Functional cooperation between the B-cell receptor and NOTCH1 in regulating metabolic reprogramming in chronic lymphocytic leukemia

Mutations in NOTCH1, which occur in similar to 10% of Chronic Lymphocytic Leukemia (CLL) patients at diagnosis, are typically associated with unmutated (UM) B-cell receptor (BCR) subsets and define patients with earlier treatment need. Using primary CLL cells classified as NOTCH1 wild-type (CLL/N-WT) or mutated (CLL/N-M), both with UM-BCR, we show that BCR stimulation activates the NOTCH1 pathway, upregulating metabolic programs and mitochondrial biogenesis, selectively in CLL/N-M. These cells display enhanced basal respiration and glycolysis, driven by higher mitochondrial mass, and further increase metabolic activity upon BCR triggering. To directly implicate NOTCH1 mutations, we engineered an MEC-1 model to generate wild-type (MEC-1/N-WT) or mutated (MEC-1/N-M) clones in a UM-BCR background. Here, NOTCH1 hyperactivation promoted mitochondrial metabolism through TFAM-dependent transcriptional control. Gene expression profiling, metabolic assays, and stable isotope tracing confirmed that MEC-1/N-M cells rely on oxidative metabolism, with increased glutamine dependency and strengthened anabolic pathways, leading to augmented proliferation compared to MEC-1/N-WT. Importantly, CLL/N-M cells exhibit a marked vulnerability to glutamine deprivation. Combined inhibition of glutamine utilization and BCL2 triggered rapid apoptosis, providing a rationale for tailored therapeutic strategies in NOTCH1-mutated CLL.