NOTCH1 dimeric signaling is essential for T-cell leukemogenesis and leukemia maintenance

• In human T-ALL, a NOTCH1 dimers - HES4 axis primes an isoform switch in the TP53 gene favouring the anti-apoptotic Δ133p53 isoform.

• NOTCH1 dimer signature links indirectly to the expression of pro-apoptotic genes and directly to markers of poor clinical outcome.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy characterized by an expansion of T-cell progenitors and DNA mutations that lead to an overactive NOTCH1 signaling in over 50% of T-ALL cases. Using synthetic models of human T-ALL, we report that NOTCH1 dimeric signaling was crucial for the leukemogenesis of human hematopoietic stem/progenitor cells (HSPCs) from cord blood. We also identified a Notch-dimerization-dependent gene signature, including HES4 transcription factor, which induced proliferative advantage in human HSPCs as well as in Notch-dimerization-dependent patient-derived xenografts (PDXs) of T-ALL. Interestingly, in human T-ALL cells, HES4 enforced the expression of D133p53 isoform with the concomitant block of pro-apoptotic p53 target genes and the induction of BCL2L1 gene expression and anti-apoptotic Bcl-xL protein. Additionally, through an integrated experimental approach including genetically modified cell lines, RNA/Chip-sequencing and single cell RNA-sequencing (scRNA-Seq) profiles of primary T-ALL samples, we revealed cell subsets with Notch-dimerization-dependent gene signature, which indirectly correlated with pro-apoptotic genes as well as directly associated with cell markers of poor clinical outcome in primary T-ALL samples. Taken together, these findings highlight the crucial role of NOTCH1 dimeric signaling in human T-cell leukemogenesis and T-ALL maintenance suggesting that a possible benefit can be obtained from a therapeutic strategy targeting NOTCH1-dimer signaling or its downstream effectors.