Combined inhibition of de novo pyrimidine synthesis and ATR promotes ATF4-mediated cell death in TP53-deficient ALL

Mutations or deletions affecting the TP53 gene predict a dismal outcome in relapsed acute lymphoblastic leukemia (ALL). Loss of p53 function compromises the response to many anti-leukemic therapies, underscoring the need for agents that are effective in TP53-deficient cells. Leukemic blasts depend on de novo pyrimidine synthesis to sustain proliferation, and the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) is essential for this pathway. While DHODH inhibitors (DHODHis) show preclinical anti-leukemic activity, the efficacy in TP53-deficient contexts has not been tested. Using both isogenic TP53-wildtype and knock-out cell line models and patient-derived xenografts (PDXs), we show that dual inhibition of DHODH and Ataxia Telangiectasia and Rad3-related (ATR) produces pronounced anti-leukemic effects, irrespective of the TP53 status. Through integrated transcriptomic and metabolomic analyses, we show that combined inhibition of DHODH and ATR leads to a reduced flux of glucose into the TCA cycle, accompanied by an increase in oxidative stress. This metabolic phenotype triggers cell death through a mechanism converging on Activating Transcription Factor 4 (ATF4), a key integrator of cellular responses to metabolic and oxidative stress, which operates largely independently of p53. Taken together, our findings identify ATF4-mediated cell death as a previously unrecognized vulnerability in TP53-deficient ALL.