Poster Presentation 30th Lorne Cancer Conference 2018

  Inhibition of MYCN transcription by M606, a novel small molecule inhibitor identified through chemical library screening (#239)

Ruby Pandher 1 , Chengyuan Xue 1 , Leanna Cheung 1 , Laura Gamble 1 , Catherine Burkhart 2 , Natalia Fedtsova 3 , Sergei Makarov 4 , Francesca Ferruci 5 , Thomas Tefler 6 , Rachel Codd 6 , David Scott 7 , Giovanni Perini 5 , Andrei Osterman 7 , Andrei Gudkov 2 , Murray Norris 1 8 , Michelle Haber 1
  1. Children's Cancer Institute, Kensington, NSW, Australia
  2. Cleveland BioLabs, Inc., Buffalo, NY, USA
  3. Roswell Park Cancer Institute, Buffalo, NY, USA
  4. Attagene, Morrisville, NC, USA
  5. Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy
  6. University of Sydney, Sydney, Australia
  7. Sanford-Burnham Medical Research Institute , La Jolla, CA, USA
  8. Centre for Childhood Cancer Research, University of New South Wales, Australia

Aberrant expression of Myc oncoproteins is a major causal factor in human cancer. The MYCN oncogene is one of the most powerful prognostic markers identified in neuroblastoma and represents a valuable target for the development of novel therapeutics. We aimed to identify MYCN inhibitors using chemical library screening. Among a number of molecules identified, M606 was found to reduce MYCN protein levels and its downstream targets in MYCN-amplified BE(2)-C cells. Analysis of signalling pathways affected by M606 using FACTORIAL™ technology (Attagene Inc) indicated that this compound inhibited Myc mediated transcription and activated the HIF1 pathway, while stability assays showed MYCN protein and mRNA stability were unaltered. siRNA-mediated knockdown of c-Myc/MYCN or HIF1A in HepG2 (hepatocellular carcinoma) and BE(2)-C cells followed by M606 treatment demonstrated that Myc downregulation and HIF1A upregulation by M606 are two independent events. Metabolomics analysis showed that the mechanism underlying the ability of M606 to inhibit MYCN involved its capacity to bind iron. Furthermore, the inhibition of MYCN promoter activity by M606 was reversed by the addition of iron. The addition of iron also reversed the effect of M606 on the levels of HIF1a and MYCN protein. Luciferase deletion assays have identified a minimal response region of M606 on the MYCN promoter. This region contains two E2F sites, the deletion of which resulted in an M606 unresponsive promoter. ChIP assays have also confirmed the presence of E2F sites on the MYCN promoter and further analysis found that iron restores the E2F-1 binding to the MYCN transcription start site following M606 treatment. Moreover, western blot analysis showed that the RB protein, which is required for E2F binding and transcriptional activation, becomes inactive due to hypophosphorylation following M606 treatment. Thus, M606 is a novel Myc inhibitor that chelates iron to directly downregulate Myc/MYCN transcription, and this mechanism of action represents a potentially valuable therapeutic approach in the treatment of cancers overexpressing Myc oncoproteins.