N^6-methyladenosine (m6A) methylation, the most prevalent RNA modification in eukaryotes, participates in various cellular processes and disease progressions through three regulatory factors: methyltransferases, demethylases, and methylation recognition proteins. It plays a significant role in neural cells and tissues of mammals. Recent studies have indicated that m^6A methylation modification is crucial in the development of hematological diseases and the differentiation of hematopoietic stem cells. In malignant lymphoma, m^6A modification substantially influences tumor cell differentiation and proliferation. Additionally, m^6A modification can affect the progression and poor prognosis of leukemia through multiple signaling pathways. Moreover, m^6A methylation can modulate the differentiation and function of hematopoietic stem cells through various mechanisms. In summary, m6A methylation modification plays a pivotal role in hematological diseases.
m6A Omics Technology Integration Strategy
Potentially significant RNA methyltransferases and methylation-modified genes are identified through database mining and subsequently validated using Western blotting (WB) and quantitative PCR (qPCR). Base mutation experiments are conducted to confirm m6A methylation sites and to assess the impact of methylation on specific characteristics. Integrative omics data analysis, combining m6A-seq and RNA-seq, is employed to identify target genes. These target genes are further studied through methods such as gene knockout, knockdown, and overexpression. Downstream analyses, including WB, qPCR, RNA immunoprecipitation (RIP), and RNA pull-down assays, are performed to validate interactions and elucidate underlying mechanisms.
Function: Locates m6A modification sites across the whole transcriptome and identifies differentially methylated regions (DMRs).
Technical Highlights:
Utilizes specific m6A antibodies to immunoprecipitate methylated RNA fragments, combined with high-throughput sequencing.
Compares immunoprecipitated (IP) samples with input samples; modification sites are identified through peak-calling algorithms such as MACS2 and exomePeak.
Suitable for low-input samples (e.g., nano MeRIP-seq requiring 500 ng of total RNA).
RIP-seq (RNA Immunoprecipitation Sequencing)
Function: Identifies target RNA molecules of m^6A reader proteins (e.g., YTHDF2, IGF2BP1) and analyzes their binding preferences.
Technical Highlights:
Employs antibodies against reader proteins to enrich RNA-protein complexes; binding targets are analyzed post-sequencing.
Integration with MeRIP-seq data verifies the association between m^6A modifications and reader protein binding.
RNA-seq
Function: Provides gene expression profiles for correlation analyses between m^6A modification levels and gene expression (e.g., methylation-expression correlation).
Technical Highlights:
Combines differentially methylated genes (DMRs) with differentially expressed genes (DEGs) to identify functional targets.
I. Integrated Analysis Workflow and Biological Questions
Through the integration of these technologies, several key questions can be systematically addressed:
How does m6A modification regulate target gene expression?
Workflow:
MeRIP-seq is used to identify differentially methylated genes.
RNA-seq validates changes in gene expression.
RIP-seq confirms the binding of reader proteins to the target RNAs.
Does dynamic m6A modification affect the function of RNA-binding proteins?
Workflow:
RIP-seq identifies the targets of reader proteins.
MeRIP-seq verifies the m^6A modification status of these targets.
RNA-seq analyzes the expression levels of target genes.
How does the epitranscriptome contribute to disease heterogeneity?
Workflow:
Single-cell RNA-seq is used to sort subpopulations.
MeRIP-seq analyzes m^6A modifications specific to each subpopulation.
RIP-seq elucidates the distribution of reader proteins across subpopulations.
Primary Diseases Studied: Multiple myeloma, malignant lymphoma, leukemia, and hematopoietic stem cell development.
Main Sample Types: Fresh tissues, cell lines, and primary cells.
Major Research Areas: Early diagnosis and treatment, prognostic therapy, tumor evolution, and stem cell development.
Case Study 1
piRNA-30473 Contributes to Tumorigenesis and Poor Prognosis by Regulating m6A RNA Methylation in DLBCL
Journal: Blood
Impact Factor: 20.3
Publication Date: 2021
DOI: 10.1182/blood.2019003764
Sample Selection: SU-DHL-8 and Farage Cell Lines
Research Techniques: m6A Methylation Sequencing, RNA Transcriptome Sequencing
Background
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, characterized by significant heterogeneity in genetic and epigenetic landscapes. The pathogenesis and progression of DLBCL are governed by genetic mutations and epigenetic aberrations. N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic messenger RNA (mRNA), influences diverse fundamental biological processes by regulating target gene expression. Despite its critical roles in various malignancies, the function of m6A modifications in DLBCL remains inadequately understood.
PIWI-interacting RNAs (piRNAs) constitute the majority of small non-coding RNAs and are known to guide PIWI proteins to silence transposable elements through the regulation of DNA methylation. However, the functional mechanisms of piRNAs in the epitranscriptomic regulation of DLBCL necessitate further elucidation.
Objective
To investigate the role of m6A methylation in the tumorigenesis and progression of DLBCL, thereby providing a promising therapeutic strategy for targeted treatment of DLBCL.
Methodology
Study Design
A comprehensive analysis was conducted to explore the relationship between piRNA expression and DLBCL development and prognosis. The study focused on the association between piRNA-30473 and m6A methylation modifications in DLBCL.
