Research Progress on N6‑Methyladenosine Editing in Cancer

Therapy

Chengxuan Zhang

Shandong Experimental High School, Ji nan, China

Keywords: N6‑methyladenosine, Cancer Therapy, Epigenetic Editing.

Abstract: One of the most prevalent internal alterations on mRNAs in eukaryotes is N6-methyladenosine (m6A). By

altering several facets of RNA metabolism, such as translation, splicing, nuclear export, stability, degradation,

and microRNA processing, m6A alteration governs and modulates gene expression. Several proteins known

as "writers," "erasers," and "readers" control m6A alterations. METTL3, METTL14, WTAP, RBM15/15B,

VIRMA, and ZC3H13 are among the writers; FTO, ALKBH5, and ALKBH3 are among the erasers; and

YTHDF1/2/3, YTHDC1/2, IGF2BP1/2/3, HNRNP, and eIF3 are among the readers. Cancer is a significant

socioeconomic and public health concern of the twenty-first century, accounting for around 22.8% of non-

communicable disease-related deaths worldwide. Nonetheless, the studies demonstrate that new cancer

treatments may be developed by adjusting the amounts of m6A alterations in particular genes. In order to help

researchers better comprehend the development, this review aims to provide an overview of the research on

m6A editing in cancer therapy.

1 INTRODUCTION

Over the past century, the severity of non-

communicable diseases (NCDs) has gradually

increased due to population growth and aging.

Among these diseases, cancer is responsible for

nearly 22.8% of global deaths from non-

communicable diseases, making it a major social

and public health issue of the twenty-first century

(Li et al. 2024). Recent findings, however, suggest

that altering the amounts of N6-methyladenosine

(m6A) alterations in particular genes may result in

the creation of novel cancer treatments in

therapeutic trials aimed at illnesses like cancer.

Translation regulation, mRNA degradation, nuclear

export, pre-mRNA splicing, 3' end processing, and

non-coding RNA (ncRNA) processing are all facets

of eukaryotic RNA metabolism that m6A is

involved in (Wang et al. 2017, Li et al. 2024). For

example, m6A modifications targeting tumor-

related genes have the ability to stop tumor cells

from replicating and spreading.

m6A regulates key features of cancer cells, and

its reversible RNA methylation affects

transcription, splicing, mRNA stability, and

translation rates (Wang et al. 2017). As a result,

both transcriptional and post-transcriptional

regulatory mechanisms related to m6A coexist

across a wide range of malignancies, impacting key

genes. The majority of species, including bacteria

and humans, exhibit the evolutionarily conserved

RNA alteration known as methylation of RNA

m6A, which is the addition of a methyl group to the

N6 position of adenosine. The post-transcriptional

modification process known as m6A RNA

modification is dynamic and reversible, and this

property makes it a key role in rapid cell

communication. Selective polyadenylation,

translation efficiency, nuclear output, mRNA

stability and splicing, and RNA metabolism are all

significantly influenced by it. Abnormal m6A

modification can lead to cell death and uncontrolled

cell proliferation, leading to the occurrence and

development of tumors (Wang et al. 2017, Li et al.

2024). These discoveries have not only made it

possible to utilize m6A-targeted therapies but have

also led to a significant increase in the success rate

of m6A-targeted therapies for human cancers.

Given the critical role of m6A in cancer,

summarizing and presenting the latest research

findings on its role in cancer is essential. This can

provide researchers with valuable insights into the

Zhang, C.

Research Progress on N6-Methyladenosine Editing in Cancer Therapy.

DOI: 10.5220/0014465400004933

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 1st International Conference on Biomedical Engineering and Food Science (BEFS 2025), pages 229-233

ISBN: 978-989-758-789-4

229

latest progress in m6A-related cancer research (Pu

et al. 2023).

