ABSTRACT
Human genetics research has discovered thousands of proteins associated with complex and rare diseases. Genome-wide association studies (GWAS) and studies of Mendelian disease have resulted in an increased understanding of the role of gene function and regulation in human conditions. Although the application of human genetics has been explored primarily as a method to identify potential drug targets and support their relevance to disease in humans, there is increasing interest in using genetic data to identify potential safety liabilities of modulating a given target. Human genetic variants can be used as a model to anticipate the effect of lifelong modulation of therapeutic targets and identify the potential risk for on-target adverse events. This approach is particularly useful for non-clinical safety evaluation of novel therapeutics that lack pharmacologically relevant animal models and can contribute to the intrinsic safety profile of a drug target. This Review illustrates applications of human genetics to safety studies during drug discovery and development, including assessing the potential for on- and off-target associated adverse events, carcinogenicity risk assessment, and guiding translational safety study designs and monitoring strategies. A summary of available human genetic resources and recommended best practices is provided. The challenges and future perspectives of translating human genetic information to identify risks for potential drug effects in preclinical and clinical development are discussed.
Subject(s)
Genome-Wide Association Study , Human Genetics , Animals , HumansABSTRACT
Purpose: This phase I/II single-arm study evaluated the safety, pharmacokinetics, pharmacodynamics, and activity of foretinib, an oral multikinase inhibitor of MET, ROS, RON, AXL, TIE-2, and VEGFR2, in the first-line setting in advanced hepatocellular carcinoma patients.Experimental Design: In the phase I part, advanced hepatocellular carcinoma patients were dose escalated on foretinib (30-60 mg) every day using the standard 3+3 design. Once the maximum tolerated dose (MTD) was determined, an additional 32 patients were dosed at the MTD in the phase II expansion cohort for assessment of efficacy and safety. Exploratory analyses were conducted to assess potential biomarkers that might correlate with clinical efficacy and survival.Results: The MTD of foretinib was established as 30 mg every day. The most frequent adverse events were hypertension, decreased appetite, ascites, and pyrexia. When dosed at 30 mg every day in the first-line setting, foretinib demonstrated promising antitumor activity. According to the modified mRECIST, the objective response rate was 22.9%, the disease stabilization rate 82.9%, and the median duration of response 7.6 months. The median time to progression was 4.2 months and the median overall survival (OS) was 15.7 months. Fifteen candidate biomarkers whose levels in the circulation were significantly altered in response to foretinib treatment were elucidated. Multivariate analyses identified IL6 and IL8 as independent predictors of OS.Conclusions: Foretinib demonstrated promising antitumor activity and good tolerability in the first-line setting in Asian advanced hepatocellular carcinoma patients. Baseline plasma levels of IL6 or IL8 might predict the response to foretinib. Clin Cancer Res; 23(10); 2405-13. ©2016 AACR.
Subject(s)
Anilides/administration & dosage , Carcinoma, Hepatocellular/drug therapy , Interleukin-6/blood , Interleukin-8/blood , Liver Neoplasms/drug therapy , Quinolines/administration & dosage , Adult , Aged , Anilides/pharmacokinetics , Biomarkers, Pharmacological/blood , Carcinoma, Hepatocellular/blood , Carcinoma, Hepatocellular/pathology , Female , Humans , Liver Neoplasms/blood , Liver Neoplasms/pathology , Male , Maximum Tolerated Dose , Middle Aged , Protein-Tyrosine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Quinolines/pharmacokinetics , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor, TIE-2/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Axl Receptor Tyrosine KinaseABSTRACT
AIM: Pyrexia is a common adverse event (AE) on dabrafenib treatment (monotherapy or combination with trametinib). Since germline SNPs and HLA alleles are implicated in drug-induced AEs, this study investigated their association with pyrexia. PATIENTS & METHODS: 1006 melanoma subjects from five dabrafenib-trametinib clinical studies underwent genotyping for genome-wide SNPs, which enabled imputation of 150 HLA alleles. SNP/HLA allele frequencies were compared between pyrexia cases (n = 218) and controls (n = 361) out of the 1006 subjects by meta-analysis. RESULTS: This analysis had adequate power to detect association of common SNPs or HLA alleles with moderate to large effects on pyrexia (odds ratio >6), but no significant association was found. CONCLUSION: The study suggests that common genetic variation or HLA polymorphisms do not contribute substantially to dabrafenib-induced pyrexia.
