Modular Domains within a Super Enhancer Determine Drug Resistance in Leukemia

Notch plays roles in T cell development and is a common oncogenic driver in T cell acute lymphoblastic leukemia. Myc is a critical target of Notch in normal and malignant pre-T cells. We and others identified a distal enhancer located >1 Mb 3' of human and murine Myc that binds Notch transcription complexes and physically interacts with the Myc proximal promoter (1, 2). Deletion of a 1 kb region surrounding this enhancer resulted in a block in T cell development and inhibited Notch induced T-ALL induction and maintenance (2). Nevertheless, this 1 kb region contains binding sites for multiple transcription factors; thus, the requirement for specific transcription factors has not been resolved. To determine the precise sequences required for function of the distal enhancer, we used CRISPR/CAS9 to specifically mutate transcription factor binding sites in the Notch-dependent Myc enhancer (NDME). Specific mutation of the RBPj binding site blocked Notch/RBPj binding and inhibited Myc expression and T-ALL cell growth/survival. Acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer (a region spanning >600 kb) that correlate with Myc expression. This broad Notch-influenced region comprises an enhancer region containing multiple domains, recognizable as discrete H3K27 acetylation peaks. Specifically mutating the single RBPj binding site abrogated H3K27Ac across this entire 600 kb region. Thus, Notch binding at a single site in the NDME is required for enhancer function and H3K27Ac across a very broad region. Furthermore, the Notch-regulated distal enhancer is comprised of individual enhancers that are uniquely sensitive to Notch or BET domain inhibitors. By selecting tumor cells resistant to each inhibitor, we show that the change in drug sensitivity results from selective use of these individual enhancers that are located within the broad Notch regulated region. These data not only provide insights into enhancer function but suggest strategies to treat T-ALL, including drug resistance, by therapeutically targeting these enhancers.

References:

1. Ohtani et al., Proc Natl Acad Sci USA 111:E4946, 2014, PMID: 25369933

2. Herranz et al., Nature Med, 20:1130, 2014, PMID: 25194570