No abstract

Bleomycin is a clinically potent anticancer drug used for the treatment of germ-cell tumors, lymphomas and squamous-cell carcinomas. Unfortunately, the therapeutic efficacy of bleomycin is severely hampered by the development of pulmonary fibrosis. However, the mechanisms underlying bleomycin-induced pulmonary fibrosis, particularly the molecular target of bleomycin, remains unknown. Here, using a chemical proteomics approach, we identify ANXA2 (annexin A2) as a direct binding target of bleomycin. The interaction of bleomycin with ANXA2 was corroborated both in vitro and in vivo. Genetic depletion of anxa2 in mice mitigates bleomycin-induced pulmonary fibrosis. We further demonstrate that Glu139 (E139) of ANXA2 is required for bleomycin binding in lung epithelial cells. A CRISPR-Cas9-engineered ANXA2 mutation in lung epithelial cells ablates bleomycin binding and activates TFEB (transcription factor EB), a master regulator of macroautophagy/autophagy, resulting in substantial acceleration of autophagic flux. Pharmacological activation of TFEB elevates bleomycin-initiated autophagic flux, inhibits apoptosis and proliferation of epithelial cells, and ameliorates pulmonary fibrosis in bleomycin-treated mice. Notably, we observe lowered TFEB and LC3B levels in human pulmonary fibrosis tissues compared to normal controls, suggesting a critical role of TFEB-mediated autophagy in pulmonary fibrosis. Collectively, our data demonstrate that ANXA2 is a specific bleomycin target, and bleomycin binding with ANXA2 impedes TFEB-induced autophagic flux, leading to induction of pulmonary fibrosis. Our findings provide insight into the mechanisms of bleomycin-induced fibrosis and may facilitate development of optimized bleomycin therapeutics devoid of lung toxicity.

Trinucleotide repeat (TNR) expansion is responsible for numerous human neurodegenerative diseases. However, the underlying mechanisms remain unclear. Recent studies have shown that DNA base excision repair (BER) can mediate TNR expansion and deletion by removing base lesions in different locations of a TNR tract, indicating that BER can promote or prevent TNR expansion in a damage location–dependent manner. In this study, we provide the first evidence that the repair of a DNA base lesion located in the loop region of a CAG repeat hairpin can remove the hairpin, attenuating repeat expansion. We found that an 8-oxoguanine located in the loop region of CAG hairpins of varying sizes was removed by OGG1 leaving an abasic site that was subsequently 5′-incised by AP endonuclease 1, introducing a single-strand breakage in the hairpin loop. This converted the hairpin into a double-flap intermediate with a 5′- and 3′-flap that was cleaved by flap endonuclease 1 and a 3′-5′ endonuclease Mus81/Eme1, resulting in complete or partial removal of the CAG hairpin. This further resulted in prevention and attenuation of repeat expansion. Our results demonstrate that TNR expansion can be prevented via BER in hairpin loops that is coupled with the removal of TNR hairpins.