Using online tools (http://www.rgenome.net/cas-offinder/), we designed a small guide RNA (sgRNA1: 5’-GCTACCACGCCTGAGCCCGT-3’; SgRNA2: 5’-tccasgRNAccttctgagactcccc-3), which targeted DNMT3A exon 6. DNMT3A-targeting sgRNAs were synthesized using the HiScribe™ T7 Quick High Yield RNA Synthesis Kit (E2050S, NEB, USA) according to the manufacturer’s instructions. After being treated with 1 µg/ml doxycycline (PHR1789, Sigma, Sweden) for one week, the SKM-1 cells were stained with anti-DNMT3A antibody, and DNMT3A negative cells were sorted as DNMT3A KO cells, using TA cloning to select double-KO cell strain.

Full-length cDNA of human DNMT3A was cloned into the EcoRI/BamHI multiple cloning site of the lentiviral vector pCDH-EF1-MCS-T2A-copGFP. DNMT3A-R882H mutant was generated using the KAPA HiFi HotStart PCR kit (KR0369–v10.17, Kapa Biosystems, USA) and confirmed via full-length DNA sequencing. After 48 to 72 h, the supernatants containing infectious lentiviruses were collected, filtered through a 0.45-µM filter, and incubated with target cells for transduction. The viral titer was determined via flow cytometry using infected SKM-1 cells.

Generation of isogenic K562 clones with the DNMT3A R882H mutation using designed TALENs, as described previously (19).

Human AML cells SKM-1 and THP-1 were obtained from the China Center for Type Culture Collection (Wuhan, China). All cell lines were tested and authenticated, utilizing short tandem repeat profiling every six months. SKM-1 and THP-1 cells were cultured in RPMI-1640 (GIBCO, Life Technologies Corporation, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 100 U/mL of penicillin-streptomycin at 37°C and 5% CO₂ and normal O₂. The CCK8 assay (A311-01/02, Vazyme, China) was used to test the proliferation of cells, according to the manufacturer’s instructions.

THP-1 monocytes were differentiated into macrophages via a 24-h incubation with 150 nM phorbol 12-myristate 13-acetate (PMA, HY-18739-1, MedChemExpress, USA). AML cells were cultured in the upper chamber of transwell inserts (HKN-3450, Corning, USA) in 6-well plates. Macrophages were placed in the lower chamber of a 6-well transwell plate (E: T; 1:1) and co-cultured for 48 h.

Cells were suspended in 1 ml TRIzol reagent (15596026, Thermo Fisher Scientific, USA), and RNA was extracted using the RNeasy Micro Kit (74004, QIAGEN, Germany) according to the manufacturer’s instructions and converted to cDNA. mRNA expression was assessed via qRT-PCR, performed using a ChamQ Universal SYBR qPCR Master Mix kit (Q711, Vazyme, China).

Supernatants were collected after a 48-h co-cultivation. Cells and apoptotic body free supernatants were concentrated using 3-kDa Amicon Ultra exclusion filters (MilliporeSigma, USA). The concentration of chemokine (C-C motif) ligands (CCL3, CCL4, IL-1β, IL-10, TGF-β) were determined in concentrated supernatants using an ELISA Kit (bioswamp, China), and quantified at 450 nm.

Macrophages were collected and blocked with 3% bovine serum albumin (BSA) for 45 min, and incubated with anti-human CD86-PE (305406, BioLegend, USA, 1:200), anti-human CD206-APC (321109, BioLegend, USA, 1:200), or anti-human CD68-PE (333808, BioLegend, USA, 1:200) according to the manufacturers’ instructions. Tumor cells were collected and incubated with Annexin V-APC/PI cell death detection kit (abs50001, Absin, China, 1:50) according to the manufacturers’ instructions for 15 min. For each sample, at least 1 × 10⁴ cells were analyzed with the Beckman CytoFLEX flow cytometry. Apoptosis rate (apoptosis%= early apoptotic cells (Upper right quadrant) + late apoptotic cells (Right lower quadrant).

Whole cells were lysed in the RIPA lysis buffer supplemented with protease inhibitor cocktail (Roche Diagnostics, USA). Samples were separated using SDS-PAGE (Bio-Rad, USA) and transferred onto nitrocellulose membranes (Bio-Rad, USA). Primary antibodies, including anti-DNMT3A (3598S, Cell Signaling, USA, 1:1000), anti-β-actin (A2066, Sigma-Aldrich, Sweden, 1:1000), as well as horseradish peroxidase-conjugated secondary antibodies (7074, Cell Signaling, USA, 1:1000), and chemiluminescent substrate (Invitrogen, USA) were used.

Xenografts tissues were fixed with paraformaldehyde, embedded in paraffin, and cut into sections for immunostaining. Mouse monoclonal anti-CD163 (sc-58965, Santa Cruz Biotechnology, USA) and Rabbit anti-CD68 (ab955, Abcam, UK) were used as the primary antibodies. After incubation with the respective primary antibodies in a humidified chamber overnight at 4°C, tissues were rinsed three times with PBS. Then incubated with secondary antibody 1h at room temperature. Finally, the nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) after washing three times with PBS. After staining, sections were observed under an inverted fluorescence microscope.

