A novel AML1-ETO/FTO positive feedback loop promotes leukemogenesis and Ara-C resistance via stabilizing IGFBP2 in t(8;21) acute myeloid leukemia

Zhou, Wei; Li, Siying; Wang, Hong; Zhou, **gfeng; Li, Shuyi; Chen, Guofeng; Guan, Wei; Fu, **anli; Nervi, Clara; Yu, Li; Li, Yonghui

doi:10.1186/s40164-024-00480-z

A novel AML1-ETO/FTO positive feedback loop promotes leukemogenesis and Ara-C resistance via stabilizing IGFBP2 in t(8;21) acute myeloid leukemia

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Published: 24 January 2024

Volume 13, article number 9, (2024)
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A novel AML1-ETO/FTO positive feedback loop promotes leukemogenesis and Ara-C resistance via stabilizing IGFBP2 in t(8;21) acute myeloid leukemia

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Wei Zhou^1,2,
Siying Li³,
Hong Wang^1,2,
**gfeng Zhou⁴,
Shuyi Li⁴,
Guofeng Chen⁵,
Wei Guan⁶,
**anli Fu⁷,
Clara Nervi⁸,
Li Yu⁴ &
…
Yonghui Li^1,4

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Abstract

Background

t(8;21)(q22;q22) is one of the most frequent chromosomal abnormalities in acute myeloid leukemia (AML), leading to the generation of the fusion protein AML1-ETO. Despite t(8;21) AML being considered as a subtype with a favorable prognosis, approximately 30–50% of patients experience drug resistance and subsequent relapse. N⁶-methyladenosine (m⁶A) is demonstrated to be involved in the development of AML. However, the regulatory mechanisms between AML1-ETO and m⁶A-related enzymes and the roles of dysregulated m⁶A modifications in the t(8;21)-leukemogenesis and chemoresistance remain elusive.

Methods

Chromatin immunoprecipitation, dual-luciferase reporter assay, m⁶A-qPCR, RNA immunoprecipitation, and RNA stability assay were used to investigate a regulatory loop between AML1-ETO and FTO, an m⁶A demethylase. Gain- and loss-of-function experiments both in vitro and in vivo were further performed. Transcriptome-wide RNA sequencing and m⁶A sequencing were conducted to identify the potential targets of FTO.

Results

Here we show that FTO is highly expressed in t(8;21) AML, especially in patients with primary refractory disease. The expression of FTO is positively correlated with AML1-ETO, which is attributed to a positive regulatory loop between the AML1-ETO and FTO. Mechanistically, AML1-ETO upregulates FTO expression through inhibiting the transcriptional repression of FTO mediated by PU.1. Meanwhile, FTO promotes the expression of AML1-ETO by inhibiting YTHDF2-mediated AML1-ETO mRNA decay. Inactivation of FTO significantly suppresses cell proliferation, promotes cell differentiation and renders resistant t(8;21) AML cells sensitive to Ara-C. FTO exerts functions by regulating its mRNA targets, especially IGFBP2, in an m⁶A-dependent manner. Regain of Ara-C tolerance is observed when IGFBP2 is overexpressed in FTO-knockdown t(8;21) AML cells.

Conclusion

Our work reveals a therapeutic potential of targeting AML1-ETO/FTO/IGFBP2 minicircuitry in the treatment for t(8;21) patients with resistance to Ara-C.

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Introduction

Acute myeloid leukemia (AML) is a highly heterogeneous disease characterized by complex cytogenetic and molecular landscape, leading to diverse prognostic outcomes [1, 2]. The t(8;21)(q22;q22) chromosomal translocation is one of the most common cytogenetic abnormalities in AML [3]. Clinically, t(8;21) AML has been categorized as a subtype with favorable prognosis; nonetheless, the outcomes of patients with this subtype still exhibit significant heterogeneity. Approximately 30–50% of t(8;21) AML patients experience relapse, resulting in poor clinical outcomes [4], suggesting the presence of genetic and clonal heterogeneity within this specific subtype of AML. Therefore, an in-depth understanding of the pathogenesis of t(8;21) AML will aid in improving personalized clinical interventions.

The AML1-ETO fusion protein, generated by the t(8;21)(q22;q22) chromosomal translocation, plays the pivotal role in the initiation of t(8;21) AML. AML1-ETO disturbs the normal biofunctions of core binding factors, blocks differentiation and induces aberrant self-renewal of hematopoietic stem cells, transforms them into preleukemic cells [5]. Cooperating gene mutations (including c-KIT, FLT3 and N/KRAS, etc.) enhance the proliferative potential of the preleukemic cells as ‘second hit’ and eventually causes full-blown leukemia [3]. Additional pathological mechanisms, such as epigenetic regulation, work in collaboration to facilitate the development of t(8;21) AML [6, 7].

As the most abundant chemical modification in eukaryotic mRNAs, N⁶-methyladenosine (m⁶A) plays a crucial role in the initiation, progression, and drug resistance of human cancer [8,9,10,11,12]. Several studies have indicated that alterations in m⁶A modifications contribute to leukemia development: m⁶A writers (i.e., methyltransferase-like 3 (METTL3) [13, 14], methyltransferase-like 14 (METTL14) [15, 16] and Wilms’ tumor 1-associating protein (WTAP) [17]), erasers (i.e., fat mass and obesity-associated protein (FTO) [18, 19] and α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5) [20, 21]), and readers (i.e., YTH domain family 2 (YTHDF2) [22, 23]) are found to act as oncogenes in AML. In particular, the m⁶A demethylase FTO is reported to be highly expressed in certain AML subtypes, including those carrying t(15;17)/PML-RARA, t(11q23)/MLL-rearrangements, NPM1, or FLT3-ITD mutations, and promote leukemogenesis by regulating its mRNA targets, especially RARA and ASB2, through m⁶A demethylation [18]. A recent study identifies that the high expression of FTO in NPM1 mutant AML patients may be attributed to an intragenic long noncoding RNA within FTO. It is a result of intragenic transcription activation occurring in the final intron of FTO, induced by a non-random epigenetic event [24]. R-2-hydroxyglutarate, produced by mutant isocitrate dehydrogenase 1/2 (IDH1/2), inhibits the proliferation of leukemic cells by suppressing the demethylase activity of FTO and its downstream CEBPA/MYC-relevant pathway [19]. Moreover, elevated expression of FTO contributes to resistance to tyrosine kinase inhibitor in AML [25]. However, the definite regulatory relationship between FTO and leukemia-initiating fusion protein AML1-ETO and the role of FTO in progression and chemoresistance in t(8;21) AML remain largely unknown.

Here, we report a positive feedback regulatory loop between AML1-ETO and FTO: AML1-ETO promotes FTO expression via transcription factor PU.1; meanwhile, FTO stabilizes AML1-ETO mRNA via m⁶A reader YTHDF2, thus upregulating its expression. Suppression of FTO expression genetically or pharmacologically inhibits cell proliferation, promotes cell differentiation and resensitizes resistant t(8;21) AML cells to Cytosine arabinoside (Ara-C), via downregulating its functional downstream target IGFBP2. Our findings suggest a therapeutic potential of targeting AML1-ETO/FTO/IGFBP2 minicircuitry in the treatment of t(8;21) AML.

