Multivariate Analysis of RNA Chemistry Marks Uncovers Epitranscriptomics-Based Biomarker Signature for Adult Diffuse Glioma Diagnostics

Abstract

One of the main challenges in cancer management relates to the discovery of reliable biomarkers, which could guide decision-making and predict treatment outcome. In particular, the rise and democratization of high-throughput molecular profiling technologies bolstered the discovery of “biomarker signatures” that could maximize the prediction performance. Such an approach was largely employed from diverse OMICs data (i.e., genomics, transcriptomics, proteomics, metabolomics) but not from epitranscriptomics, which encompasses more than 100 biochemical modifications driving the post-transcriptional fate of RNA: stability, splicing, storage, and translation. We and others have studied chemical marks in isolation and associated them with cancer evolution, adaptation, as well as the response to conventional therapy. In this study, we have designed a unique pipeline combining multiplex analysis of the epitranscriptomic landscape by high-performance liquid chromatography coupled to tandem mass spectrometry with statistical multivariate analysis and machine learning approaches in order to identify biomarker signatures that could guide precision medicine and improve disease diagnosis. We applied this approach to analyze a cohort of adult diffuse glioma patients and demonstrate the existence of an “epitranscriptomics-based signature” that permits glioma grades to be discriminated and predicted with unmet accuracy. This study demonstrates that epitranscriptomics (co)evolves along cancer progression and opens new prospects in the field of omics molecular profiling and personalized medicine.

Mass Spectrometry-Based Pipeline for Identifying RNA Modifications Involved in a Functional Process: Application to Cancer Cell Adaptation

“…tRNA, which is the most extensively modified RNA (13 modifications per molecule on average), gives the most striking separation (Figure S11). In agreement with our previous study, 15 our results show that a global epitranscriptomic profile (based on total RNA) is sufficient to differentiate cell states or culture conditions. Beyond saving experimental time, this simplified procedure offers an additional advantage: isolating total RNA (including RNA fragments) instead of purifying a RNA subtype could prevent a bias triggered by tRNA degradation in frozen samples, as pointed out by Richter et al 38 Our study shows that an 8-nucleoside signature {"m 2 G", "m 5 U", "m 3 C", "ac 4 C", "m 5 C", "m 7 G", "ncm 5 U", "mcm 5 s 2 U"} is sufficient to effectively distinguish cell samples/state according to their growth condition.…”

“…Cancer onset and evolution typically result from the accumulation of various molecular aberrations comprising genetic and epigenetic alterations. , The identification of genomic, transcriptomic, and epigenomic abnormalities is critical for precision oncology in many cancers, both to establish molecular subtypes and to offer appropriate treatment. − Likewise, the RNA mark landscape evolves along with tumor progression and represents a novel source of biomarkers for personalized medicine . To illustrate this principle, our group has recently uncovered “epitranscriptomic signatures” from glioma patients’ cohorts that could be exploited to guide glioma/glioblastoma diagnosis with unmet accuracy . Far from being mere passengers in the tumorigenic process, several of these marks participate in cancer adaptation to conventional treatments and inhospitable environment. − For instance, recent reports show that a dysregulation of 5-methylcytidine (m 5 C) players is associated with either oncogenic or tumor-suppressive functions according to cellular context. , The identification of RNA marks driving cancer cell adaptation to inhospitable environment and conventional treatment is essential to design appropriate therapeutic strategies, as emphasized by a growing drug discovery effort in the field .…”

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA

“…To gain a comprehensive understanding of the m 6,6 A modification, it is crucial to accurately determine its site and content in RNA. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is a commonly used method for analyzing nucleic acid modifications. − However, RNA is usually digested into nucleosides before LC-MS/MS analysis, resulting in the loss of site-specific information about m 6,6 A in RNA. Regarding the mapping study of m 6,6 A, the currently available method is the primer extension approach .…”

Multiplexed LC‐MS/MS quantification of salivary RNA modifications in periodontitis

“…This ultrasensitive tool currently allowed us to detect and quantify several chemically modified ribonucleosides in adult glioma. 25 Despite considerable advances in the search for biomarkers, these have not yet allowed a reliable early diagnosis of periodontitis.…”

“…Liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS) is a powerful analytical quantification technology. This ultrasensitive tool currently allowed us to detect and quantify several chemically modified ribonucleosides in adult glioma 25 …”