Up‐regulation of DDIT4 predicts poor prognosis in acute myeloid leukaemia

Abstract The mammalian target of rapamycin (mTOR) inhibitor, DNA damage inducible transcript 4 (DDIT4), has inducible expression in response to various cellular stresses. In multiple malignancies, studies have shown that DDIT4 participates in tumorigenesis and impacts patient survival. We aimed to study the prognostic value of DDIT4 in acute myeloid leukaemia (AML), which is currently unclear. Firstly, The Cancer Genome Atlas was screened for AML patients with complete clinical characteristics and DDIT4 expression data. A total of 155 patients were included and stratified according to the treatment modality and the median DDIT4 expression levels. High DDIT4 expressers had shorter overall survival (OS) and event‐free survival (EFS) than the low expressers among the chemotherapy‐only group (all P < .001); EFS and OS were similar in the high and low DDIT4 expressers of the allogeneic haematopoietic stem cell transplantation (allo‐HSCT) group. Furthermore, in the DDIT4 high group, patients treated with allo‐HSCT had longer EFS and OS than those who received chemotherapy alone (all P < .01). In the DDIT4 low group, OS and EFS were similar in different treatment groups. Secondly, we analysed two other cytogenetically normal AML (CN‐AML) cohorts derived from the Gene Expression Omnibus database, which confirmed that high DDIT4 expression was associated with poorer survival. Gene Ontology (GO) enrichment analysis showed that the genes related to DDIT4 expression were mainly concentrated in the acute and chronic myeloid leukaemia signalling pathways. Collectively, our study indicates that high DDIT4 expression may serve as a poor prognostic factor for AML, but its prognostic effects could be outweighed by allo‐HSCT.


| Patients
The first cohort included 155 de novo AML patients with DDIT4 expression data, derived from The Cancer Genome Atlas (TCGA) database (https ://cance rgeno me.nih.gov/). 16 Eighty-four patients received chemotherapy alone, whereas 71 had allogeneic haematopoietic stem cell transplantation (allo-HSCT). The baseline clinical and molecular characteristics, follow-up and survival data were publicly available from TCGA website, including gender, age, white blood cell (WBC) count, bone marrow (BM) and peripheral blood (PB) blast percentages, French-American-British (FAB) subtype, karyotype, cytogenetic risk classification, RNA and microRNA sequencing data, and gene mutation spectrum.
The second cohort contained two Gene Expression Omnibus (GEO) datasets, GSE6891 and GSE12417, including 334 and 162 patients with cytogenetically normal AML (CN-AML), respectively. This cohort was mainly used to validate the findings of the first cohort.
Affymetrix Human Genome 133 plus 2.0 and U133A gene chips were used to obtain gene expression profiles from the GSE6891 and GSE12417 datasets, and the entire process was fully compliant with the standard Affymetrix protocols. All patients' clinical, molecular and microarray data were public accessible in Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo).

| Statistical analysis
Descriptive statistics were used to summarize the clinical and molecular characteristics of the patients. Datasets were described by median and/or range. Between-group comparisons of numerical and categorical data were performed by the Mann-Whitney U test and the chi-square test, respectively. Primary endpoints were event-free survival (EFS) and overall survival (OS). The former was defined as the time from diagnosis to the first event including relapse, death, failure to achieve complete remission, or was censored at the last AML (CN-AML) cohorts derived from the Gene Expression Omnibus database, which confirmed that high DDIT4 expression was associated with poorer survival. Gene Ontology (GO) enrichment analysis showed that the genes related to DDIT4 expression were mainly concentrated in the acute and chronic myeloid leukaemia signalling pathways. Collectively, our study indicates that high DDIT4 expression may serve as a poor prognostic factor for AML, but its prognostic effects could be outweighed by allo-HSCT.

K E Y W O R D S
acute myeloid leukaemia, allogeneic haematopoietic stem cell transplantation, chemotherapy, DNA damage inducible transcript 4, prognosis follow-up. The latter was the time from diagnosis to death from any cause, or was censored at the last follow-up. Between-group comparisons of OS and EFS were performed by the Kaplan-Meier method and the log-rank test. Multivariate Cox proportional hazard models were constructed for OS and EFS using a limited backward elimination procedure. Spearman rank correlation was used to determine the associations between gene expression profile and DDIT4 expression. Multiple testing errors were assessed by false discovery rate (FDR). Gene Ontology (GO) enrichment analysis was conducted to assess enrichment of gene expression products associated with DDIT4. All tests were two-tailed. Statistical significance was defined as P < .05. All statistical analyses were performed by R software 3.5.0, SPSS software 24.0 and GraphPad Prism software 7.0.

