The Potential of N6‑Methyladenine‑Targeted Editing Tools in Cancer

Treatment

Xinfei Wu

Nanning No.36 Middle school, Nanning, China

Keywords: N6‑methyladenosine (m6A), CRISPR/Cas9, Cancer Therapy.

Abstract: With the continuous development of bioengineering technology, the study of m6A has become one of the

core fields of epitranscription, but the development of m6A editing tools is still the focus of today's research.

Increased methylation levels of m6A are highly susceptible to the development of cancer or tumors. Therefore,

this review aims to discuss the development of m6A-targeting tools in cancer therapy, and summarize the role

of m6A in the delivery system, gene editing modification in oncolytic viruses, and the markers of m6A in

breast and lung cancer, as well as its targeted editing tools. Based on the development of targeted tools and

the analysis of the defects of m6A in cancer to the prospect of the future, the new direction and vitality brought

by m6A research in cancer treatment are illustrated.

1 INTRODUCTION

RNA methylation modification is an important

example in epigenetics and an important link in the

regulation of gene expression, the most common

and abundant mRNA modification is N6-

methyladenine (m6A). m6A plays a key role in

stability, translation, splicing, transport and

localization (Lo et al. 2022). The regulation of RNA

metabolism is used to regulate the modification of

m6A. Three proteins are writing proteins, read

proteins and erasure proteins. The role of writing

proteins is reflected in promoting methylation,

including METTL3 and METTL5. Demethylases

are erasing proteins, including FTO and ALKBH5,

and methylated reader proteins in m6A include

IGF2BP1/2/3, YTHDF1/2/3, and ELAVL (Li et al.

2017). In recent years, many studies have

confirmed that the three m6A proteins mentioned

above are often not normally expressed in cancer,

and new therapeutic strategies can be developed by

regulating the m6A modification levels of specific

genes in cancer treatment research. For example,

the proliferation and metastasis of tumor cells can

be inhibited by m6A modifications targeting tumor-

associated genes. With the development of

bioengineering technology, m6A targeted editing

tools have been optimized from RNA editing

technology in CRISPR/Cas system to artificial

editing enzymes, which provides new possibilities

for regulating m6A modification, but there are still

challenges in delivery efficiency and other aspects,

and it is difficult to be truly applied in clinical

practice (Lo et al. 2022).

According to the 2022 data from the

International Agency for Research on Cancer

(IARC), lung cancer was the most diagnosed cancer

this year, with a high rate of 12.8%, and lung cancer

is one of the important causes of cancer death, with

1.8 million cases, accounting for 18.7% of cancer

deaths. Small cell lung cancer (SCLC) and non-

microcellular lung cancer (NSCLC) divide lung

cancer into two broad categories, and lung cancer is

often not detected until an advanced stage, making

it more difficult to treat. One of the prognostic

factors for lung squamous cell carcinoma (LUSC)

is M6A demethylase FTO, and a large number of

studies have found that METTL3 and FTO play a

role in lung cancer (Liu et al. 2018). Breast cancer

is the most common cancer in women, accounting

for 11.8%, becoming the second most incidenceous

cancer. In breast cancer, the m6A programming

protein plays a role by activating YAP/TAZ in the

Hippo pathway. In this review, the basic

mechanisms and regulatory principles of m6A

modification are first summarized. The current state

of development of m6A genetic engineering tools

and their potential applications in cancer treatment

are then discussed. Finally, the challenges and

Wu, X.

The Potential of N6-Methyladenine-Targeted Editing Tools in Cancer Treatment.

DOI: 10.5220/0014493300004933

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 357-361

ISBN: 978-989-758-789-4

357

future prospects of m6A-targeted editing in clinical

practice are analyzed.

2 MOLECULAR MECHANISMS

AND DYNAMIC REGULATION

OF m6A MODIFICATION

N6-methyladenosine (m6A) is an epitransc riptome

modification that is ubiquitous in eukaryotic RNA

molecules and is formed by the addition of a methyl

group (-CH₃) at the nitrogen 6 position of adenosine

(A). It is one of the most common and abundant types

of RNA modifications. m6A is dynamically

reversible and is carried out by three proteins, which

are writers, erasers and readers, and their functions

have different roles. Writers are catalyzed by

methyltransferase complexes, among which

METTL3, METTL14, and so on are more

representative, the function of METTL3 is to catalyze

the modification of m6A, and the METTL14 is to

assist METTL3 in recognizing subtraction, and

different regulator types make the modification of

m6A more diverse. The role of erasers is the opposite

of that of writers, and the more common one is FTO,

which is used to remove m6A modifications and is

now mostly being developed as a drug inhibitor. The

m6A reader protein can recognize and bind m6A

modifications that regulate gene expression by

regulating a variety of processes, and different reader

proteins will present different functions depending on

the environment, such as HNRNPC-mediated mRNA

splicing, YTHDF1 to improve translation efficiency,

and IGF2BPI1/2/3 to enhance its mRNA stability.

