Therapeutic molecular targets of SSc-ILD

Abstract

Systemic sclerosis is a fibrosing chronic connective tissue disease of unknown etiology. A major hallmark of systemic sclerosis is the uncontrolled and persistent activation of fibroblasts, which release excessive amounts of extracellular matrix, lead to organ dysfunction, and cause high mobility and motility of patients. Systemic sclerosis–associated interstitial lung disease is one of the most common fibrotic organ manifestations in systemic sclerosis and a major cause of death. Treatment options for systemic sclerosis–associated interstitial lung disease and other fibrotic manifestations, however, remain very limited. Thus, there is a huge medical need for effective therapies that target tissue fibrosis, vascular alterations, inflammation, and autoimmune disease in systemic sclerosis–associated interstitial lung disease. In this review, we discuss data suggesting therapeutic ways to target different genes in distinct tissues/organs that contribute to the development of SSc.

Introduction

Systemic sclerosis (SSc) is an idiopathic connective tissue disease in which extensive fibrosis, vascular alterations, inflammation, and autoantibodies against various cellular antigens are the principal features.

SSc has a worldwide distribution and is 3–5 times more frequent in women than in men. The age at diagnosis ranges from 30 to 50 years, although the disease can also occur in children and elderly people. ¹ Fibrosis in SSc is not restricted to a single organ system but can affect multiple tissues. The fibrotic process is most frequently observed in the skin, lungs, gastrointestinal tract, heart, tendons, and ligaments; widespread perivascular fibrosis also occurs.^2,3 Fibrotic tissue remodeling and subsequent complications such as organ failure are major courses of morbidity and mortality in SSc. ⁴

Activated fibroblasts, so-called myofibroblasts, are key effector cells of fibrosis. Myofibroblasts are characterized by the expression of contractile proteins. Myofibroblasts are a heterogeneous population of cells that can be derived from various precursor populations including resident fibroblasts, pericytes, or endothelial cells of the vascular wall, epithelial cells, and bone marrow–derived fibrocytes.^5,6 In normal wound healing, the activation of myofibroblasts is self-limited, and myofibroblast accumulations resolve after the initial damage has been repaired. In fibrotic diseases such as SSc, however, fibroblasts remain persistently activated, leading to progressive tissue remodeling that culminates in tissue fibrosis.

The molecular pathways that lead to the persistent activation of fibroblasts, however, are only partially identified. Inflammatory and vascular changes may induce profibrotic responses early in the disease course of SSc, but in later stages of the disease, fibroblast activation can be maintained by cell autonomous mechanisms and may render fibroblasts partially independent from external stimuli.^4,7 In the current review, we will discuss selected pathways of fibroblast activation in systemic sclerosis–associated interstitial lung disease (SSc-ILD). The role of inflammation is discussed elsewhere.^8–10 We will particular focus on core profibrotic pathways with high translational potential.

Coagulation and platelet activation

Platelet activation

Enhanced activation of platelets and increased tendency to aggregation have long been observed in SSc patients and are generally attributed to the concomitant dysfunction of the endothelium^11,12 and microvascular alterations, which precede fibrotic manifestations in SSc. ⁷ More than 90% of the serotonin (5-hydroxytrytophan, 5-HT) in the human body is stored in platelets. Consistent with platelet activation, the levels of circulating 5-HT are elevated in SSc patients. ¹³ 5-HT has been implicated in the pathogenesis of fibrotic tissue remodeling.^14–16 Inhibition of platelet activation or knockout of a specific 5-HT receptor, 5-HT_2B, ameliorates experimental fibrosis of the skin. Pharmacologic inactivation of 5-HT_2B also effectively prevented the onset of experimental dermal fibrosis and ameliorated established fibrosis. ¹⁴ Knockout of TPH1, the rate-limiting enzyme for the generation of 5-HT outside the central nervous system (CNS), also ameliorated experimental fibrosis.^14,17 5-HT_2B antagonism inhibited pAKT/p21 signaling pathway in human lung fibroblasts and attenuated bleomycin-induced murine pulmonary fibrosis in part by anti-proliferative effects. ¹⁸ These findings suggested that activation of the platelet is a shared mechanism in the pathogenesis of fibrotic disorders.

Coagulation

Activation of the coagulation cascade upon tissue injury has been shown to play a role in the pathogenesis of fibrotic diseases in different organs, including lung, skin, kidney, heart, and liver. ¹⁹ Thrombin is a multifunctional serine protease and a key enzyme of blood coagulation that catalyzes the conversion of fibrinogen to fibrin. ²⁰ In particular, thrombin may promote tissue fibrosis, but Factor Xa has also been reported to mediate fibroblast activation and tissue fibrosis.^21,22 Thrombin induces the expression of profibrotic and proinflammatory mediators such as connective tissue growth factor (CTGF) ²³ and CC-chemokine ligand 2 (CCL2) ²⁴ via protease-activated receptor–1 (PAR-1), stimulates proliferation of fibroblasts, and promotes transdifferentiation of resting fibroblasts into myofibroblasts and the release of extracellular matrix proteins. ²⁵ Dabigatran, a direct thrombin inhibitor, reduced contraction and type I collagen release of lung fibroblasts and ameliorated experimental pulmonary fibrosis induced by bleomycin in preventive and therapeutic regimens. ²⁵ Dabigatran also attenuated cardiac fibrosis induced by transverse aortic constriction (TAC) surgery. ²⁶ Moreover, treatment with thrombin antagonism SSR182289 reduced hepatic fibrogenesis in CCl₄-induced murine liver fibrosis. ²⁷

