Post-transplant lymphoproliferative disorder (PTLD) is a devastating and potentially life-threatening complication in transplant recipients. PTLD is associated with EBV infection and can result in malignant B cell lymphomas. We previously demonstrated that the PI3K/Akt/mTOR pathway is activated in EBV+ B cell lymphoma lines from patients with PTLD, and the mTOR inhibitor rapamycin (RAPA) inhibits proliferation of EBV+ B cell lymphomas. However, the effect of RAPA on cell proliferation was partial, and complete inhibition was not achievable at doses utilized in patients. In this study, we aim to identify molecular mediators within the PI3K/Akt/mTOR signaling axis that govern proliferation in order to establish novel therapeutic approaches that augment the efficacy of mTOR-based treatment of EBV+ B cell lymphomas in PTLD. We evaluated the efficacy of RAPA (10nM) on the proliferation of four EBV+ PTLD-derived B cell lines (AB5, MF4, VB5, and JB7). Proliferation was reduced by 51%, 66%, 29%, and 64%, respectively. Interestingly, the VB5 cell line was more resistant to RAPA-mediated inhibition compared to the other cell lines. Western blot analyses revealed constitutive Akt phosphorylation in all cell lines at Thr308 and Ser473, suggesting that both PI3K and mTORC2 are activated in EBV+ B cell lymphomas. The level of Akt phosphorylation was highest in the VB5 cell line. Because RAPA is known to target mTORC1, we next tested the effect of an mTORC1/2 inhibitor (AZD8055) and a dual PI3K/mTOR inhibitor (NVP-BEZ235) on the proliferation of EBV+ B cell lymphomas. Each of these inhibitors showed a greater anti-proliferative effect than RAPA on all of the cell lines (58-84% reduction). To clarify which PI3K isoform is responsible for Akt activation, we evaluated the expression of PI3K isoforms. PI3KΔ was highly expressed in EBV+ B cell lymphomas, whereas the expression levels of PI3KΑ and PI3KΒ were very low. The PI3KΔ specific inhibitor, CAL-101, diminished Akt phosphorylation and the proliferation of EBV+ B cell lymphomas (47-60% reduction). Furthermore, the combined use of RAPA and CAL-101 mediated synergistic anti-proliferative effects against EBV+ B cell lymphomas. Thus, targeting PI3KΔ can augment the efficacy of RAPA in inhibition of EBV+ B cell lymphoma proliferation. These results provide potential new therapeutic strategies for the treatment of EBV-associated PTLD.

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