Embryonal cancer arises from postnatally persistent embryonal remnant or “rest” cells that are uniquely characterised by the absence of p53 mutations (1). We have shown that perinatal neuroblastoma precursor cells exhibit a transiently diminished p53 response to MYCN oncoprotein stress and resistance to trophic factor withdrawal, in the TH-MYCN transgenic mouse model of neuroblastoma (2).
We performed high-throughput screening to identify novel compounds which restore the death response in MYCN-expressing precursor B lymphocytes, which demonstrates trophic factor Interleukin 7 (IL-7) independent growth in vitro and tumour growth a xenograft animal model. From an initial library of 40,000 compounds screening, we generated a short-list of 56 compounds which displayed cytotoxic activity against MYCN-expressing pre-B cells in the absence of IL-7, but had minimal effect on cells in the presence of trophic factor. Dose response curves were used to further refine the list to 5 lead hit compounds in the absence of IL-7 (IC50: 2.70 to 8.41µM), and more than 20µM for all controls including MYCN-expressing pre-B cells, normal pre-B cells in IL-7, and two normal human fibroblast cell lines. Two top compounds demonstrated marked differences in cytotoxic effect, with PB-798 causing cell cycle arrest and PB-370 inducing cell death. We have generated 14 PB-370 analogues for further investigation as PB-370 displayed the greatest difference in activities between the MYCN-expressing pre-B cells in the presence and absence of IL-7. Analysis of apoptotic genes from a PCR array and immunoblots of MYCN-expressing pre-B cells, in the presence and absence of IL-7 treated with PB370, revealed in increase expression of TP53 and Caspase 3/7/8, and decrease of MYCN protein expression. The mechanisms of cell death initiated by PB370 provide valuable insight into chemical inhibition of abnormally death persisting cells and a rationale for investigation into drug efficacy in MYCN-driven neuroblastoma mouse models.