The histone deacetylase inhibitor, romidepsin, as a potential treatment for pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive disease that usually affects elderly people. It has a poor prognosis and there are limited therapies. Since epigenetic alterations are associated with IPF, histone deacetylase (HDAC) inhibitors offer a novel therapeutic strategy to address the unmet medical need. This study investigated the potential of romidepsin, an FDA-approved HDAC inhibitor, as an anti-fibrotic treatment and evaluated biomarkers of target engagement that may have utility in future clinical trials. The anti-fibrotic effects of romidepsin were evaluated both in vitro and in vivo together with any harmful effect on alveolar type II cells (ATII). Bronchoalveolar lavage fluid (BALF) from IPF or control donors was analyzed for the presence of lysyl oxidase (LOX). In parallel with an increase in histone acetylation, romidepsin potently inhibited fibroblast proliferation, myofibroblast differentiation and LOX expression. ATII cell numbers and their lamellar bodies were unaffected. In vivo, romidepsin inhibited bleomycin-induced pulmonary fibrosis in association with suppression of LOX expression. LOX was significantly elevated in BALF of IPF patients compared to controls. These data show the anti-fibrotic effects of romidepsin, supporting its potential use as novel treatment for IPF with LOX as a companion biomarker for evaluation of early on-target effects.

Keywords: biomarkers; epigenomics; histone deacetylase Inhibitors; myofibroblasts; pulmonary fibrosis.

Figure 1. Romidepsin dose and time dependently increased acetylation of histone H3: Fibroblasts were cultured in DMEM/FBS ± TGF-β1 (5ng/ml) with the indicated concentration of romidepsin for 48-144 hours

Cell lysates were analysed by SDS-PAGE and western blotting for acetyl- and pan-histone H3. A. Representative western blot with B. semi-quantitative analysis of the dose-response of IPF fibroblasts to romidepsin at 48h in the absence (solid bars) and presence (open bars) of TGF-β1. C. Time-dependent response to 1nM romidepsin ± TGF-β1. Data are shown as mean + SD (n = 3; two-way ANOVA with Dunnett's multiple comparisons). *P < 0.05, **P < 0.01, ***P < 0.001. Where there are no lines to indicate the statistical comparison, it is between the starred bar and its equivalent baseline non-romidepsin treated control.

Figure 2. Inhibition of fibroblast proliferation by romidepsin: IPF and normal fibroblasts were cultured for up to 144h in DMEM/FBS ± TGF-β1 (5ng/ml) and ± romidepsin

Cells were formalin-fixed before being stained with methylene blue. Stained cells were eluted with a 1:1 ratio of 0.1% HCl and ethanol. Absorbance was measured at 630nm using a photometric plate reader. A. Dose response of IPF fibroblasts to romidepsin at 144h in the absence (solid symbols) or presence (open symbols) of TGF-β1. B. Comparison of the romidepsin concentration required to achieve 50% growth inhibition (IC50) of the IPF and normal fibroblasts at 144h. C. At 48h, after culturing as described, RNA was extracted using the Trizol method prior to cDNA synthesis and analysis by RTqPCR. Data were normalized to the housekeeping genes UBC/A2 using the ΔΔCT method. CDKN1A mRNA expression in response to increasing doses response of romidepsin in IPF fibroblasts without (solid bars) or with (open bars) TGF-β1. Data in B. & C. are shown as mean + SD (n = 3; two-way ANOVA with Dunnett's multiple comparisons). *P < 0.05, **P < 0.01. D. Cell cycle analysis of romidepsin treatment of 3 primary IPF fibroblasts cultures quantitatively determined by propidium iodide (PI) flow cytometric analysis. Solid bar = G1, grey bar = S, open bar = G2/M phase. Data are shown as mean + SD (n = 3; one-way ANOVA). **P < 0.01. Where there are no lines to indicate the statistical comparison, it is between the starred bar and its equivalent baseline non-romidepsin treated control.

Figure 3. Romidepsin suppressed myofibroblast differentiation: Fibroblasts were cultured in DMEM/FBS ± TGF-β1with the indicated concentration of romidepsin for 48 hours and then samples harvested into Trizol for RNA isolation, cDNA synthesis and RTqPCR analysis

Figure shows mRNA expression of A. ACTA2 B. HDAC4 and C. COL3A1 mRNA in IPF fibroblasts in response to romidepsin in the absence (solid bars) or presence (open bars) of TGF-β1. D. Inhibition of COL3A1 mRNA expression by 5nM romidepsin ± TGF-β1 in normal or IPF fibroblasts expressed as a percentage of the corresponding untreated control. Data were normalized to the housekeeping genes UBC/A2 using the ΔΔCT method. Data are presented as mean + SD (n = 3; two-way ANOVA with Dunnett's multiple comparisons). *P < 0.05, **P < 0.01. Where there are no lines to indicate the statistical comparison, it is between the starred bar and its equivalent baseline non-romidepsin treated control.

