ALBERT

Description: The presence of tumor-specific microRNAs reflects tissue of origin and tumor stage. We show that the absence of miRNAs likewise can be used to determine tumor origin (miR-155) and proliferation state because tumor suppressor miRNAs (miR-222/221, let-7 family) were significantly down-regulated in primary effusion lymphoma (PEL) and in Kaposi sarcoma (KS), an endothelial cell tumor. PEL and KS are associated with KS-associated herpesvirus infection. We identified 15 virally regulated miRNAs in latently infected, nontumorigenic endothelial cells. MiR-143/145 were elevated only in KS tumors, not virally infected endothelial cells. Thus, they represent tumor-specific, rather than virus-specific, miRNAs. Because many tumor suppressor proteins are wild-type in KS and PEL, down-regulation of multiple tumor suppressor miRNAs provides a novel, alternative mechanism of transformation.

Description: The antitumor potency of the mTOR inhibitor rapamycin (sirolimus) is the subject of intense investigations. Primary effusion lymphoma (PEL) appears as an AIDS-defining lymphoma and like Kaposi sarcoma has been linked to Kaposi sarcoma–associated herpesvirus (KSHV). We find that (1) rapamycin is efficacious against PEL in culture and in a murine xenograft model; (2) mTOR, its activator Akt, and its target p70S6 kinase are phosphorylated in PEL; (3) rapamycin inhibits mTOR signaling as determined by S6 phosphorylation; (4) KSHV transcription is unaffected; (5) inhibition of IL-10 signaling correlates with drug sensitivity; and (6) addition of exogenous IL-10 or IL-6 can reverse the rapamycin growth arrest. This validates sirolimus as a new treatment option for PEL.

Description: Background: The proto-oncogene Myc is a key regulator of cell growth and survival, and aberrant Myc expression plays a significant role in various tumors, including non-Hodgkin lymphoma (NHL). Myc-associated lymphoma is clinically aggressive, more resistant to standard therapies, and associated with a significantly higher rate of mortality. Novel treatment paradigms are needed to improve survival of patients with Myc-associated NHL. Expression of Aurora Kinase (Aurk) has been associated with Myc, and Aurk is thought to be essential for the maintenance of Myc-driven lymphoma. Aurk is required for assembly of the mitotic spindle and plays key roles in cell proliferation. Amplification and overexpression of Aurk have been observed in various human tumors, including lymphoma, and are frequently associated with tumor progression as well as resistance to chemotherapy. Inhibition of Aurk may overcome resistance to chemotherapy and improve clinical outcomes in patients with Myc-overexpressing lymphoma. Methods: Cytotoxicity assays using MTS and trypan blue were used to compare levels of drug sensitivity in lymphoma cell lines resistant or sensitive to a conventional chemotherapeutic drug cyclophosphamide. Apoptosis and cell cycle assays were performed using Annexin V and Propidium Iodide staining. The Multiplexed Inhibitor Beads and quantitative Mass Spectrometry (MIB/MS) assays were used to profile kinome changes in response to Aurk inhibition. Murine xenograft models were used to assess the efficacy and tolerability of single vs. combined therapy. Results: Two Myc-overexpressing cell lines were identified as resistant (Raji) or sensitive (Ramos) to cyclophosphamide, with IC50 of ~ 400 µM and ~ 250 µM, respectively. Raji cells were characterized by increased expression of multidrug resistant protein 1 (MDR1) and mutated p53. There were no significant differences in baseline Aurk or Myc expressions between Raji and Ramos cells. Both cell lines were sensitive to alisertib, an aurora A kinase inhibitor, with maximum cytotoxicity achieved at ~ 100 nM. Combined treatment with alisertib and cyclophosphamide induced more significant cell growth inhibition as compared to treatment with the single agent alone. The combination index (CI) values were less than 1, indicating that alisertib was synergistic to cyclophosphamide in terms of inhibitory effect on tumor cell viability. Alisertib induced apoptosis and pronounced cell cycle arrest, resulting in polyploidy, in Raji cells. Alisertib had little to no effect on Myc, p53, or the total aurora A kinase protein expression in Raji cells although p-Histone-3-Ser10, a downstream target of Aurk, and p-Src levels were significantly decreased at 24 hours of treatment in vitro. Nocodazole-treated cells had reduced p-Aurk level and increased p-Rb as well as increased Mdm2 when treated with alisertib for 24 hours. Athymic nude mice bearing Ramos or Raji lymphoma xenografts were treated with cyclophosphamide, alisertib, or the combination. As expected, all mice bearing Ramos xenograft had complete tumor regression by day 35 of treatment while all mice bearing Raji xenograft had rapid disease progression with median survival of ~ 35 days when treated with cyclophosphamide alone. In contrast, when treated with the combination of cyclophosphamide and alisertib, all mice bearing Raji xenograft had complete regression of tumor by day 35 and had significant improvement in survival (median survival not reached by day 100) compared to the single agent control (p=0.022). Lastly, kinome analysis of Raji xenograft tumors treated with alisertib showed suppression of various kinases involved in Aurk, Src, and PI3K pathways. Western blot of the Raji tumors treated with a prolonged course (25 days) of alisertib showed significant decrease in p-Src and p53 protein levels. Conclusion: Our data demonstrates that alisertib induces synthetic lethality and overcomes chemoresistance in Myc-overexpressing tumors even in the presence of MDR1 overexpression and p53 mutation. The synergistic effect was largely independent of depletion of cytoplasmic level of Myc. Alisertib, when combined with a conventional chemotherapy drug, induced apoptosis and cell cycle arrest of Myc-overexpressing tumor cells in vitro and showed promising anti-tumor activity in mice bearing chemoresistant Myc-overexpressing lymphoma. Disclosures Park: Janssen: Other: travel; Seattle Genetics: Research Funding; Teva: Research Funding.

