Abstract 4829: A purine scaffold Hsp90 inhibitor has antitumor activity in KSHV-associated malignancies by suppressing vFLIP

Abstract

Abstract Background: Kaposi's sarcoma herpesvirus (KSHV/ HHV-8), a member of the α-herpesvirus family of human DNA viruses, is the etiologic agent of several malignancies in immune-compromised individuals, such as Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). Activated NF-κB is a critical mechanism by which KSHV-infected PEL cells are protected from apoptosis. KSHV vFLIP has been identified as a viral oncogene that is responsible for NF-κB-dependent anti-apoptotic gene expression in PEL cells, and also prevents autophagy in KSHV-infected endothelial cells. In particular, the IKK signaling complex consisting of IKKα, IKKα, IKKα, vFLIP and Hsp90 was shown to be essential for survival in PEL cells. The chaperone protein Hsp90 binds client proteins involved in the regulation of cell survival and apoptosis signal transduction, including Akt, IKK complex, and KSHV vFLIP. A lack of Hsp90 causes protein misfolding, ubiquitination and degradation. The Hsp90 inhibitor geldanamycin was previously tested in PEL cells and shown to inhibit activity of the IKK complex in vitro. However, geldanamycin is of limited therapeutic potential due to its undesirable pharmacophysiology. We tested a new purine-scaffold Hsp90 inhibitor with high selectivity for tumor versus normal cell Hsp90, which is water-soluble with high oral bioavailability and excellent therapeutic window. Materials and methods: We evaluated the sensitivity of several KSHV infected and uninfected cell lines to treatment with this inhibitor, designated PU-H71. We performed viability and NF-κB reporter luciferase assays, and immunoblot and qRT-PCR analyses of cellular and viral proteins. Finally, we used a mouse PEL xenograft model and in vivo imaging to assess tumor responses to PU-H71. Results: We found all KSHV-positive PEL cell lines to be exquisitely sensitive when compared to uninfected lymphoma cells, with LC50s in the nanomolar range. PU-H71 was shown to induce PEL cell death by apoptosis and autophagy within 48 hours of treatment. PU-H71 also showed preferential toxicity in an in vitro model of KS. Pulldown demonstrated association of the compound with the active vFLIP-IKKα signalosome. Western blot analysis indicated that the IKK signaling complex was destabilized, and components vFLIP and IKKα were degraded upon PU-H71 treatment, resulting in inhibition of NF-κB signaling, as confirmed by reporter luciferase assay. qRT-PCR and reporter analysis indicated low levels of concomitant lytic reactivation. PU-H71 was further shown to inhibit progression of tumor spread and confer a significant survival advantage (p<0.02) in a mouse xenograft model of PEL, with persistence of IKKα destabilization ex vivo. Conclusions: Our findings demonstrate that Hsp90 inhibition with PU-H71 leads to reduced vFLIP levels in KSHV-infected cells, and to tumor responses. Thus, PU-H71 is a promising targeted approach for the treatment of KS and PEL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4829. doi:1538-7445.AM2012-4829