Profibrotic growth factors

Transforming growth factor β

Transforming growth factor β (TGFβ) is a master regulator of physiological and pathological tissue repair responses. ²⁸ TGFβ receptors are two “type II” and two “type I” transmembrane serine/threonine kinase receptors. Upon activation from its latent form, TGFβ binds to the TGFβRII, which subsequently dimerizes with and phosphorylates TGFβRI to activate a plethora of downstream pathways. The best studied downstream pathway is canonical TGFβ/SMAD signaling. ²⁹ Apart from SMAD signaling, TGFβ can activate multiple alternative pathways relevant for the pathogenesis of fibrosis such as mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases (ERKs), P38, and c-Jun N-terminal kinase (JNK) as well as Rho-like GTPase/Rho-associated kinase (ROCK) and AKT. ³⁰ Moreover, TGFβ signaling is highly interlinked with other growth factor and developmental pathways at multiple levels. The suitability of TGFβ downstream targets and of interactions of TGFβ signaling with other profibrotic signaling molecules is outlined below. Systemic targeting of TGFβ signaling at the level of either TGFβ isoforms themselves or at the level of its cell surface receptors is currently not considered as a prime approach: neutralization of all TGFβ isoforms, for example, with pan-TGFβ neutralizing antibodies or inactivation of TGFβR activity by genetic mutations, has been associated with the development of keratoacanthomas, low-grade rapidly growing skin tumors. ³¹ Moreover, systemic inhibition of TGFβ signaling at the ligand or receptor level may promote autoimmune responses in SSc. ³² Deregulation of TGFβ signaling in fibrotic diseases is thought to occur predominantly by increased activation of latent TGFβ from its deposits in the extracellular matrix. Inhibition of the activation of latent TGFβ may thus limit the uncontrolled activation of TGFβ signaling in fibrotic diseases. Multiple lines of evidence demonstrate that αv integrin plays a key role for the activation of latent TGFβ. ³³ Indeed, inhibition of αv integrins demonstrated potent antifibrotic effects in the mouse models of fibronectin transgenic induced dermal fibrosis, CCl₄-induced liver fibrosis, bleomycin-induced lung fibrosis, and unilateral ureteral obstruction (UUO)-induced kidney fibrosis.^34–41 Moreover, a humanized monoclonal antibody against αvβ6 (STX-100) was evaluated in phase II clinical trial in idiopathic pulmonary fibrosis (IPF), ⁴² and small molecule inhibitors targeting the integrin αv-induced activation of latent TGFβ have been developed.^34,38,39,43

Platelet-derived growth factor

Platelet-derived growth factor (PDGF) is primarily produced by platelets, vascular endothelial cells, pericytes, fibroblasts, and Kupffer cells. Four PDGF subunits, termed PDGF-A, -B, -C, and -D, have been identified. PDGFs have two types of receptor PDGFRα and PDGFRβ, which are typically expressed by mesenchymal cells across different organs. ⁴⁴ PDGFRα is commonly used to label fibroblasts, ⁴⁵ whereas PDGFRβ is more broadly expressed in cells with mesenchymal origin. ⁴⁴ Multiple PDGFR inhibitor have been studied and demonstrated antifibrotic effects in murine models of bone marrow, lung, kidney, liver, and heart fibrosis. ⁴⁴ However, clinical trials with tyrosine kinase inhibitors such as imatinib or dasatinib that encompass PDGFR inhibitory activity failed to demonstrate antifibrotic activity in phase II clinical trials in patients with SSc and ILD.^46,47 A potential reason may be that the activation of PDGFR signaling is less pronounced in SSc. ⁴⁸

Vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is predominantly known for its crucial role in angiogenesis. Aberrant VEGF signaling, however, may also promote tissue fibrosis in SSc. VEGF and its receptors VEGFR-1 and VEGFR-2 are upregulated in skin specimens of SSc patients throughout different disease stages. ⁴⁹ Mice with transgenic overexpression of VEGF (VEGF ^+/+ transgene (tg)) developed skin fibrosis in the absence of additional profibrotic stimuli. ⁵⁰ Moreover, VEGF tg mice are more sensitive to bleomycin-induced skin fibrosis. The effect was gene dose dependent with even more enhanced fibrosis in VEGF ^+/+ tg mice than in VEGF ^+/− tg mice. Tissue fibrosis was also enhanced in TSK-1/VEGF ^+/+ mice. In vitro analysis revealed that VEGF is directly inducing collagen synthesis in dermal fibroblasts. ⁵⁰ Consistently, neutralizing of all isoforms of VEGF-A by the humanized monoclonal antibody bevacizumab ameliorated hepatic fibrosis induced by CCl₄ in rats ⁵¹ and attenuated bleomycin-induced skin fibrosis. ⁵² Several VEGF inhibitors are currently evaluated in for the treatment of various types of cancer. Studies of bevacizumab have entered phase II clinical trial for the treatment of pulmonary fibrosis. (https://clinicaltrials.gov/; NCT01917877). However, the antiangiogenic effects of such an approach may further perturb angiogenesis and thus limit its usability in SSc. On the other hand, nintetanib, which also inhibits VEGF signaling along with angiogenesis-relevant pathways (see below), did not exacerbate the ulcer burden in phase III trial. ⁵³ Although patients with more than 3 ulcers were excluded from that trial, this finding provides evidence that targeting of VEGF is feasible in SSc.

Developmental signaling pathways

Several pathways that are critical for embryonic development and are silenced under most homeostatic conditions are pathologically activated in SSc and other fibrotic diseases. These developmental pathways include canonical Wnt signaling, Notch signaling, and Hedgehog signaling.

