Background: The development of agents that sensitize tumours to existing therapeutics is an attractive goal in cancer therapy. We previously used cells over-expressing the multidrug transporter protein MRP4 to screen for compounds that sensitize to the purine analogue 6-mercaptopurine (6-MP), an MRP4 substrate. We identified a compound (CCIA52) that sensitizes cells to 6-MP and inhibits neuroblastoma cell growth in vitro independent of any effects on MRP4, suggesting a novel mechanism of action.
Methods: We conducted focused library screening around CCIA52 to identify additional related compounds. In vivo activity of these compounds was assessed using the TH-MYCN transgenic mouse model of neuroblastoma. The anti-proliferative effect was investigated in vitro using GC-MS based metabolomics.
Results: We identified a series of compounds with >95% homology to the original molecule. In particular, 52-14, was identified with improved microsomal stability and comparable acid stability. In vivo, both CCIA52 and 52-14 potentiated the anti-cancer effect of 6-MP in the TH-MYCN mouse model. Surprisingly, these compounds also strongly synergized with the DNA crosslinking agent cisplatin in established tumours. CCIA52 and 52-14 also delayed tumour progression in this model as single agents. To investigate the anti-proliferative effect of these compounds, cellular metabolites were measured in neuroblastoma cells treated in vitro. Treated cells displayed significant decreases in intracellular levels of the tricarboxylic acid (TCA) cycle intermediates fumarate, malate and aspartate, compared with control cells and cells treated with an inactive analogue of CCIA52. There was no change in the upstream metabolite ketoglutarate however, suggesting that CCIA52 and 52-14 inhibit the TCA cycle between ketoglutarate and fumarate.
Conclusions: We have identified a promising new agent that sensitizes established tumours to both cisplatin and 6-MP in the TH-MYCN mouse neuroblastoma model. To elucidate its molecular targets, we are currently investigating enzymes involved in the cellular conversion of ketoglutarate to fumarate.