The adaptor protein TNF receptor-associated factor 3 (TRAF3) regulates signaling through B lymphocyte receptors, including CD40, BAFF receptor and Toll-like receptors, and also plays a critical role inhibiting B cell homoeostatic survival. Consistent with this, loss-of-function human TRAF3 mutations are common in B cell cancers, particularly multiple myeloma and B cell lymphoma. B cells of B cell-specific TRAF3-/- mice (B-Traf3-/-) display remarkably enhanced survival compared to littermate control (WT) B cells. The mechanism for this abnormal homeostatic survival is poorly understood; a key knowledge gap in selecting optimal treatments for human B cell cancers with TRAF3 deficiency. In this study, we identify novel mechanisms by which loss of TRAF3 promotes enhanced B cell survival, information highly relevant to the role of TRAF3 in B cell malignancies.
We show here that TRAF3 is a resident nuclear protein that associated with the transcriptional regulator cyclic AMP response element binding protein (CREB) in both mouse and human B cells. The TRAF-C domain of TRAF3 was necessary and sufficient to localize TRAF3 to the nucleus via a functional nuclear localization signal. CREB protein was elevated in TRAF3-/- B cells, without change in mRNA, but with a decrease in CREB ubiquitination. CREB-mediated transcriptional activity was increased in TRAF3-deficient B cells. Consistent with these findings, Mcl-1, an anti-apoptotic target of CREB-mediated transcription, was increased in the absence of TRAF3 and enhanced Mcl-1 was suppressed with CREB inhibition. Our results identify a new mechanism by which nuclear TRAF3 regulates B cell survival via inhibition of CREB stability.
We also show that TRAF3 deficiency led to induction of two proteins important for glucose metabolism, Glut1 and Hexokinase 2 (HXK2). This was associated with increased glucose uptake. In the absence of TRAF3, anaerobic glycolysis and oxidative phosphorylation were increased in B cells without changes in mitochondrial mass or reactive oxygen species. Chemical inhibition of glucose metabolism or glucose deprivation substantially attenuated the enhanced survival of TRAF3-deficient B cells, with a decrease in the pro-survival protein Mcl-1. Changes in Glut1 and Mcl-1 levels, glucose uptake and B cell number in the absence of TRAF3 were all dependent upon NF-κB inducing kinase (NIK). These results indicate that TRAF3 deficiency suffices to metabolically reprogram B cells.
Finally, loss of TRAF3 resulted in the induction of the pro-survival kinase proviral insertion in murine lymphoma 2 (Pim2) in B cells independently of non –canonical NF-κB activation. TRAF3-deficient B cells and multiple myeloma cells displayed higher susceptibility to Pim inhibition. In contrast, TRAF3 deficiency rendered cells resistant to inhibitors of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Loss of TRAF3 also led to transcription-independent c-Myc elevation that was dependent on increased Pim2 and decrease in c-Myc ubiquitination. Overexpression of c-Myc in mouse B cells resulted in Pim2 induction. TRAF3 deficiency made B cells resistant to the c-Myc inhibitor JQ1, but the drug enhanced Pim inhibitor-mediated killing.
Collectively, these findings provide insight into how TRAF3 regulates B cell survival. Our improved understanding of how loss of TRAF3 promotes development of B cell malignancies allows for the development of novel therapeutic strategies that target these mechanisms.