HMTase Inhibitors as a Potential Epigenetic-Based Therapeutic Approach for Friedreich's Ataxia

Abstract

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disorder caused by a homozygous GAA repeat expansion mutation in intron 1 of the frataxin gene (FXN), which instigates reduced transcription. As a consequence, reduced levels of frataxin protein lead to mitochondrial iron accumulation, oxidative stress, and ultimately cell death; particularly in dorsal root ganglia (DRG) sensory neurons and the dentate nucleus of the cerebellum. In addition to neurological disability, FRDA is associated with cardiomyopathy, diabetes mellitus, and skeletal deformities. Currently there is no effective treatment for FRDA and patients die prematurely. Recent findings suggest that abnormal GAA expansion plays a role in histone modification, subjecting the FXN gene to heterochromatin silencing. Therefore, as an epigenetic-based therapy, we investigated the efficacy and tolerability of two histone methyltransferase (HMTase) inhibitor compounds, BIX0194 (G9a-inhibitor) and GSK126 (EZH2-inhibitor), to specifically target and reduce H3K9me2/3 and H3K27me3 levels, respectively, in FRDA fibroblasts. We show that a combination treatment of BIX0194 and GSK126, significantly increased FXN gene expression levels and reduced the repressive histone marks. However, no increase in frataxin protein levels was observed. Nevertheless, our results are still promising and may encourage to investigate HMTase inhibitors with other synergistic epigenetic-based therapies for further preliminary studies.

Keywords: FRDA; FXN; Friedreich ataxia; GAA repeat; HMTase inhibitor; frataxin.

FIGURE 1

Cell viability analysis following 72 h HMTase inhibitor treatment. (A) BIX01294 and (B) GSK126 treatment analysis in human FRDA (GM03816 and GM04078) and normal fibroblasts (GM07492). The mean value of all data was normalized to the PrestoBlue reduction of vehicle treated cells (set at 100%). Error bars indicate SEM and values represent mean ± SEM (n = 3). Asterisks indicate statistically significant differences between drug and vehicle treated cell lines, assessed by unpaired two-tailed Student’s t-test (*P < 0.05, **P < 0.01).

FIGURE 2

qRT-PCR analysis indicating the relative FXN mRNA levels following treatment with BIX01294 and GSK126 alone or in combination in human primary fibroblasts (FRDA, GM03816 and normal, GM07492). Each result displayed is the mean of two independent experiments and the FXN mRNA levels of each sample were normalized to HPRT gene as an endogenous control. The values were expressed as a ratio to the vehicle treated samples of normal fibroblasts. Error bars indicate SEM and values represent mean ± SEM (n = 3). Asterisks indicate significant differences between drug and vehicle treated cell lines, assessed by unpaired two-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

FIGURE 3

qRT-PCR analysis indicating the relative FXN mRNA levels following combination treatment with BIX01294 (100 nM) and GSK126 (2 μM) in human primary fibroblasts (FRDA, GM03816 and normal, GM07492) for different time points. For this treatment, the cell culture medium was replaced with fresh medium supplemented with the drug every 3 days. Mean FXN mRNA levels of each sample were normalized to HPRT gene as an endogenous control. Values were expressed as a ratio to the vehicle treated samples of normal fibroblasts at the corresponding time point. Error bars indicate SEM and values represent mean ± SEM (n = 3). Asterisks indicate significant differences between drug and vehicle treated cell lines, assessed by unpaired two-tailed Student’s t-test (**P < 0.01, ***P < 0.001).

FIGURE 4

Frataxin expression levels. (A) Dipstick immunoassay of frataxin protein in primary fibroblasts (FRDA, GM03816 and normal, GM07492) following synergistic treatment with BIX01294 (100 nM) and GSK126 (2 μM) for different time points. Values are expressed as percentage of the vehicle treated samples of normal fibroblasts at the corresponding time point. (B) Western blot analysis indicating the relative frataxin protein expression levels in human fibroblasts (FRDA, GM04078 and FA3, and normal GM08399 and GM08333) following synergistic treatment with BIX01294 (100 nM) and GSK126 (2 μM) for 3 days. Values were normalized to actin levels and were expressed as percentage of the vehicle treated samples of normal fibroblasts. Representative Immunoblots are shown. Error bars indicate SEM and values represent mean ± SEM (n = 3). Asterisks indicate significant differences between drug and vehicle treated cell lines, assessed by unpaired two-tailed Student’s t-test (*P < 0.05, **P < 0.01).

FIGURE 5

HMTase enzymatic activity analysis in human fibroblasts (FRDA, GM03816 and normal, GM07492) treated individually and in combination with BIX01294 (100 nM) and GSK126 (2 μM). (A) EZH2 and (B) G9a activity levels. The values were expressed as a ratio to the vehicle treated samples of normal fibroblasts. Error bars indicate SEM and values represent mean ± SEM (n = 3). Asterisks indicate significant differences between drug and vehicle treated cell lines, assessed by unpaired two-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

FIGURE 6

Histone modification changes in the FXN 5′UTR promoter region, after synergistic treatment with BIX01294 (100 nM) and GSK126 (2 μM) for 72 h in human FRDA (GM03816) and normal (GM07492) fibroblasts. The values were expressed as a ratio to the vehicle treated samples of normal fibroblasts. Error bars indicate SEM and values represent mean ± SEM (n = 3). Asterisks indicate significant differences between drug and vehicle treated cell lines, assessed by unpaired two-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

FIGURE 7

Relative change in the expression of a panel of human endogenous control genes in primary fibroblasts (FRDA, GM03816 and normal, GM07492), following BIX01294 (100 nM) and GSK126 (2 μM) combination treatment. Values were expressed as a ratio to the vehicle treated samples of normal fibroblasts. Error bars indicate SEM and values represent mean ± SEM (n = 3). ACTB, beta-actin; B2M, beta-2-microglobulin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GUSB, beta-glucuronidase; HPRT1, hypoxanthine-guanine phosphoribosyltransferase; PPIA, cyclophillin A; RPLP, 60S acidic ribosomal protein P0; RRN18S, 18S rRNA; TBP, TATAA-box binding protein, TUBB, beta-tubulin, UCB, ubiquitin C; YWHA2, tyrosine 3/tryptophan 5-monooxygenase activation protein zeta.