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METTL3 mediates chemoresistance by enhancing AML homing and engraftment via ITGA4 - PubMed

. 2022 Nov;36(11):2586-2595.

doi: 10.1038/s41375-022-01696-w. Epub 2022 Oct 20.

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METTL3 mediates chemoresistance by enhancing AML homing and engraftment via ITGA4

Mingying Li et al. Leukemia. 2022 Nov.

Abstract

Chemoresistant leukemia relapse is one of the most common causes of death for acute myeloid leukemia (AML) patients and the homing/engraftment in bone marrow (BM) are crucial steps for AML cells to acquire chemoresistance by interacting with stromal cell components. No crosstalk between m6A modification and homing/engraftment has been reported. Here, we performed comprehensive high-throughput analyses, including RNA sequencing of CR (complete remission) and relapsed AML patients, and reverse-phase protein arrays of chemoresistant cells to identify METTL3 as a key player regulating AML chemoresistance. Then, METTL3-mediated m6A modification was proved to induce the chemoresistance in vitro and in vivo. Furthermore, AML homing/engraftment was discovered being enhanced by upregulated-METTL3 in chemoresistant cells. And the homing/engraftment and drug-resistance associated phenotypes of chemoresistant cells could be reversed by a METTL3 inhibitor. Mechanistically, METTL3 extended the half-life of ITGA4 mRNA by m6A methylation, and then, increased expression of ITGA4 protein to enhance homing/engraftment of AML cells. The results provide insights into the function of m6A modification on the interaction between AML cells and BM niches and clarify the relationship between METTL3 and AML homing/engraftment, suggesting a therapeutic strategy for the treatment of refractory/relapsed AML with METTL3 inhibitors.

© 2022. The Author(s).

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Up-regulation of METTL3 in chemoresistant AML cells.

A Flow chart for the identification and verification strategy of differentially expressed targets between chemotherapy-resistant AML cells and -sensitive AML cells. Graphical elements were adapted from the RNA-seq (GSE165430, n = 268, Log2 FC ≥ 2) and RPPA screen (LogFC > 0.25) workflow. Significant: P < 0.05; nonsignificant: P ≥ 0.05. See also Supplemental Fig. S1. B qRT-PCR analysis of METTL3 mRNA expression in CD34 + leukemia cells sorted from pretreatment patients and relapsed patients with de novo AML. Pretreatment patients who achieved CR after induction treatment with standard “7 + 3” chemotherapy were defined as the CR group (n = 17), and NR patients and relapsed patients were defined as the refractory/relapsed group (n = 20). Data are mean ± SD values. P < 0.05 was considered significant, t test. C Western blot analysis and Flow cytometry analysis for the expression of METTL3, n = 3.

Fig. 2
Fig. 2. METTL3-meditated m6A enhances the chemoresistance of AML cells.

A AML CD34+ cells from the patients shown in Fig. 1B were subjected to an m6A methylation quantification kit to assess global m6A changes (n = 13 for CR group and n = 20 for Refractory/Relapsed group). P < 0.05 was considered significant, t test. B, C m6A dot blot assays (B) and LC-MS/MS (C) for the detection of global m6A changes. MB, methylene blue staining (as a loading control). P < 0.05 was considered significant, t test. D EdU incorporation assay (upper) showing the percentage of AML cells that entered the proliferation cycle (EdU positive cells) with or without IDA pressure. Percentages after PBS treatment are shown in bold, and percentages after IDA treatment are shown in regular. Statistical analysis is shown in (lower). *P < 0.05, vs. the NC group with PBS treatment; ####P < 0.0001, vs. the NC group with IDA treatment; &, significant interaction effect; two-way ANOVA. E Colony-forming assays (upper). Bar, 500 μm. Statistical analysis is shown in (lower). ***P < 0.001, ****P < 0.0001, vs. the NC group with PBS treatment; ####P < 0.0001, vs. the NC group with IDA treatment; &, significant interaction effect; two-way ANOVA. n ≥ 3, mean ± SD values are shown for (A) and (C–E).

Fig. 3
Fig. 3. METTL3-meditated m6A reduces the sensitivity of AML cells to chemotherapeutics in vivo.

