mTOR inhibitors induce cell-cycle arrest and inhibit tumor growth in Epstein-Barr virus-associated T and natural killer cell lymphoma cells.

PURPOSE: Epstein-Barr virus (EBV) infects B cells, as well as T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoid malignancies. In various tumor cells, mTOR performs an essential function together with Akt with regard to cell growth. We investigated the effects of mTOR inhibitors on EBV-associated T- and NK-cell lymphomas. EXPERIMENTAL DESIGN: We investigated the Akt/mTOR activation pathway in EBV-positive and -negative T- and NK-cell lines (SNT13, SNT16, Jurkat, SNK6, KAI3, and KHYG1). We evaluated the antitumor effects of mTOR inhibitors (rapamycin and its analogue, CCI-779) against these cell lines in culture and in a murine xenograft model that was established by subcutaneous injection of SNK6 cells into NOG mice. RESULTS: All EBV-positive and -negative T- and NK-cell lines tested displayed activation of the Akt/mTOR pathway, and treatment with mTOR inhibitors suppressed mTOR activation. The inhibitors induced G1 cell-cycle arrest and inhibited cell proliferation in T- and NK-cell lines. Overall, T cell lines were more sensitive to rapamycin, but there were no significant differences between EBV-positive and -negative cell lines. Treatment with rapamycin did not affect lytic or latent EBV gene expression. Intraperitoneal treatment with CCI-779 significantly inhibited the growth of established tumors in NOG mice and reduced the EBV load in peripheral blood. CONCLUSION: These results suggest that inhibition of mTOR signaling is a promising new strategy for improving treatment of EBV-associated T- and NK-cell lymphoma.

Anti-CCR4 monoclonal antibody mogamulizumab for the treatment of EBV-associated T- and NK-cell lymphoproliferative diseases.

PURPOSE: Epstein-Barr virus (EBV) infects not only B cells but also T cells and natural killer (NK) cells, and T- and NK-cell lymphoproliferative diseases (T/NK-LPD) that are refractory to conventional chemotherapies may develop. To identify a molecular-targeted therapy for EBV-associated T/NK-LPDs, we investigated whether CC chemokine receptor 4 (CCR4) was expressed on EBV-infected T and/or NK cells and whether a humanized anti-CCR4 monoclonal antibody, mogamulizumab, was effective. EXPERIMENTAL DESIGN: CCR4 expression was examined in various cell lines. In vitro, the effects of mogamulizumab on cell lines were evaluated in the presence of peripheral blood mononuclear cells from volunteers. In vivo, the effects of mogamulizumab were evaluated using a murine xenograft model. CCR4 expression was examined on EBV-infected cells from patients with EBV-associated T/NK-LPDs. Ex vivo, the effects of mogamulizumab were evaluated using patient lymphocytes. RESULTS: CCR4 expression was confirmed in most EBV-positive T and NK cell lines. Mogamulizumab induced antibody-dependent cellular cytotoxicity (ADCC) activity against CCR4-positive cell lines, and inhibited the growth of EBV-positive NK-cell lymphomas in a murine xenograft model. Furthermore, CCR4 was expressed on EBV-infected cells in 8 of 17 patients with EBV-associated T/NK-LPDs. Interestingly, CCR4 was positive in 5 of 5 patients with hydroa vacciniforme, a photodermatosis caused by the clonal expansion of EBV-infected γδT cells. EBV-positive γδT cells were obtained from a patient with hydroa vacciniforme and subjected to an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. The γδT cells that were positive for CCR4 were killed by mogamulizumab via ADCC. CONCLUSIONS: These results indicate that mogamulizumab may be a therapeutic option against EBV-associated T/NK-LPDs.

Anti-tumor effects of suberoylanilide hydroxamic acid on Epstein-Barr virus-associated T cell and natural killer cell lymphoma.