Methods

Samples from patients with leukemia and healthy donors

The collection of patient samples was approved by the Medical Ethics Committee of Shenzhen University General Hospitals. Mononuclear cell (MNC) isolated from bone marrow (BM) samples from patients with t(8;21) AML (Additional file 2: Table S1) and healthy donors were obtained with informed consent and stored in TRIzol reagent (Invitrogen) until use.

Cell culture

The t(8;21) AML cell lines SKNO-1 and Kasumi-1 were maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (Solarbio). Human embryonic kidney (HEK)-293T cells were maintained in DMEM (Gibco) supplemented with 10% FBS and 1% penicillin–streptomycin.

DNA site-directed mutagenesis

The pCMV6-wild type FTO-CDS (coding region sequence) plasmid was used to construct mutant FTO-CDS carrying H231A and D233A single-point mutations. The plasmid and mutation sites of FTO-CDS were kindly provided by Dr. Chuan He and Dr. Guifang Jia. DNA site-directed mutagenesis was performed using the Mut Express II Fast Mutagenesis Kit V2 (Vazyme), following the manufacturer’s instructions. Briefly, a pair of primers was designed based on the desired mutation sites. PCR amplification of target plasmids was performed using the designed primers. Amplification products were digested with DpnI, followed by homologous recombination reaction to cyclize the products. Cyclized products were used to transform competent bacterial cells, which were plated onto selective media overnight at 37 °C. Single bacterial colonies were identified by sequencing.

Plasmid construction

Wild-type and mutant FTO-CDS were PCR-amplified from pCMV6-wtFTO and pCMV6-mutFTO, respectively, using the following primers: forward 5′-CTAGACTAGTATGAAGCGCACCCCGACTGCCGAGGAACGA-3′ and reverse 5′-ATTTGCGGCCGCCTAGGGTTTTGCTTCCAGAAGCTGA-3′, and then inserted into the pLVX lentiviral plasmid between SpeI and NotI. The DNA sequences encoding shRNAs targeting human FTO (shFTO#1 TRCN0000255405 and shFTO#2 TRCN0000255403), mouse Fto (TRCN0000277195, TRCN0000216341, and TRCN0000277143 mixed), human YTHDF2 (shYTHDF2#1 TRCN0000254336, shYTHDF2#2 TRCN0000254410), human IGFBP2 (shIGFBP2#1 TRCN0000006574 and shIGFBP2#2 TRCN0000006577), and mouse Igfbp2 (TRCN0000420942, TRCN0000422736, and TRCN0000012860 mixed) were commercially synthesized by TranSheep Bio Co. Ltd., and cloned into the pLKO.1 plasmid. Likewise, human PU.1-CDS and IGFBP2-CDS were commercially synthesized and cloned into the pTSB vector. For the experiments of silencing the three YTHDF proteins, the shRNA sequences targeting the three proteins were collectively inserted into a single pLKO.1 plasmid (DF1: TRCN0000294275; DF2: TRCN000254410; DF3: TRCN000365173 as #1 and DF1: TRCN0000286871; DF2: TRCN0000254336; DF3: TRCN0000167772 as #2).

Lentiviral infection and siRNA transfection

Lentiviruses were generated from HEK-293T cells via transfection of lentiviral expression vectors together with plp-VSV-G:psPAX2 packaging mix (Addgene) using 15 μM PEI. Supernatants containing the virus were harvested 48 and 72 h after cotransfection and were added to target cells along with polybrene (8 μg/mL, Solarbio). Selection of infected cells was performed using puromycin (Solarbio, 1.5 μg/mL) 96 h after infection.

The siRNA targeting the fusion site of the AML1-ETO mRNA (siAE#1, sense, 5′-CCUCGAAAUCGUACUGAGAAG-3′, antisense, 5′-UCUCAGUACGAUUUCGAGGUU-3′; siAE#2, sense, 5′-CCUCGAAAUCGUACUGAGATT-3′, antisense, 5′-UCUCAGUACGAUUUCGAGGTT-3′) was designed according to previous studies [26] and was commercially synthesized by Sangon Biotech. siRNAs against human FTO (sc-75002), IGFBP2 (sc-37195), YTHDF2 (sc-78661) and YTHDF3 (sc-77724) were purchased from Santa Cruz Biotechnology. siRNAs were transfected into cells using the 4D-Nucleofector System (Lonza, Cologne, Germany) with the SF Cell Line 4D-Nucleofector X Kit by DS-138 for SKNO-1 and CM137 for Kasumi-1. Cells were harvested at 48 or 72 h after transfection.

The SKNO-1-siAE cell line was kindly provided by Dr. Clara Nervi which was generated by transfected with the lentiviral vector pRRLcPPT.hPGK encoding siAE oligonucleotides to silence the expression of AML1-ETO in SKNO-1 cells [6]. Kasumi-1-siAE cells were generated by siRNA transfection using the 4D-Nucleofector system.

AML1-ETO9a-driven AML mice model

The animal experiments in our study were approved by the Ethics Committee of Shenzhen University General Hospital. BM and spleen cells isolated from AML1-ETO9a-driven AML mice were kindly provided by Dr. Lan Wang [27] and were further amplified by retransplantation into sublethally irradiated (450 cGy γ-rays) C57BL/6 recipient mice via tail vein injection (2 × 10⁴ cells/mouse). Freshly harvested BM and spleen cells were infected with pLKO.1-based lentiviral shRNA targeting mouse Fto or mouse Igfbp2, or pLKO.1-scrambled shRNA, and injected into sublethally irradiated (450 cGy) C57BL/6 recipient mice via tail vein injection (2 × 10⁴ cells/mouse). For FB23-2 treatment, FB23-2 was administered at 6 mg/kg daily for 30 days from the third day after transplantation.

Xenograft model

Four- to six-week-old female BALB/c nude mice were used and maintained in a pathogen-free environment. After 3–5 days of adaptive feeding, each mouse received subcutaneous inoculation into the right flank with 2 × 10⁷ SKNO-1 cells in 200 µL of PBS. When the tumors reached a volume of approximately 150 mm³, the mice were randomly divided into 4 groups (n = 6 in each group). Then the mice in each group received intraperitoneal injection once a day of DMSO, Ara-C (75 mg/kg/day), FB23-2 (2 mg/kg/day), or a combination of both. The mice were sacrificed by cervical dislocation under anesthesia after two-week drug injection and the harvested xenograft tumors were analyzed further.

NOD/SCID/IL2rγ^null immunodeficient mice model

The NOD/SCID/IL2rγ^null immunodeficient NSG mice (6–8 weeks old, female) were irradiated at 200 cGy and injected with GFP⁺ SKNO-1 cells (5 × 10⁶) via tail vein. The mice were randomly divided into 4 groups (n = 6 in each group), receiving intraperitoneal injection every other day of DMSO, Ara-C (75 mg/kg/day), FB23-2 (4 mg/kg/day), or a combination of both, starting one week after injection. The mice were sacrificed by cervical dislocation under anesthesia 6 weeks after AML cell injection.

Cell proliferation and colony forming unit assays

The Cell Counting Kit-8 (Do**do) was used to conduct cell proliferation assays. Cells were seeded at 30,000 cells/100 μL per well into 96-well plates in triplicate and incubated with 10 μL Cell Counting Kit-8 solution at 37 °C for 4 h. Absorbance was read using a microplate reader (Multiskan FC, Thermo Fisher) at 450 nm.

