Identification of arsenic trioxide (ATO)-rescuable p53 mutations and temperature-sensitive p53 mutations in p53-null U937 cells using a patient-derived 800 p53 mutation library

Thousands of diverse p53 mutations have been identified in cancer. The leukemia-treating drug arsenic trioxide (ATO) rescues a part of p53 mutations with high potency. To repurpose ATO in personalized p53-targeted therapy, here we quantitatively determined the frequent 800 p53 mutations, covering 95.8% p53 missense-mutation cases in cancer, for their rescue potencies of cell growth inhibitory activity and mouse tumor suppressive activity by ATO. Furthermore, we determined the temperature sensitivity of 800 mutations in mammalian U937 cells and founded that ATO preferentially rescued temperature-sensitive subtype of structural p53 mutants. Overall design: The most frequent 800 p53 missense variants recorded in IARC (R20), covering 95.8% p53 missense mutation cases, were cloned. The 800 variants, together with 5 frequent cancer-associated nonsense variants and 5 silent variants were then balanced mixed to generate the patient-derived plasmid library. The plasmid library was then packed into retroviral for p53-null U937 cell infection at a low multiplicity of infection so that the most of the infected cells expresses one p53 variant. The infected cells were then sorted by fluorescence activating cell sorter based on the co-expressed GFP, finally generating a balanced and abundant cell library. Cell growth inhibitory activities and mouse tumor suppressive activities were evaluated by the decreases of abundance for each variant in U937 cell library, as determined by the next-generation sequencing, in cultured cells and xenografted mice, respectively. To dissect the relationship between ATO-rescuable mutations and temperature-sensitive mutations, the cell library was cultured at either 32 or 37 °C for 7 days, followed by p53 variants abundance determination to determine the temperature-sensitive mutations.