Thesis presented July 15, 2021

Abstract:
Xeroderma Pigmentosium C (XP-C) is a rare autosomal recessive genodermatosis. Patients with this disorder carry a mutation in the DNA damage recognition protein XPC belonging to the nucleotide excision repair (NER) pathway. This mutation generates a pathological phenotype characterized by extreme photosensitivity and accumulation of UV-induced DNA damage without repair. XP-C and normal patient-derived cells will be used to screen a chemical library of 1280 FDA-approved drugs or a library of siRNAs aimed at decreasing the expression of all human kinases, given the involvement of kinases in different DNA repair pathways. Cells will then be irradiated with UVB to induce DNA damage, and then the phenotypic reversal will be monitored for photosensitivity or for DNA damage repair to visualize which treatment results in a decrease in the level of damage and thus repair. Further characterization of the effects of the hits will then be performed at the level of apoptosis and proliferation, by flow cytometry, and of downstream signaling via western blot. The screening procedure was performed on XPC and normal cell lines. For chemical screening, 1280 different drugs were used to treat cells in duplicate in the wells of a 96-well plate. After induction of DNA damage, the cells were also post-treated with the same drugs and then incubated for twenty-four hours to allow recovery. The viability of treated cells was measured and compared to the positive control of untreated unirradiated cells and the negative control of untreated irradiated cells. Of the drugs tested in the library, sixteen drugs resulted in an increase of more than 25% in cell viability. Then, after DNA damage labeling and quantification, two drugs, isoconazole and clemizole hydrochloride, decreased the amount of DNA damage by 20%. Further characterization of these two drugs by separating the treatment into pre-irradiation or post-irradiation treatment revealed that post-irradiation was more effective in inducing photoresistance in XPC cells. One of the two drugs, isoconazole, reduced apoptotic death of XPC cells but had no effect on their proliferation rate. The known mode of action of the two selected drugs is an antifungal for isoconazole and a histamine receptor antagonist for clemizole hydrochloride. This does not explain their effects in protecting XPC cells from irradiation-induced death. Therefore, further investigation of the possible mechanism of action is needed by comparing the protein expression profile of treated and untreated cells to identify a possible new mechanism of action for these two drugs. As for biological screening. A library of siRNAs targeting all human kinases was used to treat XPCs and normal cells, and then to detect whether a phenotypic reversal occurs. 1292 different siRNAs were used to treat XPC cells to irradiate them and allow them to recover for 24 hours before measuring their viability. Twenty-eight different siRNAs were selected based on their ability to induce a 25% increase in cell viability compared to controls transfected with non-targeted siRNA. Among them, targeting two kinases resulted in a 20% repair of induced DNA damage. One kinase in particular had an exclusive effect in photoprotection of XPC cells and no effect on normal cells. The knock down of the kinase expression induced by this siRNA was validated at the mRNA level by PCR and at the protein level by western blot. The photo-protective effect of this kinase knockdown in XPC cells was also validated in a UVB response assay where it was able to induce higher viability in transfected XPC cells compared to non-transfected cells as a function of increasing UVB doses. The regulation of downstream signaling induced by the knockdown of this kinase is also studied by western blot. XPC is an excellent model to understand skin cancer initiation, as it is accelerated in these cells. By improving this phenotype, we can therefore find ways to delay or prevent cancer initiation.

Keywords:
RNAi, screening, XPC, chemical, skin cancer