Abstract 4415: Tyrosine phosphorylation is essential for OCT2 function and a target of inhibition by TKIs
Authors: Pabla Navjotsingh , Sprowl Jason A. , Ong Su Sien , Gibson Alice A. , Du Guoqing , Lin Wenwei , Hu Shuiying , Li Lie , Chen Taosheng , Sparreboom Alex 
Published: August 2015

Abstract
Cisplatin and oxaliplatin are among the most widely used anticancer drugs in both children and adults. The clinical use of these agents is associated with dose-limiting damage to kidneys (nephrotoxicity) and peripheral nerves (neurotoxicity). Mechanistic understanding of the underlying etiology remains incomplete and preventive strategies are currently unavailable. We previously reported that cisplatin nephrotoxicity (Filipski et al, CPT 2009) and oxaliplatin neurotoxicity (Sprowl et al, PNAS 2013) are dependent on organic cation transporter-mediated uptake mechanisms and can be reduced by genetic or pharmacological inhibition of OCT2. With a goal to identify small molecule OCT2 inhibitors, that could be used to prevent these toxicities, we carried out a high-throughput chemical library screen. Multiple tyrosine kinase inhibitors (TKIs), including dasatinib and vandetanib, were identified as potent OCT2 inhibitors. Further studies showed that TKIs inhibit OCT2 through a non-competitive mechanism raising the possibility that tyrosine phosphorylation by an unknown TKI-sensitive protein kinase(s) may be essential for OCT2 function. Using in vitro cell culture models and proteomics, site-directed mutagenesis and functional assays we found that OCT2 is indeed tyrosine phosphorylated and this phosphorylation is essential for OCT2 function and sensitive to inhibition by TKIs. A subsequent RNAi kinase library screen identified the Src-family member YES1 as the OCT2-phosphorylating kinase. This was further supported by experiments where a TKI-resistant YES1 mutant was able to rescue OCT2 inhibition by dasatinib. Non-TKI inhibitors of YES1 were also found to suppress OCT2 function. Importantly, tyrosine phosphorylation of OCT2 was observed in vivo, in murine kidneys and dorsal root ganglions, major sites of OCT2 expression and toxicities of platinum-based drugs. siRNA mediated in vivo knockdown of YES1 reduced OCT2 phosphorylation and suppressed its function in the kidneys. Treatment with the YES1 targeting TKI, Dasatinib, suppressed OCT2 phosphorylation in dorsal root ganglions, reduced oxaliplatin uptake and significantly prevented oxaliplatin induced neurotoxicity in mice models. These findings have not only provided new mechanistic insights into the regulation of membrane transporters; but have also provided a rationale for the translational development of new targeted interventions using TKI pulse-exposure to mitigate the debilitating side effects associated with platinum-based chemotherapeutics.
Note: This abstract was not presented at the meeting.
Citation Format: Navjotsingh Pabla, Jason A. Sprowl, Su Sien Ong, Alice A. Gibson, Guoqing Du, Wenwei Lin, Shuiying Hu, Lie Li, Taosheng Chen, Alex Sparreboom. Tyrosine phosphorylation is essential for OCT2 function and a target of inhibition by TKIs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4415. doi:10.1158/1538-7445.AM2015-4415