MP35-09 BORTEZOMIB INTERACTS SYNERGISTICALLY WITH BELINOSTAT TO CAUSE UBIQUITINATED PROTEIN ACCUMULATION IN RENAL CANCER CELLS

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then tumor infiltrating lymphocyte was analyzed by immunohistochemistry. RESULTS: All three treatment groups ((1), (2) and (3)) showed significant decrease in tumor size compared with vehicle group. In particular, combination of PD-1 Ab and CTLA-4 Ab exhibited the most significant anti-tumor effect. The percentage of PCNA+ tumor cells were very similar amongst all therapies, but the percentage of Ki-67+ tumor cells were significantly decreased with combination of PD-1 Ab and CTLA-4 Ab. Combination of PD-1 Ab and CTLA-4 Ab demonstrated the most potent effect to trigger the infiltration of both CD4+ lymphocyte and CD8+ lymphocytes. These in vivo results indicate that combination of PD-1 Ab and CTLA-4 Ab may have a significant immunostimulatory effect to exert the anti-tumor effect to renal cell carcinoma. CONCLUSIONS: Combination therapy of anti-CTLA-4 antibody and anti-PD-1 antibody can be a potential therapeutic option for advanced kidney cancer patients.

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Leptin, HGF, HB-EGF and EGF secretions from RCC cells cultured with TKIs were measured by VersaMAP Development System. Western blot analyses were applied for detection of phosphorylated STAT3, ERK Akt, mTOR and NFkB proteins. Alterations of cell proliferation and IL-6 signaling pathway by humanized IL-6R antibody Tocilizumab was analyzed by BrdU assay, MTT assay and Western blot analysis. RESULTS: Among the measured cytokines, all the RCC cell lines secreted IL-6 when they were cultured with TKIs. IL-10, IL-23, HB-EGF and leptin were decreased by TKI treatment. Among the RCC cell lines, 786-O and Dibo showed high level of IL-6 production 1 hour after TKIs treatment. VEGF secretion was also observed when the 786O cells were treated by TKIs. In 786-O cells, Western blot analysis revealed that STAT3 and ERK were phosphorylated by TKI treatment even at a low concentration of 0.5 mM. Furthermore, Akt and mTOR were also activated by TKIs treatment. HIF2a expression and phosphorylation of NFkB which would lead to VEGF and IL-6 expression were also observed at the same concentration of TKIs. Tocilizumab treatment in combination with TKIs reduced activation of IL-6 signaling pathway and also suppressed cell proliferation. VEGF secretion following 1 hour TKIs treatment were 3-fold enhanced compared with non treated cells, and the VEGF secretion from RCC cells decreased to 40% by blocking of IL-6 receptor by tocilizumab. CONCLUSIONS: Our results indicate that TKI treatment induces IL-6 secretion in concentration under therapeutical level on RCC cells. Autocrine secretion of IL-6 activates STAT3 and Akt-mTOR pathway and it leads to VEGF secretion. IL-6 could be a possible target for overcoming the development of resistance to TKIs. Source of Funding: Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No.22591773, No.25462492)

MP35-09 BORTEZOMIB INTERACTS SYNERGISTICALLY WITH BELINOSTAT TO CAUSE UBIQUITINATED PROTEIN ACCUMULATION IN RENAL CANCER CELLS Takako Asano*, Akinori Sato, Makoto Isono, Keiichi Ito, Tomohiko Asano, Tokorozawa, Japan

Source of Funding: none

MP35-08 TYROSINE-KINASE INHIBITORS TREATMENT INDUCES IL-6 SECRETION ON RENAL CARCINOMA CELLS Kei Ishibashi*, Yoshiyuki Kojima, Fukushima, Japan; Tobias Haber, €roff, Walburgis Brenner, Mainz, Germany Joachim W. Thu INTRODUCTION AND OBJECTIVES: Tyrosine-kinase inhibitors (TKI) treatment targeted at the vascular endothelial growth factor (VEGF) pathways represents the standard of care in advanced renal cell carcinoma (RCC). Although the TKI treatment has promising effect against RCC, the development of resistance to TKIs should be solved. We have been reported that insufficient concentration of TKI treatment would lead to RCC cell proliferations. We investigated the role of IL-6 in cellular tumor biology of RCC cells treated by TKIs. METHODS: Human renal cell carcinoma cell lines 786-O, Caki-1, Caki-2, CCF-RC1, A498 and Dibo were used for this study. Sorafenib, sunitinib and pazopanib at concentrations of 0.5, 1.0, 5.0, 10.0 mM were used as TKIs. Cytokine IL-6, IL-10, IL-1 beta, IL-1 ra, IL-17, IL-19, IL-23, IL-18BPa and Cripto-1, and growth factors VEGF,

