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Background. In chronic lymphocytic leukemia (CLL), NOTCH1 mutations associate with clinical resistance to anti-CD20 immunotherapy in FCR combination (Stilgenbauer et al., Blood, 2014, Dal Bo et al., AHO, 2014), that can be ascribed to a NOTCH1 mutation-driven repression of CD20 levels by HDACs (Pozzo et al., Leukemia, 2016). Recently, novel recurrent mutations have been identified in the 3'untranslated region of NOTCH1 (3'UTR NOTCH1 mutations), determining a novel splicing event within the last NOTCH1 exon (Puente et al., Nature, 2015), leading to an impaired degradation of NOTCH1 protein. Aim. To determine if 3'UTR NOTCH1 mutations associate with low CD20 levels. Methods. NOTCH1 mutations were screened by next-generation sequencing (NGS) in exon 34 and part of 3'UTR with at least 1000X coverage. NOTCH1 (transmembrane, TM, or intracytoplasmic domain, NICD) protein levels were investigated by western blot (WB). CD20 expression was investigated by flow cytometry with a FITC-conjugated anti-CD20 antibody (L27 clone). MS4A1 (encoding for CD20 protein) transcript levels were investigated by qRT-PCR. Susceptibility to anti-CD20 rituximab and ofatumumab was investigated by complement-dependent cytotoxicity (CDC) assay. NOTCH1 signaling was inhibited by gamma-secretase inhibitor (GSI). Results. i) NOTCH1 mutational screening. In 649 CLL, NOTCH1 mutations were detected in 115 cases (17.72%), overall accounting for 127 mutations (73 c.7541-7542delCT, 11 other frameshift, 17 nonsense, 1 missense, and 25 3'UTR mutations). For analysis purposes, the 115 mutated cases were subdivided in cases with NOTCH1 coding mutations (coding NOTCH1-mut, 90 cases) and cases with 3'UTR NOTCH1 mutations (3'UTR NOTCH1-mut, 25 cases). Four cases with concomitant 3'UTR NOTCH1 mutation and coding NOTCH1 mutation were assigned to the 3'UTR group according to the substantially higher mutational burden detected for the 3'UTR mutation. ii)NOTCH1protein expression. In keeping with alternative splicing events causing an impaired NOTCH1 protein degradation, 3'UTR NOTCH1-mut cases showed higher levels than NOTCH1 wild-type (NOTCH1-wt) cases of both NOTCH1 TM and NICD by WB, and similar to coding NOTCH1-mut cases (Fig. 1a). iii) CD20 expression levels. According to FISH classification, variable CD20 levels were found by flow cytometry, with the highest levels in trisomy 12 CLL. Of note, 3'UTR NOTCH1-mut cases expressed lower CD20 levels than NOTCH1-wt cases in both trisomy 12 CLL (mean MFI in 9 3'UTR NOTCH1-mut cases = 2446.11 vs mean MFI in 66 NOTCH1-wt cases = 8254.20; p Conclusions.In keeping with an impaired NOTCH1 protein degradation, 3'UTR NOTCH1-mut cases had low CD20 expression levels, suggesting the introduction of 3'UTR NOTCH1 mutation evaluation in CLL patients undergoing anti-CD20 immuno-chemotherapy. Disclosures No relevant conflicts of interest to declare.

Venetoclax (ABT-199) is a small-molecule selective oral inhibitor of the antiapoptotic protein Bcl-2 that promotes programmed cell death of chronic lymphocytic leukemia (CLL) cells regulating the release of proapoptotic factors, such as Smac/Diablo, apoptosis-inducing factor (AIF) and cytochrome c. In April 2016, the U.S. Food and Drug Administration (FDA) granted accelerated approval to venetoclax for patients diagnosed with CLL with 17p deletion, as detected by an FDA-approved test, who have received at least one prior therapy. This review will focus on the mechanism of action, preclinical studies and clinical development of venetoclax both as a monotherapy and in combination with other drugs for CLL in the current milieu of therapy dominated by novel tyrosine kinase inhibitors such as ibrutinib and idelalisib.

