METTL3 mediates bone marrow mesenchymal stem cell adipogenesis to promote chemoresistance in acute myeloid leukaemia

Adipogenesis of bone marrow mesenchymal stem cells (MSCs) promotes chemoresistance of acute myeloid leukaemia (AML) cells. MSCs from AML patients (AML‐MSCs) display enhanced adipogenesis compared with bone marrow MSCs from healthy donors. However, the precise molecular mechanism by which adipogenesis of MSCs from AML marrow differs from normal counterparts remains obscure. We found that METTL3 significantly inhibits MSC adipogenesis. Here, we aimed to identify the molecular mechanism linking METTL3 and MSC adipogenesis. Analysis of m6A epigenetic changes in MSCs determined via RIP‐qPCR and MeRIP‐qPCR indicated that METTL3 affects AKT protein expression in MSCs by mediating m6A modification of AKT1‐mRNA. Downregulated METTL3 expression in AML‐MSCs induced an increase in AKT protein, resulting in enhanced MSC adipogenesis, thereby contributing to chemoresistance in AML cells. Therefore, targeting AKT regulation by mRNA modification in MSC adipogenesis might provide a novel therapeutic strategy to overcome AML chemoresistance.

induces the progression of leukaemia in mice transplanted with AML [15]. Meanwhile, the demethylase FTO can reduce the levels of ASB2 and RARA m 6 A-mRNA and promote oncogene-mediated malignant transformation and leukaemia [16]. In addition, m 6 A methylation regulates the process of fat formation by mediating mRNA splicing [17] and the expression of adipogenesis-related proteins [17]. Abundant METTL3 expression inhibits the adipogenesis of MSCs derived from porcine BM [18], whereas knocking out METTL3 in healthy mouse BM MSCs enhances adipogenesis [19]. However, the role of METTL3 in the adipogenesis of human AML-MSCs remains unclear.
Here, we investigated the role of METTL3 BMSC adipogenesis to deepen the understanding of the regulatory mechanisms of m 6 A methylation in adipogenesis with respect to modulating AML chemoresistance. Our results of RNA sequencing (RNA-seq), m 6 A microarray analysis and validation using clinical specimens showed that METTL3, AKT-mRNA and AKT-mRNA m 6 A were differentially expressed in MSCs from healthy donors (HD-MSCs) and AML-MSCs. These findings offer a theoretical basis for determining new therapeutic targets for AML from the perspectives of m 6 A and the BMM, which have important clinical value.

AML cell lines and primary BM MSCs
Human AML cell lines HL-60, U937 and THP-1 from Cell Bank of Type Culture Collection Chinese Academy of Sciences (Shanghai, China) were cultured and maintained in RPMI 1640 medium (HyClone, Logan, UT, USA) containing 10% FBS (Gibco, Life Technologies, Grand Island, NY, USA), 2 mM L-glutamine (Gibco), 100 units/mL of penicillin and 100 µgÁmL −1 of streptomycin (Gibco) under a humidified 5% CO 2 atmosphere at 37°C. Human AML-MSCs derived from BM specimens were obtained from the Haematology Department of the Fujian Medical University Union Hospital. This study was conducted in accordance with the Declaration of Helsinki (2013) for experiments involving humans and was approved by the ethics committee of Fujian Medical University Union Hospital. Written informed consent was obtained from all patients before participation. Table 1 shows the clinical information of the patients. BM mononuclear cells (MNCs) were isolated using Ficoll-Hypaque density centrifugation [20]. The MNCs were seeded into low-glucose Dulbecco's modified Eagle medium (LG-DMEM; HyClone) supplemented with 20% FBS, 100 units/mL of penicillin and 100 µgÁmL −1 of streptomycin, and cultured in a humidified 5% CO 2 atmosphere at 37°C for 3 days. Nonadherent cells were removed, and then, adherent MSCs were passaged at 90% confluence and expanded to passage 4 (P4). BM-derived HD-MSCs (Cyagen Biosciences, Santa Clara, CA, USA) were identified as CD34 − /CD44 + /CD45 − /CD73 + /CD90 + / CD105 + types with positive rates > 95%. The MSCs differentiated into osteocytes and adipocytes confirming their multidirectional differentiation potential.

