Abstract B193: Ibrutinib potentiates the effects of mTOR inhibitors and pazopanib in renal cell carcinoma in vitro and in vivo
Authors: Chen Jun , Hsu Jeff , Huang Yujun , James Danelle F. , Kinoshita Taisei , Chang Betty Y. 
Published: May 2016

Abstract
Introduction: Ibrutinib is a first-in-class Bruton's tyrosine kinase (BTK) inhibitor approved for treatment of multiple B-cell malignancies that also inhibits EGFR and HER2 activity (Honigberg, 2010; Gao, 2014). Ibrutinib suppressed growth of EGFR-driven NSCLC or HER2-amplified breast cancer cell lines and reduced tumor growth in murine xenograft models demonstrating the potential of anti-tumor activity in solid tumors (Gao, 2014; Elias, 2013). Ibrutinib also modulated host tumor immunity and enhanced PD-L1 Mab activity in solid tumor models from tumor cells insensitive to BTK or HER kinase inhibition (Sagiv-Barfi, 2015). These results suggest the potential of ibrutinib to be clinically active across a variety of tumors via multiple mechanisms of action (MoA). Immunotherapeutic interventions have had varying success in renal cell carcinoma (RCC), the most common type of kidney cancer. In this study, we determined the therapeutic impact of ibrutinib alone and in combination with approved therapies in RCC in vitro and in vivo and further investigated its MoA.
Methods: The effect of drugs on cell proliferation was determined with CellTiter-Glo (Promega), and apoptosis assayed with annexin-V/PI staining. Signaling pathways were evaluated with Western blotting. Single agent and combinations were studied in vivo by subcutaneous implantation of syngeneic Renca or human 786-0 RCC cell lines into mice.
Results: Treating RCC cells with everolimus alone potently inhibited p-S6, a downstream mTOR target, and significantly inhibited proliferation. Pazopanib, a multi-kinase inhibitor, similarly inhibited pAkt. As reported, these inhibitors up-regulated pAkt and/or pErk, two key pro-survival molecules, after 1h and/or 24h treatment. Such compensatory changes to signaling pathways have been suggested to attenuate the anti-tumor activity of these inhibitors (Wang, 2008; Breuleux, 2009; Grabinski, 2012; Soares, 2013). Interestingly, addition of ibrutinib antagonized up-regulation of pAkt and pErk by everolimus or pazopanib, and enhanced their anti-proliferative activities. In contrast, ibrutinib did not enhance everolimus activity in Caki-1 cells where pAkt and pErk were unaffected by the combination. Ibrutinib alone did not or only weakly inhibited RCC proliferation in cell culture despite inhibition of EGF-induced pEGFR. In contrast, pAkt and pErk, were much less affected, suggesting that EGFR is not a primary driver of proliferation in RCC cells. In vivo, ibrutinib alone showed marginal or no anti-tumor activity in Renca or 786-0 mice models. However, ibrutinib significantly reduced tumor burden in combination with sirolimus (p&lt;0.001) or everolimus (p&lt;0.05) in the Renca and 786-0 RCC models, compared to mTOR inhibitor treatments alone.
Conclusion: These results suggest that ibrutinib, a kinase inhibitor with immune modulatory properties, has anti-tumor activity against RCC when combined with mTOR inhibitors or pazopanib with different target spectrums. Although EGFR is not critical for proliferation in these untreated RCC lines, ibrutinib may prevent ErbB kinases from contributing to feedback up-regulation of Akt/Erk pathways by established drugs. These results provide a preclinical rationale for investigation of ibrutinib as a novel agent for RCC through positive interaction with mTOR inhibitors and/or pazopanib.
Citation Format: Jun Chen, Jeff Hsu, Yujun Huang, Danelle F. James, Taisei Kinoshita, Betty Y. Chang. Ibrutinib potentiates the effects of mTOR inhibitors and pazopanib in renal cell carcinoma in vitro and in vivo. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B193.