Journal of Investigative Dermatology

Skin, the bodys largest mechanosensitive organ, relies on a tension gradient across epidermal layers to maintain structure and function, but how mechanical force contributes to epidermal development and disease pathogenesis is poorly understood. By anchoring intermediate filaments (IF) to the plasma membrane, desmosomes, the most abundant intercellular junctions in the epidermis, help create a supracellular scaffolding that provides mechanical resilience to the tissue. However, the contribution of the desmosome-IF network to the epidermal response to mechanical strain remains unknown. Here we show that the desmosome-IF connection is not only required to induce a proper cellular mechano-response but is actively strengthened in response to stretch through the PP2A-mediated phospho-regulation of the cytoskeletal linker protein desmoplakin (DP). Additionally, we show in human skin dephosphorylated DP localizes to high tension layers, suggesting this mechano-response mechanism is coordinated with the epidermal tension gradient. Furthermore, in models of Carvajal syndrome, a cardio-cutaneous disorder caused by truncating DP mutations, cells lose mechano-responsive behavior and exhibit abnormal morphology in high-tension epidermal layers. Together, these findings identify the DP-IF network as a key component of the response to mechanical strain and show that its disruption compromises epidermal homeostasis and contributes to disease pathogenesis.

Introduction: There is an unmet need for effective topical anti-pruritic medications for acute itch, as there are only a few over-the-counter products that have a direct effect on itch. Tripelennamine is a first-generation antihistamine that would be useful in treating histamine-induced pruritus, however, supportive robust clinical data is lacking. Objectives: The efficacy of tripelennamine (TPA) compared to diphenhydramine (DPH) and a vehicle control cream base on histamine-induced pruritus was evaluated as the primary endpoint. Histamine-induced urticaria served as the secondary endpoint. Methods: Thirty-six healthy participants completed this single-center, double-blinded, placebo-controlled crossover clinical study. Following pretreatment with TPA1%, DPH 1% or vehicle control creams, histamine challenge occurred via iontophoresis and a visual analog scale (VAS) for pruritus was used to determine extent of itch (AUC-VAS), peak itch, and duration of itch. Results: Compared to the vehicle control, TPA reduced histamine-induced extent of itch (AUC-VAS), peak itch, and itch duration by 59%, 38% and 43%, respectively (p<0.01 all). DPH did not significantly affect these responses and TPA was superior in reducing extent of itch (48% reduction, p<0.05) and duration (38% shorter, p<0.05). TPA, but not DPH, also reduced histamine induced flare and wheal responses (secondary endpoints) by 53% and 27%, respectively. The reduction in flare responses by TPA was superior to that of DPH (45% reduction, p<0.05). Conclusion: TPA significantly attenuated histamine-induced pruritus and urticaria in a human histamine-challenge model and demonstrated greater efficacy than DPH. These findings provide strong evidence of the antipruritic activity of topical TPA and support further clinical investigation of TPA as a treatment for histaminergic itch and related dermatologic conditions.

