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Case Reports
. 2010 Jul 20;107(29):13069-74.
doi: 10.1073/pnas.1002985107. Epub 2010 Jul 6.

Efficacy of bortezomib in a direct xenograft model of primary effusion lymphoma

Kristopher A Sarosiek  1 , Lucas E Cavallin, Shruti Bhatt, Ngoc L Toomey, Yasodha Natkunam, Wilfredo Blasini, Andrew J Gentles, Juan Carlos Ramos, Enrique A Mesri, Izidore S Lossos
Affiliations
Case Reports

Efficacy of bortezomib in a direct xenograft model of primary effusion lymphoma

Kristopher A Sarosiek et al. Proc Natl Acad Sci U S A. .

Abstract

Primary effusion lymphoma (PEL) is an aggressive B-cell lymphoma most commonly diagnosed in HIV-positive patients and universally associated with Kaposi's sarcoma-associated herpesvirus (KSHV). Chemotherapy treatment of PEL yields only short-term remissions in the vast majority of patients, but efforts to develop superior therapeutic approaches have been impeded by lack of animal models that accurately mimic human disease. To address this issue, we developed a direct xenograft model, UM-PEL-1, by transferring freshly isolated human PEL cells into the peritoneal cavities of NOD/SCID mice without in vitro cell growth to avoid the changes in KSHV gene expression evident in cultured cells. We used this model to show that bortezomib induces PEL remission and extends overall survival of mice bearing lymphomatous effusions. The proapoptotic effects of bortezomib are not mediated by inhibition of the prosurvival NF-kappaB pathway or by induction of a terminal unfolded protein response. Transcriptome analysis by genomic arrays revealed that bortezomib down-regulated cell-cycle progression, DNA replication, and Myc-target genes. Furthermore, we demonstrate that in vivo treatment with either bortezomib or doxorubicin induces KSHV lytic reactivation. These reactivations were temporally distinct, and this difference may help elucidate the therapeutic window for use of antivirals concurrently with chemotherapy. Our findings show that this direct xenograft model can be used for testing novel PEL therapeutic strategies and also can provide a rational basis for evaluation of bortezomib in clinical trials.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Establishment and characterization of UM-PEL-1. (A) Mice 7 d after mock injection or injection with UM-PEL-1 cells. (B and C) H&E-stained sections of mouse tissue show a tumor nodule on the serosal surface of the bowel wall (B) and tumor cells infiltrating pancreatic parenchyma (C). (D) Tumor cells are medium to large with pleomorphic nuclear outlines, prominent nucleoli, and abundant cytoplasm and exhibit plasmacytoid differentiation. (E–J) Immunohistologic stains show that the tumor cells are positive for CD45 (E), weakly positive to absent CD20 (F), and strongly positive for CD138 (G). Tumor cells also harbor KSHV (H) and EBV RNA detected by an in situ EBER probe (I). Tumor cells lack the B-cell transcription factor PAX5 (J). (Original magnifications: B and C, 100×; D–J, 600×.)
Fig. 2.
Fig. 2.
UM-PEL-1 cells express KSHV antigens and produce infectious virions. (A) UM-PEL-1 cells stained for latent antigens LANA and Kaposin. (B and C) UM-PEL-1 cells were treated with Trichostatin A (TSA, 40 nM) for 72 h, and real-time qPCR was performed (B) as well as immunostaining of late lytic antigen K8.1 (C). (D) LANA staining of Vero cells (KSHV-negative cell line) after exposure to supernatants from untreated or TSA-treated (400 nM) UM-PEL-1 cells. (E) Quantification of LANA-positive Vero cells after supernatant treatment. Ten fields were counted for each condition, and data represent means ± SD. LANA-positive Vero cells are indicative of KSHV de novo infection. All results are representative of three independent experiments.
Fig. 3.
Fig. 3.
KSHV gene expression is microenvironment dependent. (A) Frozen-section immunostaining of PEL solid lymphomas for LANA, Kaposin, and late lytic antigen K8.1. Insets show areas of stained tumors at higher magnification. (Magnification: A, ×400; Insets, ×1,600.) (B and C) In vitro-cultured UM-PEL-1c cells have higher viral loads than ascites and solid tumors. mRNA levels of KSHV latent and lytic genes were determined by qPCR analysis. Relative expression levels were normalized to in vitro-cultured UM-PEL-1c cells (B). DNA was isolated, and KSHV viral loads were determined by qPCR using LANA-specific primers (C). All results are representative of three independent experiments.
Fig. 4.
Fig. 4.
Bortezomib potently induces apoptosis and lytic reactivation in UM-PEL-1 cells and improves overall survival of PEL mice. (A) In vitro-cultured UM-PEL-1c cells were treated with bortezomib or doxorubicin at the indicated doses. Cell viability was assayed by YO-PRO/PI staining and flow cytometric analysis at indicated time points. Results are representative of three independent experiments. (B) Mice were injected i.p. with 2 × 107 UM-PEL-1 cells. After 3 d mice were treated twice weekly for 3 wk with PBS, bortezomib (BORT) at 0.3 mg/kg, or doxorubicin (DOX) at 1.25 mg/kg. Mice were killed when moribund or showing signs of discomfort. Results are representative of two independent experiments. (C and D) UM-PEL-1 mRNA levels of latent, early, and late lytic genes were determined by qPCR and normalized to PBS control. Each group was composed of three mice. Bortezomib-treated (C) and doxorubicin-treated (D) mice were analyzed at day 1 and day 7 after treatment. Data from C and D represent means ± SD.
Fig. 5.
Fig. 5.
Bortezomib does not inhibit NF-κB or induce a terminal UPR in UM-PEL-1 cells but does induce changes in transcriptional programs. (A) Immediately after removal from lymphoma-bearing mice, UM-PEL-1 cells were treated with the NF-κB inhibitor Bay-11 (5 μM) or proteasome inhibitor bortezomib (20 nM) for the indicated time periods. Nuclear extracts were prepared and assayed by EMSA using NF-κB–specific consensus oligonucleotides as probes. Results are representative of three independent experiments. (B) UM-PEL-1c cells were treated with bortezomib for the specified time periods. Whole-cell lysates were prepared, and Western blots were performed with specified antibodies. Immunoblotting for actin served as a loading control. Normalized densitometry of the phosphorylated eIF-2α (p-eIF-2α)/eIF-2α ratio is shown. The value in untreated cells was defined arbitrarily as 1. The results are representative of three independent experiments. (C) Sets of genes whose expression is significantly repressed by treatment with bortezomib compared with PBS. The top bar (not to scale) represents the ranked list of all genes ordered by their expression in bortezomib-treated versus PBS-treated PEL. Vertical black bars represent the ranking of member genes of the indicated gene sets. All FDRs < 0.001; see also Table S2.
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