Abstract 2944: 5-azacytidine nucleosides and their derivatives: Molecular hallmarks of drug resistance & alternative therapeutic regimen

Abstract

Abstract Background: Aberrant DNA methylation turning ‘off’ the gene expression remains the consistent hallmark due to its involvement in all types of cancer. The prototypal DNA methylation inhibitors, 5-azacytidine and 2′-deoxy-5-azacytidine, are currently one of the most effective epigenetic drugs, for the treatment of blood malignancies. However, the chemotherapeutic resistance to these medicines is the major obstacle, preventing the successful epigenetic therapy. Objective: (i) To investigate the mechanism of resistance to 2′-deoxy-5-azacytidine and (ii) Design alternative therapeutic regimen. Materials and Methods: We developed several HCT116 p53 wild-type cell clones, resistant towards 2′-deoxy-5-azacytidine. Principal methods used to study the molecular alterations during the development of resistance included, flow cytometry based analyses, high throughput RNA sequencing based transcriptomics, and mass spectrometry based proteomics utilizing stable isotope labelling of amino acids in cell culture (SILAC). Further, we used MTT assays to determine the cross-resistance or sensitivity of the resistant clones towards the inhibitors of epigenetic “Readers-Writers-Erasers”. Results: Flow cytometry based studies revealed significant up-regulation of DNA and RNA synthesis. Molecular profiling of resistant clones unveiled 8010 genes and 3352 proteins, which were differentially expressed (ANOVA p<0.05) compared to parental cell line. The major affected cellular pathways were (i) Cell cycle: role of 14-3-3 proteins in cell cycle regulation, G1/S transition and initiation of mitosis (ii) Apoptosis and survival: granzyme A signaling, BAD phosphorylation, p53 dependent apoptosis (iii) Transcription: role of heterochromatin protein I family in transcriptional silencing, role of AP1 in regulation of cellular metabolism (iv) DNA damage: role of SUMO in p53 regulation. During MTT cytotoxicity assays, resistant clones exhibited cross-resistance towards all the tested epigenetic inhibitors, however, significant sensitivity was exceptionally observed for bromodomain inhibitors, which was further validated by significant down-regulation of BET bromodomains. Validation of relevant genes and/or proteins as biomarkers of drug resistance, and bromodomains as alternative therapeutic target, for re-sensitizing the cancer patients, resistant to DNA methylation inhibitors is currently ongoing. Conclusion: The present study will aid to the understanding of the molecular basis of acquired tumor resistance to 2′-deoxy-5-azacytidine and help in predicting its clinical response, as well as in designing alternative treatment regimens for overcoming resistance, hence furthering clinical development. Acknowledgement: Internal Grant Agency of Palacky University (LF-2013/016), BIOMEDREG (CZ.l.05/2.1 .00/ 01.0030) and Ministry of Industry and Trade of the Czech Republic (FR-TI4/625). Citation Format: Khushboo Agrawal, Petr Vojta, Dušan Holub, Ivo Frydrych, Petr Džubák, Miroslav Otmar, Marcela Krečmerová, Marián Hajdúch. 5-azacytidine nucleosides and their derivatives: Molecular hallmarks of drug resistance & alternative therapeutic regimen. [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 2944. doi:10.1158/1538-7445.AM2015-2944