DHODH as a Targetable Metabolic Achilles' Heel for chemo-resistant B-ALL.

Relapse remains a major barrier to survival in B-cell acute lymphoblastic leukemia (B-ALL). Both activation of B-cell signaling pathways and increased glucose consumption have been linked to chemo-resistance and relapse risk. Here, we connect these observations, showing that B-ALL cells with active signaling, marked by high phosphorylated ribosomal protein S6 (pS6+), are glucose dependent. Isotope tracing confirms that pS6+ cells are highly glycolytic and rely on glucose for de novo nucleotide synthesis. Uridine, but not other purines or pyrimidines, rescues pS6+ cells from glucose deprivation, highlighting uridine as essential for survival. Active mTOR signaling in pS6+ cells drives de novo pyrimidine synthesis by activating CAD (Carbamoyl phosphate synthetase 2, Aspartate transcarbamylase, and Dihydroorotase), which catalyzes the first steps of de novo pyrimidine synthesis. Inhibiting signaling abolishes glucose dependency and CAD phosphorylation. Primary pS6+ cells express high levels of pyrimidine synthesis proteins, including dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in pyrimidine synthesis. Increased DHODH expression correlates with relapse and poor event-free survival. Most B-ALL molecular subtypes exhibit DHODH activity. BAY-2402234, a DHODH inhibitor, effectively kills pS6+ cells in vitro, with IC50 values correlating with pS6 signaling strength across 14 B-ALL patient-derived xenografts (PDX). In vivo, DHODH inhibition prolongs survival and reduces leukemia burden in pS6+ B-ALL models. These findings link active signaling to pyrimidine dependency and relapse risk, highlighting DHODH inhibition as a promising therapeutic strategy for chemo-resistant B-ALL.