YTHDFs as radiotherapy checkpoints in tumor immunity

Radiotherapy (RT) is administered to over 50% of cancer patients and is primarily utilized to achieve localized tumor control (Liauw et al., 2013; Orth et al., 2014; Weichselbaum et al., 2017). In recent years, RT has been increasingly integrated into treatment strategies for metastatic cancers, either in conjunction with immunotherapies or through the application of targeted high-dose RT for oligometastatic conditions (Antonia et al., 2017; Brandmaier and Formenti, 2020; Kelly et al., 2021; Piper et al., 2023). Ionizing radiation (IR) eliminates tumor cells by inducing DNA damage, primarily through the formation of double-strand breaks (DSBs) (Lomax et al., 2013; Maruyama, 1969). Emerging evidence indicates that both innate and adaptive immune responses play a pivotal role in mediating the antitumor efficacy of RT (Burnette et al., 2011; Lee et al., 2009; Liang et al., 2013; Wang et al., 2024b). IR activates T cells via stimulating dendritic cells (DCs) maturation and activation (Weichselbaum et al., 2017). Moreover, IR is reported to increase the peptide repertoire and MHC-I expression as well as the release of immunostimulatory cytokines and chemokines (Dar et al., 2019; Reits et al., 2006). IR also induces immunosuppressive responses via recruiting myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), which inhibit the antitumor effects of RT (Ghosh et al., 2023; Jiménez-Cortegana et al., 2022; Liang et al., 2017; Lin et al., 2021; Muroyama et al., 2017; Schaue et al., 2012). Additionally, IR stimulates production of immunosuppressive cytokines, such as TGF-β and IL-10 (Nishigori et al., 1996; Vanpouille-Box et al., 2015; Wang et al., 2021). Recent studies have demonstrated that IR can influence RNA methylation in both tumor cells and immune cells, thereby affecting its antitumor efficacy (Wang et al., 2024a; Wang et al., 2023b; Wang et al., 2024b; Zhang et al., 2023c; Zhuang et al., 2023). These findings suggest that the RNA methylation status plays a critical role in determining the therapeutic outcomes of RT.

N⁶-methyladenosine (m⁶A) is the most common and abundant endogenous mRNA methylation in eukaryotic cells (Huang et al., 2020; Wang et al., 2014). The regulation of this modification is achieved through the coordinated action of three distinct protein groups. The “writers” (methyltransferase complex), which include METTL3, METTL14, and WTAP, are responsible for adding the m⁶A modification. In contrast, the “erasers” (demethylases), which consist of FTO and ALKBH5, remove this chemical mark. Lastly, the “readers,” a group of proteins including YTHDF1/2/3 and YTHDC1/2, recognize and bind to m⁶A-modified RNA, thereby modulating diverse RNA metabolic processes. The m⁶A reader proteins YTHDFs exhibit distinct canonical functional roles: YTHDF1 primarily boosts the efficiency of mRNA translation, YTHDF2 enhances mRNA degradation, and YTHDF3 exerts dual functions by supporting both translation and degradation of mRNA, with its role varying depending on the specific biological context (Roundtree et al., 2017; Shi et al., 2017; Wang et al., 2014; Wang et al., 2015; Zaccara et al., 2019). Recent studies have revealed that YTHDF proteins can influence the efficacy of RT through mechanisms, such as modulating DNA repair and shaping the tumor immune microenvironment (TIME) (Du et al., 2023; Shao et al., 2023; Shi et al., 2023; Wang et al., 2023a; Wang et al., 2023c; Wen et al., 2024a; Yin et al., 2023). Elucidating the functions and mechanisms of YTHDF proteins within the context of radiation biology holds significant potential for advancing therapeutic strategies in cancer RT.

This review provides an overview of recent progress in elucidating the mechanisms by which YTHDF proteins in tumor and immune cells modulate the therapeutic efficacy of RT. By synthesizing current knowledge on the functions of YTHDF proteins in the context of IR, we emphasize their indispensable role in shaping RT outcomes.

