ABSTRACT
The focus of this study, and the subject of this article, resides in the conceptually funded usability evaluation of an application of descriptive models to a specific dataset obtained from the East Slovak Institute of Heart and Vascular Diseases targeting cardiovascular patients. Delving into the current state-of-the-art practices, we examine the extent of cardiovascular diseases, descriptive data analysis models, and their practical applications. Most importantly, our inquiry focuses on exploration of usability, encompassing its application and evaluation methodologies, including Van Welie's layered model of usability and its inherent advantages and limitations. The primary objective of our research was to conceptualize, develop, and validate the usability of an application tailored to supporting cardiologists' research through descriptive modeling. Using the R programming language, we engineered a Shiny dashboard application named DESSFOCA (Decision Support System For Cardiologists) that is structured around three core functionalities: discovering association rules, applying clustering methods, and identifying association rules within predefined clusters. To assess the usability of DESSFOCA, we employed the System Usability Scale (SUS) and conducted a comprehensive evaluation. Additionally, we proposed an extension to Van Welie's layered model of usability, incorporating several crucial aspects deemed essential. Subsequently, we rigorously evaluated the proposed extension within the DESSFOCA application with respect to the extended usability model, drawing insightful conclusions from our findings.
ABSTRACT
Recent progress in targeting KRASG12C has provided both insight and inspiration for targeting alternative KRAS mutants. In this study, we evaluated the mechanism of action and anti-tumor efficacy of MRTX1133, a potent, selective and non-covalent KRASG12D inhibitor. MRTX1133 demonstrated a high-affinity interaction with GDP-loaded KRASG12D with KD and IC50 values of ~0.2 pM and <2 nM, respectively, and ~700-fold selectivity for binding to KRASG12D as compared to KRASWT. MRTX1133 also demonstrated potent inhibition of activated KRASG12D based on biochemical and co-crystal structural analyses. MRTX1133 inhibited ERK1/2 phosphorylation and cell viability in KRASG12D-mutant cell lines, with median IC50 values of ~5 nM, and demonstrated >1,000-fold selectivity compared to KRASWT cell lines. MRTX1133 exhibited dose-dependent inhibition of KRAS-mediated signal transduction and marked tumor regression (≥30%) in a subset of KRASG12D-mutant cell-line-derived and patient-derived xenograft models, including eight of 11 (73%) pancreatic ductal adenocarcinoma (PDAC) models. Pharmacological and CRISPR-based screens demonstrated that co-targeting KRASG12D with putative feedback or bypass pathways, including EGFR or PI3Kα, led to enhanced anti-tumor activity. Together, these data indicate the feasibility of selectively targeting KRAS mutants with non-covalent, high-affinity small molecules and illustrate the therapeutic susceptibility and broad dependence of KRASG12D mutation-positive tumors on mutant KRAS for tumor cell growth and survival.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Carcinoma, Pancreatic Ductal/metabolism , Cell Line, Tumor , ErbB Receptors/metabolism , Humans , Mutation/genetics , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolismABSTRACT
KRASG12C inhibitors, including MRTX849, are promising treatment options for KRAS-mutant non-small cell lung cancer (NSCLC). PD-1 inhibitors are approved in NSCLC; however, strategies to enhance checkpoint inhibitor therapy (CIT) are needed. KRASG12C mutations are smoking-associated transversion mutations associated with high tumor mutation burden, PD-L1 positivity, and an immunosuppressive tumor microenvironment. To evaluate the potential of MRTX849 to augment CIT, its impact on immune signaling and response to CIT was evaluated. In human tumor xenograft models, MRTX849 increased MHC class I protein expression and decreased RNA and/or plasma protein levels of immunosuppressive factors. In a KrasG12C -mutant CT26 syngeneic mouse model, MRTX849 decreased intratumoral myeloid-derived suppressor cells and increased M1-polarized macrophages, dendritic cells, CD4+, and CD8+ T cells. Similar results were observed in lung KrasG12C -mutant syngeneic and a genetically engineered mouse (GEM) model. In the CT26 KrasG12C model, MRTX849 demonstrated marked tumor regression when tumors were established in immune-competent BALB/c mice; however, the effect was diminished when tumors were grown in T-cell-deficient nu/nu mice. Tumors progressed following anti-PD-1 or MRTX849 single-agent treatment in immune-competent mice; however, combination treatment demonstrated durable, complete responses (CRs). Tumors did not reestablish in the same mice that exhibited durable CRs when rechallenged with tumor cell inoculum, demonstrating these mice developed adaptive antitumor immunity. In a GEM model, treatment with MRTX849 plus anti-PD-1 led to increased progression-free survival compared with either single agent alone. These data demonstrate KRAS inhibition reverses an immunosuppressive tumor microenvironment and sensitizes tumors to CIT through multiple mechanisms.
