Bone marrow samples were obtained from adults with AML (n=20; median age, 61.2; age range, 24-83; 11 male patients, 9 female patients) and non-leukemic patients (n=10; median age, 51.2; age range, 31-82; 5 male patients, 5 female patients) recruited at the Chongzuo People's Hospital of Youjiang Medical University for Nationalities (Chongzuo, China) between July 2021 and December 2022. The present study was approved (approval no. 2022-03-10) by the Ethics Committee of Youjiang Medical University for Nationalities (Baise, China). Written informed consent was obtained from all patients involved in the study.

The mRNA profiling data and clinical information of AML patients were downloaded from cBioPortal (http://www.cbioportal.org/), Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/gds), and The Cancer Genome Atlas (TCGA) (https://www.cancer.gov/tcga). The cBioPortal dataset contains data for 451 AML patients, and RNA sequencing (RNA-seq) was performed on bone marrow mononuclear cells (BMMCs) or peripheral blood mononuclear cells (PBMCs) (16). The GSE71014 dataset contains clinical information and microarray-based mRNA expression data for 104 AML patients with normal karyotypes (17). RNA-seq data and clinical information for 151 AML patients are available in the TCGA cohort. Kaplan-Meier survival analysis was used to analyze the difference in survival between patients with high- and low-expression of ZCCHC10 mRNA using the 'survival' package in R software (version 4.1.2) (https://www.r-project.org).

All chemicals and anti-bodies used in the present study are listed in Table SI. All cell lines were obtained from American Type Culture Collection (ATCC) and cultured according to ATCC instructions. All cell lines were free of mycoplasma contamination and authenticated using short tandem repeat profiling by Yubo Biological Technology Co., Ltd.

Mononuclear cells were extracted from bone marrow or peripheral blood using human lymphocyte separation medium (Applygen Technologies Inc.). Total RNA was isolated from mononuclear cells or AML cell lines using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and then reverse-transcribed into cDNA using the All-in-One™ First-Strand cDNA Synthesis Kit (GeneCopoeia, Inc.) according to the manufacturer' instructions. SYBR Green-based real-time PCR (GeneCopoeia, Inc.) was performed in an ABI 7900 thermocycler (Thermo Fisher Scientific, Inc.) according to the manufacturer' instructions. Primers were designed by PrimerBank (https://pga.mgh.harvard.edu/primerbank) and are listed in Table SII. The thermocycling conditions were as follows: Initial denaturation at 94°C for 10 min, followed by 40 cycles of denaturation at 94°C for 10 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. All reactions were examined in technical triplicate. The 18S rRNA was used as the internal reference, and the relative gene expression was estimated using 2^−ΔΔCq method (18).

Proteins were extracted with radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology, Inc.), protein concentration was determined by bicinchoninic acid. A total of 30-50 µg protein was loaded per lane and separated by 10-12% SDS-PAGE. After separation, proteins were transferred onto PVDF membranes, and the membrane were blocked with 5% non-fat milk for 1 h at room temperature. After sequential incubation with primary antibodies overnight at 4°C and appropriate horseradish peroxidase-conjugated secondary antibodies at a dilution of 1:1,0000 (cat. nos. 5450-0010 and 5220-0338, respectively; KPL, Inc.) for 1 h at room temperature, the membranes were exposed to Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.) and images were captured using a gel imaging system (Tanon 4600SF; Tanon Science and Technology Co., Ltd.). The software ImageJ (version 1.53n; National Institutes of Health) was used for densitometric analysis.

All lentiviruses were purchased from Shanghai GeneChem Co., Ltd. The lentiviruses overexpressing ZCCHC10 cDNA and expressing ZCCHC10 shRNA were previously described by the authors (10). The target sequences of SNHG1 shRNA were previously described by Sun et al (19) (shSNHG1-1: GGC CAG CAC CTT CTC TCT AAA, shSNHG1-2: GGT TTG CTG TGT ATC ACA TTT CT). Cells were infected with lentiviral particles at a multiplicity of infection of 10 for 8 h, then cultured with fresh medium for 72 h. At 72 h after infection, infected cells were selected with puromycin (2.5 µg/ml).

DNA was modified with the EpiTect bisulfite kit (cat. no. 59104; Qiagen, Inc.) for conversion of all unmethylated cytosines to uracil. The converted DNA was first amplified with an outer primer pair specific for converted DNA without distinguishing between methylated and unmethylated DNA, and then amplified with primers specific for unmethylated and unmethylated DNA. PCR products were detected by 2% agarose gel electrophoresis and visualized using ethidium bromide. Primers were designed using MethPrimer software (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi) (Table SII).