YTHDF2, the first m⁶A reader to be characterized, was identified in 2014 as facilitating the degradation of cytoplasmic mRNA targets through its interaction with the CCR4–NOT complex (Wang et al., 2014). In 2015, YTHDF1 was found to enhance the translational efficiency of m⁶A-modified mRNAs (Wang et al., 2015). Shortly thereafter, YTHDF3 was shown to function as a regulator that integrates and coordinates the activities of both YTHDF1 and YTHDF2 (Shi et al., 2017). An increasing body of evidence suggests that YTHDF proteins play a critical role in various cancer etiology and progression. Overall, YTHDF proteins have been implicated as oncogenic drivers, contributing to the initiation and progression of various cancer types. For example, elevated expression of YTHDF2 has been clinically linked to poor prognosis in glioma patients. Mechanistic studies in preclinical models reveal that YTHDF2 facilitates glioblastoma cell proliferation, invasion, and tumorigenesis by driving the m⁶A-dependent degradation of LXRA and HIVEP2 mRNAs (Fang et al., 2021). In addition, recent research highlights the pivotal roles of YTHDF proteins in modulating RT outcomes. YTHDF2 regulates the degradation of CircAFF2 in an m⁶A-dependent manner. CircAFF2 has been found to enhance the radiosensitivity of colorectal cancer (CRC) cells by interacting with CAND1 and facilitating its association with Cullin1, ultimately suppressing neddylation (covalently attaching an ubiquitin-like protein) in CRC cells (Shao et al., 2023).

RT can be administered externally using external beam through linear accelerators or internally via the implantation of radioactive sources near or within the tumor (brachytherapy). It can also involve the systemic delivery of radioisotopes, either untargeted, such as ¹³¹I, or targeted, like ¹⁷⁷Lu-PSMA. RT primarily exerts its cytotoxic effects by inducing DNA damage in tumor cells, including DSBs, single-strand breaks, base damage, and interstrand cross-links, with DSBs being the most deleterious. This cytotoxic is achieved by focusing IR on the target area, directly damaging cellular DNA and indirectly generating reactive-free radicals through energy absorption by surrounding molecules, which further interact with DNA to cause additional damage and ultimately induce cell death (Azzam et al., 2012; Lomax et al., 2013). However, DNA damage induced by RT in tumor cells often triggers the activation of DNA repair mechanisms, which can reduce the efficacy of RT (McBride et al., 2004; Wu et al., 2023). Factors capable of influencing DNA damage or DNA repair in tumor cells have the potential to impact the effectiveness of IR (Fig. 1). YTHDF1 binds to the m⁶A modification site in the 5′-UTR of PHF10 mRNA and promotes its translation, thereby contributing to the DNA damage repair in pancreatic cancer cells, as PHF10 loss-of-function impairs homologous recombination repair efficiency (Huang et al., 2022). Gene set enrichment analysis using The Cancer Genome Atlas database reveals a correlation between YTHDF1 and DNA damage repair in breast cancer. YTHDF1 promotes DNA damage repair by regulating the stability of E2F8 mRNA in breast cancer cells (Sun et al., 2022). CD133⁺ gastric cancer stem cells enhance PARP1 stability by recruiting YTHDF1 to bind to the 3′-UTR of PARP1 mRNA, thereby promoting PARP1-mediated DNA damage repair capacity (Li et al., 2022). The authors reported an uncharacterized regulatory effect of YTHDF1 on mRNA. It remains unclear whether this effect is indirect or occurs only under specific conditions. Although these findings do not directly demonstrate the impact of YTHDF1 on IR, its role in DNA damage repair suggests that YTHDF1 may influence the effectiveness of IR in pancreatic, breast, and gastric cancer cells.

