Figure S1. A. sg12 successfully edited the CDKN1A gene in SW780 with high efficiency; chromatogram shows deletion of an adenine (A). B. Histograms from TIDE analysis of single-cell clones showed that the clones have largely homogeneous populations. Figure S2. Cisplatin sensitivity was assessed in SW780 CRISPR clones 24h after a 3h pulse dose. Increased PARP cleavage (a marker of apoptosis) was observed in three p21-deficient sg12 clones compared to sgGFP. Figure S3: see separate document. Figure S4. TCC cell lines harboring p53 mutations differentially regulate p21 after cisplatin treatment (10 µM 3-hour pulse dose) Table S1: Primary antibodies used Table S2: Secondary antibodies used Table S3: sgRNA and flanking primer sequences
ARTICLE ABSTRACT
Muscle-invasive bladder cancer (MIBC) frequently harbors mutations in the CDKN1A gene, which encodes the tumor suppressor protein p21, with the majority of alterations truncating the peptide. The effect of these mutations is poorly understood. We hypothesized that after DNA-damaging events, cells deficient in p21 would be unable to halt the cell cycle and efficiently repair DNA damage, thus proceeding down the apoptotic pathway. We used synthetic CRISPR guide RNAs to ablate the whole peptide (sg12, targeting the 12th amino acid) or the C-terminal proliferating cell nuclear antigen (PCNA)-binding domain (sg109) to mimic different p21-truncating mutations compared with a negative control (sgGFP) in bladder cancer cell lines. Loss of detectable p21 and a stable truncated p21 peptide were identified in sg12 and sg109 single-cell clones, respectively. We found that p21-deficient cells (sg12) were sensitized to cisplatin, while cells harboring distally truncated p21 (sg12 clones) demonstrated enhanced cisplatin resistance. p21-deficient sg12 clones demonstrated less repair of DNA-platinum adducts and increased γ-H2AX foci after cisplatin exposure, suggesting there was persistent DNA damage after p21 loss. p21-deficient sg12 clones were also unable to prevent the activation of CDK1 after DNA damage, and therefore, continued through the cell cycle, resulting in replication fork collapse, potentially explaining the observed cisplatin sensitization. sg109 clones were neither unable to sequester PCNA nor localize p21 to the nucleus after DNA damage, potentially explaining the chemoresistant phenotype. Our findings suggest that different CDKN1A truncations have different and perhaps disparate biology, and that there may be a duality of effect on cisplatin sensitivity depending on mutation context.
Some truncating CDKN1A mutations generate a retained peptide that may have neomorphic functions and affect cisplatin sensitivity in patients with bladder cancer.
