RESUMEN
Selecting the most appropriate protein sequences is critical for precision drug design. Here we describe Haplosaurus, a bioinformatic tool for computation of protein haplotypes. Haplosaurus computes protein haplotypes from pre-existing chromosomally-phased genomic variation data. Integration into the Ensembl resource provides rapid and detailed protein haplotypes retrieval. Using Haplosaurus, we build a database of unique protein haplotypes from the 1000 Genomes dataset reflecting real-world protein sequence variability and their prevalence. For one in seven genes, their most common protein haplotype differs from the reference sequence and a similar number differs on their most common haplotype between human populations. Three case studies show how knowledge of the range of commonly encountered protein forms predicted in populations leads to insights into therapeutic efficacy. Haplosaurus and its associated database is expected to find broad applications in many disciplines using protein sequences and particularly impactful for therapeutics design.
Asunto(s)
Biología Computacional/métodos , Diseño de Fármacos , Haplotipos , Medicina de Precisión/métodos , Proteínas/genética , Diseño Asistido por Computadora , Genoma Humano/genética , Genómica/métodos , Humanos , Proteoma/genética , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
PURPOSE: Antibody-drug conjugates (ADC) utilizing noncleavable linker drugs have been approved for clinical use, and several are in development targeting solid and hematologic malignancies including multiple myeloma. Currently, there are no reliable biomarkers of activity for these ADCs other than presence of the targeted antigen. We observed that certain cell lines are innately resistant to such ADCs, and sought to uncover the underlying mechanism of resistance. EXPERIMENTAL DESIGN: The expression of 43 lysosomal membrane target genes was evaluated in cell lines resistant to ADCs bearing the noncleavable linker, pyrrolobenzodiazepine payload SG3376, in vitro. The functional relevance of SLC46A3, a lysosomal transporter of noncleavable ADC catabolites whose expression uniquely correlated with SG3376 resistance, was assessed using EPHA2-, HER2-, and BCMA-targeted ADCs and isogenic cells overexpressing or genetically inactivated for SLC46A3. SLC46A3 expression was also examined in patient-derived xenograft and in vitro models of acquired T-DM1 resistance and multiple myeloma bone marrow samples by RT-PCR. RESULTS: Loss of SLC46A3 expression was found to be a mechanism of innate and acquired resistance to ADCs bearing DM1 and SG3376. Sensitivity was restored in refractory lines upon introduction of SLC46A3, suggesting that expression of SLC46A3 may be more predictive of activity than target antigen levels alone. Interrogation of primary multiple myeloma samples indicated a range of SLC46A3 expression, including samples with undetectable levels like multiple myeloma cell lines resistant to BCMA-targeting DM1 and SG3376 ADCs. CONCLUSIONS: Our findings support SLC46A3 as a potential patient selection biomarker with immediate relevance to clinical trials involving these ADCs.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Benzodiazepinas/farmacología , Biomarcadores , Inmunoconjugados/farmacología , Maitansina/farmacología , Pirroles/farmacología , Animales , Antineoplásicos Inmunológicos/química , Benzodiazepinas/química , Línea Celular Tumoral , Modelos Animales de Enfermedad , Expresión Génica , Silenciador del Gen , Humanos , Inmunoconjugados/química , Maitansina/química , Melanoma Experimental , Ratones , Pirroles/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We examined DNA methylation throughout the endogenous murine testis-specific phosphoglycerate kinase (Pgk2) gene and in human PGK2 promoter/CAT reporter transgenes in mouse spermatogenic cells before, during, and following the period of active transcription of this gene. We observed the gradual development of a domain of demethylation beginning over the promoter and then expanding approximately 1 kilobase in each direction within the endogenous Pgk2 gene. This demethylation domain develops in the absence of DNA replication and precedes other molecular changes that potentiate tissue-specific activation of this gene. Studies with transgenes show that a signal residing in the Pgk2 core promoter directs this gene-, cell type-, and stage-specific demethylation process. These results are consistent with a model in which regulated, tissue- and gene-specific demethylation initiates a cascade of subsequent molecular events required for tissue-specific activation of transcription during spermatogenesis in vivo.
Asunto(s)
Metilación de ADN , Regulación del Desarrollo de la Expresión Génica/fisiología , Regiones Promotoras Genéticas/fisiología , Testículo/embriología , Testículo/fisiología , Animales , Islas de CpG/fisiología , Epigénesis Genética/fisiología , Isoenzimas/genética , Masculino , Ratones , Ratones Transgénicos , Fosfoglicerato Quinasa/genética , Espermatogénesis/fisiología , Activación Transcripcional/fisiología , Transgenes/fisiologíaRESUMEN
Establishment and maintenance of differential chromatin structure between transcriptionally competent and repressed genes are critical aspects of transcriptional regulation. The elements and mechanisms that mediate formation and maintenance of these chromatin states in vivo are not well understood. To examine the role of the promoter in maintaining chromatin structure and DNA methylation patterns of the transcriptionally active X-linked HPRT locus, 323 bp of the endogenous human HPRT promoter (from position -222 to +102 relative to the translation start site) was replaced by plasmid sequences by homologous recombination in cultured HT-1080 male fibrosarcoma cells. The targeted cells, which showed no detectable HPRT transcription, were then assayed for effects on DNase I hypersensitivity, general DNase I sensitivity, and DNA methylation patterns across the HPRT locus. In cells carrying the deletion, significantly diminished DNase I hypersensitivity in the 5' flanking region was observed compared to that in parental HT-1080 cells. However, general DNase I sensitivity and DNA methylation patterns were found to be very similar in the mutated cells and in the parental cells. These findings suggest that the promoter and active transcription play a relatively limited role in maintaining transcriptionally potentiated epigenetic states.