D29-08 Targeting RAD51 Mediated DNA Damage Response (DDR) Pathway for the Treatment of Pulmonary Fibrosis

Abstract Rationale Idiopathic pulmonary fibrosis (IPF) is fatal interstitial lung disease driven by apoptosis resistant fibroblasts and dysregulated energy metabolism. Although transforming growth factor-β (TGF-β) is a central profibrotic cytokine that plays a pivotal role in fibrogenic signaling, however the mechanisms that sustain their survival remain poorly understood. Our findings identify the DNA Damage Response (DDR) protein RAD51 is highly expressed in IPF lungs and may link as a crucial integrator of prosurvival and metabolic signaling in fibrotic fibroblasts. We propose that RAD51 is linked to mitochondrial metabolism and autophagy, promoting fibroblast survival via mTORC1 dependent mechanisms. Targeting RAD51 for therapeutic benefits may restore apoptotic sensitivity and facilitate the resolution of fibrosis. Methods Primary normal human lung fibroblasts (NHLFs) or IPF fibroblasts, and precision cut lung slices (PCLS) derived from both human donors and bleomycin-challenged mice were used. RAD51 expression was silenced using siRNA or inhibited pharmacologically with B02 in the presence and absence of TGF-β alone or in combination with TNF-α. Fibrotic markers (ACTA2, Col1a1), autophagic markers (LC3B, Beclin-1), apoptotic regulators, including pro-apoptotic (BAK, BAX, and cleaved caspase-3) and anti-apoptotic (BCL-2, BCL-XL) proteins were quantified by qPCR, western blotting and/or immunostaining. The mTOR signaling was assessed by evaluating the phosphorylation status of 4E-BP1, S6K, and AKT. Mitochondrial functions and bioenergetics were assessed using Seahorse analysis to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Mitopore assays and subcellular fractionation were used to evaluate mitochondrial permeability transition and cytosolic release of mitochondrial factors. Results RAD51 expression was significantly upregulated in the lungs of IPF patients and in fibrotic lung fibroblasts. TGF-β induced profibrotic and antiapoptotic responses were required RAD51 activity. Inhibition or loss of RAD51 function rendered fibroblasts more susceptible to apoptosis and autophagy while suppressing mTORC1 signaling and mitochondrial metabolism. Furthermore, pharmacological inhibition of RAD51 using B02 transitioned the profibrotic phenotype to a fibrosis-resolving state in human and bleomycin-induced mice PCLS. Conclusions We investigated the mechanism(s) by which TGF-β mediated metabolic and prosurvival regulatory network with DNA damage response pathway contributes to lung fibrosis. We provided an entirely new conceptual component for better understanding of the mechanism(s) driving fibrosis. This abstract is funded by: NHLBI, NIH