Endothelial dysfunction is a hallmark of numerous vascular pathologies and is strongly influenced by mechanobiological forces within the vascular microenvironment. While the effects of shear stress have been extensively investigated, the mechanisms by which elevated hydrostatic pressure regulates endothelial junctional organisation remain sparsely investigated. Here, we employed a microfluidic platform to investigate the combined effects of low shear stress (1.4 dyne/cm2) and elevated hydrostatic pressure (~3972 Pa) on endothelial junctional dynamics. Elevated hydrostatic pressure induced marked remodelling of VE-cadherin junctions, characterised by formation of serrated, finger-like structures accompanied by increased YAP1 nuclear localisation and reduced YAP1-VE-cadherin cytoplasmic colocalisation compared to shear stress alone conditions. Further, elevated hydrostatic pressure also demonstrated an increase in cytoplasmic accumulation of EPS8, an actin adaptor protein, and increased cytoplasmic EPS8-VE-cadherin colocalisation. These observations were accompanied by functional changes marked by increased endothelial permeability, and enhanced THP-1 monocyte adhesion, thus suggesting activation of mechanosensitive pathways linked to dynamic junctional reorganisation. Inhibition of PI3K at elevated hydrostatic pressure exhibited a thin VE-cadherin patterning and increased cytoplasmic EPS8-VE-cadherin colocalisation, thus demonstrating a prominent role for PI3K signalling in regulating the junction organisation. Interestingly, Piezo-1 activation using Yoda1 produced context-dependent effects. Under shear stress alone, Yoda1 promoted YAP1 nuclear translocation, reduced YAP1-VE-cadherin colocalisation, increased endothelial permeability but strikingly did not impact THP-1 adhesion compared to shear stress alone conditions. In contrast, under elevated hydrostatic pressure conditions, Yoda1 significantly reduced both endothelial permeability and THP-1 adhesion while increasing YAP1-VE-cadherin colocalisation and decreasing YAP1 nuclear accumulation. Collectively, these findings identify a previously underappreciated elevated hydrostatic pressure-Piezo-1-PI3K signalling axis that regulates endothelial barrier integrity and pro-adhesive endothelial activation through coordinated regulation of VE-cadherin, YAP1, and EPS8. These results highlight elevated hydrostatic pressure as a unique mechanobiological stimulus, distinct from that of shear stress alone and provide novel insights into mechanisms underlying microvascular dysfunction.

Background: Pressure ulcers often develop complex three-dimensional morphologies that extend beyond the visible wound surface. Subsurface extensions such as tunneling and undermining create hidden cavities that complicate clinical assessment and wound management. Despite their clinical relevance, the prevalence and spatial characteristics of these subsurface wound morphologies have not been well characterized at scale. Methods: We performed a registry-based analysis using data from the LIFT-OFF Pressure Ulcer Registry, which captures longitudinal clinical documentation of pressure ulcers treated in routine care. The registry included approximately 18,000 patients with 32,000 documented pressure ulcers. Spatial characteristics of tunneling and undermining were analyzed using measurements recorded during routine wound assessments, including tract length, direction, and circumferential extent. Directional and circumferential distributions of subsurface defects were examined to characterize wound geometry. Results: Tunneling was present in 764 of 14,700 full-thickness pressure ulcers (5.2%), whereas undermining occurred in 2,293 wounds (15.6%). Tunneling tracts were typically short and exhibited directional clustering relative to the wound bed. In contrast, undermining demonstrated broader circumferential distributions and frequently involved larger subsurface separations beneath the wound margin. Both morphologies demonstrated distinct spatial patterns across anatomical locations and wound stages. Conclusion: Tunneling and undermining are common subsurface features of pressure ulcers and exhibit distinct spatial geometries. Whereas tunneling manifests as directional tract-like extensions, undermining more frequently produces circumferential tissue separation beneath wound margins. Improved characterization of subsurface wound architecture may enhance assessment of wound complexity and provide information not captured by surface measurements alone. Future studies should evaluate whether these features contribute to wound severity assessment, prognosis, and risk stratification.

Keloids are benign fibroproliferative disorders majorly characterized by excessive extracellular matrix deposition, with recurrence rates exceeding 80% following conventional therapy. Although epigenetic dysregulation has been implicated in keloid pathogenesis, whether genome-wide DNA methylation actively drives pathological cellular reprogramming, and whether this state is therapeutically reversible, remains unclear. We performed genome-wide DNA methylation profiling on keloid tissues, matched primary keloid fibroblasts, and normal controls. Our analysis revealed a shared DNA hypermethylation pattern between keloid tissues and fibroblasts, which was validated by three independent public cohorts. By integrating DNA methylome and transcriptome, we demonstrated that DNA methylation-regulated genes were enriched in osteochondrogenesis-related pathways, such as cartilage and bone development pathways. Furthermore, pharmacologic inhibition of DNA hypermethylation by DNA demethylating agent decitabine reduced the expression of osteochondrogenic markers and inhibited collagen deposition and keloid growth in primary keloid fibroblasts and patient-derived xenograft (PDX) model, offering a potential therapeutic strategy of keloid.

