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Molecular targets for therapy

NOTCH1 mutations associate with low CD20 level in chronic lymphocytic leukemia: evidence for a NOTCH1 mutation-driven epigenetic dysregulation

Abstract

In chronic lymphocytic leukemia (CLL), NOTCH1 mutations have been associated with clinical resistance to the anti-CD20 rituximab, although the mechanisms behind this peculiar behavior remain to be clarified. In a wide CLL series (n=692), we demonstrated that CLL cells from NOTCH1-mutated cases (87/692) were characterized by lower CD20 expression and lower relative lysis induced by anti-CD20 exposure in vitro. Consistently, CD20 expression by CLL cells was upregulated in vitro by γ-secretase inhibitors or NOTCH1-specific small interfering RNA and the stable transfection of a mutated (c.7541-7542delCT) NOTCH1 intracellular domain (NICD-mut) into CLL-like cells resulted in a strong downregulation of both CD20 protein and transcript. By using these NICD-mut transfectants, we investigated 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 with controls, NICD-mut 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-mut 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.

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Acknowledgements

This study was supported in part by the Associazione Italiana Ricerca Cancro (AIRC), Investigator Grant IG-13227; Progetto Ricerca Finalizzata I.R.C.C.S. n. RF-2009-1469205, n. RF-2010-2307262, Progetto Giovani Ricercatori n. GR-2009-1475467, n. GR-2010-2317594, n. GR-2011-02347441, n. GR-2011-02346826, Ministero della Salute, Rome, Italy; Fondazione Cariplo (grant 2012-0689); Associazione Italiana contro le Leucemie, linfomi e mielomi (AIL), Venezia Section, Pramaggiore Group, Italy; Fondazione per la Vita di Pordenone, Italy; Ricerca Scientifica Applicata, Regione Friuli Venezia Giulia (‘Linfonet’ Project), Trieste, Italy; and ‘5x1000 Intramural Program’, Centro di Riferimento Oncologico, Aviano, Italy. FA is supported by a Beat-Leukemia fellowship.

Author Contributions

FP contributed to write the manuscript, analyzed the data and performed the research. TB performed the research. FA, PB, PM, ET, BG, FMR, RB, AZ, DB and MD contributed to perform the research. GDA, AC, FZ, GP, DR, GG., GDP and SD provided well-characterized biological samples and contributed to write the manuscript. VG and MDB designed the study, interpreted data and wrote the manuscript.

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Correspondence to V Gattei or M Dal Bo.

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Pozzo, F., Bittolo, T., Arruga, F. et al. NOTCH1 mutations associate with low CD20 level in chronic lymphocytic leukemia: evidence for a NOTCH1 mutation-driven epigenetic dysregulation. Leukemia 30, 182–189 (2016). https://doi.org/10.1038/leu.2015.182

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