AKAP9 Upregulation Predicts Unfavorable Prognosis in Pediatric Acute Myeloid Leukemia and Promotes Stemness Properties via the Wnt/β-Catenin Pathway

Abstract

Background: PRKA kinase anchor protein 9 (AKAP9) is a scaffold protein involved in various cellular processes, including cell adhesion, proliferation, differentiation, and apoptosis. Although the oncogenic role of AKAP9 in solid tumors is well elucidated, the functions and mechanisms of AKAP9 in acute myeloid leukemia (AML) are still not understood.

Methods: We used the gene expression omnibus (GEO) database (GSE2191) to determine the mRNA expression of AKAP9 in the bone marrow of pediatric AML and healthy patients. We further used the therapeutically available research to generate effective treatments (TARGET) database to elucidate the relationship between AKAP9 expression and clinical outcomes in pediatric patients with AML. In addition, cell proliferation, cell cycle, apoptosis, RT-PCR, and Western blotting assays were applied to reveal the functions of AKAP9 and the underlying mechanisms of AKAP9 silencing in THP1 and HL60 cell lines.

Results: AKAP9 is overexpressed in the bone marrow of pediatric AML patients as compared with that of healthy patients. High expression of AKAP9 was found to be a predictor of poor overall survival (OS) and event-free survival (EFS). Using univariate and multivariate survival analyses, we found that high AKAP9 expression is an independent predictor of a worse OS and EFS. Functionally, AKAP9 silencing significantly inhibited AML cell proliferation, and cell cycle progression and promoted apoptosis. Moreover, AKAP9 silencing significantly downregulated the expression of stemness markers and β-catenin.

Conclusion: AKAP9 upregulation is a predictor of unfavorable prognosis, promotes stemness, and activates the Wnt/β-catenin pathway in AML patients. AKAP9 may act as a prognostic biomarker of AML in pediatric patients and a future therapeutic target.

Keywords: PRKA kinase anchor protein 9; acute myeloid leukemia; leukemic stem cells; pediatric; prognosis.

Figure 1

The AKAP9 mRNA expression and prognosis in pediatric patients with AML. (A) The AKAP9 mRNA expression level in pediatric AML and control in the GSE2191 cohort. (B) Patients with a high AKAP9 expression had a significantly higher MRD positive rate when compared with those with a low AKAP9 expression. (C) Patients with a high AKAP9 expression showed significantly higher CR rate when compared with those with a low AKAP9 expression. (D and E) High AKAP9 mRNA expression proved to be a predictor of poor overall survival and event-free survival, as ascertained from the TARGET database. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Univariate and multivariate COX analyses of prognostic factors for OS and EFS in pediatric patients with AML. (A) The AKAP9 overexpression is an independent prognostic factor of worse OS in pediatric patients with AML. (B) AKAP9 overexpression is an independent prognostic factor of worse EFS in pediatric patients with AML.

Figure 3

Establishment of AKAP9-silenced THP1 and HL60 cell lines. (A) Fluorescence images of THP1 and HL60 cells at 48 h after transfection. (B) The mRNA and (C) protein expression levels of AKAP9 in THP1 and HL60 were reduced in AKAP9 silenced-cell lines. (D) Quantitative analysis of the AKAP9 protein expression in (C). ****P < 0.0001.

Figure 4

Effects of AKAP9 silencing on the proliferation and cell cycle of THP1 and HL60 cells. (A and B) AKAP9 silencing decreased the potential of THP1 and HL60 cells’ proliferation. (C and D) and (E and F) AKAP9 silencing inhibited the cell cycle progression of THP1 and HL60 cells. *P < 0.05, **P < 0.01.

Figure 5

AKAP9 silencing promoted the apoptosis of HL60 and THP1 cells. (A) Apoptotic cells were analyzed by flow cytometry by staining annexin APC/7-AAD. (B) The percentage of apoptosis in THP1 and HL60 cells after AKAP9 silencing. **P < 0.01, ***P < 0.001.

Figure 6

Effect of AKAP9 silencing on the stem cell-related molecular markers in HL60 and THP1 cells. (A) Western blotting of stem cell-related molecular markers—ALDH1A1 and CD133—in the control (Ctrl), sh-NC-, and sh-AKAP9-transfected THP1 and HL60 cells. (B and C) Densitometry analyses of the AKAP9 protein expression levels in THP1 and HL60. ***P < 0.001.

Figure 7

AKAP9 silencing suppressed the Wnt/β‑catenin signaling pathway in AML cells. (A) The mRNA expression level of CTTNB1 in AKAP9-high and AKAP9-low patients from the TARGET database. (B) The correlation of CTTNB1 and AKAP9 mRNA expressions in pediatric AML patients from the TARGET database. (C) Western blotting of β‑catenin in the control (Ctrl), sh-NC-, and sh-AKAP9-transfected THP1 and HL60 cells. (D) Densitometry analysis of the β‑catenin protein expression levels in (C). ***P < 0.001, **** P < 0.0001.