Background/Purpose: Systemic sclerosis (SSc) is a deadly disease characterized by immune dysregulation, vasculopathy, and fibrosis. SSc is the most lethal rheumatic disease associated with the poorest quality of life. The extent of skin fibrosis may predict internal organ involvement. The cells promoting skin fibrosis in SSc are human dermal fibroblasts (HDFs) which develop a myofibroblast (MFB)-phenotype, senescence-like-features, and resistance-to-apoptosis. We recently showed that increased genomic instability associated with double-stranded DNA breaks (DSBs) and senescence like features are present in rapidly progressive diffuse (dcSSc) HDF. In cancer, dysregulated DSBs and senescence-like-features are associated with activation of the transcription factor, forkhead box protein O (FOXO1) to promote metabolic remodelling. Therefore, we hypothesized that DSBs promotes FOXO1 activation to promote fibrosis.

Methods: Primary HDFs were generated from healthy volunteers (HC), less severe early limited (lcSSc, < 2 year disease duration), and severe dcSSc 4 mm skin biopsies (< 2 year disease duration). All our SSc patients met 2013 ACR/EULAR inclusion criteria. DSBs were quantified by measuring γ-H2AX (a DSB marker) via immunoblots (IB) and immunofluorescence/confocal microscopy (IF). Nuclear FOXO1-translocation, MFB differentiation and fibrotic markers were measured using IF, IB and qRT-PCR. HC HDFs were treated with DNA damage-inducing agents (e.g. etoposide), then nuclear FOXO1 and the MFB marker alpha-SMA were quantified using IB and IF. Pro-fibrotic signals were measured in dcSSc HDF following FOXO1 inhibition.

Results: dcSSc HDFs had the highest levels of γ-H2AX compared to HC (*p< 0.05) and lcSSc patients. They also had a substantially higher nuclear accumulation of FOXO1 which was associated with increased mRNA expression of the FOXO1 transcriptional target, pyruvate dehydrogenase kinase 4 (*p< 0.05). FOXO1 inhibition resulted in decreased fibrotic-markers and PDK4 (*p< 0.05) in dcSSc HDF. Etoposide treatment promoted FOXO1 activation, MFB differentiation, and PDH phosphorylation.

Conclusion: DSBs are more commonly present in HDF from dcSSc patients, which may promote MFB differentiation, resistance-to-apoptosis and fibrosis. We propose that FOXO1 activation may promote a downstream metabolic remodelling and an associated senescence like signal that promotes cell survival, and propagation of acquired genomic mutations with associated downstream inflammatory signals. Our findings provide mechanistic insights that may impart a deeper understanding for the role(s) of DSB-associated FOXO1 activation in promoting fibrosis in SSc. They may also have far-reaching implications related to the development of novel therapeutic strategies in SSc.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/impaired-dna-repair-responses-activate-a-novel-foxo1-dependent-metabolic-remodelling-in-patients-with-progressive-systemic-sclerosis-to-promote-fibrosis/