Khushboo Agrawal

Nucleosidic DNA demethylating epigenetic drugs – A comprehensive review from discovery to clinic

DNA methylation plays a pivotal role in the etiology of cancer by mediating epigenetic silencing of cancer-related genes. Since the relationship between aberrant DNA methylation and cancer has been understood, there has been an explosion of research at developing anti-cancer therapies that work by inhibiting DNA methylation. From the discovery of first DNA hypomethylating drugs in the 1980s to recently discovered second generation pro-drugs, exceedingly large number of studies have been published that describe the DNA hypomethylation-based anti-neoplastic action of these drugs in various stages of the pre-clinical investigation and advanced stages of clinical development. This review is a comprehensive report of the literature published in past 40 years, on so far discovered nucleosidic DNA methylation inhibitors in chronological order. The review will provide a complete insight to the readers about the mechanisms of action, efficacy to demethylate and re-express various cancer-related genes, anti-tumor activity, cytotoxicity profile, stability, and bioavailability of these drugs. The review further presents the far known mechanisms of primary and secondary resistance to azanucleoside drugs. Finally, the review highlights the ubiquitous role of DNA hypomethylating epi-drugs as chemosensitizers and/or priming agents, and recapitulate the combinatorial cancer preventive effects of these drugs with other epigenetic agents, conventional chemo-drugs, or immunotherapies. This comprehensive review analyzes the beneficial characteristics and drawbacks of nucleosidic DNA methylation inhibitors, which will assist the pre-clinical and clinical researchers in the design of future experiments to improve the therapeutic efficacy of these drugs and circumvent the challenges in the path of successful epigenetic therapy.

Cell-based DNA demethylation detection system for screening of epigenetic drugs in 2D, 3D, and xenograft models.

Aberrant DNA methylation that results in silencing of genes has remained a significant interest in cancer research. Despite major advances, the success of epigenetic therapy is elusive due to narrow therapeutic window. A wide variety of naturally occurring epigenetic agents and synthetic molecules that can alter methylation patterns exist, however, their usefulness in epigenetic therapy remains unknown. This underlines the need for effective tumor models for large‐scale screening of drug candidates with potent hypomethylation activity. In this study, we present the development of a cell‐based DNA demethylation detection system, which is amenable for high content screening of epigenetic drugs in two‐dimensional and three‐dimensional cell culture models. Additionally, the detection system also supports the in vivo monitoring of demethylation efficacy of potential lead compounds from in vitro screens in tumor xenografts. The described detection system not only permits the continuous monitoring of demethylation but also of the induced cytostatic/cytotoxic drug effects in live cells, as a function of time. The detection system is fluorescence based and exploits the dominant ability of DNA methylation to inhibit gene transcription, and utilizes FLJ32130 gene, which is silenced on account of promoter hypermethylation in human colorectal cancer. The described work will provide the researchers with an efficient tool for epigenetic drug screens on a high throughput platform and would therefore benefit academic and industrial drug discovery.

Microtubule affinity-regulating kinases are potential druggable targets for Alzheimer?s disease

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that affects normal functions of the brain. Currently, AD is one of the leading causes of death in developed countries and the only one of the top ten diseases without a means to prevent, cure, or significantly slow down its progression. Therefore, newer therapeutic concepts are urgently needed to improve survival and the quality of life of AD patients. Microtubule affinity-regulating kinases (MARKs) regulate tau-microtubule binding and play a crucial role in neurons. However, their role in hyperphosphorylation of tau makes them potential druggable target for AD therapy. Despite the relevance of MARKs in AD pathogenesis, only a few small molecules are known to have anti-MARK activity and not much has been done to progress these compounds into therapeutic candidates. But given the diverse role of MARKs, the specificity of novel inhibitors is imperative for their successful translation from bench to bedside. In this regard, a recent co-crystal structure of MARK4 in association with a pyrazolopyrimidine-based inhibitor offers a potential scaffold for the development of more specific MARK inhibitors. In this manuscript, we review the biological role of MARKs in health and disease, and draw attention to the largely unexplored area of MARK inhibitors for AD.

