Abstract LB-249: TDG, a dual genomic and epigenomic regulator, as a novel antimelanoma target

Abstract

Abstract Melanoma is an aggressive cancer resistant to treatment, whose incidence has increased over the past two decades. Although the majority of melanoma cases are cured after surgical excision of the primary tumor, metastases occur frequently, and the metastatic form of the disease has a poor prognosis and is highly resistant to all current forms of therapy. Thus, new prognostic factors and advanced therapeutic strategies are urgently needed. We recently reported that the base excision repair protein Thymine DNA Glycosylase (TDG) has dual roles in safeguarding the genome and the epigenome (Cell 146:67, 2011). TDG not only protects CpG sites from spontaneous deamination of 5-methylcytosine and cytosine (genomic stability), but importantly, at the epigenomic level, acts in a DNA demethylation pathway that converts 5-methylcytosine to cytosine (epigenomic stability). Specifically TDG removes the novel bases 5-formylcytosine and 5-carboxylcytosine, demethylation intermediates produced by the upstream TET dioxygenases. TET alterations have been recently found in melanoma and correlate with poor prognosis. Moreover, TDG sequence variants in melanoma are reported in the TCGA database. For these reasons, we began studying the functional significance of TDG in melanoma. We reasoned that the two non-redundant (genomic and epigenomic) functions of TDG may represent a vulnerability of tumor cells and be exploited as novel drug targets for cancer treatment, because targeting TDG would achieve the dual effect of impairing DNA repair and disrupting the epigenetic state of the cancer cell. We found that reduced TDG levels correlate with tumorigenic melanomas and therefore TDG inhibition might further promote aggressiveness. Unexpectedly, however, TDG knockdown in melanoma lines caused cell cycle arrest, senescence and ultimately cell death. Senescence and cell death induced by TDG knockdown occurred without apparent activation of the DNA damage response, based on absence of H2AX phosphorylation. These in vitro findings were confirmed in vivo, as TDG knockdown in melanoma lines blocked tumor formation in xenografts. Given its potential as a novel therapeutic target, we conducted a pilot high-throughput screen and identified first-generation TDG chemical inhibitors. Two compounds were confirmed to inhibit TDG repair activity in vitro by radioactive-based glycosylase assay. Importantly, both inhibitors also blocked TDG demethylase function in cells, as evidenced by increased staining intensity of 5-carboxylcytosine. Both compounds inhibited proliferation (by clonogenic, MTT and Xcelligence assays) of melanoma cell lines in the micromolar range and could synergize with alkylating agents and other anti-melanoma drugs. Thus, while reduced TDG levels may be part of the tumorigenesis process, limited levels of TDG are essential for melanoma viability. Therefore, TDG inhibition may represent a novel approach for melanoma treatment. Citation Format: Rossella Tricarico, Pietro Mancuso, Vikram Bhattacharjee, Neil Beeharry, Emmanuelle Nicolas, Margret Einarson, Laura Cosentino, Irwin Davidson, Lionel Larue, Robert W. Sobol, Timothy J. Yen, Alfonso Bellacosa. TDG, a dual genomic and epigenomic regulator, as a novel antimelanoma target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-249. doi:10.1158/1538-7445.AM2015-LB-249