Cross talk of genetic variation of m6A regulators in PAAD
After reviewing the previous literature, 25 m6A regulators were screened out in total (Fig. 2A). These m6A regulators could been classified in three types, which contained 15 readers, 8 writers, and 2 erasers (Fig. 2A). The CNV alteration frequency for the 25 m6A regulators was investigated. CNV alterations of m6A regulators were prevalent. Several genes including VIRMA, HNRNPA2B1, IGF2BP3, YTHDF3, YTHDF1 showed strong frequency of amplification in copy number (Fig. 2B). On the contrary, WTAP, YTHDF2, and RBM15B occupied a widespread frequency of CNV deletion (Fig. 2B). We immediately identified frequently mutated genes and further summarized the incidence of somatic mutations of 25 m6A regulators in PAAD. The top 30 high-frequency mutated genes were shown in Fig. 2C. These genes altered in 124 (78.48%) of 158 samples. Among them, the top 5 high-frequency mutated genes were TP53 (54%), KRAS (54%), CDKN2A (17%), SMAD4 (17%), and TTN (11%). Among 158 samples, only 8 (5.06%) experienced mutations of m6A regulators (Fig. 2D). In general, for m6A regulators, the overall average mutation frequency was low, scoping from 0.00–1.00% (Fig. 2D).
Expression difference of m6A regulators between PAAD tissues and normal tissues
To explore the effect of genetic alterations in m6A regulators expression, the expression difference of m6A regulators between PAAD tissues and normal tissues was subsequently screened out. The overall average expression level was higher, compared to normal tissues. In detail, the mRNA expressions of 8 readers including ELAVL1, HNRNPA2B1, HNRNPC, IGF2BP2, IGF2BP3 were significantly increased in PAAD tissues compared to normal tissues (P < 0.05, Fig. 3A). Moreover, 4 writers including METTL14, RBM15, VIRMA, and ZC3H13 were significantly high expressed in PAAD samples compared to normal samples (P < 0.05, Fig. 3B). The erasers mRNA (ALKBH5 and FTO) were also over expressed in tumor samples (P < 0.05, Fig. 3C). Interestingly, we found that CNV amplification of m6A regulators led to up-regulated of them in tumor tissues in comparison with normal tissues (such as VIRMA, HNRNPA2B1, IGF2BP3, YTHDF3), which suggested that CNV alterations might be one of the factors influencing the mRNA expression level of m6A regulators. IHC data containing 6 to 12 independent PAAD samples were further collected from HPA database (Fig. 3D). YTHDF1, YTHDF3, and METTL3 were not evaluated. Overall, m6A readers including HNRNPA2B1, HNRNPC, RBMX, and YTHDC1 showed high staining. But we could only observe expression of IGF2BP1, IGF2BP3, YTHDF2 in 5/12, 6/12, 2/8 samples, respectively. Almost all the writers including METTL14, RBM15, RBM15B, VIRMA, and WTAP were not detected in several samples (4/12, 2/11, 10/11, 3/11, 3/11, separately). We observed that 12/12 and 11/12 samples expressed ALKBH5, and FTO erasers m6A regulators, separately (Fig. 3D).
Cross talks among m6A regulators
We further explored m6A regulators related biological process (BPs) and KEGG signaling pathways, for better comprehending the roles of m6A regulators in PAAD. The results demonstrated that m6A regulators significantly enriched in regulation of mRNA metabolic process, mRNA processing, RNA catabolic process, RNA splicing, regulation of mRNA stability, regulation of RNA stability, regulation of mRNA catabolic process, mRNA catabolic process, RNA modification, and RNA methylation (Table S2, Fig. 4A). And the 25 m6A regulators were only regulated in two KEGG signaling pathways including spliceosome and RNA transport (Fig. 4B). These results demonstrated that m6A regulators played nonnegligible roles in m6A modification. We immediately explored the co-occurrence of genetic alterations and expression correlation among m6A regulators. Surprisingly, the result demonstrated that there was also a high association between different types of m6A regulators (Fig. 4C). For example, IGF2BP2 reader was significantly associated with eraser ALKBH5 (R = -0.62, P < 0.001, Fig. 4C). Then we constructed a PPI network of m6A regulators (Fig. 4D). Though classified into different types of m6A regulators, readers, writers, and erasers interacted with each other closely in the PPI network. We further calculated the degree of connectivity of each m6A regulators. Among all the readers, HNRNPC (degree = 17) and HNRNPA2B1 (degree = 17) showed the highest degree (Fig. 3D). VIRMA (degree = 17), WTAP (degree = 17), and METTL14 (degree = 17) showed strongest association with each other among all the writers (Fig. 4D). The two erasers (ALKBH5: degree = 15; FTO: degree = 11) also frequently associated with other m6A regulators (Fig. 4D). Summary above, we thought these m6A regulators might work with each other in PAAD.
