N6-methyladenosine-modified SENP1, identified by IGF2BP3, is a novel molecular marker in acute myeloid leukemia and aggravates progression by activating AKT signal via de-SUMOylating HDAC2

Abstract

Background: Elevated evidence suggests that the SENPs family plays an important role in tumor progression. However, the role of SENPs in AML remains unclear.

Methods: We evaluated the expression pattern of SENP1 based on RNA sequencing data obtained from OHSU, TCGA, TARGET, and MILE datasets. Clinical samples were used to verify the expression of SENP1 in the AML cells. Lentiviral vectors shRNA and sgRNA were used to intervene in SENP1 expression in AML cells, and the effects of SENP1 on AML proliferation and anti-apoptosis were detected using in vitro and in vivo models. Chip-qPCR, MERIP-qPCR, CO-IP, RNA pulldown, and dual-luciferase reporter gene assays were used to explore the regulatory mechanisms of SNEP1 in AML.

Results: SENP1 was significantly upregulated in high-risk AML patients and closely related to poor prognosis. The AKT/mTOR signaling pathway is a key downstream pathway that mediates SENP1's regulation of AML proliferation and anti-apoptosis. Mechanistically, the CO-IP assay revealed binding between SENP1 and HDAC2. SUMO and Chip-qPCR assays suggested that SENP1 can desumoylate HDAC2, which enhances EGFR transcription and activates the AKT pathway. In addition, we found that IGF2BP3 expression was upregulated in high-risk AML patients and was positively correlated with SENP1 expression. MERIP-qPCR and RIP-qPCR showed that IGF2BP3 binds SENP1 3-UTR in an m6A manner, enhances SENP1 expression, and promotes AKT pathway conduction.

Conclusions: Our findings reveal a distinct mechanism of SENP1-mediated HDAC2-AKT activation and establish the critical role of the IGF2BP3/SENP1signaling axis in AML development.

Keywords: Acute myeloid leukemia; SENP1; SUMO; m6A.

Fig. 1

Identify SENP1 as a key candidate gene for AML. A Differential gene analysis of SENPs protein families in adverse prognosis group and favorable/intermediate prognosis group using OHSU AML dataset (risk stratification based on ELN 2017, because the latest classification of the dataset is only 2017). B Survival analysis of SENPs family proteins in AML using OHSU AML dataset. (Only SENP1 results are shown, and the rest SENPs results are in the supplementary materials.) (C) Survival analysis of SENPs family proteins in AML using TARGET AML dataset. (Only SENP1 results are shown, and the rest SENPs results are in the supplementary materials.) (D) Survival analysis of SENP1 in AML using TCGA AML dataset. E Survival analysis of SENP1 in pan cancer using TCGA pan cancer data. F Clinical correlation analysis of SENP1 and AML karyotype using BLOODSPOT database. G Mononuclear cells RNA from the bone marrow of AML patients were extracted to detect the expression of SENP1 mRNA expression in AML patients with different risk levels. H Mononuclear cells protein from the bone marrow of AML patients were extracted to detect the expression of SENP1 protein expression in AML patients with different risk levels. I Mononuclear cells RNA from the peripheral blood of AML patients were extracted to detect the expression of SENP1 mRNA expression in AML patients with different risk levels. J Mononuclear cells protein from the peripheral blood of AML patients were extracted to detect the expression of SENP1 protein expression in AML patients with different risk levels

Fig. 2

Knockdown of SENP1 inhibits AML cell proliferation and resistance to apoptosis. A AML proliferation ability was detected using CCK-8 assay at different time points (0, 24, 36, 48, and 72 hours) in HL-60 and KG-1 cells after shSENP1 and shNC transduction. B The effect of silencing SENP1 on the cell cycle of AML was detected by cell cycle assay. C EDU probe was used to detect the effect of silencing SENP1 on AML proliferation rate. D Flow cytometry (representative images are shown) was used to confirm that SENP1 knockdown induced apoptosis. E The levels of cell proliferation (PCNA, C-MYC, PCNA, CYCLINA1 and CDK2) and apoptosis (cleaved caspase-3 and Bcl-2) related proteins were detected by Western blot after SENP1 silencing. F At 27 days, stripped subcutaneous tumors were observed in two different groups. G Use a vernier caliper to measure the growth curve of shSENP1 # 1 and shNC group xenografts every 6 days to draw the tumor size (width 2 × length × π/6) (Left). Subcutaneous tumors were stripped and weighed (Right). H Representative image of KI67 immunohistochemical staining in tumors resected from xenotransplantation model mice. I Representative images of Tunel staining in tumors resected from xenotransplantation model mice

Fig. 3

Overexpression of SENP1 promoted the proliferation and anti-apoptosis of AML. A Western blot was used to detect the efficiency of SENP1 overexpression. B The effect of overexpression of SENP1 on the proliferation of THP-1 was detected by the CCK8 assay. C The effect of overexpression of SENP1 on the proliferation of NB-4 was detected by the CCK8 assay. D EDU probes were used to detect the effects of overexpression of SENP1 on the proliferation rate of AML. E Apoptosis flow was used to detect the effect of overexpression of SENP1 on AML cells apoptosis. F The influence of overexpression of SENP1 on AML cells proliferating protein (PCNA) and apoptotic-related (cleaved caspase-3 and Bcl-2) protein was detected by western blot

