Background: Aberrant expression of Myc oncoproteins is a major causal factor in human cancer. In the childhood cancer neuroblastoma, the MYCN oncogene is one of the most powerful prognostic markers identified and represents a potentially valuable target for the development of novel therapeutics. We aimed to identify MYCN inhibitors using chemical library screening.
Methods: A 34,000 diverse chemical library of small molecules was screened using a cell-based assay. Signal transduction pathway analyses, PCR, Western blotting analysis, siRNA knockdown, luciferase reporter assay, GC-MS based metabolomics and Mass spectrometry (MS)were used to characterise hit compounds.
Results: Among a number of molecules identified as potential Myc inhibitors, M606 was found to reduce protein expression of MYCN and its downstream targets in MYCN-amplified neuroblastoma BE(2)-C cells. A similar effect was also observed in c-Myc over-expressing tumour cells. Analysis of signalling pathways affected by M606 using FACTORIAL™ technology (Attagene Inc) indicated that this compound inhibited Myc mediated transcription and activated HIF1 pathway. siRNA-mediated knockdown of c-Myc/MYCN or HIF1A in HepG2 and BE(2)-C cells followed by M606 treatment demonstrated that Myc downregulation and HIF1A upregulation by M606 are two independent events. Furthermore, inhibition of HIF1A prolyl hydroxylases (PHDs) by dimethyloxalylglycine resulted in downregulation of c-Myc protein independent of any HIFA upregulation. M606 was found to be structurally related to hydroxyquinoline family of compounds that can inhibit PHDs. Using MS analysis, we found M606 is able to bind to iron, which largely contributes to its inhibition of MYCN.
Conclusions:M606 represents a novel Myc inhibitor that targets Iron to downregulate Myc and upregulate HIF1A. Development of this compound may have clinical potential in the treatment of cancers overexpressing Myc oncoproteins, and also in the treatment of HIF1A-mediated disorders such as ischemia-reperfusion injury.