Targeting the DNA damage repair protein RAD51 alters fibroblast metabolism and enhances apoptosis in pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease characterized by aberrantly activated, apoptosis-resistant profibrotic lung (myo)fibroblasts. Prior research has demonstrated that lung fibroblasts from patients with IPF exhibit resistance to DNA damage, suggesting that this behavior contributes to their persistent survival and continuous proliferation. We propose that elevated levels of the DNA damage repair protein RAD51 regulate myofibroblast activation and apoptosis and provide a potential therapeutic target to impede fibrosis progression. MethodsHuman lung fibroblasts were transfected with siRNA against RAD51 or treated with RAD51-specific inhibitor B02 and markers of fibrosis, DNA damage, apoptosis, metabolic reprogramming, and mitochondrial dynamics were assessed. The preclinical efficacy of B02 was evaluated in human precision cut lung slices (PCLS) and in a mouse model of pulmonary fibrosis. FindingsRAD51 expression was significantly upregulated in the lungs and lung fibroblasts of IPF patients. Knockdown or inhibition of RAD51 in fibroblasts reduced profibrotic marker expression, suppressed mTORC1 signaling and mitochondrial function, and increased apoptosis susceptibility. Pharmacological inhibition of RAD51 shifted the profibrotic phenotype towards a fibrosis-resolving state in human and mouse PCLS, and in a bleomycin-induced mouse model of lung fibrosis. InterpretationThe inhibition of RAD51 exerts therapeutic benefits in lung fibrosis by promoting apoptosis. Our findings identify that inhibiting RAD51 with B02 in fibroblasts impairs DNA repair and induces metabolic reprogramming, making it a potential therapeutic target. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSPulmonary fibrosis (PF) is characterized by excessive fibroblast activation and subsequent deposition of extracellular matrix (ECM) proteins, which ultimately disrupt normal lung architecture. A significant contributing factor to the pathogenesis of pulmonary fibrosis is the presence of fibroblasts that are resistant to apoptosis, preventing normal wound healing. Recent studies highlight the DNA repair protein RAD51 as effective in protecting fibroblasts from death induced by chemotherapy and ionizing radiation. These finding suggested that RAD51 could have a role in fibroblast activation and apoptosis resistance in pulmonary fibrosis. Added value of this studyWe demonstrated that RAD51 is important for maintaining apoptosis-resistant fibrotic fibroblasts and their metabolic abnormalities. Our findings indicated that TGF{beta}-mediated upregulation of RAD51 reduces DNA damage, activates multiple pathways related to fibroblast activation and proliferation, and induces metabolic reprogramming, ultimately regulating apoptosis. Mechanistically, RAD51 inhibition enhanced p53 acetylation at lysine 120 and upregulated the expression proapoptotic proteins PUMA/BAK in mitochondria, promoting apoptosis. Pharmacological inhibition of RAD51 using the specific inhibitor B02 during the fibrotic phase of experimental lung disease effectively ameliorated pulmonary fibrosis. Implications of all the available evidenceOur findings establish that RAD51 plays an important role in the survival of apoptosis-resistant fibrotic fibroblasts. We propose that reducing RAD51 expression leads to the metabolic reprogramming of activated fibroblasts, resulting in decreased mitochondrial respiration, reduced ATP levels, and diminished glycolysis or glutaminolysis. These observations suggest that targeting energy metabolism through RAD51 inhibition could be a viable strategy to enhance apoptosis, thereby creating a therapeutically targetable pathway in fibrotic cells. These findings highlight the potential of RAD51 as a therapeutic target for the treatment of IPF.