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Background: Engrailed 1 (EN1), a homeodomain-containing transcription factor (TF), is transiently expressed in the developing dermis of murine embryos within a specific fibroblast lineage 1. We previously showed that EN1 is re-expressed in systemic sclerosis (SSc) in a TGFβ-dependent manner and that it amplifies the pro-fibrotic effects of TGFβ to promote fibroblast activation and fibrosis 2. EN1 might thus be a promising target for antifibrotic therapies. However, TFs are widely recognized as challenging to target. The pro-fibrotic effects of EN1 are essentially dependent on its interaction with other TF, i.p. with those from the SP family. An approach for targeting EN1 by an EN1 interference peptide (EN1-iPEP) has recently been described 3. The EN1-iPEP contains a highly conserved hexamotif WPAWVY from the EN1 homeodomain, required for binding to its interaction partners. EN1-iPEP thus prevents the effects of the endogenous EN1 by competing for binding to its interaction partners. Objectives: To evaluate the therapeutic potential of EN1-iPEP for the treatment of fibrosis. Methods: An EN1-iPEP and a control, mutated peptide (mut-iPEP) were designed; mut-iPEP is identical to the EN1-iPEP, except that the hexamotif for binding to interaction partners is mutated, allowing to evaluate the effects of EN1-iPEP that are specific to blocking the endogenous EN1. The antifibrotic effects of the administration of EN1-iPEP were evaluated in two-dimensional culture of human dermal fibroblasts, in three-dimensional human skin equivalent models, in the mouse model of bleomycin-induced skin fibrosis and in precision cut slices of SSc skin (SSc-PCSS). Bulk RNA-seq was performed from SSc-PCSS. Immunoprecipitation experiments were performed to validate the in silico predictions and confirm the mechanism of action of EN1-iPEP. Results: Treatment with EN1-iPEP, but not with mut-iPEP ameliorates the profibrotic effects of TGFβ in human dermal fibroblasts in standard two-dimensional culture, with reduced myofibroblasts differentiation and collagen production. Furthermore, treatment with EN1-iPEP prevented TGFβ-induced increases in dermal thickness, in collagen production and myofibroblasts counts in three-dimensional human skin equivalent models. In vivo treatment with EN1-iPEP ameliorates bleomycin-induced skin fibrosis in mice, with reduced myofibroblasts counts, dermal thickness and hydroxyproline content. Furthermore, EN1-iPEP partially reverses the SSc gene expression profile in SSc-PCSS. Pathway ORA and GSEA identifies de-enrichment of terms related to extracellular matrix synthesis, actin-mediated contraction and to canonical Wnt signaling. Mechanistically, EN1-iPEP prevents the formation of a transcriptional complex of EN1, β-catenin and SP1 to inhibit the aberrant Wnt/β-catenin signaling in SSc. Conclusion: By employing a series of models, including direct evaluation in SSc skin, we demonstrate that treatment with EN1-iPEP (and interference with the cooperative DNA binding of EN1) inhibits two core profibrotic pathways in SSc, TGFβ and Wnt/β-catenin in SSc, and can thus prevent fibroblast activation and collagen production. EN1-iPEP may thus offer therapeutic potential in SSc. REFERENCES: 1 Rinkevich Y, Walmsley GG, Hu MS, Maan ZN, Newman AM, Drukker M, et al. Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential. Science. 2015;348(6232):aaa2151. 2 Gyorfi AH, Matei AE, Fuchs M, Liang C, Rigau AR, Hong X, et al. Engrailed 1 coordinates cytoskeletal reorganization to induce myofibroblast differentiation. J Exp Med. 2021;218(9). 3 Sorolla A, Wang E, Clemons TD, Evans CW, Plani-Lam JH, Golden E, et al. Triple-hit therapeutic approach for triple negative breast cancers using docetaxel nanoparticles, EN1-iPeps and RGD peptides. Nanomedicine. 2019;20:102003. Acknowledgements: NIL. Disclosure of Interests: Liyan Xu: None declared, Veda Devakumar: None declared, Andrea-Hermina Györfi: None declared, Xuezhi Hong: None declared, Yanhua Xiao: None declared, Tim Filla: None declared, Yi-nan Li: None declared, Jörg Distler Stock owner of 4D Science and Scientific head of FibroCure, Actelion, Active Biotech, Anamar, ARXX, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, JB Therapeutics, Medac, Pfizer, RuiYi and UCB, Anamar, Active Biotech, Array Biopharma, aTyr, BMS, Bayer Pharma, Boehringer Ingelheim, Celgene, Galapagos, GSK, Inventiva, Novartis, Sanofi-Aventis, RedX, UCB, Alexandru-Emil Matei: None declared.
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