Matthews, Geoffrey M.

Abstract Abstract 4082 N-methyl-2-pyrrolidone (NMP) is considered an inert drug delivery vehicle that is widely used as an industrial organic solvent. NMP is found in a range of pharmaceutical preparations and bio-prosthetic cements. In particular, NMP is used to solubilize kinase inhibitors for pre-clinical testing in experimental mice. The Vk*MYC transgenic mouse is a model of multiple myeloma (MM) that recapitulates the human disease. We have adapted Vk*MYC MM using a syngeneic transplant approach to shorten latency and facilitate testing of novel therapeutics. In this model, we noted delayed disease progression and improved survival in NMP treated mice compared to alternative vehicle controls. To confirm this observation, matched cohorts of mice bearing monoclonal kappa-secreting MM were treated with 20% NMP/80% polyethylene glycol (PEG) (n = 13), PEG alone (n = 10) or PBS (n = 7) at 100uL per 20g mouse daily p.o. Mice were monitored by serial serum protein electrophoresis and quantitative kappa immunoglobulin (IgK) estimation. At the commencement of therapy, cohorts were well matched for disease burden; mean M-spikes 12%, 14% and 14% of total serum protein for NMP, PBS and PEG respectively (p=0.58 NMP vs. PBS, p=0.68 NMP vs. PEG). After 28d treatment, NMP-treated mice had lower paraproteinemia compared to PBS and PEG controls (mean M-spike NMP 31%, PBS 42% and PEG 40%; p < 0.05 NMP vs. PBS or PEG), with concordant suppression of IgK (NMP 18g/L vs. PBS 39g/L, p<0.05). Survival in NMP-treated mice was also significantly longer than that in PBS-treated controls (median survival 50d and 40d respectively; p<0.05 log rank). Survival of PEG-only treated mice did not differ from PBS controls (median survival 39.5d; p=0.49). We compared the structure of NMP to known MM therapeutics, revealing that NMP is a subunit of thalidomide and its analogues. We next tested NMP for cytotoxicity and immune-modulatory drug (IMiD) activity in vitro. NMP (1uM – 10mM) was not directly cytotoxic to healthy donor peripheral blood mononuclear cells (PBMCs) or the U266 myeloma cell line. However, treatment of PBMC with 10uM NMP significantly enhanced NK cell cytotoxicity against the NK cell-sensitive target cell line K562 from 26% to 42% (p < 0.05). Further, NMP favored a Th-1 cytokine profile without directly altering the frequency of CD4, CD8, NK or regulatory T cell subsets in vitro. As compared to the IMiD lenalidomide, NMP inhibited TNF-a production from LPS stimulated monocytes. Given the IMiD-like effects of NMP, we assessed its ability to alter the expression of NK cell ligands on myeloma targets. NMP upregulated both NKG2D and DNAM-1 ligands (MIC-A, MIC-B, ULBP-1, ULBP-2 and CD155) on U266 cells, thereby enhancing the NK cell mediated lysis of the U266 cell line. Our results demonstrate the in vivo anti-myeloma activity and IMiD-like properties of NMP, indicating that it is a putative active thalidomide subunit. The detection of IMiD-like activity was facilitated by a sensitive, immune competent MM model. NMP should no longer be considered inert for the purposes of drug delivery. These findings mandate re-evaluation of drug efficacy data where NMP has been used as the drug delivery vehicle, particularly in immune-competent cancer models. Our findings with NMP may allow the development of new IMiDs. Finally, occupational or iatrogenic exposure to NMP should be further assessed as a potential risk for immunologically mediated adverse effects. Disclosures: No relevant conflicts of interest to declare.

AbstractImmunomodulators are effective in controlling hematologic malignancy by initiating or reactivating host antitumor immunity to otherwise poorly immunogenic and immune suppressive cancers. We aimed to boost antitumor immunity in B-cell lymphoma by developing a tumor cell vaccine incorporating α-galactosylceramide (α-GalCer) that targets the immune adjuvant properties of NKT cells. In the Eμ-myc transgenic mouse model, single therapeutic vaccination of irradiated, α-GalCer–loaded autologous tumor cells was sufficient to significantly inhibit growth of established tumors and prolong survival. Vaccine-induced antilymphoma immunity required NKT cells, NK cells, and CD8 T cells, and early IL-12–dependent production of IFN-γ. CD4 T cells, gamma/delta T cells, and IL-18 were not critical. Vaccine treatment induced a large systemic spike of IFN-γ and transient peripheral expansion of both NKT cells and NK cells, the major sources of IFN-γ. Furthermore, this vaccine approach was assessed in several other hematopoietic tumor models and was also therapeutically effective against AML-ETO9a acute myeloid leukemia. Replacing α-GalCer with β-mannosylceramide resulted in prolonged protection against Eμ-myc lymphoma. Overall, our results demonstrate a potent immune adjuvant effect of NKT cell ligands in therapeutic anticancer vaccination against oncogene-driven lymphomas, and this work supports clinical investigation of NKT cell–based immunotherapy in patients with hematologic malignancies.