Subject(s)
Antineoplastic Agents/adverse effects , Fever/chemically induced , HLA Antigens/genetics , Imidazoles/adverse effects , Melanoma/drug therapy , Oximes/adverse effects , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Case-Control Studies , Fever/genetics , Genetic Association Studies , Humans , Melanoma/genetics , Polymorphism, Single Nucleotide , Proto-Oncogene Proteins B-raf/geneticsABSTRACT
TSC1 acts coordinately with TSC2 in a complex to inhibit mTOR, an emerging therapeutic target and known promoter of cell growth and cell cycle progression. Perturbation of the mTOR pathway, through abnormal expression or function of pathway genes, could lead to tumorigenesis. TSC1 and TSC2 expression is reduced in invasive breast cancer as compared with normal mammary epithelium. Because single nucleotide polymorphisms (SNPs) in regulatory genes have been implicated in risk and age at diagnosis of breast cancers, systematic SNP association studies were performed on TSC1 and TSC2 SNPs for their associations with clinical features of breast cancer. TSC1 and TSC2 haplotypes were constructed from genotyping of multiple loci in both genes in healthy volunteers. SNPs were selected for further study using a bioinformatics approach based on SNP associations with drug response in NCI-60 cell lines and evidence of selection bias based on haplotype frequencies. Genotyping for five TSC1 and one TSC2 loci were performed on genomic DNA from 1,137 women with breast cancer. This study found that for TSC1 rs7874234, TT variant carriers had a 9-year later age at diagnosis of estrogen receptor positive (ER+), but not ER-, ductal carcinomas (P = 0.0049). No other SNP locus showed an association with age at diagnosis, nor any other breast cancer phenotype. TSC1 rs7874234 is hypothesized to be functional in ER+ breast cancer because the T allele, but not the C allele, may create an estrogen receptor element (ERE) site, resulting in increased TSC1 transcription and subsequent inhibition of mTOR.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Polymorphism, Genetic , Tumor Suppressor Proteins/genetics , Alleles , Carcinoma, Ductal/genetics , Computational Biology/methods , Estrogen Receptor alpha/biosynthesis , Female , Genotype , Haplotypes , Humans , Phenotype , Time Factors , Tuberous Sclerosis Complex 1 Protein , Tuberous Sclerosis Complex 2 ProteinABSTRACT
The mechanism by which gastroesophageal reflux promotes metaplasiaâdysplasiaâcarcinoma is unknown. The aim of the study is to determine if repeated exposure to acid and bile confers a tumorigenic phenotype in a telomerase (hTERT)-immortalized benign Barrett's cell line (BAR-T). BAR-T cells were exposed to acid (pH 4) (A) and bile salt (200 µM glycochenodeoxycholic acid) (B) daily for 5 min up to 65+ weeks. The control cells were grown in parallel without any A or B treatment. Cell morphology, proliferation, transformation, and molecular changes in the gene expression for COX-2, TC22, p53 and p53 target genes were analyzed at 8-12 weeks intervals. At 46 weeks BAR-T cells exposed to (A+B) showed distinct phenotypic changes: forming clusters and acini, and at 65 weeks displayed foci in monolayer, and formed distinct colonies in soft agar. Untreated cells did not show any such changes. In A+B-treated BAR-T cells, COX-2 mRNA increased 10- to 20-fold, TC22 mRNA increased by 2- to 3-fold at 22-65 weeks, p53, MDM2, PERP, and p21mRNA increased 2.5-, 6.4-, 4-, and 2.6-fold respectively when compared to untreated cells at 34 weeks. However, at 58 weeks onward, there was a sharp decline of p53 and its target genes to the baseline level. At 65 weeks A+B-treated BAR-T cells formed tumor in nude mice whereas untreated cells did not. We demonstrate a novel in vitro model of transformation of a benign Barrett's cell line following repeated exposure to A+B over the course of 65 weeks.
Subject(s)
Barrett Esophagus/pathology , Bile/physiology , Cell Transformation, Neoplastic , Esophageal Neoplasms/etiology , Hydrochloric Acid/toxicity , Animals , Cell Line , Cell Proliferation , Cyclooxygenase 2/genetics , Esophageal Neoplasms/pathology , Genes, p53 , Humans , Mice , Time FactorsABSTRACT
A recent candidate gene association study identified a single nucleotide polymorphism (SNP) in the PPP2R2B gene (rs319217, A/G) that manifests allelic differences in the cellular responses to treatment with chemotherapeutic agents (Vazquez et al., Nat Rev Drug Discov 2008;7:979-87). This gene encodes a regulatory subunit of protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases implicated in the negative control of cell growth and division. Given the tumor suppressor activities of PP2A, here we evaluate whether this genetic variant associates with the age of diagnosis and recurrence of breast cancer in women. To investigate the linkage disequilibrium in the vicinity of this SNP, PPP2R2B haplotypes were analyzed using HapMap data for 90 Caucasians. It is found that the A variant of rs319217 tags a haplotype that appears tobe under positive selection in the Caucasian population, implying that this SNP is functional. Subsequently, associations with cellular responses were investigated using data reported by the NCI anticancer drug screen and associations with breast cancer clinical variables were analyzed in a cohort of 819 Caucasian women. The A allele associates with a better response of tumor derived cell lines, lower risk of breast cancer recurrence, later time to recurrence, and later age of diagnosis of breast cancer in Caucasian women. Taken together these results indicate that the A variant of the rs319217 SNP is a marker of better prognosis in breast cancer.