Subject(s)
Acetonitriles/antagonists & inhibitors , Antineoplastic Agents/therapeutic use , Immune Checkpoint Inhibitors/therapeutic use , Piperazines/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Pyrimidines/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation , Disease Models, Animal , Humans , Immune Checkpoint Inhibitors/pharmacology , Mice , Tumor Microenvironment/drug effectsABSTRACT
Despite decades of research, efforts to directly target KRAS have been challenging. MRTX849 was identified as a potent, selective, and covalent KRASG12C inhibitor that exhibits favorable drug-like properties, selectively modifies mutant cysteine 12 in GDP-bound KRASG12C, and inhibits KRAS-dependent signaling. MRTX849 demonstrated pronounced tumor regression in 17 of 26 (65%) KRASG12C-positive cell line- and patient-derived xenograft models from multiple tumor types, and objective responses have been observed in patients with KRASG12C-positive lung and colon adenocarcinomas. Comprehensive pharmacodynamic and pharmacogenomic profiling in sensitive and partially resistant nonclinical models identified mechanisms implicated in limiting antitumor activity including KRAS nucleotide cycling and pathways that induce feedback reactivation and/or bypass KRAS dependence. These factors included activation of receptor tyrosine kinases (RTK), bypass of KRAS dependence, and genetic dysregulation of cell cycle. Combinations of MRTX849 with agents that target RTKs, mTOR, or cell cycle demonstrated enhanced response and marked tumor regression in several tumor models, including MRTX849-refractory models. SIGNIFICANCE: The discovery of MRTX849 provides a long-awaited opportunity to selectively target KRASG12C in patients. The in-depth characterization of MRTX849 activity, elucidation of response and resistance mechanisms, and identification of effective combinations provide new insight toward KRAS dependence and the rational development of this class of agents.See related commentary by Klempner and Hata, p. 20.This article is highlighted in the In This Issue feature, p. 1.
Subject(s)
Acetonitriles/therapeutic use , Adenocarcinoma of Lung/drug therapy , Antineoplastic Agents/therapeutic use , Disease Models, Animal , Lung Neoplasms/drug therapy , Mutation , Piperazines/therapeutic use , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , Pyrrolidines/therapeutic use , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/pathology , Animals , Apoptosis , Cell Proliferation , Clinical Trials, Phase I as Topic , Female , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Middle Aged , Prognosis , Pyrimidines , Signal Transduction , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in either PKD1 or PKD2 genes, is one of the most common human monogenetic disorders and the leading genetic cause of end-stage renal disease. Unfortunately, treatment options for ADPKD are limited. Here we report the discovery and characterization of RGLS4326, a first-in-class, short oligonucleotide inhibitor of microRNA-17 (miR-17), as a potential treatment for ADPKD. RGLS4326 is discovered by screening a chemically diverse and rationally designed library of anti-miR-17 oligonucleotides for optimal pharmaceutical properties. RGLS4326 preferentially distributes to kidney and collecting duct-derived cysts, displaces miR-17 from translationally active polysomes, and de-represses multiple miR-17 mRNA targets including Pkd1 and Pkd2. Importantly, RGLS4326 demonstrates a favorable preclinical safety profile and attenuates cyst growth in human in vitro ADPKD models and multiple PKD mouse models after subcutaneous administration. The preclinical characteristics of RGLS4326 support its clinical development as a disease-modifying treatment for ADPKD.
Subject(s)
MicroRNAs/antagonists & inhibitors , Oligonucleotides/therapeutic use , Polycystic Kidney Diseases/drug therapy , Polycystic Kidney Diseases/genetics , Animals , Base Sequence , Cell Proliferation/drug effects , Disease Models, Animal , Gene Regulatory Networks/drug effects , HeLa Cells , Hematopoiesis/drug effects , Humans , Kidney Tubules/pathology , Macaca fascicularis , Male , Mice, Inbred C57BL , MicroRNAs/genetics , Oligonucleotides/pharmacokinetics , Oligonucleotides/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tissue Distribution/drug effectsABSTRACT
Hepatocellular carcinoma (HCC) is one of the most common human malignancies with poor prognosis and urgent unmet medical need. Aberrant expression of multiple members of the miR-17 family are frequently observed in HCC, and their overexpression promotes tumorigenic properties of HCC cells. However, whether pharmacologic inhibition of the miR-17 family inhibits HCC growth remains unknown. In this study, we validated that the miR-17 family was upregulated in a subset of HCC tumors and cell lines and its inhibition by a tough decoy inhibitor suppressed the growth of Hep3B and HepG2 cells, which overexpress the miR-17 family. Furthermore, inhibition of the miR-17 family led to a global derepression of direct targets of the family in all three HCC cell lines tested. Pathway analysis of the deregulated genes indicated that the genes associated with TGFß signaling pathway were highly enriched in Hep3B and HepG2 cells. A miR-17 family target gene signature was established and used to identify RL01-17(5), a lipid nanoparticle encapsulating a potent anti-miR-17 family oligonucleotide. To address whether pharmacologic modulation of the miR-17 family can inhibit HCC growth, RL01-17(5) was systemically administrated to orthotopic Hep3B xenografts. Suppression of Hep3B tumor growth in vivo was observed and tumor growth inhibition correlated with induction of miR-17 family target genes. Together, this study provides proof-of-concept for targeting the miR-17 family in HCC therapy. Mol Cancer Ther; 16(5); 905-13. ©2017 AACR.
