Combination of the mTOR Inhibitor Rapamycin with the HSP90 Inhibitor 17-AAG Has Synergistic Activity in EBV Positive 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 M. 
Published: April 2020

Abstract
PTLDs are a fatal complication of immunosuppression after organ transplantation. The majority of these lymphomas are EBV positive. Previous reports have demonstrated that the mTOR inhibitor rapamycin inhibits proliferation and induces apoptosis in PTLD. HSP90 is a chaperone protein involved in the refolding of proteins, including AKT, a member of the PI3K pathway. We hypothesized that the combination of agents that target multiple pathways that interact at the level of AKT and regulate cell cycle will lead to a synergistic activity in PTLD. 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 rapamycin 20-100nM (LC Laboratories, MA) and 17-AAG 100-600nM (provided by NCI) alone and in combination for 24 and 48 hrs. Inhibition of proliferation was measured using the MTT proliferation assay. Apoptosis was determined using Annexin V/PI analysis using the BD flow cytometer (BD Biosciences, CA). Determination of the additive or synergistic effect of the combination was calculated using the CalcuSyn software (Biosoft, MO) based on the Chou-Talalay method. Statistical analysis was performed using a two-tailed t-test. Immunoblotting was performed with antibodies against phospho-AKT, PARP, phosphoRb, and Cyclin D1 (Cell Signaling, MA). Single agents rapamycin and 17-AAG induced 20–30% inhibition of proliferation in the EBV+ cell line. The combination of 50nM rapamycin and 300nM 17-AAG induced over 80% inhibition of proliferation, (p=0.0003). The combination of the two agents demonstrated significant synergistic effects with a combination index of 0.12 according to the Chou-Talalay method. In contrast to the EBV+cell lines, 20–50nM rapamycin significantly inhibited proliferation with over 40% inhibition in the EBV-ve cell line. 17-AAG had no effect on the EBV-ve cell line and the combination did not induce further inhibition as compared to the effect of rapamycin alone. In EBV +ve cells, rapamycin 20–50nM induced 15–20% apoptosis, 17-AAG 600-1000nM induced 40-50% apoptosis and the combination markedly enhanced apoptosis with over 80% apoptotic cells (p=0.0001). Rapamycin induced a more significant effect on apoptosis in EBV-ve cells, but 17-AAG did not. Immunoblotting for candidate proteins in the PI3Kinase/AKT and HSP90 pathways was performed. Phospho-AKT was inhibited in response to 17-AAG and the combination of 17-AAG and rapamycin, but not by rapamycin alone indicating that 17-AAG interacts with the PI3K pathway at the level of AKT. Rapamycin upregulated cleaved PARP in both cell lines indicating induction of apoptosis. CyclinD1 protein expression was not changed in both cell lines treated with the agents. PhosphoRb was downregulated in response to rapamycin, 17-AAG and the combination in both cell lines. This data indicates that in EBV-ve cell lines, rapamycin used as a single agent can induce significant inhibition, while in EBV+ve cell lines, rapamycin alone is less effective. However, the combination of rapamycin and 17-AAG is synergistic in EBV+ve cell lines and induces over 80% inhibition of proliferation and induction of apoptosis. Targeting both the PI3kinase pathway and the heat shock protein response represents an attractive approach to future therapeutic options in EBV+ve PTLD. Supported in part by an ASH scholar award, a lymphoma research foundation and an ASCO YIA.