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SF3B1 mutations and NOTCH1 activation in CLL
repress the promoter activity (P=0.0244 and P=0.96, respectively) (Online Supplementary Figure S7A), in agree- ment with previous observations23 and consistent with a defective inhibitory role of altDVL2 against the NOTCH1- dependent transcriptional machinery. Similarly, after depletion of wild-type DVL2 by siRNA transfection in the SF3B1-wild-type CLL-like MEC1 cell line, NOTCH1 sig- naling was found to be increased, as demonstrated by HES1 upregulation (P=0.0176) at 24 h; in addition, CD20 expression was reduced at both protein (P=0.0062) and transcript (P=0.0129) levels (Figure 5A). Taken together, these data corroborate the inhibitory role of DVL2 on the NOTCH1 pathway, counteracted by the presence of its alternate splicing isoform.
In agreement with this observation, SF3B1-mutated CLL cases also showed high expression of the NOTCH1 target gene DTX1 (median SF3B1-mutated 0.0029, median WT 0.0013, P=0.0089; rho=0.369, P=0.0348), similar to NOTCH1-mutated cases (median NOTCH1-mutated 0.0033, median WT 0.0013, P=0.002) (Figure 5B). Notably,
in SF3B1-mutated cases, the levels of DTX1 correlated directly with the levels of altDVL2, in keeping with the hypothesis of elevated NOTCH1 signaling in the presence of a less functioning alternatively spliced DVL2 (Figure 5C).
In turn, the expression of DTX1 correlated with reduced CD20 expression; as summarized in Figure 5D and Online Supplementary Figure S7B. SF3B1-mutated and NOTCH1-mutated CLL cases showed a progressively enriched double-positive CD20dim/DTX1 population com- pared to WT cases (WT vs. SF3B1-mutated, P=0.024; WT vs. NOTCH1-mutated, P<0.001; SF3B1-mutated vs. NOTCH1-mutated, P=0.362; c2 test for proportions) (Online Supplementary Figure S7B). Accordingly, by per- forming cell sorting on nine SF3B1-mutated CLL cases, all with clonal SF3B1 mutational burden (VAF 10-50%), we isolated two populations characterized by either high or low CD20 protein and transcript expression. Evaluation of altDVL2 by next-generation sequencing in the two sub- fractions revealed an increased frequency of the alterna-
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CD
Figure 4. SF3B1 mutations induce alternative splicing of DVL2. (A) Representation of the DVL2 splicing events determine from next-generation sequencing analysis; arches indicate wild-type (blue) and altered (red) splicing, black bars indicate coverage; the position of primers and the probe for quantitative reverse transcriptase polymerase chain reaction analysis is also reported. (B) Expression of DVL2 alternative splicing (altDVL2) in unmutated (WT, n=155), SF3B1-mutated (SF3B1, n=35), or NOTCH1-mutated (NOTCH1, n=32) cases of chronic lymphocytic leukemia. Data are shown by Tukey box and whisker plots. Outliers indicate data outside the 1.5 interquartile range. ***P≤0.001, n.s. not significant, as determined by a two-sided Mann-Whitney rank-test. (C) Correlation of altDVL2 expression with SF3B1 muta- tional burden. VAF: variant allele frequency. The Spearman rank correlation coefficient (rho) and P-value are reported. (D) altDVL2 expression in relation to SF3B1 mutations according to the mutational hotspot and burden (n=35). WT cases (n=155) are reported for comparison. Diamonds indicate SF3B1-mutated cases with a VAF <10%. Horizontal marks represent the median.
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