**6. Cancer drugs: resistance and epigenetics**

Alterations in m6A and proteins responsible for its regulation on the RNA level were shown to interfere with the response to cancer treatment. Cyclophosphamide (CTX) is an alkylating chemotherapy drug used in cancer treatment; it forms adducts at the N7-guanine position, which are unstable and therefore undergo spontaneous dissociation. These features are exploited in rapidly dividing cells, which are likely to be disrupted before repair takes place [114]. Little is known about CTX effect on RNA epigenetic complexes, yet it was shown to increase m6A levels, inhibiting the gene and protein expression of FTO, YTHDF1, YTHDF2, YTHDC1, and YTHDF3 in a time- and concentration-dependent manner [115].

Cervical squamous cell carcinoma (CSCC) is an example of increased chemotherapy resistance; Zhou and colleagues [116] found that β-catenin is an FTO target: they observed FTO overexpression and reduced m6A β-catenin levels, with the effect of this change upregulation of β-catenin protein and the subsequent activation of ERCC1, a critical player in NER, which contributes to chemotherapy resistance and a poor prognosis. In case of BRCA-mutated epithelial ovarian cancers (EOC), the use of Olaparib has been clinically beneficial: it is a poly(ADP-ribose) polymerase inhibitor (PARPi), which detects and binds DNA SSBs and DSBs, using the N-terminal DNA binding domain [117, 118]. When DNA is damaged, PARP-1 can recognize damaged sites and their formation; the binding exposes the enzymatic site of PARP-1, resulting in its activation and the recruitment of XRCC1, the first protein for assembly and activation of DNA bases excision repair machinery [119]. The resistance of tumor cells to PARPi is not well-known, but in the study of Fukumoto and colleagues [120], FZD10 was observed as a receptor in Wnt/β-catenin signaling, increasing m6A modification of mRNA in resistant cells, thus stabilizing. This increase contributes to PARPi resistance by upregulating the Wnt/β-catenin pathway in BRCA-deficient EOC cells.

Xiang et al. [107] showed that FTO could be recruited to damaged γH2AX chromatin after irradiation, strengthening FTO participation in DNA damage repair. In melanoma, FTO is upregulated and promotes cell proliferation, cell migration, invasion, and cell viability. Yang et al. [121] demonstrated that FTO regulates PD-1 expression that also promotes mTOR signaling. FTO can promote resistance to anti PD-1 blockade in melanoma through m6A, mediating PD-1 (PD-1 or PDCD1 is a negative regulator of T-cell activity). This supports the anti PD-1 blockade with an anti-tumor response in advanced cancers and reduces immune-related adverse side effects, vs. with ipilimumab.

Glioblastoma is a common and aggressive primary brain tumor in adults, and is highly resistant to treatment such as surgery, irradiation, and adjuvant TMZ chemotherapy, which failed to improve the outcome. One cause of poor response to TMZ, as suggested by Visvanathan et al. [8], is that GSCs show high levels of m6A and METTL3, supporting the proposal that METTL3 is key in GSC maintenance, making those cells resistant to therapy and refractory to radiotherapy by efficient repair of DNA. Here, METTL3 alters the DNA repair efficiency and radiation sensitivity through m6A sites in SOX2–3'UTR, stabilizing it in GSC, as the recruitment of human antigen R (HuR) to m6A modified mRNA is crucial for SOX2 stabilization by METTL3. This supports a role for METTL3, shared with SOX2 in another repair pathway, mediated HR: SOX2 protects GSCs from radiation-induced cytotoxicity

by promoting HR repair, implying an oncogenic role for METTL3 and m6A. At the same time, FTO becomes a promising target to develop FTO inhibitors like Rhein, Meclofenamic acid (MA2), or its ethyl ester form, MA2 [122].

Hepatocarcinogenesis is correlated with abnormal m6A modifications, high METTL3 and YTHDF1 expression in hepatocellular carcinoma (HCC) is associated with poor prognosis, with its combination as a malignant marker, according to Zhou et al. [123]. The participation of the machinery regulating m6A was clarified by the work of Chen et al. [124]. They found a significant increase in mRNA m6A levels, supporting the role of m6A in liver cancers. When METTL3 is downregulated, it is unable to act on tumor suppressor SOCS2, being silenced by METTL3 through m6A-YTHDF2. SOCS2 transcripts are a direct target of YTHDF2 with mediated mRNA decay, promoting tumor progression. Recently, Lin et al. [125] found that METTL3 depletion leads to a resistant phenotype in HCC with sorafenib treatment through regulation of FOXO3 expression. FOXO3 m6A methylation maintains stability, but its absence accelerates degradation. The modification is read by YTHDF1, which stabilizes m6A-labeled RNA and promotes protein synthesis. These results contrast with those obtained by Taketo and colleagues [126] when they established a METTL3-KD using a pancreatic adenocarcinoma cell line. The authors concluded that these cells had higher sensitivity to gemcitabine, 5-fluorouracil, cisplatin, and irradiation. It is clear that METTL3 plays a key role in resistance to therapy, but the way this gene behaves in different types of cancer is not yet understood. In a study on colorectal cancer (CRC), it was found that c-Myc activates the YTHDF1 gene expression. YTHDF1 is overexpressed in CRC and has been associated with lymph node metastasis and poor prognosis, as the evidence in vitro with YTHDF1 knockdown indicates that cancer cells are sensitized to the exposure of 5-Fluorouracil and L-OHP (oxaliplatin) [127].
