Prognostic role of Wnt and Fzd gene families in acute myeloid leukaemia

Abstract Wnt‐Fzd signalling pathway plays a critical role in acute myeloid leukaemia (AML) progression and oncogenicity. There is no study to investigate the prognostic value of Wnt and Fzd gene families in AML. Our study screened 84 AML patients receiving chemotherapy only and 71 also undergoing allogeneic haematopoietic stem cell transplantation (allo‐HSCT) from the Cancer Genome Atlas (TCGA) database. We found that some Wnt and Fzd genes had significant positive correlations. The expression levels of Fzd gene family were independent of survival in AML patients. In the chemotherapy group, AML patients with high Wnt2B or Wnt11 expression had significantly shorter event‐free survival (EFS) and overall survival (OS); high Wnt10A expressers had significantly longer OS than the low expressers (all P < .05), whereas, in the allo‐HSCT group, the expression levels of Wnt gene family were independent of survival. We further found that high expression of Wnt10A and Wnt11 had independent prognostic value, and the patients with high Wnt10A and low Wnt11 expression had the longest EFS and OS in the chemotherapy group. Pathway enrichment analysis showed that genes related to Wnt10A, Wnt11 and Wnt 2B were mainly enriched in ‘cell morphogenesis involved in differentiation’, ‘haematopoietic cell lineage’, ‘platelet activation, signalling and aggregation’ and ‘mitochondrial RNA metabolic process’ signalling pathways. Our results indicate that high Wnt2B and Wnt11 expression predict poor prognosis, and high Wnt10A expression predicts favourable prognosis in AML, but their prognostic effects could be neutralized by allo‐HSCT. Combined Wnt10A and Wnt11 may be a novel prognostic marker in AML.


| INTRODUC TI ON
The Wingless-Int (Wnt) gene family consists of 19 structurally related genes that encode secreted lipid-modified glycoproteins.
These proteins initiate canonical and non-canonical signalling cascades through the transmission of Frizzled (Fzd) transmembrane receptors and lipoprotein-related protein (Lrp) single-transmembrane co-receptors. 1,2 Wnt signalling pathway regulates a series of cellular processes such as proliferation, differentiation, migration and apoptosis, and maintains the balance between self-renewal and differentiation of stem cells involved in the pathogenesis of multiple types of human cancers. 3,4 In haemopoietic tissues, Wnts not only play an essential role in the maturation of haemopoietic stem cells (HSCs) into mature blood cells, but also participate in myelopoiesis by regulating erythron-and thrombopoiesis. 5,6 Almost all members of the Wnt and Fzd gene families are expressed at least to some extent in different haematopoietic sits during development. 7 Therefore, Wnt signalling activity is critical in the precise regulation of normal haemopoiesis, while abnormal expression of components of Wnt-Fzd signalling pathway can induce leukaemia. 4 Acute myeloid leukaemia (AML) is a highly heterogeneous disease and belongs to clonal tumours originating from HSCs or myeloid cell precursors. It is associated with excessive proliferation of progenitor cells and differentiation of cell-cycle arrest. 8 The role of Wnt signalling in the pathogenesis of leukaemia has attracted much attention in recent years. Müller-Tidow et al first found that the activation of Wnt signalling is a common feature of several balanced translocations in AML. 9 A study showed that poor survival of AML patients is associated with the hypermethylation of Wnt genes without changes in expression levels. 10 Wnt5a, as a tumour suppressor in the Wnt gene family, exhibits decreased expression in AML cells. 11 However, Wnt4 ligand regulates leukaemic cell growth by blocking cells to G1 cell-cycle phase in an Fzd6-independent manner. 12 The overexpression of Fzd4 receptor in AML makes it highly sensitive to the activity of Wnt ligands. 13 In addition, Fzd3 expression is higher in AML patients compared with normal group. 14 Therefore, the role of Wnt and Fzd gene families in the pathogenesis, development and prognosis of AML cannot be ignored.
Gene mutation and expression profiles are associated with AML progression and prognosis. Nucleophosmin 1 (NPM1) and fms-like tyrosine kinase 3-internal duplication (FLT3-ITD) mutations occur in 50% and 30% of AML patients, respectively, and have negative impact on AML prognosis. 15 Our previous studies found that individual genes in the same gene family (FUT, DOK and PAK families) have different prognostic value in AML. [16][17][18] To investigate the prognostic significance of Wnt-Fzd signalling pathway in AML, we analysed the effects of Wnt and Fzd gene families on survival of AML patients who either received chemotherapy alone or followed by allogeneic haematopoietic stem cell transplantation (allo-HSCT) and dug deep into the underlying mechanism.
Next, plenty of clinical and molecular characteristics were collected, including age, gender, race, white blood cell (WBC) counts, bone marrow (BM) blasts, peripheral blood (PB) blasts, French-American-British (FAB) subtypes and the frequencies of known recurrent genetic mutations. We also collected follow-up data in these patients including event-free survival (EFS) and overall survival (OS) and defined them as the end points. EFS refers to the time from diagnosis to the first event including relapse and death or was censored at the last follow-up. OS refers to the time from diagnosis to death from any cause or the last follow-up. Another independent cohort (GSE12417) was used to verify the results of aforesaid cohort.

