Resumen de Discovery of synthetic lethal or viable interactions between dna repair pathways
Mammalian cells have developed a set of DNA repair pathways to protect the genomic information from mutations that could lead to cancer or cell death. Each DNA repair pathway responds to a specific kind of DNA damage. The Fanconi/BRCA pathway is one of these specialized and highly important DNA repair networks correcting interstrand cross-links that would lead to highly toxic double strand breaks if left unrepaired. Mutations in the Fanconi/BRCA pathway cause a rare blood and cancer susceptibility disease called Fanconi Anaemia. The concept of synthetic lethality is used in cancer treatment to kill specifically cancer cells. Synthetic lethality refers to the inhibition/deficiency of two gene products that leads to cell death while inhibition of either one alone does not affect viability. The treatment of BRCA-deficient breast and ovarian cancers with PARP inhibitors is an application of this concept, which proved successful in clinical trials. Fanconi genes are found regularly mutated in cancer and so far, there are a few described synthetic lethal interactions in the Fanconi pathway, which opens the possibility of exploiting this synthetic lethality for cancer treatment. The term "synthetic viability" instead, refers to a genetic interaction in which a cell that is non-viable, sensitive to a specific drug, or altered due to the presence of a genetic mutation, becomes viable with a second mutation in a different gene. The objective of the thesis was to find novel synthetic lethal or viable interactions between the Fanconi proteins FANCA and FANCD2 and other DNA repair pathways. For this a method was developed, the colour competition assay that detects synthetic lethality or viability by cytometer measurement. With the CRISPR/Cas9 method knock-out clones for FANCA and FANCD2 were generated and used with shRNA for silencing a second gene in a DNA repair pathway. Additionally, commercial or patient cell lines with known knock-outs in genes of other DNA repair pathways were employed and treated with shRNA to test for synthetic lethality or viability with FANCA or FANCD2. The colour competition assay could not discover any novel synthetic lethal interaction, it revealed a synthetic viability interaction between FANCA and FEN1 that and a second synthetic viability between FANCA and two proteins of the NER pathway, XPA and XPF. This discovery could lead to potential therapeutic applications in Fanconi anaemia treatment.
