Abstract 400: 5-azacytidine nucleosides and their derivatives: Molecular hallmarks of drug resistance
Authors: Agrawal Khushboo , Dzubak Petr , Frydrych Ivo , Holub Dusan , Vojta Petr , Krecmerova Marcela , Otmar Miroslav , Hajduch Marian 
Published: May 2015

Abstract
Background: Hypermethylation of gene promoter sequences resulting in transcriptional silencing of tumor suppressor genes has been explored as a therapeutic target in cancer for years. The cytosine analogues, 5-azacytidine and 2′-deoxy-5-azacytidine, function as DNA methyltransferase inhibitors and are currently most advanced drugs for epigenetic cancer therapies. Despite encouraging results, mechanisms of in vivo resistance to these nucleoside analogues remain unresolved and are limiting their clinical application.
Objective: The goal of this study was to investigate the mechanisms of resistance to 2’-deoxy-5-azacytidine (decitabine).
Methods: To investigate the mechanisms of resistance, we developed several HCT116 p53 wild-type cell clones resistant towards 2′-deoxy-5-azacytidine. Principal methods used during the study of resistance were MTT cytotoxicity assays, flow cytometry based analyses and molecular profiling of the resistant clones which included mass spectrometry based proteomics using stable isotope labelling of amino acids in cell culture (SILAC) and massively parallel signature sequencing (MPSS) for studies at gene and transcript level.
Results: During resistant studies, all HCT116 2′-deoxy-5-azacytidine resistant clones were &gt;100 folds more resistant to decitabine in MTT cytotoxicity assays. Resistant clones also displayed cross-resistance to other epigenetic inhibitors. Flow cytometry based studies revealed significant up-regulation of DNA and RNA synthesis. Molecular profiling of resistant clones using proteome wide analysis and transcriptomic sequencing unveiled 4343 proteins, and 8011 genes respectively which were differentially expressed (ANOVA p&lt;0.05) compared to parental cell line. The major affected cellular pathways were (i) DNA damage (ii) Transcription: role of heterochromatin protein 1 (iii) Cell cycle: regulation of G1/S transition and initiation of mitosis (iv) Apoptosis and survival: granzyme A signaling.
Conclusion: The present study will aid our 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: This work was supported by internal grant of Palacky University (LF_2013_016), BIOMEDREG (CZ.1.05/2.1.00/ 01.0030) and Ministry of Industry and Trade of the Czech Republic (FR-TI4/625).
Citation Format: Khushboo Agrawal, Petr Dzubak, Ivo Frydrych, Dusan Holub, Petr Vojta, Marcela Krecmerova, Miroslav Otmar, Marian Hajduch. 5-azacytidine nucleosides and their derivatives: Molecular hallmarks of drug resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 400. doi:10.1158/1538-7445.AM2014-400