m6A methylation affects all aspects of the mRNA

process. For m6A modification, it precisely affects

the growth, development, and other biological

functions of an individual. m6A may inhibit the

cleavage of target mRNA by splicing different

regulators, such as the deposition of TARBP2, which

improves intron retention and promotes tumor growth

(Fish et al. 2019). The more commonly known

METTL16 is to induce MAT2A intron splicing to

drive efficient splicing. The erasure and addition of

m6A methylation require additional attention to

environmental factors, which are environmentally

dependent, and in the development of human

hepatocytoma, the continuous expansion of the tumor

leads to insufficient blood supply, resulting in a

hypoxic environment. Under this condition, HIF-1α

upregulates the expression of YTHDF1 and activates

the transcription of YTHDF1, and the overall level of

m6A is upregulated (Li et al. 2021).

3 DEVELOPMENT AND

APPLICATION OF TARGETED

RNA EDITING TOOLS

Through targeted editing, m6A modification has the

ability to develop and intervene in the progression of

cancer, neurological diseases and metabolic diseases.

In the targeted modification of DNA, CRISPR/Cas9

is an extremely popular gene editing tool. In recent

years, researchers have begun to explore the

combination of CRISPR/Cas9 technology with m6A

modification to achieve more precise gene expression

regulation and disease treatment. It can complement

the required sequence through the designed single

guide RNA, allowing the enzyme associated with the

nucleic acid to enter the target cell together. Using

CRISPR/Cas9 technology to knock out METTL3,

reduce global m6A levels, inhibit tumor growth, and

achieve precise targeted regulation of m6A. CRISPR

is also one of the programmable methylation tools.

CRISPR-Cas9 is coupled with single-stranded

methyltransferase and ALKBH5/FTO to form m6A

writers and erasers, adjust the highly specific

installation of m6A on the transcript, and program the

single guide RNA. The splicing and translation

efficiency of RNA have been changed, but there is

still a more important problem that CRISPR/Cas9 can

produce off-target modifications to non-target RNA,

which needs to be solved by more researchers.

Through genetic engineering, the delivery

efficiency of m6A can be improved. It has been

demonstrated that METTL14 reduces osteoclast

bone resorption by regulating the methylation

functional site of NFATc1 upstream. In addition, in

osteoclasts, EphA2 overexpression on exosomes is

targeted delivery of METTL14 into osteoclasts,

increasing the methylation level of m6A and

inhibiting the development of osteoclasts. Exosome

delivery not only improves targeting but also

improves biocompatibility (Yang et al. 2023)).

Singleguide RNA targeting the methyltransferase

domain can reduce the rate of m6A methylation and

accelerate the apoptosis of cancer cells. For

example, ZCCHC4 was knocked out in HuCCT1

and RBE cell lines by targeting single-guide RNA,

which led to the depletion of ZCCHC4, reduced the

methylation rate of m6A, and promoted the

apoptosis of ICC cells (Chen et al. 2025). Oncolytic

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virus therapy is currently a hot topic in cancer

treatment. Oncolytic viruses can be modified by

m6A modification to improve their anti-tumor

effects. Infection with herpes simplex virus 1

(HSV-1) has the function of evading innate

immunity, and the overall level of m6A may be

reduced during early infection. By genetically

engineering oncolytic viruses (OVs), using shRNA

to knock down METTL14 to reduce the expression

of m6A, the anti-tumor activity of oHSV-1 was

enhanced, and the effect of oncolytic viruses in

treatment was better exerted (Chen et al. 2024).

4 ENGINEERING-BASED

INTERVENTIONS FOR

CANCER THERAPY

In breast cancer, METTL3 is one of the m6A writer

proteins. Experiments have shown that the mRNA,

protein, and overall m6A-methylated RNA levels of

METTL3 are lower than those in normal tissues and

MCF-10A cell lines, respectively, when monitored in

real time by CPR. Upon knocking out METTL3 in

MCF-7 and T47 cells, its deletion was found to exert

an inhibitory effect on breast tumor cells. Adriamycin

resistance is a significant challenge in the clinical

treatment of breast cancer, as its emergence often

indicates treatment failure. Elevated expression of

miR-221 in the blood of breast cancer patients serves

as a biomarker for predicting chemotherapy

resistance. Meanwhile, METTL3 reduces the m6A-

induced mRNA methylation expression mediated by

miR-221, thereby improving Adriamycin resistance

in breast cancer treatment. FTO dynamically

regulates m6A modification to influence gene

expression and holds great potential in cancer cells

through genetic engineering editing. FTO inhibitors

can be combined with immune checkpoint inhibitors.

