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F. Pozzo et al.
tively spliced isoform in the CD20low fraction compared to the CD20high fraction in most cases (P=0.0078), paralleled by a higher expression of DTX1 (P=0.05).
Finally, when challenged in a rituximab-driven comple- ment-dependent cytotoxicity assay, CLL cells from either SF3B1-mutated or NOTCH1-mutated samples displayed a lower relative lysis compared to cells from WT samples (Online Supplementary Figure S7D), in agreement with the dose-dependent response of the assay from CD20 expres- sion levels (rho=0.745, P=0.0085).
Discussion
The CD20 molecule is one of the preferred therapeutic targets for CLL as testified by the widespread use of humanized anti-CD20 antibodies in different therapeutic regimens,2,41 although a dim expression of CD20 is among the peculiar features of CLL and part of the so-called Royal Marsden score for the differential diagnosis of CLL from other B-cell lymphoproliferative disorders.25,42 This notwithstanding, very little is still known about the bio- logical instances that define CD20 expression and deter- mine the stark difference between CLL cells and normal B cells, despite a number of mechanisms having been pro- posed. Among the many, a few stand out including BCR/CXCR4 signaling,43 NF-kB signaling and epigenetic/transcription factors such as histone deacety- lases.8,44
Another of these factors is the presence of mutations of the NOTCH1 gene,5 an emerging novel predictive factor for response to anti-CD20 immunotherapy in patients treated with immuno-chemotherapeutic combina- tions.11,12 In the present study, by taking advantage of a large cohort of CLL cases, we show that mutations of the SF3B1 gene represent another factor associated with a reduction of CD20 expression through a mutation-inde- pendent activation of the NOTCH1 pathway.
In our cohort, the mutational profile of SF3B1 was con- sistent with published data, with every mutation falling inside the canonical hotpots.16-19 SF3B1 mutations are con- sistently associated with an increased rate of aberrant splicing events throughout the transcriptome with varying intensity.23 By analyzing the relative abundance of differ- ent splicing variants, we invariably detected a strong increase of aberrantly spliced transcripts on multiple genes in SF3B1-mutated cases, independently of the location of the mutated residue and with a high correlation with the mutational burden. One of these genes was DVL2, whose alternatively spliced form, altDVL2, consisted in an in- frame loss of 24 amino acids within exon 11. DVL2 is a key mediator of the Wnt pathway and has demonstrated the ability to act as a negative regulator of the NOTCH1 pathway (Online Supplementary Figure S6A) by binding the NOTCH1-related transcription factor RBPJ.39 and/or the cleaved active form of NOTCH1 (NICD) itself.38
Through co-transfection experiments of exogenous DVL2 in a NOTCH1/RBPJ-dependent luciferase reporter system, we could confirm that the in vitro induction of DVL2 is capable of silencing NOTCH1 signaling, whereas the induction of altDVL2 did not result in proficient silenc- ing, as previously suggested.23 Here, we moved further to primary CLL cells and, using GEP, showed that SF3B1-mutated cases share a gene signature with NOTCH1-mutated cases which drove an unsupervised co-
clustering of the two categories, with respect to NOTCH1/SF3B1-unmutated cases. In addition, several NOTCH1-specific gene sets were enriched in SF3B1-mutated CLL, further suggesting an active commit- ment of the NOTCH1 transcriptional machinery in this CLL subset; importantly, one of these gene sets was a cus- tom gene set derived from a CLL-specific NOTCH1 gene signature.15 Consistently, the NICD was clearly detectable in protein lysates of SF3B1-mutated cases, with expres- sion levels often comparable to those observed in NOTCH1-mutated cases. The increased NOTCH1 signal- ing positively correlated with the expression of altDVL2 and with an elevated CD20dim fraction in SF3B1-mutated CLL cases. These data were corroborated by cell sorting experiments of cell fractions with different CD20 expres- sion levels in the context of SF3B1-mutated CLL cells, and by functional experiments of DVL2 knock-out in a SF3B1-wild-type CLL cell model.
While the NOTCH1 pathway has for long been recog- nized as active in CLL,14 only few mechanisms are known to explain its activation state. NOTCH1 mutations repre- sent the main but not the sole contributor, as there is evi- dence of active NOTCH1 signaling occurring in peripheral blood cells of a fraction of NOTCH1-unmutated CLL cases,15 although without a molecular explanation. Our present study may help to sort out at least a fraction of these cases, suggesting that the constitutively activated NOTCH1 pathway can indeed occur in the context of SF3B1-mutated CLL, possibly as the result of diminished repression of the dynamic association between NOTCH1 and RBPJ45 due to higher levels of the inefficient spliced form of DVL2.15,38,39
Wang et al.23 speculated that the numerous changes induced by SF3B1 mutations in the CLL transcriptome may allow neoplastic cells to diversify their evolutionary capacity, most likely through subtle alterations of many gene transcripts rather than through a single fatal lesion. This was hinted by the fact that SF3B1 mutations, usually a later event in CLL evolution,46 may not induce per se neo- plastic transformation in B cells but rather drive a more aggressive and adaptive phenotype. This line of reasoning is in keeping with the hypothesis that the activation of the NOTCH1 pathway in CLL may reflect deregulated expression of a physiological signal, required for B lym- phocyte maturation and differentiation.15,47
The exploitation of the NOTCH1 pathway by SF3B1- mutated CLL cells may well reflect such a strategy by which the proliferative advantages that characterize NOTCH1-mutated CLL cells can be incorporated in the complex transcriptomic reshaping occurring in SF3B1-mutated CLL, thus contributing to explain the poor prognosis of this CLL subset.11,16,19,32 Also in keeping with the data presented here, it is likely that the documented activation of NOTCH1 in SF3B1-mutated CLL was prima- rily mediated by the direct involvement of the NICD through the canonical pathway.45 However, the hypothe- sis of a NICD-independent activation cannot be excluded firsthand, as Notch-independent activity of the transcrip- tion factor RBPJ has been documented.48
From a clinical standpoint, the biological similarity of NOTCH1- and SF3B1-mutated CLL could contribute to explain the poor response of these CLL subsets to chemo- immunotherapy.49,50 As documented by the CLL8 trial for NOTCH1-mutated cases, the clinical behavior of SF3B1-mutated CLL was worse that that of WT CLL,
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haematologica | 2021; 106(12)