HIV-infected cells are major inducers of plasmacytoid dendritic cell interferon production, maturation, and migration

AbstractPlasmacytoid dendritic cells (PDC), natural type-1 interferon (IFN) producing cells, could play a role in the innate anti-HIV immune response. Previous reports indicated that PDC IFN production is induced by HIV. Our results show a more robust IFN induction when purified PDC (>95%) were exposed to HIV-infected cells. This effect was not observed with non-viable cells, DNA, and RNA extracted from infected cells, and viral proteins. The response was blocked by anti-CD4 and neutralizing anti-gp120 antibodies as well as soluble CD4. IFN induction by HIV-infected cells was also prevented by low-dose chloroquine, which inhibits endosomal acidification. PDC IFN release resulted in reduced HIV production by infected CD4+ cells, supporting an anti-HIV activity of PDC. Stimulated CD4+ cells induced PDC activation and maturation; markers for PDC migration (CCR7) were enhanced by HIV-infected CD4+ cells only. This latter finding could explain the decline in circulating PDC in HIV-infected individuals

Zidovudine-based lytic-inducing chemotherapy for Epstein–Barr virus-related lymphomas

Treatment of Epstein–Barr virus (EBV)-related lymphomas with lytic-inducing agents is an attractive targeted approach for eliminating virus-infected tumor cells. Zidovudine (AZT) is an excellent substrate for EBV-thymidine kinase: it can induce EBV lytic gene expression and apoptosis in primary EBV+ lymphoma cell lines. We hypothesized that the combination of AZT with lytic-inducing chemotherapy agents would be effective in treating EBV+ lymphomas. We report a retrospective analysis of 19 patients with aggressive EBV+ non-Hodgkin lymphoma, including nine cases of acquired immune deficiency syndrome-associated primary central nervous system lymphoma (AIDSPCNSL) treated with AZT-based chemotherapy. Our results demonstrate that high-dose AZT–methotrexate is efficacious in treating highly aggressive systemic EBV+ lymphomas in the upfront setting. In primary EBV+ lymphoma cell lines, the combination of AZT with hydroxyurea resulted in synergistic EBV lytic induction and cell death. Further, AZT–hydroxyurea treatment resulted in dramatic responses in patients with AIDSPCNSL. The combination of AZT with chemotherapy, especially lytic-inducing agents, should be explored further in clinical trials for the treatment of EBV-related lymphomas

Characterization and Validation of Preclinical Models of KSHV-induced Malignancies to Elucidate Antineoplastic and Antiviral Therapeutic Approaches

Kaposi’s sarcoma (KS) and Primary effusion lymphoma (PEL) are two Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) –induced cancers that are clinically challenging to treat and often have poor prognoses. While these diseases have declined dramatically in the developed world, they are responsible for significant morbidity and mortality in the developing world. Further, current treatments are often associated with considerable toxicity. While KS and PEL are composed of cells harboring KSHV in the latent state, the paracrine hypothesis of tumor development proposes that the tumor is actually being driven by the minority of cells undergoing lytic replication. Therefore, it is believed that potential targets for therapy are the KSHV-infected cells undergoing lytic replication. Lytic replication ensures the production and spread of virions, and allows for the expression of potentially pathogenic genes which have proposed roles in the paracrine neoplasia thought to drive tumorigenesis. The lytic-inductive paradigm for therapy proposes to induce massive lytic reactivation, while concurrently administering a potent antiviral, thereby specifically causing the death of virally infected cells while abrogating the potential increase in viremia, which may lead to unwanted negative clinical sequelae. However, the studying induction of and inhibition of viral lytic replication are generally reliant upon in vitro chemical induction which may not recapitulate the temporally ordered cascade of events that is thought to occur in vivo. Facile animal models are urgently needed in which to test antineoplastic and antiviral strategies for the development of effective targeted therapies. Herein, we test the lytic-inductive paradigm in a direct xenotransplant murine model of PEL. We use two clinically approved drugs, one, Vorinostat (suberoylanilide hydroxamic acid, SAHA) is a histone deacetylase inhibitor and a known potent inducer of viral replication. The other, Velcade (Bortezomib, Btz) is an inhibitor of the 26S proteasome and has been shown in other viral models to inhibit viral replication. We found that in PEL inoculated mice, the combination of SAHA and Btz leads to significantly increased survival over mice treated with the single agents. Further, we show that there is massive apoptosis with the combination that correlates with strong lytic induction of KSHV. Importantly, we also show that a potential unwanted side effect of lytic replication, namely increased viremia in vivo, is abrogated by Btz. Indeed, Btz appears to have very potent antiviral effects in the setting of KSHV while the combination of the SAHA and Btz have very potent antitumoral effects. Our results suggest that the lytic-inductive paradigm for treating PEL, and potentially other herpesvirus-induced cancers, may be a viable option in the setting of an immunocompromised host. To test the lytic-inductive paradigm in the context of KS, a solid tumor, we needed to develop a suitable model. By employing murine bone marrow-derived endothelial-lineage cells (mEC) we have now developed two novel productively infected murine models of KS. We show that the mEC are non-tumorigenic when uninfected, but, upon infection with rKSHV.219, a recombinant replication competent virus, the KSHV-infected cells efficiently form tumors that pathologically, phenotypically and molecularly resemble Kaposi’s sarcoma. The tumors are composed of LANA positive spindle-cells and that express antigens associated with human KS spindle-cells such as podoplanin and CD31. Further, the virus is able to transcribe a wide array of viral genes representing all stages of the viral replicative cycle culminating with the in vivo production of a herpesvirus-like particle of the correct size and morphology to be rKSHV.219. Given the productive nature of the tumors, these models should provide excellent substrates in which to test the lytic-inductive paradigm for the treatment of KS along with other antineoplastic and antiviral strategies.</p