Indeed, YTHDF proteins in various tumor cells have been shown to bind m⁶A-modified transcripts, regulating their translation and degradation, and ultimately affecting the efficacy of IR (Fig. 1). The genes that are directly bound or indirectly regulated by YTHDF proteins and influence the efficacy of IR to cancer cells are mostly associated with DNA damage repair. YTHDF1 increases AURKA expression, enhancing nonhomologous end joining and promoting radioresistance in esophageal carcinoma cells (Shen et al., 2023, Preprint). YTHDF3 promotes the translation of the DNA repair proteins SETD2 in lung adenocarcinoma cells and RAD51D in cervical cancer cells, contributing to increased radioresistance (Du et al., 2023; Zeng et al., 2022). It should be noted that these studies did not utilize definitive methodologies to establish the m⁶A-dependent binding and regulatory activity of YTHDF1 and 3 on its proposed target transcripts. Therefore, the regulatory effects YTHDF1 and 3 on these targets remain to be further clarified. One study has reported that YTHDF2 enhances the stability and expression of E2F3 mRNA in breast cancer cells, ultimately promoting DNA damage repair and reducing sensitivity to X-ray radiation treatment (Wei et al., 2023). Although recent studies have shown that YTHDF2, along with YTHDF1 and YTHDF3, promotes m⁶A-mRNA degradation (Zaccara and Jaffrey, 2020; Zaccara and Jaffrey, 2024), there have been no reports demonstrating that YTHDF2 enhances the stability of its target transcripts. The direct regulatory effect of YTHDF2 on E2F3 requires further clarification. In addition, one recent interesting study has shown that YTHDF1 facilitates the translation of DDB2 transcripts through METTL14-mediated m⁶A methylation, thereby enhancing global genome repair and mitigating ultraviolet B radiation-induced skin tumorigenesis (Yang et al., 2021). Aside from DNA damage repair, YTHDF proteins have also been reported to influence the radiosensitivity of tumor cells by regulating tumor cell stemness (Wang et al., 2023e; Wei et al., 2023; Yin et al., 2023) and circular RNA (circRNAs) (Shao et al., 2023; Shi et al., 2023).

Programmed death ligand-1 (PD-L1), a critical immune checkpoint molecule, binds to the PD-1 on T cells, inhibiting T cell–mediated cytotoxicity and facilitating tumor cell immune evasion (Vesely et al., 2022). Numerous studies have demonstrated that YTHDF proteins regulate PD-L1 expression in various cancer types, including breast cancer, bladder cancer, CRC, non-small cell lung cancer, prostate cancer, nasopharyngeal carcinoma, hepatocellular carcinoma, hepatocellular carcinoma, and cholangiocarcinoma (Fig. 2) (Chen et al., 2023; Dai et al., 2024; Li et al., 2023; Luo et al., 2024; Qiu et al., 2021; Wang et al., 2024c; Wang et al., 2024d; Wen et al., 2024b; Yu et al., 2024; Zhao et al., 2022; Zheng et al., 2022). The m⁶A-modified PD-L1 mRNA is recognized by YTHDF1, leading to the upregulation of PD-L1 expression in CRC and prostate cancer cells (Li et al., 2023; Wang et al., 2024d). YTHDF2 recognizes the m⁶A modification in the 5′-UTR region of ETV5 mRNA, promoting its translation and subsequently upregulating PD-L1 expression in hepatocellular carcinoma cells (Wen et al., 2024b). The upregulated PD-L1 enables tumor cells to evade T cell cytotoxicity. Conversely, some studies have reported that YTHDF proteins can downregulate PD-L1 expression in tumor cells. ALKBH5 deficiency increases m⁶A modification in the 3′-UTR region of PD-L1 mRNA, promoting its degradation in a YTHDF2-dependent manner in cholangiocarcinoma cells, while tumor-intrinsic ALKBH5 sustains PD-L1 expression, thereby inhibiting T cell expansion and cytotoxicity (Qiu et al., 2021). In breast cancer cells, YTHDF3 recognizes and binds to PDK1 mRNA, promoting its degradation, which results in reduced PD-L1 expression and enhanced cytotoxic T lymphocyte activity (Wang et al., 2024c).

