Page 197 - 2020_01-Haematologica-web

Basal NF-κB inhibition impacts BCR responsiveness in CLL
A
B
Figure 3. Surface expression of activation-associated markers. The surface expression of several activation-associated markers was measured in 52 patients with chronic lymphocytic leukemia (CLL). (A) Surface expression levels (x-axis) of IgM, IgD, CD19, CD38 and CD43 in α-IgM-unresponsive and α-IgM-responsive cases. Individual plots and medians (gray bars) are shown. The Mann-Whitney U-test was performed for statistical analysis between the groups of patients (*P<0.05). (B) Relative response upon α-IgM stimulation (x-axis) is plotted against the surface expression [median fluorescence intensity (MFI)] expression level of IgM and CD21. Linear regression analysis was performed and R2 and P values are shown.
Collectively, these results illustrate that unresponsive cases have higher basal gene expression of several regula- tory molecules of canonical NF-kB pathway signaling.
of NF-kB inhibitory components, including TNFAIP3 and NF-kB subunit REL, upon stimulation.
Discussion
Here we aimed to study cell-intrinsic differences between unresponsive and responsive CLL, which may underlie differences in Ca2+ levels upon α-IgM stimula- tion. Based on RNA sequencing analysis several compo- nents of the canonical NF-kB pathway, especially related to NF-kB inhibition, were expressed more highly in unre- sponsive cases. Besides these inhibitors, the TNFα- induced NF-kB inhibitor A20 was also significantly more highly expressed in the BCR-unresponsive cases. Lastly we showed that upon α-IgM stimulation, the expression of these NF-kB pathway genes (especially genes coding for NF-kB pathway inhibitors but also NF-kB component REL) is upregulated in BCR-responsive cases while for the unresponsive cases the transcriptional level did not change compared to basal levels, indicating that NF-kB signaling is an important pathway for CLL cells in their ability to respond upon BCR stimulation.
Based on the lack of correlation between basal Ca2+ lev-
Upregulation of nuclear factor-kB pathway genes upon α-IgM stimulation in B-cell receptor-responsive cases
To further study expression of the NF-kB genes upon stimulation, frozen peripheral blood mononuclear cells from 21 cases (unresponsive CLL; n=11 and responsive CLL; n=10) were thawed, after which CLL cells were MACS-isolated and stimulated for 2.5 h with α-IgM (optimal stimulation was defined using normal B cells; data not shown). The 2-deltaCT values obtained after incuba- tion (α-IgM-stimulated and -unstimulated) were normal- ized by subtraction of the basal 2-deltaCT value to calculate the fold differences in expression between the groups of patients (Figure 6). BCR-responsive cases showed signifi- cant upregulation of NFKB2, REL, NFKBID, NFKBIE and TNFAIP3 after stimulation compared with unresponsive cases for which the expression of the NF-kB genes remained roughly equal.
In summary, α-IgM-unresponsive cases had high basal transcription of especially NF-kB inhibitory components, whereas the responsive cases showed clear upregulation
haematologica | 2020; 105(1)
187