Silvestrol Modulates Direct Anti-Tumor Activity Against Epstein-Barr Virus (EBV)-Associated Lymphomas While Sparing Innate and Antigen Specific Adaptive Immunity

Abstract 104

Epstein-Barr virus (EBV) is an oncogenic human herpes virus that infects more than 90% of people worldwide and is associated with a broad spectrum of malignant lymphoproliferative disorders (EBV-LPD), and nasopharyngeal and gastric carcinomas. Chemotherapy often leads to prolonged immune suppression, development of opportunistic infections, including EBV reactivation, and increased risk of relapsed disease. The poor prognosis of EBV+ diseases makes it essential to identify novel agents that can deliver direct anti-tumor activity while preserving innate and EBV-specific adaptive immune surveillance.

Silvestrol is a cyclopenta[b]benzofuran derived from the plant genus Aglaia and has been shown to possess potent anti-tumor activity against hematologic and solid tumors. Silvestrol interferes with the translation of mRNA with complex 5' untranslated regions often found on pro-survival oncoproteins. Silvestrol exhibits anti-tumor activity against malignant B-cell lines while causing minimal toxicity to peripheral blood mononuclear cells (PBMC) and resting T cells. To examine the potential selective anti-tumor activity and functionally address the effects of silvestrol on adaptive and innate immune function, we utilized in vitro and in vivo EBV lymphomagenesis models. Fully transformed EBV+ Lymphoblastoid lines (LCL) were derived from EBV-LPD tumors of severe combined immune deficient (SCID) mice engrafted with PBMC from EBV-positive donors (hu-PBL-SCID). EBV-LCLs were plated in the presence of silvestrol (2 – 50nM) and proliferation (MTS assay) and apoptosis (Annexin V/PI) was evaluated (24, 72, 120hr). While silvestrol showed potent anti-proliferative activity at these concentrations, we observed minimal cell death (IC₅₀ = 10nM). The anti-proliferative activity of silvestrol was associated with loss of LMP-1 expression, an EBV oncogene essential for B-cell transformation. Examination of LMP1 induced pathways showed decrease in pAkt levels and an increase in NFkB/p65 levels (total and phospho).

To examine the functional consequences of silvestrol on immune surveillance, we utilized a co-culture system where EBV+ LCLs are plated in the presence of autologous PBMCs. Autologous LCL/PBMC co-cultures were plated with silvestrol or DMSO vehicle control and allowed to incubate for 10 days. When EBV+ LCLs are irradiated, expansion of memory EBV-specific CD3+/CD8+ cytotoxic T cells (CTLs), capable of cytotoxicity and IFNg production, is observed. The addition of silvestrol (2–10nM) to co-cultures did not hinder CTL expansion or IFNg production. When unirradiated LCLs were plated in co-cultures, CTL expansion was seen, however, EBV+ LCLs became the dominant population in control conditions by day 10 of culture. Addition of silvestrol to unirradiated co-cultures (2–10nM) led to marked expansion of memory CD3/CD8+ CTLs as well as CD56^br NK cells. A dose dependent ablation of viable EBV-LCLs was observed in unirradiated co-cultures supporting the notion that silvestrol allowed for the expansion of both innate and adaptive immune effectors that were capable promoting anti-tumor activity. Immune effector populations that expanded in the presence of silvestrol showed preservation of direct cytotoxicity (adaptive immunity) and antibody dependent cell-mediated cytotoxicity assays (ADCC) against EBV+ LCLs (innate immunity) that was comparable to untreated effector populations.

We next examined the efficacy of silvestrol in vivo using the Hu-PBL SCID model depleted of murine NK cells. On d14, mice were randomized to receive treatment with either silvestrol (1.5mg/kg) or vehicle control by IP injection every 48hr. Human cell engraftment was measured by quantifying human IgG in blood. While silvestrol treatment did not affect cell engraftment, all treated mice showed improved survival when compared to mice treated with vehicle control (100% silvestrol vs. 22% control alive at day 140, p ≤0.001). Control mice showed decreased body weight (85% decrease) and splenomegally (spleen wt 421.6 mg vs 72.9 mg for control vs silvestrol) on necropsy at day 140, p≤.0004). There were no toxicities observed in silvestrol treated mice. Low dose silvestrol promotes direct anti-proliferative activity of EBV-transformed LCLs while sparing antigen specific adaptive and innate immune effector function that is capable of delivering potent anti-tumor activity in vitro and in vivo.