Acute myeloid leukemia (AML) refers to a kind of malignant disease originated from haemopoietic stem cells and features clonal expansion of blasts of myeloid lineage under abnormal differentiation. Upon the proliferation of immature myeloid cells, immature progenitors (blasts) are accumulated and normal hemopoiesis is impaired, resulting in the occurrence of serious infection, anemia and hemorrhage (1). AML accounts for 1.3% of newly diagnosed cancer cases in the U.S (2). The rising AML incidences are partially a result of rising prevalence of AML related to therapy, because the primary malignancy of increasing numbers of cancer patients who received cytotoxic chemotherapy have been cured (3). AML is one of the most fatal type of hematologic malignancy, with the 5-year survival rate < 30%. Hence, new prognostic biomarkers shall be urgently identified for well monitoring patient outcomes as well as more deeply explaining the AML pathogenesis.

Recent researchers have identified reversible N6-methyladenosine (m6A) RNA methylation regulators as a new way to achieve the post-transcriptional regulation (4). Geneticists confirmed that m6A was methylated in eukaryotic messenger RNA (mRNA). RNA methylation modification takes up over 60% of all the RNA modifications, of which the representative type on higher biological mRNAs is the m6A RNA methylation (5). When m6A regulators are dysregulated, the cell reproductive capacity weakens, and the self-renewal capacity losses, together with developmental defects and apoptosis (6). m6A RNA methylation regulators play roles in the cancer occurrence and development, involving liver cancer (7, 8), glioblastoma (9), osteosarcoma (10), and colorectal cancer (11).

Macrophages critically impact the remodeling of tissues in normal physiology and the way inflammation and tissue injury are resolved (12). The key step in the resolution process lies in the elimination of apoptotic cells (ACs) (13). The elimination of apoptotic cells by professional and non-professional phagocytes, a process that is essential for maintaining tissue homeostasis called “efferocytosis” (13, 14). Such process has been informed in recent studies, together with the roles it plays in maintaining tissue homeostasis and repair as well as the organism health. Our study stresses on the mechanisms regarding efferocytosis (dying cell recognition, phagocytic engulfment and homeostatic resolution), as well as explains the resulting pathological and physiological consequences upon the abrogation of the efferocytosis process (13). As we all know, m6A regulates gene expression and thus cellular processes such as cellular self-renewal, differentiation, invasion, and apoptosis (15). For example, Mettl14-mediated m6A modification could induce apoptosis of spinal cord neurons in spinal cord injury by promoting miRNA translation (16). Apoptosis and its subsequent clearance by efferocytosis occur in virtually all tissues during development, homeostasis, and disease. However, the prognostic value of m6A-related efferocytosis related-genes (ERGs) in AML has not been systematically investigated.

AML is an aggressive blood cancer among adults, and the existing techniques fail to obviously improve most patients’ survival rate. In the situation, it is necessary to find potential markers for enhancing AML patients’ diagnosis, treatment and prognosis. According to previous studies, we inferred m6A RNA methylation regulators and efferocytosis were inextricably linked to the onset and progression of AML. Our study adopts AML-related data from public database and the comprehensive biological informatics approach for mining the m6A-related ERGs in AML. The prognostic model related to m6A and ERGs was constructed to predict the prognosis of the AML patients. In addition, the exploration of potential molecular mechanisms and therapeutic approaches will contribute to the treatment and prognosis of AML.

The m6A methyltransferase METTL3 can impact the way AML is initiated and maintained. STM2457 is a high-efficiency selective first-in-class catalytic inhibitor specific to METTL3, and using it for tumor treatment can weaken the AML growth, and enhance the cell differentiation and apoptosis (36). In the study by Joselyn Cruz Cruz et al. (37), MerTK inhibition by small molecule tyrosine kinase, MRX2843, could change the leukemia microenvironment from tumor-permissive toward immune responsiveness to leukemia, as well as enhance the AML clearance mediated by immune. And the MerTK (or other vesicular cell receptors) on macrophages play an important role in mediating efferocytosis. m6A and genes related to efferocytosis can mediate the immune system, thereby affecting the AML development. Nevertheless, seldom studies have reported their joint roles. On that account, our study investigates m6A-related ERGs, aiming at contributing to new prognostic models and treatment strategies for AML patients.