In their study, Su et al. reported that FTO inhibitors

exhibit an inhibitory effect on breast cancer stem

cells, which may enhance the anti-tumor immune

response in triple-negative breast cancer (Su et al.

2020). However, m6A primarily affects cancer

progression. METTL3 is often overexpressed in

breast cancer, and inhibition of m6A modification of

LATS1 expression can lead to tumor development.

Additionally, the upregulation of METTL3 and m6A

modification inhibits tumor immune surveillance.

The aforementioned resistance is also attributed to the

reversible presence of m6A modification in cancer,

which contributes to drug resistance. This is not only

seen in Adriamycin resistance but also in the

upregulated m6A methylation of GPRC5A, which

leads to docetaxel resistance and increases the

tendency of cancer cells to metastasize to the liver

(Ou et al. 2024). All these factors may contribute to a

poor prognosis for breast cancer.

In lung cancer, the primary treatment modalities

are chemotherapy and surgery. The p53 tumor

suppressor gene, a transcription factor, regulates a

variety of key cellular functions. Specifically,

mRNA with m6A heterozygosity carrying the TP53

R273H mutant protein expression can modulate

RNA methylation function, re-enhance the efficacy

of anticancer drugs, and improve the feasibility of

cancer treatment. Similarly, the knockout of

METTL3, as mentioned earlier, also exerts an

inhibitory effect on lung cancer progression. M6A

plays a more significant role in cancer prognosis.

For instance, the YTH domain family, present in

m6A reader proteins, is predominantly involved in

m6A methylation expression during tumorigenesis.

In the treatment of small cell lung cancer (SCLC),

high expression of YTHDF1 is associated with

better prognosis and reduced resistance to

chemotherapeutic drugs (Shi et al. 2019). The

engineering intervention of M6A in cancer

treatment remains to be further explored and

refined, yet it holds great promise for improving

lung cancer prognosis. The cleavage of the miR-

143-3p/VASH1 axis by M6A promotes

angiogenesis in lung cancer, a critical aspect of

tumor progression, and may facilitate the metastasis

of cancer cells to the brain . METTL3 is also highly

likely to induce upregulation of HIF-1α via an

m6A-IGF2BP2-dependent mechanism, thereby

exacerbating ferroptosis, leading to acute lung

injury and increased lung cancer mortality.

Compared with breast cancer, drug resistance is

more prevalent in lung cancer and often leads to

rapid disease progression. SCLC is particularly

prone to developing chemotherapy resistance, and

m6A methylation negatively regulates the target

gene DCP2 of METTL3, inducing chemotherapy

resistance in SCLC (Sun et al. 2023). Thus, the

emergence of chemotherapy resistance due to m6A

methylation has become a major challenge that

needs to be addressed, and it also lays the

groundwork for the development of FTO inhibitors

for lung cancer treatment.

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359

5 CHALLENGES AND FUTURE

DEVELOPMENT DIRECTIONS

In cancer treatment, epigenetic inhibitors have shown

great advantages and good anti-cancer effects in

genetic therapy in recent years, and modification of

m6A is one of the features in cancer development and

often appears in different types of cancer. However,

only a few of the m6A modifiers can be made into

drugs, but none of them have been used in clinical

practice, and the inhibition of FTO activity after m6A

demethylation also leads to limited activity of

inhibitors, which are not suitable for clinical

treatment. The difficulty of delivering m6A's gene-

editing tools into tumor cells also leads to reduced

drug availability. For example, Chen et al.

experimentally improved the oncolytic activity of

oHSV-1 in the treatment of glioma by knocking out

METTL14, but at the same time enhanced the

transmission of HSV-1, and a balance needs to be

found between the two (Chen et al. 2024). The

transformation of inhibitor drugs modified by m6A

methylation into clinical applications still needs to go

through many levels and obstacles and large-scale

mass production needs to take into account the

production cost and regulatory approval. The clinical

trials of new drugs require the participation of a large

number of experimenters, and the protection of the

interests of experimenters and the standardization of

recruiting experimenters also involves many ethical

aspects, so the research and development technology

and clinical application of m6A-related drugs needs

to be further explored and improved.

