A NOTCH1-driven MYC enhancer promotes T cell development, transformation and acute lymphoblastic leukemia

Abstract

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. © 2014 Nature America, Inc. All rights reserved. a r t i c l e s advance online publication nature medicine Supplementary Tables 1 and 2). We identified no duplications in this region in 258 non-T-ALL hematologic tumors, and no germline copy number variant polymorphisms encompassing this area have been reported. Moreover, analysis of normal (remission) DNA confirmed the somatic origin of these copy number alterations in all four cases with available material To functionally characterize the potential role of this NOTCH1 binding site in gene regulation, we performed local ChIP analysis of chromatin regulatory factors and epigenetic histone marks in HPB-ALL T-ALL cells. These analyses confirmed high levels of NOTCH1 binding at this site and revealed bona fide active enhancer features associated with this region, including occupancy and high levels of P300 (also called EP300) and histone H3 Lys4 monomethylation (H3K4me1) with low levels of H3K4 trimethylation (H3K4me3) ( On the basis of these results, we proposed that this +1.4 Mb MYC NOTCH1-occupied enhancer-hereby named N-Me for NOTCHbound MYC enhancer-could function as an important regulatory element driving the activation of MYC downstream of NOTCH1 in T-ALL. Consistent with this hypothesis, chromatin configuration 3C (chromosome conformation capture) analysis of the MYC locus demonstrated the association of this enhancer with proximal regulatory sequences in the MYC promoter The N-Me enhancer is required for thymocyte development To test the specificity and functional relevance of the N-Me enhancer in T cell development and transformation, we used homologous recombination in mouse embryonic stem cells to generate N-Me knockout and conditional knockout mice N-Me is required for NOTCH1-induced T cell leukemogenesis Given the important role of NOTCH1-induced MYC upregulation in the pathogenesis of T-ALL, we hypothesized that deletion of the N-Me enhancer could disrupt NOTCH1-induced leukemogenesis. To test this possibility, we transplanted isogenic C57BL/6 mice with wild-type or N-Me heterozygous or homozygous knockout hematopoietic progenitors infected with retroviruses driving the expression of an To explore the pathogenic role of N-Me-mediated Myc expression in NOTCH1-induced leukemia tumor maintenance, we generated ∆E-NOTCH1-induced T-ALL tumors from wild-type (Rosa26TM-Cre N-Me +/+ ) and tamoxifen-inducible conditional heterozygous (Rosa26TM-Cre N-Me flox/+ ) and homozygous (Rosa26TM-Cre N-Me flox/flox ) N-Me knockout hematopoietic progenitors. In these experiments, mice transplanted with ∆E-NOTCH1-expressing wildtype and tamoxifen-inducible heterozygous and homozygous conditional N-Me knockout cells developed NOTCH1-induced T-ALLs with identical kinetics and immunophenotypes ( To better assess the mechanisms mediating the antileukemic effects of N-Me inactivation, we then analyzed the cellular and transcriptional phenotypes of N-Me conditional inducible knockout T-ALL cells after tamoxifen treatment. In this setting, N-Me deletion in T-ALL cells DISCUSSION NOTCH1 has a central role in the pathogenesis of T-ALL 24 and drives an oncogenic transcriptional program that promotes cell growth proliferation and survival in T-ALL lymphoblasts. Importantly, the oncogenic effects of NOTCH1 are closely linked to activation of the MYC oncogene © 2014 Nature America, Inc. All rights reserved. a r t i c l e s nature medicine advance online publication a broad regulatory area of about 100 kb located 1.7 Mb telomeric to the Myc gene, 400 kb downstream of N-Me 35 . This Myc regulatory region contains multiple enhancers that are active in myeloid cells but not the thymus and is duplicated in about 3% of acute myeloid leukemias The requirement for N-Me-mediated upregulation of Myc expression downstream of Notch1 was even more apparent in the context of leukemia initiation, where loss of one and two copies of N-Me delayed and abrogated tumor development by oncogenic NOTCH1, respectively. In addition, N-Me was also required for the maintenance of NOTCH1-induced leukemias, as secondary deletion of one copy of N-Me in established tumors resulted in a marked delay in tumor progression, and loss of two copies effectively abrogated leukemia propagation and the self-renewal capacity of leukemia-initiating cells. These results are consistent with the well-established quantitative effects of MYC expression in other tumor settings. Loss of one copy of Myc has been shown to attenuate intestinal tumorigenesis 37 , and homozygous deletion of Myc completely abrogates tumor development induced by loss of Apc in the gut Several lines of evidence support a role for loss of Myc expression as the primary driver in the developmental and tumor phenotypes associated with N-me loss. In this regard, we observed marked reductions in Myc expression in developing T cells from N-Me knockout mice and in T-ALL lymphoblasts after N-Me inactivation. Moreover, retroviral expression of Myc restored T cell lymphopoiesis from NMe-deficient hematopoietic progenitors and rescued the defects in leukemia cell growth induced by secondary deletion on N-Me in NOTCH1-induced T-ALL cells. In addition, MYC inactivation has been associated with a global decrease in transcriptional activity, with a particularly pronounced downregulation of genes involved in growth, proliferation and metabolism The generation of the N-Me conditional knockout model presented here was also useful in analyzing the specific role of this enhancer in transcriptional control. Thus, even though in some cases enhancerpromoter interactions have been implicated in the regulation of transcription by promoting the release of RNA Pol II pausing 42 , deletion of N-Me in T-ALL lymphoblasts resulted in unloading of RNA Pol II at the Myc transcription initiation site without any apparent increase in RNA Pol II pausing. Overall, our results identify the N-Me regulatory sequence as a critical mediator of NOTCH1-induced MYC expression that is required for T cell development and transformation and substantiates a pathogenic role for chromosomal duplications targeting this enhancer in the pathogenesis of T-ALL. METHODS COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. Curr. Top. Microbiol. Immunol. 360, 163-182 (2012 NOTCH1 inhibition. We inhibited NOTCH1 in JURKAT cells with 250 nM DBZ ((S)-2-(2-(3,5-difluorophenyl)acetamido)-N-((S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo [b,d]azepin-7-yl) propanamide) (Syncom) for 48 h as described previously Genomic analysis of primary T-ALL samples. A total of 160 cases of T-ALL from adult and pediatric patients referred to Saint-Louis Hospital, Paris, France were analyzed for copy number abnormalities using array-comparative genomic hybridization with informed consent under the supervision of the Institutional Review Board of the Institut Universitaire d'Hématologie, Université ParisDiderot. Sureprint G3 human CGH 180K, 244K, 400K or 1M arrays (Agilent technologies) were used, and copy number alterations were identified using Genomic Workbench software and the ADM-2 algorithm (Agilent Technologies) as described previously T-ALL oncogenic subtype was determined on the basis of gene expression profiling, as reported previousl