Recessive dystrophic epidermolysis bullosa (RDEB) is a rare inherited skin and mucous membrane fragility disorder complicated by early-onset, highly malignant cutaneous squamous cell carcinomas (SCCs). The molecular etiology of RDEB SCC, which arises at sites of sustained tissue damage, is unknown. We performed detailed molecular analysis using whole-exome, whole-genome, and RNA sequencing of 27 RDEB SCC tumors, including multiple tumors from the same patient and multiple regions from five individual tumors. We report that driver mutations were shared with spontaneous, ultraviolet (UV) light-induced cutaneous SCC (UV SCC) and head and neck SCC (HNSCC) and did not explain the early presentation or aggressive nature of RDEB SCC. Instead, endogenous mutation processes associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) deaminases dominated RDEB SCC. APOBEC mutation signatures were enhanced throughout RDEB SCC tumor evolution, relative to spontaneous UV SCC and HNSCC mutation profiles. Sixty-seven percent of RDEB SCC driver mutations was found to emerge as a result of APOBEC and other endogenous mutational processes previously associated with age, potentially explaining a >1000-fold increased incidence and the early onset of these SCCs. Human papillomavirus-negative basal and mesenchymal subtypes of HNSCC harbored enhanced APOBEC mutational signatures and transcriptomes similar to those of RDEB SCC, suggesting that APOBEC deaminases drive other subtypes of SCC. Collectively, these data establish specific mutagenic mechanisms associated with chronic tissue damage. Our findings reveal a cause for cancers arising at sites of persistent inflammation and identify potential therapeutic avenues to treat RDEB SCC.

Current gene-editing approaches for treatment of recessive dystrophic epidermolysis bullosa (RDEB), an inherited, severe form of blistering skin disease, suffer from low efficiencies and safety concerns that complicate implementation in clinical settings. We present a strategy for efficient and precise repair of RDEB-associated mutations in the COL7A1 gene. We compared the efficacy of double-strand breaks (induced by CRISPR/Cas9), single nicks, or double nicks (induced by Cas9n) in mediating repair of a COL7A1 splice-site mutation in exon 3 by homologous recombination (HR). We accomplished remarkably high HR frequencies of 89% with double nicking while at the same time keeping unwanted repair outcomes, such as non-homologous end joining (NHEJ), at a minimum (11%). We also investigated the effects of subtle differences in repair template design on HR rates and found that strategic template-nicking can enhance COL7A1-editing efficiency. In RDEB patient keratinocytes, application of double-nicking led to restoration and subsequent secretion of type VII collagen at high efficiency. Comprehensive analysis of 25 putative off-target sites revealed no off-target activity for double-nicking, while usage of Cas9 resulted in 54% modified alleles at one site. Taken together, our work provides a framework for efficient, precise, and safe repair of COL7A1, which lies at the heart of a future curative therapy of RDEB.

Mutations in the gene encoding collagen VII cause the devastating blistering disease recessive dystrophic epidermolysis bullosa (RDEB). RDEB is characterized by severe skin fragility and nonhealing wounds aggravated by scarring and fibrosis. We previously showed that TSP1 is increased in RDEB fibroblasts. Because transforming growth factor-b (TGF-b) signaling is also increased in RDEB, and TSP1 is known to activate TGF-b, we investigated the role of TSP1 in TGF-b signaling in RDEB patient cells. Knockdown of TSP1 reduced phosphorylation of smad3 (a downstream target of TGF-b signaling) in RDEB primary fibroblasts, whereas overexpression of collagen VII reduced phosphorylation of smad3. Furthermore, inhibition of TSP1 binding to the LAP/TGF-b complex decreased fibrosis in engineered extracellular matrix formed by RDEB fibroblasts, as evaluated by picrosirius red staining and analyses of birefringent collagen fibrillar deposits. We show that collagen VII binds TSP1, which could potentially limit TSP1-LAP association and subsequent TGF-b activation. Our study suggests a previously unreported mechanism for increased TGF-b signaling in the absence of collagen VII in RDEB patient skin. Moreover, these data identify TSP1 as a possible target for reducing fibrosis in the tumor-promoting dermal microenvironment of RDEB patients.