6 CONCLUSION

In general, m6A, as a research hotspot over the years,

has promoted the development of RNA dynamic

regulation and revealed development. The important

role of metabolism and immunity in disease plays a

role through the systemic regulation of writing,

erasing, and reading. In gene editing technology, it

reduces the methylation rate by modifying specific

sites of RNA. Gene editing m6A improves the

delivery efficiency of its related substances and

reversely regulates the poor prognosis of the disease

caused by the original expression of m6A into a

certain treatment method and future development

prospects. The engineering intervention of m6A has

improved the new research direction in the treatment

of breast cancer and lung cancer, and the methylation

of m6A has been found to lead to the occurrence of

drug resistance and the development of tumor

progression in cancer, which provides a new direction

and progress for the development of its inhibitors and

has also become the focus of cancer prognosis. Based

on the current research and its development trend,

m6A's gene editing tools have certain limitations in

cancer treatment, there are certain risks in the

uncontrollability of dynamic regulation, and the

immaturity of editing tools makes m6A often become

a landmark of adverse reactions in cancer treatment,

and the related inhibitory drugs modified by editing

technology have not been put into clinical treatment

and use normally, and the long-term effects of m6A

editing tools in vivo are not clear. For the

development of cancer, this review can provide a

direction for the diagnosis of diseases by studying

more accurate biomarkers and actively carrying out

experiments in phase I and phase II clinical trials of

m6A targeted therapy to verify its safety. Long-term

experiments in animal models can also be carried out

to evaluate the potential risks of m6A editing tools, so

that the m6A field can achieve its clinical

transformation through technological innovation and

interdisciplinary cooperation in the future, overcome

related shortcomings, and promote the development

of precision medicine.

REFERENCES

Chen,B., Li,L., &Huang,Y., et al. 2025. N6-

methyladenosine in 28S rRNA promotes oncogenic

mRNA translation and tyrosine catabolism. Cell

Reports 44(1):115139.

Chen,Y., Bian,S., &Zhang,J.,et al. 2024. HSV-1-induced

N6-methyladenosine reprogramming via ICP0-

mediated suppression of METTL14 potentiates

oncolytic activity in glioma. Cell Reports

43(10):114756.

Fish, L., Navickas, A., Culbertson, B. 2019. Nuclear

TARBP2 Drives Oncogenic Dysregulation of RNA

Splicing and Decay. Molecular Cell 75(5):967-981.e9.

Li, Q., Ni, Y., Zhang, L. 2021. HIF-1α-induced expression

of m6A reader YTHDF1 drives hypoxia-induced

autophagy and malignancy of hepatocellular carcinoma

by promoting ATG2A and ATG14 translation. Signal

Transduction and Targeted Therapy 6(1):76.

Li, Z., Weng, H., Su, R. 2017.FTO plays an oncogenic role

in acute myeloid leukemia as a N(6)-Methyladenosine

RNA demethylase. Cancer Cell 31:127–141.

Liu, J., Ren, D., Du, Z. 2018. m6A demethylase FTO

facilitates tumor progression in lung squamous cell

carcinoma by regulating MZF1 expression.

BEFS 2025 - International Conference on Biomedical Engineering and Food Science

360

Biochemical and Biophysical Research

Communications 502(4):456–464.

Lo, N., Xu, X., Soares, F. 2022. The Basis and Promise of

Programmable RNA Editing and Modification.

Frontiers in Genetics 13:834413.

Ou, X., Tan,Y., Xie, J. 2024. Methylation of GPRC5A

promotes liver metastasis and docetaxel resistance

through activating mTOR signaling pathway in triple

negative breast cancer. Drug Resistance Updates

73:101063.

Shi, Y., Fan, S., Wu, M. 2019. YTHDF1 links hypoxia

adaptation and non-small cell lung cancer progression.

Nature Communications 10(1):4892.

Su, R., Dong, L., Li, Y. 2020. Targeting FTO Suppresses

Cancer Stem Cell Maintenance and Immune Evasion.

Cancer Cell 38(1):79-96. e11.

Sun, Y., Shen, W., Hu, S. 2023. METTL3 promotes

chemoresistance in small cell lung cancer by inducing

mitophagy. Journal of Experimental & Clinical Cancer

Research 42(1):65.

Yang, JG., Sun, B., Wang, Z. 2023. Exosome-targeted

delivery of METTL14 regulates NFATc1 m6A

methylation levels to correct osteoclast-induced bone

resorption. Cell Death & Disease 14(11):738.

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