| Statistical analysis
Clinical characteristics of patients were displayed in descriptive statistics. The associations between Wnt and Fzd gene families were assessed by Spearman correlation analysis. The differences of two groups in numerical data and categorical data were calculated by activation, signalling and aggregation' and 'mitochondrial RNA metabolic process' signalling pathways. Our results indicate that high Wnt2B and Wnt11 expression predict poor prognosis, and high Wnt10A expression predicts favourable prognosis in AML, but their prognostic effects could be neutralized by allo-HSCT. Combined Wnt10A and Wnt11 may be a novel prognostic marker in AML.

K E Y W O R D S
acute myeloid leukaemia, allo-HSCT, chemotherapy, prognosis, Wnt-Fzd signalling pathway Mann-Whiney U test and Chi-square test, respectively. Univariate (Kaplan-Meier method and the log-rank test) and multivariate (Cox proportional hazard model) analyses were used to evaluate whether Wnt and Fzd gene expression could predict EFS and OS.
Spearman correlation analysis was further used to screen gene expression profiles related to Wnt genes with prognostic significance.
Then we used StringDB (https://strin g-db.org) for online proteinprotein interaction (PPI) analysis, used Cytoscape to visualize the results and used CytoHubba for hub gene screening. Finally, we used Metascape (http://metas cape.org/) to perform pathway enrichment analysis of gene expression profiles related to Wnt genes.
A two-tailed P < .05 was defined as statistically significant. The SPSS 24.0 statistical software and R 3.5.0 software were used for statistical analyses, and the graphics were drawn by GraphPad Prism 7.0 software.

| Prognostic value of Wnt and Fzd gene families in AML
According to the median expression levels of Wnt and Fzd family members, AML patients in chemotherapy and allo-HSCT groups were divided into high and low expression subgroups. Table 1 and Table S1 present the differences of EFS and OS between high and low Wnt and Fzd gene expression subgroups. The expression levels of all Fzd genes were not associated with EFS and OS in both groups except that the expression of Fzd1 affected EFS in the allo-HSCT group. All Wnt genes were also independent of survival in the allo-HSCT group (all P > .05). In the chemotherapy group, AML patients with high Wnt2B expression had significantly shorter EFS and OS compared with low Wnt2B expressers (all P < .05, Figure 2A,B); high Wnt11 expressers had significantly shorter EFS and OS than low expressers (all P < .001, Figure 2E,F), whereas high Wnt10A expression had favourable effect on OS (P = .043, Figure 2D).
The verified results showed that AML patients with high Fzd3 or Fzd5 expression had significantly longer OS; high expression of Wnt4, Wnt6 and Wnt7B also had positive effects on OS, while AML patients with high Wnt2B or Wnt16 expression had significantly shorter OS (all P < .05, Figure S1 and Figure S2).

