Donovan, Kathleen A.

Abstract Inflammation is a driving factor in the pathogenesis of many cancers including multiple myeloma. Myeloma is a plasma cell tumor whose early stages are characterized by cytokine dependent growth, predominantly driven by IL-6. We have demonstrated that IL-1β is a key inducer of IL-6 expressed by bone marrow stromal cells in a paracrine fashion. The P2X7 receptor (P2X7R) is an ATP-gated ion channel expressed by cells of the hematopoetic lineage that has been shown to play a critical role in IL-1β processing and secretion. We investigated the role this receptor may play in the IL-1/IL-6 cytokine pathway in multiple myeloma. A panel of multiple myeloma cell lines was evaluated by RT-PCR for the expression of P2X7R mRNA. Levels of expression in ANBL-6, MM1S, U266, RPMI-8226 and KAS-6/1 were comparable to U937, a previously characterized P2X7R positive cell line. A newly derived plasmacytoma cell line, PCYT3, showed very low levels of message expression. The ability of ATP to trigger the processing and release of IL-1β by activation of the P2X7 channel was studied using KAS-Pro, a myeloma cell line stably transfected with a pro- IL-1β gene. IL-1β, measured by ELISA, was released in a dose dependent fashion in response to increasing amounts of ATP ranging from 1mM to 5mM. Three P2X7R specific antagonists were tested for their effect on ATP stimulated IL- 1β release from KAS-Pro; all 3 compounds demonstrated inhibition in a dose dependent manner with varying degrees of potency correlating with P2X7R inhibition. In addition fresh patient samples (15 multiple myelomas/ 4 smoldering myelomas/8 MGUS) were evaluated for inhibition of IL-1β release by the P2X7R antagonists through direct measurement of IL-1β by ELISA as well as by an indirect measurement of IL-1β induced IL-6 production by normal bone marrow stromal cells. Greater than 90% inhibition of IL-1β release was demonstrated in all MM & SMM patients positive for IL-1β expression. IL-1β was not detected in any of the MGUS patients and therefore no affect of the inhibitors was observed. These results confirm the activity of functional P2X7 receptors on myeloma cell lines and in fresh patient samples and suggest a role for the use of P2X7 receptor antagonists in the therapy of myeloma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2582. doi:10.1158/1538-7445.AM2011-2582

Abstract Introduction: The eukaryotic translation initiation factor 5A (eIF5A) is the only known protein to be regulated by post-translational addition of a hypusine residue. Both hypusinated eIF5A and the enzyme that mediates eIF5A hypusination have been identified as markers of neoplastic growth and metastasis. However, recent studies have indicated that, in its unhypusinated form, eIF5A is pro-apoptotic and thus functionally distinct from hypusine-modified eIF5A. In vitro cell studies and in vivo xenograft studies have demonstrated that simultaneous suppression of eIF5A expression and overexpression of a non-hypusinable mutant of eIF5A potently induces apoptosis in multiple cancer cell types. SNS01 is a nanoparticle specifically designed for the treatment of multiple myeloma. SNS01 is comprised of three components: a DNA vector containing a B-cell-specific (B29) promoter driving expression of a pro-apoptotic mutant of eIF5A (eIF5AK50R) that cannot be hypusinated; an siRNA that targets the native hypusinated eIF5A that promotes growth of cancer cells; and a synthetic polymer called polyethylenimine (PEI). Methods: Ethidium-bromide agarose gel electrophoresis was used to detect heparin-mediated release of nucleic acids from nanoparticles. Dynamic light scattering (DLS) was used to assess the short-term stability of SNS01 by monitoring changes in size distribution, polydispersity, and zeta potential. In vitro biological activity of SNS01 was assessed using RT-qPCR to measure eIF5A and eIF5AK50R transgene expression as well as FACS detection of AnnexinV-FITC labeled apoptotic cells. Anti-tumoral activity of SNS01 was evaluated using a subcutaneous human myeloma model (KAS-6/1 cells) in SCID mice with twice-weekly intra-venous injections of SNS01. Results: SNS01 nanoparticles were found to be relatively stable for as long as 48 hours at room temperature with no significant loss in nucleic acid integrity. The nanoparticles had an average diameter of 146.2 nm, a polydispersity of 0.287 and an average zeta potential of 35.3+0.7 mV. These parameters were quite stable over 48 hours. SNS01 could also be frozen at − 80°C for at least one month with no observable loss in biological activity. Twice-weekly intra-venous injections into myeloma tumor-bearing mice of a frozen SNS01 preparation was found to have anti-tumoral activity comparable to freshly prepared SNS01 (frozen SNS01: 89 % tumor inhibition, p = 0.00158; SNS01: 84 % tumor inhibition, p = 0.00003). Conclusions: SNS01 is relatively stable at room temperature and was also found to be stable and biologically active following freezing. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A196.