The MethoCult™ H4434 Classic (StemCell Technologies) culture system was used to perform colony-forming unit assays. Briefly, 1000 cells were mixed thoroughly with 4 mL MethoCult™ medium, seeded into 35 mm dishes in triplicate, and incubated for 14 days at 37 °C and 5% CO₂ with humidification. Clusters that showed morphological hematopoietic characteristics containing more than 50 cells were counted as colonies.

Flow cytometric analysis of apoptosis, cell cycle, and differentiation

The Annexin V-FITC Apoptosis Detection kit (Do**do) was used for apoptosis assays. Cells were harvested and washed twice with cold PBS. After being suspended in 1 × Annexin V binding buffer, cells were stained with Annexin V-FITC and propidium iodide (PI) for 15 min on ice. For cell cycle assays, a DNA labeling solution (Cytognos) was used. Briefly, after collection and washing with cold PBS, cells were stained with DNA labeling solution for 15 min at 25 °C. For cell differentiation assays, human AML cells were harvested and washed with cold PBS; peripheral blood (PB), BM, and spleen cells were harvested from AML1-ETO9a AML mice, and erythrocytes were lysed using RBC Lysis Buffer (00-4333-57, Invitrogen). Human AML cells were stained with anti-human CD11b (301310, Biolegend) and isolated mice cells were stained with anti-CD11b-BV421 (101251, Biolegend) for 30 min on ice. Stained cells were analyzed using a CytoFLEX S Flow Cytometer (Beckman Coulter).

RNA extraction and quantitative RT-PCR

Total RNA was extracted using TRIzol reagent (Invitrogen), in accordance with the manufacturer’s instructions. Briefly, 1 µg of total RNA was reverse-transcribed into cDNA using the GoScript™ Reverse Transcription System (Promega). Quantitative real-time PCR (qPCR) analysis was performed using 1 µL diluted cDNA (fivefold dilution) with the SYBR Fast Universal (KAPA Biosystems) on the ABI Prism 7500 sequence detection system (Applied Biosystems). Samples were run in triplicate, with GAPDH as the endogenous control. Additional file 2: Table S3 lists the primers used.

Western blotting

Western blotting was performed with equal amounts of cell lysates (~ 20 μg), using the following antibodies: anti-FTO (#45980, 1:1000; Cell Signaling Technology), anti-AML1 (#4334, 1:1000; Cell Signaling Technology), anti-PU.1 (#2266, 1:1000; Cell Signaling Technology), anti-YTHDF2 (24744-1-AP, 1:1000; Proteintech), anti-YTHDF3 (sc-377119, 1:200; Santa Cruz Biotechnology), anti-IGFBP2 (#3922, 1:1000; Cell Signaling Technology), anti-Igfbp2 (sc-515134, 1:200; Santa Cruz Biotechnology), anti-β-actin hFAB rhodamine (12004163, 1:1000; Bio-Rad), and anti-β-actin (#4970, 1:1000; Cell Signaling Technology).

m⁶A dot blot

Polyadenylated (poly(A)) mRNA was isolated from total RNA using the GenElute™ mRNA Miniprep Kit (Sigma-Aldrich). Isolated mRNA was denatured at 95 °C for 3 min and then dotted onto an Amersham Hybond™-N⁺ membrane (RPN203B, GE Healthcare) using a Bio-Dot Apparatus (#170-6545, Bio-Rad). After UV cross-linking, the membrane was blocked for 1 h using 5% nonfat milk, incubated at 4 °C with m⁶A antibody (ab232905, 1:400; Abcam) overnight, and then incubated with HRP-linked anti-rabbit immunoglobulin G (IgG, #7074, Cell Signaling Technology) for 1 h. Finally, the membrane was imaged using a ChemiDoc Touch Imaging System (Bio-Rad). For loading control, the RNA dotted membrane was stained with 0.02% methylene blue (Solarbio) diluted with 0.5M sodium acetate (PH 5.0).

Chromatin immunoprecipitation (ChIP)

The SimpleChIP® Plus Enzymatic Chromatin IP Kit (#9005, Cell Signaling Technology) was used to conduct ChIP assays in accordance with the manufacturer’s instructions. Briefly, to crosslink proteins to DNA, cells were treated with formaldehyde (final concentration 1.5%) for 10 min. Micrococcal nuclease and sonication were used to digest chromatin to fragments of approximately 150–900 bp, which were then incubated with PU.1 antibody (#2266, Cell Signaling Technology), AML1 antibody (#sc-8563, Santa Cruz Biotechnology), ETO antibody (#sc-9737, Santa Cruz Biotechnology) or normal rabbit IgG antibody at 4 °C overnight with rotation. Finally, 1% of digested chromatin was preserved as an input sample and purified along with the immunoprecipitated complex. The primers using for ChIP-qPCR the listed in Additional file 2: Table S3.

Dual-luciferase reporter assays

The DNA fragments of different lengths of the FTO promoter were amplified from human genomic cDNA by PCR using the primers listed in Additional file 2: Table S3 and inserted into the pGL3-basic vector (Promega) between KpnI and HindIII enzyme sites. The pcDNA3.1 vector containing DNA fragments of human PU.1-CDS were synthesized by Vigene Biosciences, Inc. Total 400 ng of pcDNA3.1 vector (i.e., pcDNA3.1-mock or pcDNA3.1-PU.1-CDS) and pGL3-basic vector (i.e., PGL3-P1 to PGL3-P4), and 20 ng pRL-TK Renilla luciferase reporter vector were cotransfected into HEK293T cells in 24-well plates using the SuperFect® Transfection Reagent (Qiagen). Luciferase activity was assessed 48 h after transfection using a Dual-Luciferase Reporter Assay System (Promega). Each experiment was repeated in triplicate.

The wild-type and m⁶A-mutant IGFBP2 3′ untranslated region (3′-UTR) were directly synthesized and inserted into the psiCHECK2 vector (Promega) by Vigene Biosciences, Inc. Briefly, 400 ng wild-type or mutant psiCHECK2-IGFBP2-3′-UTR and 100 ng FTO-expressing vector (i.e., pCMV6-wtFTO, pCMV6-mutFTO, or pCMV6-mock) were cotransfected into HEK-293T cells in 24-well plates with the SuperFect® Transfection Reagent (Qiagen). Relative luciferase activity was assessed using the Dual-Luciferase Reporter Assay System (Promega) 48 h posttransfection. Each experiment was repeated in triplicate.

Gene-specific m⁶A-qPCR

The Magna MeRIP m⁶A Kit (17-10499, Millipore) was used to estimate the relative m⁶A levels in individual transcripts. Briefly, poly(A) mRNA was enriched from total RNA using the GenElute™ mRNA Miniprep Kit (Sigma-Aldrich), one-tenth of which was preserved as the input. Subsequently, mRNA was incubated overnight at 4 °C with protein A/G magnetic beads preconjugated with m⁶A antibody (MABE1006) or normal mouse IgG antibody (CS200621). The bead- immunoprecipitated methylated mRNAs were eluted by free m⁶A competition and purified using the RNeasy MiniElute Cleanup kit (#74204, Qiagen). Purified immunoprecipitated mRNAs and input sample were evaluated by qPCR using the primers listed in Additional file 2: Table S3.