Neurodegenerative diseases are increasingly associated with vascular dysfunction beyond progressive neuronal degeneration, yet how vascular pathology contributes to disease progression remains poorly understood, largely due to the lack of a neurodegenerative disease model capable of capturing neuronal pathology alongside associated vascular dysfunction. Here, we developed a microengineered 3D vascularized brain tissue model that integrates neurospheroids with a self-assembled, perfusable vascular network to recapitulate key features of the neurovascular interface. Using this model, we investigated the vascular contribution to Parkinson's disease pathology by introducing -synuclein preformed fibrils into the engineered vasculature. Intravascular -syn fibril exposure induced endothelial barrier disruption, vascular leakage, inflammation, and vascular regression. Notably, this vascular insult was accompanied by intraneuronal -synuclein aggregation within neurospheroids, suggesting that vascular dysfunction may facilitate the exposure of neural tissue to pathogenic -synuclein. Our neurodegenerative disease modeling approach establishes a versatile and tractable platform for investigating vascular contributions to neurodegenerative disease progression.

Peripheral sensory neurons innervate the skin to detect mechanical, thermal, and noxious stimuli. Within the epidermis, nerve fibers terminate beneath tight junctions, shielding them from environmental exposure. Although epidermal differentiation coordinates tight junction assembly, its role in organizing nerve terminals is poorly understood. Here, we show that activation of Notch, a master regulator of epidermal differentiation, caused near-complete loss of epidermal innervation. This was largely the result of increased contractility rather than impaired differentiation. Inducing epidermal contractility was sufficient to deplete nerve fibers with striking spatial precision, and restoring normal contractility reversed this effect. Actomyosin contractility is highest in the granular layers of the epidermis, where tight junctions form and nerve fibers terminate. Ablation of nonmuscle myosin II allowed nerve fibers to extend beyond their normal termination zone and caused touch hypersensitivity. Together, these findings demonstrate that epidermal contractility positions sensory nerve endings through spatially controlled pruning and defines a mechanical boundary established by epidermal cells that restricts neuronal outgrowth.

The role of UBE2N in myeloid cell-mediated immune suppression in cancer remains undefined. Here, we examined the function of UBE2N in myeloid cell-mediated tumor progression using a temporally inducible myeloid-specific knockout model (LysMCreERUbe2nfl/fl). Temporally induced deletion of Ube2n in myeloid cells (Ube2nMyeKO) significantly hindered growth of YUMM1.7 melanoma. This was accompanied by reduced myeloid cell burden within the tumor microenvironment. We observed altered abundance of PD-1, PD-L1, and SPP1 in the Ube2nMyeKO tumor microenvironment at the tissue level. In vitro analysis showed that knock-in expression of a catalytically deficient UBE2NC87S mutant in bone marrow-derived macrophages (BMDMs) markedly decreased expression of Spp1. We observed decreased SPP1 secretion in Ube2nMyeKO BMDM-conditioned media (CM). Treatment with Ube2nMyeKO BMDM-CM decreased co-expression of PD-1, TIM-3, and LAG-3 on chronically stimulated T cells. Antibody-mediated neutralization of SPP1 in Ube2nWT BMDM-CM decreased PD-1 expression on CD8+ T cells. Together, these findings suggest a role for myeloid UBE2N in YUMM1.7 progression.