Subject(s)
Antagomirs/administration & dosage , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/drug therapy , MicroRNAs/genetics , Animals , Antagomirs/genetics , Carcinogenesis/drug effects , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Gene Expression Regulation, Neoplastic/drug effects , Hep G2 Cells , Humans , Lipids/administration & dosage , Lipids/chemistry , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Mice , MicroRNAs/antagonists & inhibitors , MicroRNAs/chemistry , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Oligonucleotides/administration & dosage , Oligonucleotides/genetics , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: miR-122 is an important host factor for hepatitis C virus (HCV) replication. The aim of this study was to assess the safety and tolerability, pharmacokinetics, and antiviral effect of a single dose of RG-101, a hepatocyte targeted N-acetylgalactosamine conjugated oligonucleotide that antagonises miR-122, in patients with chronic HCV infection with various genotypes. METHODS: In this randomised, double-blind, placebo-controlled, multicentre, phase 1B study, patients were randomly assigned to RG-101 or placebo (7:1). We enrolled men and postmenopausal or hysterectomised women (aged 18-65 years) with chronic HCV genotype 1, 3, or 4 infection diagnosed at least 24 weeks before screening who were either treatment naive to or relapsed after interferon-α based therapy. Patients with co-infection (hepatitis B virus or HIV infection), evidence of decompensated liver disease, or a history of hepatocellular carcinoma were excluded. Randomisation was done by an independent, unblinded, statistician using the SAS procedure Proc Plan. The first cohort received one subcutaneous injection of 2 mg/kg RG-101 or placebo; the second cohort received one subcutaneous injection of 4 mg/kg or placebo. Patients were followed up for 8 weeks (all patients) and up to 76 weeks (patients with no viral rebound and excluding those who were randomised to the placebo group) after randomisation. The primary objective was safety and tolerability of RG-101. This trial was registered with EudraCT, number 2013-002978-49. FINDINGS: Between June 4, 2014, and Oct 27, 2014, we enrolled 32 patients with chronic HCV genotype 1 (n=16), 3 (n=10), or 4 (n=6) infections. In the first cohort, 14 patients were randomly assigned to receive 2 mg/kg RG-101 and two patients were randomly assigned to receive placebo, and in the second cohort, 14 patients were randomly assigned to receive 4 mg/kg RG-101 and two patients were randomly assigned to receive placebo. Overall, 26 of the 28 patients dosed with RG-101 reported at least one treatment-related adverse event. At week 4, the median viral load reduction from baseline was 4·42 (IQR 3·23-5·00) and 5·07 (4·19-5·35) log10 IU/mL in patients dosed with 2 mg/kg RG-101 or 4 mg/kg RG-101. Three patients had undetectable HCV RNA levels 76 weeks after a single dose of RG-101. Viral rebound at or before week 12 was associated with the appearance of resistance associated substitutions in miR-122 binding regions in the 5' UTR of the HCV genome. INTERPRETATION: This study showed that one administration of 2 mg/kg or 4 mg/kg RG-101, a hepatocyte targeted N-acetylgalactosamine conjugated anti-miR-122 oligonucleotide, was well tolerated and resulted in substantial viral load reduction in all treated patients within 4 weeks, and sustained virological response in three patients for 76 weeks. FUNDING: Regulus Therapeutics, Inc.
