Abstract B45: The role of combined inhibition of DNA methylation and histone deacetylation in CTCL treatment.
Authors: Rozati Sima , Cheng Phil , Fettelschoss Antonia , Belloni Benedetta , Dummer Reinhard 
Published: June 2012

Introduction: Change in the epigenetic pathways can alter level of gene expressions and trigger neoplastic transformation.1DNA methylation and histone modifications are two main epigenetic mechanisms that regulate gene expression. DNA demethylation results in reexpression of tumor suppressor genes silenced via hypermethylation of their promoter regions and HDACi, approved for cutaneous T cell lymphoma (CTCL) treatment, are shown to synergize with demethylating agents to relieve transcriptional repression2,3. We combined Romidepsin and Azacitidine treatment on CTCL primary cell cultures to investigate cell viability and apoptosis.

Methods: Human CTCL cell lines were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), glutamine (2 mM) and streptomycin (100 μg/ml) at 37°C, 5% CO2 and 95% humidity.

The CTCL cell cultures were treated with Romidepsin (0.25nM-10uM) and/or Azacitidine (500nM-10uM). After 24 to 72 hrs of combination drug treatment level of apoptosis and necrosis was measured by flow cytometry with Annexin V-FITC detection kit. With the above combination treatment the gene expression levels of cell cycle regulatory genes, HDACs (HDAC1, HDAC2, HDAC3, HDAC6,) and DNA methyltransferases (DNMT1, DNMT3a, DNMT3b) were examined by RT-PCR. The induction of Histone acetylation of H3 and H4 and cell cycle regulatory genes were analyzed by western blotting.

Results: The combination treatment showed a noticeable change of cell viability due to an increase in apoptosis and necrosis in a time and dose dependent manner. Furthermore, the RT-PCR results were notable for change in the expression of several of the genes mentioned above, suggesting that the combination of DNA demethylation and histone acetylation has a more selective effect on the induction of apoptosis in CTCL cells. We observed a more pronounced increase cell cycle arrest by induction of cell cycle regulators such as p21 and in acetylation of H3 in the combination treatment than each drug alone suggesting that the this combination treatment, counteracts loss of cell cycle control in CTCL more efficiently. This not only provides a rationale for future clinical trials with combined Romidepsin and Azacitidine treatment but also has initiated us to further explore the possible effects of this combination in HDAC inhibitor resistant CTCL.

Conclusion: In the light of our current investigation's outcome, we plan to explore the impact of the cumulative chromatin changes by this treatment combination and try to understand their specific mechanism of cell death induction in cutaneous T cell lymphomas. Further confirmation will be followed by experiments on other CTCL cell cultures and lymphocytes of mycosis fongiodes/Sezary syndrome patients.

References:

1. Sharma S, Kelly TK, Jones PA. Epigenetics in cancer. Carcinogenesis. Jan 2010;31(1):27–36.

2. Belinsky SA, Grimes MJ, Picchi MA, et al. Combination therapy with vidaza and entinostat suppresses tumor growth and reprograms the epigenome in an orthotopic lung cancer model. Cancer Res. Jan 2011;71(2):454–462.

3. Cameron EE, Bachman KE, Myohanen S, Herman JG, Baylin SB. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nature Genetics. 1999;21(1):103–107.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B45.