RESUMEN
An emerging therapeutic strategy for cancer is to induce selective lethality in a tumor by exploiting interactions between its driving mutations and specific drug targets. Here we use a multi-species approach to develop a resource of synthetic lethal interactions relevant to cancer therapy. First, we screen in yeast â¼169,000 potential interactions among orthologs of human tumor suppressor genes (TSG) and genes encoding drug targets across multiple genotoxic environments. Guided by the strongest signal, we evaluate thousands of TSG-drug combinations in HeLa cells, resulting in networks of conserved synthetic lethal interactions. Analysis of these networks reveals that interaction stability across environments and shared gene function increase the likelihood of observing an interaction in human cancer cells. Using these rules, we prioritize â¼10(5) human TSG-drug combinations for future follow-up. We validate interactions based on cell and/or patient survival, including topoisomerases with RAD17 and checkpoint kinases with BLM.
Asunto(s)
Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Redes Reguladoras de Genes/efectos de los fármacos , Genes Supresores de Tumor , Mutación , Medicina de Precisión/métodos , Mapas de Interacción de Proteínas/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Neoplasias del Cuello Uterino/tratamiento farmacológico , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Predisposición Genética a la Enfermedad , Células HeLa , Humanos , Estimación de Kaplan-Meier , Terapia Molecular Dirigida , Fenotipo , Interferencia de ARN , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/efectos de los fármacos , Mutaciones Letales Sintéticas , Factores de Tiempo , Transfección , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/mortalidadRESUMEN
We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision. From these results, we anticipate that cellular context will be critical to synthetic-lethal therapies.
Asunto(s)
Mapeo Cromosómico/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Técnicas Químicas Combinatorias , Epistasis Genética/genética , Proteínas de Neoplasias/genética , Células A549 , Línea Celular Tumoral , Células HeLa , Secuenciación de Nucleótidos de Alto Rendimiento , HumanosRESUMEN
Chemical inhibitors of the checkpoint kinases have shown promise in the treatment of cancer, yet their clinical utility may be limited by a lack of molecular biomarkers to identify specific patients most likely to respond to therapy. To this end, we screened 112 known tumor suppressor genes for synthetic lethal interactions with inhibitors of the CHEK1 and CHEK2 checkpoint kinases. We identified eight interactions, including the Replication Factor C (RFC)-related protein RAD17. Clonogenic assays in RAD17 knockdown cell lines identified a substantial shift in sensitivity to checkpoint kinase inhibition (3.5-fold) as compared to RAD17 wild-type. Additional evidence for this interaction was found in a large-scale functional shRNA screen of over 100 genotyped cancer cell lines, in which CHEK1/2 mutant cell lines were unexpectedly sensitive to RAD17 knockdown. This interaction was widely conserved, as we found that RAD17 interacts strongly with checkpoint kinases in the budding yeast Saccharomyces cerevisiae. In the setting of RAD17 knockdown, CHEK1/2 inhibition was found to be synergistic with inhibition of WEE1, another pharmacologically relevant checkpoint kinase. Accumulation of the DNA damage marker γH2AX following chemical inhibition or transient knockdown of CHEK1, CHEK2 or WEE1 was magnified by knockdown of RAD17. Taken together, our data suggest that CHEK1 or WEE1 inhibitors are likely to have greater clinical efficacy in tumors with RAD17 loss-of-function.