Chronic graft-versus-host disease (cGvHD) is a major life-threatening complication of allogeneic hematopoietic stem cell transplantation. The molecular mechanisms underlying cGvHD remain poorly understood and targeted therapies are not well established for clinical use. Here, we examined the role of the canonical WNT pathway in sclerodermatous cGvHD (sclGvHD). WNT signaling was activated in human sclGvHD with increased nuclear accumulation of the transcription factor β-catenin and WNT-biased gene expression signature in lesional skin. Treatment with highly selective tankryase inhibitor G007-LK, CK1α agonist pyrvinium or LRP6 inhibitor salinomycin, abrogated the activation of WNT signaling and protected against experimental cGvHD, without significant impact on graft-versus-leukemia effect (GvL). Treatment with G007-LK, pyrvinium or salinomycin almost completely prevented the development of clinical and histological features in the B10.D2 (H-2d)→BALB/c (H-2d) and in the LP/J (H-2b)→C57BL/6 (H-2b) model of sclGvHD. Inhibition of canonical WNT signaling reduced the release of extracellular matrix from fibroblasts and reduced leukocyte influx, suggesting that WNT signaling stimulates fibrotic tissue remodeling by direct effects on fibroblasts and by indirect, inflammation-dependent effects in sclGvHD. Our findings may have direct translational potential, as pyrvinium is in clinical use and tankyrase inhibitors are in clinical trials for other implications.

Objective Mitochondrial transcription factor A (TFAM) controls the transcription of core proteins required for mitochondrial homeostasis. This study was undertaken to investigate changes in TFAM expression in systemic sclerosis (SSc), to analyze mitochondrial function, and to evaluate the consequences for fibroblast activation. Methods TFAM expression was analyzed by immunofluorescence and Western blotting. The effects of TFAM knockout were investigated in cultured fibroblasts and in murine models of bleomycin‐induced skin fibrosis, bleomycin‐induced lung fibrosis, and skin fibrosis induced by overexpression of constitutively active transforming growth factor β type I receptor (TGFβRΙ). Results TFAM expression was down‐regulated in fibroblasts in SSc skin and in cultured SSc fibroblasts. The down‐regulation of TFAM was associated with decreased mitochondrial number and accumulation of damaged mitochondria with release of mitochondrial DNA (mtDNA), accumulation of deletions in mtDNA, metabolic alterations with impaired oxidative phosphorylation, and release of the mitokine GDF15. Normal fibroblasts subjected to long‐term, but not acute, exposure to TGFβ mimicked SSc fibroblasts, with down‐regulation of TFAM and accumulation of mitochondrial damage. Down‐regulation of TFAM promoted fibroblast activation with up‐regulation of fibrosis‐relevant Gene Ontology terms in RNA‐Seq, partially in a reactive oxygen species–dependent manner. Mice with fibroblast‐specific knockout of Tfam were prone to fibrotic tissue remodeling, with fibrotic responses even to NaCl instillation and enhanced sensitivity to bleomycin injection and overexpression of constitutively active TGFβRI. TFAM knockout fostered Smad3 signaling to promote fibroblast activation. Conclusion Alterations in the key mitochondrial transcription factor TFAM in response to prolonged activation of TGFβ and associated mitochondrial damage induce transcriptional programs that promote fibroblast‐to‐myofibroblast transition and drive tissue fibrosis.