MSC adipogenesis and Oil Red O staining
We cultured MSCs in alternating adipogenic medium A (DMEM supplemented with 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, 1 mM dexamethasone, 10 mM glutamine and 5 mgÁmL −1 insulin [Cyagen Biosciences]) for 3 days, with adipogenic medium B (DMEM supplemented with 10% FBS, 10 mM glutamine and 10 mgÁmL −1 insulin), for 1 days until adipocytes appeared. The differentiation process continued for 14 days, when lipid droplets became obvious indicating differentiation into adipocytes, which were then stained with Oil Red O (ORO; Millipore Sigma Co., Ltd., Burlington, MA, USA) [21,22]. Briefly, purified cells were fixed with methyl alcohol for 3 min and then incubated with Giemsa stain for 20 min at room temperature. The cells were washed with water and air-dried, and then stained for 10 min with a filtered working solution of 0.35% ORO stain in isopropanol to ddH 2 O (3 : 2). The cells were rinsed three times with distilled water and examined using a microscope (Leica Microsystems GmbH, Wetzlar, Germany). We quantified triglyceride accumulation by eluting ORO-stained lipids with 100% isopropanol and then measuring optical density at 450 nm by spectrometry (Thermo Fisher Scientific Inc., Waltham, MA, USA).

MSC osteogenesis and Alizarin Red S staining
The MSCs (2 × 10 5 /well) were cultured with 0.1% gelatine in six-well plates. When the ratio of fusion reached between 60% and 70%, the medium was discarded and osteogenic differentiation culture medium (2 mL) was added. The cells were fixed in 4% paraformaldehyde 21 days after the induction of differentiation for 15 min [21,23] and then stained with 1% Alizarin Red (AR; Sigma-Aldrich Corp., St. Louis, MO, USA) in 10% cetylpyridinium chloride at pH 4.2 for 5 min. Thereafter, optical density was assessed at 562 nm using a spectrometer (Thermo Fisher Scientific Inc.) [24].

Adipocyte differentiation induced using MK-2206 2HCL
MK-2206 2HCL (HY-10358, MCE) was dissolved in DMSO at a stock concentration of 10 mM and diluted to 4 μM in adipogenic media A and media B. The MSCs were incubated for 72 h with adipogenic medium A and adipogenic medium B for 24 h. Adipogenic medium A and medium B were alternated until adipocytes appeared. The differentiation process continued for 12 days before staining with ORO.

RNA m 6 A quantitation
Total RNA was isolated using TRIzol (Invitrogen Corp., Carlsbad, CA, USA) as described by the manufacturer. The quality of RNA was analysed using a NanoDrop ™ Spectrophotometer (Thermo Fisher Scientific Inc.). The m 6 A content in total RNA was quantified using EpiQuik m 6 A RNA Methylation Quantification Kits (Epigentek, Farmingdale, NY, USA) [26]. Briefly, wells were coated with 200 ng of RNA, and then, capture and detection antibodies at suitable concentrations were added separately to the wells as described by the manufacturer. The m 6 A levels were quantified by reading the absorbance of each well at 450 nm to create a standard curve [27]. All samples were assessed in triplicate.

Microarray hybridization and relative data analysis
Immunoprecipitated RNA samples of the HD-MSCs and AML-MSCs were labelled with Cy5 fluorescent dye using Super RNA Labelling Kits (Arraystar Inc., Rockville, MD, USA) and then purified using RNeasy Mini Kits. The Cy5-labelled cRNAs were fragmented and hybridised to a human mRNA and lncRNA m 6 A epitranscriptomic microarray (8 × 60 K; Arraystar) containing 44 122 mRNA and 12 496 lncRNA degenerate probes. The hybridised arrays were scanned using a G2505C Scanner (Agilent Technologies Inc., Santa Clara, CA, USA) [29]. All spots on the microarray were evaluated using Feature Extraction Software Version 11.0.1.1 (Agilent Technologies Inc.). The raw intensity of immunoprecipitated RNAs was normalised using an average of log 2scaled spike-in RNA intensities. The fold changes between the HD-MSCs/AML-MSCs were determined for each transcript, and P-values were calculated. Differentially m 6 A-methylated RNAs were identified using a cut-off of fivefold (P < 0.05). Differentially m 6 A-methylated mRNA transcripts were identified using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses and Gene Set Enrichment Analysis (GSEA) [30].
Sequence-based RNA adenosine methylation site predictor (SRAMP) database The SRAMP can predict m 6 A modification site characteristics [31]. The full-length RNA sequence of AKT1 was entered into SRAMP to predict possible positions of m 6 A modifications on AKT1.