2 CRISPR/CAS9 GENE-EDITING

TECHNOLOGY

The gene-editing technique CRISPR/Cas9 is an

effective method for locating genes that are linked to

chemoresistance, invasion, migration, and cell

proliferation (Yang et al. 2019, Cai et al. 2020, Kang

et al. 2021). The Mature CRISPR RNA (crRNA), as

well as complementary trans-activating crRNA

(tracrRNA), which are both capable of forming stable

double-RNA complex structures, are components of

the CRISPR/Cas9 system. These structures guide the

CRISPR-associated protein Cas9 to precisely target

and split the particular DNA sequences (Cong et al.

2013). When single-guide RNA (sgRNA) is

complementary to the particular DNA and contains

PAM, which is called appropriate protospacer

adjacent motif, the Cas9 protein cleaves the double-

stranded DNA at that location (Potts et al. 2020).

Cells have two different ways to fix these breaks:

homologous recombination (HR), a more accurate

process that uses a homologous template for error-

free repair, and non-homologous end joining (NHEJ),

which quickly ligates the broken DNA ends. By

leveraging these mechanisms, CRISPR/Cas9

technology can be used to delete, replace, or insert

specific gene sequences (Chiou et al. 2015).

CRISPR/Cas9 is not limited to modifying one

genomic site, and a group of sgRNAs to

simultaneously will also be employed for modifying

several different genomic sites, a process known as

multiplex editing (Gaj et al. 2013, Cai et al. 2020,

Potts et al. 2020, Kang et al. 2021). Initially,

CRISPR/Cas9 technology relied on the combination

of Cas9 ribonuclease and sgRNAs to precisely

eliminate specific genes. In 2013, researchers

successfully developed a Cas9 mutant, dCas9, which

lacks nuclease activity but retains endonuclease

activity to perform its function (Qi et al. 2021).

3 M6A MODIFICATION

One of the most prevalent internal alterations on

mRNAs in eukaryotes is m6A (Wang et al. 2020). It

regulates mRNA transcription, translation, splicing,

and degradation processes, as well as the overall

mRNA lifecycle. Additionally, m6A modulates RNA

stability and participates in various physiological and

pathophysiological processes (Pu et al. 2023, Jonas &

Frank 2024). The majority of m6A alterations take

place in RRACH sequences, where H stands for

adenine (A), cytosine (C), or uracil (U), and R for

adenine (A) or guanine (G). These alterations are

mostly seen in intronic regions and close to the 3'

untranslated region (UTR) and termination codon of

mRNAs (Wang et al. 2017, Wu et al. 2018). The

frequency of m6A distribution ranges from 0.15% to

0.6%, with approximately 25% to 60% of transcripts

carrying the modification (Pu et al. 2023). The

regulation of m6A modifications involves a diverse

set of proteins classified into three functional groups

including writers, erasers, and readers. The “writers,”

responsible for catalyzing m6A methylation, include

ZC3H13, METTL3, METTL14, WTAP, VIRMA,

and RBM15/15B. The “erasers,” which remove m6A

marks, comprise FTO, ALKBH5, and ALKBH3.

Meanwhile, the “readers,” which recognize and

interpret m6A modifications to mediate downstream

effects, consist of YTHDF1/2/3, YTHDC1/2,

IGF2BP1/2/3, HNRNP, and eIF3 (Wang et al. 2020).

The m6A mutation modifies several facets of

RNA metabolism, such as translation, alternative

splicing, nuclear export, stability, degradation, and

the processing of microRNA in order to govern and

control gene expression. These actions influence

cellular functions and physiological processes (Wu

et al. 2018, Jonas & Frank 2024). The modification

of m6A is dynamic and reversible. Specifically,

'writers' add m6A modifications, 'erasers' remove

them, and 'readers' recognize and act on the

modified RNA (Pu et al. 2023). m6A functions are

typically mediated by m6A-recognizing proteins,

such as those in the YTH family. For example,

YTHDC1 is primarily involved in pre-mRNA

processing, while YTHDF1-3 are mainly associated

with mRNA degradation (Jonas & Frank 2024). The

researchers studied m6A regulation, showing that

m6A regulation has a tight relationship to type 2

diabetes and cancer. Recent research has also found

that m6A modifications can influence tumor

progression, participate in tumor metabolism,

regulate ferroptosis (tumor cell iron death), and

alter the tumor immune microenvironment, thereby

affecting tumor immunotherapy (Pu et al. 2023).

