Abstract B44: Mithramycin exhibits dual binding mode and acts as an epigenetic switch

Abstract

Abstract The recognition of epigenetic defects in all types of cancer has led to a marked achievement in cancer research in recent years. Histone acetylation, an important post translational epigenetic modification is altered in various diseases and the balance between acetylation and deacetylation is hampered. Reversibility of such epigenetic events provides a target for chemotherapeutic intervention. The major hurdle for drugs used for brain targeting is to cross the blood brain barrier. Mithramycin (MTR), a clinically approved DNA-binding antitumor antibiotic has been found to cross the blood-brain barrier and is in its preclinical trials in Huntington's disease (HD) [Stack et al., Hum Mol Genet, 2007]. It improved altered nucleosome homeostasis in HD mice, normalizing the chromatin pattern. It has the ability to rebalance epigenetic histone modifications. The binding properties of MTR with histone proteins have been examined from the perspective of the current proposition from our laboratory to classify small DNA binding molecules in terms of their ability to bind chromosomal DNA alone (single binding mode) or both histones and chromosomal DNA (dual binding mode) [Ghosh et al., Biochim Biophys Acta, 2010; Dasgupta et al., J Biosciences, 2012]. Steady state fluorescence spectroscopy, circular dichroism (CD) spectroscopy and in vitro Histone Acetyl Transferase (HAT) assay have been employed as experimental tools. Spectrofluorimetric measurements and CD studies show that MTR binds with micromolar dissociation constant to core histone assembly and its individual histone components. A single isoelliptic point in the CD spectra indicates formation of one type of complex between MTR and histones. Complex formation leads to decrease in molar ellipticity in the far UV CD spectra of core histones. In contrast to MTR-DNA interaction, association of MTR with histones does not require the presence of bivalent metal ions. MTR thus exhibits dual binding mode since it binds to histone proteins present in chromatin besides DNA. These results led us to investigate the role of MTR as a potential epigenetic modulator. In vitro HAT assays using CREB-binding protein (CBP) HAT domain, a transcriptional coactivator, showed that MTR inhibits H3 K18 acetylation (specifically mediated mark by CBP/p300 in vivo) in H3.3 recombinant histone whereas it induces H3 K18 hyperacetylation in H3.1/H4 tetramer as well as core histones. H3 K18 acetylation is correlated with poor prognosis in patients with prostrate, pancreatic, lung, breast and kidney cancers and loss of this modification is an important event in tumor progression [Seligson et al., Nature 2005; Elsheikh et al., Cancer Res, 2009]. The interaction of MTR with histones hints at a different scenario in MTR-chromatin interaction. Experiments are in progress to address the effect of MTR on other epigenetic marks. The significance of these in vitro experiments would also be examined by performing cell line based experiments. These data would be useful in understanding the mechanism of action of anticancer drugs as well as in designing new-generation therapeutics. Citation Format: Amrita Banerjee, Chandrima Das, Dipak Dasgupta. Mithramycin exhibits dual binding mode and acts as an epigenetic switch. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B44.