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Pharmacological inhibition of porcupine induces regression of experimental skin fibrosis by targeting Wnt signalling
  1. Chih-Wei Chen1,
  2. Christian Beyer1,
  3. Jun Liu2,
  4. Christiane Maier1,
  5. Chun Li2,
  6. Thuong Trinh-Minh1,
  7. Xiaohan Xu1,
  8. Stuart H Cole2,
  9. Mindy H Hsieh2,
  10. Nicholas Ng2,
  11. Alana Althage2,
  12. Shelly Meeusen2,
  13. Shifeng Pan2,
  14. Eric C Svensson3,
  15. H Martin Seidel2,
  16. Georg Schett1,
  17. Peter Gergely4,
  18. Jennifer L Harris2,
  19. Jörg H W Distler1
  1. 1Department of Medicine 3 for Rheumatology and Clinical Immunology, University of Erlangen-Nuremberg, Erlangen, Germany
  2. 2Genomics Institute of the Novartis Research Foundation, San Diego, California, USA
  3. 3Translational Medicine Autoimmunity Novartis Institutes for Biomedical Research, Basel, Switzerland
  4. 4Translational Medicine Cardiovascular, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA
  1. Correspondence to Dr Jörg H W Distler, Department of Medicine 3 for Rheumatology and Clinical Immunology, University of Erlangen-Nuremberg, PO Box, Glücksstr.4a, Erlangen D-91054, Germany; oerg.distler{at}uk-erlangen.de

Abstract

Objectives Wnt signalling has been implicated in activating a fibrogenic programme in fibroblasts in systemic sclerosis (SSc). Porcupine is an O-acyltransferase required for secretion of Wnt proteins in mammals. Here, we aimed to evaluate the antifibrotic effects of pharmacological inhibition of porcupine in preclinical models of SSc.

Methods The porcupine inhibitor GNF6231 was evaluated in the mouse models of bleomycin-induced skin fibrosis, in tight-skin-1 mice, in murine sclerodermatous chronic-graft-versus-host disease (cGvHD) and in fibrosis induced by a constitutively active transforming growth factor-β-receptor I.

Results Treatment with pharmacologically relevant and well-tolerated doses of GNF6231 inhibited the activation of Wnt signalling in fibrotic murine skin. GNF6231 ameliorated skin fibrosis in all four models. Treatment with GNF6231 also reduced pulmonary fibrosis associated with murine cGvHD. Most importantly, GNF6231 prevented progression of fibrosis and showed evidence of reversal of established fibrosis.

Conclusions These data suggest that targeting the Wnt pathway through inhibition of porcupine provides a potential therapeutic approach to fibrosis in SSc. This is of particular interest, as a close analogue of GNF6231 has already demonstrated robust pathway inhibition in humans and could be available for clinical trials.

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Fibrosis of the skin and many internal organs is the defining hallmark of systemic sclerosis (SSc).1 Despite an urgent medical need, effective and well-tolerated therapies to treat fibrosis in SSc are not available in clinical routine.2 ,3

Intensive research over the past years has identified Wnt signalling as one of the key pathways in persistent fibroblast activation. The Wnt pathway is activated when secreted Wnt ligand protein binds to its cognate cell surface receptors, which leads to accumulation and nuclear translocation of β-catenin.4 In addition to this so-called canonical pathway, Wnt proteins can also induce intracellular responses via β-catenin-independent, non-canonical pathways, including Wnt/calcium signalling and Wnt/planar cell polarity pathways. In SSc, upregulation of Wnt ligands as well as downregulation of Wnt inhibitors activate canonical Wnt signalling and result in prominent activation of fibroblasts.5–9 Moreover, mice with active canonical Wnt signalling develop fulminant fibrosis.5 ,6 ,10 ,11 Preclinical investigations indicate that canonical Wnt signalling might be indeed an attractive target for the treatment of fibrosis in SSc. Genetic approaches to block Wnt signalling by dickkopf (DKK)-1 or small interfering RNA against evenness interrupted (EVI) blocked experimental fibrosis.5 ,12 Furthermore, small molecule inhibitors of canonical Wnt signalling, such as tankyrase inhibitors and CREB-binding protein (CBP) inhibitors, were effective in treating experimental fibrosis.5–7 ,13–16 The role of non-canonical Wnt pathways have, however, not yet been analysed.

