Targeting p53 Isoforms: A Novel Approach for Anticancer Therapy

Introduction: The tumor suppressor p53 is expressed as at least 12 distinct isoforms, each with unique functions. To identify compounds that differentially regulate these isoforms, we employed the exon-specific isoform expression reporter system (EXSISERS). By integrating three split-intein-flanked luciferases into exons 2, 4, and 7 of TP53, we quantified the de novo synthesis of the tumor-suppressive full-length (FL) p53 and ∆40p53 isoforms, relative to the pro-survival ∆133p53 and ∆160p53 isoforms.

Methods: We stably integrated EXSISERS into wild-type p53-expressing cells using CRISPR/Cas9. The effects of 4,863 anticancer compounds (MCE, HY-L025) on the differential expression profiles of p53 isoforms were assessed in a high-throughput manner. Simultaneously, the total quantity of p53 isoforms was determined using tandem mass spectrometry (MS).

Results: Through EXSISERS, we identified substances that enhanced p53-mediated tumor suppression and cell death while inhibiting pro-survival p53 isoforms. We identified 121 significant (p<0.025) p53 Tumor Suppressor Isoform Enhancers (TIEs) and 121 Pro-Survival Isoform Enhancers (PIEs). MS of isoform-specific p53 peptides, using 15N-labelled recombinant p53 as an internal standard, confirmed these findings.

Conclusion: EXSISERS enables luminescence-based analysis of protein isoforms, allowing: 1) quantification of target proteins, 2) differentiation of isoforms, 3) time-series data collection, 4) measurements in living cells, and 5) high scalability for screening. This system facilitates the identification of anti-tumor therapeutics that efficiently induce pro-apoptotic p53 isoforms.

Publication History

Article published online:
20 January 2025