Abstract NG05: Reading epigenetic landscapes: New mechanisms and therapeutic opportunities.

Abstract

Abstract Our genome is decorated with diverse chemical modifications on histone proteins that package the DNA. These modifications constitute an essential layer of information (referred to as the epigenome) above the DNA sequence to regulate genome architecture and function. It has been appreciated that histone modifications play critical roles in biological processes that are fundamental to normal development and human disease, yet how these modifications are interpreted and translated to downstream molecular events remains poorly understood. We recently discovered the ENL protein as a novel ‘reader' for histone acetylation, a chromatin mark that is generally associated with gene activation. We showed that loss of ENL induced differentiation of leukemia stem cells and inhibited leukemia maintenance in animal models. Mechanistically, ENL binds to histone acetylation via an evolutionarily conserved yet poorly understood protein domain called the YEATS domain. By determining the structure of ENL in complex with an acetylated histone peptide, we demonstrated that disrupting this interaction reduced the recruitment of ENL-associated transcriptional machinery to target genes, leading to the suppression of oncogenic programs. This study established ENL as a missing molecular link between histone acetylation and gene activation.More recently, somatic mutations in the ENL gene (MLLT1) were found in about 5 % of Wilms' tumor, making ENL one of the top genes mutated in this cancer type. These mutations are recurrent, heterozygous, and highly clustered in the ENL YEATS domain. Interestingly, these ‘hotspot' mutations all involve either small in-frame insertions or deletions. Whether and how such ENL mutations promote the formation of Wilms' tumor was unclear and the focus of our current study. Here we show that these mutations impair cell fate regulation through conferring gain of function in chromatin recruitment and transcriptional control. ENL mutants induced gene expression changes that are involved in renal differentiation and tumorigenesis. When tested in a nephrogenesis assay, ENL mutations resulted in undifferentiated blastema structures resembling those observed in human Wilms' tumor. Mechanistically, whole genome analysis showed that while mutant ENL bound to largely similar genomic loci as the wildtype, it exhibited enhanced occupancy at a subset of targets. This led to a marked increase in the recruitment and activity of transcription elongation machinery that drives aberrant upregulation of key target genes such as HOXA genes. Furthermore, ectopically expressed ENL mutant exhibited enhanced self-association and formed discrete nuclear puncta. These puncta harbor characteristics of recently described phase separation-driven biomolecular condensates in other biological contexts, an extreme form of local high-concentration hubs mediated by weak and dynamic multivalent interactions. We further dissected different regions within the ENL protein that contribute to mutation-driven self-association, and demonstrated that this increased self-association is indeed functionally linked to enhanced chromatin occupancy and gene activation. Together, this work is a remarkable demonstration of how dysregulation of chromatin reader-mediated process can act as a driving force for tumor formation. Our findings represent, to our knowledge, the first discovery that cancer-associated mutations in a chromatin reader reinforce recruitment by promoting self-association, revealing a previously unrecognized mechanism for gene control. ENL mutations are also the first example of cancer-associated mutations conferring a neomorphic property in forming biomolecular condensate-like interaction hubs, thus potentially linking this newly recognized form of biomolecular interaction to gene regulation in human cancers and opening many outstanding questions to address in the future. Together, our previous and current work uncovered a novel protein ‘reader' through which a cell interprets the epigenetic information, and revealed new mechanisms by which cancer cells hijack this process to derail normal cell fate control and fuel the initiation and growth of the tumor. These studies not only fill in an important gap in our basic understanding of chromatin regulation and how these mechanisms underlie human cancer, but also yield new therapeutic strategies for exploiting vulnerabilities of cancer cells. Ongoing efforts are aimed at developing small molecules inhibitors of ENL as valuable research tools and potential therapeutic strategies in aggressive leukemia as well as ENL mutation-driven malignancies. Citation Format: Liling Wan. Reading epigenetic landscapes: New mechanisms and therapeutic opportunities. [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr NG05.