Subject(s)
Hepatitis C, Chronic/drug therapy , MicroRNAs/antagonists & inhibitors , MicroRNAs/therapeutic use , Acetylgalactosamine , Cohort Studies , Double-Blind Method , Female , Humans , Injections, Subcutaneous , Male , MicroRNAs/pharmacokinetics , Middle Aged , Oligonucleotides , Viral Load/drug effectsABSTRACT
UNLABELLED: Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options available to cancer patients. MicroRNA 21-5p (miR-21) has been shown to be upregulated in HCC, but the contribution of this oncomiR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miRNAs) and used them to interrogate dependency on miR-21 in a panel of liver cancer cell lines. Treatment with anti-miR-21, but not with a mismatch control anti-miRNA, resulted in significant derepression of direct targets of miR-21 and led to loss of viability in the majority of HCC cell lines tested. Robust induction of caspase activity, apoptosis, and necrosis was noted in anti-miR-21-treated HCC cells. Furthermore, ablation of miR-21 activity resulted in inhibition of HCC cell migration and suppression of clonogenic growth. To better understand the consequences of miR-21 suppression, global gene expression profiling was performed on anti-miR-21-treated liver cancer cells, which revealed striking enrichment in miR-21 target genes and deregulation of multiple growth-promoting pathways. Finally, in vivo dependency on miR-21 was observed in two separate HCC tumor xenograft models. In summary, these data establish a clear role for miR-21 in the maintenance of tumorigenic phenotype in HCC in vitro and in vivo. IMPLICATIONS: miR-21 is important for the maintenance of the tumorigenic phenotype of HCC and represents a target for pharmacologic intervention.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Cell Proliferation/drug effects , Gene Regulatory Networks/drug effects , Liver Neoplasms/metabolism , MicroRNAs/metabolism , Oligoribonucleotides, Antisense/pharmacology , Animals , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Survival/drug effects , Female , Heterografts , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Mice, Nude , MicroRNAs/antagonists & inhibitors , MicroRNAs/chemistry , Neoplasm Invasiveness , Oligoribonucleotides, Antisense/therapeutic useABSTRACT
BACKGROUND: Prognosis prediction for resected primary colon cancer is based on the T-stage Node Metastasis (TNM) staging system. We investigated if four well-documented gene expression risk scores can improve patient stratification. METHODS: Microarray-based versions of risk-scores were applied to a large independent cohort of 688 stage II/III tumors from the PETACC-3 trial. Prognostic value for relapse-free survival (RFS), survival after relapse (SAR), and overall survival (OS) was assessed by regression analysis. To assess improvement over a reference, prognostic model was assessed with the area under curve (AUC) of receiver operating characteristic (ROC) curves. All statistical tests were two-sided, except the AUC increase. RESULTS: All four risk scores (RSs) showed a statistically significant association (single-test, P < .0167) with OS or RFS in univariate models, but with HRs below 1.38 per interquartile range. Three scores were predictors of shorter RFS, one of shorter SAR. Each RS could only marginally improve an RFS or OS model with the known factors T-stage, N-stage, and microsatellite instability (MSI) status (AUC gains < 0.025 units). The pairwise interscore discordance was never high (maximal Spearman correlation = 0.563) A combined score showed a trend to higher prognostic value and higher AUC increase for OS (HR = 1.74, 95% confidence interval [CI] = 1.44 to 2.10, P < .001, AUC from 0.6918 to 0.7321) and RFS (HR = 1.56, 95% CI = 1.33 to 1.84, P < .001, AUC from 0.6723 to 0.6945) than any single score. CONCLUSIONS: The four tested gene expression-based risk scores provide prognostic information but contribute only marginally to improving models based on established risk factors. A combination of the risk scores might provide more robust information. Predictors of RFS and SAR might need to be different.
