Abstract LB-249: HDAC11 regulates lysine acetylation of enolase 1
Authors: Sharma Vasundhara , Christodoulidou Agni , Yue Lanzhu , Alontaga Aileen Y. , Goodheart William E. , Hesterberg Rebecca , Zheng Xiaozhang , Martin Matthew W. , Lee Jennifer Y. , Burnette Pearlie K. , Wright Kenneth L. 
Published: August 2018

Therapeutic molecules targeting the activity of histone deacetylases (HDACs) are currently under investigation for the treatment of several malignancies. There are currently eighteen human HDACs and while histone deacetylation is associated with transcriptional repression, acetylated lysine targets are functionally diverse and include cytoplasmic, nuclear, and mitochondrial proteins. Here, we used a new class of novel small molecule inhibitors that are highly selective for HDAC11 to identify its role in the regulation of non-histone proteins. Stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry in the presence of HDAC11 selective inhibitors identified proteins with acetylation and/or expression changes after treatment and were compared to known HDAC substrates to establish a unique set of putative HDAC11 target proteins. Metabolic processes were highly enriched in this data set. Specifically, acetylated enolase 1 (ENO1, 2-phospho-D-glycerate hydrolase) which catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate (PEP) in the glycolytic pathway was highly altered after HDAC11 inhibition. Using acetylated lysine specific immunoprecipitation, we validated the hyperacetylated state of ENO1 upon HDAC11 inhibition. Functional assays confirmed that the HDAC11 inhibition lowered ENO1-mediated PEP production, and reduced proliferation and viability of hematopoietic and solid tumor cells. Similar observations were obtained in HDAC11 knock down cell lines confirming that HDAC11 is a required molecule in the regulation of ENO1-mediated metabolic regulation. The proteomics data also mapped three distinct target lysine residues of HDAC11 in ENO1 and each of these residues were substituted to either an acetylated or an un-acetylated lysine mimic to test their function in ENO1 activity and stability. We confirmed that K335 is the major target site of HDAC11 and its substitution to the acetylated mimic (glutamine) causes loss of enolase activity. Concomitantly, using proton nuclear magnetic resonance spectroscopy we identified some glycolytic intermediates upstream of ENO1 to be increased and downstream intermediates quantitatively reduced after HDAC11 inhibition suggesting that glycolysis is functionally suppressed. Glycolytic pathway disruption was associated with a compensatory increase in oxygen consumption and ATP production through oxidative phosphorylation in these oncogene transformed tumor cells, but not in their non-transformed counterparts. Suppression of fatty acid oxidation by inhibiting carnitine palmitoyltransferase 1 (CPT-1) or blocking glutamine utilization by inhibiting glutaminase (GLS1) in combination with HDAC11 inhibition resulted in a cooperative reduction in cellular ATP levels further supporting a direct role of HDAC11 in regulating glycolysis in tumor cells. For the first time, this study mechanistically and functionally defines a cytoplasmic non-histone protein regulated by HDAC11.

Citation Format: Vasundhara Sharma, Agni Christodoulidou, Lanzhu Yue, Aileen Y. Alontaga, William E. Goodheart, Rebecca Hesterberg, Xiaozhang Zheng, Matthew W. Martin, Jennifer Y. Lee, Pearlie K. Burnette, Kenneth L. Wright. HDAC11 regulates lysine acetylation of enolase 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-249.