The ChIP experiment was performed using a kit (cat. no. Bes5001) from Guangzhou BersinBio Co. Ltd. Briefly, cells were subjected to crosslinking with 1% formaldehyde and lysed, and chromatin was then sheared with sonication until DNA fragments of 200-1,000 bp were obtained. The cross-linked chromatin was precipitated with normal IgG, an anti-DNA methyltransferase (DNMT)1 antibody and an anti-DNMT3B antibody. The amount of precipitated DNA was evaluated by semi-quantitative PCR. In preliminary experiments, the thermal cycling program was selected for linear amplification. The final PCR products were separated by electrophoresis on a 1.5% agarose gel and visualized with ethidium bromide. Primers are listed in Table SII.

The RIP assay was performed using the RIP kit (cat. no. Bes5101; Guangzhou BersinBio Co. Ltd.). A total of 5×10⁷ cells were lysed using 2.5 ml lysis buffer, 0.1 ml lysates were used for input, the remaining lysates were evenly divided into three parts and precipitated with IgG, an anti-DNMT1 antibody and an anti-DNMT3B antibody, respectively. Input and precipitated RNA were purified and reverse-transcribed into cDNA. SYBR Green-based real-time PCR was then performed with specific primers (Table SII) as aforementioned.

Cells were seeded in 24-well plates at a density of 1×10³ cells/well in human methylcellulose complete medium (R&D Systems Europe, Ltd.) according to the manufacturer's instructions. After 20 days of incubation at 37°C and 5% CO₂, colonies with at least 50 cells were counted under an inverted light microscope.

Cells were cultured in 96-well plate (2,000 cells per well) at 37°C and 5% CO₂ for indicated time. Cell viability was determined according to the manufacturer's instructions using a Cell Counting Kit-8 (CCK-8) assay (cat. no. C0037; Beyotime Institute of Biotechnology). Briefly, 10 µl CCK-8 solution was added to each well and cultured at 37°C and 5% CO₂ for 4 h, and the absorbance was measured at 450 nm wavelength.

Cell cycle and apoptosis assays were performed by flow cytometry as previously described (10).

NOD/SCID mice (male, ~7-8 weeks old) were obtained from Hunan Slac Jingda Laboratory Co., Ltd. (Changsha, China) and housed in groups of 5 per cage with water and food provided ad libitum, in a specific-pathogen-free room with filtered air and controlled light/dark cycle (12/12 h), temperature (24±2°C) and relative humidity (45-65%). All mice were pretreated with an intraperitoneal injection of 20 mg/kg busulfan (APeXBIO Technology LLC) 24 h before inoculation and then injected intravenously (lateral tail vein) with 5×10⁶ cells in 100 µl serum-free medium (OCI-AML3-Vec, OCI-AML3-Zh10, OCI-AML3-shNC, or OCI-AML3-shZh10). Each treatment group included at least 10 mice; three of the mice were euthanized through cervical dislocation 30 days after inoculation, and the rest were monitored for survival. The tibias, femurs and spleens of the mice were harvested at sacrifice. Spleens, livers and kidneys were fixed with 10% formalin at room temperature for 24 h and processed for staining with hematoxylin and eosin or an anti-human CD45 antibody. Bone marrow (mixed from tibias and femurs) was minced in PBS and processed into single-cell suspensions for analysis by flow cytometry. All mice were maintained under standard conditions in accordance with institutional animal welfare guidelines. The animal experiments were approved (approval no. 2020-020) by the Animal Ethics Committee of Hunan Normal University (Changsha, China).

NOD/SCID mice were pretreated with busulfan and injected intravenously with MOLM13-Vec or MOLM13-Zh10 cells according to the aforementioned methods. Each treatment group included 10 mice. A total of eight days after inoculation, each of the aforementioned groups was divided into two subgroups (5 animals/subgroup) and treated with vehicle or venetoclax via oral gavage for three weeks (5 days/week). As recommended by Selleck Chemicals, venetoclax was prepared by the formulation in 5% DMSO, 50% PEG300 and 5% Tween-80. Mice were euthanized and dissected as aforementioned.

Statistical analysis was performed using Excel 365 software (Microsoft) and R software. The data are presented as the mean ± SD values. The difference between the means of two groups was analyzed using unpaired Student's t-test. The log-rank test was used to compare two survival curves. Relationships between two variables were evaluated using Pearson's correlation coefficient analysis, and paired Student's t-test was used to assess if the correlation was statistically significant. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR was performed to measure ZCCHC10 gene expression in BMMCs from 20 AML patients and 10 non-leukemic patients and it was found that ZCCHC10 mRNA expression was significantly decreased in AML patients compared with non-leukemic patients (Fig. 1A). Western blot analysis showed that the protein level of ZCCHC10 was significantly lower in AML (ML2, MOLM-13, OCI-AML3, OCI-AML5, and HL-60) and acute monocytic leukemia (U937) cell lines than in PBMCs (Fig. 1B and C). To assess the prognostic significance of ZCCH10 expression, Kaplan-Meier survival analysis was performed based on clinical and gene expression data from AML patients in cBioPortal, GEO and TCGA cohorts. In all three cohorts, AML patients with higher ZCCHC10 expression had longer overall survival times (Fig. 1D-F). These results suggested that ZCCHC10 expression is a favorable prognostic factor in AML.