Background: stabilizing mutations of NOTCH1 have been identified 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; Stingelbauer et al, Blood, 2014). In CLL, all NOTCH1 mutations disrupt the C-terminal PEST domain (about 80% of which are a 7544-7545delCT frameshift deletion) and cause an accumulation of an active NOTCH1 isoform. Aim: to identify molecular/biological features of NOTCH1 mutated CLL. Methods: the presence of NOTCH1 mutations was investigated by ARMS-PCR or by direct sequencing. The percentage of NOTCH1 mutated DNA in the context of the CLL clone was determined by quantitative real-time PCR (QRT-PCR) and/or by next-generation-sequencing. Gene expression profile (GEP) was performed by a one-color labeling strategy using the 4x44K platform. Specific gene/protein validations were performed by QRT-PCR, western blotting, flow cytometry and immunofluorescence. Cell proliferation was evaluated by Cell Trace Violet assay. CLL-like cell line MEC-1 was transfected with a vector either with a NOTCH1 intracellular domain (NICD) with 7544-7545delCT or with a NICD carrying a missense mutation (c.5304G>A), generating a stop codon at the beginning of the sequence, as control. Results:i) in a cohort of 588 IGHV-UM CLL, 144/588 (24.5%) cases carried NOTCH1 mutations (NOTCH1-mut cases), with a percentage of NOTCH1 mutated DNA ranging from 1% to 37%. NOTCH1-mut cases (8 cases; 11%-37% of NOTCH1 mutated DNA) showed higher NOTCH1 protein expression than NOTCH1 wild type cases (NOTCH1-wt, 11 cases) employing monoclonal antibodies either recognizing the trans-membrane (mean fold-change=3.0) or the intracellular (mean fold-change=2.1) NOTCH1 domain. ii) A GEP comparing purified cells of 5 IGHV-UM NOTCH1-mut cases (15%-37% of NOTCH1 mutated DNA) and 5 IGHV-UM NOTCH1-wt cases selected nucleophosmin-1 (NPM1) and genes codifying for several ribosomal proteins (RPS6, RPS10, RPS17, RPS28, RPSA, RPL7A, RPL18) as significantly up regulated in NOTCH1-mut CLL. A higher expression of NPM1 and of the genes codifying for 4 ribosomal proteins in NOTCH1-mut cases was validated in a wider independent series of 34 cases by QRT-PCR (18 NOTCH1-mut cases).iii) Western blot in 19 cases (8 NOTCH1-mut cases) confirmed a higher NPM1 protein expression in NOTCH1-mut cases (1.3-5.2 range of fold-change) also at protein level. When intracellular distribution and fluorescent intensity of NPM1 immunostaining were evaluated, an additional cytoplasmic staining was visible in NOTCH1-mut cases, along with a clear nucleolar staining visible in both NOTCH1-mut and NOTCH1-wt cases. NPM1 protein expression was also determined by an intra-nuclear staining by flow cytometry. By using this approach, 2 NOTCH1-mut cases were sorted according to NPM1 expression; notably, the NPM1high subpopulation showed a higher NOTCH1 mutational load than the NPM1low subpopulation. iv) MEC-1 cells transfected with mutated NICD showed higher NOTCH1 transcript and protein levels than MEC-1 cells transfected with the control vector (increments over the control: NICD transcript =2.2, NICD protein =1.3). These NICD mutated MEC-1 cells were characterized by higher NPM1 transcript and protein levels than the MEC-1 cells transfected with the control vector (increments over the control: NPM1 transcript =2.0, NPM1 protein =1.5). By performing a cell-trace-violet proliferation assay, NICD mutated MEC-1 cells were characterized by higher proliferation rates than MEC-1 cells transfected with the control vector (p=0.018). Finally, when the transfected MEC-1 cells were treated with 0.5 microM etoposide for 48 hours, NICD mutated MEC-1 cells presented higher viability rates than the MEC-1 cells transfected with the control vector, as evaluated by Annexin V/7-AAD staining (percentage of viable cells: 70% vs. 50%). Conclusions: NPM1 was constitutively overexpressed in NOTCH1-mut IGHV-UM CLL together with several ribosome-associated components. An increased activity of the ribosomal machinery/DNA-repair mechanisms (Lindstrom MS. Biochem.Res.Int. 2011) in NOTCH1-mut CLL can confer proliferative advantages and resistance to chemotherapeutic agents. Disclosures No relevant conflicts of interest to declare.