Figure 4. Romidepsin inhibited soluble collagen production and α-SMA expression in 3D pellet culture: 3D pellet cultures were treated without or with 5nM romidepsin in the absence or presence of TGF-β1 for 144h, at which point the pellet was fixed and paraffin embedded

A. assessment of soluble collagen released by the pellets under the indicated conditions and B. representative immunohistochemical staining for α-SMA. In A. data are presented as mean + SD (n = 3; one-way ANOVA with Dunnett's multiple comparisons). *P < 0.05. Where there are no lines to indicate the statistical comparison, it is between the starred bar and its equivalent baseline non-romidepsin treated control.

Figure 5. Comparison of the effect of romidepsin on alveolar type 2 cells

A. The ATII cells (solid bar), isolated from lung resections of 3 different patients, were treated with a range of concentrations of romidepsin, using IPF fibroblasts (open bar) as controls. After 72h, cell proliferation was determined by DAPI staining and direct counting of cell nuclei. B. (Upper panel) Coherent anti-Stokes Raman spectroscopy of lipids/surfactants in ATII cells alone or after treatment with 5nM romidepsin for 72h. (Lower panel) Transmission electron microscopy (TEM) of the ultrastructure of ATII cells cultured on Transwells® for 72h without or with 5nM romidepsin. C. CARS image analysis of the pixel ratio of surfactants lipid droplets (LDs) over cell area. In (A & C) data are shown as mean + SD (n = 3 donors; two-way ANOVA). * P < 0.05, *** P < 0.001.

Figure 6. Romidepsin increased histone acetylation and suppressed pro-fibrotic gene expression in bleomycin treated mice: Bleomycin (2U/kg) was instilled via the oropharyngeal route to induce lung fibrosis

Romidepsin (2mg/kg IP) or a vehicle control was administered 4 days post bleomycin treatment and lungs harvested on day 7 for analysis by western blotting and RTqPCR. A. Lungs were homogenized in normal saline with protease inhibitors and lysates analyzed for acetyl histone H3 protein by Western blotting using pan histone H3 as the loading control. mRNA expression for B. Fn1, C. Col3a1 and D. Col1a1 analyzed by RTqPCR with the gene of interest being normalized to the housekeeping gene Gapdh using the ΔΔCT method. Data are presented as mean + SD (n = 4 per group within one experiment; two-way ANOVA with Tukey's multiple comparisons). **P < 0.01, ***P < 0.001.

Figure 7. Romidepsin suppressed bleomycin induced pulmonary fibrosis in vivo: Bleomycin was instilled via the oropharyngeal route to induce lung fibrosis

Romidepsin (2mg/kg IP) or vehicle control was administered days 3, 7, 11, and 15 days post bleomycin treatment and the mice were harvested on day 21. A. & B. Representative images of left lung sections stained with Gomori's trichrome stain to visualize collagen (blue). The right lung was weighed C. and then homogenized for determination of hydroxyproline content D.. Data are presented as mean + SD (n = 6 per group within two experiments; two-way ANOVA with Tukey's multiple comparisons). *P < 0.05.

Figure 8. Inhibition of LOX expression by Romidepsin: IPF or normal fibroblasts were cultured in DMEM/FBS in the absence (solid bars) or presence (open bars) of TGF-β1 and ± romidepsin for 48h

Cells were lysed for RNA analysis by RT-qPCR and cell-conditioned media harvested for western blot analysis. A. LOX mRNA expression in the absence or presence of TGF-β1 after 48h. Data are presented as mean + SD (n = 3 per group; one-way ANOVA) B. LOX mRNA expression in IPF fibroblasts with increasing concentrations of romidepsin in the presence or absence of TGF-β1. Data were normalized to the lowest expressing fibroblast cell line from a control donor at baseline. C. & D. Western blot analysis of active LOX protein (32 kDa) secreted from IPF fibroblasts, demonstrating dose and time responses, respectively, to romidepsin. Representative blot is shown in panel C; data were normalized to total protein loaded using a Ponceau stain and expressed relative to a positive control to control for between blot variation. Data in B.-D. are presented as mean + SD (n = 4 IPF donors, each performed in duplicate). E. Semi quantitative analysis of LOX protein measured in homogenized mouse lung as prepared as previously described in Figure 7. Data are presented as mean + SD (n = 4 per group within one experiment; two-way ANOVA with Tukey's multiple comparisons). *P < 0.05, **P < 0.01, ***P < 0.001. Where there are no lines to indicate the statistical comparison, it is between the starred bar and its equivalent baseline non-romidepsin treated control.

Figure 9. LOX was increased in IPF and correlated with increased eosinophils: BALF from IPF patients and control donors were analyzed by western blot for LOX expression

A. Representative blot with B. semi-quantitative analysis of Pro LOX expression normalized to Ponceau stain for total protein, and expressed relative to a positive control. C. Regression analysis showing the correlation between BAL eosinophils and relative levels of Pro-LOX. Data are presented as mean + SD (n = 9 control and 20 IPF donors). **P < 0.01.