ZCCHC10 expression in blood leukocytes has been shown to be regulated by DNA methylation (20). Therefore, methylation-specific PCR was performed to determine the methylation status of the ZCCHC10 promoter in BMMCs from AML and non-leukemic patients. As demonstrated in Fig. 2A, methylated PCR products were more abundant than unmethylated products in 16 of the 20 AML samples (Nos. 1, 2, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 18, 19 and 20), but the ZCCHC10 promoter was hypomethylated in all nonleukemic samples except sample no. 6. Moreover, treatment with 5-aza-2′-deoxycytidine (decitabine), a DNA methylation inhibitor, significantly increased the ZCCHC10 protein level in AML cell lines (Fig. 2B). These results suggested that ZCCHC10 expression is suppressed by hypermethylation in AML. The lncRNA SNHG1 has been shown to be upregulated in AML (21,22). Accordingly, in the present study, SNHG1 was found to be overexpressed in the BMMCs of AML patients (Fig. 2C). Correlation analysis revealed that SNHG1 expression was negatively correlated with the ZCCHC10 mRNA level (Fig. 2D). In addition, the correlation between ZCCHC10 and SNHG1 was analyzed using the expression data from the cBio-Portal database and an inverse correlation was found between SNHG1 and ZCCHC10 (Fig. 2E). SNHG1 has been reported to be mainly distributed in the nucleus, where it is involved in transcriptional regulation by interacting with transcription factors (19) or histone methyltransferases (23). Therefore, it was investigated whether SNHG1 affects the expression and methylation of ZCCHC10. Compared with transduction of lentiviruses expressing scrambled shRNA, transduction of lentiviruses expressing shSNHG1-2 increased the ZCCHC10 mRNA and protein levels (Fig. 2F-I) while suppressing CpG island methylation in the ZCCHC10 gene (Fig. 2J). However, transduction of the ineffective shSNHG1-1 lentivirus had no significant effects (Fig. 2F-J). These results indicated that SNHG1 suppresses ZCCHC10 expression by promoting methylation of the ZCCHC10 promoter.

Notably, sequence analysis by BLAST identified a specific sequence (5′-AAA GUG CUG GGA UU-3′) in SNHG1 with full complementarity to five sites surrounding the CpG island in the ZCCHC10 promoter (Fig. 3A). It was hypothesized that this sequence is a potential DNA-binding motif of SNHG1. To examine whether the predicted DNA-binding motif is required for the regulation of DNA methylation of the ZCCHC10 gene, wild-type and mutant SNHG1 expression plasmids were transfected into 293 cells and the methylation status of the ZCCHC10 promoter was determined 24 h after transfection (Fig. 3B and C). The results of methylation-specific PCR showed that overexpression of wild-type SNHG1 increased the methylation of the ZCCHC10 promoter (Fig. 3B) and decreased the ZCCHC10 mRNA and protein levels (Fig. 3D and E). By contrast, overexpression of mutant SNHG1, whose DNA-binding motif was deleted, had no effect on the methylation or expression of ZCCHC10 (Fig. 3B, D and E).

DNA methylation is mediated by DNMTs, including DNMT1, DNMT3A and DNMT3B. DNMT3A is frequently mutated in AML, whereas DNMT1 and DNMT3B are usually overexpressed in AML and contribute to the hypermethylation of tumor suppressor genes (24). Therefore, a ChIP assay was performed to investigate the binding of DNMT1 and DNMT3B to the ZCCHC10 promoter in U937 acute monocytic leukemia cells. It was found that both DNMT1 and DNMT3B could bind to the ZCCHC10 promoter, but DNTM1 bound with slightly higher affinity than DNMT3B (Fig. 3F). Moreover, knockdown of SNHG1 reduced the interaction of DNMT1 and DNMT3B with the ZCCHC10 promoter (Fig. 3G). The RIP assay revealed that SNHG1 could bind to DNMT1 and DNMT3B (Fig. 3H). These results indicated that SNHG1 associates with both the ZCCHC10 promoter and DNMTs, and then recruits DNMTs to the ZCCHC10 promoter, leading to methylation of the ZCCHC10 promoter.

