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Published on: December 9, 2016
High-throughput mutagenesis identifies mutations and RNA-binding proteins controlling CD19 splicing and CART-19
Mariela Cortés-López1, Laura Schulz1, Mihaela Enculescu1
1Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany.
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.
Insights
Investigating CD19 splicing regulation in B-cell acute lymphoblastic leukaemia (B-ALL) reveals mutations and novel splice isoforms that can cause resistance to CART-19 immunotherapy, offering insights for predictive biomarkers.
Area of Science:
- Molecular Biology
- Genetics
- Immunotherapy
Background:
- Relapse in B-cell acute lymphoblastic leukaemia (B-ALL) after CART-19 immunotherapy is often linked to the loss of the CD19 epitope.
- Aberrant CD19 exon 2 processing is a key mechanism driving this epitope loss.
Purpose of the Study:
- To elucidate the regulatory code governing CD19 splicing.
- To identify genetic alterations and regulatory elements impacting CD19 expression and CART-19 therapy response.
Main Methods:
- High-throughput mutagenesis combined with mathematical modeling to analyze mutations in CD19 exons 1-3.
- Identification and characterization of novel splice isoforms and regulatory factors.
Main Results:
- Approximately 200 single point mutations were found to alter CD19 splicing, potentially conferring CART-19 resistance.
- Nearly 100 previously undescribed splice isoforms, likely encoding non-functional CD19, were identified arising from cryptic splice sites.
- Key cis-regulatory elements and trans-acting RNA-binding proteins (e.g., PTBP1, SF3B4) controlling CD19 splicing were identified and validated.
Conclusions:
- The study provides a comprehensive dataset detailing CD19 splicing regulation.
- This resource can aid in identifying predictive biomarkers for CART-19 therapy efficacy in B-ALL patients.
- Understanding CD19 splicing alterations is crucial for overcoming therapeutic resistance.