SCID mice (4 weeks old) were obtained from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Mice were randomly chosen and divided into five groups (five mice in each group): SKM-1^control, SKM-1^KO, SKM-1^NC, SKM-1^{R882H SC1}, and SKM-1^{R882H SC2}. AML cells (5 × 10⁶) were mixed with 5 × 10⁶ M1 macrophages and implanted into the armpits of the mice.

Total RNA extraction was performed with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Extraction of total cell RNA was carried out with the help of RNAiso Plus (TaKaRa, Dalian, China). 1 ~ 2 μg of total RNA per sample (3 SKM-1^WT cell samples, 3 SKM-1^KO cell samples and 3 SKM-1^{R882H SC1} cell samples) was utilized for constructing a KAPA Stranded RNA-Seq Library Prep Kit (Illumina) sequencing library. Afterwards, the constructed library was cross-checked using an Agilent 2100Bioanalyzer and quantified by qRT-PCR. Lastly, the Illumina HiSeq 4000 (service provided by Kangchen Bio-tech, Shanghai, China) was used to further sequence the libraries that had different mixed samples.

Data were represented as mean ± SEM. D ‘Agostino and Pearson omnibus normality tests were used to detect the partial normality of experimental data. When the data were not normally distributed, logarithmic transformation was performed to conform to the normal distribution. Differences between groups were tested using one-way ANOVA followed by Sidak test for multiple comparisons or a paired Student t-test as appropriate using Prism 7.0 (GraphPad Software). P ˂ 0.05 was considered statistically significant.

CRISPR-Cas9 was used to knock out DNMT3A in wild type SKM-1 cells to obtain SKM-1^KO cells. SKM-1^control is a monoclonal cell which was generated in the same conditions, but not transfected with sgRNA. DNMT3A-R882H (CGC > CAC) mutant SKM-1 cell strains (SKM-1^R882H cells) were constructed via lentiviral transfection, and two mutant monoclones (SKM-1^{R882H SC1} and SKM-1^{R882H SC2}) were selected. The negative control monoclone (SKM-1^NC cells) was transfected with an empty vector. Sanger sequencing showed that SKM-1^KO cells had -1 and -14 frameshift mutations in exon 6 of the DNMT3A allele ( Figure 1A ). Western blot analysis showed that SKM-1^KO cells did not express DNMT3A, and SKM-1^R882H mutant strains highly expressed DNMT3A-R882H ( Figures 1B–D ). After cytarabine treatment, the apoptosis rate of the experimental groups (SKM-1^KO, SKM-1^{R882H SC1}, and SKM-1^{R882H SC2} cells) was significantly lower than that of the control groups (wild type SKM-1^control cells and SKM-1^NC cells) ( Figure 1E ), and the proliferation rate of the experimental groups was not significantly different from that of the control groups ( Figure 1F ). Generation of isogenic K562 clones with the DNMT3A R882H mutation using designed TALENs (19).

RNA-seq results showed that SKM-1^{R882H SC1} and SKM-1^KO cells co-expressed 96 upregulated genes and 185 downregulated genes compared with the control group ( Figure 2A ). The overall distribution of differentially expressed genes (fold change ≥ 2 and p-Value ≤ 0.05) was reflected in the volcanic map ( Figure 2B ). Gene set pathway enrichment analysis (GSEA) revealed that the co-expressed differential genes were significantly enriched in inflammation and immune-related pathways, such as NF-κB signaling pathway (P = 0.006) and Toll-like receptor (TLR) signaling pathway (P = 0.00003) ( Figure 2C ). GSEA revealed that the TLR pathway was downregulated in SKM-1^KO (NES = -1.49, Nominal p < 0.001) and SKM-1^{R882H SC1} cells (NES = -1.87, Nominal p < 0.001) ( Figure 2D ). Transcription regulators, such as Jun proto-oncogene (JUN), Fos proto-oncogene (FOS), CCAAT enhancer-binding protein β (CEBPB), and Kruppel-like transcription factor 2 (KLF2), as well as inflammatory factors, such as interleukin-1β (IL-1β), macrophage inflammatory protein 1β (MIP-1β or CCL4 (including CCL4, CCL4L1, CCL4L2), and macrophage inflammatory protein 1α (MIP-1α or CCL3, including CCL3L1, CCL3L3), which play an important role in inflammation and immune-related pathways, were significantly downregulated in SKM-1^{R882H SC1} and SKM-1^KO cells ( Figure 2E ). We imported these differential genes into the STRING database (https://string-db.org/) for protein-protein interaction network functional enrichment analysis (PPI). The results showed that transcription regulators JUN and FOS were at the center of this interaction network, which indicated that AP-1 (a heterodimer composed of C-FOS and C-JUN) may play a key role in the downregulation of the inflammatory factors ( Figure 2F ). The trend verified via qRT-PCR was consistent with that of RNA-seq (data not shown).