The ubiquitous Epstein-Barr virus (EBV) infects not only B cells but also T cells and natural killer (NK) cells and is associated with various lymphoid malignancies. Recent studies have reported that histone deacetylase (HDAC) inhibitors exert anticancer effects against various tumor cells. In the present study, we have evaluated both the in vitro and in vivo effects of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on EBV-positive and EBV-negative T and NK lymphoma cells. Several EBV-positive and EBV-negative T and NK cell lines were treated with various concentrations of SAHA. SAHA suppressed the proliferation of T and NK cell lines, although no significant difference was observed between EBV-positive and EBV-negative cell lines. SAHA induced apoptosis and/or cell cycle arrest in several T and NK cell lines. In addition, SAHA increased the expression of EBV-lytic genes and decreased the expression of EBV-latent genes. Next, EBV-positive NK cell lymphoma cells were subcutaneously inoculated into severely immunodeficient NOD/Shi-scid/IL-2Rγnull mice, and then SAHA was administered intraperitoneally. SAHA inhibited tumor progression and metastasis in the murine xenograft model. SAHA displayed a marked suppressive effect against EBV-associated T and NK cell lymphomas through either induction of apoptosis or cell cycle arrest, and may represent an alternative treatment option.

Heat shock protein 90 inhibitors repress latent membrane protein 1 (LMP1) expression and proliferation of Epstein-Barr virus-positive natural killer cell lymphoma.

Epstein-Barr virus (EBV) LMP1 is a major oncoprotein expressed in latent infection. It functions as a TNFR family member and constitutively activates cellular signals, such as NFκB, MAPK, JAK/STAT and AKT. We here screened small molecule inhibitors and isolated HSP90 inhibitors, Radicicol and 17-AAG, as candidates that suppress LMP1 expression and cell proliferation not only in EBV-positive SNK6 Natural Killer (NK) cell lymphoma cells, but also in B and T cells. Tumor formation in immuno-defficient NOD/Shi-scid/IL-2Rγ(null) (NOG) mice was also retarded. These results suggest that HSP90 inhibitors can be alternative treatments for patients with EBV-positive malignancies.

Significance of neonatal testicular sex steroids to defeminize anteroventral periventricular kisspeptin neurons and the GnRH/LH surge system in male rats.

The brain mechanism regulating gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release is sexually differentiated in rodents. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) have been suggested to be sexually dimorphic and involved in the GnRH/LH surge generation. The present study aimed to determine the significance of neonatal testicular androgen to defeminize AVPV kisspeptin expression and the GnRH/LH surge-generating system. To this end, we tested whether neonatal castration feminizes AVPV kisspeptin neurons and the LH surge-generating system in male rats and whether neonatal estradiol benzoate (EB) treatment suppresses the kisspeptin expression and the LH surge in female rats. Immunohistochemistry, in situ hybridization, and quantitative real-time RT-PCR were performed to investigate kisspeptin and Kiss1 mRNA expressions. Male rats were castrated immediately after birth, and females were treated with EB on postnatal Day 5. Neonatal castration caused an increase in AVPV kisspeptin expression at peptide and mRNA levels in the genetically male rats, and the animals showed surge-like LH release in the presence of the preovulatory level of estradiol (E2) at adulthood. On the other hand, neonatal EB treatment decreased the number of AVPV kisspeptin neurons and caused an absence of E2-induced LH surge in female rats. Semiquantitative RT-PCR analysis showed that neonatal steroidal manipulation affects Kiss1 expression but does not significantly affect gene expressions of neuropeptides (neurotensin and galanin) and enzymes or transporter for neurotransmitters (gamma-aminobutyric acid, glutamate, and dopamine) in the AVPV, suggesting that the manipulation specifically affects Kiss1 expressions. Taken together, our present results provide physiological evidence that neonatal testicular androgen causes the reduction of AVPV kisspeptin expression and failure of LH surge in genetically male rats. Thus, it is plausible that perinatal testicular androgen causes defeminization of the AVPV kisspeptin system, resulting in the loss of the surge system in male rats.