Epigenetic Alterations In Fanconi Anemia: Role In Disease Progression and Therapeutic Potential

Abstract

Fanconi anaemia (FA) is an inherited disorder characterized by chromosomal instability, progressive bone marrow failure and increased incidence of haematological and non-haematological malignancies. Mutations in 16 FA genes have been identified which disrupt a DNA repair complex, resulting in increased chromosomal fragility. However, the phenotype is variable even amongst patients from the same family and with the same mutation. This raises the possibility that other factors, such as epigenetic modifications, may affect the disease phenotype.Epigenetic changes play an important role in oncogenesis of several haematological malignancies and solid tumours. These changes may be pharmacologically manipulated with DNA hypomethylating agents and histone deacetylase inhibitors.The aim of our project was to explore whether the epigenetic profiles in FA differ from non-FA individuals and whether these could be manipulated to alter the disease phenotype.To assess whether epigenetic patterns in FA patients differ from unaffected individuals, we quantified the expression levels of genes involved in epigenetic modifications in peripheral blood mononuclear cells (PBMNC) from 12 FA patients and 15 unaffected individuals using a 84 epigenetic gene PCR array. Compared to unaffected cells, FA patients have decreased expression levels of genes involved in DNA methylation (DNMT1, DNM3Tβ), genes encoding proteins that regulate the activity of histones by acetylation (CIITA), phosphorylation (PAK1), ubiquitination (RNF20), deacetylation (HDAC2, HDCA8, HDAC9, HDAC10, HDAC11) and methylation (SETD6).We also assessed methylation status of tumor suppressor gene promoters in one FA patient and found that the FA patient had global hypomethylation in the promoter regions of tumour suppressor genes as compared to non-FA individuals, which is an early frequent event in cancer, and correlates with severity of oncologic disease.The same epigenetic gene PCR array was used to study of the effect of Vorinostat in the expression of these genes in FA-PBMNC. Vorinostat significantly increased the expression of DNM3Tβ in these cells. Vorinostat reduced expression of CIITA and HDAC9, PAK1, USP16, all involved in different aspects of epigenetic and immune regulation.Given the ability of Vorinostat to modulate epigenetic regulator genes in FA patients, we investigated its functional effects on the FA phenotype. This was assessed by quantifying FA cell hypersensitivity towards DNA cross-linking agents following incubation with Vorinostat. FA-PBMNC pre-treated with Vorinostat showed a significant reduction of the frequency of cells with breaks induced by DEB (25,7% ± 3,5%) compared to DEB treatment alone (42,8% ± 6,5%).The effect of Vorinostat in cell cycle progression and apoptosis was evaluated by flow cytometry in FA lymphoblastoid cell lines. Vorinostat increases the number of cells in the G1 phase decreasing those in the S phase and induces apoptosis. Treatment of these cells with N-acetylcysteine protects from Vorinostat-induced apoptosis but not cell cycle arrest, suggesting reactive oxygen species may play a role in these processes.Our results identify different epigenetic patterns in FA cells relative to non-FA cells, suggesting that epigenetic changes might play a role in the pathophysiology of FA. The reduction in chromosomal breaks by Vorinostat suggests this therapeutic strategy may be useful in FA. Disclosures:No relevant conflicts of interest to declare.