Differential MYC Phosphorylation Drives the Divergent Cholangiocyte Response to Stress.

BACKGROUND & AIMS: In primary sclerosing cholangitis (PSC), some cholangiocytes undergo cell cycle arrest (senescence), whereas others proliferate (ductular reaction). Our aim was to determine the mechanisms driving this divergent response. METHODS: We analyzed PSC and control liver tissue by immunofluorescence for proliferative and senescent (sen) cholangiocytes. We used LPS to stress normal human cholangiocytes (NHCs) transfected with a senescence reporter (p16(prom)-GFP) and fluorescence-activated cell sorting (FACS)-sorted sen (GFP+) or senescent-resistant (sen-res, GFP-) fractions. We performed RNA sequencing and quantitative polymerase chain reaction (qPCR) for senescence markers and immunoblots for phospho-(p)T58- MYC and pS62-MYC, and the kinase, GSK3B. Non-phosphorylatable MYC mutant NHCs were generated, and MYC or GSK3B were depleted or inhibited to assess effects on cell fate. MYC and GSK3B inhibitors were tested in 2 PSC mouse models (DDC and Mdr2-/-). RESULTS: PSC tissue showed an overall increase in sen (∼2×), and proliferative (∼10×) cholangiocytes compared with controls, with senescence enriched in portal tracts and proliferation in parenchyma. RNA sequencing showed enrichment of MYC responsive genes in sen-res cholangiocytes (P < .001). Sen-res cholangiocytes showed increased total and pS62-MYC protein (∼3×), increased mRNA of the proliferation marker, KI67 (>2.5×), and decreased p16/p21 mRNA (∼75%). MYC inhibition in sen-res cholangiocytes promoted senescence (∼15×), whereas T58-MYC mutation reduced senescence and enhanced proliferation (∼3×). Sen cholangiocytes exhibited increased GSK3B (∼2×); GSK3B inhibition or depletion in sen-sensitive cholangiocytes reduced pT58-MYC and senescence (∼50%). In mouse models, MYC inhibition reduced, whereas GSK3B inhibition increased, cholangiocyte proliferation and fibrosis. CONCLUSION: MYC phosphorylation promotes either cholangiocyte proliferation or senescence. The results reveal kinase mediators of cholangiocyte fate and identify MYC as a stress-responsive "molecular switch."