Targeting Lung Fibrosis Using Epigenetic Therapy

PROJECT ABSTRACT Pulmonary fibrosis is a devastating disease for which there are limited treatments and no cure. There are several known causes for pulmonary fibrosis, including certain medicines, radiation, infection, allergic airway disease, and silica exposure, yet many people develop fibrosis without definite causes. Current drugs focus on reducing the inflammatory microenvironment and can be effective at preventing disease progression, but fail to resolve fibrotic injury. EZH2 is a histone methyltransferase in the Polycomb Repressive Complex 2 that is known to directly control the differentiation state of multiple cell types, including immune cells and epithelial cells, through repression of alternative cell lineage genes. The canonical mark of the PRC2 complex is histone H3 lysine 27 tri-methylation (H3K27me3). Numerous lines of evidence, including preliminary data from our laboratory, suggest that targeting EZH2 could be beneficial for fibrosis patients. Specifically, we have found a dramatic loss of the newly identified KRT8+/KRT17+ aberrant basaloid cell when EZH2 was deleted. Our Central Hypothesis is that pulmonary fibrosis can be attenuated using an EZH2 inhibitor by de-repression of FOXP2 in the epithelium, preventing accumulation of aberrant basaloid KRT8+ cells, and by decreasing IL1B secretion and signaling. Both of these changes may drive decreased accumulation of activated fibroblasts, and ameliorate fibrotic disease. Specific Aim 1: Determine if EZH2 depletion/inhibition prevents accumulation of KRT8+ cells through de-repression of FOXP2, and prevents accumulation of fibrosis through reduction in IL1B; We will leverage lung epithelial organoid cultures of both human and murine alveolar type II organoids co-cultured with fibroblasts derived from normal or fibrotic lungs. Pharmacologically, we will treat cultures with tazemetostat (EPZ-6438), and genetically we will modulate of EZH2 and FOXP2 expression using viral over-expression, knock-down and floxed alleles. Presence and abundance of KRT8+ cells and SMA+ collagen producing fibroblasts will be assessed by immunostaining and RT-qPCR. IL1B secretion, as well as additional cytokines, will be assessed and rescued with recombinant protein. ChIP-seq and RNA-seq will be used to understand how EZH2 inhibition influences chromatin state in both the epithelial and mesenchymal populations. Staining for FOXP2 and H3K27me3 in KRT8+ cells will be quantified to understand the relationship between these markers in patient samples. Specific Aim 2: Inhibit or reverse induced fibrosis in vivo with EZH2 and FOXP2 depletion/inhibition; We will use both bleomycin and AAV-TGFβ induced fibrosis models. We will treat with tazemetostat, the FDA approved EZH2 inhibitor, starting at day 1 (prevention) or day 14 (intervention). At endpoint, lung function will be tested using the flexiVent system, and lungs will be harvested for scRNAseq and histological profiling. Abundance of activated fibroblasts, KRT8+ ‘aberrant basaloid cells’ and immune cell types will be quantified. In parallel, Ezh2 floxed and Foxp2 floxed mice with lineage specific-Cre promoters will be used to understand the contributions of these transcriptional regulators in different compartments of fibrotic responses. These Aims will allow us to directly test and validate EZH2 inhibition through tazemetostat as a novel therapeutic strategy for fibrosis patients. It is possible that inhibiting EZH2 using an FDA approved orally, bio-available drug will reduce or resolve pulmonary fibrosis through two distinct cellular mechanisms. Even if one of our sub-hypotheses is disproven, we will still gain important insights into the mechanisms through which epi-drugs, which are an emerging class of small molecules, may be leveraged for lung disease. 1