title: A lentivirus-mediated genetic screen identifies dihydrofolate reductase (DHFR) as a modulator of beta-catenin/GSK3 signaling.
abstract: The multi-protein beta-catenin destruction complex tightly regulates beta-catenin protein levels by shuttling beta-catenin to the proteasome. Glycogen synthase kinase 3beta (GSK3beta), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark beta-catenin for ubiquitination and subsequent degradation. Because modulation of both beta-catenin and GSK3beta activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the beta-catenin/GSK3beta interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a beta-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3'oxime (BIO), a specific inhibitor of GSK3beta. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of beta-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear beta-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3beta and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNFalpha, IL-6, and IL-12), and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of beta-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease.
authors.affiliation: Rosetta Inpharmatics, LLC, Seattle, Washington, United States of America. richard_klinghoffer@merck.com
authors.forename: Richard A
authors.initials: RA
authors.lastname: Klinghoffer
authors.forename: Jason
authors.initials: J
authors.lastname: Frazier
authors.forename: James
authors.initials: J
authors.lastname: Annis
authors.forename: Jason D
authors.initials: JD
authors.lastname: Berndt
authors.forename: Brian S
authors.initials: BS
authors.lastname: Roberts
authors.forename: William T
authors.initials: WT
authors.lastname: Arthur
authors.forename: Raul
authors.initials: R
authors.lastname: Lacson
authors.forename: Xiaohua Douglas
authors.initials: XD
authors.lastname: Zhang
authors.forename: Marc
authors.initials: M
authors.lastname: Ferrer
authors.forename: Randall T
authors.initials: RT
authors.lastname: Moon
authors.forename: Michele A
authors.initials: MA
authors.lastname: Cleary
mesh_terms: D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D002460:Cell Line; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000378:metabolism; D018664:Interleukin-12 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D016086:Lentivirus / Q000378:metabolism*; D008727:Methotrexate / Q000494:pharmacology; D008954:Models, Biological; D010091:Oximes / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism*
chemical_list: C483321:6-bromoindirubin-3'-oxime; D000893:Anti-Inflammatory Agents; D007211:Indoles; D015850:Interleukin-6; D010091:Oximes; D014409:Tumor Necrosis Factor-alpha; D051176:beta Catenin; D018664:Interleukin-12; D013762:Tetrahydrofolate Dehydrogenase; D038362:Glycogen Synthase Kinase 3; D008727:Methotrexate