RNA-binding protein immunoprecipitation (RIP) assay
Immunoprecipitated RNA-binding protein was assayed using Magna RIP Kits as described by the manufacturer (Millipore Sigma Millipore Sigma Co., Ltd.). Harvested MSCs were lysed with RIP lysis buffer on ice and then incubated with the input anti-METTL3 (Abcam), anti-m 6 A (Synaptic Systems GmbH, Göttingen, Germany) and anti-IgG at 4°C overnight. The RNA complexes were extracted using proteinase K and phenol/chloroform/isoamyl alcohol, amplified by qRT-PCR.

Statistical analysis
Data are expressed as means AE SD of three independent experiments. All data were analysed using GRAPHPAD PRISM version 8.0 (GraphPad Software Inc., La Jolla, CA, USA). The significance of differences between groups was determined using Student t-tests, and values with P < 0.05 were considered statistically significant.

Sensitivity of AML cells to chemotherapy decreased after co-cultured with differentiated adipocytes due to the enhanced adipogenesis of MSCs
We harvested MSCs and induced their differentiation in vitro. The isolated MSCs were verified by flow cytometry as being positive for CD44, CD73, CD90 and CD105, but negative for the haematopoietic markers CD34 and CD45 (Fig. 1A). To identify the potential ability of multidirectional differentiation, MSCs were further induced into adipocytes and osteocytes, which were identified by staining with ORO and Alizarin Red S, respectively. The osteogenesis (Fig. 1B,C) and proliferation during culture MSCs and AML-MSCs. The capacity for adipogenesis was greater for AML-MSCs than HD-MSCs (Fig. 1E,F). Adipogenesis of the HD-MSCs and AML-MSCs was induced for 14 days, and then, the cells were co-cultured with different AML cells to evaluate chemoresistance of the AML cells. The results showed that chemoresistance of the AML (including HL-60, U937 and THP-1) cells was promoted more by AML-MSCs and then HD-MSCs (Fig. 1G). This indicated that the enhanced adipogenesis of MSCs promotes AML cell resistance. Therefore, understanding the molecular mechanisms affecting adipogenesis is particularly significant.

AKT1-mRNA expression was increased in AML-MSCs and promoted MSC adipogenesis
We compared RNA sequences between MSCs from three healthy donors and four patients who were newly diagnosed with AML to identify differentially expressed genes (DEGs) between HD-MSCs and AML-MSCs using principal component analysis. The results revealed distinct clustering of individual HD-MSCs and AML-MSCs ( Fig. 2A), indicating the rigour of the samples. The results of the analysis showed that 1069 genes were differentially expressed between the groups; 242 and 828 genes were, respectively, upregulated and downregulated (|fold change| ≥ 1.0; P < 0.05; Fig. 2B). Based on the DEGs, we analysed gene enrichment using KEGG pathways. We found that PI3K/AKT signal pathways were significantly upregulated in the AML-MSCs compared with HD-MSCs, which might be associated with the processes of enhancing adipogenesis (Fig. 2C). Downregulated pathways were mainly enriched in chemokine signalling, osteoclast differentiation and others pathways (Fig. 2D). The qPCR and western blotting results showed significantly upregulated AKT and p-AKT (Ser473) expression in AML-MSCs compared with HD-MSCs (Fig. 2E,F). The AKT inhibitor MK-2206 2HCL significantly reduced MSC adipogenesis (Fig. 2  G,H), indicating that AKT is essential for adipogenesis of MSCs. We then incubated AML-MSCs with MK-2206 2HCL and induced them to differentiate into adipocytes. Co-culture of these cells prevented chemoresistance in AML cells (Fig. 2I).

