicity. Ultimately the combination potential for LSD1 inhibition and ATRA will require validation in acute myeloid leukemia patients, and clinical studies to assess this are currently underway.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Differentiation/drug effects , Histone Demethylases/antagonists & inhibitors , Leukemia, Myeloid, Acute/metabolism , Tretinoin/pharmacology , Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , Benzoates/pharmacology , Caspases/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclopropanes/pharmacology , Dose-Response Relationship, Drug , Drug Synergism , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/genetics , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Treatment Outcome , Tretinoin/administration & dosageABSTRACT
Mouse syngeneic tumor models are widely used tools to demonstrate activity of novel anti-cancer immunotherapies. Despite their widespread use, a comprehensive view of their tumor-immune compositions and their relevance to human tumors has only begun to emerge. We propose each model possesses a unique tumor-immune infiltrate profile that can be probed with immunotherapies to inform on anti-tumor mechanisms and treatment strategies in human tumors with similar profiles. In support of this endeavor, we characterized the tumor microenvironment of four commonly used models and demonstrate they encompass a range of immunogenicities, from highly immune infiltrated RENCA tumors to poorly infiltrated B16F10 tumors. Tumor cell lines for each model exhibit different intrinsic factors in vitro that likely influence immune infiltration upon subcutaneous implantation. Similarly, solid tumors in vivo for each model are unique, each enriched in distinct features ranging from pathogen response elements to antigen presentation machinery. As RENCA tumors progress in size, all major T cell populations diminish while myeloid-derived suppressor cells become more enriched, possibly driving immune suppression and tumor progression. In CT26 tumors, CD8 T cells paradoxically increase in density yet are restrained as tumor volume increases. Finally, immunotherapy treatment across these different tumor-immune landscapes segregate into responders and non-responders based on features partially dependent on pre-existing immune infiltrates. Overall, these studies provide an important resource to enhance our translation of syngeneic models to human tumors. Future mechanistic studies paired with this resource will help identify responsive patient populations and improve strategies where immunotherapies are predicted to be ineffective.
Subject(s)
Neoplasms/immunology , Neoplasms/therapy , Tumor Microenvironment , Animals , CD3 Complex/metabolism , Cell Line, Tumor , Cell Proliferation , Chemokines/metabolism , Complement System Proteins/metabolism , Disease Models, Animal , Disease Progression , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Immunotherapy , Ki-67 Antigen/metabolism , Mice, Inbred BALB C , Mice, Inbred C57BL , Myeloid Cells/pathology , Neoplasm Invasiveness , Neoplasms/genetics , Neoplasms/pathology , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Treatment OutcomeABSTRACT
Purpose: Persistent androgen receptor (AR) signaling drives castration-resistant prostate cancer (CRPC) and confers resistance to AR-targeting therapies. Novel therapeutic strategies to overcome this are urgently required. We evaluated how bromodomain and extra-terminal (BET) protein inhibitors (BETi) abrogate aberrant AR signaling in CRPC.Experimental Design: We determined associations between BET expression, AR-driven transcription, and patient outcome; and the effect and mechanism by which chemical BETi (JQ1 and GSK1210151A; I-BET151) and BET family protein knockdown regulates AR-V7 expression and AR signaling in prostate cancer models.Results: Nuclear BRD4 protein expression increases significantly (P ≤ 0.01) with castration resistance in same patient treatment-naïve (median H-score; interquartile range: 100; 100-170) and CRPC (150; 110-200) biopsies, with higher expression at diagnosis associating with worse outcome (HR, 3.25; 95% CI, 1.50-7.01; P ≤ 0.001). BRD2, BRD3, and BRD4 RNA expression in CRPC biopsies correlates with AR-driven transcription (all P ≤ 0.001). Chemical BETi, and combined BET family protein knockdown, reduce AR-V7 expression and AR signaling. This was not recapitulated by C-MYC knockdown. In addition, we show that BETi regulates RNA processing thereby reducing alternative splicing and AR-V7 expression. Furthermore, BETi reduce growth of prostate cancer cells and patient-derived organoids with known AR mutations, AR amplification and AR-V7 expression. Finally, BETi, unlike enzalutamide, decreases persistent AR signaling and growth (P ≤ 0.001) of a patient-derived xenograft model of CRPC with AR amplification and AR-V7 expression.Conclusions: BETi merit clinical evaluation as inhibitors of AR splicing and function, with trials demonstrating their blockade in proof-of-mechanism pharmacodynamic studies. Clin Cancer Res; 24(13); 3149-62. ©2018 AACR.