The regulation of T cell cytotoxicity against tumor cells by YTHDF proteins involves the expression of cytokines or chemokines that recruit immunosuppressive cells to the TIME or affect T cell activity (Fig. 2) (Bao et al., 2023; Chen et al., 2024; Chen et al., 2019; Wang et al., 2023d; Yu et al., 2024; Zhang et al., 2023b; Zhang et al., 2024b), reduced tumor cell responsiveness to IFNγ (Fig. 2) (Bai et al., 2022; Jang et al., 2025; Wang et al., 2020; Yang et al., 2019), and impaired T cell–mediated recognition of tumor cells (Fig. 2) (Wu et al., 2024). YTHDF1 promotes p65 translation, leading to the upregulation of CXCL1 in CRC cells. YTHDF1 also binds to m⁶A-modified EZH2 mRNA, enhancing EZH2 translation and subsequently upregulating IL-6 expression in hepatocellular carcinoma cells. Both pathways recruit MDSCs to the TIME, ultimately leading to cytotoxic CD8⁺ T cell dysfunction (Bao et al., 2023; Wang et al., 2023d). On the contrary, in Burkitt lymphoma cells, YTHDF1 enhances TLR9 expression by promoting mRNA translation in an m⁶A-dependent manner, thereby increasing the secretion of cytokines that increase T cell–mediated cytotoxicity (Zhang et al., 2024b), implying that tumor cell–dependent context may shape the downstream effects of YTHDF1 activity. YTHDF2 reduces the mRNA stability of Stat1 and Irf1, key regulators of IFNγ signaling, thereby attenuating the response of CRC cells to IFNγ (Wang et al., 2020). The deletion of YTHDF2 in melanoma cells prolongs the lifespan of MHC-I–related genes, thereby enhancing T cell–mediated recognition and killing (Wu et al., 2024).

The effects of YTHDF proteins in myeloid cells include modulating the cross-priming capacity of DCs, reprogramming tumor-associated macrophages (TAMs), and regulating the differentiation, migration, and functions of MDSCs (Fig. 3). Studies have shown that Ythdf1 in DCs promotes tumor growth in murine cancer models. Transcripts encoding lysosomal cathepsins are marked by m⁶A and recognized by YTHDF1, which enhances their translation in DCs. Lysosomal cathepsins reduces the cross-priming capacity of DCs and CD8⁺ T cell–mediated tumor killing by degrading tumor neoantigens and impairs type I IFN (IFN-I) production through the degradation of the STING protein (Fig. 3 A) (Han et al., 2019; Wen et al., 2024a). YTHDF2 deficiency in TAMs suppresses tumor growth by reprogramming TAMs toward an antitumoral phenotype, enhancing CD8⁺ T cell–mediated antitumor immunity through the modulation of IFN-γ–STAT1 signaling (Fig. 3 B) (Ma et al., 2023). METTL3-mediated m⁶A modification of Jak1 mRNA in TAMs and MDSCs enhances JAK1 protein translation efficiency through the m⁶A–YTHDF1 axis, which subsequently phosphorylates STAT3, ultimately leading to reduced antitumor immunity by promoting protumoral gene expression (Fig. 3 B) (Xiong et al., 2022). In MDSCs, recent findings indicate that YTHDF2 directly binds to and degrades Bambi transcripts, which is a transmembrane glycoprotein with an extracellular ligand-binding domain structurally resembling TGF-βR1 and functions as an inhibitor of TGF-β signaling, thereby promoting TGF-β signaling. In addition, YTHDF2 activates NF-κB by targeting transcripts encoding negative regulators of NF-κB signaling. Together, these effects reduce antitumor immunity by promoting MDSC infiltration and enhancing their suppressive functions in the context of IR (Fig. 3 C) (Wang et al., 2023a; Wang et al., 2023c).

