B77-26 Bromodomain-containing Protein 4 (BRD4) Plays an Essential Role in Extracellular Matrix Expression in Human Precision Cut Lung Slices

Rationale

Fibrotic Interstitial Lung Diseases (fILD) are deadly disorders characterized by excessive extracellular matrix (ECM) deposition. There is a need to identify novel mechanisms of fibrogenesis to foster development of improved therapeutics. Bromodomain-containing protein 4 (BRD4) is an epigenetic reader that plays a role in reprogramming cell transcription in response to injury. We hypothesize that inhibiting BRD4 activity in a patient-derived ex vivo model of lung fibrosis, human precision cut lung slices (PCLS), would decrease markers of fibrogenesis.

Methods

We obtained lung tissue from deceased non-utilized lung transplant donation (non-fibrotic) and from patients with fILD. PCLS were created by inflating lung tissue with warm agarose, cooling, coring (10 mm), and slicing with a Leica vibratome (500 µm). PCLS were incubated overnight in DMEM supplemented with 0.01% FBS, L-glutamine, and antibiotics (huPCLS DMEM) and cultured the following day in huPCLS DMEM with vehicle (DMSO) or the highly selective BRD4 inhibitor, ZL0969 (10 µM). For non-fibrotic PCLS, 5 ng/ml TGF-β1 was added to initiate fibrogenesis. PCLS and supernatants were harvested at 120h. Supernatants were collected, cleared, and subjected to hyaluronan ELISA. PCLS were harvested for mRNA, protein, and formalin-fixed, paraffin embedded (FFPE). RT-qPCR was performed for fibronectin and collagen 1a1. Protein lysates were subjected to polyacrylamide gel electrophoresis (PAGE) followed by Western blotting. FFPE slides underwent Masson’s trichrome staining and immunostaining for fibronectin.

Results

In TGF-β1 treated non-fibrotic human PCLS (n = 4) there was robust induction of fibronectin and collagen 1a1 transcript and protein expression, along with hyaluronan content in supernatants. BRD4 inhibition with ZL0969 resulted in a significant reduction of TGF-β1-induced fibronectin and collagen 1a1 gene expression and fibronectin protein expression as assessed by IHC and PAGE/Western. ZL0969 also significantly reduced TGF-β1-induced hyaluronan secretion. In human PCLS from fibrotic lung (n = 3), there was significant variability in basal expression levels of fibronectin and collagen 1a1 mRNA. In each donor, inhibition of BRD4 with ZL0969 resulted in a decrease of fibronectin and collagen 1a1 (trend, p < 0.10). Finally, secreted hyaluronan from fibrotic PCLS were reduced by ZL0969.

Conclusion

Using a patient-derived ex vivo model to recapitulate ECM expression, we observe that BRD4 is essential for both TGF-β1- induced expression and secretion of fibronectin, collagen 1a1, and hyaluronan, and for endogenous fibronectin, collagen 1a1, and hyaluronan expression in fibrotic lung. These findings indicate that small molecule inhibitors of BRD4, such as ZL0969, have promise in disrupting lung fibrosis in patients with fILD.

This abstract is funded by: Department of Defense