BackgroundSSc is an autoimmune disease characterized by vasculopathy and tissue fibrosis. Oxidative stress is prominent in affected organs and is hypothesized to contribute to inflammatory and fibrotic damage. sGC is a heterodimeric enzyme that binds nitric oxide (NO) to a prosthetic heme group and catalyzes the production of cGMP. Released cGMP acts as a second messenger to regulate processes including tissue remodeling and immune/inflammatory response [1]. Pharmacologic modulation of sGC with a class of compounds called sGC stimulators have been shown to exert anti-fibrotic and anti-inflammatory effects in models of SSc [2]. In a phase II study in SSc, patients treated with the sGC stimulator Riociguat showed a trend towards clinical efficacy [3]. The action of sGC stimulators is dependent on NO-sGC complexes bearing a reduced heme molecule. In environments of oxidative stress, as seen in SSc, altered redox balance may limit efficacy of sGC stimulators due to heme oxidation and the formation of a pool of NO-insensitive sGC. A second class of sGC compounds, called sGC activators, are heme-independent and can act as a substitute for the heme-NO complex and in turn activate the sGC enzyme. Thus, sGC activators may show enhanced activity vs. sGC stimulators in conditions of compromised redox balance.ObjectivesTo evaluate the efficacy of the potent sGC activator BI 685509 in cellular and in vivo models of SSc pathobiology.MethodsHuman dermal microvascular endothelial cells (HDMVEC) were cultured in normoxic or 1% O2conditions in the presence of BI 685509 ranging in doses from 0.04 to 10 µM. After 48 hrs, culture supernatant was collected and the concentration of TGFβ2 was determined. Human platelet rich plasma (PRP) was isolated and activated for 5 minutes with ADP in the presence of BI 685509 or Riociguat at doses of 0.1, 1 or 10 µM. Following activation, supernatants were collected and the level of CXCL4 was measured. For the bleomycin indued skin and lung fibrosis studies, adult female C57Bl/6 mice were used. Mice received intradermal injections of bleomycin every other day for six weeks or a single intratracheal injection. Mice were treated with BI 685509, Riociguat or Nintedanib daily beginning at day 21 (dermal) or 14 (lung) after initiation of bleomycin injection. At 6 (dermal) or 4 (lung) weeks post treatment initiation, skin or lung samples were analyzed via histologic analysis for various cellular and biochemical markers of tissue fibrosis.ResultsIn HDMVEC, hypoxia induced production of the tissue remodeling factor TGFβ2 was significantly inhibited upon treatment with BI 685509 (10 µM). In the mouse model of bleomycin induced skin and lung fibrosis, activation of sGC via BI 685509 treatment at 1 mg/kg resulted in significant amelioration of skin thickness and lung fibrosis that was equivalent to mice treated with the standard of care Nintedanib (60 mg/kg) or the sGC stimulator Riociguat (1 mg/kg). Lastly, to mimic a NO deficient environment seen in SSc, human PRP was utilized. Induction of the SSc relevant chemokine CXCL4 was effectively inhibited in activated PRP treated with the sGC activator BI 685509 whereas minimal inhibition was observed in samples treated with the sGC stimulator Riociguat.Inhibition of CXCL4 production in activated human platelet rich plasma treated with the sGC activator BI 685509Figure 1.ConclusionCollectively, these results point to the use of the sGC activator BI 685509 as a novel treatment for SSc and suggests potential superior effects vs. sGC stimulators like Riociguat in this autoimmune disease.References[1]Stasch JP, et al Circulation 2011;123:2263-2273[2]Dees C, et al Ann Rheum Dis 2015;74:1621-1625[3]Khana D., et al Ann Rheum Dis 2020;79:618-625Acknowledgements:NIL.Disclosure of InterestsGerald Nabozny Employee of: Boehringer Ingelheim Pharmaceuticals Inc., Chao-Ting Wang Employee of: Boehringer Ingelheim Pharmaceuticals Inc., Leeanne Daley Employee of: Boehringer Ingelheim Pharmaceuticals Inc., David Ebenezer Employee of: Boehringer Ingelheim Pharmaceuticals Inc., Denis Delic Employee of: Boehringer Ingelheim, Tom Bretschneider Employee of: Boehringer Ingelheim, Thuong Trinh-Minh: None declared, Cuong Tran-Manh: None declared, Joerg Distler: None declared, Julia Kaufman Employee of: Boehringer Ingelheim Pharmaceuticals Inc.