One recent study has shown that YTHDF proteins are essential for maintaining suppressive function of Tregs. YTHDF2 modulates NF-κB signaling by promoting the degradation of m⁶A-modified transcripts encoding NF-κB negative regulators, thereby enhancing the suppressive function of Treg cells (Fig. 3 D) (Zhang et al., 2023a). Although no direct evidence currently indicates that YTHDF proteins in B cells influence their antitumor immune function, a recent study has shown that YTHDF2 is essential for IL-7–induced pro-B cell proliferation by regulating the suppression of specific transcripts (Zheng et al., 2020). Given the significant role of B cells in antitumor immunity, including producing tumor-specific antibodies and modulating the cytotoxicity of T cells and natural killer (NK) cells, the potential effects of YTHDF proteins in B cells on antitumor immunity warrant further investigation. Interestingly, a recently published study has revealed that YTHDF2 is dispensable for normal B cell development but facilitates immune evasion in B cell malignancies by destabilizing CD19 and HLA-DMA/B (Chen et al., 2025).

Although studies highlight YTHDF proteins as tumor immune evasion-promoting factors, some evidence supports their context-dependent immunostimulatory roles. For instance, loss of METTL3 in TAMs disrupts YTHDF1-mediated SPRED2 translation, which activates NF-κB and STAT3 via the ERK pathway, thereby reprogramming macrophages to promote tumor growth and metastasis in B16 melanoma and Lewis lung carcinoma models (Fig. 3 B) (Yin et al., 2021). USP47 deficiency in Tregs facilitates m6A-dependent c-Myc translation via YTHDF1, thereby exacerbating hyperglycolysis and impairing Treg suppressive function (Fig. 3 D) (Wang et al., 2023a). YTHDF2 is essential for enhancing CD8⁺ T cell function and suppressing tumor progression by targeting IKZF1/3 to establish an active chromatin state in MC38 colon carcinoma and B16 melanoma model (Fig. 3 E) (Zhang et al., 2024a). One study has demonstrated that YTHDF2 is essential for NK cell–mediated antitumor immunity in B16 melanoma. YTHDF2 supports IL-15–induced NK cell survival, proliferation, and effector functions by targeting Tardbp mRNA (Fig. 3 F) (Ma et al., 2021). These findings emphasize that YTHDF proteins may also facilitate immune activation, depending on specific cell types, co-regulators (such as m⁶A writers), and pro-inflammatory conditions such as IL-15 stimulation. Therefore, therapeutic targeting of YTHDF proteins in immune cells requires careful context-dependent evaluation, particularly in the setting of different therapeutic strategies, to minimize the risk of immune suppression or unintended impairment of antitumor immune responses.

Beyond its direct effect on cytotoxicity of tumor cells, RT has been shown to significantly influence the TIME (Burnette et al., 2011; Lee et al., 2009; Liang et al., 2013; Wang et al., 2024b). Radiation enhances the maturation and antigen presentation capacity of professional antigen-presenting cells, such as DCs, enabling them to present tumor antigens to T cells and activate their cytotoxic function, a process critical for IR-induced antitumor immunity. For example, IR induces DNA damage in cells, leading to the release of double-stranded DNA into the cytosol and the TIME. In DCs, double-stranded DNA from irradiated tumor cells activates cGAS-STING signaling, resulting in increased IFN-I production, which enhances DC cross-presentation capacity, primes CD8⁺ T cells, and promotes IR-induced antitumor immunity (Deng et al., 2014). However, there are immune suppressive effects induced by IR in DCs that potentially reduce the antitumor effects of IR. For instance, activation of the noncanonical NF-κB pathway by IR in DCs has been reported to negatively regulate STING-mediated IFN-I production, thereby diminishing the antitumor effects of RT (Hou et al., 2018). In addition, IR can also induce immune suppressive effects by inducing the expansion of MDSCs, enhancing suppression of CD8⁺ T cell activity in the TIME (Jiménez-Cortegana et al., 2022). Inhibiting these immunosuppressive effects further amplifies DC function and promotes the antitumor efficacy of IR.