Description: Primary effusion lymphoma (PEL) constitutes a subset of non-Hodgkin lymphoma whose incidence is highly increased in the context of HIV infection. Kaposi sarcoma–associated herpesvirus is the causative agent of PEL. The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a critical role in cell proliferation and survival, and this pathway is dysregulated in many different cancers, including PEL, which display activated PI3K, Akt, and mammalian target of rapamycin (mTOR) kinases. PELs rely heavily on PI3K/Akt/mTOR signaling, are dependent on autocrine and paracrine growth factors, and also have a poor prognosis with reported median survival times of less than 6 months. We compared different compounds that inhibit the PI3K/Akt/mTOR pathway in PEL. Although compounds that modulated activity of only a single pathway member inhibited PEL proliferation, the use of a novel compound, NVP-BEZ235, that dually inhibits both PI3K and mTOR kinases was significantly more efficacious in culture and in a PEL xenograft tumor model. NVP-BEZ235 was effective at low nanomolar concentrations and has oral bioavailability. We also report a novel mechanism for NVP-BEZ235 involving the suppression of multiple autocrine and paracrine growth factors required for lymphoma survival. Our data have broad applicability for the treatment of cytokine-dependent tumors with PI3K/mTOR dual inhibitors.

Description: Abstract 4866 MicroRNA-155 (miR-155) is overexpressed in many types of cancers including B cell lymphomas. It has also an essential function in GC development. These functions are conserved among species and miR-155 knockout (ko) mice are deficient in GC development in Peyer's patches (PP) and lymph nodes. The human tumor virus Kaposi sarcoma associated herpesvirus (KSHV) encodes an ortholog to miR-155, named miR-K12-11. MiR-155 and miR-K12-11 share 100% seed sequence homology (Nature. 2007 Dec 13;450(7172):1096-9, J Virol. 2007 Dec;81(23):12836-45). Another human herpesvirus, Epstein-Barr Virus, induces mir-155 upon infection and immortalization of naïve B cells in culture. To test the hypothesis that miR-K12-11 can complement the normal function of miR-155 in vivo, we generated transgenic mice, which express miR-K12-11, as well as the other 21 KSHV miRNAs. Using this mouse model, we show that KSHV latency locus which contains a viral ortholog of miR-155, KSHV miR-K12-11 complements B cell abnormalities associated with the lack of miR-155. Germinal center (GC) formation is rescued in spleen and PP and lowered immunoglobulin level is also augmented to normal in KSHV latency locus transgenic mice with miR-155 ko background. Furthermore, mature B cells were chronically activated, leading to hyperglobulinemia triggered by increased plasma cell frequency. Marginal zone (MZ) B cells developed hyperplasia and the mice had an augmented response to T-dependent antigen as well as the TLR4 ligand LPS in vivo. This suggests that by mimicking miR-155 (and other functions) this human tumor virus drives the expansion of infected B cells, which if left unchecked can accumulate secondary mutations leading to post-GC lymphoma and multicentric Castleman's disease. Disclosures: No relevant conflicts of interest to declare.