INTRODUCTION AND OBJECTIVES: The histone deacetylase (HDAC) inhibitor belinostat suppresses heat shock protein 90 by inhibiting HDAC6, and this increases the amount of unfolded proteins in the cell. If the proteasome functions normally, however, these unfolded proteins are ubiquitinated and rapidly degraded. We thought that combining the proteasome inhibitor bortezomib with belinostat would kill cancer cells effectively by inhibiting the degradation of these unfolded proteins and thereby causing ubiquitinated proteins to accumulate. METHODS: Renal cancer cells (769-P, 786-O, ACHN) were treated with belinostat (0.5-5 mM) and/or bortezomib (5-10 nM). Cell viability and clonogenicity were assessed by MTS assay and colony formation assay. Combination indexes (CI) were calculated using the Chou-Talalay method. Flow cytometry was used for annexin-V assay and cell cycle analysis. The pancaspase inhibitor Z-VAD-FMK was used to evaluate whether the combination-induced apoptosis was caspase dependent. Induction of the unfolded protein response and the expression of ubiquitinated proteins and cleaved poly(ADP-ribose) polymerase were evaluated by western blotting. RESULTS: Bortezomib in combination with belinostat inhibited the growth of renal cancer cells synergistically (CI <1) and colony formation significantly (P <0.05). The combination perturbed the cell cycle, leading to the accumulation of the cells in the sub-G1 fraction (up to 63.8%). The combination induced drastic apoptosis and, because ZVAD-FMK changed the number of annexin-V-positive cells (decreasing them in 769-P cells and increasing them in 786-O and ACHN cells), this apoptosis was thought to be caspase dependent. Mechanistically, the

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combination synergistically caused ubiquitinated proteins to accumulate: 10 nM bortezomib alone induced no accumulation of ubiquitinated protein but in combination with 2 mM belinostat induced marked ubiquitinated protein accumulation, whereas belinostat alone did not change the amount of ubiquitinated proteins. The combination induced an unfolded protein response evidenced by the increased expression of glucose-regulated protein 78 and heat shock protein 70, and this confirmed the accumulation of ubiquitinated unfolded proteins in the cell. CONCLUSIONS: Bortezomib in combination with belinostat synergistically induces caspase-dependent apoptosis and inhibits renal cancer growth by causing ubiquitinated protein accumulation. Source of Funding: none

MP35-10 RITONAVIR SYNERGIZES WITH CARFILZOMIB TO INDUCE ENDOPLASMIC RETICULUM STRESS AND AUTOPHAGY IN RENAL CANCER CELLS Akinori Sato*, Takako Asano, Makoto Isono, Keiichi Ito, Tomohiko Asano, Tokorozawa, Japan INTRODUCTION AND OBJECTIVES: Inducing endoplasmic reticulum (ER) stress is a novel strategy used to treat malignancies. The HIV protease inhibitor ritonavir has recently been shown to increase unfolded proteins by suppressing the function of heat shock protein 90. We postulated that combining the novel proteasome inhibitor carfilzomib with ritonavir would kill cancer cells effectively by inhibiting the degradation of the ritonavir-increased unfolded proteins and thereby inducing ER stress. METHODS: Renal cancer cells (ACHN, 769-P, 786-O) were treated with carfilzomib (20-100 nM) and/or ritonavir (25-50 mM). Cell viability and clonogenicity were evaluated by MTS assay and colony formation assay. Combination indexes were calculated using the ChouTalalay method. Apoptosis was assayed using flow cytometry and by detecting the expression of cleaved poly(ADP-ribose) polymerase. ER stress and changes in the expression of ubiquitinated proteins and the autophagy marker light chain (LC) 3 were evaluated by western blotting. RESULTS: Ritonavir in combination with carfilzomib inhibited renal cancer growth synergistically (combination indexes <1) and suppressed colony formation significantly (P <0.05). The combination induced drastic apoptosis (annexin-V fluorescence intensity increased 9.6-fold), and this apoptosis was thought to be mainly caspasedependent because the number of annexin-V-positive cells was reduced by co-treatment with the pancaspase inhibitor Z-VAD-FMK. As expected, the combination synergistically induced ER stress evidenced by increased expression of glucose-regulated protein (GRP) 78: neither 20 nor 50 nM carfilzomib alone increased GRP78 expression, but either concentration combined with 50 mM ritonavir increased GRP78 expression markedly. We also found that ritonavir-carfilzomib combinations decreased the expression of LC3-I and increased the expression of LC3-II, confirming that they induced autophagy. This autophagy was thought to be due to aggresome formation caused by extensive protein ubiquitination. CONCLUSIONS: Ritonavir combined with carfilzomib inhibits the growth of renal cancer cells by synergistically inducing ER stress and autophagy. Source of Funding: none