Wnt signaling

Canonical Wnt signaling has been implicated into the pathogenesis of multiple fibrotic diseases including SSc. Canonical Wnt signaling is activated by binding of soluble Wnt proteins to frizzled (FZ) membrane receptors and low-density lipoprotein–related protein (LRP) co-receptors, which recruit Dishevelled (DSH) to the plasma membrane. ⁵⁴ DSH destabilizes the β-catenin destruction complex consisting of AXIN, adenomatosis polyposis coli (APC) protein, casein kinase I (CKI), and glycogen synthase kinase 3β (GSK3β). Thus, upon Wnt binding to its surface receptors, β-catenin accumulates, translocates into the nucleus, and binds to transcription factors of the T cell factor/lymphoid enhancer–binding factor–1 (TCF/Lef-1) to activate the transcription of Wnt target genes. ⁵⁵ In the absence of Wnt proteins, the β-catenin destruction complex is active and GSK3β phosphorylates β-catenin, leading to its ubiquitination and subsequent proteasomal degradation. ⁵⁴ Wnt signaling was found activated in SSc with increased Wnt ligand expression such as WNT1 and WNT10b. In addition, the expression of endogenous antagonists are decreased in SSc. The levels of Dickkopf-1 (DKK1) are downregulated by TGFβ in a p38-dependent manner.^56,57 Moreover, DKK1 and secreted FZ-related protein 1 (SFRP1) are downregulated by promoter hypermethylation in SSc. ⁵⁸ Wnt inhibitory factor 1 (WIF1) is also decreased in SSc fibroblasts. ⁵⁹ Ectopic expression of WNT10b under an adipocyte promoter induces loss of subcutaneous adipose tissue and progressive skin fibrosis. Wnt10b switches differentiation of mesenchymal cells in the subcutis toward myofibroblasts. ⁶⁰ Consistent with this finding, overexpression of a constitutive active β-catenin mutant is sufficient to induce fibroblast activation and tissue fibrosis.^60–64 Moreover, inhibition of GSK3β with subsequent stabilization of β-catenin also promotes tissue fibrosis.^65,66

Multiple genetic approaches that inhibit Wnt signaling demonstrated antifibrotic effects: transgenic overexpression of DKK1 reduced experimental fibrosis, ⁵⁶ and re-expression of WIF1 also ameliorated fibroblast activation. ⁵⁹ Knockdown of tankyrases, which inactivates AXIN, by siRNA prevented experimental fibrosis, ⁶⁷ while knockdown of EVI, which is required for the release of Wnt proteins, also ameliorated experimental fibrosis. ⁶⁸ Several compounds and antibodies that target canonical Wnt signaling have been evaluated. ICG-001, a small molecule that specifically inhibits TCF/β-catenin transcription attenuates bleomycin-induced lung fibrosis in mice. ⁶⁹ The tankyrase inhibitor XAV-939 demonstrated potent antifibrotic effects in murine dermal ⁶⁷ and pulmonary fibrosis. ⁷⁰ Porcupine is an O-acyltransferase required for secretion of Wnt proteins. Inhibition of porcupine by GNF6231 was demonstrated to have potent antifibrotic effects in the mouse model of bleomycin-induced skin fibrosis, in TSK-1 mice, in murine sclerodermatous chronic graft-versus-host disease (scl-cGvHD), and in fibrosis induced by a constitutively active TGFβ receptor I. ⁷¹ Tankyrase inhibitor is currently in phase II clinical trials for breast cancer (http://clinicaltrials.gov; NCT03562832) and porcupine inhibitor is in phase II trials for advanced head and neck squamous cell carcinoma (http://clinicaltrials.gov; NCT02649530). WNT1-inducible signaling protein–1 (WISP1) was reported increased in alveolar epithelial type II (ATII) cells in both a mouse model of pulmonary fibrosis and patients with IPF. Neutralizing monoclonal antibody (mAb) specific for WISP1, which is expressed at increased levels in patients with IPF, attenuated bleomycin-induced pulmonary fibrosis. ⁷² Reactivation of DKK1 and SFRP1 expression by 5-aza-2′-deoxycytidine (5-aza) also ameliorated bleomycin-induced dermal and pulmonary fibrosis.^58,73 Thus, targeting canonical Wnt signaling consistently demonstrated potent antifibrotic effects across different models. However, chronic inhibition of canonical Wnt signaling may interfere with stem cell regeneration in rapidly cycling organs such as the intestine and may thus limit long-term application of Wnt inhibitors. Whether intermittent dosing or low-dose combination therapies may overcome those limitations in humans remains to be determined despite first promising data in mice. ⁶⁸

Notch signaling

In addition to Wnt signaling, Notch signaling is also aberrantly activated in SSc. Notch signaling is activated upon binding of membrane-bound Notch ligands to the Notch receptors on the target cell. Six different canonical Notch ligands in vertebrates (Jag-1 and 2 and Delta-like 1 to 4) and four different Notch receptors (Notch1 to 4) are described in human.^74,75 Notch signaling is activated by ligand protein binding to Notch receptors, which induces their cleavage by metalloproteases of the ADAM/TACE family and γ-secretase to release the Notch intracellular domain (NICD). The NICD translocates to the nucleus, where it recruits DNA-binding protein and co-activators to form a complex that induces transcriptional activation of Notch target genes.^76,77 Notch signaling is activated in SSc with accumulation of the NICD and increased transcription of the target gene HES-1. Moreover, ADAM-17 is also overexpressed in the skin of SSc patients in response to reactive oxygen species. ⁷⁸ Stimulation of healthy dermal fibroblasts with human Notch ligand Jagged–1 (Jag-1)-Fc chimera induced an SSc-fibroblast–like phenotype with increased release of collagen and differentiation of resting fibroblasts into myofibroblasts. Incubation with the γ-secretase inhibitor DAPT or siRNA against Notch reduced the basal collagen expression in SSc fibroblasts.^79,80 Administration of DAPT reduced the development of skin fibrosis, ⁷⁸ ameliorated experimental pulmonary arterial hypertension, ⁸¹ and alleviated chloral hydrate induced–pulmonary fibrosis. ⁸² In addition to prevention of fibrosis, targeting of Notch signaling by DAPT induced regression of established bleomycin-induced skin fibrosis ⁸³ and also reversed fibrotic remodeling in a human precision-cut lung slice of patients with IPF. ⁸⁴ Mechanistically, Notch may not only promote the transcription of profibrotic genes by Mastermind-dependent transcriptional regulation but also indirectly by promoting TGFβ/SMAD3 signaling. Notch signaling inhibits the expression of SMAD1 and SMAD2 but increases SMAD3 transcription and protein half-life to promote profibrotic SMAD3 signaling. ⁸⁵ However, chronic broad-spectrum, non-selective inhibition of Notch signaling may be complicated by gastrointestinal toxicity. ⁸⁶

