Acute Myeloid Leukaemia (AML) is one of the most genetically heterogeneous malignancies with the defining feature of inhibition of terminal myeloid development arising from a disordered development programme. To discover the most commonly dysregulated genes in this disease we compared Affymetrix GC-RMA normalized mRNA expression data of normal human hematopoietic progenitor (CD34+) cells with diagnostic samples from 223 AML patients previously enrolled in AML NCRI-MRC UK Trials. We used Mixed Model ANOVA and MetaCore™ to identify dysregulated gene networks. Processes associated with Wnt signalling were found to be the most significantly dysregulated: Wnt and cytoskeletal remodelling (p=1.16−26) and; developmental Wnt signalling (p=2.91−16). Interactome analysis identified TCF7L2 (aka TCF4) as the most significantly dysregulated Wnt factor (p=7.56−25; z-score = 19.87) which showed elevated expression in 78 percent of AML patients. The TCF7L2/β-catenin complex is a nuclear regulator of canonical Wnt signalling that is involved in the development of several cancers. To validate this result we examined the expression of TCF7L2 by RQ-PCR using matched AML samples (n=13). These data significantly correlated with the normalized array expression data (R= 0.711; p=0.006) which in turn correlated with total protein expression determined by Western blotting (n=16);R=0.598; p= 0.014 (see also below). To gain further insights into the prognostic impact of TCF7L2 expression at diagnosis in AML, we next examined a number a variables including patients' age, leukocytes count, prognostic group, gender, WHO performance in multivariate analysis. We have shown that TCF7L2 over expression was found to be independently prognostic for reduced complete remission rate after the adjustment for these co-variables (p=0.0144), OR=5.19 [95% C.I.=1.39–19.39].
The TCF7L2 gene undergoes exon splicing which yields multiple isoforms which have been reported to yield functionally distinct proteins. We therefore compared TCF7L2 mRNA isoform expression in AML patients with normal human bone marrow and progenitor cells. As expected we found extensive variability of TCF7L2 splicing particularly at the 3' end of the gene (exon 13–18). Bone marrow and progenitor cells isoforms had distinct exon composition (with respect to exon 4) suggesting developmental regulation of isoform expression. AML patients were heterogeneous in the isoform expression pattern, but aberrant exon composition (compared to normal cells) was not observed. At the protein level we detected expression of 72kDa (corresponding to predicted exon composition 1–15 and 18) and 58kDa (exons 1–13 and 18) protein isoforms. In both normal and AML cells, expression of the 58kDa form was dominant.
To determine the functional significance of TCF7L2 overexpression we employed lentiviral shRNA vectors targeting TCF7L2 isoforms in leukemic cell lines (K562 and U937) engineered to co-express a TCF-GFP reporter which enabled simultaneous analysis of the effect on TCF-dependent transcription. TCF reporter activity was inhibited by shRNA expression and the cells became non-responsive to Wnt agonists (Wnt3A and BIO) demonstrating that canonical Wnt signalling is dependent on TCF7L2 expression in these cells. Phenotypically, shRNA expression was found to strongly inhibit proliferation and to promote apoptosis indicating that TCF7L2 expression is required for the proliferation and survival of myeloid leukemia cells. In summary these data identify Wnt signalling as the most commonly dysregulated process in AML and show that one of key mediator of canonical Wnt signalling, TCF7L2, is overexpressed in AML, is associated with poor clinical outcome, and is functionally required to maintain the proliferation and viability of myeloid leukemia cells.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.