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R.W.J. Meijers et al.
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Figure 2. Phosphorylation of Syk, PLCγ2 and Akt. (A) The levels of pSyk, pPLCγ2 and pAkt were determined at baseline and upon stimulation with α-IgM and corre- lated with the α-IgM response as determined by Ca2+ signaling. Representative examples of the analysis in a case of unresponsive chronic lymphocytic leukemia (CLL) (upper histograms) and a responsive CLL case (lower histograms) are shown. After single viable cell selection, the levels of pSyk, pPLCγ2 and pAkt were determined, both at baseline (black line) and upon stimulation with α-IgM (gray line). (B) Differences in basal levels of pSYK, pPLCγ2 and pAKT (x-axis) between α-IgM unrespon- sive and responsive samples. (C) The relative response after α-IgM stimulation for pSYK, pPLCγ2 and pAKT (x-axis) in the two groups of patients. Individual plots and medians (gray bars) are shown. The Mann-Whitney U-test was performed for statistical analysis between the groups of patients (**P<0.01).
al levels of these NF-kB pathway genes also correlated with basal Ca2+ levels (Online Supplementary Figure S3). A significant correlation could only be found between basal Ca2+ levels and NFKB1 (R2=0.163, P=0.033) and NFKBIE (R2=0.234, P=0.0091) transcript levels (Online Supplementary Figure S3).
not detect statistically significant differences in expres- sion levels of genes coding for the NF-kB subunits RELA, RELB and REL between the two subgroups (data not shown), we did observe clear correlations between expres- sion levels of genes associated with NF-kB inhibition and expression levels of RELA and REL (Online Supplementary Figure S5), both involved in the canonical NF-kB. No cor- relations between inhibitor levels and levels of the non- canonical NF-kB subunit RELB were found (data not shown).
Since loss of IkBε (encoded by NFKBIE as caused by an identical 4-bp frameshift deletion in the first exon), has been associated with a progressive form of CLL,23 we determined whether patients in our cohort with low NFKBIE expression carried this identical deletion. Upon sequencing of the first exon of NFKBIE, this 4-bp deletion was not observed (data not shown).
Besides the IkB genes, we also found a difference in expression of tumor necrosis factor-α induced protein 3 (TNFAIP3; logFC=-1.70, 10log(Pvalue)=2.24) based on RNA sequencing analysis. TNFAIP3 encodes for protein A20 that is induced by TNF-α and functions as a negative reg- ulator through inhibition of NF-kB signaling.24 In addition, RQ-PCR showed significantly (P=0.017) higher expres- sion of TNFAIP3 in unresponsive cases than in responsive ones (Figure 5C).
Expression levels of genes coding for NF-kB regulators (NFKB1 and NFKB2) and coding for IkB that were expressed at lower levels in responsive cases appeared to correlate with each other (Online Supplementary Figure S4), implying that unresponsive patients show higher expres- sion of multiple NF-kB inhibitors. Even though we could
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