Hedgehog signaling

Hedgehog signaling is another developmental pathway that is activated in SSc. Three different ligands stimulate the Hedgehog signaling in vertebrates: Sonic Hedgehog (SHH), Indian Hedgehog (IHH), and Desert Hedgehog (DHH). Hedgehog ligands bind to their membrane receptor Patched (PTCH). In the absence of Hedgehog ligands, PTCH inhibits the co-receptor Smoothened (SMO). This inhibition of SMO is released, when binding of hedgehog proteins induces conformational changes of PTCH. Active SMO inhibits the degradation of GLI transcription factors (GLI1, GLI2, and GLI3) to promote the transcription of hedgehog target genes. ⁸⁷ In addition to this canonical hedgehog pathway, the transcription of hedgehog genes can also be induced by non-canonial cascades that promote accumulation of GLI proteins by alternative mechanism. These include a direct upregulation of GLI2 transcription by TGFβ. ⁸⁸ Hedgehog signaling is activated in SSc via accumulation of GLI2 in fibroblasts. Activation of hedgehog signaling, for example, by SHH, stimulates the release of collagen, promotes myofibroblast differentiation, and is sufficient to induce skin fibrosis in mice. ⁸⁹ Inhibition of SMO either by LDE223 or by siRNA prevented skin fibrosis induced by bleomycin and in TSK-1 mice ⁹⁰ as well as in murine scl-cGvHD. ⁸⁹ Antifibrotic effects were also observed with other SMO antagonists such as cyclopamine ⁹¹ and vismodegib,^88,92 which is approved for clinical use in basal cell carcinoma. Simultaneous inhibition of both canonical and non-canonical hedgehog signaling can be achieved by direct targeting of GLI proteins. Fibroblast-specific knockout of GLI2 protected mice from TBR^act-induced fibrosis. The GLI inhibitor GANT-61 also demonstrated potent antifibrotic effects in murine models of SSc. Combined targeting of canonical and non-canonical hedgehog signaling with direct GLI inhibitors exerted more potent antifibrotic effects than selective targeting of canonical hedgehog signaling with SMO inhibitors in experimental dermal and pulmonary fibrosis. ⁸⁸ The hedgehog acyltransferase (HHAT) promotes palmitoylation of SHH. Palmitoylation of SHH is required for multimerisation of SHH proteins, which promotes long-range endocrine hedgehog signaling. HHAT is upregulated in SSc in a TGFβ-dependent manner and in turn stimulates TGFβ-induced long-range hedgehog signaling to promote fibroblast activation and tissue fibrosis. Targeting of HHAT by siRNA ameliorated bleomycin-induced and topoisomerase-induced skin fibrosis and might be a novel approach to more selectively interfere with the profibrotic effects of long-range hedgehog signaling. ⁹³ Moreover, overexpression of suppressor of fused (SUFU), a negative regulator of the hedgehog pathway, inhibited the endogenous as well as the TGFβ-induced activation of SSc fibroblasts. ⁹¹ Of note, pirfenidone has also shown to block Hedgehog signaling and inhibition of Hedgehog signaling may thus contribute to its antifibrotic effects. ⁹⁴

Intracellular targets

Janus kinase/signal transducer and activator of transcription

The mammalian Janus kinase (JAK) family has four members: JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2). ⁹⁵ Upon engagement of ligand, JAKs are activated by phosphorylating each other and the intracellular part of their receptor. JAKs in turn phosphorylates and thereby activates members of the signal transducers and activators of transcription (STATs) family of transcription factors. ⁹⁶ Of interest, V362 F, one of the common variants of TYK2, is associated with SSc. ⁹⁷ Furthermore, phosphorylated JAK2 accumulates in the skin fibroblasts of patients with SSc. The activation of JAK2 was dependent on TGFβ and persisted in cultured SSc fibroblasts. ⁹⁸ The aberrant activation of JAK2, together with the activation of other upstream kinases such as SRC, c-ABL, and JNK, enhanced STAT3 signaling in SSc fibroblasts in a TGFβ-dependent manner. ⁹⁹ Furthermore, elevated levels of phosphorylated STAT3 activation were observed in lung biopsies from patients with IPF and in the lungs of mice challenged with bleomycin. ¹⁰⁰ Inhibition of JAK2 reduced basal collagen synthesis selectively in SSc fibroblasts. Moreover, inhibition of JAK2 by selective inhibitor TG101209 or by siRNA prevented the stimulatory effects of TGFβ on fibroblasts. Treatment with TG101209 not only prevented bleomycin-induced skin fibrosis but also effectively reduced skin fibrosis in TSK-1 mice. ⁹⁸ Long-term inhibition of JAK2 can result in transactivation of JAK2 by JAK1. ¹⁰¹ Combined inhibition of JAK1 and JAK2 might thus be required to overcome loss of efficacy of selective JAK2 inhibitors upon prolonged use. Indeed, treatment with the combined JAK1/JAK2 inhibitor ruxolitinib (clinical trial phase II for myelofibrosis; https://clinicaltrials.gov/; NCT03333187) or with JAK2 inhibitor in combination with HSP90 inhibitor 17-DMAG (clinical trial phase I for lymphoma; https://clinicaltrials.gov/; NCT00089271, NCT00089362, NCT00088868, NCT00803556), which destabilizes JAK2 protein, was more effective than monotherapy with JAK2 selective inhibitors (TG101209) in bleomycin-induced pulmonary fibrosis and in adTBR-induced dermal fibrosis. ¹⁰¹ Ruxolitinib was also reported to reduce bone marrow fibrosis in patients with myelofibrosis. ¹⁰²

