TRIB2 in human AML: a biological and clinical investigation

Campos, Joana Monteiro de (2016) TRIB2 in human AML: a biological and clinical investigation. PhD thesis, University of Glasgow.

Abstract

Acute myeloid leukemia (AML) involves the proliferation, abnormal survival and arrest of
cells at a very early stage of myeloid cell differentiation. The biological and clinical
heterogeneity of this disease complicates treatment and highlights the significance of
understanding the underlying causes of AML, which may constitute potential therapeutic
targets, as well as offer prognostic information. Tribbles homolog 2 (Trib2) is a potent
murine oncogene capable of inducing transplantable AML with complete penetrance. The
pathogenicity of Trib2 is attributed to its ability to induce proteasomal degradation of the
full 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 been
systematically investigated or targeted.
Across human cancers, TRIB2 oncogenic activity was found to be associated with its
elevated expression. In the context of AML, TRIB2 overexpression was suggested to be
associated with the large and heterogeneous subset of cytogenetically normal AML
patients. Based upon the observation that overexpression of TRIB2 has a role in cellular
transformation, the effect of modulating its expression in human AML was examined in a
human AML cell line that expresses high levels of TRIB2, U937 cells. Specific
suppression of TRIB2 led to impaired cell growth, as a consequence of both an increase in
apoptosis and a decrease in cell proliferation. Consistent with these in vitro results, TRIB2
silencing strongly reduced progression of the U937 in vivo xenografts, accompanied by
detection of a lower spleen weight when compared with mice transplanted with TRIB2-
expressing control cells. Gene expression analysis suggested that TRIB2 modulates
apoptosis and cell-cycle sensitivity by influencing the expression of a subset of genes
known to have implications on these phenotypes. Furthermore, TRIB2 was found to be
expressed in a significant subset of AML patient samples analysed. To investigate whether
increased expression of this gene could be afforded prognostic significance, primary AML
cells 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 a
larger cohort of well characterized AML patients would be needed to clarify TRIB2
significance in the diagnostic setting. Collectively, these data support a functional role for
TRIB2 in the maintenance of the oncogenic properties of human AML cells and suggest
TRIB2 can be considered a rational therapeutic target.
Proteasome inhibition has emerged as an attractive target for the development of novel
anti-cancer therapies and results from translational research and clinical trials support the
idea that proteasome inhibitors should be considered in the treatment of AML. The present
study argued that proteasome inhibition would effectively inhibit the function of TRIB2 by
abrogating C/EBPα p42 protein degradation and that it would be an effective
pharmacological targeting strategy in TRIB2-positive AMLs. Here, a number of cell
models expressing high levels of TRIB2 were successfully targeted by treatment with
proteasome inhibitors, as demonstrated by multiple measurements that included increased
cytotoxicity, inhibition of clonogenic growth and anti-AML activity in vivo.
Mechanistically, it was shown that block of the TRIB2 degradative function led to an
increase 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 (express
detectable levels of endogenous TRIB2 and C/EBPα) treated with the proteasome inhibitor
bortezomib (Brtz) displayed a higher cytotoxic response upon TRIB2 overexpression and
that ectopic expression of C/EBPα rescued cell death. Additionally, in C/EBPα-negative
leukaemia cells, K562 and Kasumi 1, Brtz-induced toxicity was not increased following
TRIB2 overexpression supporting the specificity of the compound on the TRIB2-C/EBPα
axis. Together these findings provide pre-clinical evidence that TRIB2- expressing AML
cells can be pharmacologically targeted with proteasome inhibition due, in part, to
blockage of the TRIB2 proteolytic function on C/EBPα p42.
A large body of evidence indicates that AML arises through the stepwise acquisition of
genetic and epigenetic changes. Mass spectrometry data has identified an interaction
between TRIB2 and the epigenetic regulator Protein Arginine Methyltransferase 5
(PRMT5). Following assessment of TRIB2‟s role in AML cell survival and effective
targeting of the TRIB2-C/EBPα degradation pathway, a putative TRIB2/PRMT5
cooperation was investigated in order to gain a deeper understanding of the molecular
network in which TRIB2 acts as a potent myeloid oncogene. First, a microarray data set
was interrogated for PRMT5 expression levels and the primary enzyme responsible for
symmetric dimethylation was found to be transcribed at significantly higher levels in AML
patients when compared to healthy controls. Next, depletion of PRMT5 in the U937 cell
line was shown to reduce the transformative phenotype in the high expressing TRIB2
AML cells, which suggests that PRMT5 and TRIB2 may cooperate to maintain the
leukaemogenic potential. Importantly, PRMT5 was identified as a TRIB2-interacting
protein by means of a protein tagging approach to purify TRIB2 complexes from 293T cells. These findings trigger further research aimed at understanding the underlying
mechanism and the functional significance of this interplay.
In summary, the present study provides experimental evidence that TRIB2 has an
important oncogenic role in human AML maintenance and, importantly in such a
molecularly heterogeneous disease, provides the rational basis to consider proteasome
inhibition 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 of
regulation to consider in AML. This work may contribute to our further understanding and
therapeutic strategies in acute leukaemias.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: AML, TRIB2, proteasome inhibitors, bortezomib, PRMT5.
• Subjects: Q Science > Q Science (General)
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences > Paul O'Gorman Leukemia Research Centre
• Funder's Name: Children's Leukaemia Research Project (CHILD-LEUK), UNSPECIFIED
• Supervisor's Name: Keeshan, Dr. Karen
• Date of Award: 2016
• Depositing User: Miss Joana Monteiro de Campos
• Unique ID: glathesis:2016-7499
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 02 Oct 2016 12:55
• Last Modified: 03 Nov 2016 08:33
• URI: https://theses.gla.ac.uk/id/eprint/7499