Distinct gene expression pattern of <i>RUNX1</i> mutations coordinated by target repression and promoter hypermethylation in acute myeloid leukemia

doi:10.1007/s11684-020-0815-4

Front. Med.

2022, Vol. 16

Issue (4) : 627-636 https://doi.org/10.1007/s11684-020-0815-4

RESEARCH ARTICLE

Distinct gene expression pattern of RUNX1 mutations coordinated by target repression and promoter hypermethylation in acute myeloid leukemia

Jingming Li¹, Wen Jin^1,², Yun Tan¹, Beichen Wang¹, Xiaoling Wang¹, Ming Zhao¹, Kankan Wang^1,²(

)

¹. Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
². CNRS-LIA Hematology and Cancer, Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

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Abstract

Runt-related transcription factor 1 (RUNX1) is an essential regulator of normal hematopoiesis. Its dysfunction, caused by either fusions or mutations, is frequently reported in acute myeloid leukemia (AML). However, RUNX1 mutations have been largely under-explored compared with RUNX1 fusions mainly due to their elusive genetic characteristics. Here, based on 1741 patients with AML, we report a unique expression pattern associated with RUNX1 mutations in AML. This expression pattern was coordinated by target repression and promoter hypermethylation. We first reanalyzed a joint AML cohort that consisted of three public cohorts and found that RUNX1 mutations were mainly distributed in the Runt domain and almost mutually exclusive with NPM1 mutations. Then, based on RNA-seq data from The Cancer Genome Atlas AML cohort, we developed a 300-gene signature that significantly distinguished the patients with RUNX1 mutations from those with other AML subtypes. Furthermore, we explored the mechanisms underlying this signature from the transcriptional and epigenetic levels. Using chromatin immunoprecipitation sequencing data, we found that RUNX1 target genes tended to be repressed in patients with RUNX1 mutations. Through the integration of DNA methylation array data, we illustrated that hypermethylation on the promoter regions of RUNX1-regulated genes also contributed to dysregulation in RUNX1-mutated AML. This study revealed the distinct gene expression pattern of RUNX1 mutations and the underlying mechanisms in AML development.

Keywords RUNX1 gene mutation acute myeloid leukemia transcriptional repression DNA methylation

Corresponding Author(s): Kankan Wang

About author:

Tongcan Cui and Yizhe Hou contributed equally to this work.

Just Accepted Date: 26 April 2021 Online First Date: 27 December 2021 Issue Date: 02 September 2022

Cite this article:

Jingming Li,Wen Jin,Yun Tan, et al. Distinct gene expression pattern of RUNX1 mutations coordinated by target repression and promoter hypermethylation in acute myeloid leukemia[J]. Front. Med., 2022, 16(4): 627-636.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-020-0815-4
https://academic.hep.com.cn/fmd/EN/Y2022/V16/I4/627

Fig.1 RUNX1 mutations are mainly located in the Runt domain and exclusive with NPM1 mutations. (A) RUNX1 mutations were exclusive with t(8;21) and inv(16) AML in 1741 patients with AML collected from three published cohorts (GEO, GSE23312; EGA, EGAS00001000275; and TCGA AML). mut, mutated; wt, wild-type. (B) Distribution of mutations in the different domains of the RUNX1 protein (NP_001745.2). Frameshift mutations are highlighted in orange. (C) Distribution of mutations in RUNX1 and 25 additional cancer genes in patients with CN-AML. Concurrent mutations are shown for patients with RUNX1 mutations (n = 66, left panel) or wild-type RUNX1 (n = 666, right panel). The starred genes are those with higher mutation frequencies compared to the other group. Sub, substitution; Ins, insertion; Del, deletion; ITD, internal tandem duplication; PTD, partial tandem duplication.

Fig.2 Patients with AML and RUNX1 mutations have a distinct gene signature. (A) Volcano plot representing the DEGs between the patients with RUNX1 mutations and wild-type RUNX1. (B) RUNX1-mutated samples were distinguished in the TCGA NPM1 wild-type CN-AML cohort. Heat map representing the expression (normalized counts) of the 300-gene signature. mut, mutated; wt, wild-type. (C) PCA results based on the 300-gene signature demonstrated the clear separation of RUNX1-mutated samples from wild-type RUNX1 samples. (D) Hierarchical clustering based on the 300-gene signature separated RUNX1-mutated samples and t(8;21) samples from others in the TCGA AML cohort (left panel, n = 144) and the GEO cohort GSE23312 (right panel, n = 269). The RUNX1-mutation-associated cluster is highlighted in pink. (E) GO analysis revealed that neutrophil-associated pathways were downregulated in RUNX1-mutated AML. (F) Four genes in the neutrophil-associated pathways were repressed in RUNX1-mutated samples of the TCGA AML cohort. P values were calculated by using the Wilcoxon signed-rank test.

Fig.3 RUNX1 target genes are generally repressed in RUNX1-mutated AML. (A) Heat maps illustrating RUNX1 binding signals in the regions near the TSSs in AML cell lines. Overlaps represent high-confidence binding peaks. (B) GSEA plot representing the repression of RUNX1 target genes. A total of 1216 RUNX1 target genes expressed in the TCGA cohort (mean RNA-seq counts>2) were used as the gene set. FDR, false discovery rate; NES, normalized enrichment score. (C) Heat map illustrating the expression of 15 RUNX1 target genes included in the gene signature in CN-AML from the TCGA cohort. (D) Direct binding of RUNX1 on the CTSG promoter in AML cell lines. The CTSG transcript shown in this plot is NM_001911.2. (E) CTSG was significantly downregulated in patients with RUNX1 mutations (Wilcoxon signed-rank test).

Fig.4 RUNX1 mutations are associated with DNA hypermethylation. (A) Heat map representing the gene expression of eight DMGs included in the gene signature of RUNX1 mutations. A total of 113 patients from TCGA were included in the analysis. DMR represents the DNA methylation status of the gene promoters. Target represents RUNX1 binding status on the promoters. mut, mutated; wt, wild-type. (B) Schematic of the hypermethylated promoter region of MS4A3. The scatter plot represents the levels of DNA methylation beta values in the TCGA AML cohort. Beta values for specific CpG residues are shown as a proportion ranging from 0 (unmethylated, 0%) to 1 (fully methylated, 100%). (C) Kaplan–Meier plot of the overall survival of patients with AML from TCGA grouped by the expression levels of MS4A3. The hazard ratio was calculated by utilizing univariate Cox regression. Gene expression levels were evaluated by using FPKM values converted from read counts by DESeq2. The cut-off value (log₂FPKM= 4.31) was selected via the method of maximum selected rank statistics provided in the survminer package.

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