STAT3-deficient fibroblasts are less sensitive to the profibrotic effects of TGFβ. Fibroblast-specific knockout of STAT3, or treatment with the STAT3 inhibitor S3I-201, ameliorates skin fibrosis in experimental mouse models of bleomycin-induced dermal fibrosis and adTBR-induced dermal fibrosis at well tolerated doses. ⁹⁹ C-188-9, another small molecule inhibitor of STAT3, also ameliorated bleomycin-induced pulmonary fibrosis. ¹⁰⁰ STAT3 inhibitors are currently in phase I clinical trials for melanoma and different types of advanced cancer (http://clinicaltrials.gov; NCT01904123 and NCT03195699). First selective inhibitors of STAT3 are currently entering clinical trials in oncology.

Inhibition of the tyrosine phosphatase SHP2 may be an alternative approach to target aberrant JAK2/STAT3 signaling. SHP2 induces dephosphorylation of JAK2 at Y570, an inhibitory site that prevents activation of JAK2. SHP2-induced dephosphorylation is thus required for effective activation of JAK2. Genetic or pharmacologic inactivation of SHP2 promotes accumulation of JAK2 phosphorylated at Y570 and reduces the levels of active JAK2 and STAT3. This translates into inhibition of TGFβ-induced fibroblast activation in vitro and in vivo with amelioration of experimental dermal and pulmonary fibrosis. ¹⁰³ Various SHP2 inhibitors are currently evaluated in clinical programs. SHP2 inhibitor JAB-3068 has recently entered first clinical trials (http://clinicaltrials.gov; NCT03518554 and NCT03565003).

Epigenetic mechanisms may also contribute to the increased activation of JAK2/STAT3 signaling in SSc. Suppressor of cytokine signaling proteins are endogenous feedback inhibitors of JAK2/STAT3 signaling. They are induced by active STAT signaling and in turn inhibit further activation of STAT3. However, persistent activation of TGFβ signaling induces the expression of the DNA methyltransferases (DNMT) 1 and 3A, which hypermethylate regulatory sites of the SOCS3 gene to silence its expression. This promotes prolonged activation of STAT3 signaling and persistent fibroblast activation. However, treatment with DNMT inhibitors 5-aza or fibroblast-specific knockout of DNMT3A reactivates the expression of SOCS3, inhibits JAK2 / STAT3 signaling, and reduces TGFβ-mediated fibroblast activation. Moreover, inhibition of aberrant DNA methylation exerted potent antifibrotic effects in bleomycin- and TGFβRI^act-induced dermal fibrosis. ¹⁰⁴

Casein kinase II

Inhibition of casein kinase II (CK2) may be another approach to reduce aberrant JAK/STAT signaling in SSc. The serine/threonine kinase CK2 is activated in SSc and is capable of activating JAK2 by phosphorylating JAK2 at the activating site Y1007/1008. ¹⁰⁵ Inhibition of CK2 by 4, 5, 6, 7-tetrabromobenzotriazole (TBB) prevented the activation of JAK2/STAT3 signaling and reduced the profibrotic effects of TGFβ in vitro and ameliorated bleomycin- and TGFβ-induced fibrosis in vivo. ¹⁰⁶ The CK2 inhibitor CX-4945 recently entered phase I clinical trials (http://clinicaltrials.gov; NCT00891280 and NCT01199718).

SRC kinase

SRC kinases are non-receptor tyrosine kinases implicated in cytoskeletal organization and cell mobility. Stimulation of fibroblasts with TGFβ and PDGF activates SRC signaling. Incubation with the SRC kinase inhibitors or overexpressed mutant SRC or of the endogenous inhibitor CSK reduced the synthesis of extracellular matrix proteins in cultured fibroblasts. Inhibition of SRC kinases prevented bleomycin-induced dermal fibrosis. ¹⁰⁷ SRC kinases are inhibited by nintedanib, which is approved for IPF and has been granted an FDA label for SSc-ILD.

ROCK

ROCKs are the major cellular mediators of Rho GTPases and play an important role in the organization of the actin cytoskeleton. Inhibition of ROCK reduced the synthesis of the major extracellular matrix (ECM) proteins at the mRNA level as well as the protein level and prevented myofibroblast differentiation. ¹⁰⁸ TGFβ activated ERK in a ROCK-dependent manner, and ERK mediated in part the stimulatory effects of ROCK on myofibroblast differentiation and reduced bleomycin-induced murine dermal fibrosis. ¹⁰⁷ ROCK inhibition ameliorated bleomycin-induced pulmonary fibrosis, ¹⁰⁹ and ROCK1- and ROCK2-deficient mice are also protected from bleomycin-induced pulmonary fibrosis. ¹¹⁰ A phase II clinical trial with the ROCK inhibitor Pravastatin suggested antifibrotic effects on radiation-induced cutaneous and subcutaneous fibrosis in patients with head and neck cancer. ¹¹¹ Moreover, topical application of the ROCK inhibitor AR-12286 is currently evaluated in a phase II clinical trial in advanced glaucoma (http://clinicaltrials.gov; NCT02173223).

