Abstract 1009: A novel sirtuin inhibitor, AS4.064, enhances apoptosis and acetylation of targets in hematological lines in combination with the novel HDACi, SelSA-1.

Abstract

Abstract Several HDAC inhibitors (HDACi) have proven preclinical and clinical activity against lymphoid malignancies. Histone deacetylases (HDACs) remove acetyl groups from histone and non-histone proteins to allow the transcriptional machinery access to the DNA. HDACs are grouped into four categories with classes I, II, and IV, constituting the zinc-dependent family members and class III comprising the NAD+ dependent members (also known as sirtuins). We have developed compounds that target either the zinc-dependent or the NAD+-dependent HDACs. SelSA-1 is a novel selenium-containing hydroxamic acid derivative HDACi and AS4.064 is a novel selenium analog of thiobarbituric acid and putative SIRT1 inhibitor. We have tested these compounds in several hematological cell lines. The cells (a Hodgkin Lymphoma line, L540; a CML line, K562; and an APL line, HL-60) showed decreased viability when treated with single agents. In combination, the drugs further diminished viability. As demonstrated by Annexin V staining, L540 cells undergo apoptosis when treated with 0.25 uM and 0.5 uM SelSA-1, while cells treated with 5 uM AS4.064 showed little Annexin V staining. The combination dramatically increased apoptosis. SelSA-1 appears to be acting as an HDACi as cells treated with the drug showed a dramatic increase in acetylation of Histone 3 lysine 9 and Histone 4 lysine 8. AS4.064 had minimal effect on these two sites, but cells treated with the combination showed a dramatic increase in acetylation at both sites. We have previously demonstrated an important role for c-Myc inhibition in the activity of HDACi in lymphoid malignancies. The combination of AS4.064 and SelSA-1 showed enhanced inhibition of c-Myc protein levels in L540 cells. The acetylation of p53 is important for apoptosis; therefore, we examined the acetylation status of several lysine residues after treatment with the drugs in L540 cells. SelSA-1 increased acetylation of lysines 320 and 382 in a dose-dependent manner, but their acetylation residues was not further enhanced by the addition of AS4.064. Acetylation of lysine 373 was relatively unaffected by either single agent, but the drug combination dramatically enhanced K373 acetylation. Lysine 120 is important for the specific activation of pro-apoptotic p53 targets. SelSA1 increased K120 acetylation and the combination further enhanced acetylation. We are currently using qRT-PCR and SuperArrays to identify other genes important for regulating the response to this drug combination. In summary, both the novel SIRT1 inhibitor, AS-64, and the novel HDACi, SelSa-1, show growth inhibitory and pro-apoptotic activity in multiple hematological lines, alone and in combination. Inhibition of all HDAC classes, including the NAD+ catalyzed deacetylases, show activity in hematological malignancies. Clinical development of these agents is warranted. Citation Format: Kimberly A. Scata, Arun K. Sharma, Dhimant Desai, Shantu Amin, Mark H. Kirschbaum. A novel sirtuin inhibitor, AS4.064, enhances apoptosis and acetylation of targets in hematological lines in combination with the novel HDACi, SelSA-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1009. doi:10.1158/1538-7445.AM2013-1009