Elucidation and Therapeutic Targeting Of The Molecular Mechanism Of TRIB2-Mediated Acute Myeloid Leukaemia
Authors: Lohan Fiona , Forde Ciaran , Salome Mara , O'Connor Caitriona , Bailey Fiona , Cahill Mary R , Delwel Ruud , Eyers Patrick , Kiely Patrick , Keeshan Karen 
Published: October 2019

The pseudokinase TRIB2 is a potent acute myeloid leukaemia (AML) oncogene, capable of inducing transplantable AML with a relatively short latency in murine models. Functionally, the oncogencity of TRIB2 has been linked to its degradation of CCAAT/enhancer binding-protein-alpha (C/EBPα), a transcription factor necessary for regulation of haematopoietic stem cells (HSC) and myeloid differentiation and is mutated in ∼10-15% of cytogenetically normal AMLs. Previously, we have demonstrated that elevated TRIB2 mRNA expression is associated with a small subset of C/EBPα dysregulated AML patients. However in our analysis of primary AML patient samples we reveal detectably high TRIB2 protein expression in a greater number of samples than predicted from mRNA studies compared to normal peripheral blood mononuclear cells.

Here, using in vivo ubiquitination assays we determined that TRIB2 exerts its effect through K48 specific ubiquitin-dependent proteasomal degradation of C/EBPα. Peptide array analysis identified the specific amino acids involved in the direct binding of these two proteins. Site-directed mutagenesis of these amino acids demonstrated that the direct binding of TRIB2 and C/EBPα was required for TRIB2-mediated C/EBPα degradation.

In order to determine if posttranslational modification of C/EBPα was a trigger for TRIB2-mediated binding and degradation, we assessed the phosphorylation of C/EBPα, often a modification involved in target substrate ubiquitination. We found that TRIB2 decreased the levels of phosphorylated Serine 21 (S21) C/EBPα through preferential binding to the phosphorylated form of S21 C/EBPα and mediating its K48 specific ubiquitin-dependent proteasomal degradation. While TRIB2 retains the canonical amino acid motifs of a kinase and the ability to bind ATP, indicative of kinase activity, the absence of phosphorylated S21 C/EBPα in the presence of TRIB2 suggests that it does not have sufficient kinase activity to enable efficient phosphotransfer. The presence of TRIB2 further blocked the ability of mitogenic stimuli to phosphorylate S21 of C/EBPα. TRIB2 thus acts to perturb the regulation and function of C/EBPα phosphorylation ultimately leading to its degradation. We propose this contributes to the leukaemic phenotype of AML cells which include increased self-renewal and proliferation. Using clinically available inhibitors of the proteasomal degradation pathway we have investigated the targeted inhibition of the TRIB2 degradation function to induce cell death in AML cells. In TRIB2 overexpressing AML cell lines, and in AML patient samples identified to have elevated levels of TRIB2, we have demonstrated that elevated TRIB2 expressing samples are more sensitive than low TRIB2 expressing samples to cell death induced by proteasomal inhibition.

Our data shows that in the presence of TRIB2 phosphorylated S21 C/EBPα is a trigger for its ubiquitin dependent degradation. We propose TRIB2 mediates is leukaemogenic effects in AML through direct protein-protein interaction, perturbation of phosphorylation signalling, resulting ultimately in proteasomal mediated degradation of its target C/EBPα. As C/EBPα plays a key role in both stem cell function and myeloid differentiation in AML, the targeted inhibition of TRIB2-mediated C/EBPα degradation may provide therapeutic avenues in AML.

Disclosures:

No relevant conflicts of interest to declare.