Inhibiting porcupine, a membrane-bound acyl-transferase, is a novel approach to target upstream Wnt signalling. Porcupine is essential for the post-translational palmitoylation of Wnt ligands, an essential step for Wnt secretion. Genetic inhibition of porcupine blocks Wnt secretion, thereby blocking both canonical and non-canonical Wnt cascades. WNT974 (also known as LGK974) is a porcupine inhibitor that is currently in clinical trials for the treatment of various cancers.17 This compound demonstrated robust pathway inhibition in both preclinical and clinical studies.17 GNF6231, used in the following study to investigate the antifibrotic activity of porcupine inhibition, is an analogue of WNT974 with similar activities.18

Materials and methods

Animal studies

Four different mouse models of SSc were used: bleomycin-induced skin fibrosis, transforming growth factor (TGF)-β-receptor I (TBR)-induced fibrosis, the B10.D2(H-2d)→BALB/c(H-2d) model of chronic-graft-versus-host disease (cGvHD) and the tight-skin-1 (Tsk-1) model as described previously.5 ,6 ,9 ,13 Animals were treated with or without GNF6231 at multiple dose levels with 0.5% methylcellulose and 0.5% Tween 80 by oral gavage twice daily.

pharmacokinetic (PK) analysis

The plasma concentrations and exposures of GNF6231 were determined by liquid chromatography/mass spectrometry.17

RNA extraction and reverse transcription-PCR analysis by TaqMan

Total RNA was isolated and processed to perform quantitative reverse transcription-PCR analysis of Axin2 mRNA expression, with Gapdh serving as an internal control.

Histology, hydroxyproline content, CD45 and myofibroblast counts

The antifibrotic effects of GNF6231 on experimental skin fibrosis were evaluated by quantification of dermal or hypodermal thickening, analyses of myofibroblast counts and assessment of hydroxyproline content as described previously.5 ,6 ,12–14 CD45 was stained and evaluated as described.13

Statistical analysis

Data were analysed with SDS_2.0 software (Applied Biosystems), statistical analysis was conducted using PRISM.

Results

Target engagement by GNF6231

To confirm the on-target activity of GNF6231, bleomycin was injected in BALB/c mice, with or without orally dosed GNF6231 at 5 milligrams per kilograms (mpk) for 3 weeks. The PK analysis of the compound showed good oral bioavailability (see online supplementary figure S1A). Further analysis of the skin demonstrated a 54% reduction of Axin2 mRNA on treatment, demonstrating effective target engagement by GNF6231 in the tissue of interest (see online supplementary figure S1B).

Bleomycin-induced dermal fibrosis

In the bleomycin model, GNF6231 reduced dermal skin thickness in a dose-dependent manner (figure 1). Of note, the 5 and 10 mpk groups stopped further fibrosis progression and reduced dermal fibrosis to levels lower than the baseline, suggesting a reversal of fibrosis. In addition to skin thickness, porcupine inhibition prevented progression and induced regression of fibrosis as assessed by the hydroxyproline content, myofibroblast counts and histology (figure 1B–E).

Figure 1

Orally dosed GNF6231 inhibited fibrosis in mouse bleomycin model. (A) The mouse study scheme. Bleomycin was injected in female C57/Bl6 aged 6 weeks for 3 weeks. The control group was injected with saline. The bleomycin-treated animals were further dosed with or without GNF6231 for another 3 weeks. (B–D) GNF6231 at 2.5, 5 and 10 mpk reduced the skin thickness, hydroxyproline content and myofibroblast count in the mouse bleomycin model. (E) Representative H&E staining of the skin tissue samples from all dosing groups shown at 100-fold magnification.

Tsk-1 model

To examine the effects of porcupine inhibition in a less inflammation-driven model of SSc fibrosis, we turned to the Tsk-1 model. Similar to the results in the bleomycin model, porcupine inhibition showed antifibrotic effects at all dose levels (figure 2). The 5 and 10 mpk groups showed evidence of reversal of fibrosis in these readouts.

Figure 2

GNF6231 showed evidence of reversal of fibrosis in tight-skin-1 (Tsk-1) model. (A) The mouse study scheme. Wild-type (WT) mice aged 5 weeks or Tsk-1 mice were dosed with or without GNF6231 for 5 weeks. (B–D) GNF6231 at 2.5, 5 and 10 mpk reduced the skin thickness, hydroxyproline content and myofibroblast count in the Tsk-1 model. (E) Representative H&E staining shown at 100-fold magnification. n.s., not significant.

Assessment of the pharmacokinetics of GNF6231 indicated good resorption with dose-dependent increases in plasma levels (see online supplementary figures S2 and S3).

TBR-induced fibrosis

We had previously shown that TGF-β activates canonical Wnt signalling by downregulation of DKK-1 and that inhibition of WNT signalling ameliorates TBR-induced fibrosis.5 We thus analysed the antifibrotic effects of 10 mpk GNF6231 in TBR-induced skin fibrosis. GNF6231 ameliorated TBR-induced fibrosis in doses of 10 mpk with reduced skin thickening, hydroxyproline content and myofibroblast counts (see online supplementary figure S4).

Experimental cGvHD

After demonstrating effects in models of skin fibrosis, we evaluated the antifibrotic effects of GNF6231 in a systemic model with internal organ involvement. Treatment with GNF6231 ameliorated skin fibrosis induced by allogeneic transplantation and pulmonary fibrosis (figure 3). ICG-001, a selective inhibitor of canonical Wnt signalling, also inhibited skin and lung fibrosis, although the effects tended to be weaker than with GNF6231.

Figure 3

GNF6231 and ICG-001 ameliorate dermal and pulmonary fibrosis in murine sclerodermatous chronic-graft-versus-host disease (cGvHD). (A–C) Dermal fibrosis: GNF6231 at 5 and 10 mpk reduced the skin thickness, hydroxyproline content and myofibroblast count. (D and E) Pulmonary fibrosis: changes in fibrotic area of the lungs and in the hydroxyproline content of the lungs. (F) Representative H&E staining of the skin and Sirius Red stainings of lungs (both at 100-fold magnification).

Tolerability

To examine potential on-target mechanism-based toxicity, body weights of all animals were monitored and minimal or no body weight changes were observed in GNF6231-treated mice at all dose levels (see online supplementary figures S5A, B). In the model of sclerodermatous cGvHD, treatment with porcupine inhibitors ameliorated the cGvHD-induced weight loss as a typical clinical feature of cGvHD (see online supplementary figure S5C). Besides body weight, mice were also monitored daily for changes in activity, body weight, texture of the fur and consistency of the stool. None of those parameters changed on treatment with GNF6231.

Effects on cytokine release and leucocyte accumulation

Wnt signalling has been shown to be capable of modulating inflammatory responses.3 We therefore monitored the effects of GNF6231 on the expression levels of interleukin-6 (IL-6), a central proinflammatory mediator in the pathogenesis of SSc, in experimental fibrosis. Treatment with GNF6231 reduced the levels of IL-6 in experimental fibrosis (see online supplementary figure S6A). In the inflammatory models of bleomycin-induced fibrosis and cGvHD, treatment with GNF6231 also reduced leucocyte counts in the skin (see online supplementary figure S6B). As Wnt and TGF-β signalling regulate each other, we also assessed effects on the levels of active TGF-β. Treatment with GNF6231 reduced the levels of active TGF-β in fibrotic skin (see online supplementary figure S6A).

