Mechanisms Underlying Augmented Lymphomagenesis by LUBAC; Possible Accumulation of AID-Mediated Somatic Mutations

Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) is characterized by constitutive activation of NF-κB signaling, where protein ubiquitination is involved in various steps. The linear ubiquitin chains specifically generated by the linear ubiquitin assembly complex (LUBAC) has recently drawn attention in NF-κB signaling. LUBAC is known to be involved in NF-κB activation and cell death protection by conjugating linear chains onto several substrates including NEMO. We have reported increased frequency of rare germline single-nucleotide polymorphisms (SNPs) of HOIP, the catalytic subunit of LUBAC in ABC-DLBCL patients (Yang et al., Cancer Discov, 2014). Although the SNPs enhanced ligase activity of LUBAC, precise mechanisms underlying augmented lymphomagenesis by LUBAC remains unknown.

In order to consolidate the roles of LUBAC in the pathogenesis of ABC-DLBCL, a mouse strain, in which wild-type murine HOIP is constitutively expressed in B cells from as early as the pre-B cell stage (CD19-cre-HOIP) is generated. In CD19-cre-HOIP, the increase of HOIP elevated the amount of trimeric LUBAC composed of HOIP, HOIL-1L and SHARPIN, augmenting NF-κB activation mildly in B cells. TLR-induced proliferation is more prominent in CD19-cre-HOIP B cells than littermate control, indicating that MYD88-dependent signaling is facilitated by LUBAC. Although some of the aged CD19-cre-HOIP mice showed splenomegaly, no CD19-cre-HOIP mice developed lymphomas as far as examined. These results indicated that enhanced expression of HOIP leads to augmentation of TLR-mediated signaling and proliferation in B cells, but it cannot induce lymphomagenesis by itself.

Importantly, majority of ABC-DLBCL found in patients with the HOIP SNPs had oncogenic MYD88 L265P mutation. To assess synergistic effects of LUBAC and MYD88 L265P, we generated two mice strains, expressing MYD88 L252P, an equivalent mutation of human L265P in mice alone (CD19-cre-MYD88LP) or MYD88 L252P and HOIP (CD19-cre-HOIP/MYD88LP) in a B-cell specific manner.

In CD19-cre-MYD88LP B cells, enhanced phosphorylation of IRAK4, degradation of IκBα, and expression of NF-κB target genes were observed as a result of constitutive activation of MYD88 signaling. Constitutive MYD88 signaling in B cells also led to the expansion of B cell population and splenomegaly since early age. As reported previously (Knittel et al., Blood, 2016), B-cell specific expression of MYD88 L252P resulted in a reduced survival and introduction of a HOIP transgenic allele significantly shortened survival of mice expressing MYD88 L252P. Since clonal B-cell lymphomas displaying characteristics of DLBCL could be found in both CD19-cre-MYD88LP and CD19-cre-HOIP/MYD88LP mice, the augmented LUBAC activity promotes active MYD88-mediated lymphomagenesis of B cells.

To clarify the mechanisms underlying LUBAC-mediated acceleration of B lymphomagenesis, whole-exome-sequencing analyses of lymphomas developed in these mice were performed. Many of the genes mutated in these lymphomas recapitulated those reported in human DLBCL, and alignment analyses suggested that majority of these mutations were identified as targets of aberrant somatic hypermutations induced by AID, which were especially prominent in the tumors developed in CD19-cre-HOIP/MYD88LP mice. These findings strongly implied that LUBAC enhances AID-mediated somatic mutations.

Induced expression of AID was unlikely the major reason for increased mutation loads by LUBAC, since AID expression was elevated in B cells of both CD19-cre-HOIP/MYD88LP, and CD19-cre-MYD88LP mice almost equally. LUBAC has been reported involved in the protection of DNA damage-induced apoptosis. AID is known to induce double strand DNA break, and we hypothesized that LUBAC may protect cells from DNA damage-induced cell death caused by AID. To probe the effect of enhanced expression of LUBAC on protection from genotoxin-mediated apoptosis, we treated Jurkat cells with cisplatin and found that enforced expression of HOIP protected cells from cisplatin-induced cell death, implying that cells with augmented LUBAC activity have higher tolerance to genotoxic stress.

In summary, the present results indicated that protection of B-cells from cell death underlies accelerating lymphomagenesis caused by augmented LUBAC activity via partial overcoming AID-mediated DNA damage, which leads to accumulation of somatic mutations.