Sensitizing tumor cells via rescue of nonsense driver mutations: a promising therapeutic strategy

Abstract

Nonsense mutations are gene mutations characterized by the substitution of a single nucleotide in the coding sequence of a gene, which causes the onset of a premature stop codon (PTC) within the reading frame of the mRNA, resulting in the formation of a truncated and non-functional protein. This type of mutation accounts for approximately 11% of genetic diseases, including conditions such as Cystic Fibrosis, Duchenne Muscular Dystrophy, and certain types of hereditary cancers involving mutations in essential tumor suppressor genes. For example, 10% of TP53 gene mutations are stop mutations. TP53 encodes a protein of 393 amino acid residues called p53, which acts mainly as a transcription factor, regulating numerous pathways such as cell cycle arrest, DNA damage repair, apoptosis, autophagy, and metabolism when cells are under certain stress conditions. Mutations of TP53 can lead to the formation of tumors due to loss of function, dominant-negative repression, or gain of oncogenic function. Today there is no therapy for the pathologies caused by this type of mutation, but an approach that has proven to be particularly effective is represented by molecules with readthrough activity (TRIDs; Translational Readthrough Inducing Drugs) which intervene on the ribosome allowing the overcoming of the PTC and the restoration of the synthesis and subsequent functionality of the protein. In this work, we investigate, specifically, the effects of TRID molecules in tumor cells that harbor the PTC R213X, the most common TP53 stop mutation, which generates a truncated and non-functional protein. We analyzed the cytotoxic effects of these molecules with an MTT assay and a vital count with Trypan blue, but also the cellular localization of p53 and its nuclear translocation after treatment and induction of DNA damage, and the protein level and rescue of the protein by Immunofluorescence assay and Western blot. After 24 hours of treatment with TRIDs, we observed that treatment with TRIDs doesn’t lead to toxic effects on cells. Furthermore, nuclear localization of p53 and an increase in protein level after the induction of DNA damage are found. This study represents a promising path for developing targeted cancer therapies against stop mutations and tumor proliferation, but also a valid alternative for therapies used against other rare genetic diseases.