With a 5-year survival rate of less than 7% and heterogeneous clinical characteristics, it is a substantial challenge to treat pancreatic adenocarcinoma(Gupta, Amanam et al. 2017). Radiotherapy and chemotherapy have little effect on PAAD treatment and surgery is the main present treatment for PAAD but with poor prognosis (Wang, Zhang et al. 2021). Therefore, new immunotherapeutic strategies are in strong need for PAAD patients. m1A regulators play a vital role in the development of many tumors, which indicates their potential as prognosis biomarkers or even novel targets for the cancerous immunotherapy (Gao, Chen et al. 2021, Wu, Chen et al. 2021). Our study comprehensively investigated the prognostic value and effects on the immune microenvironment of m1A regulators in PAAD.
We firstly found that m1A regulators were significantly correlated to prognosis in PAAD. Then we demonstrated the expression of 10 m1A regulators ALKBH1, ALKBH3, TRMT6, TRMT10A, TRMT10B, TRMT61A, YTHDC1, YTHDF1, YTHDF2, and YTHDF3 were all increased in PAAD. Meanwhile, we confirmed that all these m1A regulators had diverse gene mutations in PAAD, which might explain why their expression was increased in PAAD. Among these 10 m1A regulators, YTHDF1 and YTHDF3 had the greatest degree of variation. Both YTHDF1 and YTHDF3 are m1A binding proteins, which play important roles in RNA folding, protein translation, and RNA metabolism(Liu, Li et al. 2020, Shi, Wang et al. 2017). It was revealed that these two proteins are associated with tumor development. By binding to m6A-modified EIF3C mRNA, YTHDF1 amplifies the translation of EIF3C in an m6A-dependent manner thereby facilitating tumorigenesis and metastasis of ovarian cancer(Liu, Wei et al. 2020). The negative feedback of YTHDF3 and glycolytic lncRNA DICER1-AS1 also participated in the tumorigenesis of PAAD(Hu, Tang et al. 2022). To further explore the correlation between the specific m1A regulators and the prognosis of PAAD, we got Kaplan-Meier plots using OncoLnc database. The results showed that among these 10 m1A regulators, ALKBH1 and TRMT10B were significantly associated with better prognosis in PAAD, indicating they play an important role in PAAD. As a demethylase, ALKBH1 has key functions in reversing DNA methylation and repairing DNA damage, acting as a substantial role in cancer development (Liu, Clark et al. 2016, Liu, Clark et al. 2016, Zhang and Wang 2021). Overexpression of ALKBH1 had the capacity to reverse the suppressive impacts of overexpressed miRNA-339-5p on proliferative and metastatic abilities in gastric carcinoma (Wang, Huang et al. 2020). Additionally, depletion of ALKBH1 in glioblastoma led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility (Xie, Wu et al. 2018). In PAAD, it was reported that ALKBH1 could be involved in the development of PAAD by affecting mTOR and ErbB signaling pathways, and down-regulated ALKBH1 was correlated to the poor prognosis of PAAD patients(Zheng, Yu et al. 2021).TRMT10B is an RNA methyltransferase that has been identified as an m1A writer, and it is involved in modulating RNA stability, translation, splicing, decay, and Subcellular localization. TRMT10B was previously thought to have no impact on humans until it was proposed that TRMT10B had a restricted selectivity never seen in the Trm10 enzyme family (Howell, Jora et al. 2019). Whatever, few reports investigated the relationship between TRMT10B and tumors. We proposed for the first time that TRMT10B was related to the prognosis of PAAD patients, which was conducive to the follow-up research on the correlation between TRMT10B and other tumors.
A growing body of research suggests that immune infiltration in solid tumors is highly correlated with cancer prognosis, although the link is networked rather than absolute(Wang, Zou et al. 2021). In this study, we used R v3.6.3 to assess the relativity between m1A regulators and immune infiltration of PAAD, finding their potential to improve TME status and achieve the survival benefit of prolonging OS and progression-free survival in patients with PAAD.
