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. 2013 Sep 15;19(18):5016-26.
doi: 10.1158/1078-0432.CCR-12-3510. Epub 2013 Jul 23.

A purine scaffold HSP90 inhibitor BIIB021 has selective activity against KSHV-associated primary effusion lymphoma and blocks vFLIP K13-induced NF-κB

Ramakrishnan Gopalakrishnan  1 , Hittu Matta, Preet M Chaudhary
Affiliations

A purine scaffold HSP90 inhibitor BIIB021 has selective activity against KSHV-associated primary effusion lymphoma and blocks vFLIP K13-induced NF-κB

Ramakrishnan Gopalakrishnan et al. Clin Cancer Res. .

Abstract

Purpose: Kaposi sarcoma-associated herpes virus (KSHV)-associated primary effusion lymphomas (PEL) have extremely poor prognosis when treated with conventional chemotherapy. KSHV-encoded viral FLICE-inhibitory protein (vFLIP) K13 binds to the IkappaB kinase (IKK) complex to constitutively activate the NF-κB pathway, which has been shown to be essential for the survival and proliferation of PEL cells. The molecular chaperone HSP90 is a component of the IKK complex and is required for its activity.

Experimental design: We have analyzed the effect of HSP90 inhibitors on the survival and proliferation of PEL cells and on the activity of the NF-κB pathway.

Results: We show that BIIB021, a purine scaffold-based orally administrable HSP90 inhibitor, shows preferential cytotoxicity toward PEL cells as compared with non-PEL cells. The cytotoxic effect of BIIB021 against PEL was associated with induction of cell-cycle arrest and apoptosis. BIIB021 blocked the expression of a number of cellular proteins involved in the regulation of cell cycle and apoptosis. BIIB021 also blocked constitutive NF-κB activity present in PEL cells, in part, by blocking the interaction of vFLIP K13 with the IKK complex subunits. In a xenograft model of PEL, BIIB021 significantly reduced tumor growth.

Conclusion: BIIB021 blocks constitutive NF-κB activity in PEL and shows preferential antitumor activity against PEL in vitro and in vivo. BIIB021 may be a promising agent for treatment of PEL.

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Conflict of interest statement