JNK

TGFβ and PDGF activate JNK and stimulate the phosphorylation of its downstream target c-Jun. Incubation with CC-930, a selective JNK inhibitor, prevented the phosphorylation of c-Jun and reduced the stimulatory levels of these cytokines on the release of collagen. Inhibition of JNK by CC-930 prevented fibrosis in bleomycin-induced dermal fibrosis and in TSK-1 mice. ¹¹² CC-930 also reduced collagen expression in mouse model of dust mite–induced airway remodeling. ¹¹³ Treatment of CC-930 prevented lung fibrosis in bleomycin-induced murine pulmonary fibrosis model. ¹¹⁴ JNK1-deficient mice are protected from bleomycin-induced profibrotic gene expression and pulmonary fibrosis. ¹¹⁵ The JNK inhibitor CC-90001 and CC-930 ¹¹³ are in phase II clinical trials in IPF (http://clinicaltrials.gov; NCT03142191; NCT01203943).

Cyclin-dependent kinase 5

Cyclin-dependent kinase 5 (CDK5) is a pleiotropic member of the CDK family originally identified in neuronal cells. In contrast to other CDKs, CDK5 activity depends on its CDK5R1 subunit p35. The expression of p35 and CDK5 activity is induced by TGFβ in fibroblasts and adipocytes. The levels of p35 are elevated in SSc skin biopsies and explanted SSc fibroblasts, as well as in fibrotic murine tissues. Knockdown of CDK5 reduced the profibrotic effects of TGFβ on fibroblasts. CDK5 inhibition by roscovitine reversed TGFβ-induced fibroblast activation in conventional fibroblast cultures and in human skin organ cultures and ameliorated skin fibrosis in bleomycin- and TGFβ-driven mouse models. ¹¹⁶ Roscovitine was evaluated in a phase II clinical trial in cystic fibrosis and demonstrated anti-inflammatory effects. ¹¹⁷

Nuclear receptors

Nuclear receptors are a family of transcription factors that encompasses well-known hormone receptors but also a number of less well-studied orphan receptors that signaling in the absence of ligand binding.

The most intensively studied nuclear receptor in the context of fibrosis is peroxisome proliferator-activated receptor gamma (PPARγ). The levels of PPARγ are diminished in skin and lung biopsies from patients with SSc. ⁷⁵ PPARγ agonists such as rosiglitazone or pioglitazone reduced proliferation and increased apoptosis of SSc fibroblasts. ¹¹⁸ PPARγ ligands ameliorated fibrosis of the lung, kidney, heart, and liver.^119–123 In addition to selective PPARγ activation, the pan-PPAR agonist IVA337 also demonstrated antifibrotic effects.^124,125 However, a phase II clinical trial with the pan-PPAR agonist IVA337 failed to demonstrate clinical benefit in patients with dcSSc. ¹²⁶ Rosiglitazone and pioglitazone are approved by the Food and Drug Administration (FDA) for treating type 2 diabetes.

NR4A1 (NUR77) forms together with NR4A2 (NURR1) and NR4A3 (NOR-1) to the NR4A-family of orphan nuclear receptors. NR4A1 recruits a repressor complex comprising SP1, SIN3A, CoREST, LSD1, and HDAC1 to TGFβ target genes, thereby limiting profibrotic TGFβ effects. Knockout of NR4A1 exacerbates fibrosis, whereas NR4A1 agonist Cytosporne B inhibits experimentally induced skin, lung, liver, and kidney fibrosis in mice. ¹²⁷

Vitamin D receptor (VDR) has also been characterized as a negative regulator of TGFβ/SMAD signaling. The levels of VDR are reduced in various autoimmune diseases including SSc, and the expression of VDR is also decreased in fibrotic skin. Knockdown of VDR enhanced the stimulatory effects of TGFβ on fibroblasts and promoted collagen release and myofibroblast differentiation. In contrast, incubation with the VDR agonist paricalcitol reduced the sensitivity of fibroblasts to the activating effects of TGFβ by the formation of complexes between VDR and phosphorylated SMAD3 with subsequent inhibition of SMAD-dependent transcription. Preventive and therapeutic treatment with paricalcitol exerted potent antifibrotic effects and ameliorated bleomycin- as well as TBRI^CA-induced dermal fibrosis.^127,128 Furthermore, Vitamin D application prevented bleomycin-induced pulmonary fibrosis, ¹²⁹ and paricalcitol ameliorated experimental renal interstitial fibrosis. ¹³⁰ Paricalcitol is approved by FDA as a Vitamin D₂ analog.

In contrast to the previous nuclear receptors, activation of the constitutive androstane receptor (CAR)/NR1I3 is profibrotic. Treatment with CAR agonists such as TCPOBOP promotes TGFβ-induced fibroblast activation in cultured fibroblasts and exacerbates fibrosis in murine models of SSc such as bleomycin-induced and TβRI^CA-induced skin fibrosis. ¹³¹ The non-selective CAR agonist meclizine hydrochloride is currently evaluated for the treatment of hepatocellular carcinoma (http://clinicaltrials.gov; NCT03253289).

The liver X receptors (LXRs) may regulate fibrotic tissue remodeling by interfering with the infiltration of macrophages and their release of the profibrotic interleukin–6 rather than by direct effect on fibroblasts. ¹³² Activation of LXR signaling by the small molecule agonist such T0901317 showed antifibrotic effects in bleomycin-induced skin fibrosis and in the scl-cGvHD model. Protective roles of LXR have also been observed in murine pulmonary fibrosis.^133,134 LXR agonists have recently entered clinical trials for advanced solid tumors and lymphomas and diabetic retinopathy (http://clinicaltrials.gov; NCT02922764; NCT03403686).

Epigenetics

Several studies from different groups demonstrated that epigenetic modulations are key drivers of the persistent fibroblast activation in SSc and in other fibrotic diseases. Here, we will focus on the role of DNA methylation, histone acetylation, and microRNAs (miRNAs).

