gms | German Medical Science

Background: Persistent activation of fibroblasts with excessive release of extracellular matrix is a hallmark of fibrotic diseases such as systemic sclerosis. The ETS transcription factor PU.1 was identified as molecular checkpoint for acquisition of a “pro-fibrotic”, matrix-producing phenotype of fibroblasts (Wohlfahrt et al., Nature, 2019).

Methods: The heterocyclic diamidine DB1976 was used as therapeutic compound to inhibit PU.1. DB1976 was explored in different in vitro assays and in several mouse models of fibrosis including bleomycin-induced skin fibrosis, bleomycin-induced lung fibrosis, carbon tetrachloride (CCl4)-induced liver fibrosis, and sclerodermatous chronic graft-versus-host disease.

Results: DB1976 decreased the transcription of COL1A1, reduced the expression of type I collagen and α-SMA and inhibited the expression of F-actin in fibrotic fibroblasts at least to the levels of resting fibroblasts at non-toxic concentrations. RNA-sequencing (RNA-seq) analysis and subsequent gene set enrichment analysis (GSEA) demonstrated that incubation with DB1976 inhibited the pro-fibrotic gene signature of fibrotic fibroblasts without effects on apoptosis-related and inflammatory Gene Ontology (GO)-defined gene sets. DB1976 induced a gene expression pattern comparable to that of resting fibroblasts. Conversely, GSEA of resting fibroblasts co-transfected with PU.1 revealed upregulation of the pro-fibrotic gene set. Additional treatment with DB1976 completely blocked the pro-fibrotic effects of PU.1 overexpression. Finally, we investigated pharmacological targeting of PU.1 by DB1976 as a potential strategy to prevent uncontrolled fibrotic tissue remodelling. DB1976 showed anti-fibrotic effects in vivo in various fibrosis models and across several organs. Treatment with DB1976 not only prevented bleomycin-mediated skin fibrosis, but also induced regression of pre-established fibrosis. Treatment with DB1976 in anti-fibrotic concentrations did not affect body weight, pain and distress levels of mice. At the cellular level, we did not detect disturbance of haematopoiesis, alterations in haematopoietic and mesenchymal stem cells, defects in B cell development in the bone marrow or T cell maturation within the thymus after DB1976 treatment, and was well tolerated.

Conclusion: These findings suggest that PU.1 inhibition by DB1976 may represent a novel and effective therapeutic approach to treat a wide range of fibrotic diseases.