High-throughput library screening identifies the expression patterns, prognostic significance, and clinical correlation of SENP1

To comprehensively investigate the role of SENPs (SENP1-3 and SENP5-8) family proteins in AML, we analyzed the expression pattern and survival significance by investigating publicly available OHSU-AML datasets (Table S4). We found that SENP1 and SENP2 were highly expressed in the high-risk AML group and closely related to poor prognosis (Fig. 1A, B, and S1A ). We explored the TARGET dataset for prognostic analysis to avoid errors in single-center sample datasets. We found that both SENP1 and SENP2 were strongly associated with poor clinical outcomes (Fig. 1C and S1B). SENP1, however, is more clinically relevant. Therefore, we focus on SENP1's role in AML in this study. In the TCGA-AML dataset, SENP1 was associated with poor prognosis (Fig. 1D). Pan cancer analysis showed that tumor patients with SENP1 high expression had a shorter survival period (HR=1.2, P<0.001) than those with low SENP1 (Fig. 1E). These results suggest that SENP1 is a key molecule in AML pathology.

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

Subsequently, we analyzed the correlation between SENP1 expression and clinical features of patients with AML. We found that SENP1 expression was the highest in AML patients with P53 mutation (Figure.S2A). SENP1 expression did not significantly correlate with patient age (Figure.S2B). TP53 mutations have been identified by the 2022 ELN guidelines as markers of poor AML prognosis. Exploring the MILE dataset, a large dataset of AML showed no significant difference in the expression of SENP1 in bone marrow mononuclear cells of normal and AML patients (Figure.S2C); however, we found that SENP1 mRNA expression was significantly upregulated in AML patients with adverse prognosis (Figure.S2D). In addition, exploring the BLOODSPOT database, we found that the expression level of SENP1 in AML with adverse prognosis karyotypes, such as complex karyotypes, was significantly higher than that in AML with favorable prognosis karyotypes, such as t(15,17) (Fig. 1F) [20]. Furthermore, we found that the expression of SENP1 mRNA and protein was significantly higher in AML patients with poor prognosis than in AML patients with favorable prognosis (Fig. 1G-J). Clinical correlation analysis found that patients with high SENP1 expression had lower BM BLASTS (65.96 vs 53.26, P=0.0002), more M1 type (1 vs. 7, P=0.0015), and fewer M4 type (22 vs. 3, P=0.0026) (Table 1). Univariate and multivariate COX regression analyses suggested that SENP1 was an independent risk factor for AML (hazard ratio [HR] =1.03, P=0.03) (Table 2). These data support an important role for SENP1 in AML development.

Table 1 Correlation analysis between SENP1 and clinical characteristics of AML patients based on OHSU databaseTable 2 Univariate and multivariate analysis of the relationship between SENP1 expression and overall survival in AML patients

Subsequent pan-cancer analysis showed that SENP1 was most highly expressed in AML among all TCGA tumors (Figure.S2E). Differential gene analysis revealed that SENP1 is differentially expressed in multiple malignancies, such as BRCA and BLCA (Figure.S2F), and is closely associated with poor prognosis in malignant tumors, such as LIHC and KIRP (Figure.S2G). These data support SENP1's potential role in promoting malignancy progression.

Knockdown of SENP1 significantly inhibits AML progression in vitro and in vivo

Next, we verified SENP1's potential role in AML using in vitro and in vivo experiments. We measured SENP1 expression in four classical AML cell lines. We found that SENP1 was highly expressed in HL-60 and KG-1, and low in THP-1 and NB4 (Figure.S3A). This may be because KG-1 cells have complex karyotypes and HL-60 cells have P53 mutations. We used three independent lentivirus sequences to interfere with SENP1 expression in AML cells (HL-60 and KG-1) and found that the first and third sequences were effective in interfering with SENP1 expression (Figure.S3B). Using the CCK8 cell proliferation assay, we found that AML cell proliferation was significantly reduced after SENP1 silencing (Fig. 2A). The cell cycle assay showed that AML cells were blocked in the S1 phase after silencing of SENP1 (Fig. 2B). The EDU probe showed that the AML cell proliferation rate decreased after SENP1 silencing (Fig. 2C). The apoptosis rate were markedly increased after SENP1 silencing (Fig. 2D). Western blotting also showed that after silencing SENP1, the expression of anti-apoptosis protein (Bcl-2), proliferation protein (PCNA, C-MYC), and S phase checkpoint (CyclinA1 and CDK2) in AML cells was downregulated, and pro-apoptosis markers (Cleaved Caspase-3) were elevated (Fig. 2E). Next, we used two independent sgRNA sequences to knock out SENP1 in HL-60 and KG-1(Figure.S4A and S4B). CCK8 assay showed that the proliferation rate of AML cells decreased significantly after SENP1 knockout (Figure.S4C). The EDU probe showed that the AML cell proliferation rate decreased after SENP1 knockout (Figure.S4D). The apoptosis rate were markedly increased after SENP1 knockout (Figure.S4E). Cell cycle analysis showed that after SENP1 knockout, AML cells were blocked in S1 phase (Figure.S4F).

