Discovery of First-In-Class inhibitors of a DNA damage repair pathway

Abstract

Cancer is the second leading cause of death globally. The gold standard for cancer has historically been surgery, radiotherapy and chemotherapy. Although the efficacy of chemotherapy has been broadly confirmed, drug resistance is still one of the main causes of disease relapse and treatment failure. For this reason, new therapeutic strategies directed at targeting the key factors that transform normal cells and tissues into malignancies have been developed. Nevertheless, eventual resistance to targeted and immunological therapies has also emerged, leading to the necessity of additional targeted therapeutic approaches. Recent advances in genome sequencing have revealed vulnerabilities within cancer cells that may be exploited to selectively target the tumour with therapeutics. As one of the mechanisms that cancer cells use to develop chemo-resistance is through the acquisition of a better DNA damage capacity, several targeted disruptors of DNA damage response (DDR) pathways have been developed. This has been shown to re-sensitize cancer cells to chemotherapy, being PARP inhibitors the most well-known case. After detecting that the restoration of a specific DDR pathway is able to abolish sensitivity and lead to chemo- resistance, we decided to screen for inhibitors of this pathway by means of molecular docking and posterior cellular validation. Thanks to the availability of atomic-resolution structures of some of the proteins, we looked for compounds that disrupted a critical PPI within the pathway that resulted in a post-translational modification. The capacity of these compounds to inhibit the post-translational modification was validated in cell models and their ability to interact with one of the proteins intervening in the PPI was also tested by surface plasmon resonance. The best candidates (F27 and F2.14) were then virtually optimized in two successive rounds and also validated. F27 was able to chemo-sensitize cancer cells to cisplatin in clonogenic assays. F27 and F2.14 also showed greater cytotoxicity in cells mutated for a specific gene, what suggests that a future drug inhibitor of this pathway could be applied to specifically treat tumours with this mutation using the synthetic lethality concept. In parallel, we studied potential applications of our candidates. One of these applications was in terms of synthetic lethality. Many synthetic lethal interactions with our target DDR pathway have been reported. Initially, we validated several of them in our laboratory. Then, in order to identify new potential synthetic lethal genes, we analysed gene co-expression correlations included in cBioPortal between more than 22,000 genes and several upstream genes from this DDR pathway in different cancer types. We identified several genes that were commonly found co-expressed. Some of these synthetic lethal interactions had already been reported, and the rest represented potential new synthetic lethal genes. Furthermore, in order to estimate the potential market size of an inhibitor of this pathway we studied the annual cases of patients with solid tumours and haematologic malignancies with alterations on these synthetic lethal genes in Europe and North America. Another application that we tested was the potential synergy of our inhibitors and immunotherapy. We hypothesized that the dysregulation in several genes from this pathway that had been found altered in cancer may result in high expression of immune checkpoint ligands. In order to find correlations between inhibitory immune checkpoint ligands and these genes, we initially analysed their co-expression in different cancer types and we found several of them co-expressed with genes from this pathway. Afterwards, we validated these results using cell lines deficient for our target pathway, but we could only observe a small increase in the expression of two of these immune checkpoint ligands. In parallel, we found that F2.14 increased the expression of two immune checkpoint ligands in U2OS and HeLa cells.

Date of Award	19 Dec 2022
Original language	English
Awarding Institution	Universitat de Barcelona (UB)
Supervisor	Jordi Minguillon Pedreño (Director), Jordi Surralles Calonge (Director) & Virginia Nunes Martínez (Tutor)