Targeting TRIBBLES1 (TRIB1) Pseudokinase in GBM: A New Therapeutic Strategy

Abstract

GBM (WHO grade IV) is the most aggressive form of glioma with a 5-year survival rate of 5%. The current therapeutic regimen involves radiation therapy and concurrent and adjuvant TMZ (Stupp protocol). Due to dismal survival outcomes, 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. TRIB1 mRNA and protein have been found upregulated in several cancers (e.g. NSCLC, prostate etc.). It has also been shown to contribute towards chemotherapy resistance in NSCLC and CRC. It has also been observed that COP1, a TRIB1 associated E3-ligase is also upregulated in glioma cells. 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. For knockdown of TRIB1, a doxycycline inducible system was used. Stable cell lines were generated by puromycin selection and cell sorting. Protein levels were detected by western blotting. The global methylation analysis on a Utrecht 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 PDX GBM cell lines after radiation exposure and TMZ treatment. Overexpression of TRIB1 also increased the phosphorylation of ERK and Akt in PDX cell lines. However, in TRIB1 knockdown cell lines only phosphorylation of Akt was decreased. It was also observed that TRIB1 levels were upregulated in p53-mutant cell lines suggesting a correlation between TRIB1 and p53. TRIB1 is a potential therapeutic target for GBM therapy as targeting TRIB1 could promote radio-sensitivity of p53-mutant glioma cells by enhancing TMZ action. Targeting TRIB1 would also reduce oncogenic signaling in these cells; overall providing an additional treatment strategy for GBM.