AML-MSCs displayed decreased global m 6 A levels and expressions of METTL3 compared with HD-MSCs
The epigenetic modification RNA plays key roles in the stem cell differentiation. The m 6 A methylase METTL3 is important in the adipogenesis of BMSCs in pigs [18] and mice [19]. Therefore, we aimed to determine whether m 6 A modifications play important roles in the differentiation of human BM MSCs. Global m 6 A levels were decreased in total RNA isolated from AML-MSCs compared with HD-MSCs (Fig. 3  A). We evaluated levels of the m 6 A-related enzymes, METTL3, METTL14, WTAP, FTO and ALKBH5 in MSCs, and found significant differences in the expression of METTL3 (Fig. 3B-F). The founding of qPCR and western blotting revealed significantly decreased METTL3 expression in the AML-MSCs (Fig. 3B,G). The expression of METTL3 mRNA was obvious in the heat map of RNA-seq data. The expression of METTL3 among the five m 6 A-related enzymes was significantly lower in AML-MSCs than HD-MSCs (Fig. 3H).

Decreased METTL3 expression promoted MSC adipogenesis through an increase in AKT1
We evaluated the adipogenesis and chemosensitivity of co-cultured AML cells using gene editing to modulate METTL3 expression in AML-MSCs to determine the effects of METTL3 on MSC adipogenesis. The overexpression of METTL3 significantly inhibited AML-MSC adipogenesis (Fig. 4A,B) and increased the sensitivity of co-cultured AML cells to chemotherapy (Fig. 4C). In contrast, METTL3 knockdown promoted AML-MSC adipogenesis (Fig. 4D,E), and co-culturing AML with differentiated adipocytes knocked down METTL3-induced resistance to chemotherapy (Fig. 4F). We further investigated the molecular mechanism through which METTL3 is linked with AML-MSC adipogenesis. The overexpression and knockdown of METTL3, respectively, decreased and increased AKT1 expression at the mRNA level. The overexpression of METTL3 upregulated the protein expression of p-AKT and AKT and downregulated that of PPAR-γ (Fig. 4G,H), whereas METTL3 knockdown of exerted the opposite effects (Fig. 4I,J).
Overall, these results showed that METTL3 expression negatively regulates MSC adipogenesis and AML chemoresistance.

METTL3 mediated AKT expression by m 6 A modification to inhibit MSC adipogenesis
We investigated possible targets of METTL3 during MSC adipogenesis by profiling m 6 A-methylated RNAs in HD-MSCs and AML-MSCs using a microarray of probes for 44,122 mRNAs and 12,496 lncRNAs. We found that 127 mRNAs were differentially weakly  methylated between the HD-MSCs and AML-MSCs (Fig. 5A). In addition, the results of KEGG pathways (Fig. 5B) and GSEA (Fig. 5C) showed that the m 6 A levels in mRNAs of genes related to the PI3K/AKT signalling pathways were significantly reduced in the AML-MSCs compared with HD-MSCs. To further explore the mechanism of METTL3 mediating the regulation of AKT expression by METTL3 m 6 A to inhibit MSC adipogenesis, we overexpressed METTL3 in MSCs. Subsequent western blot findings showed that the protein expression of AKT was remarkably reduced (Fig. 5D). Further RIP assay revealed that METTL3 could bind to AKT1-mRNA in MSCs (Fig. 5E). Meanwhile, RIP data indicated that AKT1-mRNA in MSCs was modified with m 6 A (Fig. 5F). In the MSC cells, neither METTL3 bound to c-MYC nor was c-MYC present in m 6 A IPs (Fig. 5G,H). We selected c-MYC as a negative control mRNA, which could rule out false positives of METTL3/m 6 A IPs. The above results indicated that METTL3 could affect the protein expression of AKT in MSCs by mediating the m 6 A modification of AKT1-mRNA.
We also predicted m 6 A sites in AKT1-mRNA using SRAMP. The results revealed 27 m 6 A sites (Fig. 5I) of which eight, seven and three were high-, moderateand low-confidence sites ( Table 2). Most m 6 A sites were concentrated in the CDS of AKT1-mRNA.  Further investigation is needed to determine which of the predicted sites are functional.

