Translational readthrough of nonsense mutant TP53, RB1 and PTEN tumor suppressor genes as a strategy for novel cancer therapy

A nonsense mutation causes a premature termination codon in the coding sequence of an mRNA. This leads to termination of translation and release of a truncated and in most cases non-functional protein. Tumor suppressor genes normally act to prevent tumorigenesis but inactivating mutations in these genes, for example nonsense mutations, can lead to cancer. TP53, RB1 and PTEN are among the most well-known tumor suppressor genes. TP53 codes for the p53 protein that regulates cellular processes such as cell cycle arrest, metabolism and cell death by apoptosis. Around 50% of all tumors carry TP53 mutation and 11% of TP53 mutations are nonsense mutations. The Rb protein encoded by the RB1 tumor suppressor gene is a regulator of cell cycle progression. Around 25-34% of reported RB1 mutations are nonsense mutations. The PTEN gene codes for the phosphatase PTEN that controls cell proliferation and cell survival via the PI3K-AKT pathway. PTEN nonsense mutations account for around 17.3% of all somatic mutations in this gene.

Pharmacological induction of translational readthrough has been applied as a strategy to rescue different types of nonsense mutant genes including tumor suppressor genes. A number of compounds have readthrough-inducing activity, including aminoglycoside antibiotics. In this thesis, readthrough-inducing agents have been examined as single treatments and in some cases in combination treatments to induce readthrough of nonsense mutant TP53, RB1 or PTEN. In Paper I, we show that combination treatment with aminoglycosides G418 or gentamicin and Mdm2-p53 inhibitors Nutlin-3a or MI-773 or the proteasome inhibitor Bortezomib increased full-length p53 levels in a nonsense mutant TP53 background. In Paper II, we found that the chemotherapeutic drug 5-Fluorouracil can induce functional full-length p53 in TP53 R213X nonsense mutated cells via its metabolite 5-Fluorouridine that is incorporated into mRNA. We also showed induction of full-length p53 in vivo in a xenograft mouse model. In Paper III, we examined G418 as readthrough inducer of nonsense mutant RB1. We observed induction of full-length Rb by G418 and that this effect was markedly enhanced by combination treatment with the reported readthrough inducer CC-90009. Finally, in Paper IV, we performed chemical library screening and identified two novel candidate readthrough inducers, C47 and C61. We found that C47 synergizes with G418 and that C61 synergizes with CC-885/CC-90009 for induction of readthrough of R213X nonsense mutant TP53. C47 can also synergize with G418 for induction of readthrough of R130X, R233X and R335X nonsense mutant PTEN. The mechanisms of action of C47 and C61 remain unclear and require further studies.

In conclusion, this work shows that induction of translational readthrough of nonsense mutant TP53, RB1 and PTEN tumor suppressor genes is feasible. We have studied compounds already known to induce readthrough, such as G418 and CC-885/CC-90009, as well as the 5- Fluorouracil metabolite 5-Fluorouridine and the novel candidate readthrough-inducing compounds C47 and C61. Combination treatments have been shown to enhance translational readthrough in the different genes. These results may facilitate the development of nonsense mutation-targeted cancer therapy in the future.

List of scientific papers

I. Meiqiongzi Zhang*, Angelos Heldin*, Mireia Palomar-Siles, Susanne Öhlin, Vladimir J.N. Bykov and Klas G. Wiman. Synergistic Rescue of Nonsense Mutant Tumor Suppressor p53 by Combination Treatment with Aminoglycosides and Mdm2 Inhibitors. Front Oncol. (2018) 7:323. *These authors contributed equally.
https://doi.org/10.3389/fonc.2017.00323

II. Mireia Palomar-Siles, Angelos Heldin, Meiqiongzi Zhang, Charlotte Strandgren, Viktor Yurevych, Jip T. van Dinter, Sem A. G. Engels, Damon A. Hofman, Susanne Öhlin, Birthe Meineke, Vladimir J.N. Bykov, Sebastiaan van Heesch and Klas G. Wiman. Translational readthrough of nonsense mutant TP53 by mRNA incorporation of 5-Fluorouridine. Cell Death Dis. (2022) 13:997.
https://doi.org/10.1038/s41419-022-05431-2

III. Mireia Palomar-Siles, Viktor Yurevych, Vladimir J.N. Bykov and Klas G. Wiman. Pharmacological induction of translational readthrough of nonsense mutations in the retinoblastoma (RB1) gene. [Manuscript]

IV. Angelos Heldin, Matko Cancer*, Mireia Palomar-Siles*, Meiqiongzi Zhang, Susanne Öhlin, Anna Mariani, Alexander Sun-Zhang, Jianping Liu, Vladimir J.N. Bykov and Klas G. Wiman. Novel compounds that synergize with G418 or eRF3 degraders for translational readthrough of nonsense mutant TP53 and PTEN. *These authors contributed equally. [Manuscript]