Due to its dynamic regulatory function, m6A also

plays a key role in various disease processes,

including neurodegenerative diseases,

cardiovascular diseases, and metabolic diseases (Pu

et al. 2023). Traditional m6A detection methods

rely on antibodies, which can suffer from cross-

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reactivity issues. In comparison, LC-MS/MS

technology offers a more accurate method for m6A

quantification, allowing the detection of even

extremely subtle changes in m6A levels (Jonas &

Frank 2024). Single-base resolution detection of

m6A is an emerging, antibody-independent

technology that can directly detect m6A

modifications in RNA molecules, enabling single-

base resolution detection of m6A (Jonas & Frank

2024).

4 THE MECHANISMS OF M6A

ACTION

The m6A methyltransferase complex’s essential

elements, METTL3 and METTL14, combine to

create a stable heterodimer. METTL3 functions as the

catalytic subunit, and at the same time, METTL14

primarily is responsible for being an RNA-binding

structure. METTL14 as well as METTL3 will react

with WTAP to regulate the m6A RNA methylation

process (Wang et al. 2017). The m6A 'reader' family

includes YTHDF and hnRNP proteins. Among these,

YTHDF2 was the first identified m6A-binding

protein that mediates m6A-dependent RNA

degradation by targeting RNA to P-bodies (Wang et

al. 2017). YTHDF and hnRNP proteins are members

of the m6A'reader' family. Of them, YTHDF2 was the

first m6A-binding protein to be discovered. It marks

P-bodies for RNA and facilitates m6A-dependent

RNA degradation (Wang et al. 2017). m6A

modifications regulate gene expression through

multiple processes, including mRNA splicing,

transport, stability, and translation. YTH family

proteins function as key 'readers,' with YTHDC1

primarily involved in pre-mRNA processing and

YTHDF1-3 mediating mRNA degradation.

Additionally, IGF2BPs can bind to m6A-modified

mRNAs, enhancing their stability and translation

efficiency (Jonas & Frank 2024). m6A-modified

mRNAs can form condensates through phase

separation, which can further differentiate to rather

stress granules or P-bodies. This process introduces a

new dimension of m6A-dependent post-

transcriptional regulation (Jonas & Frank 2024).

5 THE ASSOCIATION BETWEEN

M6A AND CANCER

(1) The relationship between m6A modification

and cancer: Dysregulation of m6A modification has

a significant relationship with the procedure of

tumorigenesis, the development of tumor, its

metastasis, as well as prognosis in a myriad of cancer

symptoms. The breast cancer, where increased

expression of METTL3 has a positive relationship

with PIN1 expression, can be served as an example.

PIN1 stabilizes METTL3 by preventing its

ubiquitination and degradation, thereby promoting

tumorigenesis. (2) m6A modification and tumor

metabolism: m6A modification influences tumor

cell energy metabolism by regulating processes such

as glycolysis, fatty acid metabolism, and glutamine

metabolism. For example, METTL3 enhances

glycolysis in tumor cells by promoting the expression

of PDK4 and stabilizing GLUT1. (3) m6A

modification and tumor immunotherapy: m6A

modification affects the tumor immune

microenvironment by regulating the expression of

immune checkpoint proteins, such as PD-1/PD-L1.

For example, METTL3 inhibits anti-tumor T-cell

activation by enhancing PD-L1 mRNA stability and

expression in an IGF2BP3-dependent manner (Pu et

al. 2023).