Subject(s)
Antineoplastic Agents/therapeutic use , Molecular Targeted Therapy , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/metabolism , Proteins/antagonists & inhibitors , Proteins/metabolism , Alternative Splicing , Antineoplastic Agents/pharmacology , Biomarkers, Tumor , Cell Cycle Proteins , Cell Line, Tumor , Computational Biology/methods , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Male , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Prognosis , Prostatic Neoplasms, Castration-Resistant/mortality , Prostatic Neoplasms, Castration-Resistant/pathology , Protein Isoforms , RNA, Small Interfering/genetics , Receptors, Androgen/metabolism , Signal Transduction/drug effects , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Treatment OutcomeABSTRACT
BRD4 belongs to the bromodomain and extraterminal (BET) family of chromatin reader proteins that bind acetylated histones and regulate gene expression. Pharmacological inhibition of BRD4 by BET inhibitors (BETi) has indicated antitumor activity against multiple cancer types. We show that BRD4 is essential for the repair of DNA double-strand breaks (DSBs) and mediates the formation of oncogenic gene rearrangements by engaging the non-homologous end joining (NHEJ) pathway. Mechanistically, genome-wide DNA breaks are associated with enhanced acetylation of histone H4, leading to BRD4 recruitment, and stable establishment of the DNA repair complex. In support of this, we also show that, in clinical tumor samples, BRD4 protein levels are negatively associated with outcome after prostate cancer (PCa) radiation therapy. Thus, in addition to regulating gene expression, BRD4 is also a central player in the repair of DNA DSBs, with significant implications for cancer therapy.
Subject(s)
DNA End-Joining Repair , Nuclear Proteins/genetics , Oncogene Proteins, Fusion/genetics , Prostatic Neoplasms/genetics , Transcription Factors/genetics , Acetylation , Cell Cycle Proteins , Cell Line, Tumor , Chromatin/genetics , Chromatin/metabolism , DNA Damage , Gene Fusion , Gene Rearrangement , Histones/genetics , Histones/metabolism , Humans , Male , Nuclear Proteins/metabolism , Oncogene Proteins, Fusion/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Transcription Factors/metabolismABSTRACT
BACKGROUND: The amino acid response (AAR) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti-inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl-tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure. METHODS AND RESULTS: Consistent with its ability to inhibit prolyl-tRNA synthetase, halofuginone elicited a general control nonderepressible 2-dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l-proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II/phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2-dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell-derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin-1-mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/eIF2α-dependent manner. CONCLUSIONS: Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress.
