Activation Of TAK1 By MYD88 L265P Drives Malignant B Cell Growth In Non-Hodgkin Lymphomas

Massively parallel sequencing analyses have revealed a common mutation within the MYD88 gene (MYD88_L265P) occurring at high frequencies in many non-Hodgkin lymphomas (NHL) including the rare lymphoplasmacytic lymphoma, Waldenström’s macroglobulinemia (WM). Using whole exome sequencing, Sanger sequencing and allele-specific PCR, we validate the initial studies and detect the MYD88_L265P mutation in the tumor genome of 97% of WM patients analyzed (n=39). MYD88_L265P was detected at lower frequencies in other indolent lymphomas including LPL (0%), MALT (4%), nodal MZL (5%) and splenic MZL (8%); all but one MYD88_L265P was heterozygous. Due to the high frequency of MYD88 mutation in WM and other NHL, and its known effects on malignant B cell survival, therapeutic targeting of MYD88 signaling pathways may be useful clinically. However, while the effects of MYD88_L265P on the activity of IRAK1/4 and NF-κB are have been studied previously, we are lacking a thorough characterization of the role of intermediary signaling proteins such as TRAF6 and TAK1 on the biology of MYD88_L265P-expressing B cells. A better understanding of the proteins involved in MYD88_L265P signaling may lead to the development of more targeted and effective therapeutic approaches.

In an attempt to identify MYD88_L265P –specific therapeutic targets we first wanted to characterize the role of intermediary signaling proteins that facilitate the downstream activation of NF-κB. Upon activation of TLRs or IL-1b receptors, MYD88 forms a homodimer and recruits IRAK1/4 and TRAF6 into a complex resulting in association and phosphorylation of TAK1 followed by activation of NF-κB. We monitored the formation of a complex comprised of MYD88, IRAK1, IRAK4 and TRAF6 and immunoprecipitation of either endogenous IRAK4 or IRAK1 revealed constitutive association of IRAK with TRAF6 and MYD88_L265P. To assess if the formation of a MYD88_L265P/IRAK/TRAF6 complex results in downstream activation of TAK1, constitutive TAK1 phosphorylation was measured and detected in all three cell lines that express MYD88_L265P. An association between TAK1 and TRAF6, another measure of TAK1 activation, was also detectable. When a similar analysis of TAK1 was performed in DLBCL cells expressing wild-type MYD88, no phosphorylation of TAK1 was detected, nor was TAK1 associated with TRAF6. IRAK1, IRAK4, TAK1, TRAF6, and MYD88 were expressed at similar levels in all cell lines studied and therefore did not contribute the differences in MYD88 complex formation observed between cell lines. These studies were further confirmed using HEK 293T cells that were transduced with either a vector control plasmid or HA-tagged MYD88_WT or MYD88_L265P expression plasmids. Together, these studies suggest that MYD88_L265P forms a complex with IRAK and TRAF6 resulting in constitutive activation of TAK1 and NF-κB.

To confirm the significance of TAK1-mediated MYD88_L265P signaling on lymphoma cell growth, the effect of the selective TAK1 inhibitor, (5Z)-7-Oxozeaenol, on cell proliferation was tested. All MYD88_L265P-expressing cell lines were sensitive to TAK1 inhibition in a dose-dependent manner (0-10 μM). In contrast, NHL cells expressing MYD88_WT were found to be insensitive to TAK1 inhibition. We next tested the impact of the TAK1 inhibitor on a MYD88_L265P positive WM patient sample. Similar to what was seen in the WM cell lines, the TAK1 inhibitor inhibited WM cell growth and survival in a dose dependent manner. Additionally, the TAK1 inhibitor significantly reduced the level of IL-10 secreted by each of the cell lines. Together, these data suggest that MYD88_L265P drives cell proliferation and cytokine secretion through a TAK1-dependent mechanism.

In conclusion, we are the first to validate by NGS in a large patient cohort the high prevalence and specificity of MYD88_L265P in WM. Cells harboring the L265P mutation but not wild-type MYD88 exhibit constitutive signaling leading to the hyperactivation of NF-κB. We have established the role of TAK1 as an integral component of MYD88_L265P signaling in both WM and DLBCL cell. Our data suggest that targeting TAK1 clinically may be an effective strategy for the treatment of WM and other lymphomas driven by MYD88_L265P signaling.