B77-23 Pre-clinical Efficacy of the DNA Methyltransferase (DNMT) Inhibitor Zebularine in Pulmonary Fibrosis

Abstract Rationale Idiopathic pulmonary fibrosis (IPF) is a rare, chronic disease that is characterized by the accumulation of activated fibroblasts within the lung tissue. Several studies have shown that DNA hypermethylation is a key feature of fibrosis and contributes to the activated phenotype of fibrotic fibroblasts. We have previously shown that inhibitors of DNA methylation restore the expression of certain genes silenced in IPF fibroblasts. Here, we sought to test the therapeutic efficacy of the DNA methylation inhibitor zebularine in inhibiting and even reversing fibrosis in vitro and in vivo. Methods Primary lung fibroblasts derived from patients with IPF and non-fibrotic controls were isolated and expanded in 2D cell culture. In some experiments, cells were treated with zebularine (10, 25, and 50 µM) and transforming growth factor - β1 (TGF-β1) simultaneously for 72 hours before collection and analysis of α-smooth muscle actin (SMA), collagen I, and fibronectin by RT-PCR and immunoblot. In other experiments, cells were treated first with TGF-β1 for 48 hours and then zebularine (10, 25, and 50 µM) for 72 hours to assess reversal of myofibroblast differentiation. Wild-type C57bl/6 mice were treated with bleomycin or saline and zebularine was administered intraperitoneally every day from days 10-20 before they were euthanized at day 21 and assayed for hydroxyproline. Precision cut lung slices (PCLS) of IPF and non-fibrotic lung were additionally treated with zebularine in vitro. Results Both IPF and non-fibrotic control fibroblasts treated with zebularine demonstrated decreased expression of collagen I and fibronectin at both the mRNA and protein level. This was observed when treated simultaneously with TGF-β1 and even more so during reversal of myofibroblast differentiation. This effect was more pronounced in normal lung fibroblasts compared to IPF fibroblasts. This occurred independent of cell toxicity. Mice treated with zebularine demonstrated less fibrosis after bleomycin. PCLS treated with zebularine also demonstrated less collagen. Conclusions The data suggest that inhibition of DNA methylation may have the capability to inhibit and even reverse myofibroblast differentiation. DNA methylation and potentially other epigenetic modifications contribute to the activated phenotype of IPF fibroblasts. Future studies will examine which genes are differentially methylated by zebularine. This abstract is funded by: NHLBI (HL162963)