Metabolic changes induced by oncogenic drivers of cancer contribute to tumor growth and are attractive targets for cancer treatment. Here, we found that increased growth of -mutant cells was dependent on glutamine flux through the pyrimidine synthesis pathway, which created sensitivity to the inhibition of dihydroorotate dehydrogenase, a rate-limiting enzyme for pyrimidine ring synthesis. S-phase -mutant cells showed increased numbers of replication forks, and inhibitors of dihydroorotate dehydrogenase led to chromosome breaks and cell death due to inadequate ATR activation and DNA damage at replication forks. Our findings indicate that enhanced glutamine flux generates vulnerability to dihydroorotate dehydrogenase inhibition, which then causes synthetic lethality in -deficient cells due to inherent defects in ATR activation. Inhibition of dihydroorotate dehydrogenase could thus be a promising therapy for patients with -mutant cancers. We have found a prospective targeted therapy for -deficient tumors, with efficacy and in tumors derived from different tissues. This is based upon the changes in glutamine metabolism, DNA replication, and DNA damage response which are consequences of inactivation of .