High-throughput mutagenesis identifies mutations and RNA-binding proteins controlling CD19 splicing and CART-19 therapy resistance

• Published in: Nature communications 2022-09, Vol.13 (1), p.5570-5570, Article 5570
• Main Authors: Cortés-López, Mariela, Schulz, Laura, Enculescu, Mihaela, Paret, Claudia, Spiekermann, Bea, Quesnel-Vallières, Mathieu, Torres-Diz, Manuel, Unic, Sebastian, Busch, Anke, Orekhova, Anna, Kuban, Monika, Mesitov, Mikhail, Mulorz, Miriam M., Shraim, Rawan, Kielisch, Fridolin, Faber, Jörg, Barash, Yoseph, Thomas-Tikhonenko, Andrei, Zarnack, Kathi, Legewie, Stefan, König, Julian
• Format: Article
• Language: English
• Subjects: 49; 49/47; 49/91; 631/114/2163; 631/337/1645/1946; 631/67/68; Acute lymphoblastic leukemia; Alternative splicing; Binding; Biomarkers; CD19 antigen; Epitopes; Exons; Humanities and Social Sciences; Immunotherapy; Isoforms; Leukemia; Lymphocytes B; Mathematical models; mRNA; multidisciplinary; Mutagenesis; Mutation; Proteins; Regulatory sequences; RNA-binding protein; Science; Science (multidisciplinary); Splicing; Therapy
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-31818-y

Following CART-19 immunotherapy for B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the cognate CD19 epitope. Since epitope loss can be caused by aberrant CD19 exon 2 processing, we herein investigate the regulatory code that controls CD19 splicing. We combine high-throughput mutagenesis with mathematical modelling to quantitatively disentangle the effects of all mutations in the region comprising CD19 exons 1-3. Thereupon, we identify ~200 single point mutations that alter CD19 splicing and thus could predispose B-ALL patients to developing CART-19 resistance. Furthermore, we report almost 100 previously unknown splice isoforms that emerge from cryptic splice sites and likely encode non-functional CD19 proteins. We further identify cis -regulatory elements and trans -acting RNA-binding proteins that control CD19 splicing (e.g., PTBP1 and SF3B4) and validate that loss of these factors leads to pervasive CD19 mis-splicing. Our dataset represents a comprehensive resource for identifying predictive biomarkers for CART-19 therapy. Multiple alternative splicing events in CD19 mRNA have been associated with resistance/relapse to CD19 CAR-T therapy in patients with B cell malignancies. Here, by combining patient data and a high-throughput mutagenesis screen, the authors identify single point mutations and RNA-binding proteins that can control CD19 splicing and be associated with CD19 CAR-T therapy resistance.