DNA methylation

Three DNA methyltransferases (DNMT), DNMT1, DNMT3A, and DNMT3B, are capable of methylating position C5 of the pyrimidine ring of cytosine residues. ¹³⁵ Recruitment of methyl-CpG-binding domain proteins (MBDs) to methylated cytosine residues in CpG islands promotes recruitment of repressor complexes to silence the transcription of the respective target genes. ¹³⁶ Specific inhibitors of DNMTs are approved for the treatment of certain forms of leukemia. ¹³⁷ Inhibition of DNMTs has been described to inhibit fibrosis in preclinical models of SSc and other fibrotic diseases.^58,138,139 Hypoxia and aberrant TGFβ signaling may both contribute to the aberrant expression of DNMTs in SSc.^139,140 Numerous studies have demonstrated a critical role of friend leukemia virus integration 1 (FLI1), a member of the Ets transcription factor family, for fibroblast activation in SSc. FLI1 expression is constitutively suppressed in dermal fibroblasts, dermal microvascular endothelial cells, and perivascular inflammatory cells in the skin of SSc patients.^141–143 Chronic activation of TGFβ signaling inhibits FLI1 expression and activity by epigenetic and posttranslational mechanisms. TGFβ induces methylation of the FLI1 promoter^138,140 and promotes phosphorylation of FLI1 to stimulate its degradation. ¹⁴⁴ FLI1 can inhibit TGFβ signaling and limit fibroblast activation, ¹⁴⁵ thus downregulation of FLI1 facilitates aberrant TGFβ signaling in SSc. Mice with double heterozygous deficiency for FLI1 and Krüppel-like factor 5 (KLF5), another transcription factor epigenetically suppressed in SSc dermal fibroblasts, spontaneously develop three cardinal features of SSc, including autoimmunity/inflammation, vasculopathy, and tissue fibrosis of the skin and lungs. ¹⁰⁸ Treatment with DNMT inhibitors such as 5-aza or knockdown of individual DNMTs promoted the expression of FLI1 but also of other epigenetically silenced antifibrotic genes such as SOCS3, DKK1, and SFRP1 and reduced TGFβ-induced fibroblast activation and ameliorated experimental fibrosis in various tissues such as skin, lung, and kidney.^{58,138,139,146}

Histone deacetylases

Histone acetylation reduces the positive charge on lysines, thereby decreasing the affinity of histones for DNA. This leads to loosening of the chromatin structure and increases transcription. Histone acetylation is mediated by histone acetylases and can be reversed by the activity of histone deacetylases (HDACs). There are four classes of HDACs: class 1 and class 2 HDACs, class 3 NAD ⁺-dependent sirtuins (SIRTs), and HDAC-11 as the only class 4 HDAC.^147,148 The class 1 and 2 HDAC inhibitor trichostatin A (TSA) reduced the expression of collagen protein in SSc fibroblasts and ameliorated bleomycin-induced fibrosis. ¹⁴⁹ TSA also ameliorated CCl₄-induced hepatic fibrosis ¹⁵⁰ and attenuated bleomycin-induced pulmonary fibrosis in rats. ¹⁵¹ Class 1 and 2 HDAC inhibitors such as SAHA are already approved for clinical use in oncology but have not been evaluated in clinical trials in fibrotic diseases.

miRNAs

miRNAs are small non-coding RNA bind to mRNAs to inhibit their translation. Microarray analysis has identified a variety of miRNAs that are dysregulated in murine models of fibrosis and patients with SSc. ¹⁵² MiR-21 and miR-29 are two examples that have been implicated in pathogenesis of fibrotic disease across different organs. MiR-21 expression is increased in bleomycin-induced pulmonary fibrosis murine model and in patients with IPF, especially myofibroblasts. ¹⁵³ TGFβ induces the expression of miR-21, which in turn downregulates SMAD7 to promote canonical TGFβ signaling. ¹⁵³ Antagomirs against miR-21 attenuated bleomycin-induced lung fibrosis and pressure overload–induced interstitial myocardial fibrosis,^153,154 while miR-21 small hairpin RNA transfer ameliorated UUO-induced renal fibrosis. ¹⁵⁵ In contrast to miR-21, miR-29 exerts antifibrotic effects. Downregulation of miR-29 has been shown in SSc, in cardiac, renal, pulmonary, and hepatic fibrosis.^156–160 MiR-29 targets the coding region of TGFβ, thereby inhibiting TGFβ signaling and subsequent extracellular matrix deposition and remodeling in fibrotic diseases.^156,161

Polypharmacology

Nintedanib

Polypharmacology describes the simultaneous inhibition of several pathologically relevant targets by a single drug. A prominent example in the context of fibrotic diseases is the multiple tyrosine kinase inhibitor nintedanib. Nintedanib blocks PDGF receptors α and β; FGF receptors 1, 2, and 3; VEGF receptors 1, 2, and 3; TβRs; the CSF receptor; and the SRC family kinases. Nintedanib thus offers opportunity for the combined inhibition of several key profibrotic pathways. Indeed, nintedanib inhibited proliferation, migration, myofibroblast differentiation, and collagen release from cultured fibroblasts and showed antifibrotic effects in various in vivo models of SSc.^162,163 These findings translated from bench to bedside with reduction of progression of SSc-ILD in a phase III clinical trial.^164,165 Nintedanib has recently received FDA approval for clinical use in SSc-ILD.

