Biological analysis of the in-vitro effects of homologous recombination inhibition and its use in cancer treatment through synthetic lethality

Synthetic lethality represents an anticancer strategy that targets tumor specific gene defects. One of the most studied application is the use of PARP inhibitors (e.g. olaparib) in BRCA1/2-less cancer cells. In BRCA2-defective tumors, olaparib (OLA) inhibits DNA single-strand break repair, while BRCA2 mutations hamper homologous recombination (HR) repair. The simultaneous impairment of those pathways leads BRCA-less cells to death by synthetic lethality. The projects described in this thesis were aimed at extending the use of OLA in cancer cells that do not carry a mutation in BRCA2 by combining this drug with compounds that could mimic a BRCA-less environment via HR inhibition. We demonstrated the effectiveness of our “fully small-molecule induced synthetic lethality” by using two different approaches. In the direct approach (Project A), we identified a series of neo-synthesized compounds (named RAD51-BRCA2 disruptors) that mimic BRCA2 mutations by disrupting the RAD51-BRCA2 interaction and thus the HR pathway. Compound ARN 24089 inhibited HR in human pancreatic adenocarcinoma cell line and triggered synthetic lethality by synergizing with OLA. Interestingly, the observed synthetic lethality was triggered by tackling two biochemically different mechanisms: enzyme inhibition (PARP) and protein-protein disruption (RAD51-BRCA2). In the indirect approach (Project B), we inhibited HR by interfering with the cellular metabolism through inhibition of LDH activity. The obtained data suggest an LDH-mediated control on HR that can be exerted by regulating either the energy supply needed to this repair mechanism or the expression level of genes involved in DNA repair. LDH inhibition also succeeded in increasing the efficiency of OLA in BRCA-proficient cell lines. Although preliminary, these results highlight a complex relationship between metabolic reactions and the control of DNA integrity. Both the described projects proved that our “fully small-molecule-induced synthetic lethality” approach could be an innovative approach to unmet oncological needs.

Convergent bioenergetic defects in Coenzyme Q10 depleted cells by pharmacological inhibition of coq2 enzyme (p-hydroxybenzoate polyprenyl transferase) and by genome editing technology targeting the encoding gene (COQ2)

Primary CoQ10 deficiency diseases encompass a heterogeneous spectrum of clinical phenotypes. Among these, defect or mutation on COQ2 gene, encoding a para-hydroxybenzoate polyprenyl transferase, have been associated with different diseases. Understanding the functional and metabolic impact of COQ2 mutation and the consequent CoQ10 deficiency is still a matter of debate. To date the aetiology of the neurological phenotypes correlated to CoQ10 deficiency does not present a clear genotype-phenotype association. In addition to the metabolic alterations due to Coenzyme Q depletion, the impairment of mitochondrial function, associated with the reduced CoQ level, could play a significant role in the metabolic flexibility of cancer. This study aimed to characterize the effect of varying degrees of CoQ10 deficiency and investigate the multifaceted aspect of CoQ10 depletion and its impact on cell metabolism. To induced CoQ10 depletion, different cell models were used, employing a chemical and genome editing approach. In T67 and MCF-7 CoQ10 depletion was achieved by a competitive inhibitor of the enzyme, 4-nitrobenzoate (4-NB), whereas in SH-SY5Y the COQ2 gene was edited via CRISPR-Cas9 cutting edge technology.