Subject(s)
Breast Neoplasms/genetics , Genetic Predisposition to Disease , Neoplasm Recurrence, Local , Nerve Tissue Proteins/genetics , Protein Phosphatase 2/genetics , Adult , Age of Onset , Aged , Aged, 80 and over , Alleles , Antineoplastic Agents/pharmacology , Breast Neoplasms/pathology , Cell Line, Tumor , Cohort Studies , Female , Haplotypes , Humans , Linkage Disequilibrium , Middle Aged , Polymorphism, Single NucleotideABSTRACT
PURPOSE: TP53BP1 is a key component of radiation-induced deoxyribonucleic acid damage repair. The purpose of this study was to evaluate the significance of a known common single nucleotide polymorphism in this gene (rs560191) in patients treated with breast-conserving surgery and whole-breast irradiation (BCS + RT). METHODS AND MATERIALS: The population consisted of 176 premenopausal women treated with BCS + RT (median follow-up, 12 years). Genomic deoxyribonucleic acid was processed by use of TaqMan assays. Each allele for rs560191 was either C or G, so each patient was therefore classified as CC, CG, or GG. Patients were grouped as GG if they were homozygous for the variant G allele or CC-CG if they carried at least one copy of the common C allele (CC or CG). RESULTS: Of the 176 women, 124 (71%) were CC-CG and 52 (29%) were GG. The mean age was 44 years for GG vs. 38 years for CC-CG (p < 0.001). GG was more common in African-American women than white women (69% vs. 13%, p < 0.001) and more commonly estrogen receptor negative (70% vs. 49%, p = 0.02). There were no significant correlations of rs560191 with other critical variables. Despite the fact that GG patients were older, the 10-year rate of local relapses was higher (22% for GG vs. 12% for CC-CG, p = 0.04). CONCLUSIONS: This novel avenue of investigation of polymorphisms in radiation repair/response genes in patients treated with BCS + RT suggests a correlation to local relapse. Additional evaluation is needed to assess the biological and functional significance of these single nucleotide polymorphisms, and larger confirmatory validation studies will be required to determine the clinical implications.
Subject(s)
Breast Neoplasms/genetics , Intracellular Signaling Peptides and Proteins/genetics , Neoplasm Proteins/genetics , Neoplasm Recurrence, Local/genetics , Polymorphism, Single Nucleotide , Adult , Alleles , Breast Neoplasms/ethnology , Breast Neoplasms/radiotherapy , Breast Neoplasms/surgery , Combined Modality Therapy/methods , DNA Damage/genetics , DNA Repair/genetics , Female , Genes, BRCA1 , Genes, BRCA2 , Humans , Linkage Disequilibrium , Mastectomy, Segmental , Premenopause , Tumor Suppressor p53-Binding Protein 1ABSTRACT
Murine double minute 4 (MDM4) shares significant structural homology with murine double minute 2 (MDM2) and interacts and regulates transcriptional activity of the tumor suppressor p53. In tumors with wild-type p53, there is often overexpression of MDM2 or MDM4 leading to functional inactivation of p53. A single-nucleotide polymorphism (SNP) in the promoter of human MDM2 (SNP309) was shown to associate with increased MDM2 expression and increased risk of cancer. This study evaluated the association of a SNP in human MDM4 (C>T) with age of onset of breast cancer in two independent cohorts. In cohort 1 of 675 patients, the average age of diagnosis for women with estrogen receptor (ER)-positive and ER-negative breast cancers was 53.2 and 48 years, respectively. In this cohort, homozygous variant (TT) carriers developed ER-negative carcinomas at an earlier age than homozygous wild-type (CC) or heterozygous (TC) such that the age at diagnosis was accelerated by 5.0 years (P = 0.018). This association was validated in a second cohort of breast cancer patients (n = 148), where TT carriers with ER-negative cancer developed the disease 3.8 years earlier than CC carriers (P = 0.006). The effect was more pronounced in Caucasians with ER-negative ductal carcinomas with TT homozygotes developing disease 7.5 years (P = 0.031) and 6.2 years (P = 7 x 10(-5)) earlier than CC carriers in cohorts 1 and 2, respectively. No association was seen in ER-positive ductal cancers suggesting that the SNP in MDM4 only has a functional association in ER-negative breast cancer.