Bosutinib, anlotinib, vatalanib, sunitinib, and dasatinib

In addition to nintedanib, other multityrosine kinase inhibitors demonstrated efficacy in first mouse models of SSc or other fibrotic diseases. Examples include the c-ABL / SRC tyrosine kinase inhibitor bosutinib (SKI-606) with reduced fibrotic gene expression in vitro and in TBR-induced dermal and pulmonary fibrosis.^166,167 Another multitarget tyrosine kinase inhibitor anlotinib (AL3818), which has been approved for IPF treatment, attenuated bleomycin-induced pulmonary fibrosis through suppressing TGFβ signaling. ¹⁶⁸ Vatalanib decreased hepatic fibrosis in CCl4-induced liver fibrosis. ¹⁶⁹ Sunitinib inhibited PDGF and VEGF pathways and prevented HOCl-induced murine dermal fibrosis. ¹⁷⁰ Dasatinib treatment were well tolerated in SSc-ILD patients but with only 39% improver. ⁴⁷

Conclusion

Several molecular targets for the treatment of fibrosis have been identified in the last years (Table 1). Many of these findings may have direct translation relevance as drugs that modulate these pathways are either in advanced clinical development or even approved for other indications. However, given the increasing number of targets on one hand and the limited number of SSc patients with progressive fibrotic manifestations on the other hand, we may need to develop additional strategies to generate a hierarchy of drug candidates. Stringent and comparable preclinical test programs are key requirements to generate such a hierarchy. These programs should include testing of drug candidates in novel preclinical in vitro assays such as human organoids or ex vivo assays of target tissues such as precision cut slices. In addition to these in vitro assays, the preclinical programs should include testing in sets of complementary mouse models that resemble different aspects of the pathogenesis of SSc and involve different organs. In these models, drug candidates should not only be applied in a preventive manner but also whenever possible in the clinically more relevant setting of established fibrosis. Finally, several clinical trials failed because the level of target activation was modest in the selected patient population. Thus, careful evaluation of pathway activation in a large cohort of patients with different subpopulations of SSc is warranted. This may also enable enrichment of patients with particularly high level of target activation and may thus reduce the number of study subjects in a following clinical trial.

Table 1.

Targets for the treatment of fibrosis.

Compound/antibody	Target	Target organ	References
Dabigatran	Thrombin	Pulmonary fibrosis, cardiac fibrosis	Bogatkevich et al. 25 and Dong et al. 26
SSR182289	Thrombin	Liver fibrosis	Duplantier et al. 27
STX-100	αv integrin	Pulmonary fibrosis	Dupont et al. 42
ICG-001	T cell factor/β-catenin	Pulmonary fibrosis	Henderson et al. 69
XAV-939	Tankyrase	Dermal fibrosis	Distler et al. 67 and Ulsamer et al. 70
GNF6231	Porcupine	Dermal fibrosis, pulmonary fibrosis	Chen et al. 71
DAPT	Notch signaling	Dermal fibrosis, pulmonary fibrosis	Kavian et al., 78 Li et al., 81 Wang et al., 82 Dees et al., 83 Wasnick et al. 84
LDE223	Co-receptor Smoothened (SMO)	Dermal fibrosis	Horn et al. 90
Cyclopamine	SMO	Dermal fibrosis	Cao et al. 91
Vismodegib	SMO	Dermal fibrosis, pulmonary fibrosis	Liang et al. 88 and Moshai et al. 92
GANT-61	GLI	Dermal fibrosis, pulmonary fibrosis	Liang et al. 88
TG101209	Janus kinase 2 (JAK2)	Dermal fibrosis, pulmonary fibrosis	Dees et al. 98 and Zhang et al. 101
Ruxolitinib	JAK1 and JAK2	Dermal fibrosis, pulmonary fibrosis	Zhang et al. 101
17-DMAG	HSP90	Dermal fibrosis, pulmonary fibrosis	Zhang et al. 101
S3I-201	Signal transducer and activator of transcription 3 (STAT3)	Dermal fibrosis	Chakraborty et al. 99
C-188-9	STAT3	Pulmonary fibrosis	Pedroza et al. 100
TBB	Casein kinase II (CK2)	Dermal fibrosis	Zhang et al. 106
CC-930	c-Jun N-terminalkinase (JNK)	Dermal fibrosis, pulmonary fibrosis	Reich et al., 112 van der Velden et al., 113 Krenitsky et al. 114
Roscovitine	Cyclin-dependent kinase 5 (CDK5)	Dermal fibrosis	Wei et al. 116
Rosiglitazone	Peroxisome proliferator–activated receptor gamma (PPARγ) agonist	Kidney fibrosis, pulmonary fibrosis	Kiss et al. 120 and Yu et al. 123
Pioglitazone	PPARγ agonist	Liver fibrosis	Uto et al. 122
IVA337	PPARα, PPARδ, PPARγ agonist	Dermal fibrosis, pulmonary fibrosis	Ruzehaji et al. 124 and Avouac et al. 125
Cytosporne B	NR4A1 agonists	Dermal fibrosis, pulmonary fibrosis, and liver and kidney fibrosis	Palumbo-Zerr et al. 127
Paricalcitol	VDR agonist	Dermal fibrosis, kidney fibrosis	Zerr et al. 128 and Tan et al. 130
TCPOBOP	Constitutive androstane receptor (CAR)/NR1I3agonist	Dermal fibrosis	Avouac et al. 131
T0901317	Liver X receptors agonist	Dermal fibrosis, pulmonary fibrosis	Beyer et al., 132 Shichino et al., 133 Shi et al. 134
5-aza	DNA methyltransferases (DNMTs)	Dermal fibrosis, kidney fibrosis, cardiac fibrosis, and pulmonary fibrosis	Dees et al., 58 Bechtel et al., 138 Watson et al., 139 Zhao et al. 146
Trichostatin A	Histone deacetylases (HDACs)	Dermal fibrosis, liver fibrosis, and pulmonary fibrosis	Huber et al., 149 Ding et al., 150 Ye et al. 151

5-aza: 5-aza-2′-deoxycytidine; TBB: tetrabromobenzotriazole; VDR: vitamin D receptor.