Ritonavir interacts with ixazomib synergistically to cause ubiquitinated protein accumulation and endoplasmic reticulum stress in bladder cancer cells

EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 Conclusion: We propose that the combination of quisinostat and flavopiridol is a potential new therapeutic intervention for melanoma patients, independent of mutational status or acquired resistance to BRAF inhibition. No conflict of interest. 564 Ritonavir interacts with ixazomib synergistically to cause ubiquitinated protein accumulation and endoplasmic reticulum stress in bladder cancer cells A. Sato1 , T. Asano1 , M. Isono1 , K. Okubo1 , T. Asano1 . 1 National Defense Medical College, Department of Urology, Tokorozawa, Japan Background: There is no curative treatment for advanced bladder cancer. Ubiquitinated protein accumulation is a novel approach to cancer treatment. The human immunodeficiency virus protease inhibitor ritonavir suppresses heat shock protein 90 and increases the amount of unfolded proteins in the cell, which are then ubiquitinated and rapidly degraded if the proteasome functions normally. We postulated that combining the novel proteasome inhibitor ixazomib with ritonavir would kill bladder cancer cells effectively by causing unfolded ubiquitinated proteins to accumulate and thereby induce endoplasmic reticulum (ER) stress. Material and Methods: Bladder cancer cells (UMUC3, J82, 5637) were treated with ritonavir (20−40 mM) and/or ixazomib (50–100 nM). Cell viability was evaluated by MTS assay. Apoptosis was evaluated using flow cytometry for cell cycle analysis and for annexin-V assay. Induction of ER stress and the expression of ubiquitinated proteins, cell-cycle associated proteins, and acetylated histone were assessed using western blotting. Results: The combination of ritonavir and ixazomib induced drastic apoptosis (annexin-V-positive cells increased up to 98.8%) and inhibited the growth of bladder cancer cells synergistically. It decreased the expression of cyclin D1 and cyclin-dependent kinase 4, leading to the accumulation of the cells in the sub-G1 fraction (up to 51.1%). As expected, it synergistically induced ER stress evidenced by increased expression of the ER stress markers glucose-regulated protein 78, endoplasmic reticulum resident protein 44, and endoplasmic oxidoreductin-1-like protein. In UMUC3 cells the combination increased the amount of ubiquitinated protein in the soluble fraction of the cell lysate but in J82 and 5637 cells decreased it. This decrease was thought to be due to excessively accumulated ubiquitinated proteins aggregating and shifting to the detergent-insoluble fraction. The combination-induced cytotoxicity and apoptosis were markedly attenuated by co-treatment with the protein synthesis inhibitor cycloheximide, demonstrating that accumulation of newly synthesized proteins was essential for the combination’s antineoplastic activity. Interestingly, we also found that the combination induced histone acetylation synergistically. Conclusions: The combination of ritonavir and ixazomib inhibits the growth of bladder cancer cells effectively by synergistically causing ubiquitinated protein accumulation and ER stress. Histone acetylation is another important mechanism of its action. The present study provides a theoretical basis for testing the combination in patients with advanced bladder cancer. No conflict of interest. 565 PI3K blockade reverses primary resistance and adaptation to eribulin in PI3K-pathway activated breast cancer tumors ´ A. Gris-Oliver1 , C. Saura2 , M. Oliveira2 , A. Piris3 , P. Nuciforo4 , J. PerezGarc´ıa2 , J. Arribas5 , J. Baselga6 , J. Cortes ´ 2 , V. Serra1 . 1 Vall d’Hebron Institut d’ Oncologia VHIO, Experimental Therapeutics Group, Barcelona, Spain, 2 Vall d’Hebron Institut d’ Oncologia VHIO, Breast Cancer and Melanoma Group, Barcelona, Spain, 3 Vall d’Hebron Institut d’ Oncologia VHIO, Scientific Research Management, Barcelona, Spain, 4 Vall d’Hebron Institut d’ Oncologia VHIO, Molecular Pathology Group, Barcelona, Spain, 5 Vall d’Hebron Institut d’ Oncologia VHIO, Growth Factors Group, Barcelona, Spain, 6 Memorial Sloan Kettering Cancer Center, Human Oncology and Pathogenesis Program, New York, USA Background: Eribulin is an approved microtubule-targeting agent (MTA) for the management of heavily pretreated metastatic breast cancer (BC) patients. Constitutive PI3K/Akt/mTOR survival pathway activation, either via mutational activation of the PI3K catalytic subunit (PIK3CA) or via inactivation of the tumor suppressor PTEN, may confer resistance to MTAs. Hence, we hypothesized that PI3K-pathway activation limits the antitumor activity of eribulin in HER2-negative BC and that PI3K inhibition enhances the efficacy of this chemotherapeutic agent. Material and Methods: We quantified the antitumor/antiproliferative activity of eribulin in vitro in eight cell line models, and in vivo in a panel of fifteen patient-/cell line-derived xenograft models. Eribulin’s in vitro activity was further studied in an isogenic cell line pair MCF10A harboring or not the hotspot PIK3CA p.H1047R mutation. We measured proliferation by crystal violet cell staining, apoptosis induction by the percentage of SubG1 cells and cell cycle progression after double thymidine block. Results and Discussion: In ten out of fifteen xenograft models eribulin treatment resulted in disease progression, while in the five remaining models

