DOI

10.17077/etd.392lgyid

Document Type

Dissertation

Date of Degree

Spring 2018

Degree Name

PhD (Doctor of Philosophy)

Degree In

Immunology

First Advisor

Gail Bishop

First Committee Member

Siegfried Janz

Second Committee Member

Jon C. D. Houtman

Third Committee Member

Hai-Hui Xue

Fourth Committee Member

Miles August Pufall

Abstract

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.

Pages

xii, 130 pages

Bibliography

Includes bibliographical references (pages 110-130).

Copyright

Copyright © 2018 Nurbek Mambetsariev

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