Renee M. McKay

Meibomian gland dysfunction (MGD) is the leading cause of Dry Eye Disease (DED), accounting for approximately 90% of DED cases worldwide. The transcription factor KROX20 has been shown to mark stem cells that play a critical role in Meibomian gland (MG) development and homeostasis, however, the molecular mechanisms underlying these processes are not well understood. In this report, we used multiple Krox20 lineage tracing and ablation studies to investigate lineage commitment during MG morphogenesis and homeostasis. KROX20 marks MG stem cell pools, and regulates cellular contributions to the glands, as loss of Krox20 leads to MG atrophy, while its overexpression results in MG enlargement. We also found that Notch1 signaling is regulated by KROX20 and that ablation of the Notch1 gene in KROX20-expressing cells leads to MG atrophy, while Notch1 overexpression partially rescues MG atrophy caused by the loss of Krox20. Together, these results reveal a critical Krox20-Notch1 regulatory pathway in MG stem cells. Activation of Notch1 signaling may offer a novel approach to preventing or treating DED caused by depletion of the MG stem cell pool that can occur with ageing.

Mammalian skin is a vital barrier with the epidermis serving as its protective outer layer, continually undergoing renewal. Given that loss of the epidermis or its barrier function is lethal for mammals, multiple stem cell populations likely exist for the interfollicular epidermis (IFE), enhancing evolutionary survival. Here, we demonstrate that transcription factor KROX20 marks a heterogeneous stem cell population in the upper and middle mouse hair follicle (HF), partially overlapping with known HF stem cell markers in those regions. Lineage tracing in mice using different reporter lines shows that Krox20 -lineage cells migrate from the HF to the IFE, contributing to both basal and suprabasal layers during adulthood. Spatial transcriptomics data corroborate our findings. Depletion of epithelial Krox20 -expressing cells leads to epidermal hyperplasia and a disruption of stratification during homeostasis. Our study highlights the contribution of hair follicle Krox20 -lineage cells to the IFE and the regulation of epidermal homeostasis.

Epidermal stem cells control homeostasis and regeneration of skin and hair. In the hair follicle (HF) bulge of mammals, populations of slow-cycling stem cells regenerate the HF during cyclical rounds of anagen (growth), catagen (regression), and telogen (quiescence). Multipotent epidermal cells are also present in the HF above the bulge area, contributing to the formation and maintenance of sebaceous gland and upper and middle portions of the HF. Here, we report that the transcription factor KROX20 is enriched in an epidermal stem cell population located in the upper/middle HF. Expression analyses and lineage tracing using inducible Krox20-CreERT showed that Krox20 -lineage cells migrate out of this HF region and contribute to the formation of the bulge in the HF, serving as ancestors of bulge stem cells. In vivo depletion of these cells arrests HF morphogenesis. This study identifies a marker for an epidermal stem cell population that is indispensable for hair homeostasis.

Neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene that encodes neurofibromin, a RAS GTPase–activating protein. Inactivating NF1 mutations cause hyperactivation of RAS-mediated signaling, resulting in the development of multiple neoplasms, including malignant peripheral nerve sheath tumors (MPNSTs). MPNSTs are an aggressive tumor and the main cause of mortality in patients with NF1. MPNSTs are difficult to resect and refractory to chemo- and radiotherapy, and no molecular therapies currently exist. Immune checkpoint blockade (ICB) is an approach to treat inoperable, undruggable cancers like MPNST, but successful outcomes require an immune cell–rich tumor microenvironment. While MPNSTs are noninflamed “cold” tumors, here, we converted MPNSTs into T cell–inflamed “hot” tumors by activating stimulator of IFN genes (STING) signaling. Mouse genetic and human xenograft MPNST models treated with a STING agonist plus ICB exhibited growth delay via increased apoptotic cell death. This strategy offers a potential treatment regimen for MPNSTs.

Neurofibromatosis type 1 (NF1) is one of the most common tumor-predisposing genetic disorders. Neurofibromas are NF1-associated benign tumors. A hallmark feature of neurofibromas is an abundant collagen-rich extracellular matrix (ECM) that constitutes more than 50% of the tumor dry weight. However, little is known about the mechanism underlying ECM deposition during neurofibroma development and treatment response. We performed a systematic investigation of ECM enrichment during plexiform neurofibroma (pNF) development and identified basement membrane (BM) proteins, rather than major collagen isoforms, as the most upregulated ECM component. Following MEK inhibitor treatment, the ECM profile displayed an overall downregulation signature, suggesting ECM reduction as a therapeutic benefit of MEK inhibition. Through these proteomic studies, TGF-β1 signaling was identified as playing a role in ECM dynamics. Indeed, TGF-β1 overexpression promoted pNF progression in vivo. Furthermore, by integrating single-cell RNA sequencing, we found that immune cells including macrophages and T cells produce TGF-β1 to induce Schwann cells to produce and deposit BM proteins for ECM remodeling. Following Nf1 loss, neoplastic Schwann cells further increased BM protein deposition in response to TGF-β1. Our data delineate the regulation governing ECM dynamics in pNF and suggest that BM proteins could serve as biomarkers for disease diagnosis and treatment response.