Loss of G9a preserves mutation patterns but increases chromatin accessibility, genomic instability and aggressiveness in skin tumours

Mutations in, and the altered expression of, epigenetic modifiers are pervasive in human tumours, making epigenetic factors attractive antitumour targets. The open-versus-closed chromatin state within the cells-of-origin of cancer correlates with the uneven distribution of mutations. However, the long-term effect of targeting epigenetic modifiers on mutability in patients with cancer is unclear. Here, we increased chromatin accessibility by deleting the histone H3 lysine 9 (H3K9) methyltransferase G9a in murine epidermis and show that this does not alter the single nucleotide variant burden or global genomic distribution in chemical mutagen-induced squamous tumours. G9a-depleted tumours develop after a prolonged latency compared with their wild-type counterparts, but are more aggressive and have an expanded cancer progenitor pool, pronounced genomic instability and frequent loss-of-function p53 mutations. Thus, we call for caution when assessing long-term therapeutic benefits of chromatin modifier inhibitors, which may promote more aggressive disease

Epigenomic characterization of human keratinocyte cancers and their cellular origin

Keratinocyte cancers (KC) are the most common malignancy in fair-skinned populations, with millions of cases diagnosed yearly. Although historically overlooked by cancer registries, their continuously rising incidence and the economic burden they pose on health systems have put KC in the spotlight. KC arise from epidermal keratinocytes and comprise two main tumor types: basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC). The latter mostly originates from pre-cancerous dysplasias named actinic keratosis (AK) or in situ carcinomas known as Bowen’s disease (BD). Two subclasses of AK/cSCC were previously identified based on their DNA methylation profiles, reflecting two distinct cells-of-origin. This notion was further explored in this thesis by epigenomically characterizing 102 epidermal samples using Infinium MethylationEPIC BeadChips. This cohort included healthy controls, AK, cSCC, BD, BCC, and non-cancerous senile warts (seborrheic keratosis; SK), thus comprising the full range of malignancy in epidermal tumors. Methylation patterns at keratinocyte-specific enhancers stratified the samples into two subclasses with epidermal stem cell-like or keratinocyte-like profiles. While SK and BCC samples predominantly displayed keratinocyte-like profiles, cSCC and its precursors displayed both, indicating potential clinical implications. Further analyses indicated distinct cell division rates and invasive features between cell-of-origin subclasses. Lastly, single-cell methylomics and transcriptomics validated the cell-of-origin-based stratification of epidermal tumors. Dermal fibroblasts are a heterogeneous cell population with essential roles in maintaining skin architecture and function. However, their diversity in human skin and its functional significance are still relatively unclear. In this work, fibroblast heterogeneity was systematically analyzed using the single-cell transcriptomes of more than 5,000 fibroblasts from sun-protected healthy human skin. Four distinct subpopulations with specific localization and primed to exert differential secretory, mesenchymal, and pro-inflammatory roles were identified. Importantly, this priming was substantially reduced upon intrinsic aging. Collectively, this work provides important insight into the cellular origin of KC, establishes new opportunities for robust patient risk assessment, and represents a detailed analysis of human dermal fibroblast heterogeneity and their intrinsic aging

Functionally distinct cancer-associated fibroblast subpopulations establish a tumor promoting environment in squamous cell carcinoma

Abstract Cutaneous squamous cell carcinoma (cSCC) is a serious public health problem due to its high incidence and metastatic potential. It may progress from actinic keratosis (AK), a precancerous lesion, or the in situ carcinoma, Bowen’s disease (BD). During this progression, malignant keratinocytes activate dermal fibroblasts into tumor promoting cancer-associated fibroblasts (CAFs), whose origin and emergence remain largely unknown. Here, we generate and analyze >115,000 single-cell transcriptomes from healthy skin, BD and cSCC of male donors. Our results reveal immunoregulatory and matrix-remodeling CAF subtypes that may derive from pro-inflammatory and mesenchymal fibroblasts, respectively. These CAF subtypes are largely absent in AK and interact with different cell types to establish a pro-tumorigenic microenvironment. These findings are cSCC-specific and could not be recapitulated in basal cell carcinomas. Our study provides important insights into the potential origin and functionalities of dermal CAFs that will be highly beneficial for the specific targeting of the cSCC microenvironment

Differentiation‐related epigenomic changes define clinically distinct keratinocyte cancer subclasses

Abstract Keratinocyte cancers (KC) are the most prevalent malignancies in fair‐skinned populations, posing a significant medical and economic burden to health systems. KC originate in the epidermis and mainly comprise basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC). Here, we combined single‐cell multi‐omics, transcriptomics, and methylomics to investigate the epigenomic dynamics during epidermal differentiation. We identified ~3,800 differentially accessible regions between undifferentiated and differentiated keratinocytes, corresponding to regulatory regions associated with key transcription factors. DNA methylation at these regions defined AK/cSCC subtypes with epidermal stem cell‐ or keratinocyte‐like features. Using cell‐type deconvolution tools and integration of bulk and single‐cell methylomes, we demonstrate that these subclasses are consistent with distinct cells‐of‐origin. Further characterization of the phenotypic traits of the subclasses and the study of additional unstratified KC entities uncovered distinct clinical features for the subclasses, linking invasive and metastatic KC cases with undifferentiated cells‐of‐origin. Our study provides a thorough characterization of the epigenomic dynamics underlying human keratinocyte differentiation and uncovers novel links between KC cells‐of‐origin and their prognosis

Resolving the spatial architecture of myeloma and its microenvironment at the single-cell level

Abstract In multiple myeloma spatial differences in the subclonal architecture, molecular signatures and composition of the microenvironment remain poorly characterized. To address this shortcoming, we perform multi-region sequencing on paired random bone marrow and focal lesion samples from 17 newly diagnosed patients. Using single-cell RNA- and ATAC-seq we find a median of 6 tumor subclones per patient and unique subclones in focal lesions. Genetically identical subclones display different levels of spatial transcriptional plasticity, including nearly identical profiles and pronounced heterogeneity at different sites, which can include differential expression of immunotherapy targets, such as CD20 and CD38. Macrophages are significantly depleted in the microenvironment of focal lesions. We observe proportional changes in the T-cell repertoire but no site-specific expansion of T-cell clones in intramedullary lesions. In conclusion, our results demonstrate the relevance of considering spatial heterogeneity in multiple myeloma with potential implications for models of cell-cell interactions and disease progression