NOTCH1 Mutations Are Associated with High CD49d Expression in Chronic Lymphocytic Leukemia
Authors: Zucchetto Antonella , Caldana Chiara , Pozzo Federico , Rasi Silvia , Ciardullo Carmela , Benedetti Dania , Tissino Erika , Rossi Francesca Maria , Bomben Riccardo , Zaja Francesco , Pozzato Gabriele , Di Raimondo Francesco , Del Poeta Giovanni , Dal Bo Michele , Rossi Davide , Gaidano Gianluca , Gattei Valter 
Published: October 2019

Abstract
Mutations of NOTCH1 have emerged as one of the most frequent somatic alterations in chronic lymphocytic leukemia (CLL), affecting up to 10–15% of patients. These mutations (~80% are 7544-7545delCT frameshift deletions) generate a truncated protein that accumulates in the cell and activates the downstream NOTCH1 signaling which is implicated in apoptosis resistance and increased survival of CLL cells.
CD49d (α4 integrin chain) is one of the most relevant negative prognosticator in CLL, expressed by ~40% of CLL cases, and associated with aggressive/accelerated clinical courses, whose key role in CLL cell microenvironmental interactions has been thoroughly investigated.
Literature data indicate that NOTCH1 has a role in activating the integrin signaling in several cell models. Given the higher CD49d expression characterizing trisomy 12 CLL, a CLL subset in which the NOTCH1 pathway is more often activated by NOTCH1 mutations, this study was aimed at investigating the contribution of NOTCH1 in the regulation of CD49d expression in CLL.
The 7544-7545delCT NOTCH1 mutations were investigated by ARMS-PCR in 1027 CLL cases, all characterized for CD49d expression and for the cytogenetic profiles by FISH. NOTCH1 mutated cases were 158/1027 (15%), with a higher prevalence (36.7% of NOTCH1 mutated cases) in the trisomy 12 cytogenetic group.
Analysis of CD49d expression highlighted a very strong association between the presence of NOTCH1 mutations and CD49d expression (p<0.0001). In particular, high CD49d expression (>30% of positive cells) was found in 102/158 (64.5%) NOTCH1 mutated cases as compared to 285/869 (32.8%) NOTCH1 wild-type cases. Of note, excluding trisomy 12 CLL, again NOTCH1 mutated CLL (100/865, 11.6%) displayed a significantly higher frequency of CD49d+ cases (52%) as compared to NOTCH1 wild-type CLL (25.7%) (p<0.0001).
We next analyzed the percentage of mutated NOTCH1 DNA in the context of the CLL clone by a quantitative real-time PCR (QRT-PCR) approach set up by us to quantify the delCT NOTCH1 mutation. Using the 10% cut-off value to discriminate between cases with high (high NOTCH1) and low (low NOTCH1) mutation burden, 90/138 (67.7%) and 43/138 (32.3%) CLL cases were classified as low NOTCH1 and high NOTCH1, respectively. A higher prevalence of CD49d+ cases was found in the high NOTCH1 group (79%) as compared to the low NOTCH1 group (58%) (p=0.03). Moreover, a significant association between CD49d expression and a high NOTCH1 mutation burden was observed also excluding trisomy 12 CLL, with 69% of CD49d+ cases in the high NOTCH1 group, compared to 41% of CD49d+cases in the low NOTCH1 group (p=0.03).
The association between NOTCH1 mutations and CD49d expression was next confirmed by next-generation sequencing results using the flow cytometrically sorted (>99% purity) CD49d- and CD49d+ components from 8 CLL cases characterized by both CD49d bimodal expression, and the presence of 7544-7545delCT NOTCH1 mutations at the subclonal level. In 7/8 cases, the CD49d+ component showed a higher NOTCH1 mutation burden compared to the CD49d- component, this difference reaching statistical significance in 4/7 cases. Of note, a similar clustering of mutations could not be observed in the CD49d- and CD49d+ components of other CLL cases characterized by bimodal CD49d expression and subclonal mutations of SF3B1 (n=1), BIRC3 (n=2) or TP53 (n=2).
To verify whether NOTCH1 accumulation, as occurring in NOTCH1 mutated CLL, may influence CD49d expression, MEC-1 cells were transfected with a vector containing either a NOTCH1 intracellular domain (NICD) with 7544-7545delCT or a NICD carrying a missense mutation (c.5304G>A) generating a stop codon at the beginning of the sequence, as control. The higher levels of both NOTCH1 transcript (fold increase over control=2.2) and protein (fold increase over control=1.3) characterizing mutated-NICD MEC-1 cells, was paralleled by higher levels of CD49d expression (mean fluorescence intensity=23.300 versus 12.400) in these cells.
Altogether our data demonstrate a direct correlation between NOTCH1 mutations and CD49d expression also outside the trisomy 12 CLL group, and suggest that accumulation of NOTCH1 may be directly or indirectly responsible for the up-regulation of CD49d expression.

Disclosures
No relevant conflicts of interest to declare.