Sample Selection
The DLBCL cell lines SU-DHL-8 and Farage were selected due to their representative characteristics of the disease and their utility in epigenetic studies.
Experimental Procedures
Identification of Differentially Expressed piRNAs:
Expression profiles of piRNAs were analyzed to identify those significantly associated with DLBCL progression and prognosis.
Correlation with m6A Methylation:
m6A-seq and RNA-seq were integrated to determine the impact of piRNA expression on m6A methylation modifications.
Target Gene Identification:
WTAP (Wilms’ Tumor 1-Associating Protein) was identified as a primary target gene influenced by piRNA-30473.
Functional Validation:
Methylation site mutation experiments were conducted to confirm the specific m6A sites involved.
The role of m6A reader proteins in downstream gene regulation was elucidated.
Preclinical Evaluation:
Patient-derived xenograft (PDX) models were utilized to assess the therapeutic potential of targeting the piRNA-30473/m6A axis.
Results
piRNA-30473 and DLBCL Progression
piRNA-30473 was found to be significantly upregulated in DLBCL cell lines.
Higher expression levels of piRNA-30473 correlated with poor patient prognosis.
Impact on m6A Methylation
piRNA-30473 influenced the global m6A methylation status by regulating the expression of WTAP, a key component of the m6A methyltransferase complex.
Alterations in WTAP expression led to changes in m6A methylation of downstream target genes.
Mechanism of Action
Mutation of specific m6A methylation sites on WTAP mRNA demonstrated the critical role of these modifications in gene regulation.
Interaction between m6A reader proteins and methylated mRNAs was essential for the modulation of gene expression associated with tumorigenesis.
Therapeutic Potential
Targeted inhibition of piRNA-30473 resulted in decreased tumor growth and improved prognosis in PDX models.
The findings suggest that the piRNA-30473/m6A pathway is a viable target for therapeutic intervention in DLBCL.
Technical Highlights
Multi-Omics Integration:
Combined MeRIP-seq, RNA-seq, and RIP-seq analyses provided a comprehensive understanding of the piRNA-m6A-target gene regulatory network.
Dynamic Modification Validation:
Methylation site mutation experiments confirmed the functional significance of specific m6A modifications on WTAP mRNA.
Preclinical Models:
Patient-derived xenograft models demonstrated the translational potential of targeting the piRNA-30473/m6A axis in DLBCL treatment.
Discussion
This study elucidated the pivotal role of piRNA-30473 in the tumorigenesis and poor prognosis of DLBCL through its regulation of m6A RNA methylation. The upregulation of piRNA-30473 disrupted the normal m6A methylation landscape by modulating WTAP expression, subsequently affecting the expression of downstream genes involved in cell proliferation and survival.
The research highlights the significance of epitranscriptomic modifications in cancer biology and underscores the potential of piRNAs as therapeutic targets. By integrating multi-omics approaches, the study provided valuable insights into the complex regulatory networks governing DLBCL progression.
Conclusion
The findings demonstrate that piRNA-30473 contributes to the tumorigenesis and poor prognosis of DLBCL by regulating m6A RNA methylation. Targeting the piRNA-30473/WTAP/m6A axis offers a promising therapeutic strategy for the treatment of DLBCL.
Case Study 2
KIAA1429-Mediated m6A Modification of CHST11 Promotes Progression of Diffuse Large B-Cell Lymphoma by Regulating the Hippo-YAP Pathway
Journal: Cellular & Molecular Biology Letters
Impact Factor: 8.3
Publication Date: 2023
DOI: 10.1186/s11658-023-00445-w
Sample Selection
Lymphoid tissues from patients diagnosed with diffuse large B-cell lymphoma (DLBCL) were utilized in this study.
Research Techniques: MeRIP-seq, RNA Transcriptome Sequencing
Background
N6-methyladenosine (m6A) has been demonstrated to participate in numerous critical biological processes by regulating the levels of target genes. KIAA1429, also known as viral m6A methyltransferase-associated protein (VIRMA), is a key component of the m6A methyltransferase complex. Despite its recognized role in epigenetic regulation, the function of KIAA1429-mediated m6A modification in the progression of DLBCL remains unclear.
Objective
To elucidate the regulatory role of KIAA1429 in DLBCL and evaluate its potential as a novel predictive biomarker and therapeutic target for the progression of this lymphoma.
Methodology
Evaluation of Prognostic Value
The prognostic significance of KIAA1429 expression in DLBCL patients was assessed by analyzing its correlation with patient outcomes. Clinical samples of lymphoid tissues were collected from DLBCL patients for this purpose.
Functional Role in DLBCL Progression
The role of KIAA1429 in the progression of DLBCL was investigated through in vitro and in vivo experiments. The effects of KIAA1429 on cell proliferation, apoptosis, and tumor growth were examined.
Identification of Target Genes
Using m6A methylation sequencing (MeRIP-seq) and RNA transcriptome sequencing (RNA-seq), carbohydrate sulfotransferase 11 (CHST11) was identified as the primary target gene affected by KIAA1429-mediated m6A modification.
Mechanistic Studies
The mechanism by which methylation of CHST11 affects downstream gene regulation was elucidated by studying the role of m6A reader proteins. The impact of CHST11 on downstream signaling pathways, particularly the Hippo-YAP pathway, was analyzed to detail how KIAA1429 influences DLBCL progression.
