Dysregulation of the Cancer-Associated Glycan Polysialic Acid in Systemic Sclerosis: A Novel Biomarker and Anti-Fibrotic Target

Objectives Diffuse cutaneous systemic sclerosis (dcSSc) is a life-limiting inflammatory disease characterized by progressive fibrosis, vasculopathy and immune dysfunction. Fibroblasts (FB) are the key drivers of fibrosis and adapt epigenetic changes that promote their activation, resistance to apoptosis, and release of pro-fibrotic mediators. Autologous stem cell transplantation (ASCT) is a disease-modifying therapy that improves fibrosis in some dcSSc patients - although its effects on restoring FB function(s) are unknown. We recently identified the cancer-associated glycan, polysialic acid (polySia), in skin sections from patients with dcSSc and polySia levels correlated with fibrosis which normalized post-ASCT.[1] We also showed that dermal sections from patients with dcSSc had increased genomic instability, double-stranded DNA breaks (DSB) and epigenetic activation of the transcription factor FOXO1. We hypothesized that FOXO1 may promote polySia expression to enhance fibrosis, and that targeting polySia may provide anti-fibrotic effects. Methods We measured polySia levels in sera from 22 patients with SSc using a specific polySia ELISA and correlated polySia levels with the severity of skin fibrosis using the modified Rodnan skin score (mRSS). We also used primary dermal FB from skin biopsies of healthy controls (HC, age/sex matched), less severe limited cutaneous SSc (lcSSc), dcSSc and post-ASCT patients (N=4-6/per group) and measured the frequency of DSBs, active (nuclear) FOXO1, and polySia levels using immunofluorescence/confocal microscopy and/or immunoblot (IB). We also quantified polySia, ST8SIA2 levels or pro-fibrotic markers (fibronectin and CTGF) using qRT-PCR and/or IB following FOXO1 pharmacological inhibition, ST8SIA2 knockdown via siRNA, or treatment with a polySia elongation inhibitor, 8-keto-Neu5Ac. Results Total polySia levels correlated with mRSS (p=0.007, rho=0.560) in SSc patients – highlighting its direct link with fibrosis in SSc. We also observed that FB from dcSSc had a 2-3-fold induction in polySia levels and ST8SIA2 expression compared to FB from lcSSc and age/sex matched HC (p=0.02). Importantly, post-ASCT FB had a substantial reduction in ST8SIA2 (p=0.04) and polySia levels. This was associated with increased DSBs and FOXO1 activation exclusively in dcSSc FB. Finally, FOXO1 inhibition, ST8SIA2 siRNA knockdown, or treatment with 8-keto-Neu5Ac resulted in a 1.9-fold reduction in pro-fibrotic markers and total polySia levels (Figure 1). Figure 1: A. Graphical abstract of proposed mechanism. In dcSSc fibroblasts, double-stranded DNA breaks activate the transcription factor FOXO1. Upon activation, FOXO1 upregulates the gene expression of the polySia synthetic enzyme ST8SIA2 , polySia, and fibrotic mediators such as CTGF and fibronectin, thereby promoting fibrotic remodelling and fibrosis in dcSSc. B. Confocal images showing polySia (green), cis-golgi marker GM130 (red), nucleus (DAPI, blue) in fibroblasts from HC, dcSSc and post-ASCT. C. Spearman correlation analysis comparing serum polySia to mRSS in SSc patients (n = 22). Conclusion Our study identifies a novel DSB/FOXO1/polySia pathway as a key driver of fibrosis in SSc. This axis may serve as a biomarker for disease progression and may be indicative of restoration of FB functions post-ASCT. We postulate that targeting the FOXO1/polySia pathway may provide a novel therapeutic approach in dcSSc. References [1.] Khan L. J Autoimmun 2023;140:103110.