6 THE ROLE OF M6A IN HUMAN

CANCER

(1) Glioblastoma: Tumorigenesis and cell

proliferation in glioblastoma are linked to the

presence of m6A in RNA. METTL3 or METTL14

knockdown increases the expression of certain

oncogenes, including EPHA3, ADAM19, and KLF4.

Glioblastoma stem cells (GSCs) have high levels of

ALKBH5, and GSC growth is inhibited by its

silencing. (2) Acute Myeloid Leukemia (AML): By

controlling the expression of target genes (such as

RARA and ASB2), FTO, a m6A demethylase,

promotes leukemogenesis as well as leukemia

oncogene-mediated cell transformation. ATRA-

induced AML cell differentiation is also inhibited by

it. (3) Lung Adenocarcinoma (LUAD): EGFR and

TAZ are two mRNAs that METTL3 stimulates to

translate in LUAD. Target mRNA translation is

improved by METTL3 through the recruitment of

eIF3 to the translation initiation complex. (4) Breast

Cancer (BRC): Knocking down METTL14 in HCC

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can increase tumor metastasis and lower the amount

of m6A in RNA. Hypoxia-dependent overexpression

of ALKBH5 decreases m6A levels in NANOG

mRNA, which in turn increases the stability of

NANOG mRNA and the amounts of NANOG protein

in breast cancer stem cells (BCSCs).

7 CANCER-SPECIFIC M6A

DYNAMICS

(1) Glioblastoma: METTL3 promotes glioma stem

cell maintenance and radiation resistance by

enhancing SOX2 stability. ALKBH5 promotes

glioma stem cell proliferation by demethylating

FOXM1 mRNA. (2) Cancers of the Female

Reproductive System: In endometrial cancer,

reduced m6A methylation correlates with METTL14

mutations or decreased METTL3 expression, and

AKT pathway activation drives tumorigenesis. In

cervical cancer, FTO promotes chemoresistance and

radiation resistance by demethylating β-catenin

mRNA. (3) Pancreatic Cancer: Expression changes

in FTO and ALKBH5 correlate with pancreatic

cancer invasion and chemotherapy resistance. (4)

Nasopharyngeal Carcinoma: METTL3 promotes

the development of nasopharyngeal carcinoma by

inhibiting ZNF750 and FGF14 expression. (5) Lung

Cancer: Through the enhancement of the MALAT1-

miR-1914-3p-YAP axis and the promotion of YAP

mRNA translation, METTL3 causes treatment

resistance and metastasis in non-small cell lung

cancer (NSCLC). (6) Hepatocellular Carcinoma:

the defective prognosis in hepatocellular carcinoma is

related to high levels of METTL3 and YTHDF1

expression. By controlling SOCS2 m6A alteration,

METTL14 prevents the advancement of

hepatocellular carcinoma. (7) Colorectal Cancer:

METTL3 stimulates the proliferation of colorectal

cancer and its migration via IGF2BP2-dependent

mechanism. METTL14 inhibits the growth and

migration of colorectal cancer cells by regulating the

miR-375/SP1 and miR-375/YAP1 signaling cascade.

(8) Bladder Cancer: METTL3 stimulates the

AFF4/NF-κB/MYC signaling network, which

accelerates the development of bladder cancer. (9)

Prostate Cancer: METTL3 promotes prostate

cancer progression by regulating GLI1 expression.

(10) Breast Cancer: METTL3 promotes breast

cancer progression by enhancing m6A modification

of BCL-2 mRNA. (11) Renal Cancer: Changes in

m6A modification levels are significantly correlated

with the deterioration of clinical parameters in renal

cancer. (12) Gastric Cancer: Decreased m6A

modification levels activate the WNT/PI3K-AKT

signaling pathway, thereby promoting the

development of gastric cancer.