Single-cell technologies have generated cell censuses of tissues, however, how tissue geometry reflects functional needs remains poorly characterized. The human pilosebaceous unit offers a tractable model, a prenatally-formed complex mini-organ combining hair and sebum production with a stem cell reservoir. Using histomorphology, spatial transcriptomics, and single-cell multiomics on the same human prenatal scalp skin samples (8-19 post-conception weeks), integrated and analyzed using machine learning approaches, we built a spatiotemporal map of pilosebaceous unit development. We demonstrate that epithelial-mesenchymal interactions coordinate cellular fate and organogenesis, using an in vitro hair-bearing skin organoid model to validate this tissue-patterning. In addition, we show sebaceous gland developmental programmes are overcome during tumor formation. Our large-scale multi-modal analysis provides a unique framework for understanding form and function of tissues with applications in tissue engineering and pathology.

Chemoradiotherapy (CRT) is the standard-of-care therapy for many solid malignancies, yet predictive biomarkers of treatment response remain limited. We identified a germline single nucleotide polymorphism (SNP) in an intrinsically disordered region of the lysine demethylase KDM3C/JMJD1C (p.S464T) that is associated with CRT outcomes in locally advanced rectal cancers (LARC) and head and neck squamous cell carcinoma (LA-HNSCC). In silico modeling with AlphaFold predicted S464T substitution influenced interaction between phosphorylated KDM3C and RNF8 FHA domain. In cellular models, conversion of S464 to T464 increased sensitivity to DNA-damaging agents. S464T substitution impaired damage-induced MDC1-RAP80 signaling and downstream RAP80-BRCA1 colocalization. SNP carrying cells impaired DNA repair causing genotoxic stress that is associated with increased cGAS-cGAMP innate immune signaling and increased apoptosis. Population analyses with the SNP highlighted an increase incidence of UV-induced skin and other cancers, linking inherited variation in the chromatin regulatory gene KDM3C to genome instability, cancer risk, and therapeutic vulnerability.

Keratin gene mutations are often associated with inflammatory skin disorders, in which the ensuing immunopathology is generally attributed to disrupted barrier integrity and consequent microbial invasion. Here, we challenge this paradigm by demonstrating a role for keratin 76 (Krt76) in thymic central tolerance to skin-targeting autoimmune responses. We show that transfer of Krt76-/- thymic lobes under the kidney capsules of athymic recipients is sufficient to induce expansion of effector T cells in the secondary lymphoid organs, T cell skin infiltration, and autoantibody reactivity to both skin and oral mucosa tissue. Mechanistically, we demonstrate that loss of thymic Krt76 expression disrupts canonical differentiation of the thymic medulla and impacts the development of post-AIRE-expressing keratinocyte-like mimetic medullary epithelial cells (termed CorneoTECs). Notably, Krt76-expressing CorneoTECs differentially express a specific skin and oral mucosa-associated gene signature, including skin-specific tissue self-antigens (TSAs). Importantly, in the absence of Krt76 this skin and oral mucosa TSA signature is almost entirely lost, and T cell negative selection is affected. Collectively, these data highlight a heretofore unanticipated role for Krt76 in thymic central tolerance to skin and oral mucosatargeting T cells and suggest that loss-of-keratin-associated skin disorders could also include autoimmune pathologies.

The immune checkpoint protein PD-L1 plays a pivotal role in tumor immune evasion by binding to PD-1 on immune cells, including T lymphocytes. While the expression and function of PD-L1 have been well studied, the importance of its spatial organization on the cell surface of tumor cells remains poorly understood. In this study, we used super-resolution microscopy combined with biochemical perturbations to investigate the factors regulating PD-L1 clustering and its effects on PD-1 binding and T cell inhibition. We found that PD-L1 is organized into nanoscale clusters at the plasma membrane, with distinct regulatory roles for the actin cytoskeleton, galectin-3, and cholesterol. Disruption of cortical actin increased PD-L1 cluster size, while galectin-3 promoted smaller, denser clusters and increased PD-L1 lateral mobility. Cholesterol depletion reduced PD-L1 cluster size and number and impaired PD-1 binding. These findings indicate that PD-L1 surface organization is collectively regulated by the actin cytoskeleton, galectin-3, and membrane cholesterol within the plasma membrane of tumour cells. Our results provide new insights into the dynamic regulation of PD-L1 and its potential as a therapeutic target in cancer immunotherapy.

