Efficacious proteasome/HDAC inhibitor combination therapy for primary effusion lymphoma
Shruti Bhatt, … , Izidore S. Lossos, Juan Carlos Ramos
Shruti Bhatt, … , Izidore S. Lossos, Juan Carlos Ramos
Published May 1, 2013
Citation Information: J Clin Invest. 2013;123(6):2616-2628. https://doi.org/10.1172/JCI64503.
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Efficacious proteasome/HDAC inhibitor combination therapy for primary effusion lymphoma

Abstract

Primary effusion lymphoma (PEL) is a rare form of aggressive B cell lymphoma caused by Kaposi’s sarcoma-associated herpesvirus (KSHV). Current chemotherapy approaches result in dismal outcomes, and there is an urgent need for new PEL therapies. Previously, we established, in a direct xenograft model of PEL-bearing immune-compromised mice, that treatment with the proteasome inhibitor, bortezomib (Btz), increased survival relative to that after treatment with doxorubicin. Herein, we demonstrate that the combination of Btz with the histone deacetylase (HDAC) inhibitor suberoylanilidehydroxamic acid (SAHA, also known as vorinostat) potently reactivates KSHV lytic replication and induces PEL cell death, resulting in significantly prolonged survival of PEL-bearing mice. Importantly, Btz blocked KSHV late lytic gene expression, terminally inhibiting the full lytic cascade and production of infectious virus in vivo. Btz treatment led to caspase activation and induced DNA damage, as evidenced by the accumulation of phosphorylated γH2AX and p53. The addition of SAHA to Btz treatment was synergistic, as SAHA induced early acetylation of p53 and reduced interaction with its negative regulator MDM2, augmenting the effects of Btz. The eradication of KSHV-infected PEL cells without increased viremia in mice provides a strong rationale for using the proteasome/HDAC inhibitor combination therapy in PEL.

Authors

Shruti Bhatt, Brittany M. Ashlock, Ngoc L. Toomey, Luis A. Diaz, Enrique A. Mesri, Izidore S. Lossos, Juan Carlos Ramos

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Figure 5

SAHA-induced acetylation of p53 led to dissociation from MDM2, contributing to cell apoptosis.

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SAHA-induced acetylation of p53 led to dissociation from MDM2, contribut...
(A and B) Tumor-bearing mice were treated with a single dose of DMSO, SAHA, Btz, or Btz/SAHA, and cell protein lysates were prepared from peritoneal effusions. (A) Immunoblotting for acetylated p53 (Lys-382) and H3 at 2 hours and 24 hours after treatment. (B) Immunoblots for MDM2 and p53 in whole cell lysates prior to immunoprecipitation (left). Immunoblots showing MDM2 and p53 in complexes captured by immunoprecipitation using indicated antibodies (right top). Complex-free p53 in flow through supernatant not captured in MDM2 antibody resin bead complexes (right bottom). (C–F) UM-PEL-1 cells stably expressing p53-specific shRNA or nonsilencing (N.S.) vectors were passaged as xenografts in mice. Mice (n = 2 per group) were treated with a single dose of DMSO, Btz, SAHA, or Btz/SAHA, and at 24 hours, cells were harvested from peritoneal effusions. (C and D) shRNA-mediated knockdown of p53 at the mRNA level in untreated UM-PEL-1 cells and partial abrogation of Btz-induced p53 levels measured by qRT-PCR and immunoblotting. (E) In vivo apoptosis of UM-PEL-1 cells examined by YO-PRO-1/PI staining, followed by flow cytometry. Data represent values relative to normalized DMSO-treated control mice. (F) Immunoblotting of p21 and activated caspase-3. (G) UM-PEL-1 cells harvested from p53 shRNA– or nonsilencing vector–expressing xenograft mice were treated in culture with 2.5 nM Btz, 0.5 μM SAHA, 7 μM nutlin-3a, or indicated combinations for 24 hours. The percentage of dead cells was measured by annexin V/PI staining. Data represent values relative to normalized untreated cells. (A–C) Experiments were repeated twice in triplicates. Error bars represent SEM.