TRIB2 in human AML: a biological and clinical investigation

[摘要] Acute myeloid leukemia (AML) involves the proliferation, abnormal survival and arrest ofcells at a very early stage of myeloid cell differentiation. The biological and clinicalheterogeneity of this disease complicates treatment and highlights the significance ofunderstanding the underlying causes of AML, which may constitute potential therapeutictargets, as well as offer prognostic information. Tribbles homolog 2 (Trib2) is a potentmurine oncogene capable of inducing transplantable AML with complete penetrance. Thepathogenicity of Trib2 is attributed to its ability to induce proteasomal degradation of thefull length isoform of the transcription factor CCAAT/enhancer-binding protein alpha(C/EBPα p42). The role of TRIB2 in human AML cells, however, has not beensystematically investigated or targeted.Across human cancers, TRIB2 oncogenic activity was found to be associated with itselevated expression. In the context of AML, TRIB2 overexpression was suggested to beassociated with the large and heterogeneous subset of cytogenetically normal AMLpatients. Based upon the observation that overexpression of TRIB2 has a role in cellulartransformation, the effect of modulating its expression in human AML was examined in ahuman AML cell line that expresses high levels of TRIB2, U937 cells. Specificsuppression of TRIB2 led to impaired cell growth, as a consequence of both an increase inapoptosis and a decrease in cell proliferation. Consistent with these in vitro results, TRIB2silencing strongly reduced progression of the U937 in vivo xenografts, accompanied bydetection of a lower spleen weight when compared with mice transplanted with TRIB2-expressing control cells. Gene expression analysis suggested that TRIB2 modulatesapoptosis and cell-cycle sensitivity by influencing the expression of a subset of genesknown to have implications on these phenotypes. Furthermore, TRIB2 was found to beexpressed in a significant subset of AML patient samples analysed. To investigate whetherincreased expression of this gene could be afforded prognostic significance, primary AMLcells with dichotomized levels of TRIB2 transcripts were evaluated in terms of their xenoengraftment potential, an assay reported to correlate with disease aggressiveness observed in humans. A small cohort of analysed samples with higher TRIB2 expression did not associate with preferential leukaemic cell engraftment in highly immune-deficient mice,hence, not predicting for an adverse prognosis. However, further experiments including alarger cohort of well characterized AML patients would be needed to clarify TRIB2significance in the diagnostic setting. Collectively, these data support a functional role forTRIB2 in the maintenance of the oncogenic properties of human AML cells and suggestTRIB2 can be considered a rational therapeutic target.Proteasome inhibition has emerged as an attractive target for the development of novelanti-cancer therapies and results from translational research and clinical trials support theidea that proteasome inhibitors should be considered in the treatment of AML. The presentstudy argued that proteasome inhibition would effectively inhibit the function of TRIB2 byabrogating C/EBPα p42 protein degradation and that it would be an effectivepharmacological targeting strategy in TRIB2-positive AMLs. Here, a number of cellmodels expressing high levels of TRIB2 were successfully targeted by treatment withproteasome inhibitors, as demonstrated by multiple measurements that included increasedcytotoxicity, inhibition of clonogenic growth and anti-AML activity in vivo.Mechanistically, it was shown that block of the TRIB2 degradative function led to anincrease of C/EBPα p42 and that response was specific to the TRIB2-C/EBPα axis.Specificity was addressed by a panel of experiments showing that U937 cells (expressdetectable levels of endogenous TRIB2 and C/EBPα) treated with the proteasome inhibitorbortezomib (Brtz) displayed a higher cytotoxic response upon TRIB2 overexpression andthat ectopic expression of C/EBPα rescued cell death. Additionally, in C/EBPα-negativeleukaemia cells, K562 and Kasumi 1, Brtz-induced toxicity was not increased followingTRIB2 overexpression supporting the specificity of the compound on the TRIB2-C/EBPαaxis. Together these findings provide pre-clinical evidence that TRIB2- expressing AMLcells can be pharmacologically targeted with proteasome inhibition due, in part, toblockage of the TRIB2 proteolytic function on C/EBPα p42.A large body of evidence indicates that AML arises through the stepwise acquisition ofgenetic and epigenetic changes. Mass spectrometry data has identified an interactionbetween TRIB2 and the epigenetic regulator Protein Arginine Methyltransferase 5(PRMT5). Following assessment of TRIB2‟s role in AML cell survival and effectivetargeting of the TRIB2-C/EBPα degradation pathway, a putative TRIB2/PRMT5cooperation was investigated in order to gain a deeper understanding of the molecularnetwork in which TRIB2 acts as a potent myeloid oncogene. First, a microarray data setwas interrogated for PRMT5 expression levels and the primary enzyme responsible forsymmetric dimethylation was found to be transcribed at significantly higher levels in AMLpatients when compared to healthy controls. Next, depletion of PRMT5 in the U937 cellline was shown to reduce the transformative phenotype in the high expressing TRIB2AML cells, which suggests that PRMT5 and TRIB2 may cooperate to maintain theleukaemogenic potential. Importantly, PRMT5 was identified as a TRIB2-interactingprotein by means of a protein tagging approach to purify TRIB2 complexes from 293T cells. These findings trigger further research aimed at understanding the underlyingmechanism and the functional significance of this interplay.In summary, the present study provides experimental evidence that TRIB2 has animportant oncogenic role in human AML maintenance and, importantly in such amolecularly heterogeneous disease, provides the rational basis to consider proteasomeinhibition as an effective targeting strategy for AML patients with high TRIB2 expression.Finally, the identification of PRMT5 as a TRIB2-interacting protein opens a new level ofregulation to consider in AML. This work may contribute to our further understanding andtherapeutic strategies in acute leukaemias.

[发布机构] University:University of Glasgow;Department:Institute of Cancer Sciences

[关键词] AML, TRIB2, proteasome inhibitors, bortezomib, PRMT5.