RNA immunoprecipitation (RIP) assay

The Magna RIP RNA-Binding Protein Immunoprecipitation Kit (17–700, Millipore) was used to perform RIP assays following the manufacturer’s instructions. Briefly, cell lysates were incubated at 4 °C overnight with magnetic beads preconjugated with antibodies against FTO (#31687, Cell Signaling Technology), YTHDF1 (#57530, Cell Signaling Technology), YTHDF2 (24744-1-AP, Proteintech), YTHDF3 (sc-377119, Santa Cruz Biotechnology), or mouse IgG. One-tenth of cell lysate was saved as an input sample. RNAs were eluted using proteinase K at 55 °C for 30 min. Eluted RNAs were purified using the RNeasy MiniElute Cleanup Kit (#74204 Qiagen). qPCR was performed to evaluate the interaction between YTHDF2 and mRNA transcripts. Primers used are listed in Additional file 2: Table S3.

RNA stability

Cells were incubated with actinomycin D (final concentration 5 mg/mL) and harvested after 1, 1.5, or 3 h of incubation. Total RNA was extracted using the TRIzol reagent (Invitrogen), followed by qPCR. The mRNA degradation rate was estimated in accordance with previous studies [18] using the formula: In(C/C₀) = − K_decayt. The mRNA half-life of the decay t_1/2 was calculated as follows: t_1/2 = In2/K_decay.

RNA-seq and m⁶A-seq

Wild-type FTO-overexpressing and control Kasumi-1 cell lines were used in triplicate to conduct RNA-seq (RNA sequencing) and m⁶A-seq (m⁶A sequencing). RNA-seq and m⁶A-seq were performed by LC-BIO Bio-tech Ltd. (Hangzhou, China). Briefly, total RNA was extracted from wild-type FTO-overexpressing (wtFTO) and control (mock) cells using the TRIzol reagent. The RNA integrity number (RIN) of total RNA was assessed using an Agilent Bioanalyzer 2100 with an RNA 6000 Nano LabChip Kit (Agilent) with RIN ≥ 7. Poly(A) mRNA was enriched from total RNA using poly-T oligo-attached magnetic beads (Invitrogen) and then fragmented using divalent cations at elevated temperatures. Fragmented mRNAs were incubated at 4 °C with m⁶A antibody (#202003, Synaptic Systems) for 2 h in IP buffer, and then incubated with protein-A beads for another 2 h at 4 °C. The mixture was eluted with elution buffer (6.7 mM m⁶A in IP buffer) and precipitated using 75% ethanol. The eluted IP and input mRNAs were used to construct a cDNA library with strand-specific library preparation using the dUTP method. Then 2 × 150 bp paired-end sequencing was performed on an Illumina Novaseq 6000 platform (Illumina). The accession number of these data in the Sequence Read Archive (SRA) database is PRJNA876028.

RNA pull-down and mass spectrometry analysis

Biotin-labelled RNA oligonucleotides containing m⁶A or adenosine were directly synthesized by Sangon Biotech. The Pierce Magnetic RNA-Protein Pull-Down Kit (20164, Thermo Fisher Scientific) was used to perform RNA pull-down assays, following the manufacturer’s instructions. Briefly, up to 50 pmol of biotin-labelled RNAs was mixed with 50 μL streptavidin beads at 25 °C for 30 min and then incubated together with up to 200 μg of protein lysates with rotation for 1 h at 4 °C. After washing thrice, streptavidin beads were boiled, separated by 10% SDS-PAGE, and detected by western blot analysis or silver staining. Mass spectrometry analysis was performed by Qinglian Biotech Co. Ltd. Briefly, in-gel proteins were digested with trypsin. Peptides were then used for LC–MS/MS analysis on an EASY nLC 1200 ultra-high-pressure system (Thermo Fisher Scientific) with a nano-electrospray ion source attached to a quadrupole Orbitrap mass spectrometer (Q Exactive HF-X, Thermo Fisher Scientific).

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository [Full size image

Considering the m⁶A "eraser" role of FTO, we only regarded the mRNA transcripts that carried m⁶A peaks with decreasing abundance, namely m⁶A hypomethylated peaks, upon FTO overexpression as potential targets of FTO. Combined with RNA-seq data, we found 372 hypomethylated peaks in FTO-overexpressing cells relative to control cells, among which 255 mRNA transcripts were significantly upregulated (hypo-up, fold change ≥ 1.2; p < 0.05), whereas 117 were downregulated (hypo-down, fold change ≤ 0.83; p < 0.05, Fig. 5D), suggesting that the majority of m⁶A hypomethylated mRNA transcripts (68.5%) were associated with upregulated mRNA levels in FTO-overexpressing cells. GSEA analysis [41] indicated that the hypo-up transcripts were significantly enriched with target genes of SOX2 and NANOG, BRCA1 and CHEK2 network, and many important biological processes, such as cell cycle, immune system and RNA metabolism (Additional file 1: Figure S5C). Hypo-down transcripts were significantly enriched with genes involving the BRCA1 network (Additional file 1: Figure S5D).

We further analyzed the expression correlations between FTO and the m⁶A hypomethylated genes in AML patient samples from the TCGA dataset. We found that 11 hypo-up (CEP57, FTX, ZC3H11A, TERF2, PAX6, IGFBP2, BTBD3, NOA1, VKORC1L1, GCC2, and YAF2) and 7 hypo-down (SSU72, FBXW5, SERF2, Rab3A, TCEA3, TSPAN14, and GGT1) genes demonstrated a significantly positive and negative correlation in expression, respectively, with FTO in TCGA AML datasets (Fig. 5E and Additional file 2: Table S2). In agreement, gene-specific m⁶A qPCR assays confirmed that among the 7 m⁶A-hypo genes (PAX6, TERF2, TCEA3, Rab3A, CEP57, YAF2, and FBXWS), 6 were associated with a significant reduction in the m⁶A levels (86%), whereas 2 non-m⁶A-hypo genes (DNMT1 and DNMT3A) in m⁶A-seq data did not demonstrate a significant decrease in m⁶A abundance (Fig. 5F). This verifies the reliability of our m⁶A-seq data.

IGFBP2 is directly targeted by FTO via its m⁶A demethylase activity

We further found that the high expression of IGFBP2 (insulin-like growth factor binding protein 2), which was listed in Fig. 5E, was significantly upregulated in patients with t(8;21) AML (n = 30) compared with normal control (n = 9, GSE30285 and GSE34814, Additional file 1: Fig. S5E). Moreover, IGFBP2 was associated with significantly worse event-free survival and overall survival in patients with AML (GSE6891, n = 344, log-rank test, p < 0.001 and p = 0.006, respectively; Additional file 1: Fig. S5F) and t(8;21) AML (n = 26, log-rank test, p = 0.045 and p = 0.061, respectively; Fig. 5G). The primary BM cells from AML patients at diagnosis with poor response to Ara-C had a significantly higher level of IGFBP2 mRNA compared with high responders (GSE97393; Fig. 5H). Moreover, IGFBP2 has been shown to support the migration and survival of AML cells [42]. We observed that IGFBP2 manifested a significant positive association with the expression of FTO in AML cohorts from the TCGA database (Additional file 1: Fig. S5G) and our in-house t(8;21) AML cohorts (Fig. 5I). Our m⁶A-seq and RNA-seq data indicated that overexpression of FTO caused a significant reduction of m⁶A levels in the 3′ untranslated region (3′-UTR) of IGFBP2 mRNA (fold change = 0.72, p = 0.001, Fig. 5J) and significant increase level of IGFBP2 transcripts (fold change = 2.22, p < 0.001; Fig. 5K). We thus aimed at IGFBP2 as a potential target of FTO for further investigation.