YTHDF proteins have been reported to influence tumor occurrence and progression by modulating the TIME. For instance, YTHDF1 upregulates CXCL1 via p65 in CRC and promotes the EZH2–IL-6 signaling axis in hepatocellular carcinoma, both contributing to MDSC recruitment and subsequent impairing antitumor immunity (Bao et al., 2023; Wang et al., 2023d). Numerous preclinical and clinical studies have demonstrated that MDSCs can inhibit the efficacy of RT. Based on these findings, it can be inferred that YTHDF proteins either act on tumor cells to induce changes in MDSCs or directly regulate MDSCs, ultimately affecting the efficacy of RT. Indeed, the recent study of Wang and colleagues has demonstrated that IR induces YTHDF2 expression in MDSCs, which subsequently impairs the antitumor effects of IR by regulating MDSC migration and suppressive function (Fig. 4 A) (Wang et al., 2023a; Wang et al., 2023c). IR promotes the expansion of MDSCs and upregulates YTHDF2 expression in both murine models and human samples. In response to IR, the loss of Ythdf2 in MDSCs enhances antitumor immunity and increases tumor radiosensitivity by reshaping MDSC differentiation, reducing their infiltration, and suppressing their immunosuppressive activity. Mechanistically, IR-induced YTHDF2 expression relies on NF-κB signaling, while YTHDF2, in turn, activates NF-κB by binding to and degrading transcripts encoding negative regulators of NF-κB, thereby establishing an IR–YTHDF2–NF-κB feedback loop (Wang et al., 2023a). In addition, IR-induced YTHDF2 directly binds to and degrades Bambi transcripts, an antagonist of TGF-β signaling, in an m⁶A-dependent manner in MDSCs. This degradation enhances MDSC migration and suppressive function by activating TGF-β signaling, ultimately leading to radioresistance (Wang et al., 2023c).

Similar to IR-induced immune suppressive mechanisms, such as activation of the noncanonical NF-κB pathway in DCs and MDSC expansion, recent studies suggest that upregulation of YTHDF1 in DCs after IR may represent a compensatory immune evasion mechanism that attenuates the pro-immunogenic effects of RT (Fig. 4 B) (Wen et al., 2024a). To evaluate YTHDF1 expression in patients undergoing RT, Wen and colleagues measured YTHDF1 levels in peripheral blood mononuclear cells from metastatic non-small cell lung cancer patients enrolled in a clinical trial at our institution (Concurrent or sequential ipilimumab, nivolumab, and stereotactic body radiotherapy [COSINR] study, NCT03223155), who received sequential or concurrent stereotactic body RT combined with immune checkpoint blockade therapy. They observed a significant increase in YTHDF1 expression in DCs after RT compared with matched pre-RT samples, while no significant changes were detected in other immune cell populations tested. In a melanoma mouse model, RT similarly elevated YTHDF1 levels in tumor-infiltrating DCs. Greater YTHDF1 expression was associated with poorer survival outcomes. To assess the functional role of YTHDF1, they generated the DC-specific Ythdf1 deletion mice by breeding Cd11c^Cre mice with Ythdf1^fl/fl mice (Ythdf1-cKO mice). RT significantly suppressed tumor growth in Ythdf1-cKO mice compared with controls in both the MC38 colon carcinoma and B16-SIINFEKL(OT-I)-ZsGreen (B16-OZ) melanoma models. Enhanced antitumor effects were linked to improved cross-priming ability of DCs and increased CD8⁺ T cell activation. Mechanistically, IR induced YTHDF1 expression in DCs via the STING/IFN-I/STAT2 axis. Their findings reveal that lysosomal cathepsins are key targets of YTHDF1 in DCs under IR conditions, with STING undergoing cathepsin-mediated degradation upon activation. Ythdf1 deletion in DCs reduces cathepsin levels, stabilizes STING, and enhances IFN-I production and DC function during IR. These findings highlight the IR–YTHDF1–STING–IFN-I feedback loop in DCs as a critical regulator of radiation-induced immunity and suggest targeting YTHDF1 could improve antitumor efficacy. Given that increased antigen presentation by DCs is essential for unleashing the full antitumor effects of IR and the loss of Ythdf1 in DCs inhibits tumor growth by impairing DC-mediated antigen presentation, it is reasonable that the interaction between IR and YTHDF1 is primarily observed in DCs but not in other immune cell types.

