Abstract B051: Alterations in SIRT2-H3K18Ac identify increased P300 activity in circulating tumor cells from patients with CRPC
Authors: Filon Mikolaj , Yang Bing , Schehr Jennifer , Singh Anupama , Bigarella Marcelo , Denu John , Lang Joshua , Jarrard David 
Published: June 2023

Background: Histone modifying enzymes (HMEs) hold significant promise as targets for drug discovery in cancer. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) dictate the presence of acetyl marks, which regulate the openness of chromatin and transcriptional activation. We previously developed a novel histone array on a chip to measure HME dysregulation with the development of castration resistance (ACS Chem Biol 2017). This approach identified decreased SIRT2 expression and increased p300 activity leads to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18) in CRPC. This alteration was subsequently identified in a subset of castration-resistant tumors (BMC Cancer 2017). The goal of the current study was to determine if the isolation of circulating tumor cells (CTCs) could be used as a biomarker to identify patients with altered p300 acetylation activity. Methods: Peripheral blood was collected from 13 patients (15 samples) with advanced PC after informed written consent under the University of Wisconsin IRB-approved protocol. Peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll-paque PLUS, and lymphocytes were depleted using pre-conjugated, anti-CD45 magnetic beads. CTCs were then captured with EpCAM labeled Sera-Mag streptavidin-coated magnetic beads (SM). Cells bound to SMs were isolated with ESP technology using an automated magnetic PipetMax pipetting robot. SM-bound cells were moved through staining and washing wells prior to imaging at 20x with a Nikon Eclipse Ti fluorescent microscope and NIS-Elements AR 4.10 software, and finally images were analyzed with NIS Elements software. Results: We first optimized the staining of acetyl-p300, total p300, H3K18ac, and SIRT2 in prostate cancer cell lines. Employing a selective inhibitor of p300 acetylation (A-485) reduced levels of p300 and H3K18 acetylation. We then confirmed that CTCs from patient samples were successfully captured by the EpCAM antibody and show clear expression of acetyl-p300, acetyl-H3K18, SIRT2, and CK (cytokeratin). No expression of CD45+ cells (lymphocyte exclusion) is noted. Increased acetylation of p300 and H3K18 and decreased SIRT2 is found in 6/10 CRPC samples compared to HSPC patients. Paired samples collected from one patient show a marked increase in p300 activity, H3K18 hyperacetylation, and a decrease in SIRT2 expression in the later developed CRPC sample compared to the HSPC sample. Another CRPC subject, decreased acetyl p300 and histone acetyl-H3k18, and increased SIRT2 staining is noted several months after beginning enzalutamide administrated. Analysis in CTCs indicates a positive correlation between acetyl-p300 and H3K18ac (R=0.61) and between SIRT2 and H3K18ac (R=-0.59). Conclusions: This study suggests that acetyl-p300, H3K18ac, and SIRT2 are epigenetic biomarkers that increase in a subset of CTC from CRPC. These biomarkers could potentially be used for monitoring outcomes and predicting tumor responses to developing p300 acetylation inhibitors.

Citation Format: Mikolaj Filon, Bing Yang, Jennifer Schehr, Anupama Singh, Marcelo Bigarella, John Denu, Joshua Lang, David Jarrard. Alterations in SIRT2-H3K18Ac identify increased P300 activity in circulating tumor cells from patients with CRPC [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B051.