Due to the abnormal expression of macrophage-related inflammatory proteins in DNMT3A-mutated cells, we co-cultured AML cells with THP-1-derived macrophages. After PMA induction, THP-1 monocytes became adherent. Under 400× magnification, the cells were polynuclear with antenna formation ( Figure 3A ). The expression of recognized macrophage markers, cluster of differentiation 68 (CD68), was significantly increased (65.4% vs 6.94%, P < 0.001) in THP-1-derived macrophages ( Figure 3B ). These results indicated that THP-1 cells successfully differentiated into macrophages. CD86, IL-6, inducible nitric oxide synthase (iNOS), and CC-chemokine receptor 7 (CCR7) were used as markers of M1 macrophages, while macrophage mannose receptor (CD206), peroxisome proliferator-activated receptor (PPAR-γ) as markers of M2 macrophages. Co-culturing THP-1-derived macrophages and AML cells for 48 h. The results showed that the mRNA expression of M1 macrophage-related markers was significantly downregulated (P < 0.05) in macrophages of the experimental group (SKM-1^KO and SKM-1^R882H cells, K562^clone143 and K562^clone368) ( Figures 3C, D ), while the mRNA expression of M2 macrophage-related markers was significantly increased compared with the control group (SKM-1^control cells and SKM-1^NC cells, K562^control cells) (P < 0.01) ( Figures 3C, D ). Flow cytometry verified the above trends compared with the control group, CD86 expression was downregulated (P < 0.01) and CD206 expression was upregulated (P < 0.01) in macrophages of the experimental group ( Figures 3E–H ). To amplify the killing effect of macrophages on AML cells, we co-cultured AML cells with M1 macrophages. After 48 h, the apoptosis rate of the experimental group was significantly lower than that of the control group (P < 0.05) ( Figures 3I–L ).

We mixed SKM-1 cells with M1 macrophages and injected them subcutaneously into the mice ( Figure 4A ). We found that the growth rate of the experimental group was significantly larger than that of the control group (P < 0.05) ( Figures 4B, C ). Immunofluorescence showed that the proportion of CD68+ and CD163+ double-positive cells (M2 macrophages) infiltrating the tumor tissue of the experimental group was significantly higher than that in the control group ( Figures 4D, E ).

Co-cultivation promoted the expression of the inflammatory factors in AML cells. However, the rise in the expression of MIP-1α, MIP-1β and IL-1β in the experimental group was significantly lower than that in the control group (P < 0.01), while IL-10 and TGF-β expression in the experimental group was not significantly different from that in the control group (P > 0.05) ( Figures 5A, B ).

In the supernatant of the co-culture system, the concentration of inflammatory factors MIP-1α, MIP-1β, and IL-1β in the experimental group was significantly lower than that in the control group, as revealed using ELISA (P < 0.05). Moreover, the concentration of immunosuppressive factors IL-10 and TGF-β was significantly higher than that of the control group (P<0.05). This indicated that in the experimental group microenvironment, anti-tumor immunity was suppressed compared with the control group ( Figures 5C, D ).

AP-1, an intracellular transcriptional activator, is a heterodimer composed of C-FOS and C-JUN. Considering that AP-1 is at the center of our protein-protein interaction network, we selected resistin, an AP-1 agonist, to verify the key regulatory role of AP-1. The results showed that resistin significantly increased the expression of MIP-1α, MIP-1β, and IL-1β in AML cells (P < 0.001) (nearly ten times) ( Figures 6A, B ). After treating AML cells with resistin for 12 h, we co-cultured them with macrophages. It narrowed the difference in the expression levels of MIP-1α, MIP-1β, and IL-1β between mutant and control groups after the co-culture, and even promoted the mutant group to further express MIP-1α, MIP-1β and IL-1β ( Figures 6C, D ). Still, there was no significant difference in the expression of anti-inflammatory factors IL-10 and TGF-β between mutant and control groups ( Figures 6C, D ). As to the co-cultured macrophages, the results showed that the mRNA expression of M1 macrophage-related markers in the mutant group was not lower than that in the control group, while the mRNA expression of M2 macrophage-related markers was significantly decreased compared with that in the control groups (P < 0.05) ( Figures 6E, F ). Flow cytometry verified the above trends; CD86 expression in the mutant group was not significantly different from that in the control group, and CD206 expression was downregulated (P < 0.01) in macrophages of the experimental group ( Figures 6G–J ). We also co-cultured the resistin-treated AML cells with M1 macrophages for 48 h. There was no significant difference between the apoptosis rate of the mutant groups and that of the control group ( Figures 7A–D ). We detected the concentration of inflammatory factors in the supernatant of macrophages and AML cells treated with drugs. The results showed that after co-cultivation the concentration of pro-inflammatory factors MIP-1α, MIP-1β, and IL-1β in the supernatant of the experimental group was no longer lower than that in the control group (even slightly higher), and the concentration of immunosuppressive factors IL-10 and TGF-β was no longer higher than that in the control group (or even slightly lower) ( Figures 7E, F ).