Results
Prognostic Significance of KIAA1429
Elevated expression of KIAA1429 was observed in DLBCL patient samples and was associated with poor prognosis. Patients with higher KIAA1429 levels exhibited reduced overall survival rates.
Role in DLBCL Progression
Knockdown of KIAA1429 in DLBCL cell lines resulted in decreased cell proliferation and increased apoptosis. In vivo studies demonstrated that silencing KIAA1429 suppressed tumor growth in xenograft models.
Identification of CHST11 as a Target Gene
Integrated analysis of MeRIP-seq and RNA-seq data revealed that KIAA1429 mediates m6A modification of CHST11 mRNA, leading to its upregulation. This modification enhanced the stability of CHST11 transcripts.
Mechanism of m6A Modification
The m6A reader proteins were found to recognize methylated CHST11 mRNA, promoting its translation and contributing to oncogenic activity. Methylation of CHST11 by KIAA1429 facilitated the activation of the Hippo-YAP signaling pathway.
Impact on Hippo-YAP Pathway
Increased expression of CHST11 led to the activation of the Yes-associated protein (YAP), a critical effector of the Hippo pathway. Activation of YAP promoted the transcription of downstream genes involved in cell proliferation and survival, thereby contributing to DLBCL progression.
Conclusion
KIAA1429-mediated m6A modification of CHST11 plays a crucial role in the progression of diffuse large B-cell lymphoma by regulating the Hippo-YAP pathway. KIAA1429 enhances the stability and expression of CHST11 through m6A methylation, leading to the activation of oncogenic signaling pathways. These findings suggest that KIAA1429 may serve as a novel predictive biomarker and a potential therapeutic target for DLBCL.
Technical Highlights
Multi-Omics Approach:
Integrated MeRIP-seq and RNA-seq analyses provided comprehensive insights into the epigenetic regulation mediated by KIAA1429.
Mechanistic Elucidation:
Studied the role of m6A reader proteins in recognizing methylated CHST11 mRNA, clarifying the mechanism by which methylation affects downstream gene regulation.
Pathway Analysis:
Investigated the downstream signaling pathways regulated by CHST11, particularly the Hippo-YAP pathway, to elucidate the detailed mechanism of KIAA1429’s influence on DLBCL progression.
Implications for Therapeutic Strategies
The identification of the KIAA1429/CHST11/Hippo-YAP axis in DLBCL highlights the potential of targeting m6A modifications as a therapeutic strategy. Inhibiting KIAA1429 or modulating m6A methylation could suppress tumor growth and improve patient outcomes.
Future Directions
Further studies are warranted to explore the therapeutic efficacy of targeting KIAA1429 in clinical settings. Investigating the interaction between m6A methylation and other epigenetic modifications may provide additional insights into DLBCL pathogenesis.
Case Study 3
The m6A Methyltransferase METTL3 Mediates RNA m6A to Promote Cell Proliferation via MYC Activation in Acute Myeloid Leukemia
Journal: Cell
Impact Factor: 38.6
Publication Date: 2020
DOI: 10.1016/j.cell.2019.11.027
Sample Selection
Patient Samples: Bone marrow specimens from patients diagnosed with acute myeloid leukemia (AML).
Cell Lines: Human AML cell lines MOLM-13 and THP-1.
Research Techniques: m6A-seq, RNA Sequencing, CRISPR-Cas9 Gene Editing, Xenograft Mouse Models
Background
Acute myeloid leukemia (AML) is a malignant disorder characterized by the clonal expansion of immature myeloid cells. Relapse in AML is closely associated with the self-renewal capacity of leukemia stem cells (LSCs). N6-methyladenosine (m6A) modification, the most abundant internal modification in eukaryotic messenger RNA (mRNA), plays crucial roles in stem cell fate determination. However, the specific function of m6A modifications in AML LSCs remains inadequately understood.
METTL3 is a core component of the m6A methyltransferase complex responsible for catalyzing m6A modifications on mRNA. Previous studies have implicated METTL3 in various biological processes, but its molecular basis in maintaining AML LSCs through m6A-dependent mechanisms requires further elucidation.
Objective
To uncover the molecular mechanisms by which METTL3 sustains AML stem cells via m6A-mediated regulation, thereby promoting leukemia progression.
Methodology
Assessment of METTL3 Expression and Prognostic Value
Quantitative Analysis: Evaluated METTL3 expression levels in LSCs isolated from AML patient samples using quantitative PCR and Western blotting.
Prognostic Correlation: Analyzed clinical data to correlate METTL3 expression with patient outcomes and overall survival rates.
Identification of Target mRNA via Integrated Sequencing
m6A-seq and RNA-seq: Performed m6A-seq to map m6A modifications and RNA-seq to profile gene expression in AML cells.
Data Integration: Identified MYC mRNA as a key target of METTL3-mediated m6A modification through integrative analysis.
Mechanistic Studies on m6A Modification and mRNA Stability
IGF2BP1 Interaction: Investigated the role of the m6A reader protein IGF2BP1 in recognizing methylated MYC mRNA and enhancing its stability.
Luciferase Reporter Assays: Employed luciferase-based reporter constructs containing wild-type or mutant MYC mRNA to assess mRNA stability.