Endothelial senescence is increasingly recognized as a driver of vascular pathology, while immunoglobulin G (IgG) has recently been reported to accumulate in aging tissues and induce senescence in macrophages and microglia. In cerebral cavernous malformations (CCMs), IgG accumulation has been obviously observed in CCM lesions, but the contribution of IgG to endothelial injury remains unclear. Using multi-omic profiling, endothelial models, and CCM mice, we identified IgG-secreting plasma cells enriched in lesions associated with endothelial senescence, hemorrhage, and disease severity. CCM loss-associated mTOR activation impaired lysosomal acidification and IgG processing, promoting intracellular IgG accumulation. IgG, in turn, induced NF-kB-dependent endothelial senescence. In vivo, BCMA-mediated plasma cell depletion attenuated lesion progression, whereas IgG supplementation partially restored disease severity. Anti-CD38 treatment likewise reduced IgG accumulation, endothelial senescence, hemorrhage, and lesion progression. These findings identify lysosomal dysfunction-mediated IgG as a pathogenic trigger of endothelial senescence and support targeting the plasma cell-IgG axis in CCM.

Tumor-promoting myofibroblastic carcinoma-associated fibroblasts (myCAFs) are induced by activation of transforming growth factor-{beta} signaling. However, the molecular basis of myCAF-specific transcriptional programs regulated by TGF-{beta} signaling remains poorly understood. Using a meta-analysis of single-cell RNA-seq data from 132 human breast tumor and non-tumor tissues, we show that myCAFs activate gene regulatory programs relevant to skeletal and cardiovascular development that are associated with poorer outcomes in breast cancer patients. Of note, distal-less homeobox 5 (DLX5), a master transcription factor for skeletal development, is activated in human breast myCAFs at both epigenetic and transcriptional levels. DLX5 expression is also initiated by TGF-{beta}1 treatment in human mammary fibroblasts. Immunoprecipitation and CUT&RUN assays using DLX5-expressing fibroblasts demonstrate that DLX5 interacts with Smad2/3/4 proteins, enabling their cooperative occupancy at shared genomic binding sites of target genes, thereby promoting canonical TGF-{beta} signaling and the myCAF state. DLX5-primed myCAFs also enhance paracrine TGF-{beta} signaling and neuropilin-2 expression to promote collective tumor invasion. Our findings indicate that DLX5 induces myCAF formation and promotes breast tumor progression in collaboration with canonical TGF-{beta} signaling.

Background: Spatial organization is increasingly recognized as a key determinant of tumor-immune interactions in head and neck squamous cell carcinoma (HNSCC). The GSE300147 Xenium spatial transcriptomic resource generated by McCord and colleagues established a framework for mapping spatially coordinated T-cell states in HNSCC. However, how tumor-enriched epithelial immune states relate to metabolic, redox, and stress-adaptive transcript programs remains incompletely defined. Methods: A secondary, data-driven reanalysis of GSE300147 was performed, focusing on 17 confirmed HNSCC Xenium sections after exclusion of a non-HNSCC ameloblastoma specimen. A total of 1,148,244 cells were analyzed, including 558,867 EpCAM+ tumor-enriched epithelial cells. Tumor-enriched epithelial cells were classified into Hot, Intermediate, and Cold states using a Composite Hotness framework integrating T-cell inflammatory signature score, checkpoint-associated signaling, CD274 expression, IFN/antigen-presentation signature score (IFN/AP), and tumor-immune proximity. Six metabolic ecosystem states, neighborhood profiling, spatial permutation testing, and an integrated Immune-Metabolic-Redox Ecosystem Score (IMRES) were then applied. Results: Immune activation was spatially heterogeneous across HNSCC sections. Immune-hot tumor-enriched epithelial regions showed not only inflammatory, checkpoint-associated, and antigen-presentation signature scores, but also coordinated metabolic, oxidative-redox, and stress-response transcript programs. IMRES, derived from available immune, metabolic, redox, and stress-response transcript components represented in the Xenium panel, increased progressively from Cold to Intermediate to Hot tumor-enriched epithelial states and was associated with NFE2L2, GDF15, HLA-DRA, CD274, KEAP1, and MDM2. Integrating IMRES with Composite Hotness identified a distinct Hot+IMREShigh ecosystem comprising 106,874 tumor-enriched epithelial cells. This state showed the strongest immune-active and stress-adaptive features and was positioned closer to immune populations than expected by random assignment. An alternative rank-based robustness analysis reproduced the IMRES-associated ecosystem axis and correlated with the original module-based score (Spearman r = 0.597). Conclusions: This secondary reanalysis extends the original spatial T-cell framework by defining a complementary tumor-centered immune-metabolic-redox ecosystem in HNSCC. IMRES provides a transcript-derived framework for identifying Hot+IMREShigh neighborhoods where immune activation, checkpoint signaling, metabolic remodeling, and stress adaptation converge, providing a hypothesis-generating framework for studying immune resistance and therapeutic vulnerability.