Productively infected murine Kaposi's sarcoma-like tumors define new animal models for studying and targeting KSHV oncogenesis and replication.

Kaposi's sarcoma (KS) is an AIDS-defining cancer caused by the KS-associated herpesvirus (KSHV). KS tumors are composed of KSHV-infected spindle cells of vascular origin with aberrant neovascularization and erythrocyte extravasation. KSHV genes expressed during both latent and lytic replicative cycles play important roles in viral oncogenesis. Animal models able to recapitulate both viral and host biological characteristics of KS are needed to elucidate oncogenic mechanisms, for developing targeted therapies, and to trace cellular components of KS ontogeny. Herein, we describe two new murine models of Kaposi's sarcoma. We found that murine bone marrow-derived cells, whether established in culture or isolated from fresh murine bone marrow, were infectable with rKSHV.219, formed KS-like tumors in immunocompromised mice and produced mature herpesvirus-like virions in vivo. Further, we show in vivo that the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat) enhanced viral lytic reactivation. We propose that these novel models are ideal for studying both viral and host contributions to KSHV-induced oncogenesis as well as for testing virally-targeted antitumor strategies for the treatment of Kaposi's sarcoma. Furthermore, our isolation of bone marrow-derived cell populations containing a cell type that, when infected with KSHV, renders a tumorigenic KS-like spindle cell, should facilitate systematic identification of KS progenitor cells

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

Abstract 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. Disclosures: No relevant conflicts of interest to declare

Preclinical Activity of Brentuximab Vedotin (SGN-35) in Primary Effusion Lymphoma (PEL)

Abstract Abstract 3728 Primary effusion lymphoma (PEL) is a distinct and aggressive subtype of non-Hodgkin lymphoma (NHL) commonly presenting with pleural, peritoneal, or pericardial malignant effusions usually without a contiguous tumor mass. PEL is most commonly diagnosed in HIV-positive patients, accounting for 4% of all NHLs in this population, yet may also develop in immunosuppressed HIV-negative individuals. While Human Herpes Virus 8 (HHV8 or Kaposi's sarcoma-associated herpesvirus) is directly implicated in the oncogenesis of this lymphoma, most PEL cases are also associated with Epstein-Barr virus and the combination of the two may facilitate transformation. The tumor cells exhibit plasmablastic features and express CD45, CD38, CD138, HHV8 and CD30. PEL is an aggressive tumor characterized by a short median survival of only 6 months with current therapeutic approaches underscoring the urgent need for development of new therapeutics. Brentuximab vedotin (SGN-35) is an antibody-drug conjugate (ADC) comprised of an anti-CD30 monoclonal antibody cAC10 conjugated by a protease-cleavable dipeptide linker to a potent cell killing agent monomethyl auristatin E (MMAE). Following binding to CD30, brentuximab vedotin is rapidly internalized and is transported to lysosomes, where the peptide linker is selectively cleaved allowing binding of the released MMAE to tubulin and leading to cell cycle arrest and apoptosis. Brentuximab vedotin was recently reported to have promising antitumor activity in CD30 expressing tumors, such as Hodgkin and Anaplastic large cell lymphomas. Since PEL tumors are reported to express CD30, we have hypothesized that brentuximab vedotin might be effective in the treatment of this NHL subtype. Initially, we have confirmed by flow cytometry the expression of CD30 on PEL cell lines (UM-PEL 1, UM-PEL 3, BC-1 and BC-3), and by review of immunohistochemistry and flow cytometry results in patients with previous diagnosis of PEL at our institution. To examine in vitro potency of brentuximab vedotin, UM-PEL 1, UM-PEL 3, BC-1 and BC-3 PEL cell lines were treated with brentuximab vedotin at concentration ranging from 0–100 micrograms/ml. Staining with YO-PRO and Propidium Iodide (PI) demonstrated dose dependent cell apoptosis and death in all the cell lines at 72 hours post treatment. In contrast, control IgG conjugated with MMAE failed to induce apoptosis and cell death of PEL cell lines confirming specific brentuximab vedotin cytotoxicity. Furthermore, brentuximab vedotin decreased proliferation of PEL cells at 48 hours leading to a complete proliferation arrest at 72 hours, as measured by MTS assay. These effects were absent after equivalent doses of control IgG conjugated drug treatment. Supportive to this, labeling of cells with PI to detect active DNA content by flow cytometry showed that bretuximab vedotin induced growth arrest in G2/M phase. To further establish the anti-tumor potential of brentuximab vedotin in vivo, we used the direct xenograft UM-PEL 1 model, established in our laboratory (Sarosiek, PNAS 2010), which mimics human PEL tumors. UM-PEL 1 bearing mice were injected intraperitoneally 3 times a week with brentuximab vedotin or control IgG conjugated MMAE for 4 weeks. Brentuximab vedotin treatment markedly prolonged overall survival of UM-PEL-1 bearing mice compared to controls (p Disclosures: No relevant conflicts of interest to declare

