Dual-trigger model of CD20 escape: NONO regulation and cryptic splicing induced by transcript overload in pediatric B-ALL.

[INTRODUCTION] The B-cell-specific marker CD20 is expressed in various B-cell malignancies, including B-cell acute lymphoblastic leukemia (B-ALL) and serves as a key target for immunotherapies. Reduced or absent CD20 expression has been associated with diminished responses to anti-CD20 antibodies and CD20 directed CAR T-cells. Antigen loss may arise from alternative splicing or transcriptional downregulation of , the gene coding for CD20, a processes influenced by RNA- and DNA-binding proteins. , a non-POU domain-containing octamer-binding protein implicated in several cancers, regulates CD20 surface expression.

[METHODS] To explore factors associated with heterogeneous CD20 expression, we quantified transcript levels, profiled messenger RNA (mRNA) isoforms, and analyzed mRNA in pediatric B-ALL samples. In addition, we used an CRISPR/Cas9 knockout model to assess the effects of loss on transcript abundance, isoform distribution, and transcript stability. Plasmid-based overexpression of was used to examine its effect on splicing.

[RESULTS] Loss of was associated with increased transcript levels without detectable changes in isoform distribution or stability, and mRNA expression was negatively associated with mRNA expression in CD20-positive blasts. At diagnosis, two mRNA isoforms were detected in CD20-positive blasts: The wild-type (WT-CD20) and a shorter variant (D393-CD20), a Δ4-6 multi-exon-skipped isoform that yields a truncated intracellular protein inaccessible to CD20-directed immunotherapies. Although WT-CD20 was the dominant splice isoform, the D393/WT-CD20 ratio correlated positively with overall transcript abundance. High WT-CD20 transcript abundance further biased splicing toward the D393-CD20 isoform, indicating involvement of cryptic splice sites and potential re-splicing events at the level of mature mRNA.

[DISCUSSION] Together, these findings are consistent with a model in which expression and transcript-level dynamics of are associated with CD20 heterogeneity in pediatric B-ALL. These observations may contribute to understanding variability in CD20 expression and antigen availability in pediatric B-ALL.