Conflict of Interest: The authors declare no competing financial interests

Figures

Figure 1
Figure 1
HSP90 inhibitors efficiently target KSHV associated primary effusion lymphoma. A, Chemical structures of BIIB021, 17-DMAG and NVP-AUY922. B, The indicated PEL and non-PEL cell lines were treated with increasing concentrations of BIIB021, 17-DMAG and NVP-AUY922 for 72 h and cell viability was measured using an MTS assay as described in materials and methods section. A grey circle shows preferential toxicity of BIIB021 on PEL cell lines at 100 nM. C, BC-1, BC-3, BJAB and Namalwa cells were treated with increasing concentrations of BIIB021 for 24 and 48 h and cell viability was measured by MTS assay. The values shown are mean± SEM of two independent experiments performed in triplicate.
Figure 2
Figure 2
BIIB021 induces cell cycle arrest and apoptosis in PEL cells. A, left, cell-cycle analysis of control and BIIB021 treated BC-1 and BC-3 cell lines. BIIB021 (200 nM for 48 h) results in G1 arrest in BC-1 cells and G2/M arrest in BC-3 cells. Cells were stained with propidium iodide (PI) and analyzed by flow cytometry. The data is a representative of three individual experiments. Right, Western blot analysis showing the effect of BIIB021 on the expression of HSP90 client proteins involved in cell-cycle regulation. Lack of effect on Cyclooxygenase-2 (COX-2), a protein that is not an HSP90 client, shows the specificity of the observed effect. B, left, BC-1 and BC-3 cells were treated with 200 nM BIIB021 or DMSO control for 48 h. Cells were then stained with Hoechst 33258 and photographed. Right, BC-1 and BC-3 cells were treated with 200 nM BIIB021 or DMSO control for 48 h, stained with Annexin-V-FITC/PI and analyzed for apoptosis by flow cytometry. C, Western blot showing the effect of BIIB021 on the cleavage of PARP, activation of caspases and expression of BCL-2 family members and p53. D, Western blot showing the effect of BIIB021 (100 nM, 48h), 17-DMAG (100 nM, 48h) and NVP-AUY922 (50 nM, 48h) treatment on the cleavage of PARP in the indicated cell lines. FL – Full length; Cl - Cleaved.
Figure 3
Figure 3
Inhibition of NF-κB pathway by BIIB021in PEL cells. A, left, BC-1/NF-κB-Luc and BC-3/NF-κB-Luc cells were treated with increasing concentrations of BIIB021 or vehicle control for 15 h and cell lysates used for measurement of luciferase activity. Values shown are the mean ± SEM from one representative experiment out of three performed in duplicate. Asterisks (***) and (****) indicate significance at levels of p≤0.001 and p≤0.0001, respectively. Right, an ELISA-based NF-kB binding assay showing reduced p65/RelA DNA-binding activity in the nuclear extracts of BC-1 and BC-3 cells treated with BIIB021 (200 nM for 48 h). Values shown are the mean ± SEM from one representative experiment out of three performed in duplicate. Asterisks (****) indicate significance at levels of p≤0.0001. B, BIIB021 blocks IL-6 secretion. IL-6 levels were measured using ELISA in the supernatants of BC-1 and BC-3 cells treated with 200 nM of BIIB021 or DMSO control for 24, 48 and 72 h. The values (mean ± SEM) shown are from a representative of three independent experiments performed in triplicate. C, Western blots showing the effect of BIIB021 on the expression of phospho-IκBα and total IκBα, IKKα/β and NEMO, and NF-κB target proteins A20 and XIAP1. D, PathScan ELISA for phospho-IKKα (Ser176/180) and phospho-IKKβ (Ser177/181) showing inhibition of IKKα/β phosphorylation by BIIB021 (200 nM for 48 h) in BC-1 and BC-3 cells. A representative of two independent experiments performed in triplicate. Statistically significant differences are shown by asterisks (***) at a level of p≤0.001 and (****) at level of p≤ 0.0001.
Figure 4
Figure 4
BIIB021 down-regulates vFLIP K13 expression and blocks K13-induced NF-κB activation. A, left, Western blots showing a reduction in K13, LANA and vCyclin protein levels in BC-1 and BC-3 cells treated for 48 h with the indicated concentrations of BIIB021. Right, qRT-PCR analysis showing a decline in K13, LANA and v-Cyclin mRNA expression in BC-1 and BC-3 cells following treatment with 200 nM BIIB021 for 24 h. Real-time PCR reactions were performed in triplicate and the data is presented as fold change in target gene expression (mean±SEM) from a representative of two independent experiments. B, Effect of BIIB021 on the protein stability. BC-1 and BC-3 cells were treated with vehicle or BIIB021 in the presence of 5 µg/ml cycloheximide (CHX) for 0, 3, 6, 12, 18 and 24 hours, respectively. Whole cells lysates were immunoblotted for indicated proteins. Semi-quantitative analysis of the immunoblots is presented in Supplementary Fig.1. C, upper panel, luciferase-based reporter assay showing inhibition of K13-induced NF-κB transcriptional activity by HSP90 inhibitors. 293-pSLIK-TO-K13/NF-κB-Luc cells were treated with increasing concentrations of BIIB021, 17-DMAG and NVP-AUY922 for 2 h prior to induction of K13 expression by addition of doxycycline (DOX, 500 ng/ml). After 15 h, cell lysates were prepared to measure the NF-κB luciferase activity. The values (mean±SEM) shown are from a representative of three independent experiments performed in triplicate. Asterisks indicate statistical significance as explained for Figure 3D, ns- not significant. Lower panel, Western blot analysis from the cell lysates confirmed that inhibition of NF-κB activity by BIIB021 is accompanied by inhibition of IκBα phosphorylation and downregulation of NF-κB target gene (i.e. A20) expression but is not due to a block in K13 expression.
Figure 5
Figure 5
Effect of HSP90 inhibitors on K13-IKK complex interaction and KSHV lytic genes. A, left, co-IP assay showing disruption of K13-IKK complex interaction by HSP90 inhibitors.293-pSLIK-TO-K13/NF-κB-Luc cells were treated with BIIB021 (200 nM), 17-DMAG (200nM), NVP-AUY922 (100nM) and DMSO control for 2 h followed by treatment with doxycycline (DOX, 500 ng/ml for 15 h) to induce K13 expression. Whole cell lysates (WCL) were immunoprecipitated (IP) using a control antibody (C) or FLAG antibody (F) and subjected to SDS-PAGE and immunoblotting (IB) with the indicated antibodies. UT- Untreated. Right, a co-IP assay showing reduced interaction between K13 and IKK complex in FLAG-tagged K13 expressing BCBL-1 cells upon treatment with 200 nM BIIB021 for 24 h. B, a co-IP assay showing K13 interacts with HSP90 in wild-type Jurkat cells but not in NEMO-deficient Jurkat cells. Whole cell lysates (WCL) were prepared from wild-type Jurkat cells and NEMO-deficient Jurkat cells expressing FLAG-K13 and immunoprecipitated (IP) using a control antibody (C) or FLAG antibody (F) and immunoblotted (IB) with the indicated antibodies. C, Western blots showing down-regulation of RELB and p100 expression and p100 to p52 processing in PEL cells upon treatment with BIIB021 for 48 h. D, BIIB021 fails to induce expression of KSHV lytic proteins RTA and vIL6 and blocks their expression induced by TPA (12-O-tetradecanoylphorbol-13-acetate). BCBL-1 cells were treated with the indicated doses of BIIB021 for 2 h followed by treatment withTPA (100ng/ml) for 48 h. Cell lysates were subjected to Western blot analysis using the indicated antibodies. NS- Non Specific.
Figure 6
Figure 6
BIIB021 impairs in vivo growth of PEL in a mouse xenograft model. A, Athymic NCr-nu/nu mice with established subcutaneous BC-1 tumors were treated with BIIB021 (100 mg/kg body weight; n=7) or vehicle control (n=6) for 14 days. Tumor volumes are presented as mean±SEM from each group. Asterisks indicate significance at levels of p≤0.05. Tumor weight (in grams) of animals treated with either vehicle control or BIIB021 on day 14. Asterisks indicate significance at levels of p≤0.05. Circulating levels of hIL-6 on day 14 in the plasma of animals treated with either vehicle control or BIIB021. Asterisks indicate significance at levels of p≤0.05. B, Gross representative images of mice, tumor and spleen from groups of animals treated with either vehicle control or BIIB021. C, Immunohistochemical staining showing decreased expressions of IKKα/β, LANA along with positive staining for cleaved caspase-3, indicative of apoptosis, in the tumors of mice treated with BIIB021. D, TUNEL staining showing increased apoptosis in tumor tissues extracted from animals treated with BIIB021. Asterisks (***) indicate significance at levels of p≤0.001.
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