Discussion

Robust evidence indicates Wnt signalling is activated in SSc and promotes fibroblast activation and tissue fibrosis.5–7 ,16 Although inhibition of canonical Wnt signalling by modulation of β-catenin-driven activities strongly reduces fibrosis in experimental models,6 ,12–15 these preclinical results have not yet been translated into proof-of-concept trials in SSc. Major reasons for the delayed transfer from bench to bedside include the lack of compounds with suitable pharmacokinetics and concerns on adverse events caused by chronic inactivation of β-catenin-dependent pathways.4

In the present study, we demonstrate potent antifibrotic effects and good tolerability of a novel approach with an alternative mode of action. In contrast to previously tested drug candidates, inhibition of porcupine does not directly target downstream Wnt-β-catenin pathway components, but inhibits the post-translational acylation of Wnt proteins, thereby preventing the secretion of Wnt proteins.

Inhibition of porcupine offers several theoretic advantages: (1) while most other Wnt inhibitors only reduce the progression of fibrosis in preventive setting, we demonstrate that the porcupine inhibitor GNF6231 has robust activity in the reversal of pre-established fibrosis in murine models of SSc. This is particularly remarkable as all models used here represent different stages and different subpopulations of SSc. GNF6231 was effective in inflammation-driven as well as in inflammation-independent models, in preventive and in therapeutic settings and in lungs and skin as the two most commonly affected tissues. Porcupine inhibition has also been shown to be effective in renal fibrosis;19 (2) preliminary, unpublished evidence suggests that non-canonical Wnt signalling is activated in SSc and stimulates the release of collagens via c-Jun N-terminal kinase-dependent pathways. Other Wnt inhibitors, including the tankyrase inhibitor XAV939 or the CBP inhibitor ICG-001, selectively target β-catenin-dependent activities, but do not inhibit the profibrotic effects of non-canonical Wnt pathways.13 ,14 Simultaneous targeting of all Wnt pathways by porcupine inhibition might thus provide additional benefit. This hypothesis is indirectly supported by the very potent antifibrotic effects of GNF6231 as well as our recent findings on knockdown of EVI, a chaperone protein that is also required for secretion of Wnt proteins;12 (3) porcupine inhibitors with favourable pharmacokinetics and pharmacodynamics activity in humans are available for clinical use. Porcupine inhibition by WNT974, a close analogue of GNF6231, has demonstrated manageable safety profile and robust pathway inhibition with downregulation of Wnt target genes in the skin of human patients.20 Although long-term data and careful consideration of the pre-existing intestinal manifestations of patients with SSc are required, these clinical data provide evidence that Wnt signalling can be inhibited by porcupine inhibition.

Acknowledgments

We thank Rita Weinkam, Regina Kleinlein, Katja Dreißigacker and Rossella Mancuso, PhD, for excellent technical assistance.

References

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Footnotes

  • Handling editor Tore K Kvien

  • JL, JLH and JHWD are co-corresponding authors.

  • Contributors Design of the study: C-WC, CB, JL, JLH and JHWD. Acquisition of data: C-WC, CB, JL, CM, CL, TT-M, XX, SHC, MHH, NN, AA, SM, SP, ECS, HMS, JLH and JHWD. Interpretation of data: CWC, CB, JL, CM, DL, TTM, XX, SHC, MHH, NN, AA, SM, SP, ECS, HMS, JLH and JHWD. Manuscript preparation: C-WC, CB, JL, CM, JLH and JHWD.

  • Funding Grant support was provided by the Erlanger Leistungsbezogene Anschubfinanzierung und Nachwuchsförderung (ELAN), grants J29 and A40 of the Interdisciplinary Center of Clinical Research (IZKF) in Erlangen and grants DI 1537/1-1, DI 1537/2-1, DI 1537/4-1, AK 144/1-1, BE 5191/1-1 and SCHE 1583/7-1 from the Deutsche Forschungsgemeinschaft. In addition, the study was supported by the Career Support Award of Medicine of the Ernst Jung Foundation (to JHWD).

  • Competing interests JHWD has consultancy relationships and/or has received research funding from Actelion, Pfizer, Ergonex, BMS, Celgene, Bayer Pharma, JB Therapeutics, Sanofi-Aventis, Novartis, Array Biopharma and Active Biotec in the area of potential treatments of systemic sclerosis and is stock owner of 4D Science. JL, CL, SHC, MHH, NN, AA, MS, SM, SP, ECS, HMS, PG and JLH are employees of Novartis.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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