Thesis presented October 13, 2023

Abstract:
Xeroderma pigmentosum group C (XPC) is a multifunctional DNA damage recognition protein involved in the global genome nucleotide excision repair (GG-NER) pathway. Mammalian cells employ this pathway to eliminate bulky DNA lesions caused by mutagens such as UV radiation. Loss-of-function mutations in the XPC gene lead to various malignancies, including skin cancers. Photosensitivity and the buildup of DNA damage can both define the phenotype of XP-C patients’ cells. We carried out, for the first time, XPC gene editing in several human immortalized skin cells (keratinocytes, fibroblasts, and melanocytes) using ribonucleoprotein CRISPR-Cas9 technology, where we had a tremendous editing efficacy. Characterization of the XPC gene mutation was validated at the mRNA and protein expression levels, and at the DNA level via sequencing. We showed that human skin cells edited for the XPC gene recapitulate the phenotypes of XPC mutations: photosensitivity and impairment of UV-damage repair.
Following that, wild-type and XPC KO keratinocytes were utilized to quantify the kinases phosphorylation activity at basal state and at an early UVB irradiation exposure period. One hour post UVB irradiation could dysregulate 104 protein tyrosine kinases in XPC KO keratinocytes versus wild type. To better decipher the signaling pathways implicated, mass spectrometry-based quantitative proteomics analyzed the total proteomes of both wild-type and XPC KO cells subjected or not to UVB irradiation at a late period of time exposure.
Following bioinformatic analysis for all the significant datasets and following western blot validation, we identified a unique and complete dysregulation of type 1 interferon response mediated by JAK/STAT signaling in XPC KO keratinocytes. To identify novel hits for therapeutics, a library of siRNAs targeting all the human phosphatases was used to screen XPC KO keratinocytes and then to detect whether a phenotypic reversal occurs by means of increasing the photoresistance following UVB irradiation. Out of 894 distinct siRNAs treated, 27 different siRNAs were selected based on their ability to increase cellular viability compared to controls by means of robust z-score value (>2). Indeed, part of these hits seem to be negative regulators of the EGFR-MAPK pathway, and their knockdown tends to induce a proliferation rescue. Furthermore, preliminary results on a kinase screening identified two kinase hits LATS1 and PIK3C3 (Farah Kobaisi PhD) that can ameliorate the repair potential of XPC mutated fibroblasts. One kinase, PIK3C3, had no effect on normal cells and only had an impact on the photoprotection of XPC-mutated fibroblasts. Results were confirmed on XPC KO keratinocytes. Finally, to better mimic the niche of this disease, XPC KO keratinocytes, fibroblasts, and melanocytes were utilized to construct a 3D reconstructed XPC KO skin model. This model exhibited an inflammatory behavior and UVB exposure was capable of causing invasions from the epidermal to the dermal part and extensive epidermal differentiation. Based on our data and observations, XP-C disease is an excellent model for understanding skin cancer initiation, as it is accelerated in these cells. By understanding the etiology, we can therefore find ways to delay or prevent cancer initiation.

Keywords:
CRISPR-Cas9, Cell signaling pathways, Xeroderma Pigmentosum C, siRNA screening, Proteomics and kinases activity, 3D XPC skin model