8 PROSPECTS FOR CANCER

THERAPY TARGETING M6A

EDITING

(1) FTO Inhibitors: Rhein and Meclofenamic Acid

(MA) are FTO inhibitors that increase m6A levels in

mRNAs by pairing with the active site of FTO,

thereby inhibiting it from interacting with m6A

substrates. (2) ALKBH5 Inhibitors: Citrate and

IOX3 can inhibit ALKBH5, thereby maintaining the

tumorigenicity of glioblastoma stem cells. (3) SAM-

Dependent Methyltransferase Inhibitors: S-

Adenosylhomocysteine (SAH) in this reaction is the

competitive inhibitor of SAM-dependent

methyltransferases. Additionally, 3-deazoadenosine

(DAA), an inhibitor of SAH hydrolase, prevents the

incorporation of m6A into mRNA substrates (Wang

et al. 2017).

9 THE POTENTIAL OF M6A

MODIFICATION AS A

CANCER THERAPEUTIC

TARGET

(1) FTO Inhibitors: Inhibitors targeting FTO, such

as R-2HG, have been developed to inhibit AML cell

proliferation and promote apoptosis. (2) Melanoma

and FTO Knockdown: In melanoma, knockdown of

FTO increases m6A modification, decreases the

expression of tumor-associated genes, and increases

sensitivity to immune checkpoint inhibitors (Wang et

al. 2020). (3) Changes in m6A in Cancer:

According to recent research, m6A quantity is

significantly reduced in bladder cancer tissues,

possibly due to an imbalance in the composition of

the chemical complex of m6A methyltransferase. (4)

METTL3 Therapeutic Target: METTL3, the main

“writer” of m6A, is investigated as a possible drug

target in various cancers. In acute myeloid leukemia

(AML), STM2457, a small molecule inhibitor of

METTL3, which can decrease tumor phenotype of

leukemia cells, performs functions to increase the life

of mice. Similar researches show that METTL3

inhibitors have therapeutic influences on a variety of

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solid tumors. (5) Dual Roles of METTL3: In many

tumors, METTL3 has a variety of functions,

functioning as a tumor suppressor to stop tumor

growth and as an oncogene to encourage tumor

development. (6) m6A-Dependent Condensates as

Therapeutic Targets: Targeting m6A-dependent

pathophysiological condensates may provide a more

tumor-selective therapeutic strategy. For example,

inhibiting the interaction of m6A-reading proteins

with modified targets through small molecules or

peptides may prevent the formation of oncogenic

condensates or promote their dissociation. (7)

Potential of METTL3 Inhibition in Cancer

Immunotherapy: Recent studies have shown that

METTL3 inhibitors can enhance anti-tumor immune

responses. For example, STM3006 inhibitors

increase the anti-tumor immunity through removing

m6A modification levels, inducing double-stranded

RNA (dsRNA) formation, and activating intracellular

interferon responses (Jonas & Frank 2024).

10 CONCLUSION

In this review, as a new way of therapy, the editing

m6A shows its multiple functions. m6A

modifications regulate gene expression through

multiple processes, including mRNA splicing,

transport, stability, and translation. In the

meantime, CRISPR gene editing technology is a

powerful tool. For identifying genes associated with

cell proliferation, migration, invasion, and

chemoresistance. In 2013, the researchers

successfully developed a Cas9 mutant, dCas9,

which lacks nuclease activity but retains

endonuclease activity to perform its function. This

will greatly promote the subsequent application of

gene editing in various aspects, especially in gene

editing m6A, which will greatly improve its

efficiency and accuracy. m6A modification

influences tumor cell energy metabolism by

regulating processes such as glycolysis, fatty acid

metabolism, and glutamine metabolism. m6A

modification affects the microenvironment of

tumor immune by controlling the gene of immune

checkpoint proteins expressions. According to

current research, m6A is served as a factor which is

responsible for a number of different cancers.

Although it is not clear whether gene editing m6A

is more efficient enough to treat tumors, according

to the current development, gene editing m6A as a

potential regulatory mechanism will play a

significant role in the future.

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