Differential Regulation of Methylation-Regulating Enzymes by Senescent Stromal Cells Drives Colorectal Cancer Cell Response to DNA-Demethylating Epi-Drugs

The advanced-stage colon cancer spreads from primary tumor site to distant organs where the colon-unassociated stromal population provides a favorable niche for the growth of tumor cells. The heterocellular interactions between colon cancer cells and colon-unassociated fibroblasts at distant metastatic sites are important, yet these cell-cell interactions for therapeutic strategies for metastatic colon cancer remain underestimated. Recent studies have shown the therapeutic potential of DNA-demethylating epi-drugs 5-azacytidine (AZA) and 5-aza-2′-deoxycytidine (DAC) for the treatment of solid tumors. While the effects of these epi-drugs alone or in combination with other anticancer therapies are well described, the influence of stromal cells and their secretome on cancer cell response to these agents remain elusive. In this study, we determined the effect of normal and senescent colon-unassociated fibroblasts and their conditioned medium on colorectal cancer (CRC) cell response to AZA and DAC using a cell-based DNA demethylation reporter system. Our data show that fibroblasts accelerate cell proliferation and differentially regulate the expression of DNA methylation-regulating enzymes, enhancing DAC-induced demethylation in CRC cells. In contrast, the conditioned medium from senescent fibroblasts that upregulated NF-κB activity altered deoxycytidine kinase levels in drug-untreated CRC cells and abrogated DAC effect on degradation of DNA methyltransferase 1. Similar to 2D cultures, senescent fibroblasts increased DNA demethylation of CRC cells in coculture spheroids, in addition to increasing the stemness of CRC cells. This study presents the first evidence of the effect of normal and senescent stromal cells and their conditioned medium on DNA demethylation by DAC. The data show an increased activity of DAC in high stromal cell cocultures and suggest the potential of the tumor-stroma ratio in predicting the outcome of DNA-demethylating epigenetic cancer therapy.

Abstract 4101: Stromal cell-induced alterations in the response of colorectal cancer cell to demethylating agents

Epigenetic mechanisms such as DNA hypermethylation, that results in gene silencing, is closely associated with resistance to platinum-based chemotherapy for colorectal cancer (CRC). DNA methyltransferase inhibitors (DNMTi) that reactivate and induce reexpression of silenced gene have the potential to improve the outcome of platinum-based and other therapies in CRC. In fact, non-cytotoxic concentrations of azacitidine, a clinically used DNMTi, has been reported to resensitize platinum-resistant ovarian cells to carboplatin. Hypomethylating agents, such as azacitidine and decitabine have shown significant promise in hematologic malignancies; however, studies on their application to CRC and other solid tumors have yielded ambivalent results potentially due to the complexities of the tumor microenvironment that result from the numerous cell type interactions. In this study, we analyzed the impact of different tumor-stroma ratios on cancer cell response to azacitidine, decitabine, and two lead compounds identified by in vitro screens in two-dimensional (2D) and three-dimensional (3D) co-cultures of colorectal HCT116 carcinoma demethylation reporter cells and adipose-derived mesenchymal stromal cells (MSCs) isolated from healthy individuals undergoing elective lipoaspiration. Each subject provided informed consent. Activity of hypomethylating drugs was examined in 2D and 3D cultures of different tumor-stroma ratios using confocal microscopy, flow cytometry, and MTT analyses. Our preliminary flow cytometry results show an altered response of hypomethylating agents in cocultures of HCT116 and MSCs compared to monocultures of HCT116 cells alone. There was an increase in demethylation of HCT116 cells in 3D cultures containing a higher ratio of stromal cells following treatment with low concentration of decitabine. Furthermore, confocal analysis 3D co-cultures at various z-plane heights revealed increased demethylation at low concentrations of all drugs in cultures with high stromal cell numbers. These results indicate that intratumoral stroma may alter the sensitivity of cancer cells to demethylating agents and warrant further studies. Citation Format: Viswanath Das, Svetlana Skolekova, Khushboo Agrawal, Jan Gursky, Lucia Kucerova, Marian Hajduch. Stromal cell-induced alterations in the response of colorectal cancer cell to demethylating agents. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4101.