Subject(s)
Amino Acids/metabolism , Enzyme Inhibitors/pharmacology , Fibroblasts/drug effects , Heart Failure/prevention & control , Myocytes, Cardiac/drug effects , Piperidines/pharmacology , Protein Synthesis Inhibitors/pharmacology , Quinazolinones/pharmacology , Stress, Physiological , Amino Acids/deficiency , Amino Acyl-tRNA Synthetases/antagonists & inhibitors , Amino Acyl-tRNA Synthetases/metabolism , Animals , Autophagy/drug effects , Cells, Cultured , Disease Models, Animal , Dose-Response Relationship, Drug , Fibroblasts/metabolism , Fibroblasts/pathology , Fibrosis , Heart Failure/metabolism , Heart Failure/pathology , Heart Failure/physiopathology , Humans , Hypertrophy, Left Ventricular/metabolism , Hypertrophy, Left Ventricular/pathology , Hypertrophy, Left Ventricular/physiopathology , Hypertrophy, Left Ventricular/prevention & control , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Male , Mice, Inbred C57BL , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Protein Serine-Threonine Kinases/metabolism , Time Factors , Ventricular Function, Left/drug effects , Ventricular Remodeling/drug effectsABSTRACT
Importance: Amyotrophic lateral sclerosis (ALS) is a common adult-onset neurodegenerative disease characterized by selective loss of upper and lower motor neurons. Patients with ALS have persistent peripheral and central inflammatory responses including abnormally functioning T cells and activated microglia. However, much less is known about the inflammatory gene profile of circulating innate immune monocytes in these patients. Objective: To characterize the transcriptomics of peripheral monocytes in patients with ALS. Design, Setting, and Participants: Monocytes were isolated from peripheral blood of 43 patients with ALS and 22 healthy control individuals. Total RNA was extracted from the monocytes and subjected to deep RNA sequencing, and these results were validated by quantitative reverse transcription polymerase chain reaction. Main Outcomes and Measures: The differential expressed gene signatures of these monocytes were identified using unbiased RNA sequencing strategy for gene expression profiling. Results: The demographics between the patients with ALS (mean [SD] age, 58.8 [1.57] years; 55.8% were men and 44.2% were women; 90.7% were white, 4.65% were Hispanic, 2.33% were black, and 2.33% were Asian) and control individuals were similar (mean [SD] age, 57.6 [2.15] years; 50.0% were men and 50.0% were women; 90.9% were white, none were Hispanic, none were black, and 9.09% were Asian). RNA sequencing data from negative selected monocytes revealed 233 differential expressed genes in ALS monocytes compared with healthy control monocytes. Notably, ALS monocytes demonstrated a unique inflammation-related gene expression profile, the most prominent of which, including IL1B, IL8, FOSB, CXCL1, and CXCL2, were confirmed by quantitative reverse transcription polymerase chain reaction (IL8, mean [SE], 1.00 [0.18]; P = .002; FOSB, 1.00 [0.21]; P = .009; CXCL1, 1.00 [0.14]; P = .002; and CXCL2, 1.00 [0.11]; P = .01). Amyotrophic lateral sclerosis monocytes from rapidly progressing patients had more proinflammatory DEGs than monocytes from slowly progressing patients. Conclusions and Relevance: Our data indicate that ALS monocytes are skewed toward a proinflammatory state in the peripheral circulation and may play a role in ALS disease progression, especially in rapidly progressing patients. This increased inflammatory response of peripheral immune cells may provide a potential target for disease-modifying therapy in patients with ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/blood , Disease Progression , Gene Expression Profiling , Inflammation/blood , Monocytes/metabolism , Female , Humans , Male , Middle Aged , Phenotype , Sequence Analysis, RNAABSTRACT
The EZH2 methyltransferase silences gene expression through methylation of histone H3 on lysine 27 (H3K27). Recently, EZH2 mutations have been reported at Y641, A677, and A687 in non-Hodgkin lymphoma. Although the Y641F/N/S/H/C and A677G mutations exhibit clearly increased activity with substrates dimethylated at lysine 27 (H3K27me2), the A687V mutant has been shown to prefer a monomethylated lysine 27 (H3K27me1) with little gain of activity toward H3K27me2. Herein, we demonstrate that despite this unique substrate preference, A687V EZH2 still drives increased H3K27me3 when transiently expressed in cells. However, unlike the previously described mutants that dramatically deplete global H3K27me2 levels, A687V EZH2 retains normal levels of H3K27me2. Sequencing of B-cell-derived cancer cell lines identified an acute lymphoblastic leukemia cell line harboring this mutation. Similar to exogenous expression of A687V EZH2, this cell line exhibited elevated H3K27me3 while possessing H3K27me2 levels higher than Y641- or A677-mutant lines. Treatment of A687V EZH2-mutant cells with GSK126, a selective EZH2 inhibitor, was associated with a global decrease in H3K27me3, robust gene activation, caspase activation, and decreased proliferation. Structural modeling of the A687V EZH2 active site suggests that the increased catalytic activity with H3K27me1 may be due to a weakened interaction with an active site water molecule that must be displaced for dimethylation to occur. These findings suggest that A687V EZH2 likely increases global H3K27me3 indirectly through increased catalytic activity with H3K27me1 and cells harboring this mutation are highly dependent on EZH2 activity for their survival.
