Combined Targeting 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), with limited curative treatment options available. Due to their highly proliferative state, ALL cells are strongly dependent on nucleotide synthesis. Inhibitors of dihydroorotate dehydrogenase (DHODH), a pivotal enzyme in de novo pyrimidine biosynthesis, have demonstrated promising single-agent efficacy in some ALL and acute myeloid leukemia (AML) preclinical models. Using both isogenic TP53 cell line models and patient-derived xenografts (PDX), 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 to 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 independent of p53. Taken together, our findings identify ATF4-mediated cell death as a previously unrecognized vulnerability in TP53-deficient ALL.