CTP sensing and Mec1ATR-Rad53CHK1/CHK2 mediate a two-layered response to inhibition of glutamine metabolism.

Glutamine analogs are potent suppressors of general glutamine metabolism with anti-cancer activity. 6-diazo-5-oxo-L-norleucine (DON) is an orally available glutamine analog which has been recently improved by structural modification for cancer treatment. Here, we explored the chemogenomic landscape of DON sensitivity using budding yeast as model organism. We identify evolutionarily conserved proteins that mediate cell resistance to glutamine analogs, namely Ura8CTPS1/2, Hpt1HPRT1, Mec1ATR, Rad53CHK1/CHK2 and Rtg1. We describe a function of Ura8 as inducible CTP synthase responding to inhibition of glutamine metabolism and propose a model for its regulation by CTP levels and Nrd1-dependent transcription termination at a cryptic unstable transcript. Disruption of the inducible CTP synthase under DON exposure hyper-activates the Mec1-Rad53 DNA damage response (DDR) pathway, which prevents chromosome breakage. Simultaneous inhibition of CTP synthase and Mec1 kinase synergistically sensitizes cells to DON, whereas CTP synthase over-expression hampers DDR mutant sensitivity. Using genome-wide suppressor screening, we identify factors promoting DON-induced CTP depletion (TORC1, glutamine transporter) and DNA breakage in DDR mutants. Together, our results identify CTP regulation and the Mec1-Rad53 DDR axis as key glutamine analog response pathways, and provide a rationale for the combined targeting of glutamine and CTP metabolism in DDR-deficient cancers.