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R.W.J. Meijers et al.
Figure 6. Transcriptional levels of nuclear factor-kB related genes upon α-IgM stimulation. Upon α-IgM stimulation, real-time quantitative polymerase chain reac- tions were performed to examine the transcriptional levels of multiple nuclear factor (NF)-kB pathway genes (NFKB1, NFKB2, REL, NFKBIB, NFKBID, NFKBIE and TNFAIP3) that were differently expressed at baseline between responsive and unresponsive cases of chronic lymphocytic leukemia (CLL). The 2-deltaCT value upon α- IgM stimulation was subtracted from the 2-deltaCT value at baseline and divided by the 2-deltaCT value of the unstimulated condition corrected by the 2-deltaCT value at baseline to calculate the net increase or decrease upon α-IgM stimulation. α-IgM unresponsive CLL cases (n=11, white bars) were compared to α-IgM responsive CLL cases (n=10, black bars) and statistical analysis was performed using the Mann-Whitney U-test (*P<0.05, **P<0.01).
between CD21 expression and the responsive capacity upon α-IgM stimulation is striking. In other immune- related diseases, such as rheumatoid arthritis, common variable immunodeficiency25 and Sjögren syndrome,26 patients had increased populations of CD21low B cells compared to healthy individuals.25 These CD21low B cells were found to represent unresponsive cells expressing autoreactive BCR which failed to respond, as determined from Ca2+ levels upon BCR stimulation.25,26 CD21low CLL cells were not found to be autoreactive and are associated with a poor prognosis.27 Unfortunately we had no access to patients’ longitudinal data and we were therefore unable to evaluate progression of the CLL.
RNA sequencing analysis showed that especially genes coding for regulatory molecules involved in NF-kB inhibi- tion are differentially expressed between BCR-responsive and -unresponsive cases. Several studies have shown that CLL cells have higher basal NF-kB levels compared to nor- mal B cells and that they are continuously activated.28 In addition, it has been shown that NF-kB signaling is important for preventing apoptosis by multiple mecha- nisms, including CD40L-mediated signaling.28-30
We found that the unresponsive cases had higher basal gene expression of several components of the canonical NF-kB pathway, especially those involved in inhibition. Genes coding for the p105/p50 (NFKB1) and p100/p52 (NFKB2) subunits were expressed more highly in unre- sponsive CLL. Both are potential inhibitors and allow functional NF-kB activation in which p105/p50 is involved in the canonical NF-kB pathway and p100/p52 in the alternative (non-canonical) NF-kB pathway.31 In addition, we found that genes coding for IkB were more highly expressed in unresponsive cases. IkBε (coded by NFKBIE), which is an important regulator of B-cell prolif- eration,32 was found to be mutated in patients with CLL.23,33 In particular, a recurrent 4-basepair frameshift deletion resulting in functional loss of IkBε and leading to continuous NF-kB activation was detected in progressive forms of CLL 23 as well as in other B-cell malignancies.34 However, we could not identify this identical deletion as
a possible cause for the lower NFKBIE gene expression in the responsive cases.
Besides BCR stimulation, the canonical NF-kB pathway can be activated by TNF receptor stimulation.31 It might thus be that NF-kB signaling in BCR-unresponsive cases is more dependent on TNF-mediated activation. Higher TNFAIP3 expression, a negative feedback regulator of NF- kB signaling induced by TNFα, as we noted in unrespon- sive cases, provides a basis for this theory. From B-cell lymphoma patients it is known that increased and sus- tained NF-kB activation of especially the proto-oncogene c-REL promotes TNFα-induced cell survival.35 Through this feedback loop mechanism, secretion and uptake of TNFα might result in NF-kB-induced survival of (anergic) CLL cells, independently of BCR signaling. Foa et al.36 reported that CLL cells continuously produce TNFα, especially cells from patients with an indolent form of the disease compared to patients with a progressive form.36
Genomic aberrations in the TNFAIP3 gene resulting in the loss of A20 are linked with autoimmune disease with a humoral component as well as several B-cell lym- phomas.37 In B cells from aged mice it was demonstrated that selective loss of A20 increases the activation thresh- old and enhances proliferation and survival of B cells causing an inflammatory condition and inducing autoim- munity.38 Such a loss of A20 caused by genetic aberrations of TNAIP3 has not been associated with human CLL.39
Even though the focus of our study was mostly on those genes that were expressed at higher levels in unre- sponsive cases, multiple genes, including SYK, were found to be expressed more highly in responsive cases. Although SYK was differently expressed based on the RNA sequencing analysis between the two groups of patients in the extended cohort of patients with CLL, we did not find a difference in SYK protein level (by phos- pho-flow analysis; data not shown). Another gene of inter- est that emerged from our analysis is Early B-cell Factor 1 (EBF1), a transcription factor important in B-cell differen- tiation, which was expressed at higher levels by the responsive cases.40 Seifert et al. had earlier shown that
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haematologica | 2020; 105(1)