p53-dependent and -independent mechanisms of p53-targeting small molecules

Tumor suppressor p53 (Tp53) is mutated in around half of human cancers, while in wild type p53 cells its activity is continuously inhibited by MDM2 through proteasome degradation resulting in the loss of its function. Currently, cancer treatments with small molecules based on reactivation of wild type p53 and restoration of mutant p53 have moved to clinical trials and exhibited promising anti-cancer effects. Our lab previously found a small molecule RITA which reactivates p53 and has strong anti-cancer effect without affecting normal cells. However, small molecules always have multiple targets and those should be validated for either predicting potential side effects or evaluating their efficacy in different types of cancers.

In this thesis, we addressed a p53-independent mechanism of RITA along with two other anti-cancer compounds Aminoflavone and Oncrasin-1. Using thermal proteome profiling (TPP) approach, we found that transcription machinery is commonly inhibited by these three compounds in a reactive oxidative species (ROS)-dependent manner. Global transcription inhibition results in massive downregulation of the majority of oncogenes as well as genes that are involved in homologous recombination (HR). By taking advantage of that, we performed combination treatments of these three compounds with PARP-1 inhibitors Olaparib and talazoparib. The combination treatments displayed clear synergistic anti-cancer effects in several cancer lines as well as in primary ovarian and breast cancer patient samples.

Moreover, we found that mRNA translation is also inhibited by RITA through activation of eIF2α phosphorylation, in a p53-independent manner. Complementary to these findings, we discovered a potent downregulation of MDM2 by RITA. Using different approaches, we confirmed that MDM2 is not inhibited by RITA through proteasome degradation, autophagy or microRNAs-mediated translation inhibition. In addition, the inhibition of MDM2 is not the cause of cell death since both MDM2 overexpression and MDM2 KO could not rescue RITA killing effect. We conclude that, RITA dramatically inhibits RNA processing in cancer cells, leading to inhibition of transcription and translation, resulting in cell death.

Reactivation of p53 also has dark sides which are related to p53-mediated growth arrest or apoptosis in normal tissues. We investigated the mechanism of action of the well-known p53 inhibitor PFT-α and found that PFT-α cannot prevent p53 activation-induced growth repression in several cancer cell lines but can attenuate post-translational modifications (PTMs) of p53 and by that differentially inhibit p53 target genes. Although we found that PFT-α exhibits strong intracellular antioxidant activity through activation of AHR/NRF2 pathway, we cannot link the antioxidant activity to its capacity to attenuate PTMs of p53. Worth to note, both PFT-α and NAC can promote primary fibroblasts growth per se. Therefore, PFT-α rescued Nutlin-3-induced growth repression in primary fibroblasts. Our findings suggest that caution needs to be taken when using PFT-α to study p53 signaling cascade, since it is not a pan-p53 inhibitor as it is described. The phenomenon we observed with PFT-α in primary fibroblasts also indicates the clinical potential of combining p53 reactivators with PFT-α in cancer therapies.

List of scientific papers

I. Sylvain Peuget, Jiawei Zhu, Gema Sanz Santos, Madhurendra Singh, Massimiliano Gaetani, Xinsong Chen, Yao Shi, Amir Ata Saei, Torkild Visnes, Mikael Lindström, Ali Rihani, Lidia Moyano-Galceran, Joseph Carlson, Elisabet Hjerpe, Ulrika Joneborg, Kaisa Lehti, Johan Hartman, Thomas Helleday, Roman Zubarev, and Galina Selivanova. Thermal proteome profiling identifies oxidative-dependent inhibition of the transcription of major oncogenes as a new therapeutic mechanism for select anticancer compounds. Cancer Research. 2020 Feb 4. pii: canres.2069.2019.
https://doi.org/10.1158/0008-5472.CAN-19-2069

II. Jiawei Zhu, Madhurendra Singh, Galina Selivanova and.Sylvain Peuget. Pifithrin-α alters p53 post-translational modifications pattern and differentially inhibits p53 target genes. Scientific Reports. 2020 Jan 23;10(1):1049.
https://doi.org/10.1038/s41598-020-58051-1

III. Johannes Ristau, Vincent van Hoef, Sylvain Peuget, Jiawei Zhu, Bo-Jhih Guan, Shuo Liang, Maria Hatzoglou, Ivan Topisirovic, and Galina Selivanova & Ola Larsson. RITA requires eIF2α-dependent modulation of mRNA translation for its anti-cancer activity. Cell Death Dis. 2019 Nov 7;10(11):845.
https://doi.org/10.1038/s41419-019-2074-3