Subject(s)
Adenocarcinoma/mortality , Adenocarcinoma/pathology , Biomarkers, Tumor/analysis , Colonic Neoplasms/mortality , Colonic Neoplasms/pathology , Transcriptome , Adenocarcinoma/chemistry , Adult , Aged , Antineoplastic Agents/therapeutic use , Area Under Curve , Colonic Neoplasms/chemistry , Disease-Free Survival , Female , Fixatives , Formaldehyde , Gene Expression Regulation, Neoplastic , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Neoplasm Recurrence, Local/mortality , Neoplasm Recurrence, Local/pathology , Neoplasm Staging , Paraffin , Predictive Value of Tests , Prognosis , Proportional Hazards Models , ROC Curve , Randomized Controlled Trials as Topic , Risk Assessment , Risk Factors , Tissue Array Analysis , Treatment FailureABSTRACT
The clinical efficacy of anti-angiogenic therapies has been difficult to predict, and biomarkers that can predict responsiveness are sorely needed in this era of personalized medicine. CVX-060 is an angiopoietin-2 (Ang2) targeting therapeutic, consisting of two peptides that bind Ang2 with high affinity and specificity, covalently fused to a scaffold antibody. In order to optimize the use of this compound in the clinic the construction of a predictive model is described, based on the efficacy of CVX-060 in 13 cell line and 2 patient-derived xenograft models. Pretreatment size tumors from each of the models were profiled for the levels of 27 protein markers of angiogenesis, SNP haplotype in 5 angiogenesis genes, and somatic mutation status for 11 genes implicated in tumor growth and/or vascularization. CVX-060 efficacy was determined as tumor growth inhibition (TGI%) at termination of each study. A predictive statistical model was constructed based on the correlation of these efficacy data with the marker profiles, and the model was subsequently tested by prospective analysis in 11 additional models. The results reveal a range of CVX-060 efficacy in xenograft models of diverse tissue types (0-64% TGI, median = 27%) and define a subset of 3 proteins (Ang1, EGF, Emmprin), the levels of which may be predictive of TGI by Ang2 blockade. The direction of the associations is such that better efficacy correlates with high levels of target and low levels of compensatory/antagonizing molecules. This effort has revealed a set of candidate predictive markers for CVX-060 efficacy that will be further evaluated in ongoing clinical trials.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Angiopoietin-2/antagonists & inhibitors , Biomarkers, Pharmacological/metabolism , Neoplasms/drug therapy , Peptides/pharmacology , Angiopoietin-1/genetics , Angiopoietin-1/metabolism , Angiopoietin-2/genetics , Angiopoietin-2/metabolism , Animals , Basigin/genetics , Basigin/metabolism , Cell Line, Tumor , Disease Models, Animal , Epidermal Growth Factor/genetics , Epidermal Growth Factor/metabolism , Female , Gene Expression , Gene Expression Profiling , Humans , Mice , Molecular Targeted Therapy , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Polymorphism, Single Nucleotide , Predictive Value of Tests , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Figitumumab (CP-751,871), a potent and fully human monoclonal anti-insulin-like growth factor 1 receptor (IGF1R) antibody, has been investigated in clinical trials of several solid tumors. To identify biomarkers of sensitivity and resistance to figitumumab, its in vitro antiproliferative activity was analyzed in a panel of 93 cancer cell lines by combining in vitro screens with extensive molecular profiling of genomic aberrations. Overall response was bimodal and the majority of cell lines were resistant to figitumumab. Nine of 15 sensitive cell lines were derived from colon cancers. Correlations between genomic characteristics of cancer cell lines with figitumumab antiproliferative activity revealed that components of the IGF pathway, including IRS2 (insulin receptor substrate 2) and IGFBP5 (IGF-binding protein 5), played a pivotal role in determining the sensitivity of tumors to single-agent figitumumab. Tissue-specific differences among the top predictive genes highlight the need for tumor-specific patient selection strategies. For the first time, we report that alteration or expression of the MYB oncogene is associated with sensitivity to IGF1R inhibitors. MYB is dysregulated in hematologic and epithelial tumors, and IGF1R inhibition may represent a novel therapeutic opportunity. Although growth inhibitory activity with single-agent figitumumab was relatively rare, nine combinations comprising figitumumab plus chemotherapeutic agents or other targeted agents exhibited properties of synergy. Inhibitors of the ERBB family were frequently synergistic and potential biomarkers of drug synergy were identified. Several biomarkers of antiproliferative activity of figitumumab both alone and in combination with other therapies may inform the design of clinical trials evaluating IGF1R inhibitors.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/genetics , Receptor, IGF Type 1/antagonists & inhibitors , Cell Line, Tumor , Cell Proliferation/drug effects , Cluster Analysis , DNA Copy Number Variations , Drug Synergism , Gene Expression Profiling , Humans , Inhibitory Concentration 50 , Mutation , Quantitative Trait Loci , Receptor, IGF Type 1/metabolism , Signal TransductionABSTRACT