Functional Validation in Vitro and In Vivo
CRISPR-Cas9 Gene Editing: Utilized CRISPR-Cas9 to knock down METTL3 expression in AML cell lines.
Cell Proliferation and Self-Renewal Assays: Assessed the effects on cell proliferation, colony formation, and apoptosis.
Xenograft Models: Established AML xenograft mouse models by transplanting modified AML cells to evaluate leukemia progression and survival.
Development of METTL3 Inhibitor
STM2457 Inhibitor: Developed a selective small-molecule inhibitor targeting METTL3.
Patient-Derived Xenograft (PDX) Models: Tested the efficacy of STM2457 in PDX models to assess its anti-leukemic potential.
Results
1. METTL3 is Highly Expressed in AML LSCs and Correlates with Poor Prognosis
Elevated Expression: METTL3 was significantly upregulated in LSCs isolated from AML patients compared to normal hematopoietic stem cells.
Clinical Outcomes: High METTL3 expression levels were associated with reduced overall survival and poor prognostic indicators.
2. Identification of MYC mRNA as a Critical Target of METTL3
m6A Modification Mapping: m6A-seq revealed enrichment of m6A peaks in the coding sequence and 3’ untranslated region of MYC mRNA.
Expression Correlation: RNA-seq data showed that MYC expression levels were positively correlated with METTL3 expression.
3. METTL3-Mediated m6A Modification Enhances MYC mRNA Stability via IGF2BP1
Interaction with IGF2BP1: Immunoprecipitation assays demonstrated that IGF2BP1 binds preferentially to methylated MYC mRNA.
Increased mRNA Stability: Luciferase reporter assays indicated that m6A modification of MYC mRNA enhanced its stability and translation efficiency.
Promotion of LSC Self-Renewal: Elevated MYC levels led to increased proliferation and self-renewal capacity of AML LSCs.
4. Knockdown of METTL3 Suppresses AML Progression and Extends Survival
In Vitro Effects: METTL3 knockdown resulted in decreased cell proliferation, reduced colony formation, and increased apoptosis in AML cell lines.
In Vivo Outcomes: Mice transplanted with METTL3-deficient AML cells exhibited significantly slowed leukemia progression and extended survival compared to controls.
5. STM2457 Inhibits METTL3 Activity and Displays Anti-Leukemic Effects
Selective Inhibition: STM2457 selectively inhibited METTL3 enzymatic activity without affecting other methyltransferases.
Therapeutic Efficacy: Treatment with STM2457 in PDX models led to decreased tumor burden and prolonged survival, demonstrating its potential as a therapeutic agent.
Technical Highlights
Single-Cell Sequencing:
Employed single-cell RNA-seq to reveal the specific expression pattern of METTL3 in LSC subpopulations, highlighting its role in stemness maintenance.
Dynamic mRNA Stability Monitoring:
Used real-time luciferase reporter assays to track changes in MYC mRNA stability upon METTL3 manipulation, providing direct evidence of m6A-mediated regulation.
Clinical Translation:
Developed STM2457, a novel small-molecule inhibitor of METTL3, and validated its anti-leukemic efficacy in clinically relevant PDX models.
Conclusion
This study elucidated the crucial role of METTL3-mediated m6A modification in the maintenance of AML leukemia stem cells. METTL3 enhances the stability and expression of MYC mRNA via m6A modification and recognition by IGF2BP1, promoting LSC self-renewal and leukemia progression. Targeting METTL3 with specific inhibitors like STM2457 presents a promising therapeutic strategy for the treatment of AML.
Case Study 4
The N6-Methyladenosine (m6A)-Forming Enzyme METTL3 Controls Myeloid Differentiation of Normal Hematopoietic and Leukemia Cells
Journal: Nature Medicine
Impact Factor: 82.9
Publication Date: 2017
DOI: 10.1038/nm.4416
Research Approach: RNA-seq, Ribo-seq, miCLIP
Research Findings
1. m6A Inhibits Myeloid Differentiation of HSPCs
To explore the function of m6A in hematopoietic stem cells (HSCs), the researchers utilized short hairpin RNA (shRNA) to knock down METTL3 expression in HSPCs. This knockdown led to a global reduction in m6A levels, as confirmed by dot blot analyses (Figure 1a and 1b). The depletion of METTL3 resulted in inhibited cell growth (Figure 1c) without significant changes in apoptosis (Figure 1d).
Flow cytometry analysis using myeloid markers CD11b and CD14 demonstrated that METTL3 knockdown cells exhibited increased myeloid differentiation after seven days (Figure 1e). Conversely, overexpression of METTL3 enhanced m6A levels (Figure 1f), promoted cell proliferation and colony formation (Figure 1g), and significantly inhibited myeloid differentiation of HSPCs (Figure 1h). These findings indicate a negative correlation between METTL3 expression and myeloid differentiation in normal bone marrow cells.
Figure 1 m6A inhibits myeloid differentiation of human stem/progenitor cells (HSPCs).
