Discovery Histone Deacetylase (HDAC)6 Specific Proteomic Biomarkers In Multiple Myeloma (MM) Using Stable Isotope Labeling By Amino Acids In Cell Culture (SILAC)

ACY-1215 is a novel and selective inhibitor of HDAC6 currently in Phase 1b clinical trials in combination with bortezomib (Velcade) or lenalidomide (Revlimid) in relapsed/refractory MM. HDAC6 has been to be involved in misfolded protein clearance, stress response and cytoskeleton functions. This activity is in part due to the regulation of the acetylation status of α-tublin, a critical component of microtubules and a specific substrate for the deacetylase activity of HDAC6. ACY-1215 in combination with inhibitors of the major protein clearance pathway, the proteasome, has shown synergistic effects leading to enhanced MM cell death. However, except for acetylated α-tubulin (lysine 40), there are few other HDAC6 specific biomarkers to illustrate additional cellular functions of targeting HDAC6 in the observed combination activity. In the present work, we undertook the identification of specific acetylated lysine (AcK) containing peptides caused by HDAC6 inhibition in MM cells using a quantitative mass spectrometry method, Stable Isotope Labeling by Amino acids in Cell culture (SILAC).

The MM cell line MM.1S was used to identify HDAC6 specific AcKs when exposed to the HDAC6 inhibitors ACY-1215, ACY-775 or Tubastatin A at 2 µM, a biologically active concentration on MM cells. ACY-775 and Tubastatin A are both HDAC6 inhibitors with much greater selectivity of HDAC6 over class I HDACs (500-1000x) than ACY-1215 (11x) and were used as reference compounds to identify HDAC6 specific AcK biomarkers. A total of 2,558 unique AcKs were detected for all three treatments. Specifically, for each treatment group, ACY-1215, ACY-775 and Tubastatin A, 1,367, 1,859 and 1,123 AcKs were detected, respectively. Of these 427, 186 and 322 AcKs were significantly changed from DMSO treated cells (>1.3 fold) for ACY-1215, ACY-775 and Tubastatin A, respectively. In addition the ACY-775 treated MM cell lysate was further fractionated into three subcellular fractions, cytoplasm, soluble nuclear and insoluble nuclear, for AcK quantification. In the total unique AcKs detected 1,868, 1,093, 1,338 and 716 were identified from cytoplasm, soluble nuclear and insoluble nuclear, respectively, and less than 10% (173) of these showed either elevated (86) or reduced (87) levels of AcKs. Surprisingly, since HDAC6 has been recognized as a predominately a cytoplasmic deacetylase, most changes were found in the nuclear fraction (soluble and insoluble) than those in cytoplasm. We also identified changes in absolute levels of peptides in ACY-775 treated MM cells. Of the 4,007 quantified peptides, 109 peptides were increased and 78 were decreased following treatment compared to controls. These peptides identified several proteins involved in acetylation, methylation, ubiquitination, DNA repair, stress response, apoptosis and transcription. Orthogonal confirmation of some of the acetylated and un-acetylated peptides identified is underway using alternative (acetyl-) peptide approaches for example immunoblot studies. In conclusion the AcK peptides identified in MM cells by the SILAC approach utilizing selective inhibitors of HDAC6, confirmed the critical function of HDAC6 in protein folding, ubiquitination, degradation, cytoskeleton structure and apoptosis, and also suggested other new functional targets for HDAC6 inhibition. These AcK peptide biomarkers will expand our knowledge on the role of HDAC6 inhibition particularly in combination with other MM therapeutic agents and assist in the development of predictive biomarkers of ACY-1215 activity in MM patients.