Abstract 5845: Identification of proteosome pathway and a novel serine threonine kinase DCLK3: Potential therapeutic targets for innately radiation resistant glioblastoma cells

Abstract

Abstract INTRODUCTION Glioblastoma resistance and recurrence is attributed to the presence of innately Radiation Resistant (RR) cells present in the heterogeneous parent tumour. However, targeting these cells has been impossible due to inaccessibility of these cells. METHODOLOGY We therefore recapitulated clinical scenario of resistance in a cellular model developed from fresh primary GBM patient samples and cell lines. The model allowed us to capture 1) Parent cells 2) innately Radiation Resistant cells - less than 10% of the parent population and 3) Relapse (R) cells. To identify the targetable proteins governing the survival of RR cells, we performed iTRAQ based quantitative proteomic analysis on all the three populations from GBM cell line (SF 268). RESULTS The proteomic data analysis identified 34 proteins as differentially present in RR population of which 22 were upregulated and 12 were downregulated. A GENE STRING analysis of all the differential proteins in RR population revealed putative interaction of a novel serine threonine kinase DCLK3 with 14-3-3 zeta. The increased expression of DCLK3 and 14-3-3 zeta was confirmed by western blot in RR cells of two GBM cell lines and 8 patient samples. Meta-analysis of 242 tumor samples from COSMIC database showed DCLK3 overexpression in 232 tumors. Furthermore, it harbours 8 missense deleterious mutations, 6 of which were in the kinase domain, indicating towards an important kinase function of this protein. We hypothesized that DCLK3 mediated interaction and phosphorylation of 14-3-3 zeta modulated 14-3-3 zeta functions facilitating RR cell survival. For this, first we wanted to see if DCLK3 and 14-3-3 zeta can interact together. In silico docking of DCLK3 with 14-3-3 zeta did show interaction of 14-3-3 zeta and the kinase domain of DCLK3 kinase. This interaction was confirmed by in vitro immunoprecipitation studies. Further studies are ongoing to understand the importance of these proteins and their interaction in GBM recurrence. Additionally, pathway analysis of differentially upregulated proteins in RR cells revealed deregulation of the proteins involved in Ubiquitin-Proteasome System. Indeed, proteasome activity assay showed increased proteosome activity in the RR population of GBM cell lines and Patient samples. Accordingly, Bortezomib, a proteosome inhibitor induced significant apoptosis in the RR population at a concentration significantly lower than that required for inducing apoptosis in the parent cells. SIGNIFICANCE In conclusion this is the first study to identify a proteome signature of innately radiation resistant cells of GBM and identify proteosome pathway and a novel serine-threonine kinase DCLK3 in RR cells as a potential therapeutic target to inhibit GBM radioresistance and recurrence. Citation Format: Jacinth Rajendra, Keshava Datta, , Sheikh Burhan Ud Din Farooqee, Raja Reddy, Nilesh Gardi, Ekjot Kaur, Ketaki Patkar, Aliasgar Moiyadi, Prasanna Venkataraman, Kakoli Bose, Amit Dutt, Harsha Gowda, Shilpee Dutt. Identification of proteosome pathway and a novel serine threonine kinase DCLK3: Potential therapeutic targets for innately radiation resistant glioblastoma cells [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 5845. doi:10.1158/1538-7445.AM2017-5845