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TIRAP, a novel familial lymphoma risk gene
variant in activating NF-kB leading to enhanced B-cell proliferation and survival.
INF-kB signaling mediated by TIRAP is important for B-cell survival
To study the effect of TIRAP on cell survival, we per- formed a siRNA-mediated knockdown of endogenous TIRAP in PBMCs isolated from available family members. Overall, TIRAP knockdown efficiency was 60% at the mRNA level (Online Supplementary Figure S4A). TIRAP knockdown significantly diminished the number of living B-cells (Figure 6A and B). This effect was independent of the p.R81C, indicating that TIRAP is an important deter- minant for B-cell survival. Furthermore, we profiled the expression of genes important for cell survival and prolif- eration including NF-kB target genes (Figure 6C). Silencing TIRAP strongly reduced the expression of the NF-kB target genes (NFKB1, IL6 and MYC) in PBMCs, indicating that both WT and p.R81C TIRAP mediate signal through the NF-kB pathway (Figure 6D and Online Supplementary Figure S4). However, downregulation of NF-kB target genes was more pronounced in TIRAP p.R81C PBMCs suggesting that these cells particularly rely on the NF-kB pathway (Figure 6E and Online Supplementary Figure S4). Consistent with the reduced B-cell survival, CASP9 expression increased following TIRAP silencing in PBMCs to a higher extent in p.R81C mutated cells (Figure 6D and E). Interestingly, NF-kB signature was further reduced fol- lowing stimulation with LPS (Online Supplementary Figure S5), supporting the concept that TIRAP transduces signals from TLR4.20
TIRAP p.R81C drives NF-kB pathway activity and reduces stress-induced cell death
To evaluate the functional consequence of TIRAP p.R81C, 293T cells were transfected with bidirectional plasmids encoding for EGFP and TIRAP p.R81C, TIRAP WT or empty vector (control), respectively. Under home- ostatic conditions, we did not observe any changes in cell viability 24 hours (h) post transfection (Online Supplementary Figure S6). However, gene expression analy- sis on GFP-positive transfected cells revealed increased expression of the NF-kB target genes NFKB1, BCL2L1, CDKN2A and MYC by TIRAP p.R81C compared to WT (Figure 7A). Thus, we tested whether these transcriptional changes could affect cell viability upon stress-induced challenge. Therefore, sorted GFP-positive cells were cul- tured in minimal starving media for 48 h. Surprisingly, cell viability was significantly reduced in control or TIRAP WT transfected cells (Figure 7B). Remarkably, our data indicate that TIRAP p.R81C variant is an upstream activa- tor of NF-kB which leads to a better cell survival/prolifer- ation via enhanced NF-kB activity and decreased stress- induced cell death.
Discussion
The etiology of DLBCL is poorly understood. Familial clustering of lymphoma is reported to increase disease risk, indicating a role for genetic factors.15,16 Although familial lymphoma cases are rare, studying such pedigrees might identify disease-causing variations and lead to a bet- ter understanding of lymphomagenesis. A Finnish family with 3 siblings affected by PMBL and a cousin with extra-
nodal DLBCL has been described.19 These lymphomas segregate with the p.H1845N mutation in MLL. The role of this variant in lymphomagenesis has not been corrobo- rated by functional studies, and it is to the best of our knowledge the only DLBCL/PMBL predisposing mutation that has been described so far.
Here, we studied a Swiss/Japanese family with 2 sisters affected by B-cell lymphomas in the mediastinum. Although at initial diagnosis their lymphomas were con- sidered, according to the current WHO classification,1 as distinct DLBCL subtypes, the characterization of the somatic lesions by WES and aCGH revealed noticeable molecular similarities. Their somatic landscape is marked by multiple alterations affecting important players of the JAK-STAT signaling cascade which collectively lead to constitutive pathway activity, known to be crucially implicated in lymphomagenesis.14 Shared gains of 9p24/JAK2 and 12q13 (STAT2 and STAT6) were detected by aCGH. Furthermore, we identified missense hotspot mutations in STAT6 in both lymphomas (p.N417 and p.D417). A significant enrichment of STAT6 mutations in PMBL has been described, with mutations being present in more than 30% of cases.9,22 9p24/JAK2 gains are also recurrent genetic alterations in PMBL, but are not strictly confined to this subtype.13,40
Constitutive activation of the NF-kB pathway is a hall- mark of both ABC-DLBCL and PMBL, and promotes sur- vival of malignant cells. Somatic oncogenic mutations in components of the B-cell receptor signaling pathways including CD79A/B and CARD11 activate NF-kB.14 Gain- of-function mutations in MYD88 have been described in 29% of ABC-DLBCLs.14 Moreover, TNFAIP3, which nega- tively regulates the NF-kB pathway, is somatically inacti- vated in one-third of ABC-DLBCLs and PMBLs.14 Interestingly, germline mutation in TNFAIP3 and CARD11 have been described in lymphomas complicating primary Sjögren syndrome and congenital B-cell lymphocytosis, respectively.41,42 We searched for possible risk alleles asso- ciated with the lymphomas in the family investigated by WES, and discovered germline variants in TIRAP and IL1R1 (detailed data on IL1R1 not shown). The latter was not among the final candidate genes, as the homozygous mutation was present in all family members. Nevertheless, the presence of germline variants in two upstream regulators of NF-kB in a PMBL family is an inter- esting finding that confirms the importance of the path- way in lymphogenesis. Of note, our data support the co- operation between rare germline variants and constitutive pathway activation in malignant lymphomas.43
The predicted damaging effect of TIRAP p.R81C variant occurred at a highly conserved amino acid in close prox- imity to the functional TIR domain that is stabilized by two internal disulfide bonds.44,45 Therefore the substitution of an arginine by a cysteine might have implications on the TIRAP protein interaction with downstream signaling proteins. Of note, an arginine to cysteine mutation in MYD88, another adapter molecule involved in NF-kB signaling, diminished its interaction with TIRAP.46
In mice, deletion of Tirap reduced B-cell proliferation in response to TLR4 signaling.20 In agreement with this, we observed a direct correlation between B-cell proliferation and TIRAP expression. In this family, a high expression of TIRAP correlated with the p.R81C genotype. In 420 pri- mary DLBLC cases, high TIRAP levels correlated with a poor survival and were significantly increased in high-risk
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