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the tumors shrunk upon treatment. Among the eribulin-resistant models, four harbored PIK3CA hotspot mutations; two, an AKT1-p.E17K mutation; and one, a PIK3CG-splice site mutation. We observed a similar pattern in vitro, whereby the activity of eribulin or paclitaxel was reduced in PIK3CA-mutant cell lines, but not that of DNA-damaging agents such as cisplatin or doxorubicin. MCF10A PIK3CA-wt/wt cells were sensitive to eribulin by induction of a G2/M-phase mitotic arrest. Compared to wt/wt cells, MCF10A PIK3CA-wt/H1047R exhibited an accumulation of S-phase cells and minor G2/M-phase arrest upon eribulin treatment. Accordingly, PIK3CA-wt/H1047R cells expressed higher levels of the cyclin-dependent kinase inhibitor p21 than the parental counterpart, and exhibited delayed cell cycle progression, potentially promoting resistance to eribulin. Furthermore, shRNA against p21 and PI3K inhibitors reduced p21 levels and sensitized PIK3CA-wt/H1047R to eribulin in vitro. In vivo, eribulin plus PI3K-blockade resulted in tumor regression in three out of four PIK3CAmutant models. Noteworthy, eribulin induced adaptive Akt phosphorylation in vitro, in xenograft models and in patient tumors, a compensatory effect that may provide an escape mechanism through increased p21. This crosstalk was reverted by PI3K blockade and resulted in tumor regression/stabilization in the four models tested. Conclusion: We highlight a potential role for PI3K-pathway activation inducing baseline or treatment-induced cell-cycle delay and resistance to MTA. These results support the clinical development of therapeutic regimens combining PI3K-inhibitors with eribulin. No conflict of interest. 566 Retargeting of anti-viral immune responses to solid tumors using bispecific adapters 1 1 ¨ . Hannover J. Niemann1 , N. Woller1 , M.P. Manns1 , S. Kubicka1 , F. Kuhnel Medical School, Clinic for Gastroenterology- Hepatology and Endocrinology, Hannover, Germany

Background: Oncolytic adenoviruses are promising tools for the treatment of solid cancers but induce strong antibody-based immune responses against the virus thus limiting the success of viral oncolysis and preventing readministration of the same vector. On the other hand it has been recognized that immunogenicity of viral vectors contributes to therapy by induction of antitumoral immune responses. In this study we investigated the redirection of virus-directed humoral immune responses to malignant cells using molecular adapters. Material and Methods: To enable retargeting of virus-specific antibodies to polysialic acid (polySia) which is selectively expressed on the cell surface of several clinically relevant tumors (such as glioblastoma and small cell lung cancer) a bifunctional adapter protein was established, consisting of a polySiaspecific single chain Fv fragment (scFv) and a recombinant adenoviral B-cell antigen as antibody-binding domain. Functional analyses were performed in a subcutaneous (s.c.) tumor mouse model, using a polySia expressing variant of the lung cancer cell line CMT64 (CMTpSia). For investigations in an orthotopic metastasis model CMTpSia cells were injected intravenously. Results: Experiments using serum of mice after immunization with adenovirus serotype 5 showed that the DE1 domain of the viral capsid protein hexon is a major target of adenovirus-specific IgGs. Fusion of the DE1 domain to a polySia-specific scFv resulted in the adapter protein DE1scFv-pSia. The effective binding of DE1scFv-pSia to virus-specific IgGs as well as to polySiapositive cells could be confirmed in vitro. In a proof of principle experiment expression of the adapter DE1scFv-pSia by polySia-positive tumors led to a significant and specific inhibition of tumor growth in immunized mice but not in naive mice. Studies with purified adapter protein revealed the intravenous (i.v.) injection as more effective compared with intratumoral application, leading to significant growth inhibition of s.c. tumors, as well as lung metastases. FACS analyses of s.c. tumors showed infiltration of NK cells after adapter application. Consequently, depletion experiments proved that NK cells, in cooperation with CD8 cells, are required for the anti-tumoral effects observed after tumor retargeting of antiviral antibodies. Conclusions: Our results demonstrate a successful retargeting of Ad5 specific antibodies to polySia expressing tumor cells. In the future, combination of virotherapy and application of those adapters could lead to an additional therapeutic benefit by the use of the anti-viral immune response. No conflict of interest. 567 Development of a drug discovery platform focused on SENPs as therapeutic targets for oncology Y. Ofir-Rosenfeld1 , N. Arnaudo1 , F. Enjalbert1 , J. Harrigan1 , M. Woodrow1 , A. Jones1 , M. Kemp1 , H. Robinson1 , X. Jacq1 . 1 Mission Therapeutics Ltd, Babraham Research Campus, Cambridge, United Kingdom SUMO is a ubiquitin-like protein which is covalently linked to a large number of proteins, controlling their function and localisation in a process termed SUMOylation. SUMOylation is a dynamic event that is reversed by Sentrin/SUMO-specific proteases (SENPs). There are nine SENPs in humans, which can be divided into three distinct structural classes and display