Overexpression of wild-type FTO upregulated the expression of IGFBP2 at both the mRNA and protein levels compared with those in control and mutant FTO cells, whereas silencing the endogenous expression of FTO resulted, as expected, to an opposite pattern (Fig. 6A–D). In addition, treatment with FB23-2 also resulted in a dose-dependent reduction in the expression of IGFBP2 (Fig. 6E, F). RIP assay demonstrated the direct binding of FTO to IGFBP2 transcripts (Fig. 6G). Gene-specific m⁶A qPCR assays further confirmed that forced expression of wild-type FTO substantially reduced the levels of m⁶A in IGFBP2 mRNA transcripts (Fig. 6H); opposite results were shown when the endogenous FTO was knocked down (Fig. 6I). To further validate that FTO targets the 3′-UTR of IGFBP2 mRNA transcripts, we created reporter vectors baring the coding sequence of IGFBP2 3′-UTR or corresponding m⁶A consensus mutant sequences (Additional file 1: Fig. S6A) and found that compared with the empty and mutant FTO vectors, luciferase activity was significantly promoted by wild-type FTO when cotransfected with the reporter vector containing the wild-type IGFBP2 3′-UTR fragment with intact m⁶A consensus, whereas this effect was abrogated when cotransfected with reporter vectors containing mutant m⁶A consensuses (Fig. 6J).

RNA stability assays indicated that FTO knockdown noticeably shortened the half-life of IGFBP2 mRNA transcripts in SKNO-1 and Kasumi-1 cells (Fig. 6K). Likewise, the half-life of IGFBP2 mRNA transcripts manifested a dose-dependent reduction when the two t(8;21) AML cells were treated with FB23-2 (Fig. 6L), suggesting that upregulation of IGFPB2 is at least partially attributed to the increased stability of its transcripts mediated by FTO-induced m⁶A demethylation.

To screen specific m⁶A readers targeting the 3′-UTR of IGFBP2, we performed an RNA pull-down assay using 4 pairs of m⁶A methylated or unmethylated single-stranded RNAs carrying part of the 3′-UTR sequence of IGFBP2 (~ 20 bp in length) as baits, followed by mass spectrometry analysis. Each pair of RNA baits contained one of the 4 putative m⁶A consensus motifs in the 3′-UTR of IGFBP2 (Additional file 1: Fig. S6B). We detected that the m⁶A reader YTHDF2 was dramatically enriched in all 4 m⁶A-modified RNA baits, YTHDF1 and YTHDF3 were markedly enriched in 3 of the 4 m⁶A-methylated baits compared with the unmethylated control, whereas other known readers were enriched in no more than 1 bait (YTHDC2, IGF2BP2/3, HNRNPA2B1 and HNRNPC were enriched in only 1 baits and YTHDC1/3, IGF2BP1 were enriched in none of the 4 baits; Additional file 1: Fig. S6C). RIP assays further confirmed that YTHDF1, YTHDF2 and YTHDF3 bound to IGFBP2 transcripts directly (Fig. 6M–O). The expression of IGFBP2 was barely altered when either YTHDF2 or YTHDF3 was knocked down (Additional file 1: Fig. S6D and S6E). However, when the three YTHDF proteins were simultaneously knockdown by shRNA, the expression of IGFBP2 were greatly increased (Fig. 6P, Q), suggesting that the YTHDFs proteins work together to mediate the decay of IGFBP2 mRNA.

IGFBP2 is a functionally important target gene of FTO in t(8;21) AML

The pathological role of IGFPB2 in t(8;21) AML was then investigated. When endogenous IGFBP2 was knocked down in the two t(8;21) AML cell lines (Fig. 7A), we observed a substantial repression on cell proliferation (Fig. 7B) and colony-forming capacity (Additional file 1: Fig. S7A) and a promotion on G1 cell cycle arrest (Additional file 1: Fig. S7B), cell apoptosis (Fig. 7C and Additional file 1: Fig. S7C) and cell differentiation (Fig. 7D, E), which was largely similar to the effect of the FTO knockdown. Moreover, IGFBP2 knockdown notably decreased the IC₅₀ value of Ara-C in the two Ara-C resistant cell lines (Fig. 7F).

In the AML1-ETO9a AML mouse model, we found that Igfbp2 knockdown prolonged the overall survival of recipient mice (log-rank test, p < 0.001; Fig. 7G, H). Leukemic infiltration in BM, liver and spleen was also significantly suppressed when Igfbp2 was knocked down (Fig. 7I and Additional file 1: Fig. S7D–E). Moreover, Igfbp2 knockdown decreased the abundance of leukemic cells (Fig. 7J–L) and the proportion of CD11b-stained leukemic cells (Fig. 7M–O) in PB, BM, and spleen samples.

Rescue experiments demonstrated that when IGFBP2 was forced to be overexpressed in FTO-knockdown t(8;21) AML cells (Fig. 8A), the restoration of cell proliferation (Fig. 8B), colony-forming capacity (Fig. 8C) and regain of Ara-C tolerance (Fig. 8D and Additional file 1: Fig. S8A) were observed. Rescue experiments conducted with AML1-ETO9a AML mouse model also revealed a significant restoration in the infiltration of leukemic cells in PB, BM, liver and spleen when Igfbp2 was overexpressed in Fto-knockdown cells (Fig. 8E–G and Additional file 1: Fig. S8B–C). Force expression of Igfbp2 greatly prevented differentiation of leukemic cells in PB, BM and spleen mediated by Fto knockdown (Additional file 1: Fig. S8D). Taken together, our data suggest that IGFBP2 is a functionally critical target of FTO and is essential for FTO-mediated t(8;21) AML progression and Ara-C resistance.

Discussion

In the present study, we demonstrate a positive regulatory loop between the fusion protein AML1-ETO and FTO, which exerts a critical role in promoting leukemogenesis and resistance of t(8;21) AML cells to Ara-C by modulating the expression of its mRNA targets, such as IGFBP2, via m⁶A demethylation (see the proposed model in Fig. 8H).

Although the overexpression of FTO are found in several AML subtypes, and it has been reported that MLL fusions may upregulate the expression of FTO by directly targeting it in MLL-rearranged AML cells [18], the underlying molecular mechanism driving FTO overexpression in t(8;21) AML remains to be determined. AML1-ETO has been shown to promote or silence the expression of various oncogenes or cancer suppressor genes via genetic or epigenetic regulation [6, 7]. In the present study, although no evidence was found to indicate a direct interaction between the AML1-ETO protein and FTO gene, the transcription factor PU.1, which is critical during myelopoiesis, might play a bridging role between AML1-ETO and FTO. PU.1 has been reported to strongly promote differentiation of myeloid lineages [43, 44], acting as a tumor suppressor gene in AML [45]. Moreover, PU.1 is known to be expressed at a low level in t(8;21) AML [34, 46]. AML1-ETO has been reported to inhibit the transcriptional capacity of PU.1 by expelling the coactivator c-Jun from the β₃β₄ region of PU.1 [34]. Our findings indicate that AML1-ETO can also repress the expression of PU.1 via binding to its promoter, thereby inhibiting the PU.1-mediated transcriptional repression of FTO. The AML1-ETO/PU.1 axis may be responsible for the high expression of FTO in t(8;21) AML.

