Qing Wang1, Mélanie Sagniez3,4, Anshul Budhraja3,4, Bastien Paré4, Claire Fuchs4, Shawn M. Simpson4, Maxime Caron4, Elizabeth Snell5, Etienne Rainmondeau5, Vincent-Philippe Lavallée4,6, Thai Hoa Tran4,6, Alain Bataille4, Daniel Sinnet3,4 , Martin A. Smith1,2,3,4
1UNSW Sydney, School of Biotechnology and Biomolecular Sciences, Sydney, NSW, Australia
2UNSW Sydney, RNA Institute, Sydney, NSW, Australia
3Universiy of Montreal, Department of Biochemistry and Molecular Medicine, Montreal, Quebec, Canada
4CHU Sainte-Justine Research Centre, Montreal, Quebec, Canada
5Oxford Nanopore Technologies, Oxford, United Kingdom
6Universiy of Montreal, Department of Pediatrics, Montreal, Quebec, Canada
Acute lymphoblastic leukemia (ALL) remains a significant challenge in precision medicine due to its molecular heterogeneity and complex regulatory mechanisms. Long non-coding RNAs (lncRNAs) have emerged as critical regulators of gene expression and potential diagnostic biomarkers, yet their functional roles and mechanisms of action remain largely unexplored. Our previous analysis of nanopore cDNA and native RNA transcriptomes identified several lncRNAs as key diagnostic biomarkers in leukemia, emphasizing the need for deeper investigation into their regulatory roles.
Using CRISPR interference (CRISPRi) and Cas13-mediated knockdowns, we systematically perturbed candidate lncRNAs identified from patient-derived transcriptomic analyses. We then performed high-depth direct RNA sequencing on RNA004 PromethION flowcells to capture both transcriptomic and epitranscriptomic changes at single-base resolution. In parallel, we used single-molecule fluorescence in situ hybridization (smFISH) to resolve the subcellular localization of selected lncRNAs.
This integrative analysis revealed that knockdown of specific lncRNAs led to coordinated changes in the expression of key pathways, including cell differentiation as well as alterations in RNA methylation patterns. We identified genes with significant m⁶A changes independent of transcription abundance, suggesting a regulatory role beyond transcriptional control. These differentially modified genes were enriched in pathways such as B cell differentiation and cytokine production. These findings provide direct evidence linking m⁶A modification dynamics to the functional activity of disease-associated lncRNAs, establishing a framework for functional epitranscriptomics in leukemia.
This multi-omic approach integrates differential gene expression and RNA modifications, offering a robust resource for novel biomarker discovery and personalized therapeutic targeting in leukemia. Our findings highlight the potential of real-time clinical epitranscriptomics to advance cancer diagnostics and treatment strategies.