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.

Purpose: In chronic lymphocytic leukemia (CLL), TP53 mutations are associated with reduced survival and resistance to standard chemoimmunotherapy (CIT). Nevertheless, the clinical impact of subclonal TP53 mutations below 10% to 15% variant allele frequency (VAF) remains unclear. Experimental Design: Using a training/validation approach, we retrospectively analyzed the clinical and biological features of TP53 mutations above (high-VAF) or below (low-VAF) the previously reported 10.0% VAF threshold, as determined by deep next-generation sequencing. Clinical impact of low-VAF TP53 mutations was also confirmed in a cohort (n = 251) of CLL treated with fludarabine-cyclophosphamide-rituximab (FCR) or FCR-like regimens from two UK trials. Results: In the training cohort, 97 of 684 patients bore 152 TP53 mutations, while in the validation cohort, 71 of 536 patients had 109 TP53 mutations. In both cohorts, patients with the TP53 mutation experienced significantly shorter overall survival (OS) than TP53 wild-type patients, regardless of the TP53 mutation VAF. By combining TP53 mutation and 17p13.1 deletion (del17p) data in the total cohort (n = 1,220), 113 cases were TP53 mutated only (73/113 with low-VAF mutations), 55 del17p/TP53 mutated (3/55 with low-VAF mutations), 20 del17p only, and 1,032 (84.6%) TP53 wild-type. A model including low-VAF cases outperformed the canonical model, which considered only high-VAF cases (c-indices 0.643 vs. 0.603, P < 0.0001), and improved the prognostic risk stratification of CLL International Prognostic Index. Clinical results were confirmed in CIT-treated cases (n = 552) from the retrospective cohort, and the UK trials cohort. Conclusions: TP53 mutations affected OS regardless of VAF. This finding can be used to update the definition of TP53 mutated CLL for clinical purposes.

Key Points CD49d, a negative prognosticator with a key role for microenvironmental interactions in CLL, is near universally expressed in trisomy 12 CLL. CD49d overexpression in trisomy 12 CLL is regulated by a methylation-dependent mechanism.

functional annotation was done by gene set enrichment analysis (GSEA). Results: Genes downregulated after ETS1 silencing by siRNAs in 5 ABC DLBCL cell lines (TMD8, HBL-1, U2932, OCI-Ly10, SU-DHL-2) belonged to the signaling of BCR (NES 2.45 FDR <0.001), CD40 (NES 2.67 FDR <0.001), and NFKB/TNFA (NES 2.03 FDR 0.01), as well as to inflammatory response (NES 2.14 FDR 0.005) and differentiation (NES 2.5 FDR <0.001). Three candidate genes were selected for further studies according to the following educated guess criteria: i) potential biologic role; and ii) presence of binding sites for ETS factors in their promoter regions. HCST, RGS1 and FAIM3 were confirmed to be downregulated, by qPCR, also after silencing with shRNA. Their expression in clinical specimens was confirmed in two large series of 181 and 233 DLBCL cases, respectively. FAIM3 expression was higher in ABC DLBCL cases than germinal center type (GCB) DLBCL in both series (P < 0.0001), and a similar pattern was confirmed at protein level in cell lines derived from ABC (n = 6) or GCB DLBCL (n = 8). Conclusions: ETS1 positively regulates the expression of fundamental pathways in ABC DLBCL cells, including the BCR signaling. FAIM3, which codes for a high affinity IgM FC receptor overexpressed in chronic lymphocytic leukemia, appeared as a novel potential ETS1 transcription target, differentially regulated between ABC and GCB DLBCL, and requires additional investigation. Introduction: Stabilizing mutations of NOTCH1 occur in about 10% of chronic lymphocytic leukemia (CLL) cases at diagnosis, with a higher frequency in unmutated IGHV (IGHV-UM), immuno-chemorefractory or advanced disease phase CLL, and have been associated with particularly unfavourable prognosis (Rossi et al., Blood, 2012; Del Poeta et al., Br J Haematol, 2013; Stilgenbauer et al., Blood, 2014). In CLL, NOTCH1mutations cause accumulation of the active NOTCH1 iso-form, resulting in a sustained pathway activation. We therefore aimed at identifying molecular/biological features of NOTCH1 mutated (NOTCH1-mut) CLL. Methods: NOTCH1 mutations were investigated by NGS. Gene expression profile (GEP) was performed on a one-color 4x44K platform. Validations were performed by QRT-PCR, western blotting, flow cytometry. Cell proliferation was evaluated by CellTrace assay. Results: i) A GEP comparing 10 IGHV-UM CLL cases (5 NOTCH1-mut; 15%-37% of NOTCH1-mut alleles) selected nucleophosmin-1 (NPM1) and genes codifying for ribosomal proteins (RNPs) as up-regulated in NOTCH1-mut cases. Results were validated in an independent series of 188 cases (76 NOTCH1-mut). ii) Western blotting in 11 CLL cases (5 NOTCH1-mut) confirmed a higher NPM1 protein expression in NOTCH1-mut cases,with a direct correlation with NOTCH1 expression (r = 0.814). In NOTCH1-mut cases, the NPM1 high subpopulation, isolated by cell sorting, showed higher mutational load than the NPM1 low subpopulation. Iii) EDTA treatment of 12 CLL cases (6 NOTCH1-mut), activated NOTCH1 signaling, as by HES1 and DTX1 induction, and up-regulated NPM1 and othe...