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F. Pozzo et al.
reduction of CD20 expression in NOTCH1-mutated CLL cells could be due to a NOTCH1 mutation-driven epige- netic dysregulation involving histone deacetylases.5,13 Although genetic mutations represent the main contribu- tor to the aberrant activation of the NOTCH1 pathway in CLL,14 a mutation-independent activation was also report- ed for a significant fraction of NOTCH1-unmutated cases.15 In that setting, however, nothing is known regard- ing the mechanism(s) behind the activation of NOTCH1 and the concomitant modulation of CD20 expression.
Another recurrently mutated gene in CLL is the RNA splicing factor 3b subunit 1 (SF3B1), found mutated in about 10% CLL cases.16-19 SF3B1 is a key component of the splicing machinery, responsible of recognizing the branch-point sequences in proximity of the 3′ splice site (acceptor site) allowing intron removal from precursor messenger RNA. Mutations are predicted to alter the pro- tein’s tertiary structure, hampering the correct high-affini- ty recognition of the branch-point sequences and resulting in the selection of alternative 3ʹ splice sites. This leads to aberrantly spliced transcripts, gain/change/loss-of-func- tion variants, novel stop codons or downregulation of gene expression through nonsense-mediated decay.20-23
In CLL, mutations of SF3B1 have been shown to induce transcriptome-wide alterations of splicing patterns, result- ing in an increased frequency of alternative 3′ splice site selection, with functional consequences on several path- ways such as DNA damage, telomere maintenance and, possibly, NOTCH1 signaling,18,19,23,24 although the actual impact of SF3B1 mutations in the pathobiology of CLL remains to be fully elucidated.
In the present study, by taking advantage of a large cohort of primary CLL cases, we demonstrated that SF3B1-mutated CLL have features of NOTCH1 pathway activation and NOTCH1-dependent CD20 downregula- tion.
Methods
Primary chronic lymphocytic leukemia cells
This study is part of a comprehensive CLL characterization approved by the Internal Review Board of the Centro di Riferimento Oncologico di Aviano (approval n. IRB-05-2010 and IRB-05-2015) upon informed consent in accordance with the Declaration of Helsinki, and included peripheral blood samples from 537 CLL patients (Online Supplementary Table S1).1,25 Primary CLL cells were separated by Ficoll-Hypaque (GE Healthcare, Uppsala, Sweden) density gradient centrifugation and either used directly or cryopreserved until use. All studies were performed on highly purified cells (>95% pure). CLL cases were characterized for IGHV mutational status, the main cytogenetic abnormalities, CD49d expression and mutational status of TP53 as described elsewhere.26,27 In 382/537 cases, time-to-first-treatment (TTFT) data were available (median TTFT 30 months, 95% confidence interval: 27-34 months).
Next-generation sequencing
Mutational status of NOTCH1 and SF3B1 was assessed by next-generation sequencing. An amplicon-based strategy, with at least 2000X coverage, was used for NOTCH1, covering the whole of exon 34 and part of the 3′ untranslated region.9 SF3B1 muta- tional status was assessed using the Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA) covering exons 10 to 17. For the purpose of the present study, CLL samples were considered
mutated for NOTCH1 and SF3B1 if the variant allele frequency (VAF) exceeded 1%9 or 5%, respectively; in the presence of con- current mutations, cases were assigned to the SF3B1-mutated cat- egory.
Evaluation of CD20 expression
CD20 expression was investigated in the whole cohort by flow cytometry, using an anti-CD20 PE-Cy7 antibody (clone L27, BD Biosciences, Milan, Italy) in the neoplastic CD19+/CD5+ and in the normal CD19+/CD5- compartments. Samples were acquired on a FACSCanto II cell analyzer calibrated with CS&T calibration beads (BD Biosciences) and processed with FACSDiva (BD Biosciences) or FlowJo software (FlowJo LLC, Ashland, OR, USA). A complementary analysis of CD20 distribution was implement- ed as follows: (i) a linear gate (P1) spanning from the peak value of the histogram and comprising the whole CD20bright population was defined; and (ii) the percentage of the P1-gated population was subtracted from the residual (100-%P1) population.28
Gene expression profiling
For gene expression profiling (GEP) experiments, total RNA was labeled, hybridized on oligonucleotide microarray slides (SurePrint G3 Human GE v2 8x60K) and analyzed as previously described.29 Microarray data are available at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession num- ber GSE137024.
Statistical analyses
Statistical analyses were performed using Medcalc (Medcalc Software Ltd., Ostend, Belgium) and R (www.r-project.org). Data were compared using a two-sided Mann-Whitney rank-test and are presented with Tukey box-and-whisker plots. For correlation analyses, the Spearman rho correlation coefficient was employed. A P-value smaller than 0.05 was considered statistically significant and is represented with asterisks: *P≤0.05, **P≤0.01, ***P≤0.001.
Further details are provided in the Online Supplementary Methods. Results
SF3B1 and NOTCH1 mutational status in chronic lymphocytic leukemia
In a cohort of 537 unselected CLL cases, we investigated the mutational status of SF3B1 and NOTCH1 by next- generation sequencing. SF3B1 mutations were present in 48/537 cases (8.9%), with a VAF ranging from 5.0 to 53.0% (mean 32.0%) (Online Supplementary Table S1) and all clustered in the previously reported canonical hotspots.16,19,23 The residues surrounding Lys700 were the most frequently mutated (23/48, 48%), followed by Gly742 (13/48, 27%) (Figure 1A). Multiple mutations were rare, with one case having two concomitant mutations and one case having three of them (cases #3647 and #3301, respectively) (Online Supplementary Table S1). SF3B1 mutations were associated with TP53 mutations (c2 P=0.0313) and with unmutated IGHV status (P=0.0006), but not with specific IGHV alleles. At differ- ence from previous reports,30,31 SF3B1 mutations did not show significant associations with classical chromosomal abnormalities (Online Supplementary Table S2).
NOTCH1 mutations were present in 89/537 cases (16.6%) and concomitant mutations were more common, with 19 cases (19/89, 21.0%) bearing more than one lesion, increasing the overall mutation frequency to 21.6% (116 mutations in 537 cases). The VAF ranged from 1.6 to
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