Abstract B040: Imiquimod initiates tumor specific overload of the ER stress response in Tasmanian devil facial cancer cells

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Abstract Background: The potent immune modifier imiquimod (R-837) induces apoptosis of various tumor cell lines by mechanisms that are not fully understood. As a topical immunotherapy, imiquimod successfully activates tumor-specific cytotoxic responses via stimulation of TLR7 signaling pathways and antagonism of adenosine receptor mediated regulatory pathways. A role for imiquimod-induced tumor apoptosis in augmenting this response has not been defined, however it remains plausible that molecular changes may render the cells more susceptible to an anti-tumor response. An improved understanding of the pathways regulated by imiquimod in tumor cells will reveal whether this is likely the case. Devil facial tumor disease (DFTD) is a clonal transmissible cancer threatening the world's largest marsupial carnivore, the Tasmanian devil. DFTD evades both non-self and anti-cancer immune defenses to survive in new hosts, providing an ideal model for studying immunotherapeutic mechanisms. As we have previously shown that DFTD cells are sensitive to treatment with imiquimod, investigation of these effects could reveal new therapeutic strategies for DFTD, and will improve our understanding of this drug in cancer immunotherapy. Methods: To investigate the mechanisms of imiquimod action in tumor cells, DFTD cell lines and control fibroblast cell lines were treated in culture with imiquimod. Changes to cell viability were monitored by proliferation and cell death assays. Molecular changes to DFTD cells were assessed through RNA sequencing and proteomic mass spectrometry, and bioinfomatic techniques were employed to identify biological processes regulated by imiquimod. Major findings were confirmed by quantitative PCR. Results: Imiquimod treatment induces complete apoptosis of DFTD cells, but not fibroblasts, over a period of days. Transcriptomic and proteomic analysis revealed high constitutive expression of ER stress genes in DFTD cell lines, which were further augmented in response to imiquimod treatment. Up-regulation of genes required for ER-stress mediated apoptosis was also detected, suggesting that imiquimod induces apoptosis through exhaustion of the ER stress response. Molecular pathways associated with tumor growth and survival were modulated in the dying cells, and damage-associated alarmins were highly expressed. Conclusions: Our study provides the first whole transcriptome and proteome analysis of any imiquimod treated tumor cell line. By using DFTD cells as a tumor model, we have shown that imiquimod directly increases the apoptotic potential of tumor cells through augmentation of ER stress, while producing molecular changes consistent with reduced tumorigenicity. These effects were not replicated in fibroblasts, suggesting that constitutive activation of stress responses in tumor cells may impact their ability to cope with additional stressors. Our findings suggest that ER stress pathways are ideal targets for therapy against DFTD in our endangered Tasmanian devil. These data can also be applied for more rational use of imiquimod as an immunotherapeutic agent in human cancer. Citation Format: Amanda L. Patchett, Terry L. Pinfold, Cesar Tovar, Bruce Lyons, Gregory M. Woods. Imiquimod initiates tumor specific overload of the ER stress response in Tasmanian devil facial cancer cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B040.