MP35-11 POINT MUTATIONS IN MTOR LEAD TO CONSTITUTIVE ACTIVATION OF MTOR IN RENAL CELL CARCINOMA Arindam Ghosh*, Richard Kirkman, Eun-young Kho, Eun-hee Shim, Sunil Sudarshan, Birmingham, AL INTRODUCTION AND OBJECTIVES: The serine/threonine kinase mTOR (mechanistic target of rapamycin) is activated by diverse

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stimuli to control cell growth and proliferation. Deregulation of multiple elements of the mTOR pathway (PI3K amplification/mutation, PTEN loss of function, AKT overexpression, and S6K1, 4EBP1 and eIF4E overexpression) have been reported in many types of cancers. Although some types of cancer are accompanied by the hyperactivation of mTOR, it remains unclear if mTOR hyperactivation drives tumorigenesis. Recently, mTOR inhibitors(temsirolimus and everolimus) have been approved for use in the setting of metastatic RCC. Despite the established anti-tumoral efficacy of mTOR inhibitors in RCC, little is known about mTOR in the context of renal carcinogenesis. Hence determining the mechanisms by which mTOR is activated in RCC and the critical pathways that are affected, are of biologic and clinical interest. METHODS: A variety of approaches including deletion analyses, yeast genetic screening and mining of human cancer genome databases have identified point mutations in mTOR in about 6% of clear cell renal carcinomas and these mutations are clustered in the FRAP/ ATM/TRRAP (FAT), FKBP12-rapamycin binding (FRB) and kinase domains of mTOR. In this study we focused a “hot spot” of mutations reported in the FAT domain of mTOR that are highly represented in RCC. Point mutations in the FAT domain of mTOR leading to single amino acid substitutions were generated using a Site-directed mutagenesis kit (Stratagene). The mutations were sequence verified and HEK293T cells were transfected with wild-type mTOR or with mutant mTOR constructs. Activation of downstream targets of mTOR was assessed in these mutants in the presence or absence of nutrients and the mTOR inhibitor rapamycin. RESULTS: Our preliminary data suggest that these mutations lead to constitutive activation of mTOR even in the absence of nutrients as evidenced by phosphorylation of its downstream targets p70S6K and 4EBP-1. Our data also reveal that FAT domain mutants demonstrate continued mTOR signaling in the presence of rapamycin. CONCLUSIONS: We have identified a “hot spot” of mutations in the FAT domain of mTOR in RCC that leads to constitutive activation of mTOR and a selective increase mTORC1 kinase activity. These mutations may be a potential cause of resistance to rapamycin therapy in metastatic RCC. Future studies will focus on the mechanism by which these mutations promote mTOR activation and complex assembly. Additionally, we will test the in vivo response to mTOR inhibition in the setting of hyperactivating FAT domain mutations. Source of Funding: This work was supported by a postdoctoral grant from the Urology Care Foundation.

MP35-12 THE HSP90 INHIBITOR GANETESPIB SYNERGIZES WITH THE MET KINASE INHIBITOR CRIZOTINIB IN BOTH CRIZOTINIBSENSITIVE AND CRIZOTINIB-RESISTANT MET-DRIVEN RENAL TUMOR MODELS Naoto Miyajima*, Tomoshige Akino, Kunihiko Tsuchiya, Satoru Maruyama, Takashige Abe, Nobuo Shinohara, Katsuya Nonomura, Sapporo, Japan; Len Neckers, Bethesda, MD INTRODUCTION AND OBJECTIVES: The proto-oncogene MET is aberrantly activated via overexpression or mutation in numerous cancers including papillary renal carcinoma, making it a prime anticancer molecular target. However, the clinical success of MET-directed tyrosine kinase inhibitors (TKI) has been limited due, in part, to mutations in the MET kinase domain that confer therapeutic resistance. Circumventing this problem remains a key challenge to improving durable responses in patients receiving MET-targeted therapy. Small molecule HSP90 inhibitors cause HSP90 to dissociate from its client proteins, resulting in their destabilization and eventual degradation. HSP90 inhibitors have been validated in numerous preclinical tumor models and have shown promising activity in several clinical trials. Given the distinct mechanisms of action of TKI and HSP90 inhibitors, we evaluated the synergistic effects of these two drugs on both TKIsensitive and TKI-resistant MET-driven tumor models.