Exploring the functional role of EZH2-dependent autocrine signaling in T-cell leukemia
Worldwide Cancer Research
Casa Sollievo della Sofferenza
BACKGROUND: T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) is a malignancy with refractory/relapsed cases, presumably due to the ineffective targeting of leukemia initiating cells (LICs). DNA mutations, leading to the hyperactivation of Notch1 signaling, are found in >50% of human T-ALL. NOTCH1 binding induces the eviction of PRC2, which promotes the repressive H3K27me3 chromatin modification. Moreover, we observed that in NOTCH1-induced mouse T-cell leukemias, the shRNA-mediated knockdown of EZH2 (a functional component of PRC2) stimulates cell growth/survival by inducing an autocrine IGF-like signaling, critical for LIC activity.  OBJECTIVE/HYPOTHESIS: This proposal intends to characterize the functional role of EZH2 in the NOTCH1-driven malignant transformation of human cells and to define the EZH2-dependent autocrine signaling involved in T-ALL pathogenesis. We hypothesize that the silencing of EZH2 modulates functional pathways of T-cell transformation and LIC activity underlying the different outcomes of relapsed/recurrent leukemias.  SPECIFIC AIMS: Determine whether the loss of EZH2 promotes the malignant transformation of hematopoietic stem/progenitors(HSPCs) from human cord blood; Characterize the EZH2-dependent autocrine signaling in human NOTCH1-induced HSPCs; Define the functional role of EZH2 in the LIC activity of human T-ALL.     STUDY DESIGN: We propose to use novel models of human T-ALL in which HSPCs from cord blood are transformed into clonal T-ALL-like malignant cells, after transduction with lentiviral vectors encoding NOTCH1-ΔE (a constitutively active NOTCH1 variant) alone or in combination with three known T-ALL oncogenes, and subsequent injection into immunocompromised mice. We will perform gain/loss-of-function assays to assess the EZH2 contribution to leukemogenesis and LIC-related phenotypes. We will employ ELISA/Luminex analyses, combined with RNA-Seq, ChIP-Seq and TaqMan gene expression assays for characterizing the EZH2-dependent autocrine signaling. Conventional EZH2 inhibitors and patient-derived xenograft(PDX) leukemias will be also included.  SIGNIFICANCE: This study will outline relevant pathways of leukemia maintenance, development and chemoresistance and provide rationale for drug design by identifying novel molecular targets.
2.1 Biological and endogenous factors