HDACi mediate UNG2 depletion, dysregulated genomic uracil and altered expression of oncoproteins and tumor suppressors in B- and T-cell lines

Journal of Translational Medicine

Kristian Lied Wollen¹  Tobias S. Iveland²  Mirta M. L. Sousa³  Antonio Sarno³  Nina Beate Liabakk³  Animesh Sharma⁴  Lars Hagen⁴  Geir Slupphaug⁴ 

[1] Department of Clinical and Molecular Medicine, Faculty of Medicine and Health, Norwegian University of Science and Technology, 7491, Trondheim, Norway;Department of Clinical and Molecular Medicine, Faculty of Medicine and Health, Norwegian University of Science and Technology, 7491, Trondheim, Norway;Cancer Clinic, St. Olav’s Hospital, Trondheim, Norway;Department of Clinical and Molecular Medicine, Faculty of Medicine and Health, Norwegian University of Science and Technology, 7491, Trondheim, Norway;Clinic of Laboratory Medicine, St. Olav’s Hospital, Trondheim, Norway;Department of Clinical and Molecular Medicine, Faculty of Medicine and Health, Norwegian University of Science and Technology, 7491, Trondheim, Norway;Clinic of Laboratory Medicine, St. Olav’s Hospital, Trondheim, Norway;Proteomics and Modomics Experimental Core, PROMEC, at NTNU and the Central Norway Regional Health Authority, Stjørdal, Norway;

关键词: Histone deacetylase inhibitors;    HDACi;    Lymphoma;    PCLAF;    SILAC;    Uracil-DNA glycosylase;    UNG2;    Pyrimidine metabolism;    HIV-1;   

DOI  :  10.1186/s12967-020-02318-8

来源: Springer

【 摘 要 】

BackgroundHDAC inhibitors (HDACi) belong to a new group of chemotherapeutics that are increasingly used in the treatment of lymphocyte-derived malignancies, but their mechanisms of action remain poorly understood. Here we aimed to identify novel protein targets of HDACi in B- and T-lymphoma cell lines and to verify selected candidates across several mammalian cell lines.MethodsJurkat T- and SUDHL5 B-lymphocytes were treated with the HDACi SAHA (vorinostat) prior to SILAC-based quantitative proteome analysis. Selected differentially expressed proteins were verified by targeted mass spectrometry, RT-PCR and western analysis in multiple mammalian cell lines. Genomic uracil was quantified by LC–MS/MS, cell cycle distribution analyzed by flow cytometry and class switch recombination monitored by FACS in murine CH12F3 cells.ResultsSAHA treatment resulted in differential expression of 125 and 89 proteins in Jurkat and SUDHL5, respectively, of which 19 were commonly affected. Among these were several oncoproteins and tumor suppressors previously not reported to be affected by HDACi. Several key enzymes determining the cellular dUTP/dTTP ratio were downregulated and in both cell lines we found robust depletion of UNG2, the major glycosylase in genomic uracil sanitation. UNG2 depletion was accompanied by hyperacetylation and mediated by increased proteasomal degradation independent of cell cycle stage. UNG2 degradation appeared to be ubiquitous and was observed across several mammalian cell lines of different origin and with several HDACis. Loss of UNG2 was accompanied by 30–40% increase in genomic uracil in freely cycling HEK cells and reduced immunoglobulin class-switch recombination in murine CH12F3 cells.ConclusionWe describe several oncoproteins and tumor suppressors previously not reported to be affected by HDACi in previous transcriptome analyses, underscoring the importance of proteome analysis to identify cellular effectors of HDACi treatment. The apparently ubiquitous depletion of UNG2 and PCLAF establishes DNA base excision repair and translesion synthesis as novel pathways affected by HDACi treatment. Dysregulated genomic uracil homeostasis may aid interpretation of HDACi effects in cancer cells and further advance studies on this class of inhibitors in the treatment of APOBEC-expressing tumors, autoimmune disease and HIV-1.

【 授权许可】

CC BY