Identification And Quantification Of Isoforms Of Eukaryotic Initiation Factor 4e As Biomarker In Mnk Inhibitor-Treated Mouse Model By Capillary-Based Immunoassay Platform

Elevated levels of the phosphorylated m 7 G cap binding protein, eukaryotic initiation factor 4E (eIF4E) are associated with neoplasia. An observed oncogenic signaling effect upon activation of Erk1/2 or p38 MAPKs in cells is the phosphorylation of eIF4E specifically at Serine 209 by downstream kinase MAP kinase-interacting kinase 1 and 2 (Mnks). Therefore, inhibiting Mnks could be a potential therapeutic approach that selectively targets cancer cells without introducing toxicity to normal cells. To measure the target engagement and efficacy of Mnk inhibitors, we have established a sensitive method to quantitatively detect phospho-eIF4E and non-phospho-eIF4E. Here, we report using NanoPro 100 system, an automated capillary-based immunoassay platform that could separate the phosphorylated and non-phosphorylated eIF4E isoforms based on different isoelectric points. Primary antibodies against both non-phospho eIF4E and Serine 209 phosphorylated eIF4E were used for identification and verification of eIF4E isoforms in protein samples obtained from cell lysate, blood samples and tumor samples. Two major isoforms of eIF4E with isoelectric point at 5.38 for phospho-eIF4E and 5.88 for non-phospho-eIF4E were identified in eIF4E-overexpressing K562 cells. siRNA knockdown of eIF4E expression reduced both isoforms, while treatment with Mnk inhibitor only reduced phospho-eIF4E level. The measurement and quantification of the relative changes of phosphorylated eIF4E by this method can potentially be used in monitoring the efficacy of Mnk inhibitors in xenograft and future clinical trials. Citation Format: Zhiyuan Ke, Sifang Wang, Vithya Manoharan, Susmitha Vuddagiri, Esther Ong, Sharon Lim, Sin Tiong Ong, Kanda Sangthongpitag, Nacro Kassoum, Jeffrey Hill, May Ann Lee. Identification and quantification of isoforms of eukaryotic initiation factor 4E as biomarker in Mnk inhibitor-treated mouse model by capillary-based immunoassay platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3819. doi:10.1158/1538-7445.AM2017-3819