Background: Reliable melanoma classification requires models that capture both local dermoscopic morphology and broader contextual patterns while maintaining auditable, leakageaware internal validation. Objectives: To develop and internally validate an EfficientNetB0-Swin Transformer Tiny ensemble for classifying histopathologically verified dermoscopic images as benign melanocytic lesions or malignant melanoma. Methods: This retrospective diagnostic model-development and internal validation study screened 552,869 ISIC Archive records; filtering and dermatologist review yielded 1,199 uniquepatient and unique lesion images (578 benign and 621 malignant). Images were the predictors and histopathology was the reference. ImageNet pretrained EfficientNetB0 and Swin-T features were fused. Patient independent five fold validation used weighted sampling, mixup, label smoothing, AdamW, early stopping, and five view test time augmentation. Results: Mean accuracy was 0.89325 {+/-} 0.03179, mean receiver operating characteristic area under the curve (ROC-AUC) was 0.96348 {+/-} 0.01695, and mean support weighted F1-score was 0.89300 {+/-} 0.03220. The fold level 95% confidence intervals were 0.8538-0.9327 for accuracy and 0.9424-0.9845 for ROC-AUC. Pooled counts were 526 true negatives, 52 false positives, 76 false negatives, and 545 true positives, yielding 87.76% sensitivity and 91.00% specificity. Qualitative Grad-CAM review showed peripheral artifact activation in two false positives and lesion centered activation in two correctly classified cases; these observations were not systematically scored. Limitations: The validation folds were also used for early stopping and checkpoint selection. Device stratified analysis, systematic interpretability scoring, calibration, and independent external validation were unavailable. Conclusions: The ensemble showed high internal discrimination and is intended only as a clinician facing adjunct. The error audit workflow enables targeted retrospective review, but external validation is required before clinical use or generalizability claims.

Orthodontic tooth movement relies on a tightly regulated pro-inflammatory and pro resorptive mechanoresponse of local periodontal ligament fibroblasts (PdLFs). Dysregulation is linked to complications such as root resorption and tooth loss. Hyperlipidemic conditions promote excessive PdL mechanoresponses, with growth differentiation factor 15 (GDF15) acting as potential regulator. This study examined the contribution of the inflammasome/pyroptosis pathway as underlying mechanism for dysregulated mechanoresponses. Human PdLFs were treated with palmitic acid (PA) or oleic acid (OA) for six days before 24 hours of compressive loading. PA increased CASP1, CASP4, and CASP3 activity, secretion of IL-1{beta}, IL-18, and HMGB1, and LDH release. Pharmacological blockade and siRNA-mediated knockdown of inflammasome- and pyroptosis-related targets revealed that NLRP3, CASP1, CASP4, and GSDMD partially contributed to monocyte and osteoclast overactivation. Silencing PA-increased GDF15, partially normalized the phenotype, at least in part by inflammasome/pyroptosis regulation. GDF15 acted through extracellular, and a nuclear signaling route, each accounting partially to this phenotype. Together, GDF15 partially regulates the PA-induced, pyroptosis-associated overactivated mechanoresponse alongside pyroptosis-independent mechanisms suggesting it as an interesting target for potential clinical interventions.