Abstract 4453: Molecular hallmarks of drug resistance to DNA methylation inhibitors and alternative therapeutic regimen for overcoming resistance

Aberrant DNA methylation perturbing the epigenetic machinery has been continuously underlined as the major hallmark involved in all types of cancer. The archetypal DNA methylation inhibitor, 2’-deoxy-5-azacytidine (5-AZA-CdR) have been approved by FDA as the effective epigenetic drug for the treatment of blood malignancies, over a decade. However, the successful epigenetic therapy remains impeded owing to its chemotherapeutic resistance. To investigate the molecular mechanism of resistance to 5-AZA-CdR, we developed several HCT116 p53 wild-type cell clones, resistant towards 5-AZA-CdR, and principally studied the molecular alterations during the development of resistance, using high throughput RNA sequencing based transcriptomics and mass spectrometry based proteomics. The molecular profiling of HCT116 cells sensitive to 5-AZA-CdR treatment versus 5-AZA-CdR resistant cell clones distinguished the differential expression of various genes and the proteins coded by the genes, compared with untreated HCT116 wild-type cells (ANOVA p Citation Format: Khushboo Agrawal, Petr Vojta, Dusan Holub, Rastislav Slavkovsky, Ivo Frydrych, Petr Dzubak, Marcela Krecmerova, Marian Hajduch. Molecular hallmarks of drug resistance to DNA methylation inhibitors and alternative therapeutic regimen for overcoming resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4453.

Abstract B72: Cell-based DNA demethylation detection system for screening of epigenetic drugs in 2D, 3D and xenograft models

Aberrant DNA methylation that turns ‘off’ the gene expression has remained a significant interest in cancer research. Despite major advances, the success of epigenetic therapy is prevented due to narrow therapeutic window. A wide variety of naturally occurring epigenetic agents and artificially synthesized molecules which can alter methylation patterns exists, but their usefulness in epigenetic therapy remains unknown. This underlines the need for large scale screening of epigenetic drugs with potent hypomethylation activity. In this ‘quest’ for more potent drugs targeting methylation, we present a cell-based DNA demethylation detection system, suitable for high content screening of epigenetic drug libraries in two-dimensional and three-dimensional culture systems. The detection system is fluorescence based and exploits the dominant ability of DNA methylation to inhibit gene transcription, and utilizes FLJ32130 gene which was discovered to be silenced on account of promoter hypermethylation in human colorectal cancer. In addition to demethylation screening, the detection system further incorporates the ability to monitor induced cytostatic/cytotoxic drug effects, in live cells, in a time dependent manner. Furthermore, our detection system also supports the in vivo determination of demethylation drug efficacy of potential hits selected on in vitro screening. Here, we describe the development of our cell-based DNA demethylation detection system, the anticipated results on screening of well-established DNA methylation inhibitors in two-dimensional versus three-dimensional cell cultures, and as well the efficacy of the system for monitoring demethylation in xenograft models. Acknowledgements The authors are grateful to Dr. Toshikazu Ushijima, Carcinogenesis Division, National Cancer Center Research Institute for his kind gift of FLJ32130 targeting vector. This study was supported by the grants awarded by the Ministry of Industry and Trade of the Czech Republic (MPO TIP FR-TI4/625), Ministry of Education, Youth and Sports (CZ09 / 7F14009 and LO1304; CZ.1.07/2.3.00/30.0041 to VD), Grant Agency of the Czech Republic (14-02652S and 14-03636S), and the Internal Grant Agency of Palacky University (IGA UP LF-2013/016). Citation Format: Khushboo Agrawal, Vishwanath Das, Miroslav Otmar, Marcela Krecmerova, Petr Dzubak, Marian Hajduch. Cell-based DNA demethylation detection system for screening of epigenetic drugs in 2D, 3D and xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B72.

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

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA 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, Dusan Holub, Ivo Frydrych, Petr Džubak, Miroslav Otmar, Marcela Krecmerova, Marian Hajduch. 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

Abstract 400: 5-azacytidine nucleosides and their derivatives: Molecular hallmarks of drug resistance

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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 >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<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