The pivotal role of AML1-ETO in the pathogenesis of t(8;21) AML makes it a promising candidate for therapeutic intervention [47]. Moreover, the copy number of AML1-ETO transcripts has been regarded as an indicator of relapse in t(8;21) AML [48]. Given the challenge of directly targeting AML1-ETO, it would be worthwhile to investigate the molecular mechanisms that govern its expression. Our findings show that AML1-ETO is also a downstream target of FTO; this regulation is achieved at least partially due to the inhibition of AML1-ETO mRNA decay mediated by YTHDF2. Of note, a study reported that AML1-ETO can repress the transcription of YTHDF2 via targeting its promoter [23], while our results reveal that YTHDF2 inhibits the expression of AML1-ETO in a m⁶A-dependent manner. Thus, these results suggest a negative feedback loop between AML1-ETO and YTHDF2 that maintains the stable expression level of the two proteins.

Although the oncogenic role of FTO has been reported in leukemia, the functional significance of FTO in some leukemic cells, such as the human erythroleukemic cell line K562, appears to be less essential, possibly due to a lower level of endogenous expression in these cells [18]. The higher level of FTO which may be attributed to the AML1-ETO/FTO feedback loop and the in vitro and in vivo evidence further validate the essential role of FTO in promoting leukemogenesis and progression of t(8;21) AML. Furthermore, the involvement of FTO in Ara-C resistance is also investigated in the present study. Besides the high level of FTO mRNA in patients with primary refractory t(8;21) AML, our data demonstrate an intriguing finding that indicates sustained high levels of FTO mRNA in patients with c-KIT mutation even after complete remission. It is well appreciated that t(8;21) patients with c-KIT mutations had a significantly higher rate of relapse [29]. In addition, the acquired drug resistance and relapse is largely associated with the genetic heterogeneity within cancer cell clones. It is plausible to speculate that the post-chemotherapy surviving c-KIT mutations harboring clones, which are responsible for later relapse, express high levels of FTO. All these findings provide evidence of a strong relevance between FTO and chemoresistance in t(8;21) AML. Our present findings validate a substantial Ara-C resistance in the t(8;21) AML cell lines SKNO-1 and Kasumi-1, as evidenced by the IC₅₀ values of approximately 1000-fold higher compared to the Ara-C sensitive cell lines HL-60 and HEL, which exhibited IC₅₀ values of around 10^–1 μM [49]. Suppression of FTO genetically or pharmacologically significantly sensitizes the two Ara-C-resistant cell lines to Ara-C, suggesting a potentially druggable target of FTO in the treatment of Ara-C resistance in t(8;21) AML. In addition to FB23-2, the newly developed small-molecule FTO inhibitors CS1 and CS2 have been reported to display effective antitumor effects even at low nanomolar levels [50]. Selective inhibition of FTO can break the AML1-ETO/FTO feedback loop, inhibit progression and resensitizes resistant cells to Ara-C, highlighting a great therapeutic potential for the efficacious small-molecule FTO inhibitors, in combination with routine chemotherapy, in the treatment of t(8;21) AML.

Accumulating evidence indicates that high expression of IGFBP2 promotes tumorigenesis, progression, and chemotherapy resistance in many malignant tumors [51,52,53]. Studies in AML also demonstrate that IGFBP2 supports the activity of hematopoietic stem cells (HSCs) and promotes the proliferation of AML cells by suppressing PTEN expression and activating Akt [42, 54]. High expression of IGFBP2 is also found to be an independent predictive factor for primary refractory AML, suggesting a potential relationship between IGFBP2 and chemoresistance in AML [54]. Concordantly, our study reveals that IGFBP2, as an important target of FTO, exerts a crucial role in both cell proliferation and Ara-C resistance in t(8;21) AML. In addition, the Akt activation has been reported to be implicated in drug resistance in multiple cancers [55]. It might serve as a potential mechanism underlying IGFBP2-mediated Ara-C resistance, which requires further confirmation.

It is worth noting that nearly two-thirds of mRNA transcripts with hypomethylated m⁶A peaks after overexpression of FTO demonstrate upregulated mRNA levels in t(8;21) AML cells, contradicting the results in the MONOMAC-6 AML cell line in a previous study [18]. This indicates the heterogeneity of FTO-dependent m⁶A modification patterns in different AML subtypes. It also suggests that FTO-mediated m⁶A demethylation in t(8;21) AML cells tends to enhance the mRNA stability of the majority FTO target genes, the process of which is likely to be mediated by YTHDF2/YTHDF3 [35, 56]. Our data demonstrate that the half-life of IGFBP2 mRNA is shortened when FTO is knocked down. However, neither silencing YTHDF2 nor YTHDF3 significantly alters the expression of IGFBP2, although we show the direct binding of these 2 readers to the 3′-UTR of IGFBP2 mRNA. Interestingly, a recent study reports that YTHDFs family proteins (including YTHDF1, YTHDF2, and YTHDF3) tend to bind the same m⁶A-methylated mRNAs and function together to promote mRNA degradation. The ability to regulate mRNA decay becomes evident when all 3 YTHDFs proteins are simultaneously depleted [57]. Similarly, our data indicate that the three YTHDFs, rather than other known m⁶A readers, can be enriched by the m⁶A methylated single-stranded RNA of the IGFBP2 3′-UTR. The expression of IGFBP2 is upregulated when the three YTHDFs are knockdown simultaneously, indicating that YTHDFs proteins work together to mediate the decay of IGFBP2 mRNA in t(8;21) AML.

Conclusion

In summary, we reveal a positive feedback loop between the leukemia-initiating fusion protein AML1-ETO and the m⁶A demethylase FTO in t(8;21) AML. AML1-ETO upregulates the expression of FTO through PU.1; FTO in turn promotes the expression of AML1-ETO by inhibiting YTHDF2-mediated AML1-ETO mRNA decay through m⁶A demethylation. The use of FTO selective inhibitors effectively represses t(8;21) AML progression, promotes cell differentiation and sensitizes resistant cells to Ara-C. The AML1-ETO/FTO/IGFBP2 minicircuitry holds potential as a therapeutic target in t(8;21) AML, particularly for the Ara-C tolerant patients.

Availability of data and materials

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.

Abbreviations

AML:: Acute myeloid leukemia
m⁶A:: N⁶-methyladenosine
FTO:: Fat mass and obesity-associated protein
YTHDF2:: YTH domain family 2
IGFBP2:: Insulin-like growth factor binding protein 2
BM:: Bone marrow
PB:: Peripheral blood
TSS:: Transcription start site
shNS:: Scramble shRNA
3′-UTR:: 3′ Untranslated region
IgG:: Immunoglobulin G
RIP:: RNA immunoprecipitation
m⁶A-seq:: m⁶A sequencing
RNA-seq:: RNA-sequencing
ChIP-seq:: Chromatin immunoprecipitation sequencing
CR:: Complete remission
TCGA:: The Cancer Genome Atlas

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Acknowledgements

The authors thank Dr. Lan Wang from the CAS Key Laboratory of Tissue Microenvironment and Tumor, Chinese Academy of Sciences, for providing the AML1-ETO9a-driven AML mouse model. The authors also thank Dr. Yisheng Li for assistance with bioinformatics analysis.

