Abstract
Members of the MYC family are the most frequently deregulated oncogenes in human cancer and are often correlated with aggressive disease and/or poorly differentiated tumors. Since patients with MYCN-amplified neuroblastoma have a poor prognosis, targeting MYCN using small molecule inhibitors could represent a promising therapeutic approach. We have previously demonstrated that the small molecule 10058-F4, known to bind to the c-MYC bHLHZip dimerization domain and inhibiting the c-MYC/MAX interaction, also interferes with the MYCN/MAX dimerization in vitro and imparts anti-tumorigenic effects in neuroblastoma tumor models with MYCN overexpression. Our previous work also revealed that MYCN-inhibition leads to mitochondrial dysfunction resulting in accumulation of lipid droplets in neuroblastoma cells. To expand our understanding of how small molecules interfere with MYCN, we have now analyzed the direct binding of 10058-F4, as well as three of its analogs; #474, #764 and 10058-F4(7RH), one metabolite C-m/z 232, and a structurally unrelated c-MYC inhibitor 10074-G5, to the bHLHZip domain of MYCN. We also assessed their ability to induce apoptosis, neurite outgrowth and lipid accumulation in neuroblastoma cells. Interestingly, all c-MYC binding molecules tested also bind MYCN as assayed by surface plasmon resonance. Using a proximity ligation assay, we found reduced interaction between MYCN and MAX after treatment with all molecules except for the 10058-F4 metabolite C-m/z 232 and the non-binder 10058-F4(7RH). Importantly, 10074-G5 and 10058-F4 were the most efficient in inducing neuronal differentiation and lipid accumulation in MYCN-amplified neuroblastoma cells. Together our data demonstrate MYCN-binding properties for a selection of small molecules, and provide functional information that could be of importance for future development of targeted therapies against MYCN-amplified neuroblastoma.
Highlights
The MYC family members c-MYC, MYCN and L-MYC are transcription factors crucial for the regulation of normal cellular functions including proliferation, cell growth, differentiation, metabolism and apoptosis
We have previously shown that this compound interferes with the MYCN/MAX interaction leading to cell cycle arrest, apoptosis, and neuronal differentiation in MYCN-overexpressing NB cell lines [40]
In order to verify the folding of the protein domains following purification under denaturing conditions and refolding, circular dichroism (CD) was performed
Summary
The MYC family members c-MYC, MYCN and L-MYC are transcription factors crucial for the regulation of normal cellular functions including proliferation, cell growth, differentiation, metabolism and apoptosis. C-MYC and MYCN (hereafter MYC), exert their functions mainly through transcriptional modulation of their target genes. In normal tissue the expression pattern of these two proteins differ significantly [5,6]. MYCN is expressed in certain tissues including the central and peripheral nervous systems, lung and spleen, whereas in adults its expression is very low or absent. C-MYC is expressed in all proliferating cells in adults [6,7,8,9]
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