Targeting TRIBBLES1 (TRIB1) Pseudokinase as a New Therapeutic Approach in GBM

Abstract

Glioblastoma (GBM) (WHO grade IV) is the most aggressive form of glioma with a 5-year survival rate of 5%. The current therapeutic regimen involves maximal safe surgical resection followed by radiation therapy and concurrent and adjuvant TMZ (Stupp protocol). Due to dismal survival, there is an urgent need to identify druggable therapeutic targets in GBM for drug development. In this study we identified TRIB1, a Ser/Thr pseudokinase that acts as a scaffold protein to initiate Ubiquitin Proteasome System-mediated degradation of target proteins in the cell. It has also been shown to activate MAPK and Akt signaling in various cell types. Reports suggest that TRIB1 also contributes towards chemotherapy resistance in NSCLC and CRC.We utilized a patient-centered reverse translational approach to identify novel therapeutic targets. TRIB1 was identified by statistical association (Cox regression analysis) and logic-based network analyses of the patient-derived methylation data generated using EPIC methylation array. TRIB1 was functionally validated in vitro by overexpression and knockdown approaches. Stable cell lines were generated by puromycin selection and cell sorting. Site-directed mutagenesis was used to create specific amino acid mutations. Co-immunoprecipitation was performed to identify protein-protein interaction. Protein levels were detected by western blotting.The global methylation analysis on an institutional GBM cohort revealed that TRIB1 promoter methylation was associated with better OS of GBM patients (HR = 0.81 (0.62-1.05); P = 0.11). We also observed that overexpression of TRIB1 caused a decrease in apoptosis of patient derived GBM cell lines after radiation and TMZ treatment. Overexpression of TRIB1 also increased the phosphorylation of ERK in patient derived cell lines. TRIB1 directly bound to MEK1 in these cell lines and the disruption of its biding by site-directed mutation led to reduced TRIB1-MEK1 interaction.Our data suggest that TRIB1 is a potential therapeutic target for GBM therapy as targeting TRIB1 could promote treatment sensitizing glioma cells to RT/TMZ. Targeting MEK1 binding site on TRIB1 could also reduce MAPK oncogenic signaling in these cells thereby providing an additional treatment strategy for GBM.K. Singh: None. C. Han: None. J.L. Fleming: None. J. McElroy: None. E.H. Bell: None. P. Robe: None. S.J. Haque: None. A. Chakravarti: None.