Abstract
During 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 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 enhanced CD19 mis-splicing. Our dataset represents a comprehensive resource for potential prognostic factors predicting success of CART-19 therapy.
Highlights
Mutations in relapsed CART-19 patients lead to CD19 mis-splicing
High-throughput mutagenesis uncovers ~200 single point mutations with a potential role in CART-19 therapy resistance
Many mutations generate non-functional CD19 proteins by activating cryptic splice sites
RNA-binding proteins such as PTBP1 are key to the expression of properly spliced, CART-19 immunotherapy-sensitive isoforms
Competing Interest Statement
A.T.-T. has an interest in intellectual property "Discovery of CD19 Spliced Isoforms Resistant to CART-19". This interest does not meet the definition of a reviewable interest under Children's Hospital of Philadelphia's (CHOP's) conflict of interest policy and is therefore not a financial conflict of interest. Furthermore, this intellectual property has not been licensed or otherwise commercialised to date. However, should this technology be commercialised in the future, A.T.-T. would be entitled to a share of royalties earned by CHOP per its patent policy. The other authors have no competing interests.
