High-throughput mutagenesis identifies mutations and RNA-binding proteins controlling CD19 splicing and CART-19 therapy resistance
Mariela Cortés-López, Laura Schulz, Mihaela Enculescu, Claudia Paret, Bea Spiekermann, Mathieu Quesnel-Vallières, Manuel Torres-Diz, Sebastian Unic, Anke Busch, Anna Orekhova, Monika Kuban, Mikhail Mesitov, Miriam M. Mulorz, Rawan Shraim, Fridolin Kielisch, Jörg Faber, Yoseph Barash, Andrei Thomas‐Tikhonenko, Kathi Zarnack, Stefan Legewie, Julian König
Abstract
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.