RESUMEN
While acute myeloid leukemia (AML) comprises many disparate genetic subtypes, one shared hallmark is the arrest of leukemic myeloblasts at an immature and self-renewing stage of development. Therapies that overcome differentiation arrest represent a powerful treatment strategy. We leveraged the observation that the majority of AML, despite their genetically heterogeneity, share in the expression of HoxA9, a gene normally downregulated during myeloid differentiation. Using a conditional HoxA9 model system, we performed a high-throughput phenotypic screen and defined compounds that overcame differentiation blockade. Target identification led to the unanticipated discovery that inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) enables myeloid differentiation in human and mouse AML models. In vivo, DHODH inhibitors reduced leukemic cell burden, decreased levels of leukemia-initiating cells, and improved survival. These data demonstrate the role of DHODH as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML.
Asunto(s)
Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Terapia Molecular Dirigida , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Diferenciación Celular , Dihidroorotato Deshidrogenasa , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento , Proteínas de Homeodominio/genética , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Células Mieloides/patología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Pirimidinas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.
Asunto(s)
Descubrimiento de Drogas , Bibliotecas de Moléculas Pequeñas , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , National Institutes of Health (U.S.) , Estados UnidosRESUMEN
Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFß-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.
Asunto(s)
Glutatión Peroxidasa/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Cadherinas/metabolismo , Muerte Celular , Línea Celular Tumoral , Linaje de la Célula , Transdiferenciación Celular , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal , Humanos , Hierro/metabolismo , Peróxidos Lipídicos/metabolismo , Masculino , Melanoma/tratamiento farmacológico , Melanoma/enzimología , Melanoma/metabolismo , Melanoma/patología , Mesodermo/efectos de los fármacos , Mesodermo/enzimología , Mesodermo/metabolismo , Mesodermo/patología , Neoplasias/genética , Neoplasias/patología , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteómica , Proteínas Proto-Oncogénicas B-raf/genética , Reproducibilidad de los Resultados , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genéticaRESUMEN
Changes in cellular gene expression in response to small-molecule or genetic perturbations have yielded signatures that can connect unknown mechanisms of action (MoA) to ones previously established. We hypothesized that differential basal gene expression could be correlated with patterns of small-molecule sensitivity across many cell lines to illuminate the actions of compounds whose MoA are unknown. To test this idea, we correlated the sensitivity patterns of 481 compounds with â¼19,000 basal transcript levels across 823 different human cancer cell lines and identified selective outlier transcripts. This process yielded many novel mechanistic insights, including the identification of activation mechanisms, cellular transporters and direct protein targets. We found that ML239, originally identified in a phenotypic screen for selective cytotoxicity in breast cancer stem-like cells, most likely acts through activation of fatty acid desaturase 2 (FADS2). These data and analytical tools are available to the research community through the Cancer Therapeutics Response Portal.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Aflatoxinas/química , Aflatoxinas/farmacología , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Simulación por Computador , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Estructura Molecular , Análisis de Componente Principal , Reacción en Cadena en Tiempo Real de la PolimerasaAsunto(s)
Investigación Biomédica/métodos , Difusión de la Información/ética , Sondas Moleculares/química , Bibliotecas de Moléculas Pequeñas/química , Investigación Biomédica/instrumentación , Humanos , Propiedad Intelectual , Internet , Sondas Moleculares/farmacología , Peso Molecular , Sensibilidad y Especificidad , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Chordoma is a primary bone cancer with no approved therapy1. The identification of therapeutic targets in this disease has been challenging due to the infrequent occurrence of clinically actionable somatic mutations in chordoma tumors2,3. Here we describe the discovery of therapeutically targetable chordoma dependencies via genome-scale CRISPR-Cas9 screening and focused small-molecule sensitivity profiling. These systematic approaches reveal that the developmental transcription factor T (brachyury; TBXT) is the top selectively essential gene in chordoma, and that transcriptional cyclin-dependent kinase (CDK) inhibitors targeting CDK7/12/13 and CDK9 potently suppress chordoma cell proliferation. In other cancer types, transcriptional CDK inhibitors have been observed to downregulate highly expressed, enhancer-associated oncogenic transcription factors4,5. In chordoma, we find that T is associated with a 1.5-Mb region containing 'super-enhancers' and is the most highly expressed super-enhancer-associated transcription factor. Notably, transcriptional CDK inhibition leads to preferential and concentration-dependent downregulation of cellular brachyury protein levels in all models tested. In vivo, CDK7/12/13-inhibitor treatment substantially reduces tumor growth. Together, these data demonstrate small-molecule targeting of brachyury transcription factor addiction in chordoma, identify a mechanism of T gene regulation that underlies this therapeutic strategy, and provide a blueprint for applying systematic genetic and chemical screening approaches to discover vulnerabilities in genomically quiet cancers.
Asunto(s)
Cordoma/metabolismo , Proteínas Fetales/metabolismo , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/metabolismo , Proliferación Celular/efectos de los fármacos , Cordoma/genética , Cordoma/patología , Quinasas Ciclina-Dependientes/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Genes Esenciales , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
UNLABELLED: Identifying genetic alterations that prime a cancer cell to respond to a particular therapeutic agent can facilitate the development of precision cancer medicines. Cancer cell-line (CCL) profiling of small-molecule sensitivity has emerged as an unbiased method to assess the relationships between genetic or cellular features of CCLs and small-molecule response. Here, we developed annotated cluster multidimensional enrichment analysis to explore the associations between groups of small molecules and groups of CCLs in a new, quantitative sensitivity dataset. This analysis reveals insights into small-molecule mechanisms of action, and genomic features that associate with CCL response to small-molecule treatment. We are able to recapitulate known relationships between FDA-approved therapies and cancer dependencies and to uncover new relationships, including for KRAS-mutant cancers and neuroblastoma. To enable the cancer community to explore these data, and to generate novel hypotheses, we created an updated version of the Cancer Therapeutic Response Portal (CTRP v2). SIGNIFICANCE: We present the largest CCL sensitivity dataset yet available, and an analysis method integrating information from multiple CCLs and multiple small molecules to identify CCL response predictors robustly. We updated the CTRP to enable the cancer research community to leverage these data and analyses.
Asunto(s)
Biología Computacional/métodos , Resistencia a Antineoplásicos/genética , Ensayos de Selección de Medicamentos Antitumorales , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias/genética , Bibliotecas de Moléculas Pequeñas , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Análisis por Conglomerados , Conjuntos de Datos como Asunto , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Mutación , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
To address questions of protein stability, researchers have increasingly turned to combinatorial approaches that permit the rapid analysis of libraries of protein variants. Phage-display has proved to be a powerful tool for analyzing protein stability due to the large library size and the robustness of the phage particle to a variety of denaturing conditions. With the B1 domain of protein G (GB1) and a camelid heavy chain antibody as model systems, we are using phage-display libraries to experimentally address questions that have generally been addressed in silico, either through computational studies or statistical analysis of known protein structures. One effort has focused on identifying novel solutions to repacking the hydrophobic core of GB1, while maintaining stability comparable to the wild type protein. In a second study, a small set of substitutions in complimentarity-determining region 3 was found to stabilize the framework of the camelid antibody. Another major focus has been to obtain quantitative data on beta-sheet stability determinants. We have successfully adapted a phage-display method for quantitating affinities of protein variants (shotgun alanine scanning) to analysis of GB1 stability. Using this method, we have analyzed the energetic contributions of cross-strand side chain-side chain interactions. Finally, we discuss parameters to consider in using phage-display to discriminate subtle stability differences among fully folded variants. Overall, this method provides a fast approach for quantitatively addressing biophysical questions.