A novel MYD88 mutation, L265RPP, in Waldenström macroglobulinemia activates the NF-κB pathway to upregulate Bcl-xL expression and enhances cell survival

Waldenström macroglobulinemia (WM) is a distinct clinicopathological entity defined by a low-grade B-cell lymphoma, lymphoplasmacytic lymphoma, infiltrating the bone marrow and producing a monoclonal immunoglobulin of the IgM class. Recently, a distinct mutation in MYD88, L265P, has been found in more than 90% of patients with WM. On the other hand, various MYD88 mutations have been found in about 40% of cases with activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), with MYD88 L265P constituting 75–80% of these mutations. However, MYD88 L265P represents almost the sole MYD88 mutation reported so far in WM and in 50–60% of patients with the IgM-type monoclonal gammopathy of undetermined significance, which may precede WM. This mutation has been found in o10% of patients with other low-grade B-cell neoplasms showing plasmacytic differentiation and producing a monoclonal IgM protein, such as splenic marginal zone lymphoma, chronic lymphocytic leukemia and IgM-secreting multiple myeloma. Thus, detection of this mutation is valuable for the differential diagnosis of WM from these low-grade B-cell neoplasms, leading to the development of highly sensitive allele-specific PCR (AS-PCR) methods to detect specifically the MYD88 L265P mutation. MYD88 is an adaptor protein that mediates Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) signaling regulating diverse immune responses. MYD88, as well as TLRs and IL-1R, possesses a Toll–IL-1 receptor (TIR) domain and is recruited to these receptors upon stimulation of their ligands through homophilic interaction between the respective TIR domains. MYD88 then interacts with and activates the IRAK1 and IRAK4 serine/threonine kinases, ultimately leading to NF-κB activation via IκBα phosphorylation. The L265P mutation in the TIR domain has been postulated to change the structure of MYD88 to induce constitutive formation of the MYD88/IRAK complex to activate the NF-κB pathway to stimulate proliferation and to suppress apoptosis of B cells. MYD88 L265P has also been reported to promote NF-κB activation by binding and activating BTK and to activate the Jak-STAT3 pathway as additional downstream effects. Through these mechanisms, MYD88 mutations have been implicated in the pathogenesis and development of therapy resistance in B-cell neoplasms, mainly WM and ABC-DLBCL. Thus, MYD88 mutants, as well as their downstream effectors, may represent promising therapeutic targets for these diseases and need to be further investigated in detail. We have developed a very simple AS-PCR method that could detect the MYD88 L265P mutation when present in 41% of cells and examined the bone marrow cells from five patients with WM (Supplementary Materials). We failed to detect this mutation in one out of the five patients examined (Supplementary Figure S1), although this patient showed typical clinical features of WM (Supplementary Materials) and abnormal plasmacytic cells constituted 45% of the bone marrow cells. Thus, we directly sequenced the PCR products coding for MYD88 exon 5 derived from genomic DNA, as well as cDNA from the bone marrow cells of the patient. Because a region around the sequences coding for MYD88 L265 gave mixed and ambiguous signals (data not shown), we next sequenced and analyzed the subcloned full-length MYD88 RT-PCR products, as well as genomic DNA PCR products, and found a novel mutation of MYD88, c.792_794delACTins GCGGCCCCC (Figure 1a), causing the substitution of L265 with RPP (L265RPP) (Figure 1a). Because the MYD88 L265RPP mutation has never been reported in WM or in any other B-cell malignancies, we analyzed the effects of this novel mutation comparably with MYD88 L265P by constructing retroviral expression plasmids for both of these mutants, as well as for wild-type MYD88, tagged N-terminally with the FLAG epitope in the pMIG vector. We infected the BJAB cells, a model lymphoma cell line with low endogenous NF-κB activity used in studies for MYD88 mutations, with these retroviral vectors and selected infected cells by sorting cells expressing GFP. As shown in Figure 1b, western blot analysis revealed that MYD88 L265RPP and, to a lesser extent, MYD88 L265P were expressed at lower levels as compared with wild-type MYD88 in BJAB cells, which expressed comparable levels of GFP (data not shown), thus suggesting that these mutants may be unstable in these cells. To address this possibility, we treated these BJAB cells with cycloheximide to shut down translation and examined its effect on the expression levels of these MYD88 proteins. As shown in Figure 1c, expression levels of MYD88 L265RPP and, to a lesser extent, MYD88 L265P were more rapidly reduced by cycloheximide as compared with that of wild-type MYD88, thus indicating that these mutants are more rapidly degraded in cells as compared with wild-type MYD88. To explore the mechanisms involved in degradation of these mutant MYD88 proteins, we next examined the effects of the proteasome inhibitor bortezomib, which is used also for treatment of WM, on expression of the MYD88 proteins. As shown in Figure 1d, the expression level of MYD88 L265RPP was more significantly increased by bortezomib as compared with that of wild-type MYD88, whereas that of MYD88 L265P did not show any significant change. These results suggest that MYD88 L265RPP may be rapidly degraded through the proteasome pathway sensitive to inhibition by bortezomib in a different manner as compared with MYD88 L265P. Next, we examined the possible activation of the MYD88IRAK1/4 and NF-κB pathway in BJAB cells expressing MYD88 L265RPP, as well as MYD88 L265P. First, we failed to detect the physical association of MYD88 L265RPP or MYD88 L265P with IRAK1 by coimmunoprecipitation experiments (negative data not shown). However, when stabilized by treatment with bortezomib, MYD88 L265RPP, but not MYD88 L265P or wild-type MYD88, was shown to associate prominently with slowly migrating IRAK1 species, which should represent IRAK1 hyper-phosphorylated by IRAK4 (Figure 2a). Moreover, in BJAB cells expressing MYD88 L265RPP, IκBα was phosphorylated to a much higher extent than in cells expressing wild-type MYD88 or MYD88 L265P, whereas its expression level was slightly decreased in cells expressing MYD88 L265RPP, as expected from its enhanced degradation after phosphorylation (Figure 2b). In agreement with this, NF-κB p65 Citation: Blood Cancer Journal (2015) 5, e314; doi:10.1038/bcj.2015.36