ABSTRACT
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.
Subject(s)
Chromatin/genetics , Drug Resistance, Neoplasm , Methyltransferases/metabolism , Molecular Targeted Therapy , Pancreatic Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Small Molecule Libraries/pharmacology , Animals , Apoptosis , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Proliferation , Female , Histocompatibility Antigens/genetics , Histocompatibility Antigens/metabolism , Histone Deacetylases/chemistry , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , MAP Kinase Kinase 1/antagonists & inhibitors , MAP Kinase Kinase 1/genetics , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 2/antagonists & inhibitors , MAP Kinase Kinase 2/genetics , MAP Kinase Kinase 2/metabolism , Methyltransferases/antagonists & inhibitors , Methyltransferases/genetics , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Pyridones/pharmacology , Pyrimidinones/pharmacology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
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.
