RESUMEN
BRAF is the most frequently mutated gene in melanoma. Constitutive activation of mutant BRAFV600E leads to aberrant Ras-independent MAPK signaling and cell transformation. Inhibition of mutant BRAF is a current frontline therapy for such cases, with improved survival compared with chemotherapy. Unfortunately, reactivation of MAPK signaling by several mechanisms has been shown to cause drug resistance and disease recurrence. In this work, we describe the co-occurrence of an in-frame deletion within an amplified BRAFV600E locus and a missense point mutation of the transcriptional repressor BCORL1 in vemurafenib-resistant A375 melanoma cells. Functional data confirmed that truncated p47BRAFV600E and mutant BCORL1Q1076H both contribute to resistance. Interestingly, either endogenous BCORL1 silencing or ectopic BCORL1Q1076H expression mimicked the effects of a CRISPR/Cas9-edited BCORL1Q1076H locus, suggesting a complex mixture of loss- and gain-of-function effects caused by the mutation. Transcriptomic data confirmed this hypothesis. Finally, we show that the pan-RAF inhibitor sorafenib is not affected by expression of BRAF deletion variant and effectively synergizes with vemurafenib to block resistant cells, suggesting a possible intervention for this class of mutants.
Asunto(s)
Melanoma/tratamiento farmacológico , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Represoras/genética , Vemurafenib/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/efectos de los fármacos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Mutación Missense/efectos de los fármacos , Mutación Missense/genética , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Colorectal carcinomas (CRC) harbor well-defined genetic abnormalities, including aberrant activation of ß-catenin (ß-cat) and KRAS, but independent targeting of these molecules seems to have limited therapeutic effect. In this study, we report therapeutic effects of combined targeting of different oncogenes in CRC. Inducible short hairpin RNA (shRNA)-mediated silencing of ß-cat, ITF2, or KRAS decreased proliferation by 88%, 72%, and 45%, respectively, with no significant apoptosis in any case. In contrast, combined blockade of ß-cat and ITF2 inhibited proliferation by 99% with massive apoptosis. Similar effects occurred after combined shRNA against ß-cat and KRAS. In vivo, single oncogene blockade inhibited the growth of established tumors by up to 30%, whereas dual ß-cat and ITF2 targeting caused 93% inhibition. Similar tumor growth suppression was achieved by double ß-cat/KRAS shRNA in vivo. Our findings illustrate an effective therapeutic principle in CRC based on a combination targeting strategy that includes the ITF2 oncogene, which represents a novel therapeutic target.