TRAF3 is a novel tumor suppressor gene identified in a variety of human B lymphoma, including splenic marginal zone lymphoma, B cell chronic lymphocytic leukemia and mantle cell lymphoma, as well as multiple myeloma (MM). To explore the role of TRAF3, we recently generated a new genetically modified mouse model that has the TRAF3 gene specifically deleted in B cells (B-TRAF3-/- mice). We found that TRAF3 deletion results in prolonged survival of mature B cells, which eventually leads to spontaneous development of B lymphomas in mice. This proposal aims to understand how TRAF3 inactivation promotes oncogenic B cell survival. We first found that the mechanism of aberrant B cell survival mediated by TRAF3 inactivation is fundamentally different from that of normal B cell survival induced by physiological stimuli. To identify novel targets of TRAF3 inactivation, we performed microarray analysis to compare the global gene expression profiles of splenic B cells purified from young, tumor-free B-TRAF3-/- mice and littermate control mice. Strikingly, a number of genes identified in our microarray analysis are implicated in subcellular organelle or vesicle trafficking, including Rhbdf1, Rasgrp3, Ehd1, and Kif11, etc. Current evidence indicates that many survival proteins are trafficking between two important organelles, endoplasmic reticulum (ER) and mitochondria, and that the balance between mitochondrial levels of pro-survival proteins and anti-survival proteins controls the threshold of cell death. Interestingly, or preliminary results demonstrated that mitochondrial levels of the anti-survival protein Bak were decreased, and mitochondrial levels of the pro- survival protein Mcl-1 were increased in premalignant TRAF3-/- B cells. However, total cellular levels of Bak and Mcl-1 were not altered in these cells. Based on our new findings, we will test the central hypothesis that TRAF3 inactivation modulates the trafficking of Bak and Mcl-1 between ER and mitochondria to increase the threshold of cell death in B cells. To address this, we propose complementary studies using B-TRAF3-/- mice and human patient-derived multiple myeloma cell lines with TRAF3 deletions or mutations as model systems.
In Aim 1, using microarray analysis and a stringent prioritization scheme, our preliminary results led us to focus on a novel target of TRAF3, Rhbdf1, which is implicated in ER protein trafficking. To delineate the causal role of Rhbdf1 in oncogenic B cell survival, we will employ lentiviral shRNA vector-mediated knockdown, ectopic overexpression, and mutagenesis of Rhbdf1.
In Aim 2, we will perform extensive biochemical fractionation, confocal microscopic imaging, and co-immunoprecipitation studies to understand how TRAF3 inactivation and Rhbdf1 modulate the trafficking of Bak and Mcl-1 between ER and mitochondria. Furthermore, we will interrogate how TRAF3 signaling interplays and cross-talks with ER stress responses. In summary, this study will provide new insights into the exact mechanisms of TRAF3 inactivation-mediated oncogenic B cell survival, and will open up new avenues for the treatment of B lymphoma and MM.

Public Health Relevance

Lymphoma is the 5th most common human cancer with approximately 21,000 annual deaths in the United States. TRAF3 is a novel tumor suppressor gene identified in a variety of human B lymphoma, including splenic marginal zone lymphoma, B cell chronic lymphocytic leukemia and mantle cell lymphoma, as well as multiple myeloma. This proposal aims to investigate a novel target of TRAF3, Rhbdf1, and a new signaling mechanism that connect TRAF3 inactivation to oncogenic B cell survival. To address the gaps in knowledge, we propose complementary studies using a new mouse B lymphoma model with the TRAF3 gene specifically deleted in B lymphocytes and human patient-derived multiple myeloma cell lines with TRAF3 deletions or mutations. Information gathered from the proposed studies will advance the understanding of the pathological mechanisms underlying B cell oncogenesis, and will open up new avenues for the development of treatment strategies to combat B lymphoma and multiple myeloma.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA158402-01A1
Application #
8372542
Study Section
Cancer Molecular Pathobiology Study Section (CAMP)
Program Officer
Howcroft, Thomas K
Project Start
2012-09-04
Project End
2017-06-30
Budget Start
2012-09-04
Budget End
2013-06-30
Support Year
1
Fiscal Year
2012
Total Cost
$336,206
Indirect Cost
$116,463
Name
Rutgers University
Department
Anatomy/Cell Biology
Type
Schools of Arts and Sciences
DUNS #
001912864
City
New Brunswick
State
NJ
Country
United States
Zip Code
08901
Zhu, Sining; Jin, Juan; Gokhale, Samantha et al. (2018) Genetic Alterations of TRAF Proteins in Human Cancers. Front Immunol 9:2111
Lalani, Almin I; Zhu, Sining; Xie, Ping (2018) Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay. J Vis Exp :
Woolaver, Rachel A; Wang, Xiaoguang; Dollin, Yonatan et al. (2018) TRAF2 Deficiency in B Cells Impairs CD40-Induced Isotype Switching That Can Be Rescued by Restoring NF-?B1 Activation. J Immunol 201:3421-3430
Lalani, Almin I; Zhu, Sining; Gokhale, Samantha et al. (2018) TRAF molecules in inflammation and inflammatory diseases. Curr Pharmacol Rep 4:64-90
Zhang, Rui; Zhang, Guoying; Xiang, Binggang et al. (2017) TRAF3 negatively regulates platelet activation and thrombosis. Sci Rep 7:17112
Eyo, Ukpong B; Peng, Jiyun; Murugan, Madhuvika et al. (2016) Regulation of Physical Microglia-Neuron Interactions by Fractalkine Signaling after Status Epilepticus. eNeuro 3:
Edwards, Shanique K E; Han, Yeming; Liu, Yingying et al. (2016) Signaling mechanisms of bortezomib in TRAF3-deficient mouse B lymphoma and human multiple myeloma cells. Leuk Res 41:85-95
Moore, Carissa R; Edwards, Shanique Ke; Xie, Ping (2015) Targeting TRAF3 Downstream Signaling Pathways in B cell Neoplasms. J Cancer Sci Ther 7:67-74
Lalani, Almin I; Moore, Carissa R; Luo, Chang et al. (2015) Myeloid cell TRAF3 regulates immune responses and inhibits inflammation and tumor development in mice. J Immunol 194:334-48
Lalani, Almin I; Luo, Chang; Han, Yeming et al. (2015) TRAF3: a novel tumor suppressor gene in macrophages. Macrophage (Houst) 2:e1009

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