To elucidate the role of ZCCHC10 in AML, the lentivirus-mediated expression system was used to generate AML cell lines with stable overexpression or knockdown of ZCCHC10 gene expression (Fig. 4A). Cell proliferation and anchorage-independent growth were investigated using a CCK-8 assay and a methylcellulose colony formation assay, respectively. The CCK-8 assay revealed that overexpression of ZCCHC10 decreased the viability, whereas suppression of ZCCHC10 increased the viability (Fig. 4B). According to the results of the methylcellulose colony formation assay, cells stably expressing ZCCHC10 formed significantly less and smaller colonies than the cells expressing empty vector, whereas cells expressing ZCCHC10 shRNA formed more and larger colonies than those expressing scrambled shRNA (Figs. 4C and S1). Flow cytometric analysis showed that the proportion of G1 phase cells was increased among cells overexpressing ZCCHC10 whereas it was decreased among cells with ZCCHC10 knockdown, indicating that ZCCHC10 led to cell cycle arrest in the G1 phase (Figs. 4D and S2). In addition, ZCCHC10 promoted apoptosis (Figs. 4E and S3).

It was previously demonstrated that ZCCHC10 plays a role in stabilizing the p53 protein in lung cancer (10). Consistent with this finding, the present study demonstrated that overexpression of ZCCHC10 increased the levels of p53, Bax and p21, but decreased the level of Bcl2; the effect of ZCCHC10 knockdown on p53 and its targets (p21, Bax and Bcl2) was opposite to that of ZCCHC10 overexpression (Fig. 4F). This suggested that ZCCHC10 activates the p53 pathway in AML cells.

NOD/SCID mice were injected with 5×10⁶ cells (OCI-AML3-Vec, OCI-AML3-Zh10, OCI-AML3-shNC or OCI-AML3-shZh10) via the lateral tail vein, and the leukemia burden and survival of the NOD/SCID mice were compared. Kaplan-Meier survival analysis demonstrated that mice injected with OCI-AML3-Zh10 cells survived longer than mice injected with OCI-AML3-Vec cells. By contrast, mice injected with OCI-AML3-shZh10 cells had shorter survival times than mice injected with OCI-AML3-shNC cells (Fig. 5A). Leukemia burden was determined by assessing the spleen size and human hematopoietic (hCD45) chimerism in spleen and bone marrow. The mice bearing OCI-AML3-Zh10 cells had smaller and lighter spleens than the mice bearing OCI-AML3-Vec cells, but the mice bearing OCI-AML3-shZh10 cells had larger and heavier spleens than the mice bearing OCI-AML3-Vec cells (Fig. 5B and C). IHC assays and flow cytometric analysis demonstrated that overexpression and knockdown of ZCCHC10 decreased and increased, respectively, the number of hCD45⁺ cells in the spleen (Fig. 5D) and bone marrow (Fig. 5E and F). These results indicated that ZCCHC10 can suppress the growth of AML cells in hematopoietic tissues in a xenograft mouse model.

It has been reported that the antiapoptotic protein BCL2 is overexpressed in AML and that targeting BCL2 may be a successful therapeutic strategy in AML patients (25). A BCL2-specific inhibitor, venetoclax (ABT-199), has been approved by the FDA for use in AML. The aforementioned results revealed that overexpression of ZCCHC10 increased cleavage of PARP-1 and caspase 3, while treatment with venetoclax enhanced the effects of ZCCHC10 (Fig. 6A and B). Knockdown of ZCCHC10 decreased the cleavage of PARP-1 and caspase 3, but venetoclax treatment reversed the effect of ZCCHC10 knockdown (Fig. 6A and B). In addition, the effects of ZCCHC10 on venetoclax sensitivity were examined in vitro and in vivo. Overexpression of ZCCHC10 decreased the half maximal inhibitory concentration (IC₅₀) values of venetoclax in ML2 and AML3 cells, but knockdown of ZCCHC10 increased the venetoclax IC₅₀ values in AML3 cells (Fig. 6C), suggesting that ZCCHC10 enhanced sensitivity to venetoclax. In addition, sensitivity to venetoclax was examined in vivo in AML xenograft mice. Venetoclax treatment significantly decreased splenomegaly (Fig. 6D and E) and engraftment of leukemic cells in the peripheral blood (Figs. 6F and S4), bone marrow (Figs. 6G and S4), spleen, liver, and kidney (Fig. 6H) in mice bearing MOLM-13-Vec and MOLM13-Zh10 cell-derived xenografts. Moreover, venetoclax had a stronger anti-AML effect in mice bearing MOLM13-Zh10 cell-derived xenografts than in the corresponding control xenograft mice (Fig. 6D-H). These results indicated that ZCCHC10 enhances sensitivity to venetoclax in AML in vitro and in vivo.