An 8-month-old patient was found to have an adrenal cortical carcinoma (ACC) characterized by a TP53 c.758C>T (p.T253I) mutation in the TP53 tumor suppressor gene. p53 protein levels were overexpressed in the patient’s ACC, and molecular profiling of the tumor suggested suppression of the remaining wild-type (WT) TP53 allele. As this type of malignancy has been reported to occur in Li-Fraumeni Syndrome (LFS) patients with germline inheritance of loss-of-function TP53 mutations, we evaluated the patient’s TP53 gene in non-tumor tissue and found evidence of a constitutional heterozygous germline TP53 c.758C>T (p.T253I) variant. Since the mutation has not yet been linked to LFS, we sought to characterize the functionality of the T253I mutation. We acquired p53 -/- HEK293 cells and stably transduced them with GFP-tagged wild type (T253) or T253I p53 as well as two established pathogenic p53 mutants (C176Y and R213X). Compared to p53 WT, levels of T253I p53 increased while MDM2 levels decreased, suggesting a loss of MDM2-mediated regulation of T253I p53. Additionally, T253I showed a reduction in DNA damage responsive events, diminished DNA binding capabilities, and blunted transactivation capacity. These experimental data, coupled with clinical observations, lead us to conclude that T253I represents a pathologic variant in TP53 that may predispose to LFS-associated tumors.

Acquired resistance to first-line treatments in various cancers both promotes cancer recurrence as well as limits effective treatment. This is true for epidermal growth factor receptor (EGFR) mutations, for which secondary EGFR mutations are one of the principal mechanisms conferring resistance to the covalent inhibitor osimertinib. Thus, it is very important to develop a deeper understanding of the secondary mutational resistance mechanisms associated with EGFR mutations arising in tumors treated with osimertinib to expedite the development of innovative therapeutic drugs to overcome acquired resistance. This work uses all-atom molecular dynamics (MD) simulations to investigate the conformational variation of two reported EGFR mutants (L858R/L718Q and L858R/L792H) that resist osimertinib. The wild-type EGFR kinase domain and the L858R mutant are used as the reference. Our MD simulation results revealed that both the L718Q and L792 secondary mutations induce additional hydrogen bonds between the residues in the active pocket and the residues with the water molecules. These additional hydrogen bonds reduce the exposure area of C797, the covalent binding target of osimertinib. The additional hydrogen bonds also influence the binding affinity of the EGFR kinase domain by altering the secondary structure and flexibility of the amino acid residues in the domain. Our work highlights how the two reported mutations may alter both residue-residue and residue-solvent hydrogen bonds, affecting protein binding properties, which could be helpful for future drug discovery.