Mapping and targeting 3D regulatory elements in leukemia and lymphoma - PROJECT SUMMARY/ABSTRACT Despite cancer being typically depicted as a genetic disease, aberrations in epigenetics and gene expression play a determinant role in transformation and response to therapies. Derailed from their developmental paths, malignant cells may arrest their differentiation fate and activate alternative programs. Aberrant chromatin plasticity gives a survival advantage by expanding the repertoire of epi-clones and allowing them to react to the immune system, environmental stress, or exogenous treatments. Investigating which genetic and epigenetic aberrations alter chromatin homeostasis and how they shape tumor heterogeneity is thus a critical challenge to designing effective targeted approaches and predicting disease risk. Our laboratory dedicated extensive efforts to trying to understand the non-genetic drivers of leukemia, focusing on epigenetic modifications, long non- coding RNAs, and the study of 3D chromatin architecture. We previously investigated changes in large 3D structures in T-cell acute lymphoblastic leukemia (T-ALL), including chromosomal compartments (A vs B) and topological associated domains (TADs). More recently, using H3K27ac HiChIP analysis of enhancer-promoter interactions in T-ALL, we mapped and characterized the biological role of 3D “hubs” as DNA elements that interact with multiple other loci. We hypothesized that hubs represent regulatory units responsible for the transcription of key genes, serving as 'headquarters' of cell identity during development but also coordinating the tumorigenic program and shaping therapy responses. What we propose in this application is to expand our analysis and provide a complete characterization of 3D hub interactions in both immature T cell malignancies (T-ALL) and mature T cell neoplasms (focusing on T cell lymphoma), creating a continuum that spans normal T cells, their progenitors and malignant counterparts at distinct stages of differentiation. Our work will highlight how nuclear topologies and related gene networks evolve during T-cell development or get rewired to promote malignancy, providing new candidates to target therapeutically and deriving associations with clinical outcomes. This is of particular interest for T cell lymphoma, an aggressive and heterogeneous disease that represents a clinical emergency, given the lack of robust targeted therapies or immunotherapies. By leveraging the mapping of 3D hubs at multiple levels, we expect to define better DNA elements that are rewired by transformation and contribute to tumor progression or response to drug treatment. In this grant, we propose to a) extensively map 3D hubs in primary PTCL human specimens, b) perform CRISPRi and CRISPRko screens to identify biologically important hubs, c) characterize hub heterogeneity at the single cell level, and d) target them using both genetic and pharmacological tools. These experiments will test the hypothesis that 3D DNA hubs are elements that control transformation, tumor progression, and response to drug treatment.
