Abstract 2618: DNMT1 as a marker of differential sensitivities to epigenetic therapy of a Kras mutant and Kras wild type human non small cell lung cancer cell line

Abstract

Abstract INTRODUCTION: Epigenetic therapy with a demethylating agent and an histone deacetylase inhibitor (HDACi) is now being pursued in hematological as well as solid tumor malignancies. Clinical responses have been varied with complete responses in some patients and no efficacy of the drugs in others. We investigated the effect of this therapy on two targets, DNA methyltransferase1 (DNMT1) and H3K4 modified histones, respectively. METHODS: Two human lung adenocarcinoma cell lines, A549 (Kras mutant) and H838 (Kras wild type) were pre-exposed for 72 hours with daily treatment of 500nM azacitidine alone or 72 hours of daily 500nM azacitidine followed subsequently with 10 nM, 100nM, 1000nM entinostat at 96 hours. DNMT1 and H3K4 protein levels were assayed by Western blot analysis at various time points after treatment. Cells were also re-plated after treatment to assess differential growth characteristics. RESULTS: In vitro growth of H838 cells is sensitive to azacitidine alone and in combination with entinostat whereas growth of the A549 cell line is only sensitive to azacitidine and entinostat in combination. Treatment with azacitidine, severely depleted DNMT1 protein expression in H838 cells both when given alone and in combination with most dose levels of entinostat. This decrease was transient and within 4 days, the protein was fully replenished. Azacitidine alone did not cause an appreciable decrease in DNMT1 protein expression of A549 cells but, when combined with increasing levels of entinostat, DNMT1 levels were significantly depleted and did not replenish for 7 days. Re-plating after treatment showed growth inhibition versus mock of all regimens containing azacitidine in the H838 cell line only whereas growth inhibition in the A549 cells was observed only in the combinatory therapy arms. Furthermore, in the A549 cell line, H3K4 protein levels (a marker of active gene expression) peaked at 7 days after treatment versus a peak in the first 24 hours in the H838 cell line. CONCLUSIONS: Differing sensitivities to epigenetic therapy in lung adenocarcinoma cell lines may reflect underlying differences in molecular phenotypes. Large decreases in DNMT1 expression are associated with sensitivity to azacitidine. In a cell line insensitive to azacidine alone, the addition of entinostat may accentuate DNMT1 protein depletion and delay its reexpression. This effect on DNMT1 may render this latter cancer cell line more sensitive to epigenetic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2618. doi:10.1158/1538-7445.AM2011-2618