Synthetic rescue of XPC phenotype via PIK3C3 downregulation

Abstract

Xeroderma Pigmentosum C is a dermal hereditary disease. It is caused by a mutation in the DNA damage recognition protein XPC that belongs to the Nucleotide excision repair pathway. XPC patients exhibit a photosensitive phenotype and fail to repair UV induced DNA lesions leading to their accumulation and ultimate conversion to mutations and carcinomas. In an attempt to normalize this phenotype, we screened a library of siRNAs targeting the human kinases, given their role in different DNA repair pathways. WT and XPC immortalized patient fibroblasts were transfected with the library then irradiated with UVB to induce DNA damage. XPC phenotypic reversal was monitored by the quantification of decreased photosensitivity and increased DNA damage repair. Out of the 1292 kinase siRNAs tested, twenty-eight were selected cellular survival compared to cells transfected with non-targeting siRNA in XP-C irradiated cells. Out of the selected hits, two kinases, PIK3C3 and LATS1, induce more than 20% repair of 6-4PP DNA lesions. The down regulation of autophagy-related protein PIK3C3 alone had an exclusive photo protective effect on XP-C irradiated cells validated these effects also on primary XP-C patient fibroblasts and CRISPR-Cas9 generated XPC-KO keratinocytes. PIK3C3 knock down in XP-C cells ameliorated in UVB dose response analysis, decreased apoptosis and lowered phosphorylation of P53 with no effect on proliferation. More importantly, PIK3C3 knock down induced an increase in UVRAG expression, a previously reported cDNA conveying lower photosensitivity in XP-C cells. Attempts to improve the XPC photosensitive and deficient repair phenotype using PIK3C3 inhibitors could thus pave a way for new therapeutic approaches delaying or preventing tumor initiation.