Abstract LB-294: In vivo evaluation of Ames negative HDAC6 inhibitor in melanoma model

Abstract

Abstract Histone deacetylases (HDAC), originally known for their ability to modify histones, have also been found to modulate a multitude of cellular processes unrelated to chromatin modification, including the regulation of cellular pathways involved in anti-tumor immune responses. Our group and others have shown that the selective inhibition of HDAC6 impairs in vitro and in vivo tumor growth in a number of cancer models. Selective HDAC6 inhibitors (HDAC6i) such as Tubastatin A have proven to be very useful in cellular models. However, due to their mutagenic characteristics (Ames positive), usage has been limited only to preclinical research and a few types of cancer. We report that the novel and selective HDAC6i SS-2-08 is negative in the Ames assay and shows promising in vivo anti-tumor activity. SS-2-08 was obtained via five synthetic steps in 20% overall yield and 99% purity. It was incubated with two strains of Salmonella typhimurium (TA98 and TA1537) in the presence and absence of mammalian microsomal enzymes (S9 mix) for Ames assessment. Murine SM1 melanoma cells were treated with SS-2-08 in vitro to evaluate the expression of HDAC6-target genes by qRT-PCR, immunoblot and flow cytometry. CellTox Green Cytotoxicity® and HDAC-Glo I/II® assays were used to evaluate viability and HDAC activity, respectively. Cellular pathway analysis was performed by Cignal™ Reporter. In vivo studies were performed with SM1 cells injected into C57/BL/6 mice. Tumors were grown until they reached 5-8 mm in diameter and then treated with vehicle control, 25 mg/kg, and 50 mg/kg of SS-2-08 three times a week. Tumor measurements were recorded twice a week. At the experimental endpoint, tissue samples were collected for analysis. SS-2-08 shows nanomolar level potency against HDAC6 and over 400-fold selectivity over HDAC1. In the Ames assay, no significant number of reverting colonies was found for either strain, thus supporting the lack of mutagenicity of SS-2-08. Preliminary in vitro work revealed that SS-2-08 has low cytotoxicity and selective HDAC inhibitory properties, an observation verified in multiple cancer cell types. The selectivity and potency of this novel compound was similar to or better than other conventional HDAC6i as demonstrated by comprehensive molecular profiling including previously characterized HDAC6 targets such as IL-10, STAT3 and PD-L1. Additionally, in vivo experiments using murine SM1 melanoma tumors showed an important reduction in tumor growth at a dose of 25 mg/kg. Acetylated tubulin was increased in the end-point tumor samples in the treatment group, indicating that SS-2-08 reached the tumor site in vivo. Finally, we observed a down regulation of PD-L1 and PD-L2 in tumor samples from the treated group previously reported for other HDAC6i. These results provide evidence for the effectiveness of this new Ames negative-HDAC6i in vivo. SS-2-08 and derivatives have potential for use as therapeutic agents to treat cancer and other diseases where the inhibition of HDAC6 could be used as a therapeutic option. Citation Format: Melissa Hadley, Sida Shen, Debarati Banik, Jennifer Kim, Jayakumar Nair, Tessa Knox, Vincent Gallub, Shannon Kirkland, Katherine B. Chiappinelli, Eduardo Sotomayor, Alan Kozikowski, Alejandro Villagra. In vivo evaluation of Ames negative HDAC6 inhibitor in melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-294. doi:10.1158/1538-7445.AM2017-LB-294