Abstract Background: IL-6 is a central myeloma growth factor and in vitro abnormal production of IL-1beta in the myeloma microenvironment stimulates the generation of paracrine IL-6. Mimicking in vitro observations, we have shown that responsive early stage myeloma patients at risk for progression to active myeloma who were treated with IL-1 inhibitors demonstrated decreases in the myeloma proliferative rate and C-reactive protein (IL-6 surrogate marker) leading to a chronic disease state with an improved progression free survival. In this study we investigated the effect of XOMA 052, a humanized anti-IL-1 antibody to inhibit stromal cell IL-6 production induced by either recombinant IL-1beta or supernatants generated from unsorted bone marrow cells from patients with smoldering multiple myeloma. Methods: Bone marrow stromal cells were incubated with varying concentrations of recombinant IL-1β (100, 10, 1, 0.1 pg/ml) in the absence or presence of anti-IL-1 antibody, anti-TNF antibody, or dexamethasone. In a similar fashion, supernatants from unsorted bone marrow cells from six patients with smoldering myeloma were co-cultured with bone marrow stromal cells and the effect of the humanized anti-IL-1β antibody was tested. Results: The anti-IL-1β antibody inhibited the IL-1 induced IL-6 production in vitro by > 85% at 100 pg/ml of IL-1 and > 90% at 10, 1, and 0.1 pg/ml of IL-1; specifically, using 10 pg/ml of IL-1, IL-6 production was inhibited from 149 down to 5.2 ng/ml in the presence of anti-IL-1 antibody. Anti-TNF antibody had minimal effect. At 10 pg/ml of IL-1 beta, a physiologically relevant amount of IL-1 in vitro in bone marrow samples from myeloma patients, the anti-IL-1 antibody was superior to dexamethasone at inhibition of IL-6 production. In in vitro testing of patient supernatants from unsorted bone marrow cells, three patients were high inducers and three patients low inducers of paracrine IL-6 production. The results demonstrate > 85% reduction of IL-6 production in all patients' samples tested. Importantly, the anti-IL-1β antibody was highly effective in samples from the three patients who were high inducers of paracrine IL-6 production (IL-6 production was inhibited from 191 down to 12 ng/ml; 157 to 13 ng/ml and 320 to 11 ng/ml). In comparison, the anti-TNF antibody had marginal effects. Conclusion: The humanized anti-IL-1beta antibody is highly effective in vitro at the inhibition of IL-6 generated by supernatant cultures from patients with early stage myeloma or by recombinant IL-1. Combination therapy with IL-1 inhibitors and apoptosis inducing agents may be useful in patients with active myeloma that have elevated IL-6 levels and a high growth rate at diagnosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2449.

Abstract Multiple myeloma (MM) and primary systemic amyloidosis (AL) remain incurable disorders, and new treatments targeted to the malignant plasma cells are needed. Alemtuzumab is a humanized monoclonal antibody to CD52 and has activity in chronic lymphocytic leukemia. We examined the CD52 expression on CD45+ and CD45- plasma cell populations to evaluate the potential for using alemtuzumab for these disorders. Bone marrows from 61 patients (29 AL, 23 MM, and 9 MGUS [monoclonal gammopathies of undetermined significance]) were studied using 3-color (CD38/45/52) flow cytometry. Among those with MGUS, MM, and AL, 67%, 52%, and 35%, respectively, were positive for CD52 expression. The CD52 expression was predominantly confined to the clonal CD38+/CD45+ plasma cell fraction with median expression of 68%, 88%, and 82% in MGUS, MM, and AL, respectively, compared with 18%, 6%, and 9% among the CD45- plasma cell population. Clinical trials are warranted in these diseases to learn the therapeutic benefit of anti-CD52 immunotherapy.

Live attenuated measles virus (MV-Edm) has potent oncolytic activity against myeloma xenografts in mice. Therapy of multiple myeloma, a disseminated plasma cell malignancy, would require systemic administration of the virus. Thus, the virus should ideally be targeted to infect only myeloma cells to minimize collateral damage to normal tissues: viral binding to its natural receptors must be ablated and a new specificity domain that targets entry into myeloma cells be added. This study covers 2 critical steps toward generating such a retargeted virus: (1) a new specificity domain against the plasma cell marker CD38 was constructed in the form of a single-chain antibody (scFv) and (2) display of that scFv on the measles viral envelope glycoprotein successfully redirected virus entry through CD38 expressed on target cells devoid of the natural MV receptors. The anti-CD38 scFv was tethered to the C-terminus of the hemagglutinin (H) glycoprotein of MV-Edm through a Factor Xa protease cleavable linker. Immunoblot analysis demonstrated that the scFv was efficiently incorporated into recombinant viral particles. Replication of MV-αCD38 was not hindered by the scFv, reaching titers comparable to MV-Edm. Chinese hamster ovary (CHO) cells were resistant to infection by MV-Edm and MV-αCD38. In contrast, CHO cells expressing CD38 became susceptible to infection by MV-αCD38 but not MV-Edm. Removal of the displayed scFv rendered MV-αCD38 noninfectious on CHO-CD38 cells. Tumorigenicity of CHO-CD38 cells in immunocompromised mice was significantly attenuated by MV-αCD38, resulting in enhanced survival of these mice compared with the control group.