N6-Methyladenosine RNA Methylation Regulators Contribute to Malignant Progression and Survival Prediction in Chronic Lymphocytic Leukemia

Introduction

N⁶-methyladenosine (m⁶A) RNA methylation is the most abundant epitranscriptomic modification, dynamically installed by the m⁶A methyltransferases (termed as "writers"), reverted by the demethylases (termed as "erasers"), and recognized by m⁶A binding proteins (termed as "readers"). Emerging evidence suggests that m⁶A RNA methylation regulates RNA stability, and participates in the pathogenesis of multiple diseases including cancers. Nevertheless, the role of m⁶A RNA methylation in chronic lymphocytic leukemia (CLL) remains to be unveiled. Herein, we hypothesized that m⁶A RNA methylation contributed to the tumorigenesis and maintenance of CLL. Moreover, the risk-prediction model integrated with the m⁶A regulators could serve as a novel and effective prognostic indicator in CLL. This study aimed to identify robust m⁶A RNA methylation-associated fingerprints for risk stratification in patients with CLL.

Methods

A total of 714 de novo CLL patients from 4 cohorts (China, Spain, Germany and Italy) were enrolled with informed consents. EpiQuik m⁶A RNA methylation colorimetric quantification assay was utilized to assess m⁶A RNA methylation levels. LASSO Cox regression algorithm was performed to calculate m⁶A RNA methylation-associated risk score (short for "m⁶A risk score") in R software. Besides, Kaplan-Meier survival analysis with log-rank test, univariate and multivariate Cox regression analyses and ROC curve analysis of overall survival (OS) were conduct to explore the prognostic value of m⁶A signature in CLL. Furthermore, RNA-seq, MeRIP-seq, Ribo-seq, functional enrichment analyses in silico and preclinical experiments ex vivo were applied to confirm the biological mechanism of the m⁶A regulators in CLL.

Results

In the present study, we performed a comprehensive analysis to dissect the role of m⁶A RNA methylation regulators in CLL. Compared with normal B cells from healthy donors, obvious decreased level of m⁶A RNA methylation was observed in primary CLL cells (p<0.01; Figure 1A). In addition, down-regulated m⁶A RNA methylation was also detected in CLL cell lines MEC1 and EHEB (p<0.05; Figure 1A).

Then, we further investigated the association of the m⁶A RNA methylation regulators with clinical outcomes of CLL patients. By LASSO Cox regression analysis in 486 CLL patients, the m⁶A risk score was established with the coefficients of fourteen m⁶A regulators at the minimum lambda value of 0.00892 (Figure 1B-C). Based on the median risk score as the cut-off value, a clear distribution pattern was delineated in CLL patients (Figure 1D).

Kaplan-Meier curves showed stratified high-risk patients presented significantly shorter OS versus the low-risk group (HR=4.477, p<0.001; Figure 2A). Besides, m⁶A risk score also predicts inferior prognosis in stable subgroup (HR=3.097, p=0.037; Figure 2B), and progressed/ relapsed subgroup (HR=3.325, p=0.001; Figure 2C). Moreover, univariate, multivariate cox regression analyses and ROC curve confirmed high m⁶A risk score as an independent survival predictor in CLL patients (p<0.001; Figure 2D-E).

Thereafter, the clinicopathological relevance and underlying mechanism of m⁶A risk score were explored. Significant elevated m⁶A risk score was detected in patients with unfavorable treatment responses compared with stable status (p<0.001; Figure 3A). Furthermore, CLL patients with advanced Binet stage, positive ZAP-70 and unmutated IGHV present increased m⁶A risk score (p<0.05; Figure 3B-C). Intriguingly, we also observed the significantly negative correlation between highrisk score and 13q14 deletion, in accordance with patients' inferior outcome (p=0.047; Figure 3D). Moreover, Pearson correlation analysis, STRING interactive network and functional enrichment analyses deciphered that the m⁶A regulators exerted crucial roles in CLL progression potentially via modulating RNA metabolism and oncogenic pathways (Figure 4A-C).

Conclusion

To date, our study provides evidence for the first time that reduced m⁶A RNA methylation contributes to the tumorigenesis of CLL. Distinct m⁶A risk scoreis demonstrated as an efficient tool facilitating prognosis evaluation in CLL patients. However, validation of the signature in more independent cohorts are warranted. Further interrogations will be elucidated on the biological mechanism of m⁶A regulators, highlighting insights into pathogenesis and therapy strategy of CLL.

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