A Representative in vivo pseudocolor bioluminescence images of NSG mice transplanted with control or METTL3/METTL3-CD THP-1 cells by detecting up the GFP signal. Unit of radiance is photons/second/cm2/steradian. B The distribution of THP-1 cells in BM and spleen of the NSG mice with or without IDA treatment was measured by flow cytometry at the end point. *P < 0.05, **P < 0.01, ***P < 0.001, vs. the NC group with PBS treatment; ###P < 0.001, ####P < 0.0001, vs. the NC group with IDA treatment; &, significant interaction effect; two-way ANOVA. C Kaplan–Meier survival curves (n = 10 for each group) showing the effects of forced METTL3 and METTL3-CD expression on the progression of human AML cells in NSG mice with or without IDA treatment. ***P < 0.001, ****P < 0.0001; NS nonsignificant. n ≥ 5, mean ± SD values are shown for (B).

Fig. 4
Fig. 4. METTL3 enhances AML cell adhesion/migration in vitro and homing/engraftment in vivo.

A GSEA analyses of changed genes from RNA-seq and m6A-seq analysis of METTL3-overexpressing and control THP-1 cells (n = 3). B Images (left) and corresponding statistical results (right) showing that METTL3-overexpressing AML cells significantly improved the migration (upper panel) and adhesion (lower panel) of AML cells. P < 0.05 was considered significant, vs. the NC group, t test. Bar, 250 μm. C Flow cytometry was carried out on the BM and spleen of the xenograft recipient mice 16 h (homing) or 42 days (engraftment) after tail vein injection. P < 0.05 was considered significant, vs. the NC group, t test. n ≥ 3, mean ± SD values are shown for (B) and (C).

Fig. 5
Fig. 5. A METTL3 inhibitor reverses AML chemoresistance and homing/engraftment capacity.

A Statistical results showing the improvement of migration (upper) and adhesion (lower) in THP-1/IDA and Kasumi-1/IDA cells can be reversed by STM2457 treatment of 48 h. *P < 0.05, **P < 0.01; ***P < 0.001, NS nonsignificant; t test. B The Statistical analysis of EdU incorporation assay showing the improvement of AML chemoresistance can be reversed by the pretreatment of STM2457. *P < 0.05, **P < 0.01; ***P < 0.001, NS nonsignificant. C The homing and engraftment of THP-1&THP-1/IDA cells with or without STM2457 treatment in the BM and spleen measured by flow cytometry 16 h and 42 days after tail vein injection. *P < 0.05, **P < 0.01; ***P < 0.001, ****P < 0.0001, NS nonsignificant; t test. n ≥ 3, mean ± SD values are shown for (A–C).

Fig. 6
Fig. 6. METTL3 mediates AML chemoresistance by regulating ITGA4.

A ITGA4 appears at a high frequency in multiple migration and adhesion pathways. B The improvement of migration and adhesion in METTL3-overexpressing AML cells can be partly reversed by an ITGA4 inhibitor (TR-14035). ***P < 0.001, ****P < 0.0001, vs. the NC group under DMSO treatment; #P < 0.05, ##P < 0.01, ###P < 0.001, vs. the NC group under TR-14035 treatment; Ω, significant TR-14035 treatment effect; &, significant interaction effect; two-way ANOVA. Bar, 200 μm. C EdU incorporation assay showing that the improvement in the percentage of METTL3-overexpressing AML cells entering the proliferation cycle with IDA pressure can be partly reversed by TR-14035. *P < 0.05, **P < 0.01; ***P < 0.001; t test. n ≥ 3, mean ± SD values are shown for (B) and (C).

Fig. 7
Fig. 7. METTL3 regulates stability of ITGA4 mRNA.

A The m6A abundances of ITGA4 mRNA transcripts in METTL3-overexpressing and control THP-1 cells as detected by m6A-seq (n = 3). Dotted boxes indicate regions for qPCR in (B). B Increasing m6A modification in specific regions of ITGA4 transcripts upon METTL3 overexpression as tested by gene-specific m6A-qPCR assays of THP-1 and Kasumi-1 cells. C Dual-luciferase reporter assays showing the effect of METTL3 and METTL3-CD on ITGA4 reporters with either wild-type or mutated m6A sites. *P < 0.05, **P < 0.01; t test. n ≥ 3, mean ± SD values are shown for (B) and (C).

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