Given the significant role of YTHDF proteins in various diseases, some compounds targeting YTHDF are gradually being developed. Salvianolic acid C (SAC) (Zou et al., 2023), tegaserod (Hong et al., 2023), and ebselen (Micaelli et al., 2022) have recently been reported to disrupt YTHDF1 activity. 17 small-molecule ligands have been identified that compete with m⁶A for binding to the m⁶A-reader domain of YTHDF2 (Nai et al., 2022). CCI-38 and DC-Y13–27 have been recognized as effective YTHDF2 inhibitors (Chen et al., 2025; Wang et al., 2023a). The recent study of Wang and colleagues conducted fluorescence polarization-based high-throughput screening using an in-house compound library and identified DC-Y13–27 as a selective YTHDF2 inhibitor (Wang et al., 2023a). The compound demonstrated selective inhibition of YTHDF2 binding to m⁶A-containing RNA with an IC50 of 21.8 ± 1.8 μM, whereas it inhibited YTHDF1–m⁶A interaction with an IC50 of 165.2 ± 7.7 μM. DC-Y13–27 inhibited NF-κB activation in MDSCs, leading to reduced MDSC migration and suppression of their immunosuppressive activity, ultimately enhancing the antitumor effects of IR, similar to the effects observed in YTHDF2 knockdown or knockout cells. Notably, a triple-combination therapy comprising DC-Y13–27, IR, and anti–PD-L1 significantly slowed MC38 tumor growth compared with monotherapy or double-combination therapies.

A selective YTHDF1 inhibitor SAC has recently been identified by using a fluorescence polarization-based high-throughput screening of an in-house small molecule library. The results revealed that SAC inhibited the interaction of YTHDF1 with m⁶A-containing RNA with an IC50 value of 1.4 ± 0.2 µM compared with 29.6 ± 3.7 µM for YTHDF2, as measured by the AlphaScreen assay (Zou et al., 2023). SAC treatment reduced the expression of cathepsins A and B and enhanced IR-induced IFN-I production, a manner similar to the effects observed in Ythdf1-deficient DCs. Wen and colleagues developed a prototype DC vaccine using either Ythdf1-deficient DCs (Ythdf1-KO DC vaccines) or SAC-treated BMDCs (SAC DC vaccines). Similar to the Ythdf1-KO DC vaccines, the SAC DC vaccines significantly enhanced IR-induced antitumor efficacy compared with WT DC vaccines. Interestingly, a triple-combination therapy consisting of Ythdf1-KO DC vaccines, IR, and anti–PD-L1 achieved nearly complete tumor regression in mice (Wen et al., 2024a).

While currently available YTHDF1 and YTHDF2 inhibitors exhibit IC50 values in the micromolar range, these compounds have already demonstrated proof of concept by validating target engagement and functional relevance in preclinical settings. This supports the druggability of YTHDF proteins and offers a solid foundation for further medicinal chemistry efforts. Nevertheless, future development will require enhanced specificity and potency to meet clinical standards. In addition, targeting a single YTHDF protein may not be sufficient in certain biological contexts. Although current studies predominantly focus on inhibiting YTHDF1 or YTHDF2, further investigation is needed to determine whether pan-YTHDF inhibitors or combinatorial approaches could yield superior therapeutic outcomes. These possibilities warrant systematic exploration across diverse tumor types and immune environments.