PURPOSE: We aimed to assess the biologic activity of PF-03084014 in breast xenograft models. The biomarkers for mechanism and patient stratification were also explored. EXPERIMENTAL DESIGN: The in vitro and in vivo properties of PF-03084014 were investigated. The mRNA expressions of 40 key Notch pathway genes at baseline or after treatment were analyzed to link with the antitumor efficacy of PF-03084014 in a panel of breast cancer xenograft models. RESULTS: In vitro, PF-03084014 exhibited activity against tumor cell migration, endothelial cell tube formation, and mammosphere formation. In vivo, we observed apoptosis, antiproliferation, reduced tumor cell self-renewal ability, impaired tumor vasculature, and decreased metastasis activity after the treatment of PF-03084014. PF-03084014 treatment displayed significant antitumor activity in 10 of the 18 breast xenograft models. However, the antitumor efficacy in most models did not correlate with the in vitro antiproliferation results in the corresponding cell lines, suggesting the critical involvement of tumor microenvironment during Notch activation. In the tested breast xenograft models, the baseline expressions of the Notch receptors, ligands, and the cleaved Notch1 failed to predict the antitumor response to PF-03084014, whereas several Notch pathway target genes, including HEY2, HES4, and HES3, strongly corresponded with the response with a P value less than 0.01. Many of the best molecular predictors of response were also significantly modulated following PF-03084014 treatment. CONCLUSIONS: PF-03084014 showed antitumor and antimetastatic properties via pleiotropic mechanisms. The Notch pathway downstream genes may be used to predict the antitumor activity of PF-03084014 and enrich for responders among breast cancer patients.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Biomarkers, Tumor/metabolism , Breast Neoplasms/metabolism , Tetrahydronaphthalenes/pharmacology , Valine/analogs & derivatives , Amyloid Precursor Protein Secretases/metabolism , Antineoplastic Agents/administration & dosage , Biomarkers, Tumor/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Cell Line, Tumor , Cluster Analysis , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Neoplasm Metastasis/drug therapy , Receptors, Notch/metabolism , Signal Transduction/drug effects , Tetrahydronaphthalenes/administration & dosage , Valine/administration & dosage , Valine/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Targeting cancers with amplified or abnormally activated c-Met (hepatocyte growth factor receptor) may have therapeutic benefit based on nonclinical and emerging clinical findings. However, the eventual emergence of drug resistant tumors motivates the pre-emptive identification of potential mechanisms of clinical resistance. We rendered a MET amplified gastric cancer cell line, GTL16, resistant to c-Met inhibition with prolonged exposure to a c-Met inhibitor, PF-04217903 (METi). Characterization of surviving cells identified an amplified chromosomal rearrangement between 7q32 and 7q34 which overexpresses a constitutively active SND1-BRAF fusion protein. In the resistant clones, hyperactivation of the downstream MAPK pathway via SND1-BRAF conferred resistance to c-Met receptor tyrosine kinase inhibition. Combination treatment with METi and a RAF inhibitor, PF-04880594 (RAFi) inhibited ERK activation and circumvented resistance to either single agent. Alternatively, treatment with a MEK inhibitor, PD-0325901 (MEKi) alone effectively blocked ERK phosphorylation and inhibited cell growth. Our results suggest that combination of a c-Met tyrosine kinase inhibitor with a BRAF or a MEK inhibitor may be effective in treating resistant tumors that use activated BRAF to escape suppression of c-Met signaling.
Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Nuclear Proteins/metabolism , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Pyrazines/pharmacology , Recombinant Fusion Proteins/metabolism , Triazoles/pharmacology , Cell Line, Tumor , Drug Resistance, Neoplasm , Endonucleases , Humans , Nuclear Proteins/genetics , Proto-Oncogene Proteins B-raf/genetics , Recombinant Fusion Proteins/geneticsABSTRACT
Several linkage studies provided evidence for the presence of the hereditary prostate cancer locus, HPCX1, at Xq27-q28. The strongest linkage peak of prostate cancer overlies a variable region of ~750 kb at Xq27 enriched by segmental duplications (SDs), suggesting that the predisposition to prostate cancer may be a genomic disorder caused by recombinational interaction between SDs. The large size of SDs and their sequence similarity make it difficult to examine this region for possible rearrangements using standard methods. To overcome this problem, direct isolation of a set of genomic segments by in vivo recombination in yeast (a TAR cloning technique) was used to perform a mutational analysis of the 750 kb region in X-linked families. We did not detect disease-specific rearrangements within this region. In addition, transcriptome and computational analyses were performed to search for nonannotated genes within the Xq27 region, which may be associated with genetic predisposition to prostate cancer. Two candidate genes were identified, one of which is a novel gene termed SPANXL that represents a highly diverged member of the SPANX gene family, and the previously described CDR1 gene that is expressed at a high level in both normal and malignant prostate cells, and mapped 210 kb of upstream the SPANX gene cluster. No disease-specific alterations were identified in these genes. Our results exclude the 750-kb genetically unstable region at Xq27 as a candidate locus for prostate malignancy. Adjacent regions appear to be the most likely candidates to identify the elusive HPCX1 locus.