F I G U R E 1
Heatmap of correlation between Wnt and Fzd gene families in patients who received chemotherapy (A) and allogeneic haematopoietic stem cell transplantation (B). Blue represents significant positive correlation, red represents significant negative correlation and a darker colour indicates a stronger correlation; blank represents no statistically significant correlation

| Association of Wnt2B/10A/11 expression with clinical and molecular characteristics in the chemotherapy group
As shown in Table 2

| Multivariate analysis of possible prognostic factors in the chemotherapy group
In order to further evaluate independent prognostic value of and MLL; mutated vs. wild) were included in multivariate analysis. As presented in Table 3, high Wnt11 expression was an independent risk factor for EFS and OS, along with age ≥ 60, BM blasts ≥ 70% and mutations in DNMT3A, RUNX1, TP53 and MLL (all P < .05); high Wnt2B expression and WBC count ≥ 15×10 9 /L were independent risk factors for EFS (all P < .05), whereas high Wnt10A expression was an independent favourable factor for EFS (HR = 0.0560, P = .038) and OS (HR = 0.514, P = .018).   and 'mitochondrial RNA metabolic process' signalling pathways ( Figure 4E).

| D ISCUSS I ON
In this retrospective study, we found significant correlations be- Wnt signalling pathway has crucial importance in the pathogenesis of AML. There is heterogeneity in Wnt signalling activation in AML, even in groups with the same genotype. 26

TA B L E 2 (Continued)
and FLT3-ITD mutations. The small sample size and the influence of other regulatory factors may be responsible for this phenomenon.
This also makes it possible for these three Wnt genes to serve as independent prognostic indicators.
Wnt2B is one of the Wnt ligands that stimulate the canonical Wnt pathway. 5 Wnt2B is associated with clinical stage, T stage and cervical lymph node metastasis in patients with nasopharyngeal carcinoma. 32 Wang et al reported that silencing Wnt2B can decrease the capacity of metastatic dissemination for ovarian cancer cells, and the decreased Wnt2B expression can inhibit cancer cell survival and promote cell apoptosis after chemotherapy treatment. 33 We found that elevated Wnt2B expression is associated with worse survival in AML patients with chemotherapy only. This is similar to previous findings that Wnt2B expression is significantly higher in human pancreatic cancer tissues than in normal pancreatic tissues, and patients with high Wnt2B expression have worse outcomes. 34 Therefore, Wnt2B expression may be used as a new prognostic indicator for AML.
As a member of the canonical Wnt/β-catenin pathway, Wnt10A overexpression is associated with poor survival in patients with idiopathic pulmonary fibrosis. 35 38 We found that high Wnt10A expression is associated with good outcomes in AML patients with chemotherapy only. This may be due to the heterogeneity of the expression and function of the same gene in different tumours.
Wnt11 activates both canonical and non-canonical Wnt signalling pathways and plays controversial role in different cancers.
On the one hand, Wnt11, as a tumour promoter, is involved in the proliferation, migration and invasion of various cancers, such as breast cancer, colon cancer, prostate cancer and leukaemia. [39][40][41][42] On the other hand, Wnt11 is considered to be a tumour-suppressor protein that inhibits tumour cell migration, invasion and adhesion in ovarian cancer, endometrial cancer and liver cancer. [43][44][45] We found that high Wnt11 expression indicates poor outcomes in AML patients with chemotherapy only, suggesting that high Wnt11 expression may be involved in leukaemogenesis as a tumour promoter.
We found that the prognostic value for Wnt2B is still signifi-  pathways. The specific joint mechanism of them in AML needs to be further clarified.

ACK N OWLED G EM ENTS
This work was supported by grant from Xinjiang Joint Fund of National Natural Science Foundation of China (U1903117)

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.