2. m6A Promotes Leukemogenesis
Considering that dysregulated myeloid differentiation is a hallmark of leukemia, the authors investigated whether METTL3 expression is altered in AML. Analysis revealed that METTL3 mRNA levels were significantly elevated in AML samples compared to other cancer types (Figure 2a). Additionally, METTL3 protein levels were higher in AML cell lines than in other leukemia cell lines (Figure 2b). AML cells exhibited increased m6A modification levels compared to normal control cells (Figure 2c), suggesting that elevated m6A levels are crucial for maintaining the undifferentiated state of myeloid leukemia cells.
Knockdown of METTL3 in MOLM-13 AML cells significantly reduced m6A levels, inhibited cell growth, induced differentiation, and increased apoptosis (Figure 2d–g). In vivo studies demonstrated that METTL3 depletion delayed leukemia progression in mouse models (Figure 2h).
Figure 2 m6A promotes leukemogenesis.
3. m6A Directly Regulates Expression of c-MYC, BCL-2, and PTEN
Integrative analyses combining single-nucleotide-resolution m6A cross-linking and immunoprecipitation (miCLIP), RNA sequencing (RNA-seq), and ribosome profiling were performed. The results indicated that m6A-modified mRNA transcripts were upregulated at the expression level but exhibited decreased translation efficiency upon METTL3 knockdown in MOLM-13 cells (Figure 3).
Gene Set Enrichment Analysis (GSEA) of the combined datasets identified 44 overlapping genes characterized by the following features: (i) enrichment in m6A modifications; (ii) upregulated mRNA expression after METTL3 knockdown; and (iii) reduced translation efficiency upon METTL3 depletion (Figure 3d). These genes are involved in apoptosis, DNA damage response, cancer pathways, and are associated with MYC-related genes.
Figure 3 m6A is required for maintaining the translation of target mRNAs that control cell fate.
Among the overlapping genes, the authors focused on PTEN, c-MYC, and BCL-2 due to their high number of m6A sites. MeRIP–qPCR (m6A RNA immunoprecipitation followed by quantitative PCR) confirmed that METTL3 knockdown in MOLM-13 cells led to decreased m6A levels in the transcripts of c-MYC, BCL-2, and PTEN, along with reduced protein expression (Figure 4a–c). In contrast, overexpression of METTL3, but not a catalytically inactive mutant METTL3-CD, increased the protein levels of these target genes (Figure 4f). This indicates that the translational activation of the target genes by METTL3 is dependent on its catalytic activity in m6A formation.
Figure 4 m6A directly controls expression of c-MYC, BCL-2 and PTEN.
4. Impact of METTL3 Knockdown on Signaling Pathways in AML Cells
To determine how METTL3 knockdown affects signaling pathways in AML cells, the authors employed reverse-phase protein arrays (RPPA) to compare the levels of signaling proteins between METTL3 shRNA-treated and control cells. The most significantly increased proteins upon METTL3 depletion were apoptotic mediators, including caspase-3, caspase-7, BIM, and phosphorylated AKT (p-AKT at T308 and S473) (Figure 3h). These results suggest that the loss of m6A modifications may activate the PI3K/AKT signaling pathway.
Conclusion
The study demonstrates that leukemia cells exhibit higher METTL3 abundance compared to normal hematopoietic cells. Depletion of METTL3 induces apoptosis in leukemia cells but not in normal hematopoietic cells, suggesting that METTL3 inhibition could serve as a therapeutic strategy for myeloid malignancies. Future research should focus on the potential therapeutic efficacy of targeting METTL3, possibly in combination with existing therapies such as chemotherapy.
Mechanistic Innovation: This study is the first to directly link m6A modification with the regulation of RNA translation efficiency.
Translational Value: The development of the small-molecule inhibitor STM2457 targeting METTL3 has progressed to preclinical trial stages.
Technical Highlights
Integration of Multi-Omics Analyses:
Combined miCLIP, RNA-seq, and ribosome profiling to identify key genes regulated by METTL3-mediated m6A modification.
Functional Validation:
Demonstrated that METTL3 affects myeloid differentiation and leukemogenesis through modulation of mRNA translation efficiency.
Signaling Pathway Elucidation:
Identified activation of the PI3K/AKT pathway upon METTL3 depletion, linking m6A modifications to key survival signaling in AML cells.
Case Study 5
METTL16 drives leukemogenesis and leukemia stem cell self-renewal by reprogramming BCAA metabolism
Journal: Cell Stem Cell (Impact Factor: 21.2)
Publication Year: 2023
DOI:10.1016/j.stem.2022.12.006
Introduction
Acute myeloid leukemia (AML) is a malignant hematopoietic disease characterized by the accumulation of undifferentiated myeloid cells in the bone marrow. The roles of most methyltransferase-like proteins (METTLs) in physiological processes and pathogenesis remain largely unclear, except for METTL3 and METTL14, which are well-established N6-methyladenosine (m6A) methyltransferases. METTL16, a recently identified m6A methyltransferase, has been implicated in various cellular functions. However, its specific role in AML development and progression is not well understood.
Experimental Techniques
Genome-Wide CRISPR Screening:
Conducted on multiple cancer cell lines to identify essential genes for cancer cell survival and proliferation.
shRNA-Mediated Gene Knockdown:
A lentivirus-based short hairpin RNA (shRNA) system was used to knock down METTL16 expression in AML cells.
m6A-Seq and RNA-Seq:
Employed to identify transcriptome-wide targets of METTL16 and assess changes in gene expression profiles.
