Homozygous deletions of methylthioadenosine phosphorylase in human biliary tract cancers

Abstract

The p16(INK4A)/CDKN2A gene on chromosome 9p21 is a site of frequent allelic loss in human cancers, and in a subset of cases, homozygous deletions at this locus encompass the telomeric methylthioadenosine phosphorylase (MTAP) gene. The MTAP gene product is the principal enzyme involved in purine synthesis via the salvage pathway, such that MTAP-negative cancers are solely dependent on de novo purine synthesis mechanisms. Inhibitors of the de novo pathway can then be used to selectively blockade purine synthesis in cancer cells while causing minimal collateral damage to normal cells. In this study, we determine that 10 of 28 (35%) biliary tract cancers show complete lack of Mtap protein expression. In vitro analysis using a selective inhibitor of the de novo purine synthesis pathway, L-alanosine, shows robust growth inhibition in MTAP-negative biliary cancer cell lines CAK-1 and GBD-1 accompanied by striking depletion of intracellular ATP and failure to rescue this depletion via addition of exogenous methylthioadenosine, the principal substrate of the MTAP gene product; in contrast, no significant effects were observed in MTAP-expressing HuCCT1 and SNU308 cell lines. Colony formation studies confirmed that L-alanosine reduced both number and size of CAK-1 colonies in soft agar assays. Knockdown of Mtap protein by RNA interference in L-alanosine-resistant HuCCT1 cells conferred sensitivity to this agent, confirming that intracellular Mtap protein levels determine response to L-alanosine. Inhibitors of de novo purine synthesis can be a potential mechanism-based strategy for treatment of biliary tract cancers, one third of which show complete loss of MTAP function.