Abstract Introduction:The histone deacetylase inhibitor (HDACi) Panobinostat (LBH-589, Novartis Pharmaceutical Corp) belongs to a new class of chemotherapeutics with demonstrated anticancer activities in laboratory studies and within the clinic. Using pre-clinical models of leukemia and lymphoma we have demonstrated that HDACi mediate their therapeutic effects via the intrinsic apoptosis pathway. Here we investigated the ability of Panobinostat to induce apoptosis in human multiple myeloma (MM) cell lines, alone and in combination with agents that target pro-survival Bcl-2 proteins (ABT-737) and the extrinsic apoptosis pathway (rhTRAIL). Moreover, we have used the Vk*myc murine model of MM to demonstrate the therapeutic efficacy of Panobinostat and its ability to induce apoptosis in MM cells in vivo. Methods: JJN3, OPM-2, RPMI-8226 and U266 MM cell lines were incubated with Panobinostat, ABT-737 or rhTRAIL alone or in combination. Cell viability and cell cycle were assessed using flow cytometry. Histone acetylation was assessed by western blot. C57BL/6 mice (n = 32) bearing transplanted Vk*myc MM (2 months post-transplant) were treated with Panobinostat (25mg/kg, 4 days; 15mg/kg 5 days/week, 3 weeks) or vehicle (D5W). Serum paraprotein levels were assessed weekly in all mice. Mice were sacrificed at various time points to measure apoptotic and therapeutic effects. Results: Treatment of MM cell lines with Panobinostat resulted in time- and dose-dependent induction of apoptosis associated with G1 phase arrest and histone H3/H4 acetylation. Combinations of Panobinostat with ABT-737 or rhTRAIL synergised to mediate robust and rapid apoptosis in the human MM cells. Serum paraprotein in mice bearing Vk*myc MM cells was significantly reduced after 4 days of high dose (25mg/ml) Panobinostat compared with vehicle treated mice (p&lt;0.01) and remained at this reduced level for the treatment period (≤60% reduction compared with vehicle treated mice). This was associated with a significant induction of apoptosis and a reduction in CD-138+ plasma cells within the bone marrow. Removal of treatment was associated with increased paraprotein levels, progression of extramedullary disease and death endpoints. Overall, Panobinostat induced a significant survival advantage compared with vehicle (median=36.5 vs. 16 days, p&lt;0.01). Conclusions: These results suggest that the HDACi Panobinostat has single agent activity against MM cell lines in vitro and provides a significant therapeutic benefit to mice bearing syngeneic MM tumors. Moreover, combination therapies consisting of HDAC inhibitors with agents that target pro-survival Bcl-2 proteins or the TRAIL apoptosis pathway may provide a rational approach to the treatment of MM. Future studies will investigate the molecular events underpinning the effects mediated by Panobinostat, ABT-737 and TRAIL in the Vk*myc MM model. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B176.

Abstract The attrition rate for anticancer drugs entering clinical trials is unacceptably high. For multiple myeloma (MM), we postulate that this is because of preclinical models that overemphasize the antiproliferative activity of drugs, and clinical trials performed in refractory end-stage patients. We validate the Vk*MYC transgenic mouse as a faithful model to predict single-agent drug activity in MM with a positive predictive value of 67% (4 of 6) for clinical activity, and a negative predictive value of 86% (6 of 7) for clinical inactivity. We identify 4 novel agents that should be prioritized for evaluation in clinical trials. Transplantation of Vk*MYC tumor cells into congenic mice selected for a more aggressive disease that models end-stage drug-resistant MM and responds only to combinations of drugs with single-agent activity in untreated Vk*MYC MM. We predict that combinations of standard agents, histone deacetylase inhibitors, bromodomain inhibitors, and hypoxia-activated prodrugs will demonstrate efficacy in the treatment of relapsed MM.