Replacing BACK36 in mECK36 cells for rKSHV.219 generates the tumorigenic cells, mECK^null.rK.

<p>(A) Graph depicting mECK36 tumor kinetics in athymic nu/nu mice. 3×10⁶ mECK36 cells were subcutaneously injected into the hind flanks of 3 athymic nu/nu mice. Concurrently, 3×10⁶ mECK^null cells were subcutaneously injected into another group of 10 athymic nu/nu mice. Within 4–6 weeks solid mECK36 tumors were palpable and growth was monitored by caliper measurement (open squares). Error bars represent the standard deviation between 3 different tumors. mECK^null cells did not form tumors (black dots). (B) Fluorescence microscopy of mECK^null.rK. <i>In vitro</i>, mECK^null.rK express GFP constitutively, indicating rKSHV.219 infection, and maintain tight latency as determined by the absence of RFP expression. (C) Cell cultures of mECK^null.rK were prepared for immunofluorescence for the KSHV LANA which exhibited the classic punctate nuclear pattern of the protein.</p

rKSHV.219 lytic replication <i>in vivo</i> results in productively infected tumors culminating in the formation of virus-like particles.

<p>(A) rKSHV.219 lytic gene expression increases <i>in vivo</i> relative to mECK^null.rK133 cells in culture during tumorigenesis. RNA was isolated from tumors and cells in culture for analysis of rKSHV.219 gene expression by qRT-PCR. A representative comparative analysis is shown; error bars represent the SD of experimental duplicates. (B) RT-PCR analysis of rKSHV.219 transcripts was performed as a preliminary confirmation that the virus was able to express genes representative of the entire viral replicative cycle. RNA was isolated from tumors, reverse transcribed and run on a 3% agarose gel. Gene expression <i>in vivo</i> reveals the presence of transcripts that span the entire KSHV genome and replicative potential. Reverse transcriptase negative and non-template controls were run to confirm the absence of contamination. (C) Transmission electron microscopy (TEM) analysis of tumors: Tumors were excised and fixed in gluteraldehyde. TEM revealed the presence of herpesvirus-like particles (100 nm–200 nm) <i>in vivo</i>.</p

rKSHV.219 infected cells and viral DNA can be detected throughout the murine host.

<p>(A) Graph depicts viral DNA copy number per 500 ng total DNA in 10×10⁶ cells purified from bone marrow and in 500 µl whole blood of tumor bearing mice. (B) rKSHV.219 infected cells were detected in murine lymph nodes. Cells from murine lymph nodes were dissociated into single cell suspensions and plated in chamber slides and fixed for fluorescence microscopy for GFP expression. The top panels depict IgG control antibody and native GFP expression, which is quite dim. GFP expression was enhanced with an antibody directed against GFP in the middle and bottom panels. (C) rKSHV.219 infected cells are present in murine spleen. Spleen from a tumor bearing mouse was excised, dissociated with collagenase IV and cultured in puromycin containing selective medium. GFP expressing LANA positive cells grew from the splenic cell culture.</p