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F. Diop et al.
367-438 and amino acids 537-564. BIRC3 variants were predicted to generate aberrant truncated transcripts caus- ing the elimination or truncation of the C-terminal RING domain of the BIRC3 protein. The RING domain of
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BIRC3 harbors the E3 ubiquitin ligase activity that is essential for proteasomal degradation of MAP3K14, the central activating kinase of non-canonical NF-κB signaling. This observation points to non-canonical NF-κB activation
Figure 1. The non-canonical nuclear factor-κB pathway is active in BIRC3- mutated chronic lymphocytic leukemia cell lines and primary samples. (A) Disposition of BIRC3 mutations across the protein. The mutations identified by Landau et al.6, Puente et al.7 and from a public CLL mutation catalogue (COSMIC v85) are plotted in gray. Individual BIRC3 mutations identified in the cur- rent studied cohort and in our previous study13 are plotted in red. (B) Western blot analysis of BIRC3 protein expres- sion and NF-κB2 activation and process- ing in the splenic marginal zone lym- phoma (SMZL) cell lines SSK41, VL51 and in the chronic lymphocytic leukemia (CLL) cell line MEC1, carrying wildtype (wt) or disrupted BIRC3. The MAVER-1 and Z-138 cell lines were used as posi- tive controls of non-canonical NF-κB activation, harboring genetic activation of non-canonical NF-κB signaling. The JEKO-1 and HEK 293T cell lines were used as negative controls for non- canonical NF-κB signaling. α-actin was used as a loading control. Color codes indicate the gene status in each cell line. The aberrant BIRC3 band expressed in MEC1 and VL51 cell lines corresponds in size to the predicted BIRC3-truncated protein, encoded by the mutant allele. (C) Western blot analysis showing BIRC3 expression and NF-κB2 processing in purified primary tumor cells from five CLL and SMZL patients carrying wildtype or disrupted BIRC3. Color codes indicate the gene status in each cell line. The aberrant BIRC3 bands in patients 09321, 14462 and 12603 correspond in size to the predicted BIRC3-truncated protein encoded by the mutant allele. α-actin was used as a loading control. (D) Western blot of whole cell extract, cyto- plasmic or nuclear fractions of the SMZL and CLL cell lines probed for the NF-κB2 subunits p100 and p52. The MAVER-1 and Z-138 cell lines served as positive controls while the JEKO-1 and HEK 293T cell lines were used as nega- tive controls. β-tubulin and BRG1 served as controls for the purity of the cytoplasmic and nuclear fractionations, respectively. (E) Gene set enrichment analysis score and distribution of non- canonical NF-κB target genes along the rank of transcripts differentially expressed in the SMZL cell lines SSK41, VL51 and in the CLL cell line MEC1. The JEKO-1 cell line was used as a negative control. (F) Validation of expression of non-canonical NF-κB target genes in the same SMZL and CLL cell lines as deter- mined by quantitative real-time poly- merase chain reaction analysis. Changes of gene expression were nor- malized to GAPDH expression; relative quantities were log2 normalized to con- trol samples (the mantle cell lymphoma cell line, JEKO-1).
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haematologica | 2020; 105(2)