Discussion
The BMMs of AML are remodelled to ensure that AML cells survive and resist the effects of chemotherapy. Adipocytes in BM are mainly differentiated from MSCs. The enhanced adipogenesis of AML-MSCs can prevent chemotherapy from killing AML cells. Modification of RNA by m 6 A is the most abundant RNA modification in eukaryotic mRNAs, but the role of m 6 A-mRNA in tumorigenesis and tumour development has not been investigated from the perspective of the tumour microenvironment of MSCs. To utilise the restructured BMM that controls the differentiation of MSCs into specific lineages for clinical AML treatment, deeper understanding of the molecular mechanism involved in specific lineage differentiation is essential. The present study identified an important mechanism that promotes the differentiation of pluripotent MSCs into adipocytes. We found that METTL3 mediates the m 6 A modification of AKT1-mRNA, leading to increased AKT1-mRNA and protein expression, which renders MSCs more likely to differentiate into adipocytes, thus changing the BMM and causing changes in AML chemoresistance (Fig. 6).
The dynamic and reversible m 6 A modification of RNA is the most abundant internal RNA modification in eukaryotes [32], and it plays a key role in regulating the proliferation, metastasis, pluripotency and immunity of tumour stem cells. The m 6 A methylase, METTL3, has two-way effects in different cancers. For example, abundant METTL3 expression in AML [15,33,34], liver cancer [26] and glioblastoma [35] promotes the occurrence and development of tumours. In contrast, METTL3 can serve as a tumour suppressor to inhibit the growth and invasion of ovarian [36] and prostate [37] cancer. Abundant METTL3 expression inhibits MSC adipogenesis in pigs and mice. However, the effects of METTL3 on human MSCs have not been investigated in detail. The present findings showed more adipogenesis and lower METTL3 expression in AML-MSCs than HD-MSCs. The negative impact of METTL3 on MSC adipogenesis implied that METTL3 m 6 A-dependently regulates the differentiation of MSCs.
The PI3K/AKT pathway plays important roles in mediating the proliferation, apoptosis and differentiation of cells [38]. Overactivation of the PI3K/AKT pathway results in aberrant cell cycle progression, altered cell adhesion and motility, inhibition of apoptosis and the induction of angiogenesis [38,39]. Neprilysin accelerates adipogenesis in the MSC line C3H10T1/2 by enhancing PI3K/AKT activation [40]. Collectively, our results indicated that AKT plays a regulatory role in MSC adipogenesis and that m 6 A-dependently interacts with METTL3. The preliminary results of RNA-seq and the m 6 A microarray showed reduced m 6 A activity in the PI3K/AKT signalling pathway. The m 6 A modification of mRNAs associated with PI3K/AKT signalling pathways was significantly upregulated, which might have promoted the adipogenic differentiation of MSCs. However, the precise molecular mechanisms require further exploration. Decreases in METTL3 regulate AKT activities and promote the proliferation and tumorigenicity of endometrial cancer, and m 6 A methylation regulates AKT pathways [41]. However, METTL3 regulation of PI3K/AKT signalling pathways in the adipogenic differentiation of MSCs has not been assessed. The effects of m 6 A modification on mRNA transcription are mediated by specific m 6 A-binding proteins called m 6 A readers [42]. The YTH domain family of proteins bind as m 6 A in mammals [43]. For instance, YTHDF2 recognises and destabilises mRNA containing m 6 A [44]. Because m 6 A modifications and the YTH domain family are widespread in eukaryotes and play regulatory roles in various biological processes, we propose that m 6 Abinding proteins play specific roles in m 6 A-mediated adipogenesis. Amount of AKT protein expression negatively correlated with levels of m 6 A-modified mRNA.
We plan to further investigate whether YTHDF2 promotes AKT degradation and inhibits MSC differentiation. Due to a limited sample size, further study of a larger sample is required to verify the present finding.
We found that METTL3 is associated with the adipogenesis of human MSCs. Decreased METTL3 expression in AML-MSCs significantly reduced the amount of m 6 A modification of mRNA associated with PI3K/AKT signalling pathways. Activation of the PI3K/AKT signalling pathways might promote MSC adipogenesis, which could potentially mediate AML chemoresistance. The present findings provide a theoretical foundation that should help to determine new targets of AML treatment from the perspective of BMMs and provide important insights that will lead to novel clinical strategies for treating AML.