The role of complement in antibody therapy of cancer is in general poorly understood. We used the EL4 syngeneic mouse model of metastatic lymphoma to investigate the role of complement in immunotherapy directed against GD2, a target of clinical relevance. IgG2a and IgM anti-GD2 therapy protected EL4-challenged mice from metastases and prolonged survival. Expression of CD59, an inhibitor of direct complementmediated cytotoxicity (CMC), effectively protected EL4 cells from CMC in vitro but did not affect the outcome of monoclonal antibody therapy. Protection by IgG therapy was also unaffected in mice deficient in C3 or complement receptor 3 (CR3) but was almost completely abrogated in Fc;R I/III-deficient mice. These data indicate a crucial role for antibody-dependent cell-mediated cytoxicity (ADCC). However, at lower doses of IgG, therapeutic effect was partially abrogated in C3-deficient mice, indicating complement-mediated enhancement of ADCC at limiting IgG concentration. In contrast to IgG, the therapeutic effect of IgM was completely abrogated in C3-deficient mice. High level expression of CD59 on EL4 did not influence IgM therapy, suggesting IgM functions by complement-dependent cell-mediated cytotoxicity (CDCC), a mechanism thought to be inactive against tumor cells. Thus, IgG and IgM can operate via different primary mechanisms of action, and CDCC and complement-dependent enhancement of ADCC mechanisms are operative in vivo. The effects of complement can be supplemental to other antibody-mediated mechanisms and likely have increased significance at limiting antibody concentration or low antigen density. (Cancer Res 2005; 65(22): 10562-8)

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Objective: Niclosamide, a salicyclamide derivative and FDA approved teniacide, exhibits potent effects against ovarian cancer in vitro by inhibiting the Wnt/β-catenin, STAT3, and mTOR pathways. Unfortunately, its low bioavailability as a chemotherapeutic agent in vivo necessitates investigation of analogs with improved pharmacokinetics and pharmacodynamics. The objective of this study was to investigate the role of targeting the Wnt/β-catenin and STAT3 pathways with niclosamide and niclosamide analogs (C-11 and C-32) in platinum-sensitive and -resistant ovarian cancer cell lines, primary ovarian cancer ascites cell populations, and tumor tissues. In addition, carboplatin and/or LLL12, a novel STAT3 inhibitor effective against ovarian cancer, were used in combination with niclosamide and its analogs to test for synergistic cytotoxicity. Methods: Two ovarian cancer cell lines A2780, SKOV3ip1 and their chemo-resistant derivatives, A2780.cp20 and SKOV3ip2.TRip2, were treated with niclosamide or C-11 or C-32 (0.1 to 5 μM) alone or in combination with carboplatin (5 to 150 μM) or LLL12 (0.1 nM - 10 µM). Tumorspheres were isolated from the ascites of ovarian cancer patients who underwent surgery. Tumor slices were prepared from freshly obtained ovarian cancer specimens. ATPLite assay was performed to measure cytotoxicity in cell lines, tumorspheres, and tumor slices. Western blot was used to assess expression of proteins implicated in the Wnt, mTOR, and STAT3 pathways. β-catenin signaling activity was measured using the TOPflash reporter assay. Results: All four cell lines were sensitive to niclosamide, C-11, and C-32 with similar IC50 values. Additive cytotoxicity was seen when niclosamide or its analogs were combined with carboplatin or LLL12 in the cell lines, tumorspheres, and tumor tissues. Ex vivo and in vitro samples from the same patient demonstrated similar cytotoxicity with niclosamide, C-11, and C-32 treatment. Synergism was also seen between carboplatin and LLL12 in vitro. The TOPflash assay showed that the combination of niclosamide (0.4 μM) and LLL12 (0.25 μM) decreased β-catenin activity more than either agent alone, suggesting that there is crosstalk between the STAT3 and Wnt/β-catenin pathways. Wnt/β-catenin, STAT3, and mTOR pathway target proteins (LRP6, pLRP6, Axin2, and Survivin) were downregulated with niclosamide and C-32 treatment, and to a greater degree with C-11. Conclusion: Combination treatment with niclosamide, or its analogs, with carboplatin or LLL12 induced greater cytotoxicity than either agent alone in ovarian cancer cell lines, primary tumorspheres, and tumor slices. The synergistic cytotoxicity seen in platinum-resistant cell lines with niclosamide, or its analogs, in combination with carboplatin suggest that targeting the Wnt/ β-catenin and STAT3 pathways may prove effective in overcoming chemoresistance in patients with platinum resistant ovarian cancer. Citation Format: Rebecca C. Arend, MD, Abhishek Gangrade, Christen L. Walters Haygood, MD, Chandrika Kurpad, Brandon J. Metge, Rajeev S. Samant, PhD, Pui-Kai Li, PhD, Yonghe Li, PhD, Deepak Bhasin, PhD, Charles Landen, MD, Ronald Alvarez, MD, J. Michael Straughn, MD, Donald J. Buchsbaum, PhD. Overcoming platinum resistance in ovarian cancer with niclosamide [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1402.

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