Investigating Epigenetic Vulnerabilities in MYCN-Amplified Neuroblastoma

BACKGROUND: Neuroblastoma (NB) is a childhood cancer often arising from the adrenal glands. The MYCN transcription factor (TF) is amplified in 40% of high-risk cases. MYCN-amplified (MNA) NB is associated with aggressive behaviour and poor prognosis. The ‘undruggable’ property of MYCN has been an impediment to effective treatment for MNA NB. MYCN is known to be involved in epigenetic regulation and mitochondrial metabolism (1-3). We hypothesized that there is a crosstalk between mitochondrial metabolism and epigenetic regulation in MNA NB, and we can exploit this crosstalk for the therapy of MNA NB. AIMS: Here, I aimed to identify 1) changes in histone modifications by targeting mitochondrial complex I with diphenyleneiodonium chloride (DPI); 2) anti-proliferative epigenetic modifiers (EMs) from an epigenetic small interfering RNA (siRNA) screen; and 3) synergistic drug combinations targeting the mitochondria and/or epigenetic function in MNA NB. METHODS: The chromatin fraction and histone mixture pellet were extracted from the 24 hours DPI-treated Be(2)-C cells to identify changes in histone modifications by total proteomics. Then, MNA and non MNA NB cells were transfected with selected siRNAs for 72 hours, and cell viability was measured. Lastly, ATP-citrate lyase (ACLY) inhibitor and epigenetic drugs were used to identify IC50s in Be(2)-C and then pairwise drug combinations including DPI were conducted in MNA NB cell lines. The cell viability was measured and synergy of the tested drug combinations was calculated. RESULTS: DPI treatment of Be(2)-C cells resulted in changes in histone modifications. Six anti-proliferative EMs were identified in MNA NB cell lines. Selected chemical inhibitors targeting these anti-proliferative EMs (afimoxifene, SB431542 and fulvestrant) also decreased the viability of Be(2)-C. Synergistic drug combinations were discovered; these include the combination of SB431542 with fulvestrant and BMS303141 with fulvestrant in Be(2)-C and DPI with fulvestrant in IMR-32 cells. CONCLUSIONS: The mitochondrial inhibitor DPI indirectly regulates epigenetic changes, confirming a crosstalk between mitochondrial metabolism and epigenetics in MNA NB. Combined targeting of the mitochondria and epigenetic function is a promising approach for MNA NB.