Subject(s)
Histones/metabolism , Mutation , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Cell Line, Tumor , Cluster Analysis , Enhancer of Zeste Homolog 2 Protein , Gene Expression , Gene Expression Profiling , Gene Silencing , Heterozygote , Humans , Lysine/metabolism , Methylation , Models, Molecular , Molecular Sequence Data , Polycomb Repressive Complex 2/chemistry , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/mortality , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein Conformation , Sequence Alignment , Substrate Specificity , Transcriptional ActivationABSTRACT
Trimethylation of histone H3 on lysine 27 (H3K27me3) is a repressive posttranslational modification mediated by the histone methyltransferase EZH2. EZH2 is a component of the polycomb repressive complex 2 and is overexpressed in many cancers. In B-cell lymphomas, its substrate preference is frequently altered through somatic mutation of the EZH2 Y641 residue. Herein, we identify mutation of EZH2 A677 to a glycine (A677G) among lymphoma cell lines and primary tumor specimens. Similar to Y641 mutant cell lines, an A677G mutant cell line revealed aberrantly elevated H3K27me3 and decreased monomethylated H3K27 (H3K27me1) and dimethylated H3K27 (H3K27me2). A677G EZH2 possessed catalytic activity with a substrate specificity that was distinct from those of both WT EZH2 and Y641 mutants. Whereas WT EZH2 displayed a preference for substrates with less methylation [unmethylated H3K27 (H3K27me0):me1:me2 k(cat)/K(m) ratio = 9:6:1] and Y641 mutants preferred substrates with greater methylation (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1:2:13), the A677G EZH2 demonstrated nearly equal efficiency for all three substrates (H3K27me0:me1:me2 k(cat)/K(m) ratio = 1.1:0.6:1). When transiently expressed in cells, A677G EZH2, but not WT EZH2, increased global H3K27me3 and decreased H3K27me2. Structural modeling of WT and mutant EZH2 suggested that the A677G mutation acquires the ability to methylate H3K27me2 through enlargement of the lysine tunnel while preserving activity with H3K27me0/me1 substrates through retention of the Y641 residue that is crucial for orientation of these smaller substrates. This mutation highlights the interplay between Y641 and A677 residues in the substrate specificity of EZH2 and identifies another lymphoma patient population that harbors an activating mutation of EZH2.
Subject(s)
Alanine/genetics , DNA-Binding Proteins/genetics , Histones/metabolism , Lymphoma, B-Cell/enzymology , Lymphoma, B-Cell/genetics , Lysine/metabolism , Mutation/genetics , Transcription Factors/genetics , Amino Acid Sequence , Base Sequence , Binding Sites , Cell Line, Tumor , DNA Mutational Analysis , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Enhancer of Zeste Homolog 2 Protein , Gene Expression Regulation, Neoplastic , Glycine/genetics , Heterozygote , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/chemistry , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , Methylation , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Polycomb Repressive Complex 2 , Substrate Specificity , Transcription Factors/chemistry , Transcription Factors/metabolismABSTRACT
BACKGROUND: Globally, gastric cancer is the second most common cause of cancer-related death, with the majority of the health burden borne by economically less-developed countries. METHODS: Here, we report a genetic characterization of 50 gastric adenocarcinoma samples, using affymetrix SNP arrays and Illumina mRNA expression arrays as well as Illumina sequencing of the coding regions of 384 genes belonging to various pathways known to be altered in other cancers. RESULTS: Genetic alterations were observed in the WNT, Hedgehog, cell cycle, DNA damage and epithelial-to-mesenchymal-transition pathways. CONCLUSIONS: The data suggests targeted therapies approved or in clinical development for gastric carcinoma would be of benefit to ~22% of the patients studied. In addition, the novel mutations detected here, are likely to influence clinical response and suggest new targets for drug discovery.