Description: Key PointsKaposi sarcoma associated herpesvirus miRNAs and latent proteins drive B-cell proliferation. Viral miRNAs and latent proteins induce BCR and TLR hyper-responsivness in transgenic mice.

Description: Abstract 4786 Gamma-herpes viruses are the etiologic cause of a significant portion of human lymphomas. Epstein Barr virus (EBV) is virtually found in nearly all cases of endemic Burkitt lymphoma (BL), AIDS-related Hodgkin's lymphoma, and primary CNS lymphoma (PCNSL). EBV also affects 30-60% of AIDS related BL cases, and the great majority of immunoblastic lymphomas (IBLs) and primary effusion lymphomas (PELs). PELs are usually also co-infected with human herpes virus 8 (HHV-8), which is thought to drive the proliferation of this tumor. A hallmark of viral-related lymphomas is the high constitutive expression of NF-κB, a key molecule which activates anti-apoptotic pathways. Investigators have demonstrated that pharmacologic blocking of NF-κB disrupts viral latency inducing apoptosis in EBV and HHV-8 lymphomas. In a recent study, the combination of arginine butyrate and gancyclovir (GCV) was found to be efficacious in patients with EBV-related lymphoproliferative diseases. The investigators postulated that butyrate induces EBV lytic replication and expression of the viral thymidine kinase, which in turn phosphorylates the nucleoside analog GCV which gets incorporated into DNA. We recently reported that azidothymidine (AZT) alone inhibited NF-κB and disrupted EBV latency inducing apoptosis in primary low-passage EBV+ Burkitt lines, while GCV did not exert this effect. BL displays a restricted EBV latency pattern (Type I) and lack the expression of latent membrane proteins (LMPs), while primary EBV+ IBL lines express LMP-1. This EBV-encoded oncoprotein activates NF-κB and enforces latency. While IBLs are generally resistant to the effects of AZT alone, we recently found that adding hydroxyurea sensitized them to AZT. Hydroxyurea is a ribonucleotide reductase inhibitor, which increases the intracellular levels of AZT monophosphate. The use of AZT in targeting gamma-herpes virus tumors is an attractive concept given that this drug is preferentially phosphorylated intracellularly thus exploiting viral kinases potentiating its own cytostatic and anti-tumor effects. We have recently opened to accrual a phase II study evaluating the combination of EBV lytic-inducing chemotherapy drugs (methotrexate, doxorubicin, and AZT/hydroxyurea) for relapsed EBV-related NHL. So far, an HIV+ patient suffering from EBV+ and HHV-8+ solid PEL variant has been treated, and undergone a complete remission with this therapy. We are actively investigating new combinations of lytic inducing agents in our established primary EBV+ BL and IBL cell lines, including the proteasome inhibitor bortezomib, DNA methyltransferase (DNMT) inhibitors such as 5-azacytidine (5-AZA), and histone-deacteylase (HDAC) inhibitors. Here we report, that sub lethal doses of 5-AZA and HDAC-inhibitors (sodium butyrate, and suberoylanilide hydroxamic or SAHA) potently synergize to induce EBV lytic proteins Zebra and EAD in both primary BL and IBL lines. Sodium butyrate by itself appeared to be more potent than 5-AZA or SAHA at inducing EBV lytic replication in all cell lines tested, and also synergized with the nucleoside analog AZT. Experiments are currently ongoing testing the ability of these biological agents to sensitize EBV+ lymphoma lines to nucleoside analogs. Our goal is to continue the development of promising therapeutic approaches for gamma-herpes virus lymphomas. These data will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1115 A small subset of genes is transcribed during latency in Kaposi sarcoma-associated herpesvirus (KSHV) lymphomas. To better understand viral lymphomagenesis we generated a transgenic mouse expressing all viral latent genes including LANA, vFLIP, vCyc, kaposin and all viral miRNAs. All viral miRNAs were expressed in B cells. The complete latency locus induced activation of mature B cells more robustly than a single LANA protein transgenic mouse. It led to the specific expansion of marginal zone (MZ) B cells. The transgenic mice showed an augmented primary response to T-dependent antigen leading to acute MZ activation and germinal center (GC) activation. Furthermore, they exhibited an augmented response to TLR4 stimulation. Our data indicates that the KSHV latency locus fuels transformation processes as a chronic antigenic stimulator. These data supports a model of lymphomagenesis, in which virally infected B cells become hyperresponsive to B cell stimulation, thus providing an expanded pool for secondary genetic alterations. Disclosures: No relevant conflicts of interest to declare.