Dottorato in BIOLOGIA UMANA E GENETICA MEDICA

Titolo della tesi: The functional role of EZH2 inactivation in the pathogenesis of T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a hematological malignancy, characterized by frequent loss-of-function mutations in EZH2 gene. EZH2 is a histone-lysine N-methyltransferase enzyme and in mouse, its inactivation leads to the initiation and progression of T-cell leukemia. Here, we characterized the in vitro phenotypes associated with a long-term expansion of human CD34+ hematopoietic stem and progenitor cells (HSPCs) in response to CRISPR/Cas9-mediated inactivation of EZH2 alone or in combination with the constitutive expression of known T-ALL oncogenes after lentiviral transduction. It is known that early T cell leukemias with inactivating EZH2 mutations are closely associated with the upregulation of HOXA9 and LYL1 oncogenes. In our in vitro model EZH2 knockout in LYL1 or HOXA9-transduced HSPCs, expanded in vitro up to 31 days, exhibited an increased leukemia clonogenic capacity and promoted an enrichment of early progenitor T cells at the double negative 1 (DN1) development stage. Furthermore, EZH2-null LYL1 and EZH2-null HOXA9-transduced cells demonstrated high penetrance leukemia in vivo experiments. RNA-seq data from ETP-ALL-like LOUCY cells revealed that EZH2 knockout overexpressing HOXA9 or LYL1 resulted in a gene expression signature of immature T-ALL, stem/immature immunophenotypic stage, increased replication status, and enhanced dependency on the inflammation response. Lastly, RNA-seq data analysis from a large pediatric cohort of T-ALLs revealed that patients with Loss of Function (LoF) mutations in EZH2 gene shared a stem cell-associated transcriptional program and were statistically enriched for the immature HOXA cluster and ETP subtype in human T-ALL leukemia.