­­­­­­N-Me, a Long Range T-Cell Specific Oncogenic Enhancer in T-ALL

Over the last years, numerous studies have dissected the mutational landscape of T-cell acute lymphoblastic leukemia (T-ALL) resulting in the identification of numerous oncogenes and tumor suppressors implicated in T-cell transformation. However, most genetic abnormalities found in cancer are located in intergenic regions, whose role in cancer development, if any, remains poorly understood. Here we hypothesized that recurrent cancer-associated intergenic mutations, amplifications and deletions may implicate strong transcriptional regulatory sequences responsible for the activation of key oncogenic factors in the pathogenesis of T-ALL. To address this question, we first used ChIPseq analysis to map the genomic landscape of enhancer sequences controlled by NOTCH1, a critical T-ALL oncogene activated by mutations in over 60% of human T-ALLs. In addition, we performed high resolution aCGH analysis of somatic chromosomal amplifications and deletions in a comprehensive series of 160 T-ALL samples. These analyses revealed recurrent focal duplications at chromosome 8q24 in 8/160 (5%) T-ALL cases in an area devoid of protein-coding genes containing a prominent 1 kb NOTCH1-binding peak. Strikingly, this putative oncogenic element is located +1,427 kb 3’ from the MYC locus and chromatin configuration 3C analysis demonstrated its direct association with the MYC proximal promoter. Multispecies DNA sequence alignment revealed remarkable conservation of this region in mammals, birds and reptiles and local ChIP analysis revealed bona fide active enhancer features including P300 occupancy and high levels of H3K4me1 with low levels of H3K4me3. In addition, detailed analysis of epigenetic marks across 64 hematopoietic and non-hematopoietic cell lines and tissues revealed that his regulatory element is located within a major superenhancer specifically active in T-cells. Based on these results, we proposed that this regulatory sequence, hereby named N-Me for NOTCH-bound MYCenhancer, could function as an important regulatory element driving the activation of MYC downstream of NOTCH1 in T-ALL. Consistently, luciferase reporter assays showed strong, orientation-independent and NOTCH-dependent activation of reporter constructs containing N-Me in association with a -2.5 kb MYC proximal promoter in JURKAT T-ALL cells.

Next and to formally test the functional relevance of this enhancer in T-cell development and transformation we generated and characterized N-Me knockout and conditional knockout mice. N-Me null animals were viable and showed a marked and selective reduction in thymus size and cellularity as their only developmental alteration. Detailed immunophenotypic analysis of N-Me knockout thymocytes demonstrated accumulation of double negative 3 (DN3) T cells and marked reductions in double positive and CD4+ and CD8+ single positive cells. Mechanistically, this phenotype was associated with a marked reduction in Myc expression in DN3, DN4 and ISP cells. Moreover, transplantation of N-Me knockout bone marrow hematopoietic progenitors demonstrated that this phenotype is cell autonomous, and can be rescued upon retroviral expression of MYC. Next, we analyzed the role of N-Me in the induction of NOTCH-driven leukemias by transplanting mice with N-Me wild-type and knockout hematopoietic progenitors infected with retroviruses expressing a mutant constitutively active form of NOTCH1 (ΔE-NOTCH1). In this context, mice transplanted with ΔE-NOTCH1-infected N-Me wild type cells developed T-ALL with 100% penetrance 6 weeks after transplant. In contrast, mice transplanted with ΔE-NOTCH1-N-Me knockout cells showed complete resistance to NOTCH1-induced transformation remaining 100% leukemia-free at 15 weeks post-transplant. In addition, secondary deletion of N-Me in established NOTCH1 induced leukemias from tamoxifen-inducible N-Me conditional knockout mice (Rosa26TMCre N-Me^flox/flox) induced profound antileukemic effects with extended survival and almost complete suppression of leukemia initiating cell activity. Altogether, these results identify N-Me as the first long range oncogenic enhancer directly implicated in the pathogenesis of human leukemia.