Quantitative Acetylomics Uncover Acetylation-Mediated Pathway Changes Following Histone Deacetylase Inhibition in Anaplastic Large Cell Lymphoma

Abstract

Histone deacetylases (HDACs) target acetylated lysine residues in histone and non-histone proteins. HDACs are implicated in the regulation of genomic stability, cell cycle, cell death and differentiation and thus critically involved in tumorigenesis. Further, HDACs regulate T-cell development and HDAC inhibitors (HDACis) have been approved for clinical use in some T-cell malignancies. Still, the exact targets and mechanisms of HDAC inhibition in cancer are understudied. We isolated tumor cell lines from a transgenic mouse model of anaplastic large cell lymphoma (ALCL), a rare T-cell lymphoma, and abrogated HDAC activity by treatment with the HDACis Vorinostat and Entinostat or Cre-mediated deletion of Hdac1. Changes in overall protein expression as well as histone and protein acetylation were measured following Hdac1 deletion or pharmacological inhibition using label-free liquid chromatography mass spectrometry (LC-MS/MS). We found changes in overall protein abundance and increased acetylation of histones and non-histone proteins, many of which were newly discovered and associated with major metabolic and DNA damage pathways. For non-histone acetylation, we mapped a total of 1204 acetylated peptides corresponding to 603 proteins, including chromatin modifying proteins and transcription factors. Hyperacetylated proteins were involved in processes such as transcription, RNA metabolism and DNA damage repair (DDR). The DDR pathway was majorly affected by hyperacetylation following HDAC inhibition. This included acetylation of H2AX, PARP1 and previously unrecognized acetylation sites in TP53BP1. Our data provide a comprehensive view of the targets of HDAC inhibition in malignant T cells with general applicability and could have translational impact for the treatment of ALCL with HDACis alone or in combination therapies.

Epigenetic Alterations in Canine Malignant Lymphoma: Future and Clinical Outcomes

“…For example, functional dysregulation of histone acetyltransferases has been associated with cancer development, including B-cell [45,46] and T-cell lymphomas [47,48]. In humans, histone modifier gene mutations have been associated with inferior progression-free survival time in patients with T-cell lymphomas [48], and it has been demonstrated that HDAC regulates the expression of BCL6, involved in cell survival and/or differentiation in B-cell lymphoma [49], and HDAC inhibitors have been tried as therapy in humans [50][51][52]. In CML, repression of p16 tumour suppressor gene is regulated by histone H3 acetylation in vitro [53].…”

HDAC1 acts as tumor suppressor in ALK-positive anaplastic large-cell lymphoma: Implications for HDAC inhibitor therapy

“…Additionally, HDACs target non-histone proteins and changes in overall protein acetylation might contribute to the observed phenotype. Indeed, in a previous study we identified several hundred differentially acetylated proteins, including chromatin modifying proteins and transcription factors, in mouse NPM-ALK tumor cell lines depleted for HDAC1, using quantitative acetylomics 15 .…”

“…Snap-frozen tissues were lysed in Hunt buffer and processed for SDS-PAGE and Western blot analysis as previously described 15 . Antibodies and buffers are listed in Supplementary Methods.…”

“…In ALCL, the proapoptotic gene BIM can be epigenetically silenced through the recruitment of the SIN3a corepressor complex, where HDAC1/2 act as catalytic core 14 . In addition to histone proteins, HDACs can deacetylate non-histone proteins and signaling proteins 15 , like STAT3, which is a key signal transmitter in ALCL 4,16 .…”

AggressiveKRASmutations direct TGF-β response towards partial EMT in patient-derived colorectal cancer tumoroids

“…Post-collection, the pellets were snap-frozen and subsequently stored at -80°C. Protein isolation and western blotting was carried out as described earlier (51), with minor deviations: 10 µg Protein was used for each blot and BSA was used for blocking of membranes. Antibodies used are provided in SI supplementary table 3.…”