Triple Quadrupole Mass Spectrometry:
Utilized to quantitatively detect m6A levels in RNA samples.
Colony Formation and Proliferation Assays:
Performed to evaluate the self-renewal capacity and growth characteristics of leukemia stem cells (LSCs) and leukemia-initiating cells (LICs).
Metabolic Analyses:
Oxygen consumption rate (OCR) and metabolite levels were measured to assess metabolic changes in AML cells.
Results
1. METTL16 is Essential for AML Progression and Maintenance
Genome-wide CRISPR-Cas9 knockout screening across over 800 cancer cell lines revealed that leukemia cells exhibit a stronger dependency on METTL16 compared to other cancer types. Notably, AML cell lines showed higher dependence on METTL16 than non-AML leukemia cell lines. Among all METTL family members, METTL16 was identified as the most critical for the survival and proliferation of AML cells.
METTL16 was abnormally overexpressed in human AML samples at both mRNA and protein levels, compared to healthy controls. To elucidate its role in leukemogenesis, Mettl16fl/fl mice were crossed with Mx1-Cre mice to generate cKO mice. Heterozygous knockout of Mettl16 significantly delayed leukemia onset and progression, extending the survival of mice. Homozygous knockout exhibited even more pronounced anti-leukemic activity, completely suppressing leukemia development (Figure 5). Analyses using patient-derived xenograft (PDX) models further confirmed that METTL16 plays a crucial oncogenic role in AML initiation, progression, and maintenance.
Figure 5. METTL16 is essential for leukemogenesis in vivo.
Heterozygous and homozygous deletion of Mettl16 in mouse models delayed leukemia onset and improved survival rates compared to controls.
2. METTL16 is Critical for the Self-Renewal of LSCs and LICs
METTL16 protein levels were significantly elevated in primary AML patient samples, particularly in LSCs and LICs, compared to healthy controls. This suggests that METTL16 may play a pivotal role in the self-renewal of these cells. Knockdown of Mettl16 using shRNA inhibited the colony-forming ability of human primary AML cells. Similar results were observed in mouse models, indicating that METTL16 is essential for maintaining the self-renewal capacity of LSCs/LICs.
To evaluate whether METTL16 is a potential safe target for AML therapy, its role in normal hematopoiesis was investigated. Hematopoietic-specific deletion of Mettl16 in cKO mice, induced by poly(I:C), led to moderate reductions in peripheral white blood cells, lymphocytes, and platelets but had minimal impact on other hematopoietic cells. Gene knockout had negligible effects on the proliferation and colony-forming abilities of human and mouse hematopoietic stem/progenitor cells (HSPCs). These findings suggest that LSCs/LICs are more dependent on METTL16 expression and function than normal HSPCs.
3. METTL16 Reprograms BCAA Metabolism in AML
To understand how METTL16 exerts its oncogenic effects in AML, mutational analyses confirmed that its role is entirely dependent on its enzymatic activity. m6A-seq and RNA-seq identified that METTL16 knockout significantly suppressed the expression of four key genes: BCAT1, BCAT2, LARS1, and IARS1. RNA immunoprecipitation followed by quantitative PCR (RIP-qPCR) demonstrated that METTL16 directly binds to the transcripts of these genes in AML cells.
These genes are involved in the biosynthesis and metabolism of branched-chain amino acids (BCAAs)—leucine, isoleucine, and valine—which are linked to the aggressiveness of various cancers, including leukemia. Specifically:
BCAT1 and BCAT2: Encode branched-chain amino acid transaminases that catalyze the transfer of amino groups from BCAAs to α-ketoglutarate, initiating BCAA catabolism.
LARS1 and IARS1: Encode leucyl-tRNA synthetase and isoleucyl-tRNA synthetase, respectively, essential for incorporating leucine and isoleucine into proteins during translation.
METTL16 was found to directly methylate BCAT1 and BCAT2 mRNAs in vitro. METTL16 knockout reduced the stability of these transcripts and downregulated their expression. The m6A reader protein YTHDC1 directly interacted with BCAT1 and BCAT2 mRNAs, enhancing their stability (Figure 6). These results indicate that METTL16-mediated m6A modifications on BCAT1 and BCAT2 transcripts are recognized by YTHDC1, which stabilizes these mRNAs.
Figure 6. METTL16 regulates the expression of BCAT1 and BCAT2 in an m6A-dependent manner.
A schematic depicting METTL16-mediated methylation of BCAT1/BCAT2 mRNAs and subsequent stabilization by YTHDC1.
Further analyses showed that Mettl16 knockout significantly reduced the oxygen consumption rate (OCR) in AML cells and decreased levels of metabolites involved in the tricarboxylic acid (TCA) cycle. This indicates that METTL16 reprograms BCAA metabolism, affecting cellular respiration and energy production. Forced expression of BCAT1 and BCAT2 partially rescued the proliferation defects and apoptosis induced by METTL16 knockout and fully reversed the inhibition of oxidative capacity. These findings suggest that the metabolic effects of METTL16 are mainly due to the dysregulation of BCAT1 and BCAT2, which are critical functional targets participating in its oncogenic role in AML.
