Post-transplant lymphoproliferative disease (PTLD) describes a morphologically heterogeneous complication of solid organ and bone marrow transplantation that ranges from benign B cell hyperplasia to malignant lymphoma. The hallmark of PTLD is the development of Epstein Barr Virus (EBV)-associated B cell lymphomas. Currently, there are no established therapies for PTLD and reported mortality rates commonly exceed 50%. Multiple strategies have been utilized to treat patients with PTLD such as anti-viral drugs, B cell specific antibodies including anti-CD20 (Rituximab) and adoptive cellular immunotherapy. However, each of these approaches has distinct limitations. Our broad, long term objective has been to determine the cellular and viral proteins that are essential to the growth and survival of PTLD-associated EBV+ B cell lymphomas with the intent to identify novel candidates for therapeutic targeting. We hypothesize that EBV co-opts host cellular pathways to promote growth and survival of B cell tumors. In support of this hypothesis, we have defined a novel pathway by which the latent cycle EBV protein, latent membrane protein 1 (LMP1) induces production of human IL-10 (hIL-10) in infected B cells. hIL-10 acts as a potent autocrine growth factor in a variety of EBV+- associated malignancies including PTLD. We have also determined that the spleen tyrosine kinase (Syk), activated by the viral protein latent membrane protein 2A (LMP2A), is critical to the survival of PTLD- associated B cell lines. Moreover, our preliminary data indicate that a small molecule inhibitor of Syk significantly inhibits the growth and survival of PTLD-derived cell lines. Finally we demonstrate that LMP1 subverts host immunity through an NF:B-dependent pathway by conferring resistance to key death-receptor (DR) effector mechanisms utilized by CTL and NK cell to eliminate virally-infected and transformed cells. Our findings implicate the cellular protein, cFLIP in the resistance to DR-induced apoptosis. In this proposal we will target identified signal transduction pathways, and cellular proteins, induced by the key viral proteins LMP1 and LMP2a with the goal of inhibiting growth of PTLD-associated B cell lymphomas. To achieve these objectives we propose three specific aims.
Specific Aim 1 will investigate the requirement for Syk in survival of PTLD-associated EBV+ B cell lymphomas and test, in a NOD/SCID model, the value of targeting Syk as a potential therapeutic strategy.
Specific Aim 2 will define the LMP1-mediated activation of the PI3K/AKT signaling axis and its role in hIL-10 induction. Further, strategies to intervene in this pathway will be tested as a means to inhibit tumor growth in vivo.
In Specific Aim 3, we will target the LMP1-induced cellular protein, cFLIP with the intent to boost the ability of host CTL to eliminate EBV-infected B cell lymphomas. We anticipate that these approaches will reveal novel, and effective, therapeutic options for the treatment of PTLD.7.

Public Health Relevance

The development of Epstein Barr Virus-associated B cell lymphomas remains a serious, and often fatal, complication of solid organ and bone marrow transplantation. Currently, there are no established therapies for treatment of this disease. Thus, studies that identify and define novel targets for inhibiting tumor growth could lead to new diagnostic and therapeutic agents for prevention of this devastating disease.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Transplantation, Tolerance, and Tumor Immunology (TTT)
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Kehn, Patricia J
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Stanford University
Schools of Medicine
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Harris-Arnold, A; Arnold, C P; Schaffert, S et al. (2015) Epstein-Barr virus modulates host cell microRNA-194 to promote IL-10 production and B lymphoma cell survival. Am J Transplant 15:2814-24
Hatton, Olivia L; Harris-Arnold, Aleishia; Schaffert, Steven et al. (2014) The interplay between Epstein-Barr virus and B lymphocytes: implications for infection, immunity, and disease. Immunol Res 58:268-76
Hatton, O; Lambert, S L; Phillips, L K et al. (2013) Syk-induced phosphatidylinositol-3-kinase activation in Epstein-Barr virus posttransplant lymphoproliferative disorder. Am J Transplant 13:883-890
Furukawa, S; Wei, L; Krams, S M et al. (2013) PI3K? inhibition augments the efficacy of rapamycin in suppressing proliferation of Epstein-Barr virus (EBV)+ B cell lymphomas. Am J Transplant 13:2035-43
Hatton, Olivia; Martinez, Olivia M; Esquivel, Carlos O (2012) Emerging therapeutic strategies for Epstein-Barr virus+ post-transplant lymphoproliferative disorder. Pediatr Transplant 16:220-9
Hatton, Olivia; Lambert, Stacie L; Krams, Sheri M et al. (2012) Src kinase and Syk activation initiate PI3K signaling by a chimeric latent membrane protein 1 in Epstein-Barr virus (EBV)+ B cell lymphomas. PLoS One 7:e42610
Wei, L; Wang, M; Qu, X et al. (2012) Differential expression of microRNAs during allograft rejection. Am J Transplant 12:1113-23
Hatton, Olivia; Phillips, Lori K; Vaysberg, Maria et al. (2011) Syk activation of phosphatidylinositol 3-kinase/Akt prevents HtrA2-dependent loss of X-linked inhibitor of apoptosis protein (XIAP) to promote survival of Epstein-Barr virus+ (EBV+) B cell lymphomas. J Biol Chem 286:37368-78
Harris, A; Krams, S M; Martinez, O M (2010) MicroRNAs as immune regulators: implications for transplantation. Am J Transplant 10:713-9
Vaysberg, M; Lambert, S L; Krams, S M et al. (2009) Activation of the JAK/STAT pathway in Epstein Barr virus+-associated posttransplant lymphoproliferative disease: role of interferon-gamma. Am J Transplant 9:2292-302

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