Funding

This work was supported by grants from National Natural Science Foundation of China (82070161 to YL, 81870134 to YL, 82030076 to LY, 81970151 to LY, and 82100164 to WG), China Postdoctoral Science Foundation (2021TQ0217 to WZ), Science, Technology and Innovation Commission of Shenzhen Municipality (JCYJ20220531103003007 to YL), Natural Science Foundation of Guangdong Province (2022A1515012465 to YL), Medicine Plus Project of Shenzhen University (2023YG005 to YL), Natural Science Foundation of Shenzhen University General Hospital (SUGH2020QD008 to YL), Shenzhen Key Laboratory Foundation (ZDSYS20200811143757022 to LY), Sanming Project of Shenzhen (SZSM202111004 to LY), Italian Ministry of University and Research (PRIN-2017WWB99Z to CN) and Sapienza University of Rome (RM12117A8691212C to CN).

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Authors and Affiliations

Central Laboratory, Shenzhen University General Hospital, Shenzhen University, Shenzhen, 518055, Guangdong, China
Wei Zhou, Hong Wang & Yonghui Li
Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, National-Regional Key Technology Engineering Laboratory for Medical Ultrasound, School of Biomedical Engineering, Shenzhen University Medical School, Shenzhen, 518060, Guangdong, China
Wei Zhou & Hong Wang
School of Medicine, South China University of Technology, Guangzhou, 511400, Guangdong, China
Siying Li
Department of Hematology and Oncology, International Cancer Center, Shenzhen Key Laboratory of Precision Medicine for Hematological Malignancies, Shenzhen University General Hospital, Shenzhen University, Shenzhen, 518055, Guangdong, China
**gfeng Zhou, Shuyi Li, Li Yu & Yonghui Li
Department of Endoscopy, Tian** Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Molecular Cancer Epidemiology of Tian**, Key Laboratory of Cancer Prevention and Therapy, Tian**’s Clinical Research Center for Cancer, Tian**, 300060, China
Guofeng Chen
Senior Department of Hematology, The Fifth Medical Center of PLA General Hospital, Bei**g, 100039, China
Wei Guan
Department of Pathology, Shenzhen University General Hospital, Shenzhen, 518055, Guangdong, China
**anli Fu
Department of Medical and Surgical Sciences and Biotechnologies, University of Roma La Sapienza, 04100, Latina, Italy
Clara Nervi

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**gfeng Zhou
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Shuyi Li
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Guofeng Chen
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Wei Guan
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**anli Fu
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Clara Nervi
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Li Yu
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Yonghui Li
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Contributions

YL, and WZ designed the research; WZ performed the majority of experiments with help from HW on RNA stability assay and m⁶A-qPCR assay and SiL, JZ and ShuL and on mice experiments; XF performed H&E stain assay; GC and WG helped with or advised on experiments; WZ analyzed the experimental data; CN and LY commented on the paper; WZ and YL wrote the paper and YL supervised the work.

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Correspondence to Yonghui Li.

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This research was conducted in accordance with international guidelines and the ethical standards outlined in the Declaration of Helsinki. This study was approved by the Medical Ethics Committee of Shenzhen University General Hospital and written informed consent was obtained from each patient for the use of their bone marrow samples. All animal experiments were conducted in compliance with the institutional guidelines for the Care and Use of Laboratory Animal by International Committees. All animal procedures were approved by the Experimental Animal Ethics Committee of Shenzhen University General Hospital.

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The authors declare no competing interests.

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Supplementary Information

Additional file 1: Fig. S1.