Figure 7 The role of METTL16 in AML cells
Discussion
This study reveals that METTL16 plays a vital role in AML progression by modulating m6A-dependent expression of key metabolic enzymes involved in BCAA metabolism. The METTL16/m6A/BCAT1/BCAT2/BCAA axis contributes to leukemogenesis and the maintenance of LSCs/LICs by reprogramming metabolic pathways crucial for leukemia cell survival and proliferation.
Importantly, METTL16 appears to be dispensable for normal hematopoiesis, suggesting that targeting METTL16 could selectively affect leukemia cells while sparing normal cells. This highlights the potential of METTL16 as a therapeutic target in AML.
Technical Highlights
Multi-Omics Integration:
Combined metabolomics and m6A sequencing analyses revealed a regulatory axis linking m6A modifications to metabolism, specifically in the context of AML.
Clinical Relevance:
Patients with high METTL16 expression exhibited a 50% increase in chemotherapy resistance rates, indicating that METTL16 levels could serve as a prognostic biomarker and influence treatment strategies.
Conclusion
The findings highlight METTL16 as a crucial regulator of AML progression and maintenance through m6A-dependent metabolic reprogramming. By stabilizing the transcripts of key enzymes in BCAA metabolism, METTL16 promotes the survival and self-renewal of LSCs/LICs. Developing small-molecule inhibitors targeting METTL16 holds significant therapeutic potential and could improve outcomes for patients with AML.
Case Study 6
Decoding the m6A RNA Methylome Identifies PRMT6-Regulated Lipid Transport Promoting AML Stem Cell Maintenance
Journal: Cell Stem Cell (Impact Factor: 21.2)
Publication Date: 2022
DOI: 10.1016/j.stem.2022.12.003
Background
Acute myeloid leukemia (AML) is a malignant hematopoietic disorder characterized by the blockade of myeloid progenitor differentiation and uncontrolled proliferation of immature myeloid cells. The accumulation of genetic and epigenetic alterations transforms normal hematopoietic stem/progenitor cells (HSPCs) into leukemia stem cells (LSCs), initiating AML development and progression. Despite advancements in combined chemotherapy regimens, AML remains a significant clinical challenge, with poor prognosis and a five-year overall survival rate of less than 30%. Therefore, it is imperative to explore the underlying mechanisms of AML pathogenesis, identify novel therapeutic targets, and develop innovative treatment strategies.
N6-methyladenosine (m6A) modification of RNA is a critical epitranscriptomic regulator involved in various biological processes, including stem cell maintenance and differentiation. Decoding the dynamic changes of m6A modifications during AML development may reveal key regulatory mechanisms and potential therapeutic targets.
Objective
To decode the m6A RNA methylome during AML development, identify key molecules such as PRMT6 that regulate AML progression and LSC function, and elucidate the underlying mechanisms, thereby providing potential therapeutic strategies for clinical treatment of AML.
Methodology
1. Decoding the m6A Methylome in Leukemia-Initiating Cells
MeRIP-seq:
Performed MeRIP-seq on leukemia-initiating cells (LICs) to profile the m6A methylome.
Identified 3,587 high-confidence m6A modification sites.
Comparative Analysis:
Compared the m6A methylome of LICs with that of normal HSPCs.
Integrated m6A modifications with changes in gene expression levels.
2. Functional Analysis of m6A Reader Proteins
RNA Immunoprecipitation Sequencing (RIP-seq):
Analyzed the targets of different m6A reader proteins in LICs.
Focus on IGF2BP2:
Identified insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) due to its high expression in AML patient samples and association with poor prognosis.
Investigated the role of IGF2BP2 in regulating the stability of m6A-modified mRNAs.
3. Identification of PRMT6 as a Key Molecular Target
Expression Analysis:
Determined that PRMT6 (protein arginine methyltransferase 6) is highly expressed in AML samples.
Functional Studies:
Assessed the impact of PRMT6 on LSC maintenance and AML progression.
Utilized loss-of-function and gain-of-function approaches to elucidate PRMT6’s role.
4. Mechanistic Studies on PRMT6-Mediated Regulation
Chromatin Immunoprecipitation Sequencing (ChIP-seq):
Conducted H3R2me2a ChIP-seq to identify genomic regions modified by PRMT6.
Integration with RNA-seq Data:
Identified downstream target genes regulated by PRMT6, focusing on lipid transport molecules like MFSD2A.
5. Therapeutic Evaluation of PRMT6 Inhibition
Pharmacological Intervention:
Tested the efficacy of the PRMT6 inhibitor EPZ020411 in inhibiting LSC function and AML progression.
Functional Assays:
Assessed cell viability, apoptosis, and LSC maintenance upon PRMT6 inhibition.
Results
1. m6A Modifications Target Lipid Metabolism-Related Genes in LSCs
Enrichment in Stem Cell Self-Renewal Genes:
m6A modifications in LICs positively regulated the expression of genes associated with stem cell self-renewal.
Implication in LSC Stemness:
The data suggest that m6A modifications contribute to the acquisition and maintenance of stemness in LICs during AML development.
2. IGF2BP2 Regulates PRMT6 mRNA Stability via m6A Recognition
IGF2BP2 Expression Correlates with Poor Prognosis:
High expression levels of IGF2BP2 in AML patient samples were associated with unfavorable outcomes.
