The roles and therapeutic implications of messenger RNA internal N⁷‐methylguanosine and N⁶‐methyladenosine modifications in chemoresistance

Resistance to chemotherapy is responsible for the death of most cancer patients. RNA modifications are key players in post-transcriptional gene regulation and their dysregulations contribute to tumor initiation, progression, and resistance to chemotherapy. Despite the identification of over 170 chemical modifications in nearly all types of RNAs, the biological functions and underlying mechanisms of the vast majority of RNA modifications remain elusive in the context of tumorigenesis and drug resistance. N6-methyladenosine (m6A) is the most prevalent internal modification in eukaryotic messenger RNAs (mRNAs). The characterization of proteins that deposit, remove, and recognize mRNA m6A marks, as writers, erasers and readers, respectively, has revealed the profound roles of m6A in determining mRNA fates during both physiological and pathological processes. Besides m6A, another commonly observed positively charged modification is N7-methylguanosine (m7G), which is ubiquitously located at the 5′ cap of mRNAs. This m7G cap is fundamental to mRNA stability, export, and translation. Meanwhile, multiple independent studies have revealed that m7G modification could also be introduced internally onto mRNAs by the METTL1/WDR4 methyltransferase complex.1, 2 In eukaryotic cells, the internal m7G/G ratio in mRNAs ranges from 0.02% to 0.05%, roughly at 5%−10% of the level of mRNA m6A/A ratio. However, unlike m6A, the functions of mRNA internal m7G remain largely unexplored. As the role of internal m7G is mediated by its reader proteins, identification of such readers is pivotal for us to understand the function of internal m7G. Our recent work discovered the Quaking (QKI) protein (including three isoforms, QKI5, QKI6 and QKI7) as the first reader for internal m7G modification.3 Utilizing multiple high-throughput sequencing approaches, we demonstrated that QKI7 regulates the stability and translation efficiency of a subset of internal m7G-modified transcripts under stress conditions, rendering cancer cells more responsive to chemotherapy drugs in vitro and in vivo. Tumours are complex ecosystems that dynamically adapt to various types of external stress stimuli. Evidence is emerging that RNA modifications serve as critical regulatory mechanisms in cancer cells to respond to external stress, including chemotherapeutics, and are critical for tumorigenesis and drug resistance. Consistently, the expression of their regulators (i.e., writers, erasers, and readers) is frequently dysregulated in tumours. Notably, many genes involved in drug resistance are decorated with m6A on their transcripts, including drug-metabolizing enzymes (e.g. CYP2C8), multidrug efflux transporters (e.g., ABCG2, ABCC9 and ABCC10), and DNA damage repair genes (e.g. p53, BRCA1).4 Additionally, a recent transcriptome-wide profiling study of internal m7G revealed a significantly lower internal m7G level on ABC transporter-encoded transcripts (key players in multidrug resistance) in drug-resistant acute myeloid leukaemia (AML) cells than in regular AML cells.5 Therefore, targeting the dysregulated m6A/m7G machinery appears to be a promising strategy to overcome cancer chemoresistance (Figure 1). Stress conditions including chemotherapy drugs induce the formation of stress granules (SGs), the membraneless cytoplasmic ribonucleoprotein particles in cells. The formation of SGs requires certain RNA binding proteins such as G3BP1 to act as a molecular hub to trigger SG assembly.6 Current evidence supports the implication of SGs in chemoresistance. For instance, G3BP1 depletion sensitizes glioblastoma cell lines to Bortezomib treatment by suppressing SG assembly and glutamine deprivation overcomes chemoresistance in pancreatic cancer through inhibition of SG formation. However, the capacity of SG to confer cancer cells chemoresistance and survival advantage relies on the recruitment of a set of target RNAs to influence their expression/function. Until recently, the mechanism behind such recruitment has been mostly unknown. In 2020, Fu et al. reported that YTHDF proteins, as m6A readers, recruit m6A-modified mRNAs into SGs by promoting phase separation.7 Our recent work revealed that, via direct interaction with the SG core protein G3BP1, QKI7 could selectively shuttle internal m7G-modified mRNAs into SGs under stress.3 Importantly, the clinical relevance of this finding was demonstrated in cancer cells treated with doxorubicin, a first-line chemotherapy drug that induces SG assembly. Further in vitro and in vivo models showed that forced expression of QKI7 sensitizes cancer cells to doxorubicin treatment, as associated with suppressed translation of a subset of m7G-modified transcripts, including those in the Hippo signaling pathway (e.g. GSK3B and TEAD1) (Figure 1). Furthermore, clinical data from TCGA database showed a significant positive correlation between QKIs and several SG markers in various types of cancers, suggesting a broad role of QKIs as mRNA internal m7G-binding proteins in regulating cancer cells’ stress response and drug resistance. Given the critical roles of RNA modifications in cancer chemoresistance, researchers have enthusiastically explored the potential of targeting these RNA modification modulators to enhance the efficacy of chemotherapeutics. Some encouraging results have been achieved for small molecules targeting m6A regulators in different cancer types. For example, meclofenamic acid, an FTO inhibitor, restores gefitinib sensitivity in non-small cell lung cancer, and the combination of rhein (another FTO inhibitor) with tyrosine kinase inhibitors (TKIs) may benefit tyrosine kinase inhibitor-resistant cancer patients.8 In terms of m7G regulators, while our work highlights the significance of QKIs as internal m7G readers in drug resistance, several additional studies underscore the importance of m7G writers METTL1/WDR4 in chemoresistance.9 However, no small molecules targeting QKIs or METTL1/WDR4 have been reported so far. Thus, the development of potent therapeutic agents targeting m7G regulators represents a major area of research and potential advancement in cancer treatment. While targeting RNA modifications holds promise as a novel therapeutic strategy to combat chemoresistance, our understanding of the fundamental molecular mechanisms underlying the functions of epitranscriptomic modifications, especially those other than m6A, in drug resistance is still in its infancy. Future efforts to fully elucidate the mechanism through which mRNA modifications (e.g. m7G and m6A) regulate the response of cancer cells to chemotherapy drugs, along with the development of novel potent therapeutic agents guided by these mechanisms and validated through clinical trials, are imperative. These endeavours may offer valuable insights into effectively overcoming drug resistance in clinical settings. The authors are supported in part by The U.S. National Institute of Health (NIH) grants R01 CA271497, R01 CA236399, R01 CA243386 and R01 CA280389 (to Jianjun Chen), The Simms/Mann Family Foundation (to Jianjun Chen), The AASLD Foundation PNC22-261362 (to Rui Su) and Leukemia Research Foundation (to Rui Su). The authors declare no conflict of interest.