Subject(s)
High-Throughput Nucleotide Sequencing/methods , Mutation/genetics , Precision Medicine , Stomach Neoplasms/genetics , Cell Cycle Proteins/genetics , DNA Copy Number Variations/genetics , DNA, Neoplasm/genetics , Epithelium/metabolism , Epithelium/pathology , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genotype , Humans , Molecular Targeted Therapy , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Phenotype , RNA, Neoplasm/genetics , Reproducibility of Results , Sequence Analysis, DNA , Signal Transduction/genetics , Stomach Neoplasms/drug therapy , Stomach Neoplasms/enzymology , Wnt Proteins/genetics , Wnt Proteins/metabolismABSTRACT
Polo-like kinases are a family of serine threonine kinases that are critical regulators of cell cycle progression and DNA damage response. Predictive biomarkers for the Plk1-selective inhibitor GSK461364A were identified by comparing the genomics and genetics of a panel of human cancer cell lines with their response to a drug washout followed by an outgrowth assay. In this assay, cell lines that have lost p53 expression or carry mutations in the TP53 gene tended to be more sensitive to GSK461364A. These more sensitive cell lines also had increased levels of chromosome instability, a characteristic associated with loss of p53 function. Further mechanistic studies showed that p53 wild-type (WT) and not mutant cells can activate a postmitotic tetraploidy checkpoint and arrest at pseudo-G(1) state after GSK461364A treatment. RNA silencing of WT p53 increased the antiproliferative activity of GSK461364A. Furthermore, silencing of p53 or p21/CDKN1A weakened the tetraploidy checkpoint in cells that survived mitotic arrest and mitotic slippage. As many cancer therapies tend to be more effective in p53 WT patients, the higher sensitivity of p53-deficient tumors toward GSK461364A could potentially offer an opportunity to treat tumors that are refractory to other chemotherapies as well as early line therapy for these genotypes.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Chromosomal Instability , Mutation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Thiophenes/pharmacology , Tumor Suppressor Protein p53/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Humans , Immunoblotting , Inhibitory Concentration 50 , Mitosis/drug effects , Mitosis/genetics , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Polyploidy , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Tumor Suppressor Protein p53/metabolism , Polo-Like Kinase 1ABSTRACT
With genome-wide cancer studies producing large DNA sequence data sets, novel computational approaches toward better understanding the role of mutations in tumor survival and proliferation are greatly needed. Tumors are widely viewed to be influenced by Darwinian processes, yet molecular evolutionary analysis, invaluable in other DNA sequence studies, has seen little application in cancer biology. Here, we describe the phylogenetic analysis of 353 cancer cell lines based on multiple sequence alignments of 3,252 nucleotides and 1,170 amino acids built from the concatenation of variant codons and residues across 494 and 523 genes, respectively. Reconstructed phylogenetic trees cluster cell lines by shared DNA variant patterns rather than cancer tissue type, suggesting that tumors originating from diverse histologies have similar oncogenic pathways. A well-supported clade of 91 cancer cell lines representing multiple tumor types also had significantly different gene expression profiles from the remaining cell lines according to statistical analyses of mRNA microarray data. This suggests that phylogenetic clustering of tumor cell lines based on DNA variants might reflect functional similarities in cellular pathways. Positive selection analysis revealed specific DNA variants that might be potential driver mutations. Our study shows the potential role of molecular evolutionary analyses in tumor classification and the development of novel anticancer strategies.
Subject(s)
Cell Line, Tumor , Neoplasms/genetics , Algorithms , Cluster Analysis , Evolution, Molecular , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Mutation , Nucleotides/genetics , Oligonucleotide Array Sequence Analysis , Phylogeny , Polymorphism, Single Nucleotide , RNA, Messenger/metabolism , Sequence Analysis, DNAABSTRACT
HER2-directed therapies, such as trastuzumab and lapatinib, are important treatments for breast cancer. However, some tumors do not respond or develop resistance to these agents. We isolated and characterized multiple lapatinib-resistant, HER2-positive, estrogen receptor (ER)-positive breast cancer clones derived from lapatinib-sensitive BT474 cells by chronic exposure to lapatinib. We show overexpression of AXL as a novel mechanism of acquired resistance to HER2-targeted agents in these models. GSK1363089 (foretinib), a multikinase inhibitor of AXL, MET, and vascular endothelial growth factor receptor currently in phase II clinical trials, restores lapatinib and trastuzumab sensitivity in these resistant cells that exhibit increased AXL expression. Furthermore, small interfering RNA to AXL, estrogen deprivation, or fulvestrant, an ER antagonist, decreases AXL expression and restores sensitivity to lapatinib in these cells. Taken together, these data provide scientific evidence to assess the expression of AXL in HER2-positive, ER-positive patients who have progressed on either lapatinib or trastuzumab and to test the combination of HER2-targeted agents and GSK1363089 in the clinic.