Functional Role of IGF2BP2:
Knockdown of IGF2BP2 induced apoptosis, impaired LSC function, and inhibited AML progression.
Stabilization of PRMT6 mRNA:
IGF2BP2 recognizes m6A-modified PRMT6 mRNA, enhancing its stability and expression.
3. PRMT6 Maintains LSC Function and Promotes AML Progression
Elevated PRMT6 Expression in AML:
PRMT6 was overexpressed in AML samples compared to normal controls.
Essential for LSC Maintenance:
PRMT6 is critical for maintaining the self-renewal capacity of LSCs.
Inhibition of PRMT6 Suppresses AML:
Genetic deletion or pharmacological inhibition of PRMT6 impaired LSC function and delayed AML progression.
4. PRMT6 Suppresses MFSD2A Expression by Catalyzing H3R2me2a
Identification of MFSD2A as a Key Downstream Target:
ChIP-seq and RNA-seq analyses identified MFSD2A (major facilitator superfamily domain-containing protein 2A) as a direct target of PRMT6.
Epigenetic Regulation:
PRMT6 catalyzes asymmetric dimethylation of histone H3 at arginine 2 (H3R2me2a) at the MFSD2A promoter, leading to transcriptional repression.
Impact on Lipid Transport:
Suppression of MFSD2A reduces the uptake of fatty acids such as docosahexaenoic acid (DHA), affecting lipid metabolism.
5. Dysregulated Lipid Metabolism Contributes to LSC Stemness
Dependence on Fatty Acid Oxidation:
LSCs rely on fatty acid oxidation due to impaired lipid transport caused by decreased MFSD2A expression.
Restoration of MFSD2A Function:
Overexpression of MFSD2A or supplementation with DHA attenuated LSC stemness and inhibited AML progression.
Therapeutic Potential of PRMT6 Inhibition:
The PRMT6 inhibitor EPZ020411 effectively impaired LSC function and suppressed AML development in preclinical models.
Conclusion
This study decoded the m6A RNA methylome during AML development and identified the PRMT6-MFSD2A signaling axis as a critical regulator of LSC maintenance. IGF2BP2-mediated stabilization of PRMT6 mRNA enhances PRMT6 expression, which in turn represses MFSD2A through H3R2me2a-mediated epigenetic modification. This repression disrupts lipid transport, leading to metabolic reprogramming that promotes LSC stemness and AML progression. Targeting the PRMT6-lipid metabolism axis, particularly with the PRMT6 inhibitor EPZ020411, offers a promising therapeutic strategy for AML treatment.
Technical Highlights
Epigenetic Crosstalk:
Demonstrated the interplay between histone modifications (H3R2me2a) and RNA methylation (m6A) in gene regulation.
Revealed how epigenetic modifications influence metabolic pathways critical for LSC maintenance.
Novel Therapeutic Strategy:
Proposed targeting the PRMT6-lipid metabolism axis as an innovative approach for AML therapy.
Validated the efficacy of the PRMT6 inhibitor EPZ020411 in suppressing LSC function and AML progression.
Technological Trends and Future Directions
Single-Cell m6A Analysis
Integrating single-cell sequencing technologies, such as single-cell m6A sequencing (scm6A-seq), enables the analysis of hematopoietic stem cell (HSC) heterogeneity. This approach elucidates the spatiotemporal specificity of m6A modifications in cell fate determination, providing insights into the dynamics of gene regulation at the single-cell level.
AI-Driven Target Prediction
Utilizing databases such as RMBase and m6A2Target facilitates the screening of m6A-related targets in hematological malignancies. By combining this information with machine learning algorithms, it becomes possible to predict therapeutic responses, enhance target validation, and accelerate drug discovery processes.
Epitranscriptomic and Metabolomic Crosstalk
Investigating m6A-regulated metabolic enzymes, including branched-chain amino acid transaminase 1 (BCAT1) and isocitrate dehydrogenase 1 (IDH1), sheds light on their roles in leukemic metabolic reprogramming. This research avenue aims to develop dual-pathway therapeutics that target both metabolic pathways and m6A modifications, offering a novel strategy for leukemia treatment.
Future Research Directions
Dynamic Modification Profiling
Employ single-cell m6A sequencing techniques to track the dynamics of m6A modifications during hematopoietic differentiation and leukemic transformation. This approach will enhance the understanding of how m6A modifications contribute to cell fate decisions and disease progression.
Development of Targeted Therapies
Design specific inhibitors targeting key enzymes involved in m6A modification, such as methyltransferase-like 3 (METTL3), fat mass and obesity-associated protein (FTO), and protein arginine methyltransferase 6 (PRMT6). Developing these inhibitors holds potential for modulating aberrant m6A methylation in leukemia.
Regulation of the Immune Microenvironment
Explore the impact of m6A modifications on the functions of tumor-associated macrophages (TAMs) and T cells. Understanding how m6A influences immune cell behavior may reveal new strategies for modulating the immune microenvironment in hematological malignancies.
Clinical Translational Validation
Advance small-molecule inhibitors, such as STM2457 and EPZ020411, into clinical trials to evaluate their therapeutic efficacy and safety profiles. Clinical validation of these inhibitors will be crucial for translating epigenetic research findings into effective treatments for leukemia.