Targeting m⁶A readers, such as YTHDF proteins, may provide greater specificity and controllability in cancer therapy, as they directly determine the fate of m⁶A-modified mRNAs. Recent discovery of IR-induction of YTHDF1 and YTHDF2 expression in immune cells suggest that they are targetable checkpoint for RT. In this review, we summarize the role of YTHDF proteins in regulating DNA damage repair pathways in tumor cells, thereby influencing their radiosensitivity. Additionally, we explore how YTHDF proteins impact the recruitment and function of immune cells within the TIME, highlighting their distinct roles in RT. These findings suggest that YTHDF proteins play a dual role in regulating both tumor-intrinsic and immune-mediated responses to RT, presenting opportunities to enhance RT efficacy through targeted modulation of YTHDF proteins. The context-dependent role of YTHDF proteins need to be elucidated in relation to their effects on DNA repair and the immune system, which is an area of future investigation.

The clinical application of immune checkpoint inhibitors targeting PD-1/PD-L1 and CTLA4 is now prevalent. However, some clinical trials combining immune checkpoint inhibitors with single-site RT have not demonstrated improved outcomes (Lim et al., 2022; Mahmood et al., 2021; Masini et al., 2022; McBride et al., 2021; Tao et al., 2023; Theelen et al., 2019; Welsh et al., 2020). Current preclinical studies have demonstrated that a triple-combination therapy involving the YTHDF1 or YTHDF2 inhibitor, IR, and anti-PD-L1 leads to nearly complete tumor regression in mice. Therefore, the development of effective inhibitors targeting YTHDF proteins presents a promising avenue and a focus of intensive investigation for augmenting the clinical effectiveness of the RT and its synergistic application with immunotherapy.

Although the majority of studies have demonstrated that YTHDF proteins are implicated as oncogenic drivers, their functions have been shown to be highly context dependent (Zou and He, 2024). Posttranslational modifications, such as O-GlcNAcylation and phosphorylation—both of which can be influenced by ROS—can modulate the functional activity of YTHDF proteins (Chen et al., 2018; Lennicke and Cochemé, 2021; Zou and He, 2024). Moreover, studies have shown that under oxidative stress, YTHDF protein–bound mRNAs become enriched in stress granules (Fu and Zhuang, 2020). These findings suggest that IR, through the generation of ROS, may alter the posttranslational modification landscape and subcellular localization of YTHDF proteins, thereby impacting their regulatory functions. These mechanisms warrant further investigation. Current findings in the RT context are largely confined to the interaction of YTHDF proteins with m⁶A-modified mRNA. Notably, YTHDF proteins can also bind to other types of RNA, such as noncoding RNAs, and act on RNA modification sites beyond m⁶A, such as m⁵C (Chen et al., 2025; Liu et al., 2023). Exploring alternative targets of YTHDF may open new avenue in enhancing RT and immunotherapy.

This work was funded by National Institutes of Health R01 grants R01CA262508 to R.R. Weichselbaum and RM1HG008935 to C. He, the Chicago Tumor Institute, and an endowment from the Ludwig Cancer Research Foundation to R.R. Weichselbaum and C. He. C. He is an investigator of the Howard Hughes Medical Institute.

Author contributions: C. Wen: visualization and writing—original draft, review, and editing. E.Z. Naccasha: writing—original draft. C. He: conceptualization and writing—review and editing. H. Liang: conceptualization, investigation, project administration, resources, supervision, validation, and writing—review and editing. R.R. Weichselbaum: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, supervision, visualization, and writing—review and editing.