Fig. 4

SENP1 promotes AML progression through AKT signal. A The changes in AKT signal, YAP1, β-catenin and p-P65 protein expression levels were detected by Western blot after SENP1 was silenced. B Immunofluorescence double labeling shows silencing of SENP1, impairing AKT and mTOR phosphorylation signals. C Comparison of proliferation and apoptosis related markers detected by Western Blot in shNC, shSENP1 and AKT activator groups. After using AKT phosphorylation activator, the expression of PCNA, cleaved caspase-3 and Bcl-2 returned. D CCK8 assay showed that activating AKT can restore the effect of silencing SENP1 on AML proliferation. E Apoptosis probes showed that activating AKT can restore the apoptotic effect of silencing SENP1 on AML

Fig. 5

HDAC2 is the downstream sumo target of SENP1. A Conduct PPI analysis based on the bioGRID database to identify HDAC2 as a potential downstream target for SENP1. B Forward and reverse CO-IP identified HDAC2 and SENP1 interactions. C Dual immunofluorescence assay showed that SENP1 and HDAC2 were partially co-located in the nucleus. D The HDAC2 sumo sites reported or predicted in previous literature were identified based on the GPS SUMO database, the protein structure was downloaded from the PDB database, and Pymol software was used for protein structure visualization. E SUMO probe found that SENP1 can de-sumo modify HDAC2. F CO-IP identifies the specific domain of HDAC2 to which SENP1 binds. G HDAC2 enzyme activity probe was used to detect the effect of silencing SENP1 on HDAC2 activity

Fig. 6

HDAC2 mediates SENP1 regulation of AKT signaling. A Exploring the CitromeDB database (CistromeDB: 93), HDAC2 has PEAK enrichment in the EGFR promoter. B After silencing SENP1, the ability of HDAC2 to bind to the EGFR promoter decreases. PCR products were used for gel electrophoresis for data visualization. C Overexpression of HDAC2 can restore the effect of silencing SENP1 on AKT phosphorylation signaling, EGFR, cleaved caspase-3 and Bcl-2. D Overexpression of HDAC2 can reverse the effect of silencing SENP1 on the proliferation of AML cells. E Overexpression of HDAC2 promotes AML (HL-60) growth in vivo. F Immunohistochemistry suggests overexpression of HDAC2, which can increase the expression of KI67, EGFR and pAKT (S473), and decrease cleaved caspase-3 expression. G Tunel staining showed that overexpression of HDAC2 reduced the apoptosis of AML cells in mice

Fig. 7

IGF2BP3 drives SENP1 expression in an m6A dependent manner. A Analyzing the RMVar database, it was found that IGF2BP3 binds to the SENP1 3-UTR region and there is a m6A site near the peak. B The effect of silencing IGF2BP3 on SENP1 mRNA expression was detected by RT-PCR. C The effect of silencing IGF2BP3 on the expression of SENP1 protein in HL-60 was detected by western blot. D The effect of silencing IGF2BP3 on the expression of SENP1 protein in KG-1 was detected by western blot. E The co-localization of SENP1 mRNA and IGF2BP3 protein was detected by FISH combined with immunofluorescence. F MeRIP-qPCR showed that there was m6A modification in the 3-UTR region of SENP1. PCR products were used for gel electrophoresis for data visualization. G RIP-PCR showed IGF2BP3 binding in the SENP1 3UTR region in a m6A manner. PCR products were used for gel electrophoresis for data visualization. H RNA pulldown assay detected that IGF2BP3 could bind SENP1 mRNA in an m6A-dependent manner. I mRNA attenuation experiment showed that silencing IGF2BP3 significantly promoted the degradation of SENP1 mRNA. J After mutating the IGF2BP3 binding m6A site identified by the aforementioned RMVar database, double luciferase reporter gene experiment showed that IGF2BP3 could not regulate mutant SENP1 3-UTR

Fig. 8

SENP1 mediates IGF2BP3's ability to regulate AKT/mTOR pathway activity and AML proliferation and anti-apoptosis. A After silencing IGF2BP3, EGFR and SENP1 expression were decreased, and AKT/mTOR pathway activity was down-regulated in HL-60. B After silencing IGF2BP3, SENP1 expression was decreased, and AKT/mTOR pathway activity was down-regulated in KG-1. C CCK8 showed that Over-expression of SENP1 could recover the effects of silencing IGF2BP3 on the proliferation of HL-60. (D) CCK8 showed that Over-expression of SENP1 could recovery the effects of silencing IGF2BP3 on the proliferation of KG-1. E Western blot assays showed that over-expression of SENP1 could reverse the effect of silencing IGF2BP3 on AKT pathway activity in AML cells. F Apoptosis flow cytometry showed that over-expression of SENP1 could reverse the effect of silencing IGF2BP3 on apoptosis of AML cells. G Research mechanism diagram