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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10990/591

Autori: Pozzo, Federico
Supervisore afferente all'Università: PUCILLO, CARLO ENNIO MICHELE
Titolo: NOTCH1 mutations are associated with low CD20 expression in Chronic Lymphocytic Leukemia: evidences for a NOTCH1-mediated epigenetic regulatory mechanism
Abstract (in inglese): Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with highly variable clinical courses and survivals ranging from months to decades. Recently, it has been reported that stabilizing mutations of NOTCH1 are recurrently associated with CLL, being identified in about 10% of CLL at diagnosis and with higher frequencies in chemorefractory CLL, CLL in advanced disease phases, and in Richter Syndrome. All NOTCH1 mutations disrupt the C-terminal PEST domain, causing an accumulation of a stabilized NOTCH1 isoform. By taking advantage of a large retrospective cohort of CLL cases (n=463), for a subset of which clinical data was available, we confirm that NOTCH1 mutations behave as independent prognosticator, identifying a high-risk subset characterized by unfavourable prognosis and poor overall survival. We further demonstrate that the presence of NOTCH1 mutations also identifies a CLL subset which does not benefit from addition of rituximab in the context of a maintenance therapy after first-line treatment with fludarabine. These results are in keeping with recently published data, reporting that the NOTCH1 mutated subset of CLL patients does not benefit from the addition of rituximab to chemotherapeutic treatment with fludarabine plus cyclophosphamide. As the reasons for this different clinical behaviour remained to be elucidated, considering that the response to rituximab treatment in B cell neoplasms directly depends upon CD20 expression, we investigated whether NOTCH1 mutations could affect CD20 expression in CLL. By taking advantage of a wide CLL series (n=692), we demonstrated that NOTCH1 mutated CLL cells (87/692) were characterized by lower CD20 expression and lower relative lysis induced by rituximab in-vitro. Consistently, CD20 expression by CLL cells was up-regulated in-vitro by exposure to γ-secretase inhibitors, and the stable transfection of the NOTCH1 intracellular domain (NICD) into CLL-like cells resulted in a strong downregulation of both CD20 protein and transcript. By using these NICD transfectants, we investigated the protein interactions of RBPJ, a transcription factor acting either as activator or repressor of NOTCH1 pathway when respectively bound to NICD or histone deacetylases (HDACs). Compared to controls, NICD transfectants had RBPJ preferentially complexed to NICD, and showed higher levels of HDACs interacting with the promoter of the CD20 gene. Finally, treatment with the HDAC inhibitor valproic acid upregulated CD20 in both NICD transfectants and primary CLL cells. In conclusion, NOTCH1 mutations are associated with low CD20 levels in CLL and are responsible for a dysregulation of HDAC-mediated epigenetic repression of CD20 expression. In conclusion, in the present thesis we i) confirm that NOTCH1 mutations are an independent prognosticator of overall survival in CLL; ii) identify a CLL subset, characterized by the presence of NOTCH1 mutations, that do not benefit from addition of rituximab to chemotherapeutic treatment; iii) provide a proof of concept that NOTCH1 mutations responsible for a truncated NOTCH1 protein are associated with low CD20 expression levels in CLL by a dysregulated HDAC-dependent repression mechanism. This low CD20 levels may be, in turn, responsible for the specific immunoresistance to rituximab-based treatments, such as FCR, of NOTCH1 mutated CLL.
Parole chiave: Immunoresistance; NOTCH1; Chronic Lymphocytic Leukemia
MIUR : Settore BIO/11 - Biologia Molecolare
Lingua: eng
Data: 10-apr-2015
Corso di dottorato: Dottorato di ricerca in Scienze biomediche e biotecnologiche
Ciclo di dottorato: 27
Università di conseguimento titolo: Università degli Studi di Udine
Luogo di discussione: Udine
Altre informazioni: Co-supervisori: Valter Gattei, Michele Dal Bo - Struttura di aggregazione: Unità di Oncoematologia Clinico-Sperimentale, Centro di Riferimento Oncologico, Aviano (IT)
Citazione: Pozzo, F. NOTCH1 mutations are associated with low CD20 expression in Chronic Lymphocytic Leukemia: evidences for a NOTCH1-mediated epigenetic regulatory mechanism. (Doctoral Thesis, Università degli Studi di Udine, 2015).
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