Abstract 3358: Tribbles 1 (TRIB1) pseudokinase forms a complex with and activates Akt in GBM cells
Authors: Karnika Singh, Chunhua Han, Heather Manring, Saikh Jaharul Haque, Arnab Chakravarti
Journal: Cancer Research (2024)

Abstract Glioblastoma (GBM; WHO grade 4) is the most aggressive form of glioma with a dismal 5-year survival rate. The current therapies are largely ineffective and therapy resistance is commonly encountered. We have recently shown that TRIB1, a Ser/Thr pseudokinase contributes to therapeutic resistance in GBM cells by activating both ERK and Akt pathways. Here we show that, increased expression of TRIB1 contributed to RT/TMZ resistance by activating pro-survival Akt signaling resulting in increased viability of GBM cells. High level of Akt phosphorylation/activation is correlated with poor prognosis of GBM patients. Many pan Akt inhibitors have been developed which are currently in various phases of clinical trials. The three Akt isoforms (1, 2 and 3) are known to play a role in cancer during proliferation, transformation and metastasis. A previous report has shown that TRIB1 interacts with Akt1 in Drosophila and blocks its activation. In this study we show that Akt binds and forms a complex with TRIB1 in GBM cells and this interaction could be isoform and cell line specific. We utilized patient derived xenograft (PDX) GBM cell lines and established GBM cell lines in this study. Stable TRIB1 overexpressing cell lines were generated by antibiotic selection. Co-immunoprecipitation (Co-IP) was performed utilizing Anti-FLAG or anti-c-Myc magnetic agarose beads to evaluate protein-protein interactions. Western blotting was done to detect protein levels. The FLAG tagged TRIB1 deletion mutants were a generous gift from Dr. Takuro Nakamura (Division of Carcinogenesis, The Cancer Institute, Tokyo, Japan). Cells were transfected using Lipofectamine 2000 reagent. Co-IP studies showed that TRIB1 formed a complex with Akt in PDX GBM cell lines. We then transiently transfected respective TRIB1 deletion mutants lacking either N and C terminal residues in HEK 293 cells and performed Co-IP with Anti-FLAG magnetic agarose beads. We observed that all TRIB1 deletion mutants except TRIB1-ΔN2 (lacking first 160 amino acids) were able to pull down Akt. The western blot results showed that each Akt isoform had a variable protein expression across different cell lines. Co-IP studies in the PDX cell lines showed that TRIB1 interacted with all three Akt isoforms in T08-387 cells whereas TRIB1 consistently interacted with only Akt3 but not Akt1/2 in GBM30 cells that lack wild-type p53 expression. Our data suggest that TRIB1 forms a complex with Akt in PDX GBM cell lines. The potential Akt binding site on TRIB1 lies between 90-160 amino acids. TRIB1 can interact with different Akt isoforms in a cell type dependent manner thereby playing a role in their activation and consequent tumorigenic processes. Therefore, targeting TRIB1 mediated Akt activation could be considered a better therapeutic option over targeting Akt directly, which may decrease Akt’s oncogenic activities without risking the toxicity associated with available Akt- specific inhibitors. Citation Format: Karnika Singh, Chunhua Han, Heather Manring, Saikh Jaharul Haque, Arnab Chakravarti. Tribbles 1 (TRIB1) pseudokinase forms a complex with and activates Akt in GBM cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3358.