Identification of a Novel Inhibitor That Selectively Targets NF-κB Activity in KSHV-Infected Lymphoma Cells

Abstract 160

Kaposi's sarcoma herpesvirus (KSHV/HHV-8), a member of the γ-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 lymphoma cells infected by KSHV are protected from apoptotic stress. vFLIP is the viral protein largely responsible for the constitutive NF-κB activity, and is essential for PEL cell survival. Using a subclone of the BC3 PEL cell line that stably expresses firefly luciferase under the control of an NF-κB promoter (BC3/NFκB-luc), we performed high throughput screening (HTS) of compound libraries to identify inhibitors of vFLIP/NF-κB in PEL cells.

Assay optimization was done using the NCI Training set, following which screening of the NIH diversity set at 5μM picked up 60 hits (with >50% inhibition) from a total of ∼2000 compounds. To further test these compounds, we introduced a renilla luciferase controlled by a constitutive promoter (retroviral LTR) to obtain a double reporter cell line (BC3-REN#3). Three compounds showed preferential inhibition of firefly luciferase in the double-reporter cell line, and/or preferential inhibition of the BC3/NFκB-luc cell line when compared to a control non-lymphoid cell line with constitutive luciferase expression (U251-pGL3). Analogs of these three compounds were further tested in BC3-REN#3 cells, and three analogs were identified with increased activity/specificity for NF-κB.

One of the compounds identified so far, here designated NSC-E, has shown dose-dependent inhibition of NF-κB in PEL cells, as evaluated independently by reporter and electrophoretic mobility shift assays (EMSA). Nuclear translocation of the NF-κB active subunit p65, but not inactive p50 is inhibited by this compound, although it does not alter upstream events in the NF-κB pathway, such as IKKα/β phosphorylation or IκBα degradation. Studies are ongoing to elucidate the direct molecular target of NSC-E.

Inhibition of NF-κB by NSC-E occurs only in PEL cell lines, and not in other lymphoma cell types. In addition, this compound preferentially inhibits viability of KSHV-infected lymphoma cells, at LC₅₀s ranging from 18nM to ∼200nM in various PEL cell lines tested. In contrast, an effect on EBV-infected lymphoma cell viability is only apparent at much higher doses (LC₅₀s in the 20–80μM range). Hence, ∼300X the KSHV-effective dose of NSC-E would be required to achieve inhibition in EBV-associated or virus-negative lymphomas, suggesting this inhibitor is very KSHV-specific.

Finally, NSC-E was evaluated in a traceable reporter xenograft mouse model of KSHV-associated PEL using in vivo imaging, and found to be highly efficacious against both incipient and well-established tumors, with 100% tumor responses and no apparent toxicity. Thus, we have identified a novel, specific, and highly effective inhibitor of KSHV-associated lymphoma that demonstrates clear pre-clinical promise for therapeutic applications.