A long-range MYC enhancer is duplicated in human T-ALL and is required for T cell development and NOTCH1-induced leukemogenesis. Efforts to identify and annotate cancer driver genetic lesions have been focused primarily on the analysis of protein-coding genes; however, most genetic abnormalities found in human cancer are located in intergenic regions. Here we identify a new long range–acting MYC enhancer controlled by NOTCH1 that is targeted by recurrent chromosomal duplications in human T cell acute lymphoblastic leukemia (T-ALL). This highly conserved regulatory element, hereby named N-Me for NOTCH MYC enhancer, is located within a broad super-enhancer region +1.47 Mb from the MYC transcription initiating site, interacts with the MYC proximal promoter and induces orientation-independent MYC expression in reporter assays. Moreover, analysis of N-Me knockout mice demonstrates a selective and essential role of this regulatory element during thymocyte development and in NOTCH1-induced T-ALL. Together these results identify N-Me as a long-range oncogenic enhancer implicated directly in the pathogenesis of human leukemia and highlight the importance of the NOTCH1-MYC regulatory axis in T cell transformation and as a therapeutic target in T-ALL.

In NOTCH-induced T cell acute lymphoblastic leukemia, the resistance to anti-NOTCH therapy conferred by loss of the Pten tumor suppressor is linked to reversal of the effects of NOTCH inhibition on leukemic cell metabolism. Activating mutations in NOTCH1 are common in T cell acute lymphoblastic leukemia (T-ALL). Here we identify glutaminolysis as a critical pathway for leukemia cell growth downstream of NOTCH1 and a key determinant of the response to anti-NOTCH1 therapies in vivo. Mechanistically, inhibition of NOTCH1 signaling in T-ALL induces a metabolic shutdown, with prominent inhibition of glutaminolysis and triggers autophagy as a salvage pathway supporting leukemia cell metabolism. Consequently, inhibition of glutaminolysis and inhibition of autophagy strongly and synergistically enhance the antileukemic effects of anti-NOTCH1 therapy in mice harboring T-ALL. Moreover, we demonstrate that Pten loss upregulates glycolysis and consequently rescues leukemic cell metabolism, thereby abrogating the antileukemic effects of NOTCH1 inhibition. Overall, these results identify glutaminolysis as a major node in cancer metabolism controlled by NOTCH1 and as therapeutic target for the treatment of T-ALL.

Activated B cells reshape their primary antibody repertoire after antigen encounter by two molecular mechanisms: somatic hypermutation (SHM) and class switch recombination (CSR). SHM and CSR are initiated by activation-induced cytidine deaminase (AID) through the deamination of cytosine residues on the immunoglobulin loci, which leads to the generation of DNA mutations or double-strand break intermediates. As a bystander effect, endogenous AID levels can also promote the generation of chromosome translocations, suggesting that the fine tuning of AID expression may be critical to restrict B cell lymphomagenesis. To determine whether microRNAs (miRNAs) play a role in the regulation of AID expression, we performed a functional screening of an miRNA library and identified miRNAs that regulate CSR. One such miRNA, miR-181b, impairs CSR when expressed in activated B cells, and results in the down-regulation of AID mRNA and protein levels. We found that the AID 3′ untranslated region contains multiple putative binding sequences for miR-181b and that these sequences can be directly targeted by miR-181b. Overall, our results provide evidence for a new regulatory mechanism that restricts AID activity and can therefore be relevant to prevent B cell malignant transformation.