In this study, we identified 7 m6A-DE-ERGs (CXCR4, PPARG, SGK1, WNK1, DNM1L, ADAM17, HIF1A), 27 miRNAs, and 7 lncRNAs, which were used to construct the lncRNA-miRNA-mRNA network. Some studies have reported the close association between SGK1, CXCR 4, PPARG, and ADAM17 and HIF1A and AML (38–42). DLEU1 can only exert impact on HIF1A through regulating hsa-miR-381-3p, while HIF1A can be impacted by various miRNAs. According to Abdul-Aziz AM et al. (42), PARP14 regulated the expression of HIF1A, thereby enhancing AML cell growth and glycolysis. On that account, applying miRNAs and 7 lncRNAs to regulate m6A-related DE-ERGs can impact the AML development.

Amino acids not only constitute proteins, but also serve as the intermediate metabolites for various biosynthetic pathways. The study by Yoko Tabe et al. (43) summed up the amino acid metabolism occurring in hematologic malignancy, and assisted in reclassifying amino acid-depleting enzymes into targeted therapeutic agents. In our study, amino acid metabolic pathways significantly impact AML development. According to Courtney L Jones et al. (44), the leukemia stem cell (LSC) population presented elevated amino acid uptake, steady-state levels, and catabolism, and drugs targeting LSC metabolic vulnerabilities could serve for eradicating LSCs in clinical practice. Hence, m6A-related DE-ERGs may regulate the AML occurrence and development through various amino acid metabolic pathways. Our study also reveals the close association between chemokine signaling pathway and AML. Chemokine refers to a family of small cytokines with chemotactic properties, consisted by 8-10 kilodaltons. Chemokine can traffic and regulate the proliferative, migratory, differentiative and homing activities of immune cells. The CXCR4 chemokine receptor can enhance the survival rate of various cell types. According to the study of Kimberly N Kremer et al. (45), CXCR4 chemokine receptor signaling regulated many of the Bcl-2 family members (Bcl-XL, Noxa, and Bak), thereby inducing AML cell apoptosis. Taken together, m6A-related DE-ERGs may be involved in AML progression by downregulating chemokine signaling pathway.

In the 14 m6A-related ERGs related-subtypes analysis, different subpopulations present obviously different monocytes, plasma cells and naive B cells, with more infiltrated in the worse prognosis cluster1. Clinical experiments in the study confirmed the significant role of monocytes, plasma cells and naive B cells in the AML pathogenesis, and its close association with myeloid tumor cell progression. Monocyte is the innate immune cell in the mononuclear phagocyte system and can remarkably regulate tumor development and progression. Plasma cell that secretes antibody serves as the core pillar of the humoral immunity, generated during the basic cellular restructuration from the naive B cells-antigen contact. When naive B cells are differentiated into extrafollicular B cells, antibody-secreting cells with weaker affinity and short life can be generated. Claire E Olingy et al. (46) made a comprehensive explanation of the monocyte heterogeneity in the homeostasis process, highlighted the role played by monocyte in the cancer development, as well as gave effective monocyte-targeted cancer treatment strategies. In the study by Maartje C A Wouters et al. (47), substantial evidences, by virtue of a comprehensive PubMed search, had proved that plasma cells positively impact the antitumor immunity, and it is suggested to enhance these responses in designing cancer immunotherapies. Helmink BA et al. (48) used mass cytometry for interrogating various surface proteins, finding the existence of naive B cells, memory B cells, activated memory B cells, and plasmablasts. Therefore, we speculated that different subtypes suggested the possible mediating roles played by monocytes, plasma cells and naive B cells in the prognosis of patients with different AML subtypes.

Our study constructed the prognosis model and the prognostic genes of UCP 2, DOCK 1, SLC14A1, and SLC25A1. Uncoupling protein 2 (UCP2), and mitochondrial uncoupling proteins belong to the family of mitochondrial anion carrier proteins (MACP). AML patients showed elevated UCP2 expression. Dongxu Gang et al. (49) found that UCP2 inhibition could lead to weakened AML cell line proliferation, cell cycle alternation, and apoptosis enhancement in vitro. Dedicator of cytokinesis 1(DOCK1) is the dedicator of cytokinesis proteins and the guanine nucleotide exchange factors specific to small Rho family G proteins. According to Sze-Hwei Lee et al. (50), highly expressed DOCK1 led to worse AML prognosis, and higher DOCK1 expression exhibited an obvious relevance to older age, higher platelet and peripheral blast counts, intermediate-risk cytogenetics, FLT3-ITD, MLL-PTD and PTPN11, NPM1, RUNX1, ASXL1 and DNMT3A mutations. Solute carrier family 14 member 1 (SLC14A1) is a gene that encodes a protein that mediates urea transport in erythrocytes (51). Through targeting SLC14A1, ARHGAP5 and PIK3CA, miR-10a-3p may be involved in the development of FLT3 mutation in adult AML (52). Solute carrier family 25 member 1(SLC25A1) is a mitochondrial carrier that facilitates the flow of citrate/isocitrate in mitochondria in exchange for the entry of malate in the cytoplasm (53, 54). According to a report, prognostic signature associated with SLC25A1 denotes AML patients worse prognosis (55). On that account, UCP 2, DOCK 1, SLC14A1, and SLC25A1 show important prognostic value in AML, but subsequent studies are still needed to explore their functions in AML.

