Analyse der Rolle von zirkulären RNAs für die Pathogenese und Etablierung von Therapieresistenz des ALK-positiven anaplastisch-großzelligen Lymphoms
Kinder- und Jugendmedizin
Zusammenfassung der Projektergebnisse
Anaplastic large-cell lymphoma (ALCL) is an aggressive pediatric lymphoma of the T-cells. The characteristic driver of this disease is an oncogenic fusion between the nucleophosmin (NPM) and anaplastic lymphoma kinase (ALK) gene. While the response to frontline chemotherapy is usually favorable, one in three patients does not respond to therapy. Understanding the reasons for therapy resistance is in the focus of current research. We and others could previously show that regulatory RNAs, like microRNAs play an important role in resistance formation. Circular RNAs are regulatory RNAs that are highly stable and have been shown to be important for gene expression control, especially by interacting with other RNAs or proteins. This project aimed to explore the link of circular RNAs (circRNAs) with therapy resistance in ALK+ ALCL. By analyzing a cohort of ALK+ ALCL biopsies of patients sensitive or resistant to treatment by deep total RNA sequencing, we unveiled that globally circRNAs were higher expressed in resistant cases. Differential expression analysis identified a signature of circRNAs that was associated with a bad response to treatment. Most of the circRNA candidates could be successfully validated in vitro in ALK+ ALCL cell lines, which confirms our detection pipeline. Inhibition of ALK/STAT3 signaling, the major oncogenic pathway in ALK+ ALCL, by the ALK inhibitor crizotinib or siRNA showed that circRNA expression was under the control of this pathway. We created cell models with modified expression of circRNAs and tested their effect on treatment response with crizotinib. By this approach, we discovered the effect of a circRNA on sensitivity to ALK inhibition. To understand the mechanism of action we performed a specific RNA pulldown assay that we recently adapted to identify the interaction partners. This led to a strong enrichment of the circRNA. The analysis of enriched proteins and RNAs by mass spectrometry and RNA sequencing is currently ongoing. Further, to completely unveil the function of detected circRNAs it is important to know the full-sequence, which is not confidently possible by short-read sequencing. We therefore adapted a workflow using the Oxford Nanopore long-read sequencing platform. This allowed us to sequence circRNAs in full-length and unveil their whole sequence. This will be used to predict interactions in silico and complement the interaction data obtained by the pulldown. Lastly, the increased stability of circRNAs makes them suitable as clinical biomarkers in liquid biopsies. We established a workflow to analyze circRNAs in cell-free RNA from ALK+ ALCL patients by qRT-PCR and as a proof of principle detected selected candidates in patients, but not in healthy donors. We obtained funding to analyze a large clinical cohort of serum samples of ALK+ ALCL patients. Currently, low input total RNA sequencing of selected samples is ongoing to identify those candidates that correlate with resistance. Those will be analyzed in more samples and correlated with the clinical course of patients. Together the results of this study and ongoing analyses may lead to the discovery of new therapeutic angles and clinical biomarkers in ALK+ ALCL. Especially the circRNA candidates that are most promising in the analysis of liquid biopsies should be included in future clinical trials to test their potential as predictive biomarkers and make them available for the benefit of the patients.
Projektbezogene Publikationen (Auswahl)
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From circRNAs to fusion circRNAs in hematological malignancies. JCI Insight, 6(21).
Babin, Loelia; Andraos, Elissa; Fuchs, Steffen; Pyronnet, Stéphane; Brunet, Erika & Meggetto, Fabienne
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Neuroblastoma Risk Assessment and Treatment Stratification with Hybrid Capture-Based Panel Sequencing. Journal of Personalized Medicine, 11(8), 691.
Szymansky, Annabell; Kruetzfeldt, Louisa-Marie; Heukamp, Lukas C.; Hertwig, Falk; Theissen, Jessica; Deubzer, Hedwig E.; Willing, Eva-Maria; Menon, Roopika; Fuchs, Steffen; Thole, Theresa; Schulte, Stefanie; Schmelz, Karin; Künkele, Annette; Lang, Peter; Fuchs, Jörg; Eggert, Angelika; Eckert, Cornelia; Fischer, Matthias; Henssen, Anton G. ... & Schulte, Johannes H.
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Generation of full-length circular RNA libraries for Oxford Nanopore long-read sequencing. PLOS ONE, 17(9), e0273253.
Fuchs, Steffen; Babin, Loélia; Andraos, Elissa; Bessiere, Chloé; Willier, Semjon; Schulte, Johannes H.; Gaspin, Christine & Meggetto, Fabienne
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Defining the landscape of circular RNAs in neuroblastoma unveils a global suppressive function of MYCN. Nature Communications, 14(1).
Fuchs, Steffen; Danßmann, Clara; Klironomos, Filippos; Winkler, Annika; Fallmann, Jörg; Kruetzfeldt, Louisa-Marie; Szymansky, Annabell; Naderi, Julian; Bernhart, Stephan H.; Grunewald, Laura; Helmsauer, Konstantin; Rodriguez-Fos, Elias; Kirchner, Marieluise; Mertins, Philipp; Astrahantseff, Kathy; Suenkel, Christin; Toedling, Joern; Meggetto, Fabienne; Remke, Marc ... & Schulte, Johannes H.