AML1-ETO promotes expression of FTO via PU.1. (A) Comparison of overall survival of patients with de novo t(8;21) AML (n = 26) using the Kaplan–Meier method grouped by the expression of FTO (high vs. low). p value was evaluated using the log-rank test. (B) Kaplan–Meier analysis of event-free survival (left) and overall survival (right) of patients with AML (n = 344, data from GSE6891) based on the expression of FTO. (C) Comparison of the expression of FTO in Kasumi-1 cells with or without AML1-ETO knockdown (shAE vs. shNS) detected by RNA-seq in the GSE115121 data set. (D) ChIP-seq of GSE65427 depicting FTO loci in Kasumi-1 cells targeting C- terminus of ETO (upper panel) and N-terminus of AML1(lower panel), representing AML1-ETO peaks on FTO. (E) Schematic diagrams showing the amplified regions on the promoter of SPI1 for the ChIP-qPCR showed in Fig. 1I and Fig. S1F. The location of targeted amplified region (named ‘Target’) and negative control site (NC) are indicated with blue horizontal lines. The red triangle indicates the location of peak summit of AML1-ETO on the promoter of SPI1 detected by the ChIP-seq of GSE65427. (F) ChIP-qPCR assays showing no direct binding of AML1 or ETO within 200 bp upstream of the SPI1 promoter in SKNO-1-siAE cells. (G and H) Pearson correlation of the expression of FTO and SPI1 in (G) normal blood tissues from the Genotype-Tissue Expression Project (GTEx, n = 444) or (H) BM samples of patients with AML from TCGA database (n = 173). (I) Sequences of the 4 FTO promoter fragments (the P1 to P4 showed in Fig. 1P) and putative PU.1 binging sites (sites 1, 2, and 3). Fig. S2. FTO upregulated AML1-ETO in a m⁶A-dependent manner. (A–C) The level of AML1-ETO mRNA detected by qPCR in SKNO-1 and Kasumi-1 cells (A) transduced with wild-type FTO (wt-FTO), mutant FTO (mut-FTO), or mock vectors; (B) transduced with FTO-knockdown (shFTO#1 and shFTO#2) or scramble shRNA (shNS) vectors; (C) treated with DMSO or FB23-2 treatment for 72 h. Fig. S3. Oncogenic role of FTO in t(8;21) AML cells and AML1-ETO9a driven AML mice. (A) Effects of forced expression or knockdown of FTO on cell cycle in SKNO-1 and Kasumi-1 cells. (B) Effects of forced expression or knockdown of FTO on colony-forming capacity of Kasumi-1 cells. (C) The effect of FTO knockdown on differentiation of Kasumi-1 cells. The percentage of CD11b⁺ cells was quantified (right panel). (D) Wright-Giemsa staining of Kasumi-1 cells with or without FTO knockdown. (E) Spleen size in AML1-ETO9a-driven AML mice with or without Fto knockdown or treatment with DMSO or FB23-2 (6 mg/kg) 7 weeks after transplantation (n = 6 for each group). (F) Spleen weight of AML1-ETO9a-driven AML mice from (E). (G) Wright-Giemsa staining of bone marrow of AML1-ETO9a-driven AML mice. (H–J) Percentage of GFP⁺ AML1-ETO9a AML cells in the (H) peripheral blood (PB), (I) bone marrow (BM), and (J) spleen (SP) of the mice treatment with DMSO or FB23-2 by flow cytometric analysis. (K–M) Flow cytometric analysis of the distribution of anti-CD11b-stained GFP + AML1-ETO9a AML cells in PB (K), BM (L), and SP (M) of mice treatment with DMSO or FB23-2. Fig. S4. Suppression of FTO resensitizes resistant cells to Ara-C in vitro and in vivo. (A and B) Apoptosis measured by flow cytometry for SKNO-1 (A) and Kasumi-1 (B) cells treated with DMSO, 30 µM Ara-C alone, 10 µM FB23-2 alone or combination of Ara-C and FB23-2 for 48 h with FB23-2 pretreatment for 6 h. (C and D) Percentage of GFP⁺ AML cells in peripheral blood (C) and spleen (D) of NOD/SCID/γ_c^null immunodeficient mice injected with SKNO-1 cells through tail vein treated with DMSO, Ara-C, FB23-2, or a combination of Ara-C and FB23-2 (n = 6 for each group). Fig. S5. Transcriptome-wide identification of FTO targets in t(8;21) AML. (A and B) Proportion of the distribution of m⁶A peaks in exon, intron, and intergenic regions across entire mRNA transcripts (A) or in the 5′-UTR, first exon, other exon, and 3′-UTR of mRNA transcripts (B) detected by m⁶A-seq assays in Kasumi-1 cells transduced with wild-type FTO or empty vector. (C and D) Gene set enrichment analysis (GSEA) of genes with a significant decrease in m⁶A levels as well as a significant increase (Hypo-up) or decrease (Hypo-down) in overall transcript levels in FTO-overexpressing Kasumi-1 cells. (E) Comparison of IGFBP2 expression between human primary AML cases with t(8;21) (n = 30, data from GSE30285) or normal controls (NC) (n = 9, data from GSE34814). (F) Kaplan–Meier analysis of event-free survival (left) and overall survival (right) of patients with AML (n = 344, data from GSE6891) based on the expression of IGFBP2. (G) Pearson correlation of the expression of FTO and IGFBP2 in BM samples of patients with AML from TCGA database (n = 173). Fig. S6. Luciferase reporter construction and identification of specific m⁶A readers targeting the 3′-UTR of IGFBP2 mRNA. (A) Construction of luciferase reporter vectors. Synthesized wildtype (wt) or mutant (mut) 3′ coding sequences of IGFBP2 were inserted into the XhoI and NotI site of the psiCHECK2 luciferase reporter. Putative m⁶A consensus motifs are shown in bold, whereas mutation sites (A to T mutation) are shown in red. (B and C) Identification of m⁶A specific binding proteins on 3′-UTR of IGFBP2 by RNA pull-down using 4 pairs of single-stranded RNA (ssRNA) baits containing the 4 m⁶A consensus motif on the 3′-UTR sequence of IGFBP2 respectively, with methylated (green) or unmethylated (red) adenosine (B). The iBAQ value of previously reported m⁶A readers (including YTHDFs, YTHDCs, IG2FBPs and hnRNPs) enriched by the 4 pairs of ssRNA probes detected by mass spectrometry analysis are shown (C). The YTHDC1, YTHDC3, and IGF2BP1 proteins that could not be enriched by all 4 pairs of ssRNA probes are not shown in the Figure (see Additional file 3: Table S4). (D and E) Western blot analysis of the expression of IGFBP2 with or without silencing of YTHDF2 (D) or YTHDF3 (E) by siRNA in SKNO-1 or Kasumi-1 cells. siNS, scramble siRNA. Fig. S7. Functional role of IGFBP2 in t(8;21) AML. (A and B) Effects of IGFBP2 knockdown on colony-forming capacity (A) and cell cycle (B) in SKNO-1 and Kasumi-1 cells. (C) Western blot analysis of silencing IGFBP2 by siRNA in SKNO-1 and Kasumi-1 cells. siNS, scramble siRNA. (D and E) External views (D) and weight (E) of the spleens from AML1-ETO9a-driven AML mice with or without Igfbp2 knockdown (n = 6 for each group) 7 weeks after transplantation. Fig. S8. FTO regulates leukemogenesis and sensitivity of t(8;21) AML cells to Ara-C through IGFBP2. (A) Effects of FTO knockdown with IGFBP2 overexpression after Ara-C treatment on colony-forming capacity of SKNO-1 and Kasumi-1 cells. (B–D) External views (B), weight of the spleens (C) and flow cytometric analysis of CD11b⁺ AML cells in PB, BM, and SP (D) of AML1-ETO9a-driven AML mice in Fig. 8E–G.

Additional file 2: Table S1.

The clinical characteristics, morphological and genetic features of patients with de novo t(8;21) AML at diagnosis (n = 26) and relapse (n = 1). Table S2. List of 18 genes that show significant decrease abundance in m⁶A peak and significant change in the levels of corresponding mRNA transcript between FTO-overexpressing and control Kasumi-1 cells, and are also significantly positively or negatively correlated with FTO in expression in TCGA AML database. Table S3. (Related to Methods) List of oligonucleotides.

Additional file 3: Table S4

. (Related to Fig. S6C) Results of mass spectrometry analysis for the proteins obtained in RNA pull down assay.

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Zhou, W., Li, S., Wang, H. et al. A novel AML1-ETO/FTO positive feedback loop promotes leukemogenesis and Ara-C resistance via stabilizing IGFBP2 in t(8;21) acute myeloid leukemia. Exp Hematol Oncol 13, 9 (2024). https://doi.org/10.1186/s40164-024-00480-z

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Received: 06 September 2023
Accepted: 18 January 2024
Published: 24 January 2024
DOI: https://doi.org/10.1186/s40164-024-00480-z

A novel AML1-ETO/FTO positive feedback loop promotes leukemogenesis and Ara-C resistance via stabilizing IGFBP2 in t(8;21) acute myeloid leukemia

Abstract

Background

Methods

Results

Conclusion

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Introduction

Methods

Samples from patients with leukemia and healthy donors

Cell culture

DNA site-directed mutagenesis

Plasmid construction

Lentiviral infection and siRNA transfection

AML1-ETO9a-driven AML mice model

Xenograft model

NOD/SCID/IL2rγnull immunodeficient mice model

Cell proliferation and colony forming unit assays

Flow cytometric analysis of apoptosis, cell cycle, and differentiation

RNA extraction and quantitative RT-PCR

Western blotting

m6A dot blot

Chromatin immunoprecipitation (ChIP)

Dual-luciferase reporter assays

Gene-specific m6A-qPCR

RNA immunoprecipitation (RIP) assay

RNA stability

RNA-seq and m6A-seq

RNA pull-down and mass spectrometry analysis

IGFBP2 is directly targeted by FTO via its m6A demethylase activity

IGFBP2 is a functionally important target gene of FTO in t(8;21) AML

Discussion

Conclusion

Availability of data and materials

Abbreviations

References

Acknowledgements

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Supplementary Information

Additional file 1: Fig. S1.

Additional file 2: Table S1.

Additional file 3: Table S4

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NOD/SCID/IL2rγ^null immunodeficient mice model

m⁶A dot blot

Gene-specific m⁶A-qPCR

RNA-seq and m⁶A-seq

IGFBP2 is directly targeted by FTO via its m⁶A demethylase activity