Myc super-enhancer dynamics in T-ALL

Abstract Mutations in non-coding regulatory elements are increasingly recognized as critical drivers of cancer development and progression. Among these, structural alterations such as duplications of super-enhancers (SEs), large clusters of enhancers that regulate key oncogenes, have emerged as significant contributors to oncogenesis. In T-cell acute lymphoblastic leukemia (T-ALL), a particularly aggressive hematologic malignancy, SE duplications have been identified in approximately 5% of human cases. These duplications frequently involve a non-coding region located approximately 1.3 megabases downstream of the Myc oncogene, a region known as N-Me. Despite the growing recognition of the role of SE duplications in T-ALL, functional studies to determine their molecular origins and contributions to leukemogenesis have been limited, largely due to the absence of suitable and genetically tractable preclinical models. This gap in knowledge hinders both our understanding of disease pathogenesis and our ability to design targeted therapeutic interventions. Here, we report the generation and characterization of a novel genetically engineered mouse model of T-ALL that spontaneously develops oncogenic SE duplications at the Myc locus. This model offers the first spontaneous in vivo system to investigate the origin, regulation, and oncogenic function of SE duplications. This model was created by conditionally stabilizing β-catenin in CD4⁺CD8⁺ double-positive (DP) thymocytes in combination with targeted deletion of the transcription factor HEB. This combination is sufficient to drive the development of aggressive T-cell leukemias that consistently harbor duplications of the N-Me super-enhancer. This finding recapitulates the genomic alteration observed in a subset of human T-ALL and provides a unique platform for mechanistic studies. We further demonstrate that the molecular events leading to SE duplication and oncogenic Myc activation unfold in a stepwise manner. At the preleukemic stage, stabilization of β-catenin alone is sufficient to enhance recruitment of HEB to the N-Me region. The simultaneous loss of HEB greatly increased chromatin accessibility at the N-Me enhancer. This altered chromatin landscape permits aberrant binding of transcription factors including TCF-1, which plays a critical role in T-cell development and leads to N-Me SE activation associated with significant three-dimensional chromatin reorganization. Chromosome conformation analyses in the leukemic cells reveal the formation of a novel chromatin loop that brings the activated N-Me SE into close spatial proximity with the Myc promoter, enabling oncogenic Myc transcription. These findings uncover a novel mechanistic pathway by which β-catenin and HEB interact to reshape the enhancer landscape and three-dimensional chromatin architecture, ultimately promoting SE duplication and aberrant Myc expression in T-ALL. β-catenin appears to play a dual role, first facilitating HEB recruitment under physiological conditions and then, upon HEB loss, permitting the reprogramming of enhancer activity through chromatin opening and ectopic transcription factor binding. Our data suggest that the cooperative interplay between signaling pathways and transcriptional regulators at enhancer elements can initiate structural genomic changes that drive malignancy. This work provides a long-sought preclinical model that faithfully mirrors key features of human T-ALL. Our findings open new avenues for understanding how non-coding regulatory mutations arise and exert oncogenic effects and have the potential to provide insights that may extend beyond T-ALL to other cancers driven by enhancer dysregulation. Moreover, by revealing molecular intermediates such as β-catenin, HEB, and TCF-1 in the regulation of enhancer activity, our findings identify potential therapeutic targets to disrupt the oncogenic enhancer-promoter interactions that underlie Myc-driven leukemogenesis.