Histone Profiling in Leukemia Using LC-MS/MS

It has become increasingly evident that epigenetic changes, including aberrant histone modifications, play a crucial role in malignant transformation and may contribute to relapsed and refractory disease. There are over 160 known histone modifications, most of which remain poorly explored. Post-translational histone modifications (PTMs) have been shown to be altered in hematologic malignancies, and the protein network that regulates these PTMs offers potential therapeutic opportunities through pharmacologic inhibition. Prominent examples include aberrant histone acetylation in multiple malignancies, mutations in the histone acetyl transferase CEBBP in relapsed acute lymphoblastic leukemia (ALL), and activation mutations of the H3K27 lysine methyltransferase (KMT) EZH2 in lymphoma. Several histone deacetylase (HDAC) and KMT inhibitors are currently in clinical trials. Most mutations in the histone modifying enzymes have been identified by whole genome sequencing efforts. More recently, mass spectrometry has been proposed as a technique to identify patterns of histone modifications in cancer. Jaffe et al (Nature Genetics, 2013) developed a mass spectrometry approach to global chromatin profiling using leukemia cell lines. Their initial analysis focused on lysine methylation and acetylation, including some of the best characterized residues (H3K4, H3K9, H3K27, H3K36, H3K79). This proof of principle study showed that histone profiles matched what would be predicted based on our knowledge of genetic mutations. More importantly, profiles could serve as leads to discover previously unknown mutations in chromatin modifying enzymes.

We adapted the methods established by Jaffe et al to histone modification profiling using small amounts of primary material from leukemia patients. Our work flow involves enrichment of blasts by ficoll centrifugation or fluorescent activated cell sorting, isolation highly purified histones from patient samples using a resin column followed by gel electrophoresis, digestion with ArgC and Trypsin, and nanoscale liquid chromatography followed by tandem mass spectrometry (nano LC-MS/MS). We established a leukemia cell line panel labeled with heavy (13C6) arginine and heavy (13C6) lysine for SILAC based internal standardization. Initially focusing our analysis on H3K27 and H3K36 methylation and using published retention times and m/z ratios, we were able to identify almost all major combinatorial acetylation and methylation states for the two residues (Table 1). We also identified methylation/acetylation states for H3K9 (ac), H3K14 (ac), H3K18 (ac, me), H3K23 (ac), and H3K79(me, me2) (data not shown). Preliminary experiments using a cell line with an activating NSD2 mutation correctly identified increased H3K36 dimethylation and decreased H2K27 trimethylation compared to the SILAC standard. We will use this method to correlate histone profiles of patient leukemia samples with clinical and molecular information as a means of interrogating the potential role for a number of known histone modifying enzymes in a single sample. In the future, we hope to extend our analysis to less well characterized histone PTMs.