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

Subcutaneous tumorigenesis experiments in nude mice showed that SENP1 promoted AML cell proliferation in vivo (Fig. 2F, 2G, and S5A). Immunohistochemical results showed that the subcutaneous tumor proliferation marker KI67 decreased after SENP1 was silenced (Fig. 2H). Immunofluorescence showed that after SENP1 silencing, the abundance of proliferating PCNA protein in the subcutaneous tumors decreased (Figure.S5B). Immunofluorescence also showed that after SENP1 silencing, the expression of anti-apoptotic markers (Bcl-2) in subcutaneous tumors decreased (Figure.S5B). TUNEL staining revealed an increase in apoptosis in the silent SENP1 group (Fig. 2I). Western blotting also showed that after silencing of SENP1, PCNA expression was downregulated (Figure.S5C).

Overexpression of SENP1 promoted the proliferation and anti-apoptosis of AML

Next, we overexpressed SENP1 in THP-1 and NB4 cells (Fig. 3A). CCK8 assay showed that overexpression of SENP1 promoted AML cell proliferation (Fig. 3B and C). EDU showed an increased the proliferation rate of AML cells after increasing SENP1 expression (Fig. 3D). The apoptosis rate of AML cells was markedly reduced by increasing SENP1 expression (Fig. 3E). Western blotting showed that the expression of anti-apoptotic proteins (BCL-2) and proliferative proteins (PCNA) was upregulated, and the expression of pro-apoptotic protein (cleaved caspase-3) was downregulated after overexpression of SENP1 (Fig. 3F).

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

We aimed to determine whether SENP1 regulates AML progression in a sumo enzyme-dependent manner. We overexpressed two SENP1 enzyme-inactivated mutants (R630L and K631M) in AML cells (Figure.S3A). CCK8, EDU, and cell cycle assays showed that the SENP1 mutant did not affect AML cell growth (Figure.S6B-E).

SENP1 promotes AML progression through AKT signaling pathway

Next, we investigated the mechanism by which SENP1 regulates AML progression. To predict the downstream signaling pathways of SENP1, we conducted differential gene analysis based on SENP1 expression and identified 4746 DEGs between high-SENP1 and low-SENP1 expression groups. GO annotation was performed on 4746 DEGs (Figure.S7A and Table.S5). GO annotation showed that these genes were related to biological processes, such as mitotic division, nuclear division, and regulation of cell proliferation (Figure.S7B). GSEA and GSVA were performed on 4746 DEGs (Figure.S7C and S7D, Tables S6 and S7). The intersection of the GSEA and GSVA results was used to avoid errors caused by a single analysis method. These identified common signaling pathways, such as the adherent junction, cell cycle, and AKT/mTOR signaling pathways (Figure.S7E). Abnormal activation of the AKT/mTOR signaling pathway has been identified as a key feature of AML progression [21].

We then examined the changes in the expression of key molecules (p-AKT, p-mTOR, YAP1, p-P65, and β-catenin), which AML common signaling pathways in AML, after SENP1 knockdown. We found that AKT/mTOR signaling was significantly downregulated after SENP1 silencing (Fig. 4A). Next, we used the previous subcutaneous tumor tissue sections for double-fluorescence labeling. We found that silencing SENP1 significantly weakened AKT and mTOR phosphorylation (Fig. 4B). Addition of the AKT activator SC-79 restored the effect of SENP1 silencing on the expression of AML cell proliferation and apoptosis markers (Fig. 4C). CCK8 and apoptosis flow probes showed that SC-79 reversed the effect of SENP1 interference on AML cell proliferation and apoptosis (Fig. 4D and E).

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

SENP1 regulates HDAC2 activity in a sumo-dependent manner

To further investigate the mechanism by which SENP1 activates AKT signaling, we excavated the BioGRID database and conducted protein-protein interaction network analysis (PPI analysis) [22]. Interestingly, we observed potential interactions between HDAC2 and SENP1 (Fig. 5A). HDAC2 was also located in the central region of the network, suggesting that it may play a crucial role in SENP1 function. Studies have reported that sumo1 modification of HDAC2 may inhibit HDAC2 function [23]. HDAC2 plays a key role in AKT activation by transcriptionally activating EGFR expression [17]. Importantly, HDAC2 has also been proven to be a critical regulatory molecule in AML development [18]. The CO-IP assay revealed that SENP1 was combined with HDAC2 (Fig. 5B). Fluorescence colocalization showed that SENP1 and HDAC2 were partially colocalized in the AML nucleus (Fig. 5C). We identified a large number of sumo modification sites in HDAC2 using the GPS SUMO database (Fig. 5D). The SUMO probe showed that after overexpression of SENP1, HDAC2 sumo modification was suppressed (Fig. 5E). HDAC2 has both deacetylase and non-deacetylase protein domains, and SENP1 mainly binds to the HDAC2 deacetylase protein domain (Fig. 5F). These data support the regulation of HDAC2 deacetylase activity by SENP1 in a sumo enzyme–dependent manner. We found that silencing SENP1 did not affect HDAC2 expression but significantly reduced HDAC2 activity (Figure.S8A and 5G).

