ESTRO 2024 - Abstract Book

S5428

Radiobiology - Tumour biology

ESTRO 2024

Keywords: Nanomedicine, radiosensitizer, prostate cancer

2769

Poster Discussion

TRIBBLES1 (TRIB1) pseudokinase- mediated modulation of p53 function through COP1 and/or HDAC1 in GBM

Karnika Singh, Chunhua Han, Heather Manring, S. Jaharul Haque, Arnab Chakravarti

The Ohio State University Comprehensive Cancer Center, Radiation Oncology, Columbus, USA

Purpose/Objective:

Glioblastoma (GBM, WHO Grade 4) has a low 5-year survival rate and is highly aggressive. TP53 mutations and deletions are observed in 87% and 28-35% of GBM cases respectively. We have previously identified TRIB1 which correlates with worse overall survival of GBM patients. It is a Ser/Thr pseudokinase that acts as a scaffold to bind substrates for either degradation by ubiquitin proteasome system or activating downstream oncogenic signaling. COP1 E3 ubiquitin ligase is one such protein that binds TRIB1 to degrade C/EBPα, a characterized substrate of TRIB1. COP1 is highly expressed in GBM and also known to degrade p53. However, it is not known if TRIB1 is required for COP1-p53 interaction. Therefore, in this study we evaluated the interaction between TRIB1 and p53 and how this may modulate p53 function.

Material/Methods:

Patient derived xenograft (PDX) cell lines stably overexpressing TRIB1 were created by antibiotic selection. Cell viability was detected by MTS assay. Immunoprecipitation was done to evaluate protein-protein interactions. Western blotting was utilized to detect protein levels. Transient transfection was used to express TRIB1 deletion mutant lacking COP1 binding site. COP1 was knocked down using shRNA.

Results:

We observed that overexpression of TRIB1 caused a decrease in viability of PDX GBM cell lines after radiation and TMZ treatment in vitro. Immunoprecipitation showed that TRIB1 formed a complex with p53 and COP1 independent of TP53 mutation status. Knockdown of COP1 led to increased p53 protein levels but overall decreased p53 pull down with TRIB1. To further evaluate the role of COP1 in p53 binding to TRIB1, we utilized a TRIB1 deletion mutant lacking the C-terminal binding site for COP1. We observed that COP1 binding was abolished to TRIB1 and COP1 protein levels were decreased. However, p53 was pulled down with the TRIB1 deletion mutant in two PDX cell lines. We also observed that TRIB1 formed a complex with HDAC1, which is known to deacetylate p53.

Conclusion: