Identification of novel genes and compounds for the development of precision therapeutics for dystrophic epidermolysis bullosa and associated cutaneous squamous cell carcinoma
2021-08
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Identification of novel genes and compounds for the development of precision therapeutics for dystrophic epidermolysis bullosa and associated cutaneous squamous cell carcinoma
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2021-08
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Abstract
Dystrophic epidermolysis bullosa (DEB) is a skin blistering disease caused by dominant (DDEB) or recessive (RDEB) mutations in the COL7A1 gene, with the latter being more severe. COL7A1 encodes type VII collagen (C7), which aggregates into structures called anchoring fibrils that maintain skin integrity by securing the epidermis to the dermis. DEB is characterized by shearing and blistering of the skin (and various mucosae) at the level of the superficial dermis. In severe cases of RDEB, this leads to fibrosis, scarring, and aggressive cutaneous squamous cell carcinoma (RDEBSCC). Using a combination of high-throughput sequencing and genome-wide screening tools we identified novel (1) genes and compounds that increase C7 production in wild-type and RDEB keratinocytes, (2) genes that become progressively dysregulated with RDEB disease progression, and (3) genes that drive or inhibit proliferation in wild-type, RDEB, and RDEBSCC keratinocytes. In Chapter 2, we start by describing the creation and validation of a keratinocyte C7 reporter cell line, in which C7 production is linked to tdTomato fluorescence. We used this reporter line to perform a genome wide CRISPR activation (CRISPRa) screen to identify genes that increase C7 production in keratinocytes. There were 1544 CRISPRa single guide RNAs (sgRNAs), targeting 1464 distinct genes, whose abundance was substantially increased in the top 10% of tdTomato-expressing cells relative to the plasmid DNA library. Validation of the top two candidates identified in this screen, DENND4B and TYROBP, showed that CRISPRa-mediated upregulation of these genes significantly increased tdTomato fluorescence and C7 protein production, but not COL7A1 mRNA. Pathway analysis of the 1464 gene targets identified significantly enriched upstream regulators, signaling pathways, and biological functions. We performed a targeted drug screen using compounds that act on some of these upstream regulators and signaling pathways and found that kaempferol, a plant flavonoid, was able to increase COL7A1 mRNA and C7 protein in both wild-type and RDEB keratinocytes.
In Chapter 3, we investigate potential mechanisms of RDEBSCC development. We performed RNA-sequencing on nine sibling pairs of wild-type and RDEB keratinocytes (WTK, RDEBK) and six RDEBSCC cell lines and identified numerous genes whose expression progressively increased or decreased from WTKs to RDEBKs to RDEBSCCs, suggesting that these genes could be involved in disease progression. To identify inhibitors of proliferation in keratinocytes and RDEBSCCs, we performed CRISPRi proliferation screens in one WTK (NTERT) and three RDEBSCC (RDEBSCC2, 53, and 70) cell lines. There were 53 sgRNAs that were enriched in all four CRISPRi screens, including all three sgRNAs targeting the TAFA3 gene. To identify drivers of proliferation in keratinocytes, we performed CRISPRa proliferation screens in one RDEBK (RDEBK8) and two WTK (NTERT and WTK1) cell lines. No sgRNAs were enriched in all three screens, but there was some overlap across each of the three possible pairs. We performed competition assays to validate some of the top hits from the CRISPRa and CRISPRi screens and found that inhibition of PTK2B, QPRT, STAT2, or TAFA3 expression and upregulation of ADAM2, CDYL2, CSF3R, DENND4B, FSTL1, GCSAM, ITGB1, ITGB3, KLHDC8A, KRT33B, SEMA5A, or TYROBP expression significantly increased proliferation in NTERT keratinocytes.
Using a combination of RNA-sequencing and genome wide CRISPRai screens, we identified (1) genes that promote C7 production, (2) a novel strategy for increasing C7 production in RDEB skin, (3) genes that were progressively dysregulated from WTK to RDEBK to RDEBSCC, (4) genes that drive proliferation in wild-type and/or RDEB keratinocytes, and (5) genes that inhibit proliferation in keratinocytes and RDEBSCC cells. We also created and validated a keratinocyte C7 reporter cell line that could be used in future DEB research. Overall, this research has led to the discovery of numerous previously unexplored avenues of investigation in dystrophic epidermolysis bullosa research and a multitude of new gene targets for the development of novel targeted therapeutics.
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University of Minnesota Ph.D. dissertation. 2021. Major: Molecular, Cellular, Developmental Biology and Genetics. Advisor: Jakub Tolar. 1 computer file (PDF); vi, 134 pages.
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Pickett-Leonard, Michael. (2021). Identification of novel genes and compounds for the development of precision therapeutics for dystrophic epidermolysis bullosa and associated cutaneous squamous cell carcinoma. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/224980.
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