Alternative Super-Enhancer States Determine MYC Sensitivity to Notch and Brd4 Inhibitors in T Lymphoblastic Leukemia/Lymphoma

Gain-of-function NOTCH1 mutations are oncogenic drivers in a high fraction of T-cell lymphoblastic leukemia/lymphoma (T-LL). These mutations variously cause increased production or stabilization of the free intracellular domain of NOTCH1, which regulates gene expression by forming a transcription complex with the DNA-binding factor RBPJ and coactivators of the MAML family. Using expression profiling and ChIP-seq, we have shown that NOTCH1/RBPJ complexes activate most target genes by binding to super-enhancers, large regulatory elements that switch on transcription through long-range interactions with gene promoters. MYC is a critical target of Notch in normal and malignant pre-T cells, but how Notch regulates MYC is unknown. To understand which regulatory element(s) regulate MYC expression, we used chromatin conformation capture (3C) assays to test the interaction between putative enhancer(s) and the MYC promoter in T-LL cell lines, and reporter gene assays to confirm enhancer function of candidate sites. We identified a distal site located >1 Mb 3’ of human and murine MYC termed the Notch-dependent MYC enhancer (NDME) that binds Notch transcription complexes and physically interacts with the MYC proximal promoter. An ~1 kb DNA fragment containing this site activates a luciferase reporter gene in a Notch-dependent fashion in T-LL cells but not in heterologous cell types. The Notch binding site lies within a large enhancer region (>600 kb in breadth) containing multiple discrete H3K27ac peaks. Remarkably, acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer region and the MYC promoter that correlate with NOTCH1/RBPJ complex binding and MYC expression. T-LL cells selected for resistance to gamma-secretase inhibitors (GSIs) exhibit epigenetic silencing of the NDME and loss of NDME looping interactions with the MYC promoter, yet maintain MYC expression. 3C analysis of GSI resistant cells shows preferential interaction between the MYC promoter and a more 3’ enhancer element recently described as a BRD4-dependent regulator of MYC expression in acute myeloid leukemia cells. In line with this observation, BRD4 antagonists are potent inhibitors of MYC expression in GSI resistant T-LL cells but not GSI-sensitive cells. We also studied a case of Notch-mutated early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL). ChIP-Seq analysis of the leukemic blasts revealed an “AML-like” MYC enhancer chromatin state, and as predicted from our analysis of cell lines, the blasts rapidly down-regulated MYC in response to BRD4 inhibitor but not in response to GSI. These findings suggest that specific MYC chromatin states predict responsiveness to Notch and BRD4 inhibitors, and provide a rationale for use of Notch and BRD4 inhibitor combinations in Notch-mutated leukemias.