| Differences in clinical and molecular characteristics between different DDIT4 expression groups
In order to evaluate the prognostic significance of DDIT4 in AML, the first cohort was divided into the chemotherapy-only group and the allo-HSCT group. Within each group, the respective median DDIT4 expression level was used to divide the group into high and low expression subgroups, and the clinical and molecular characteristics of subgroups were compared (Table 1).
In the chemotherapy-only group, compared with the DDIT4 low subgroup, the DDIT4 high subgroup had more patients ≥60 years old were not statistically different between the two subgroups.

| Prognostic value of DDIT4 expression in AML
In the TCGA cohort, high DDIT4 expressers generally had significantly shorter OS and EFS than the low expressers (all P < .001; Figure 1A,B). Then, patients were further stratified according to the treatment modality and the median DDIT4 expression levels in the different treatment subgroups. For the high expressors (n = 77), those treated with allo-HSCT had significantly better survival than those who received chemotherapy alone (all P < .01, Figure 1C,D). For the low expressors (n = 78), treatment modality did not have outstanding influence on survival (all P > .05, Figure 1C,D). Kaplan-Meier analysis demonstrated that in the chemotherapy-only group, high DDIT4 expressers had significantly shorter OS and EFS than the low expressers (all P < .001, Figure 2A,B), whereas the survival was similar in the high and low expressors of the allo-HSCT group (all P > .05, Figure 2C,D).
In the chemotherapy-only group, high DDIT4 expression was an independent risk factor for both EFS and OS, along with age ≥ 60, BM blasts ≥ 70% and TP53 mutation (all P < .05). In addition, DNMT3A mutation was an independent risk factor for OS (P = .038). In the allo-HSCT group, FLT3-ITD was an independent risk factor for OS and EFS (all P < .05), and WBC count ≥ 15 × 10 9 /L and TP53 mutation were independent risk factors for EFS (P = .039) and OS (P = .004), respectively, but DDIT4 expression was not an independent factor for survival.

| Associations between genome-wide gene expression profile and DDIT4 expression
To elucidate the possible mechanism for the influence of DDIT4 in AML, DDIT4-associated gene expression profiles were summarized utilizing the high-throughput sequencing information from TCGA database. Three hundred and sixty-eight up-regulated and 171 down-regulated genes that were significantly associated with DDIT4 expression (P < .05, fold change = 1.5, Figure 3A) were screened. Eventually, with a more rigorous analysis (fold change = 2), 359 genes were excluded, and the remain- GO enrichment analysis suggested that the genes related to DDIT4 expression were mainly concentrated in "acute and chronic myeloid leukaemia," "bladder cancer," "hedgehog signalling pathway," "endometrial cancer," and "basal cell carcinoma" signalling pathways ( Figure 3C).

| Validation of the prognostic value of DDIT4 expression in AML
In the two other large CN-AML cohorts from the GEO database, high DDIT4 expression was also related to significantly shorter OS. Combining the data with the TCGA cohort, results consistently showed that up-regulated expression of DDIT4 had deleterious effect on survival of AML patient (all P < .01, Figure 4). adenocarcinoma, 13 indicating its role as a tumour promotor. DDIT4

| D ISCUSS I ON
is heavily involved in the PI3K-Akt-mTOR signalling pathway, a crucial pathway that regulates cell growth, motility, proliferation, apoptosis and one of the most commonly altered pathways in cancer. 21 It is a downstream effector of PI3K-Akt-mTOR. By collaborating with other proteins, it is responsible for prostate cancer cells' invasive behaviour. 22  between DDIT4 and NOTCH1 expression, and both of them tend to highly express in high-risk AML patients. 24 These results indicate that DDIT4 expression may explain some of the aggressive features of AML by involving in the above pathways, though the exact role of DDIT4 in leukaemogenesis requires further study. To summarize, our results indicated that enhanced DDIT4 expression could be a poor prognostic factor for AML patients treated It is reasonable to envision it as a marker for risk stratification and guidance for treatment in AML. Our study was limited by its small, retrospective nature, and the results would need to be verified by a larger prospective population.

ACK N OWLED G EM ENTS
This study was supported by grants from the National Natural

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest. Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo) databases. We did not involve direct interaction with patients. All analyses during this study were included in this article.