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

HDAC2 mediates SENP1 to regulate AKT-mTOR signaling in AML

By exploring the Cistrome DB database, we found that HDAC2 has peak enrichment in the EGFR promoter based on a previous ChIP-seq dataset (Fig. 6A) [24]. After silencing of SENP1, the ability of HDAC2 to bind to the EGFR promoter decreased (Fig. 6B). Overexpression of HDAC2 partially restored the effect of silencing SENP1 on EGFR, AKT phosphorylation signaling, and anti-apoptosis-related markers in AML cells (Fig. 6C and S9A). CCK8 experiments showed that overexpression of HDAC2 partially restored the silencing effect of SENP1 on AML cell proliferation (Fig. 6D). In the subcutaneous tumor experiment in nude mice, overexpression of HDAC2 promoted AML growth in vivo (Fig. 6E). Immunohistochemistry suggested overexpression of HADC2, enhancing KI67, EGFR, and p-AKT, and reducing apoptosis proteins (cleaved caspase-3) (Fig. 6F). TUNEL staining showed that HDAC2 overexpression reduced apoptosis (Fig. 6G). These results suggest that SENP1 activates the AKT pathway and promotes AML progression, at least in part dependent on HDAC2.

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

SENP1, which is regulated by IGF2BP3 mediated m6A, is highly expressed in high-risk groups of AML

Next, we explored the upstream regulatory mechanism of SENP1 in high-risk AML patients. In our previous study, we found that m6A is abnormally modified in the high-risk group of AML and systematically analyzed that the m6A reading protein IGF2BP3 is highly expressed in high-risk groups of AML and promotes AML development [4]. Therefore, we speculated that m6A may participate in SENP1 regulation in AML. By exploring the RM2Target database, we found that 14 RNA m6A regulators could potentially regulate SENP1 [25]. In order to screen the core factors, we conducted differential gene analysis between high-risk and low-risk AML groups and found that only YTHDF1, IGF2BP3, and FTO were significantly different (Figure.S10A). Survival analysis based on the TARGET and TCGA databases revealed that only IGF2BP3 was positively associated with poor prognosis in AML and pan-cancer patients (Figure.S10B-D). Correlation analysis revealed a positive correlation between IGF2BP3 and SENP1 expression (Figure.S10E and S10F). These findings suggest that IGF2BP3 is a key regulator of SENP1 expression. To determine the binding mechanism between IGF2BP3 and SENP1, we searched the RMVar database. The results showed that IGF2BP3 has the potential to bind to the SENP1 mRNA 3-UTR region (Fig. 7A) [26]. We found that SENP1 protein and mRNA levels decreased significantly after silencing IGF2BP3 (Fig. 7B-D). It is known that the IGF2BP1/2/3 protein family can bind m6A and contribute to the regulation of mRNA stability, so what role does IGF2BP1/2 play when it comes to SENP1. The results showed that silencing IGF2BP1/2 did not affect the regulation of SENP1 expression (Figure.S10G and S10H). These results suggested that SENP1 is specifically regulated by IGF2BP3.

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

Immunofluorescence combined with FISH revealed a large amount of colocalization of SENP1 mRNA and IGF2BP3 protein in AML cells (Fig. 7E). RIP experiments confirmed that IGF2BP3 and m6A could bind to the 3-UTR region of SENP1 mRNA (Fig. 7F and G). RNA PULL DOWN experiments also showed that IGF2BP3 binds to SENP1 mRNA in an m6A-dependent manner (Fig. 7H). mRNA half-life experiments showed that after silencing IGF2BP3, SENP1 mRNA decay accelerated (Fig. 7I). Mutation at the m6A site predicted by the aforementioned database and a double luciferase assay revealed that IGF2BP3 does not regulate mut-SENP1 (Fig. 7J).

SENP1 mediates IGF2BP3's ability to regulate AKT/mTOR pathway activity and AML proliferation and anti-apoptosis

Next, we explored the role of SENP1 in IGF2BP3 regulated AML progression and signaling pathways. By re-analyzing our previous RNA-seq data of IGF2BP3 silencing in HL-60 [4], we found that IGF2BP3 function was significantly enriched in the PI3K/AKT/mTOR signaling pathway (Figure.S11A). Finally, we found that after silencing IGF2BP3, the expression of SENP1 and the activity of the AKT/mTOR pathway were significantly reduced (Fig. 8A and B). Functional recovery experiments showed that overexpression of SENP1 could reverse the effect of silencing IGF2BP3 on the proliferation, anti-apoptosis, and AKT pathway activities of AML cells (Fig. 8C-F).

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