B cell progenitor acute lymphoblastic leukemia (B-ALL) treatment has been revolutionized by T cell-based immunotherapies—including chimeric antigen receptor T cell therapy (CAR-T) and the bispecific T cell engager therapeutic, blinatumomab—targeting surface glycoprotein CD19. Unfortunately, many patients with B-ALL will fail immunotherapy due to ‘antigen escape’—the loss or absence of leukemic CD19 targeted by anti-leukemic T cells. In the present study, we utilized a genome-wide CRISPR–Cas9 screening approach to identify modulators of CD19 abundance on human B-ALL blasts. These studies identified a critical role for the transcriptional activator ZNF143 in CD19 promoter activation. Conversely, the RNA-binding protein, NUDT21, limited expression of CD19 by regulating CD19 messenger RNA polyadenylation and stability. NUDT21 deletion in B-ALL cells increased the expression of CD19 and the sensitivity to CD19-specific CAR-T and blinatumomab. In human B-ALL patients treated with CAR-T and blinatumomab, upregulation of NUDT21 mRNA coincided with CD19 loss at disease relapse. Together, these studies identify new CD19 modulators in human B-ALL. Aifantis and colleagues use leukemic cell lines to identify modulators of CD19 expression that have the potential to improve therapeutic strategies. 

NUDT21 limits CD19 levels through alternative mRNA polyadenylation in B cell acute lymphoblastic leukemia

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. HighlightsO_LIMutations in relapsed CART-19 patients lead to CD19 mis-splicing C_LIO_LIHigh-throughput mutagenesis uncovers ~200 single point mutations with a potential role in CART-19 therapy resistance C_LIO_LIMany mutations generate non-functional CD19 proteins by activating cryptic splice sites C_LIO_LIRNA-binding proteins such as PTBP1 are key to the expression of properly spliced, CART-19 immunotherapy-sensitive isoforms C_LI

https://www.biorxiv.org/content/biorxiv/early/2021/10/08/2021.10.08.463671.full.pdf

Laura Schulz

Papers

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