Differential epigenetic modulation of TGF-β target genes in fibroblasts from DMD-patient skeletal muscle by the HDAC inhibitor givinostat


Non-Gene Targeting Therapies

Poster Number: 130


Monica Forino, PhD, Valeria Spadotto, PhD, Christian Steinkuhler, PhD, Gianluca Fossati, PhD, Sara Cazzaniga, M.Sc.


1. Italfarmaco S.p.A., 2. Italfarmaco S.p.A., 3. Italfarmaco S.p.A., 4. Italfarmaco S.p.A., 5. Italfarmaco S.p.A.

Lack of dystrophin in Duchenne Muscular Dystrophy (DMD) leads to degeneration of muscles fibers progressively substituted by fibrous tissue. In DMD, TGF-β has a recognized pro-fibrotic role and levels of TGF-β are elevated in plasma and in muscles of patients and correlate with increased fibrosis. In vitro, TGF-β is highly expressed in DMD myotubes and in muscle-derived fibroblasts. Inactive TGF-β is bound to the latent TGF-β binding protein (LTBP) and in DMD patients, four LTBP4 SNPs are predictive of the age at onset of loss of ambulation. The LTBP4 IAAM variant releases less TGF-β and is associated with older age at loss of ambulation and less TGF-β signaling. Histone deacetylases (HDACs) are involved in fibrogenesis and reduction of HDAC activity hinders TGF-β induced fibrosis. Our HDAC inhibitor givinostat significantly reduced fibrosis in mdx mice and in DMD patients. Here, we described the effects of givinostat on TGF-β induced gene expression in fibroblast from DMD-patient skeletal muscle. TGF-β modulated pro-fibrotic and pathway specific genes at various extent. Givinostat had a different effect depending on the gene analyzed. Thus, it reduced the expression of collagen III, but had a lower effect on αSMA. A different regulation of genes in the TGF-β pathway also occurred. TGF R1 was upregulated by the cytokine and downregulated by givinostat while TGF R2 was downmodulated by TGF-β and upregulated by givinostat. Increased acetylation of histones and tubulin indicated target engagement. Furthermore, givinostat induced histone crotonylation in agreement with the recently described de-crotonylase activity of class I HDAC. These data emphasize how a different epigenetic regulation of key fibrotic genes could takes place in DMD-patient cells. They also highlight the importance of having an in vitro model to study the specific pathways that can be targeted in diseased cells to more accurately advance drug candidates into in vivo studies.