Periodic Reporting for period 4 - TRANSREAD (Restoration of tumor suppressor function by induction of translational read-through of premature termination codons - a strategy for improved cancer therapy)
Reporting period: 2021-03-01 to 2022-02-28
Cancer is a major public health problem around the world. According to statistics from the World Health Organization (WHO), there were 18.1 million new cases of cancer and 9.6 million cancer deaths in 2018. Thus, improved cancer therapy is urgently needed. Novel anti-cancer drugs have been developed that can inhibit certain activated kinases in tumor cells, and so called immunocheckpoint inhibitors can unleash the immune response against tumors. Yet despite significant progress in diagnosis and therapy, efficient treatment is still lacking for many cancer patients. The TP53 tumor suppressor gene is inactivated by mutation in around 50% of all tumors. We and others have explored the possibility of restoring normal function to mutant p53 protein and thereby trigger tumor cell death and eliminate the tumor. This type of strategy could potentially be applied to a large number of cancer patients worldwide. We and others have previously identified small molecules that targets missense mutant p53 and trigger tumor cell death. However, around 10% of TP53 mutations in tumors are nonsense mutations in which a premature stop codon in the coding sequence leads to expression of a truncated inactive p53 protein. Nonsense mutations will clearly require an entirely different strategy for reactivation. This project is aimed at exploring whether expression of full length p53 can be restored by pharmacological induction of translational readthrough across a premature termination codon and whether this strategy can also be applied to nonsense mutations in other tumor suppressor genes, e.g. RB1, PTEN and APC. Our overall objective with this project is to develop novel cancer therapy by pharmacological induction of readthrough of nonsense mutant tumor suppressor genes.
We have studied aminoglycosides antibiotics known to induce readthrough of nonsense mutations. We have confirmed results from others showing that they can induce full length active p53 protein, leading to upregulation of p53 target genes and induction of tumor cell death. Moreover, we have demonstrated that the Mdm2 inhibitor Nutlin3a synergizes with G418 in inducing tumor cell death. This suggests that combination treatment with Mdm2 inhibitors could allow lower doses of aminoglycosides and therefore reduced toxicity. However, since aminoglycosides have severe side effects that make them less attractive for clinical use as anti-cancer agents, we have focused on the identification and characterization of novel molecules with higher potency and lower toxicity.
We have screened chemical libraries and analyzed available data in a major database to identify novel molecules that can induce efficient translational readthrough of nonsense mutant TP53 including R213X, the most common nonsense mutation in TP53 in human tumors. We have identified several candidate molecules and have characterized these further with the aim of in vivo studies in mice to confirm readthrough and anti-tumor effect. One compound has also shown potent activity on nonsense mutant PTEN. Furthermore, we have found that the chemotherapeutic drug 5-fluorouracil (5-FU) can induce readthrough of nonsense mutant TP53 via one of its metabolites. This produces full-length p53 that retains wild type activity as transcription factor as assessed by upregulation of p53 target genes and induction of tumor cell death. In addition, we have generated a new mouse model with a Trp53 R210X nonsense mutant knock-in allele (mouse R210X corresponds to human R213X) that will allow further in vivo studies with selected compounds. Since 5-FU is used for treatment of colon cancer and other types of cancer, our results may have important clinical implications.
We have screened chemical libraries and analyzed available data in a major database to identify novel molecules that can induce efficient translational readthrough of nonsense mutant TP53 including R213X, the most common nonsense mutation in TP53 in human tumors. We have identified several candidate molecules and have characterized these further with the aim of in vivo studies in mice to confirm readthrough and anti-tumor effect. One compound has also shown potent activity on nonsense mutant PTEN. Furthermore, we have found that the chemotherapeutic drug 5-fluorouracil (5-FU) can induce readthrough of nonsense mutant TP53 via one of its metabolites. This produces full-length p53 that retains wild type activity as transcription factor as assessed by upregulation of p53 target genes and induction of tumor cell death. In addition, we have generated a new mouse model with a Trp53 R210X nonsense mutant knock-in allele (mouse R210X corresponds to human R213X) that will allow further in vivo studies with selected compounds. Since 5-FU is used for treatment of colon cancer and other types of cancer, our results may have important clinical implications.
We have identified several candidate compounds and are currently characterizing them with regard to translational readthrough of nonsense mutant TP53, biological effect and potential synergy with other agents. We are also accumulating data on their effect on other TP53 nonsense mutations. In parallel, translational readthrough of three different PTEN nonsense mutations will be further studied in terms of biological effect and in vivo activity. We also hope to achieve an understanding of the molecular mechanism of action, which may facilitate the design of compounds with more potent readthrough activity. The compounds will be further explored for induction of translational readthrough of nonsense mutant RB1 and APC tumor suppressor genes. Our finding that a metabolite of 5-FU can induce translational readthrough of nonsense mutant TP53 is the most clinically relevant output from the project. This is new knowledge beyond state of the art with potential significance for cancer therapy.