Differential Response to Proteosome Inhibition in EBV Positive and Negative Lymphoma Cell Lines: Clinical Implications in Post Transplant Lymphoproliferative Disorders (PTLD).
Authors: Alsayed Yazan , Singha Ujjal , Francis Lanie , Huston Alissa , Lu Ganwei , Anderson Judy , Lentzsch Suzanne , Roodman G. David , Ghobrial Irene 
Published: April 2020

Abstract
PTLDs represent fatal complications of immunosuppression in patients undergoing organ transplantation. Although the EBV virus is implicated in the development of most of these lymphomas, EBV-ve cases occur in 20-30% of the cases. The latent membrane protein (LMP1) of EBV leads to the activation of the nuclear factor-kB (NF-kB) transcription factor that leads to proliferation in these lymphomas. Therefore, targeting the NF-kB pathway may represent a rational approach. Our objective was to test the effect of bortezomib in EBV +ve and EBV-ve cell lines. EBV+ve B lymphoblastic cell lines, IM-9 and Daudi (ATCC) and EBV-ve lymphoma cell line, Pfeiffer (ATCC) were exposed to serial dilutions of bortezomib 2.5-10nM (Millenium, MA; supplied by the NCI) for 16, 24 and 48 hrs. Inhibition of proliferation was measured using the MTT proliferation assay. Apoptosis was determined using Annexin V/PI flow cytometry analysis (BD Biosciences, San Jose, CA). Immunoblotting was performed at 16 hrs with antibodies against PARP, Bcl-2, and phosphoNF-kB (Cell Signaling, MA). To determine potential mechanisms of resistance of the cell lines to bortezomib, we performed proteomic analysis using the nanoscale BD Clontech antibody-based protein microarray. IM-9 cells were treated with bortezomib (2.5nM for 16 hrs) or vehicle (DMSO). The antibody microarray is a technique that detects differences in protein abundance between the treated and control sample by hybridizing fluorescently labeled (Cy3 and Cy5) protein mixtures onto slides spotted with 512 human monoclonal antibodies. The slides were scanned using the Axon GenePix 4000B scanner. Two ratios were generated from the spot images for each protein target. The mean of the ratios of Cy5/Cy3 of both slides were analyzed using Clontech software and used to calculate an Internally Normalized Ratio (INR = Ratio1/Ratio2) for each spot. Proteins whose expression was greater than 1.3 fold relative to control were determined. Bortezomib induced significant inhibition of proliferation (75–90%) in EBV-ve cell lines. However it only had 15–19% inhibition of proliferation in EBV+ve cells. In addition, bortezomib 2.5nM induced more than 60% apoptosis in EBV-ve cells, and only 15% apoptosis in EBV+ve cells. These results were confirmed by immunoblotting where cleaved PARP was not affected in EBV+ve cells indicating no effect on apoptosis. However, the anti-apoptotic Bcl-2 and phosphoNF-kB were inhibited in both cell lines in response to low doses of bortezomib indicating that other signaling pathways induce resistance to apoptosis in the EBV+ve cell lines. Using proteomics analysis in EBV+ve cells, bortezomib increased expression of several proteins that may induce resistance to apoptosis including cell cycle regulatory proteins such as cyclin D-1 and CDK2; kinases such as JNKK1, PKC lambda and ERK2; anti-apoptotic protein p53 related protein such as 53BP2; and heat shock protein HSP70. These results indicate that bortezomib exerts a significant growth inhibitory effect on EBV-ve but not EBV+ve cell lines. The mechanisms of resistance to this agent may be due to upregulation of anti-apoptotic proteins. Future use of inhibitors of these proteins such as the use of heat shock protein inhibitors may overcome resistance to bortezomib in EBV+ve cells. In addition, selective use of bortezomib in EBV-ve PTLD may be warranted. Supported in part by an ASCO YIA and ASH scholar award.