Ubiquitinomic profiling of CYLD cutaneous syndrome skin tumours identifies an insulin signalling axis

CYLD cutaneous syndrome (CCS) is an inherited hair follicle tumour syndrome driven by biallelic loss of function pathogenic variants in the deubiquitinase (DUB) CYLD. CCS tumour cells hence represent a patient relevant model to gain insights into the ubiquitinomic consequences of loss of function of this important DUB. We utilised ubiquitinomic profiling to detect enriched ubiquitin tagged substrates in CCS tumours. We complemented these data with CCS tumour keratinocyte proteome and single cell transcriptome data to develop a multi-omic model which encapsulated the CCS tumour microenvironment. These data led us to discover an altered insulin signalling axis in CCS tumours.

CCS tumour tissue and healthy skin control samples were collected and flash frozen for ubiquitinomic analysis. Tissue slices were lysed, and trypsin digested with the resulting peptides undergoing di-glycine immuno-purification. Additionally, we processed fresh tumour tissue and control skin, by enzymatic digestion and flow cytometric sorting. We assessed the proteome of CCS tumour initiating CD45- CD200+ keratinocytes and prepared CD45- cells for single cell transcriptomic analysis using the 10X workflow. Tryptic peptides were analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) on a Bruker Tims-ToF HT mass spectrometer, utilising a data independent acquisition method. Peptide identification was performed by searching MS data against an in-silico neural network derived spectral library with di-glycine modification.

We identified 1273 ubiquitination sites across 649 proteins in CCS tumour and control samples through ubiquitinomic analysis. We found 305 ubiquitination sites that were either unique to their respective condition or differentially regulated after performing t-tests (p<0.05). Analysis of ubiquitinated peptide sequences uncovered cell surface receptors with increased ubiquitination in CCS tumour samples including the insulin receptor which had ubiquitination modifications at its kinase domain. Immunofluorescence staining of cylindroma sections revealed localisation of the insulin receptor to central regions of cylindroma "islands". We interrogated a CCS single cell transcriptomics dataset (n=3 tumours; k=19,754) and a CCS CD45- CD200+ proteomic dataset (n=5 tumours) to delineate insulin signalling. Across these datasets we demonstrated evidence of an insulin signalling axis, with increased expression of IGF1 in CCS tumour associated fibroblasts, and IGF1R, INSR, IGFBP3, and MMP7 in CCS tumour keratinocytes. Our findings delineate the ubiquitinome of CCS keratinocytes and highlight the insulin receptor as a putative target of CYLD in the CCS tumour microenvironment.