RESUMEN
Molecular mechanisms underlying adaptive targeted therapy resistance in pancreatic ductal adenocarcinoma (PDAC) are poorly understood. Here, we identify SETD5 as a major driver of PDAC resistance to MEK1/2 inhibition (MEKi). SETD5 is induced by MEKi resistance and its deletion restores refractory PDAC vulnerability to MEKi therapy in mouse models and patient-derived xenografts. SETD5 lacks histone methyltransferase activity but scaffolds a co-repressor complex, including HDAC3 and G9a. Gene silencing by the SETD5 complex regulates known drug resistance pathways to reprogram cellular responses to MEKi. Pharmacological co-targeting of MEK1/2, HDAC3, and G9a sustains PDAC tumor growth inhibition in vivo. Our work uncovers SETD5 as a key mediator of acquired MEKi therapy resistance in PDAC and suggests a context for advancing MEKi use in the clinic.
Asunto(s)
Cromatina/genética , Resistencia a Antineoplásicos , Metiltransferasas/metabolismo , Terapia Molecular Dirigida , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Apoptosis , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Proliferación Celular , Femenino , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/metabolismo , Histona Desacetilasas/química , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/antagonistas & inhibidores , MAP Quinasa Quinasa 2/genética , MAP Quinasa Quinasa 2/metabolismo , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Piridonas/farmacología , Pirimidinonas/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Improved treatments for chronic HCV infections remain a challenge, and new chemical strategies are needed to expand the current paradigm. The HCV RNA polymerase (RdR(P)) has been a target for antiviral development. For the first time we show that the boranophosphate (BP) modification increases the substrate efficiency of ATP analogs into HCV NS5BΔ55 RdRP-catalyzed RNA. Boranophosphate nucleotides contain a borane (BH3) group substituted for a non-bridging phosphoryl oxygen of a normal phosphate group, resulting in a class of modified isoelectronic DNA and RNA mimics capable of modulating the reading and writing of genetic information. We determine that HCV NS5BΔ55, being a stereospecific enzyme, incorporates the Rp isomer of both ATPαB and the two boranophosphate analogs: 2'-O-methyladenosine 5'-(α-P-borano) triphosphate (2'-OMe ATPαB, 5a) and 3'-deoxyadenosine 5'-(α-P-borano) triphosphate (3'-dATPαB, 5b). The R(p) diastereomer of ATPαB (6), having no ribose modifications, was found to be a slightly better substrate than natural ATP, showing a 42% decrease in the apparent Michaelis-Menten constant (K(m)). The IC50 of both 2'-O-Me and 3'-deoxy ATP was decreased with the boranophosphate modification up to 16-fold. This "borano effect" was further confirmed by determining the steady-state inhibitory constant (K(i)), showing a comparable potency shift (21-fold). These experiments also indicate that the boranophosphate analogs 5a and 5b inhibit HCV NS5B through a competitive mode of inhibition. This evidence, together with previous crystal structure data, further supports the idea that HCV NS5B (in a similar manner to HIV-1 RT) discriminates against the 3'-deoxy modification via lost interactions between the 3'-OH on the ribose and the active site residues, or lost intramolecular hydrogen bonding interactions between the 3'-OH and the pyrophosphate leaving group during phosphoryl transfer. To our knowledge, these data represent the first time a phosphate modified NTP has been studied as a substrate for HCV NS5B RdRP.