Subject(s)
Chromosomes, Human, X/genetics , DNA, Neoplasm/genetics , Genetic Loci , Prostatic Neoplasms/genetics , Autoantigens/genetics , Chromosome Mapping , Chromosomes, Human, X/chemistry , DNA Mutational Analysis , Family , Female , Genetic Linkage , Genetic Predisposition to Disease , Humans , Male , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Prostate/metabolism , Prostate/pathology , Prostatic Neoplasms/diagnosis , Recombination, Genetic , Saccharomyces cerevisiae/genetics , Segmental Duplications, GenomicABSTRACT
PF-03814735 is a novel, reversible inhibitor of Aurora kinases A and B that finished a phase I clinical trial for the treatment of advanced solid tumors. To find predictive biomarkers of drug sensitivity, we screened a diverse panel of 87 cancer cell lines for growth inhibition upon PF-03814735 treatment. Small cell lung cancer (SCLC) and, to a lesser extent, colon cancer lines were very sensitive to PF-03814735. The status of the Myc gene family and retinoblastoma pathway members significantly correlated with the efficacy of PF-03814735. Whereas RB1 inactivation, intact CDKN2A/p16, and normal CCND1/Cyclin D1 status are hallmarks of SCLC, activation or amplification of any of the three Myc genes (MYC, MYCL1, and MYCN) clearly differentiated cell line sensitivity within the SCLC panel. By contrast, we found that expression of Aurora A and B were weak predictors of response. We observed a decrease in histone H3 phosphorylation and polyploidization of sensitive lines, consistent with the phenotype of Aurora B inhibition. In vivo experiments with two SCLC xenograft models confirmed the sensitivity of Myc gene-driven models to PF-03814735 and a possible schedule dependence of MYC/c-Myc-driven tumors. Altogether our results suggest that SCLC and other malignancies driven by the Myc family genes may be suitable indications for treatment by Aurora B kinase inhibitors.
Subject(s)
Biomarkers, Tumor/genetics , Heterocyclic Compounds, 3-Ring/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Animals , Antineoplastic Agents/pharmacology , Aurora Kinase A , Aurora Kinase B , Aurora Kinases , Biomarkers, Tumor/metabolism , Blotting, Western , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Gene Expression Profiling , Genomics/methods , Histones/metabolism , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Mice, Nude , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Small Cell Lung Carcinoma/drug therapy , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/metabolism , Xenograft Model Antitumor AssaysABSTRACT
It is a well-established fact that the tRNA genes in yeast can function as chromatin barrier elements. However, so far there is no experimental evidence that tRNA and other Pol III-transcribed genes exhibit barrier activity in mammals. This study utilizes a recently developed reporter gene assay to test a set of Pol III-transcribed genes and gene clusters with variable promoter and intergenic regions for their ability to prevent heterochromatin-mediated reporter gene silencing in mouse cells. The results show that functional copies of mouse tRNA genes are effective barrier elements. The number of tRNA genes as well as their orientation influence barrier function. Furthermore, the DNA sequence composition of intervening and flanking regions affects barrier activity of tRNA genes. Barrier activity was maintained for much longer time when the intervening and flanking regions of tRNA genes were replaced by AT-rich sequences, suggesting a negative role of DNA methylation in the establishment of a functional barrier. Thus, our results suggest that tRNA genes are essential elements in establishment and maintenance of chromatin domain architecture in mammalian cells.
Subject(s)
Epigenesis, Genetic , Gene Silencing , Genes, Reporter/genetics , RNA, Transfer/genetics , RNA, Transfer/metabolism , Animals , Base Sequence , Cell Line, Tumor , Chromatin Immunoprecipitation , DNA Methylation , DNA Polymerase III/genetics , Heterochromatin/metabolism , Mice , Multigene Family , Promoter Regions, Genetic , Sequence Analysis, DNA , Transcription, GeneticABSTRACT
BACKGROUND: Human cancer is caused by the accumulation of tumor-specific mutations in oncogenes and tumor suppressors that confer a selective growth advantage to cells. As a consequence of genomic instability and high levels of proliferation, many passenger mutations that do not contribute to the cancer phenotype arise alongside mutations that drive oncogenesis. While several approaches have been developed to separate driver mutations from passengers, few approaches can specifically identify activating driver mutations in oncogenes, which are more amenable for pharmacological intervention. RESULTS: We propose a new statistical method for detecting activating mutations in cancer by identifying nonrandom clusters of amino acid mutations in protein sequences. A probability model is derived using order statistics assuming that the location of amino acid mutations on a protein follows a uniform distribution. Our statistical measure is the differences between pair-wise order statistics, which is equivalent to the size of an amino acid mutation cluster, and the probabilities are derived from exact and approximate distributions of the statistical measure. Using data in the Catalog of Somatic Mutations in Cancer (COSMIC) database, we have demonstrated that our method detects well-known clusters of activating mutations in KRAS, BRAF, PI3K, and beta-catenin. The method can also identify new cancer targets as well as gain-of-function mutations in tumor suppressors. CONCLUSIONS: Our proposed method is useful to discover activating driver mutations in cancer by identifying nonrandom clusters of somatic amino acid mutations in protein sequences.