Tissue mechanics influence diverse biological processes, yet directly linking stiffness measurements to spatially resolved molecular states in intact tissues remains challenging. Here we developed a paired-surface spatial mechanomics approach to map Young's modulus by nanoindentation on a fresh tissue surface and co-register the stiffness grid with 10x Genomics Visium HD spatial transcriptome bins from the immediately adjacent, parallel surface. Applied to the mouse ovary, which has spatially distinct compartments and undergoes extracellular matrix remodeling with cycle and age, the workflow generated >2,900 matched measurements across 21 regions of interest. Nanoindentation at 50-m grid spacing enabled millimeter-scale stiffness maps while balancing acquisition time in fresh tissues, with ~92 4-m transcriptome bins assigned to each stiffness value. Global and compartment-specific analyses associated stiffer regions with lower elastic fiber programs and higher inflammatory signaling, with age-dependent differences. This correlative strategy integrates experimentally measured mechanics with spatial omics in fresh tissues.

DNMT3A is a de novo DNA methyltransferase whose recruitment to chromatin regulates its function. Missense mutations within the chromatin-binding PWWP domain are associated with diverse human disorders, yet how mutations in the same domain produce distinct phenotypes remains unclear. Here we systematically characterise 19 clinically reported mutations in the PWWP domain of DNMT3A that are associated with Heyn-Sproul-Jackson syndrome (HESJAS), paraganglioma (PG) and clonal haematopoiesis (CH). We show that all PWWP-domain mutations associated with HESJAS abolished interaction with H3K36me2 modified nucleosomes, defining this as a consistent biochemical feature of HESJAS. In contrast, mutations from all disease classes differentially altered DNA binding of the PWWP domain, driven by alterations in the net charge of the domain. However, these effects are largely overcome by inclusion of the DNNMT3A1 N-terminal region, which is absent from its embryonic isoform, suggesting that PWWP mutations may differentially affect DNMT3A function through development. Changes in the thermal stability of the isolated PWWP domain mutants did not directly translate into altered stability of full-length DNMT3A1 in cells. We show that HESJAS mutations can affect the intramolecular interaction between the PWWP and adjacent ADD domain, an interaction proposed to contribute to the autoinhibitory function of the ADD domain. However, not all mutations behaved in the same way, suggesting that multiple factors govern the intramolecular autoinhibition of DNMT3A. Together, this study advances our understanding of the molecular mechanisms by which DNMT3A PWWP-domain mutations are mechanistically heterogeneous, providing a biochemical framework that contributes to distinct disease phenotypes.

The ability to longitudinally track clonal evolution non-invasively would transform cancer interception strategies, long before late-stage disease when most cancer genomes are analysed. Here, we demonstrate that repeated hair sampling from the same individual followed by exome sequencing enables tracking of somatic evolution in vivo over several months after chemically induced skin carcinogenesis. We found that hair follicles accumulate a higher mutation burden than spatially-matched skin and harbour mutations that spread into surrounding epidermis and persist throughout tumour progression. DNA-damaged follicles enter sustained quiescence that delays replication and repair, creating a reservoir for long-lived mutations. During premalignant progression, carcinogen-associated mutations become enriched as follicular clones expand into adjacent skin. Mutation tracking identified genes that may govern tumour predisposition and initiation, many of which are mutated at high incidence in human cutaneous squamous cell carcinoma cohorts. Hair follicles therefore provide a non-invasive readout to forecast the early development of skin cancer, enabling patient risk stratification.