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

Abstract

Background: HDAC 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 Band T-lymphoma cell lines and to verify selected candidates across several mammalian cell lines. Methods: Jurkat 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. Results: SAHA 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. Conclusion: We 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

A possible link to uracil DNA glycosylase in the synergistic action of HDAC inhibitors and thymidylate synthase inhibitors

“…The newfound ability to disrupt uracil base excision repair with HDAC inhibitors has immense potential for cancer treatment. UNG2 levels have been pharmacologically reduced with HDAC inhibitors in diverse cell lines that include HT29, HeLa, HEK293, Jurkat, SUDHL5, DLD1, HaCaT, A549, HCT116, and HAP1 [1,9,10]. We note that several of these cell lines also lack normal p53 activity and are resistant to TS inhibitors unless they are sensitized pharmacologically or genetically (e.g., HT29 cells which harbor mutant p53) [5,9].…”

“…Indeed, Iveland et al also measured reduced TS protein levels in cells treated with HDAC inhibitors. It is quite notable that HDAC inhibitors can reduce protein levels of both TS and UNG2, which are critical components of a common pathway governing pyrimidine metabolism and DNA repair [1,2], and that reduction of TS and UNG2 occur through both transcriptional and post-translational mechanisms [1,9]. We note that HDAC inhibitors can also impact the expression of additional proteins involved in pyrimidine metabolism, such as thymidine phosphorylase, which regulates the efficacy of other fluoropyrimidine anti-metabolites [15].…”

“…We note that several of these cell lines also lack normal p53 activity and are resistant to TS inhibitors unless they are sensitized pharmacologically or genetically (e.g., HT29 cells which harbor mutant p53) [5,9]. Providing further pharmacologic specificity is the fact that the selective HDAC inhibitor MS275, which targets HDACs 1-3 [16], reduced UNG2 protein to similar levels as non-selective pan-HDAC inhibitors [1]. This presents a clear path for co-targeting specific HDAC proteins and TS in p53 deficient cells, and evaluating toxicity associated with deficient uracil base excision repair.…”

“…Classically, HDAC inhibitors are thought to alter gene transcription by modifying the acetylation state of lysine residues on histone tails, thereby altering chromatin structure and transcription factor access. Transcriptional changes presumably alter the levels of critical proteins that affect cell viability or proliferation [1]. Alternatively, HDACs are known to have many non-histone protein substrates whose acetylation state is also enhanced by HDAC inhibitors, and the acetylation of these proteins can affect their function and stability [6].…”

“…The reduction of UNG2 was impressively rapid after treatment with a chemically diverse set of molecules (SAHA, MS275, valproate, and sodium butyrate). The reason for reduced UNG2 levels is that HDAC inhibitors induce hyperacetylation of UNG2 [1], which can facilitate its interaction with a ubiquitin ligase [10], thereby resulting in UNG2′s degradation by the proteasome. Cellular UNG2 levels are important for the toxicity of TS inhibitors, which elevate genomic uracil content by depleting dTTP pools in favor of dUTP [2].…”