Subject(s)
Cluster Analysis , Models, Statistical , Mutation , Neoplasms/genetics , Genes, ras/genetics , Genome, Human , Humans , Proto-Oncogene Proteins B-raf/genetics , beta Catenin/geneticsABSTRACT
The role of repetitive DNA sequences in pericentromeric regions with respect to kinetochore/heterochromatin structure and function is poorly understood. Here, we use a mouse erythroleukemia cell (MEL) system for studying how repetitive DNA assumes or is assembled into different chromatin structures. We show that human gamma-satellite DNA arrays allow a transcriptionally permissive chromatin conformation in an adjacent transgene and efficiently protect it from epigenetic silencing. These arrays contain CTCF and Ikaros binding sites. In MEL cells, this gamma-satellite DNA activity depends on binding of Ikaros proteins involved in differentiation along the hematopoietic pathway. Given our discovery of gamma-satellite DNA in pericentromeric regions of most human chromosomes and a dynamic chromatin state of gamma-satellite arrays in their natural location, we suggest that gamma-satellite DNA represents a unique region of the functional centromere with a possible role in preventing heterochromatin spreading beyond the pericentromeric region.
Subject(s)
Chromatin/chemistry , DNA, Satellite/genetics , Epigenesis, Genetic , Gene Silencing , Transgenes/physiology , Animals , Binding Sites , CCCTC-Binding Factor , Centromere/genetics , Chromatin/genetics , Chromatin Immunoprecipitation , Chromosomes, Human/genetics , DNA, Satellite/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Genetic Vectors , Humans , Ikaros Transcription Factor/genetics , Ikaros Transcription Factor/metabolism , Leukemia, Erythroblastic, Acute/genetics , Leukemia, Erythroblastic, Acute/metabolism , Luciferases/metabolism , Mice , Phylogeny , Promoter Regions, Genetic , Repetitive Sequences, Nucleic Acid/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Tumor Cells, CulturedABSTRACT
Mismatch repair detection (MRD) was used to screen 93 matched tumor-normal sample pairs and 22 cell lines for somatic mutations in 30 cancer relevant genes. Using a starting amount of only 150 ng of genomic DNA, we screened 102 kb of sequence for somatic mutations in colon and breast cancer. A total of 152 somatic mutations were discovered, encompassing previously reported mutations, such as BRAF V600E and KRAS G12S, G12V, and G13D, as well as novel mutations, including some in genes in which somatic mutations have not previously been reported, such as MAP2K1 and MAP2K2. The distribution of mutations ranged widely within and across tumor types. The functional significance of many of these mutations is not understood, with patterns of selection only evident in KRAS and BRAF in colon cancer. These results present a novel approach to high-throughput mutation screening using small amounts of starting material and reveal a mutation spectrum across 30 genes in a large cohort of breast and colorectal cancers.
Subject(s)
Breast Neoplasms/genetics , Colorectal Neoplasms/genetics , DNA Mismatch Repair , DNA Mutational Analysis/methods , Mutation , Base Sequence , Cell Line, Tumor , DNA, Neoplasm/genetics , Female , Humans , MaleABSTRACT
The sperm protein associated with nucleus in the X chromosome (SPANX) genes cluster at Xq27 in two subfamilies, SPANX-A/D and SPANX-N. SPANX-A/D is specific for hominoids and is fairly well characterized. The SPANX-N gave rise to SPANX-A/D in the hominoid lineage approximately 7 MYA. Given the proposed role of SPANX genes in spermatogenesis, we have extended studies to SPANX-N gene evolution, variation, regulation of expression, and intra-sperm localization. By immunofluorescence analysis, SPANX-N proteins are localized in post-meiotic spermatids exclusively, like SPANX-A/D. But in contrast to SPANX-A/D, SPANX-N are found in all ejaculated spermatozoa rather than only in a subpopulation, are localized in the acrosome rather than in the nuclear envelope, and are expressed at a low level in several nongametogenic adult tissues as well as many cancers. Presence of a binding site for CTCF and its testis-specific paralogue BORIS in the SPANX promoters suggests, by analogy to MAGE-A1 and NY-ESO-1, that their activation in spermatogenesis is mediated by the programmed replacement of CTCF by BORIS. Based on the relative density of CpG, the more extended expression of SPANX-N compared to SPANX-A/D in nongametogenic tissues is likely attributed to differences in promoter methylation. Our findings suggest that the recent duplication of SPANX genes in hominoids was accompanied by different localization of SPANX-N proteins in post-meiotic sperm and additional expression in several nongonadal tissues. This suggests a corresponding functional diversification of SPANX gene families in hominoids. SPANX proteins thus provide unique targets to investigate their roles in the function of spermatozoa, selected malignancies, and for SPANX-N, in other tissues as well.