Survival landscape of m6A regulators in PAAD
To investigate the association between the m6A regulator expression and prognosis of PAAD, survival analysis was further conducted. Overall, half of the m6A regulators (13/25) showed significant correlation with PAAD patient survival by using TCGA data (Fig. 3E). In detail, all the 13 genes showed oncogenic features, PAAD patients in high expression group of these genes occupied obviously worse survival compared to these in low expression group (Fig. 4E). Seven m6A regulators including 5 readers (FMR1 (TCGA: P = 0.029; ICGC: P = 0.003), IGF2BP2 (TCGA: P < 0.001; ICGC: P = 0.028), IGF2BP3 (TCGA: P < 0.001; ICGC: P = 0.005), RBMX (TCGA: P = 0.016; ICGC: P = 0.028), YTHDC1 (TCGA: P = 0.013; ICGC: P < 0.001)) and 2 writers (METTL14 (TCGA: P = 0.028; ICGC: P = 0.017), VIRMA (TCGA: P = 0.001; ICGC: P = 0.016)) were validated to show the similar correlation with PAAD patient survival by using ICGC data (Fig. 4F). Unfortunately, none of erasers showed significant association with PAAD patient survival (Fig. 4E-F), which pointed out that types of m6A regulators might affect their roles in prognosis of PAAD patients.
m6A modification patterns defined via 25 m6A regulators
Based on R package “ConsensuClusterPlus”, unsupervised hierarchical clustering of the 179 tumors with matched m6A regulator expression profiles from TCGA data was obtained. Finally, as shown in Fig. 5A, 179 tumors were classified into two distinct modification patterns (80 samples in m6Acluster A, 99 samples in m6Acluster B). PCA demonstrated that there was significant distinction existed between different m6A modification patterns (Fig. 5B). Further analysis suggested that there was prognosis difference between m6A cluster A and m6Acluster B. PAAD patients in m6Acluster A occupied better survival compared to m6Acluster B (P = 0.02, Fig. 5C). We immediately explored the expression difference of m6A regulators between different m6A clusters, the results were shown as a heatmap (Fig. 5D). Both the two erasers were higher expressed in m6AclusterA compared to m6Acluster B (ALKBH5: P < 0.0001; FTO: P < 0.05). Several readers including IGF2BP1 (P < 0.0001), IGF2BP2 (P < 0.0001), and IGF2BP3 (P < 0.0001) were significantly up-regulated in m6Acluster B while RBMX (P < 0.05), YTHDC1 (P < 0.05), YTHDC2 (P < 0.05), and YTHDF1 (P < 0.05) were obviously down-regulated in m6Acluster B. The expressions of writers were consistent between different m6A modification patterns. In detail (Fig. 5D), METTL14, METTL3, RBM15B, and WTAP occupied a significant down-regulation in m6Acluster B across different clusters.
Association between m6A modification patterns and tumor microenvironment
As shown in Fig. 6A, after measuring the relative abundance of 22 TME-infiltrating cells, a stacked bar plot was used to show the relative proportion of these cell types in each PAAD sample. The results demonstrated that the relative proportion of the 22 cells were quite different across the PAAD samples. Obviously, some TME-infiltrating cells showed strong correlation with each other (Fig. 6B). For example, B cells memory showed negatively association with T cells CD4 naïve. T cells CD8 had strong correlation with Macrophages M0, positively. More relationships between different types of TME-infiltrating cells could be explored in Fig. 6B. What surprised us was that the immune infiltration levels of several immune cells were quite different between different m6A clusters (Fig. 6C). Macrophages M0 (P < 0.05), and Macrophages M1 (P < 0.05) were significantly enriched in m6Acluster B group, compared to m6Acluster A group (Fig. 6C). In addition, Monocytes, and T cells CD8 were enriched in m6Acluster A group when compared with m6Acluster B group (Fig. 6C). We further calculated immune scores and stromal scores of PAAD samples. Unfortunately, we could not explore significant relationship between these scores and m6A modification patterns. Not only immune scores (Fig. 6D) but also stromal scores (Fig. 6E) did not show significant expression difference between different m6A clusters. We further explored the influence of immune/stromal score on prognosis prediction by m6A modification patterns. In PAAD patients of low immune score, we found PAAD patients in m6Acluster A occupied better survival compared with these in m6Acluster B (Fig. 6F, P = 0.038). Furthermore, there was no survival difference between patients in different m6A modification patterns in PAAD patients of high immune score, which indicated that immune score could affect the survival possibility of PAAD patients in different m6A modification patterns. Unfortunately, subsequent analysis demonstrated that stromal score could not affect the survival probability of patients in different m6A modification patterns (Fig. 6G, P = 0.087).