Subject(s)
Age of Onset , Breast Neoplasms/genetics , Carcinoma, Ductal/genetics , Nuclear Proteins/genetics , Polymorphism, Single Nucleotide , Proto-Oncogene Proteins/genetics , Adult , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/physiopathology , Carcinoma, Ductal/metabolism , Carcinoma, Ductal/pathology , Carcinoma, Ductal/physiopathology , Cell Cycle Proteins , Cohort Studies , Female , Genetic Predisposition to Disease , Humans , Middle Aged , Receptors, Estrogen/metabolism , Tumor Suppressor Protein p53/geneticsABSTRACT
TOPORS is the first example of a protein that possesses both ubiquitin and SUMO E3 ligase activity. The ubiquitination activity maps to a conserved RING domain in the N-terminal region of the protein, which is not required for sumoylation activity. Similar to other E3 ligases, it is likely that the ubiquitin and sumoylation activities of TOPORS are regulated by post-translational modifications. Therefore, we employed mass spectrometry to identify post-translational modifications of TOPORS. Several putative phosphorylated regions were identified in conserved regions of the protein. We investigated the role of phosphorylation of serine 98, which is adjacent to the RING domain, in both cells and in vitro. Mutation of serine 98 to aspartic acid resulted in an increase in the ubiquitin ligase activity of TOPORS both in cells and in vitro. In addition, this mutation increased the binding of TOPORS to the E2 enzyme UbcH5a both in vitro and in cells. Conversely, a phospho-deficient mutant (S98A) exhibited little change in ubiquitin ligase activity compared to wild-type TOPORS, both in cells and in vitro. Neither of the mutants affected the localization of TOPORS to punctate nuclear regions. In addition, neither mutant affected the SUMO ligase activity of TOPORS in cells or in vitro. Molecular modeling studies support a role for serine 98 in regulating TOPORS-E2 interactions. Our findings indicate that phosphorylation of serine 98 regulates the ubiquitin but not the SUMO ligase activity of TOPORS, consistent with a potential binary switch function for TOPORS in protein ubiquitination versus sumoylation.
Subject(s)
Neoplasm Proteins/metabolism , Nuclear Proteins/metabolism , Serine , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Binding Sites , Cell Line, Tumor , Humans , Mass Spectrometry , Mutation, Missense , Phosphorylation , Protein Binding/genetics , Protein Processing, Post-Translational , Small Ubiquitin-Related Modifier Proteins/metabolismABSTRACT
Clinically relevant resistance to the currently approved camptothecins, irinotecan and topotecan, is poorly understood but may involve increased expression of ATP-dependent drug transporters such as ABCG2 (breast cancer resistant protein, BCRP). Gimatecan (ST1481) is a lipophilic 7-substituted camptothecin derivative that exhibits potent anti-tumor activity in a variety of preclinical cancer models and is under investigation in the clinic. Previous studies reported that gimatecan cytotoxicity was not affected by expression of ABCG2. To confirm and extend this finding, we assessed the cytotoxicity of gimatecan in pairs of isogenic cell lines consisting of transfectants expressing either ABCG2 (including wild-type, R482T, or R482G mutants), ABCB1 (P-glycoprotein), ABCC1 (MRP1), ABCC2 (MRP2), or ABCC4 (MRP4). Expression of wild-type or mutant ABCG2 in human cell lines conferred resistance to topotecan but not to gimatecan. Similarly, intracellular accumulation of gimatecan was unaffected by expression of wild-type ABCG2. Furthermore, expression of P-glycoprotein or MRP2 did not alter gimatecan cytotoxicity. Whereas expression of MRP1 had a minor effect on gimatecan cytotoxicity, expression of ABCC4 was found to significantly reduce the anti-proliferative effects of this drug. Cells containing resistance-conferring mutations in topoisomerase I were also resistant to gimatecan. These results suggest that gimatecan may be more effective than irinotecan or topotecan in cancers that express ABCG2, but not in cancers that express high levels of ABCC4 or contain certain topoisomerase I (TOP1) mutations.
Subject(s)
Antineoplastic Agents/pharmacology , Camptothecin/analogs & derivatives , Drug Resistance, Neoplasm , Multidrug Resistance-Associated Proteins/metabolism , Animals , Camptothecin/pharmacology , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Topoisomerases, Type I/genetics , DNA Topoisomerases, Type I/metabolism , Dogs , Gene Expression , Humans , Irinotecan , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/genetics , Mutation , Topotecan/pharmacology , TransfectionABSTRACT
TOPORS is the first example of a protein with both ubiquitin and SUMO-1 E3 ligase activity and has been implicated as a tumor suppressor in several different malignancies. To gain insight into the cellular role of TOPORS, a proteomic screen was performed to identify candidate sumoylation substrates. The results indicate that many of the putative substrates are involved in chromatin modification or transcriptional regulation. Transfection studies confirmed mammalian Sin3A as a sumoylation substrate for TOPORS. These findings suggest that TOPORS may function as a tumor suppressor by regulating mSin3A and other proteins involved in chromatin modification.