Kinome siRNA screening for the treatment of the XPC cancer-prone disease

Xeroderma Pigmentosium C is a rare autosomal recessive genodermatosis. Patients with this disorder carry a mutation in DNA damage recognition protein XPC belonging to the nucleotide excision repair (NER) pathway. This generates a phenotype characterized by extreme photosensitivity and accumulation of UV-induced DNA damage without a repair potential. XP-C and normal patient-derived cells were used to screen a library of siRNAs aimed at decreasing the expression of all human kinases, given their involvement 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 decreased photosensitivity or for DNA damage 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. 1292 different siRNAs were used to treat XPC cells out of which 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, PIK3C3, had an exclusive effect in the photoprotection of XPC cells and not normal cells. The knockdown of the kinase expression was validated by PCR and western blot. The photoprotective 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 was 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.