Construction of the m6A gene signature
We immediately tried to construct a m6A gene signature which significantly associated with prognosis of patients with PAAD. As shown in Fig. 7A, 146m6A phenotype-related DEGs which differently expressed between m6Acluster A and m6Acluster B were firstly screened out in total, via the standards we set before (adjusted P value < 0.001 and |log2FC | ≥ 1.5). The detail information for these DEGs were shown in Table S3. Among the 146 m6A-related genes, 48 genes were significantly high expressed and 98 genes were obviously low expressed in m6Acluster A compared to m6Acluster B (Fig. 7B). Then univariate Cox regression analysis was used to identify prognosis-related gene from the 146 DEGs. 75 prognosis-related genes including 25 favorable factors and 50 risk factors were further desired. As the alluvial diagram showed, all the 25 favorable factors of prognosis belonged to the 48 down-regulated DEGs in m6Acluster B meanwhile 50 risk factors of prognosis were included in the 98 up-regulated DEGs in m6Acluster B (Fig. 7C). Furthermore, as shown in Fig. 7D, based on the 75 prognosis-related factors, unsupervised clustering algorithm was conducted and the 179 PAAD patients were further classified into two different genomic subtypes (m6A gene cluster A: 90 patients; m6A gene cluster B: 89 patients). Survival analysis indicated that PAAD patients in m6A gene cluster A subtype had worse overall survival (OS) compared with patients in m6A gene cluster B (P = 0.005, Fig. 7E). All of these outcomes indicated that m6A methylation modification acted as an indispensable role in the prognosis of patients with PAAD. Nonetheless, the above results were only in view of the patient population, which meant those might not play significant roles in m6A methylation modification pattern predication for individual patients. Therefore, based on the 75 prognosis-related factors, we further set up a consolidated scoring system for quantifying the m6A modification pattern of individual PAAD patients. By combining HR of each prognosis-related factor (Table S4) and PCA, we calculated m6A-score for each PAAD patient with the formula we mentioned above. The value of m6A-score in predicting outcome of PAAD patients was immediately explored. Based on the median value of m6A-score, patients were split into high m6A-score group (n = 89) and low m6A-score group (n = 90). Survival analysis demonstrated that PAAD patients in low m6A-score group had better OS compared with these in high m6A-score group (P < 0.001, Fig. 7F). We further compared the m6A-score level between different m6A modification patterns and genomic subtypes. As Fig. 7G showed, the m6A-score levels of patients in m6Acluster A group were higher than these of patients in m6Acluster B group (P < 0.001, Fig. 7G). Similarly, difference of m6A-score levels also existed between patients in different m6A gene related patterns (P < 0.001, Fig. 7H). Furthermore, an alluvial diagram was plotted to show the changes of m6Acluster, survival status, m6A gene cluster and m6A-score (Fig. 7I).
Role of m6A modification patterns in immunotherapeutic benefit prediction
Immunotherapy has been proved to be an effective and safe cancer treatment. Among these therapeutic methods, PD-L1 and PD-1 blockade represents a quantum leap for medical science. In IMvigor210, PAAD patients in high m6A-score group (n = 94) occupied significantly shorter survival (P < 0.001, Fig. 8A), compared with patients in low m6A-score group (n = 204). The predictive value of the m6A-score to anti-PD-L1 immunotherapy was further confirmed by using IMvigor210 (Fig. 8B-D). Patients in higher m6A-score group were more likely to benefit from anti-PD-L1 treatment (Fig. 8B-C), which was validated by Kruskal-Wallis test (P = 0.023, Fig. 8D). Next ROC analysis (based on IMvigor210) was used to evaluate the efficacy of anti-PD-L1 treatment by m6A-score, which indicated that m6A-score was a predictive biomarker to immunotherapeutic benefits (AUC: 0.782, Fig. 8E). In addition, we also tried to find some relationship between m6A-score and anti-PD-1 treatment via GSE78220 cohort. But unfortunately, m6A-score was not significantly related to OS (P = 0.830, Fig. 8F) and response to anti-PD-1 immunotherapy (Fig. 8G-I, Kruskal-Wallis test: P = 0.510). Further ROC analysis also demonstrated that m6A-score might not be an appropriate predictive tool to anti-PD-1 treatment benefits (AUC: 0.583, Fig. 8J). Talked together, the present study obviously demonstrated that m6A methylation modification patterns was associated with response to anti-PD-L1 immunotherapy. Thus, the constructed m6A modification signature might effectively give play to the prediction of response to anti-PD-L1 treatment.