Subject(s)
Anilides/pharmacology , Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Drug Resistance, Neoplasm/genetics , Genes, erbB-2 , Oncogene Proteins/biosynthesis , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , Quinolines/pharmacology , Receptor Protein-Tyrosine Kinases/biosynthesis , Anilides/therapeutic use , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Humanized , Antineoplastic Agents/therapeutic use , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Clone Cells , Drug Antagonism , Drug Resistance, Neoplasm/drug effects , Female , Humans , Lapatinib , Oncogene Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins , Quinazolines/therapeutic use , Quinolines/therapeutic use , RNA, Small Interfering , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptors, Estrogen/antagonists & inhibitors , Receptors, Estrogen/biosynthesis , Trastuzumab , Axl Receptor Tyrosine KinaseABSTRACT
Measles, mumps, and rubella are three common viral childhood diseases that can have serious complications. Active immunization against these diseases became possible with the development of live attenuated virus vaccines in the late 1960s. Vaccines against these three diseases were combined into trivalent (Priorix, GlaxoSmithKline Biologicals and M-M-R(II), Merck & Co., Inc.) or tetravalent vaccines including the addition of a live attenuated VZV Oka strain (Priorix-Tetra, GlaxoSmithKline Biologicals and ProQuad, Merck & Co., Inc.). In this study, we report the complete nucleotide sequence of the vaccine strain genomes of the measles (Schwarz and attenuated Edmonston Enders), mumps (RIT 4385 and JL1 component of Jeryl Lynn), and rubella (Wistar RA 27/3) viruses included in the two tetravalent vaccines. Sequencing analysis of the individual virus components in the commercially distributed tetravalent vaccine lots showed that there are no nucleotide differences between the measles, mumps (JL1 component), and rubella vaccine strain genomes of Priorix-Tetra and ProQuad. The observed genetic identity of the individual strains in both vaccines is consistent with their clinical profiles; Priorix-Tetra and ProQuad are both well tolerated and elicit protective immune responses against these three childhood diseases.
Subject(s)
Genome, Viral , Measles virus/genetics , Measles-Mumps-Rubella Vaccine/genetics , Mumps virus/genetics , Rubella virus/genetics , Sequence Analysis, DNA , Vaccines, Attenuated/geneticsABSTRACT
Two-metal binding HIV-1 integrase inhibitors (INIs) are potent inhibitors of HIV-1 in vitro and in patients. We report here for the first time the kinetics of inhibition of integrase-catalyzed strand transfer. First, the IC(50) values for each of six structurally distinct INIs decreased when a preincubation was included: S-1360 (1.3 microM vs 0.12 microM), L-731,988 (130 nM vs 9 nM), L-870,810 (130 nM vs 4 nM), raltegravir (300 nM vs 9 nM), elvitegravir (90 nM vs 6 nM), and GSK364735 (90 nM vs 6 nM). When reactions with these INIs were initiated with integrase, progress curve analyses indicated time-dependent inhibition, which could be fitted to a two-step mechanism of binding. Overall fitted K(i) values matched the IC(50) values measured with a preincubation: S-1360 (0.17 microM), L-731,988 (34 nM), L-870,810 (2.4 nM), raltegravir (10 nM), elvitegravir (4.0 nM), and GSK364735 (2.5 nM). To begin to understand the mechanism for this slow onset of inhibition and its possible impact on drug resistance, studies of resistance mutations were initiated. T66I/M154I exhibited little if any time-dependent inhibition by any of the six INIs, as measured by differences in potency upon preincubation or by progress curve analysis. These data demonstrate that slow binding is a signature of two-metal binding INIs, and that the second slow step is required for full potency. We discuss a possible structural explanation of the second slow step of inhibition and also the relationship between loss of time-dependent inhibition and drug resistance of this important new class of HIV-1 antiretroviral drugs.