ALKBH1 and TRMT10B could significantly upregulate infiltration levels of tumor-associated immune cells, particularly CD4+T cells. This appeared to share a common pathway with the immune signature of yes-associated protein 1, a recently discovered PAAD prognostic biomarker (Yang, Zheng et al. 2020). Previous personalized neoantigen targets selected for PAAD vaccines have mostly focused on CD8+T cells but with little success, which may be interpreted by the deficient CD8+ T cells’ infiltration and a predominance of M2 macrophages in the TME of PAAD(Balachandran, Łuksza et al. 2017, Yi, Hong et al. 2022, Balachandran, Beatty et al. 2019, Ren, Cui et al. 2018, Shi and Gao 2022). However, increasing evidence indicates that the selection of MHC-II neoantigen-mediated CD4+ T cells is also critical in killing tumors even distinct from CD8+ T cells. Also, their independent prognostic effect is better than TMB in many solid tumors such as bladder cancer and mammary adenocarcinoma.(Yi, Hong et al. 2022) Therefore, the new biomarkers ALKBH1 and TRMT10B that elevate CD4+T cells can be hopefully more sensitive clinical covariates to patients’ prognosis. CD4 helper T cells can also induce DCs migrating to lymph nodes, enhancing the presentation of tumor antigens (Cohen, Giladi et al. 2022). What’s more, gemcitabine-resistance in PAAD patients was ascertained to be associated with certain miRNA targeting that alters the ratio of memory CD4+ T cells(Gu, Zhang et al. 2020). These facts all suggest that ALKBH1 and TRMT10B, in addition to being potential targets in immunotherapy, may simultaneously provide synergy for reversing resistance to chemotherapeutic agents and enhancing the therapeutic efficacy of ICIs. They are as well promising a fresh perspective to address the conceptual challenges posed by negative outcomes of current PAAD mono-immunotherapies.
Due to the lack of studies on CD4+ T cells alterations with clinical relevance in long-term PAAD survivors, we firstly demonstrated the survival benefits of PAAD patients with high expression of ALKBH1 and TRMT10B might be associated with the increase in recruited CD4+ T cells and the adjusted ratio of tumor-infiltrating lymphocytes in TME.
ICIs have emerged as a powerful therapeutic strategy for multiple cancers, stimulating the innate and adaptive immune system, but with a poor prognosis in PAAD(Balachandran, Beatty et al. 2019). Our study found that high ALKBH1 expression downregulated the expression of immunomodulators CD276, TMEM173, and TNFSF9. TRMT10B could affect the expression of immunomodulators CD160, CD276, TMEM173, CD70, NT5E, TNFSF4, and the MHC molecule B2M. NO previous studies have demonstrated the signaling pathways of ALKBH1 and TRMT10B in the PAAD immune network. (Chen, Deng et al. 2021). Here, we firstly explored the interaction of immunomodulators closely related to ALKBH1 and TRMT10B with the immune system and their eventual relevance to the PAAD patient’s prognosis. The immunomodulator CD160 is an IgG-like glycoprotein expressed on the membranes of most peripheral blood circulating natural killer cells and γδT cells(Liu, Zhang et al. 2020). One study confirmed that infiltrating CD8+ T cells in PAAD highly expressed a CD160 subpopulation. Therefore, our study found that TRMT10B can elevate CD160 expression, thus ensuring a direct positive in-vitro antitumor effect and providing new ideas for CAR-T therapy. CD276, an immune checkpoint from the B7 superfamily, is highly expressed in TAMs and functionally suppressed DCs of TME. It can inhibit proliferation and activation of CTL, thus closely affecting tumor progression and metastasis(Geng, Huang et al. 2022). Therefore, our study suggested that ALKBH1 might improve the prognosis of PAAD patients by downregulating CD276 expression. TMEM173-dependent DNA sensor pathway was demonstrated to promote macrophage migration, activation, and malignant infiltration in PAAD, which indicated that TRMT10B could block the development of TMEM173-driven PAAD(Dai, Han et al. 2020). Other immunomodulators that we find could also be used as potential targets for immunotherapy. Our research indicated that ALKBH1 and TRMT10B signatures can reflect and improve TME features, thus holding hope for optimizing immunotherapy outcomes of PAAD.
To further determine the biological functions of ALKBH1 and TRMT10B in PAAD development, we performed GO and KEGG enrichment analysis(Chen, Zhang et al. 2017). The results indicated ALKBH1 and TRMT10B were enriched in the regulation of transcription from RNA polymerase II promoter, protein binding, RNA polymerase II core promoter sequence-specific DNA binding, and Herpes simplex virus 1 infection pathway. More studies have implicated that RNA polymerase II is significant in the development and treatment of PAAD(Ferdoush, Karmakar et al. 2017, Karmakar, Dey et al. 2018, Karmakar, Rauth et al. 2020, Liu, Gao et al. 2020). For example, regulating the nuclear localization of RNA polymerase II in pancreatic cancer cells represents a potential therapeutic target(Zhang, Li et al. 2021). Therefore, we speculate that ALKBH1 and TRMT10B may influence the development of PAAD by regulating RNA polymerase II. However, it needs further in vitro and in vivo validation in the direction of our future research.
Finally, we confirmed that ALKBH and TRMT10B were conducive to the prognosis of PAAD, and revealed their role in the tumor immune environment. Our study demonstrated that ALKBH1 and TRMT10 worked as prognosis biomarkers as well as potential immunotherapeutic targets for PAAD.