TP53 is the most frequently mutated gene in human cancer, yet the clinical utility of p53 mutation status remains limited as different mutation types produce highly variable biological effects. This challenge is especially pronounced for rare TP53 splice mutations, which remain poorly characterised despite emerging evidence that they may be associated with worse clinical outcomes. One such variant, the X126 splice mutation, has been linked to reduced disease‐free survival, but its functional consequences are not well understood.
To investigate the biological impact of this mutation, we used CRISPR‐Cas9 to generate A549 cell lines carrying defined X126 genotypes: wild‐type (WT; 0/4 mutant alleles), single‐mutant allele (SMA; 1/4), and triple‐mutant allele (TMA; 3/4). Mutant cells showed altered p53 isoform expression, with both SMA and TMA cells exhibiting increased levels of p53α, Δ40p53α, and an additional unconfirmed isoform, and with TMA cells showing the strongest induction. Despite this elevated isoform expression, SMA and TMA cells displayed a dose‐dependent reduction in canonical p53 target proteins, including p21 and MDM2, indicating dysregulated p53 signaling in the presence of X126 alleles.
To understand the underlying mechanism, RNA‐seq analysis was performed. In TMA cells— and to a lesser extent in SMA cells—TP53 transcripts lacked 21 nucleotides at the 5′ end of exon 5. Predictive modeling suggested that this would remove seven amino acids from the p53 DNA‐binding domain, a change that could explain the impaired activation of downstream targets such as p21 and MDM2. Additionally, Δ40p53α expression was elevated in mutant cells, and this isoform has been reported to inhibit p53α‐mediated transcriptional activation. Either mechanism, or their combined effects, could contribute to dysfunctional p53 activity in X126 mutant cells.
Beyond p53 signaling, RNA‐seq revealed differential regulation of genes involved in cell adhesion, angiogenesis, and immune modulation. Notably, KRT19 expression was markedly increased in SMA and TMA cells. Elevated KRT19 has been associated with enhanced invasion and metastasis, poor clinical outcomes, and degradation of p21, further supporting a shift toward a more aggressive phenotype.
Taken together, these findings provide new mechanistic insights into how the X126 splice mutation alters tumor biology. With further functional characterisation, this work may support the development of new therapeutic strategies and improve the clinical utility of this splice mutation as a prognostic and predictive biomarker.