The HLA correlation analysis revealed the highly expressed HLA in the high-risk group, which had a worse prognosis, and the two risk groups presented obvious difference in the expressions of the 14 immune checkpoints, 20 HLA family genes, and IPS. Immune checkpoint molecules, inhibitory and stimulatory, refer to ligand-receptor pairs that inhibit or stimulate the immune responses (56). Luca Vago et al. (57) conducted studies to test the latest immunotherapies for the specific targeting of AML cells (antibody therapy and cellular therapy, etc.) or the broader reactivation of antileukemia immunity (vaccines and checkpoint blockade, etc.), which combines complementary immunotherapeutic strategies with chemotherapeutics or other pharmacotherapies. Rikako Tabata et al. (58) demonstrated the underlying clinical benefits exhibited by immuno-oncology (IO) therapy specific to AML and ICIs with or without conventional chemotherapy. These prove the certain efficacy of immunotherapy in AML. Immune checkpoint, HLA and IPS are different in the two risk groups, suggesting their mediating roles in AML prognosis. Hence, the immune microenvironment of AML offers theoretical basis for immunotherapy of AML.

In the study, the predicted drugs are Erlotinib, Dasatinib, BI.2536, and Bortezomib, etc. Erlotinib, as a type of tyrosine kinase inhibitor, did not achieve a good response in AML patients in pilot study (59). Dasatinib is a type of kinase inhibitor, and has the function of inhibiting BCR-ABL, Src family kinases, c-Kit, and platelet-derived growth factor receptor kinase. Due to the inhibitory effect on BCR-ABL, it is usually used for treating chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia. A subpopulation of AML patients show BCR-ABL expression. Patients with unselected AML present remarkably mixed clinical responses to dasatinib, which shall be validated in larger-scale studies (60). BI.2536 is a newly discovered Plk inhibitor capable of inducing mitotic arrest and apoptosis. According to the randomized, open-label, phase I/II trial, clinical activity in patients treated with single-agent BI 2536 provides the first evidence of the potential therapeutic value of targeted Plk in patients with relapsed refractory AML (61). Bortezomib, proteasome inhibitor, is the mainstream drug for treating various myeloma and mantle cell lymphoma. AML patients give a series of different clinical responses to the chemotherapy regimens that combine bortezomib, and some cases show a complete remission rate over 80% (62). On these account, dasatinib, BI.2536, and bortezomib may be applicable for treating AML, which shall be more deeply concerned in future studies.

In summary, using bioinformatic methods, this study has identified prognostic genes for AML and constructed a prognostic model associated with m6A and ERGs. Differential analyses were conducted between high and low-risk groups, evaluating immune cell infiltration, immune therapy response, functional enrichment, and drug sensitivity. However, the study has discernible limitations. The analysis predominantly relies on a constrained number of samples from public databases, highlighting the imperative need for an expanded sample size. While gene expression levels have been validated through qRT-PCR, the requisite further verification and elucidation of potential molecular mechanisms necessitate animal experiments. Moreover, the analyses pertaining to immune therapy and drug sensitivity in the study require clinical validation to ascertain their clinical value further. Such exploration will constitute the main thrust of our ensuing research efforts. Ultimately, our findings furnish researchers with a novel theoretical framework for delving deeper into the relationship between m6A regulatory factors, efferocytosis, and AML, thereby providing new targets for enhancing the prognosis and treatment of AML.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

The studies involving humans were approved by Seventh Affiliated Hospital, Sun Yat-Sen University. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. The manuscript presents research on animals that do not require ethical approval for their study.

YW: Validation, Writing – original draft. TB: Resources, Writing – review & editing. JT: Conceptualization, Data curation, Writing – review & editing. X-JX: Formal analysis, Writing – review & editing. CL: Project administration, Software, Writing – original draft. BL: Funding acquisition, Investigation, Writing – review & editing. T-TS: Methodology, Visualization, Writing – review & editing.