Subject(s)
Drug Resistance, Viral/genetics , HIV Integrase Inhibitors/pharmacology , HIV Integrase/drug effects , HIV-1/drug effects , Mutation , Base Sequence , DNA Primers , HIV-1/genetics , KineticsABSTRACT
Varicella-zoster virus (VZV) is a herpesvirus and is the causative agent of chicken pox (varicella) and shingles (herpes zoster). Active immunization against varicella became possible with the development of live attenuated varicella vaccine. The Oka vaccine strain was isolated in Japan from a child who had typical varicella, and it was then attenuated by serial passages in cell culture. Several manufacturers have obtained this attenuated Oka strain and, following additional passages, have developed their own vaccine strains. Notably, the vaccines Varilrix and Varivax are produced by GlaxoSmithKline Biologicals and Merck & Co., Inc., respectively. Both vaccines have been well studied in terms of safety and immunogenicity. In this study, we report the complete nucleotide sequence of the Varilrix (Oka-V(GSK)) and Varivax (Oka-V(Merck)) vaccine strain genomes. Their genomes are composed of 124,821 and 124,815 bp, respectively. Full genome annotations covering the features of Oka-derived vaccine genomes have been established for the first time. Sequence analysis indicates 36 nucleotide differences between the two vaccine strains throughout the entire genome, among which only 14 are involved in unique amino acid substitutions. These results demonstrate that, although Oka-V(GSK) and Oka-V(Merck) vaccine strains are not identical, they are very similar, which supports the clinical data showing that both vaccines are well tolerated and elicit strong immune responses against varicella.
Subject(s)
Chickenpox Vaccine/genetics , DNA, Viral/chemistry , DNA, Viral/genetics , Genome, Viral , Herpesvirus 3, Human/genetics , Amino Acid Substitution/genetics , Base Sequence , Genes, Viral , Molecular Sequence Data , Polymorphism, Genetic , Sequence Analysis, DNAABSTRACT
We performed multilocus sequence typing on 203 invasive disease isolates of Streptococcus pneumoniae to assess the clonal compositions of isolates from two provinces in Belgium and to determine the relationship between clones and antibiotic nonsusceptibility, particularly nonsusceptibility to two or more classes of antibiotics. The frequency of multiclass nonsusceptibility (MCNS) was higher in the province of West Flanders (38%) than in Limburg (21%). This difference was largely attributable to five clonal complexes (CC156, CC81, CC143, CC193, and CC1848), which contained high proportions of isolates with MCNS (>47%) and which were circulating at higher frequencies in West Flanders. The S. pneumoniae population changed over time, as CC156 and CC81 declined in frequency from 1997 to 1999 to 2001 to 2004. Over the same time period, the frequency of pneumococcal conjugate vaccine 7 (PCV7) serotypes dropped from 69% to 41%. In contrast, the nonvaccine serotype 19A increased in frequency from 2.1% to 6.6%. None of these changes can be attributed to PCV7 vaccine, as it was not in use in Belgium during the time period studied. There was evidence that MCNS clones flowed from West Flanders to Limburg.
Subject(s)
Drug Resistance, Multiple, Bacterial , Pneumococcal Infections/epidemiology , Streptococcus pneumoniae/classification , Streptococcus pneumoniae/drug effects , Adolescent , Adult , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Typing Techniques , Belgium/epidemiology , Child , Child, Preschool , DNA, Bacterial/analysis , Drug Resistance, Multiple, Bacterial/genetics , Humans , Infant , Infant, Newborn , Microbial Sensitivity Tests , Pneumococcal Infections/microbiology , Polymerase Chain Reaction , Serotyping , Streptococcus pneumoniae/geneticsABSTRACT
BACKGROUND: The thymidine kinase (tk) mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV) replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1. METHODS: A panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol) within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases. RESULTS: Although HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAAr5 exhibited a 2-4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed significant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA structure. CONCLUSIONS: This study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a) mutations may be modulated by other viral polypeptides cooperating with Pol, and (b) the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2.