Synergistic Preclinical Activity of Bortezomib with Suberoylanilide Hydroxamic Acid (SAHA) in Primary Effusion Lymphoma (PEL)

Abstract 1650

Primary effusion lymphoma (PEL) is an aggressive subtype of non-Hodgkin lymphoma typically presenting as effusions in the serous body cavities without a contiguous tumor mass. PEL may develop in elderly immunosuppressed HIV-negative individuals but more commonly affects HIV-positive patients, accounting for 4% of all lymphomas in this population. Kaposi's sarcoma-associated herpesvirus (KSHV) is directly implicated in the pathogenesis of PEL, however in most patients the malignant B cells are also coinfected with Epstein-Barr virus which may facilitate transformation. Current chemotherapeutic approaches result in dismal outcome of PEL patients with a median survival of only 6 months. Consequently, development of new therapeutic approaches is urgently needed. Recently we reported development of the UM-PEL1 direct xenograft mice model reproducing human PEL (Sarosiek, PNAS 2010) in which bortezomib (BORT) induced virus lytic reactivation leading to malignant B cell death and transient remission of the PEL in vivo. Further improvement on this monotherapy is warranted. Recent studies have shown that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor is a highly effective viral lytic-cycle inducer. As herpesviruses are dependent on the proteasome for replication and mature viral production, induction of lytic replication with concomitant inhibition of the proteasome may provide a highly targeted strategy for eradicating KSHV infected cells without leading to increased viremia. Consequently, we hypothesized that combining BORT with SAHA may act synergistically in PEL tumors. Incubation of human PEL cell lines, UM-PEL1, BC1, BC3 and BC5 with BORT-SAHA resulted in increased apoptotis compared to individual treatment with BORT or SAHA, as assayed by flow cytometry using YO-PRO/PI staining. Concordantly, a statistically significant decrease in UM-PEL1 cell proliferation and viability, as examined by an MTT assay, was observed at 48 and 72 hours following combination therapy as compared to untreated cells or cells treated individually with BORT or SAHA. Cell cycle analysis demonstrated that BORT-SAHA combination induced more pronounced G1 cell cycle arrest and apoptosis as compared to individual treatments. SAHA induced a more robust KSHV lytic reactivation compared to BORT. Intriguingly, the BORT-SAHA combination led to an increased expression of the master lytic transactivator RTA and thymidine kinase, however the late lytic gene, K8.1, showed reduced mRNA expression relative to the individual SAHA treatment. These findings were further confirmed by immunofluorescence staining of the K8.1 protein suggesting that BORT could inhibit mature virion production in lytically reactivated malignant B-cells. To comprehensively examine the activity of the BORT-SAHA combination compared to individual BORT or SAHA treatments in vivo, we used UM-PEL1 direct xenograft model. Mice receiving intraperitoneal BORT-SAHA combination showed statistically significant prolonged survival compared to all the control treatments (p<0.001). Since PEL cells are known to be highly dependent on NF-κB for survival, we examined whether the apoptosis induced by the combination treatment was due to the inhibition of this pro-survival pathway. In contrast to our previous observations that individual BORT treatment did not alter NF-κB activity, the in vivo addition of SAHA led to NF-κB inhibition as demonstrated by gel shift assay. Moreover, Western blotting demonstrated downregulation of anti-apoptotic genes, upregulation of pro-apoptotic genes along with the rise in the p53, p21 and increased acetylation of histone 3 in the combination treated mice versus BORT alone. Further, RTA and early lytic gene expression confirmed our in vitro findings that KSHV lytic reactivation is enhanced in the BORT-SAHA treated mice compared to individual treatments. However, transcription of all late lytic genes tested (gB, K8.1, gM, ORF38, ORF67, ORF68) was uniformly inhibited in the animals treated with the BORT-SAHA as compared to SAHA alone, suggesting that the virus was unable to complete the full replicative cycle. In conclusion, this study demonstrates strong pre-clinical activity of the combination of proteasome inhibitor with HDAC inhibitor as a potent anti-PEL therapy that triggers apoptosis by prompting KSHV lytic reactivation without increasing infectious virus production.