Quantitative analysis of histone deacetylase-1 selective histone modifications by differential mass spectrometry.
Inhibitors of class 1 and class 2 histone deacetylase (HDAC) enzymes have shown antitumor activity in human clinical trials. More recently, there has been interest in developing subtype-selective HDAC inhibitors designed to retain anticancer activity while reducing potential side effects. Efforts have been initiated to selectively target HDAC1 given its role in tumor proliferation and survival. The development of HDAC1-specific inhibitors will require the identification of HDAC1-selective pharmacodynamic markers that correlate closely with HDAC1-inhibition in vitro and in vivo. Existing histone markers of HDAC target engagement were developed using pan-HDAC inhibitors and do not necessarily represent robust readouts for isoform-specific inhibitors. Therefore, we have initiated a proteomic approach to identify readouts for HDAC1 inhibition. This approach involves the use of differential mass spectrometry (dMS) to identify post-translational changes in histones by profiling histone-enriched cellular fractions treated with various HDAC inhibitors. In this study, we profiled histones isolated from the HCT116 human colon cancer cell line that have been treated with compounds from multiple chemical classes that are specific for HDAC1; HDAC1 and 3; and HDAC1, 3, and 6 enzymes. In two independent experiments, we identified 24 features that correlated with HDAC1-inhibition. Among the peptides modulated by HDAC1-selective inhibitors were Ac-H2B-K5 from histone H2B, and Ac-H3-K18 from histone H3. Commercially available antibodies to specific histone acetyl-lysine residues were used to confirm that these peptides also provide pharmacodynamic readouts for HDAC1-selective inhibitors in vivo and in vitro. These results show the utility of dMS in guiding the identification of specific readouts to aid in the development of HDAC-selective inhibitors.
Lee
Anita Y H
AY
Department of Proteomics, Merck Research Laboratories, Merck & Co. Inc., Rahway, New Jersey 07065, USA.
Paweletz
Cloud P
CP
Pollock
Roy M
RM
Settlage
Robert E
RE
Cruz
Jonathan C
JC
Secrist
J Paul
JP
Miller
Thomas A
TA
Stanton
Matthew G
MG
Kral
Astrid M
AM
Ozerova
Nicole D S
ND
Meng
Fanyu
F
Yates
Nathan A
NA
Richon
Victoria
V
Hendrickson
Ronald C
RC
D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods; D005189:False Positive Reactions; D015971:Gene Expression Regulation, Enzymologic; D006655:Histone Deacetylases / Q000378:metabolism*; D006657:Histones / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D020033:Protein Isoforms; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012372:ROC Curve; D021241:Spectrometry, Mass, Electrospray Ionization
D000970:Antineoplastic Agents; D006657:Histones; D010455:Peptides; D020033:Protein Isoforms; D020543:Proteome; D006655:Histone Deacetylases