Leveraging mosaic genodermatoses to discover genetic and molecular mechanisms of comedogenesis - PROJECT SUMMARY The formation of comedones, referred to as comedogenesis, describes the production of keratinized plug that occludes the opening of the hair follicle. Comedones are often observed in common cutaneous diseases like acne vulgaris, with up to 85% of individuals experiencing comedonal lesions in their lifetime. Comedones are also seen in rare, severe diseases such as nevus comedonicus (NC), a disease involving comedones that progress into painful, inflammatory cysts. We have shown that NC is caused by somatic mutations in the NEK9 gene. Remarkably, whole exome sequencing (WES) of a common comedonal disorder, the epidermal inclusion cyst, revealed identical mutations in NEK9, suggesting NEK9 mutations may drive comedogenesis across different manifestations. While NEK9 has been well studied in the context of mitosis and cell cycle progression, sporadic associations of NEK9 with primary cilia are also seen in the literature. The primary cilium, an immotile finger-like projection from the cell with critical functions in signal transduction, houses many of the signaling pathways related to proper development and maintenance of hair follicles. Because comedones arise from follicular structures, these mutations in NEK9 provide an unmatched opportunity to elucidate the genetic and biologic mechanisms underlying comedogenesis. To accomplish this, we have generated in vitro and in vivo models of NEK9 mutations identified in NC and EIC. Our preliminary data show NEK9 mutations result in shorter cilia and less ciliation in vitro. Hedgehog signaling, a key follicular signaling pathway that acts through the primary cilium, is diminished in NEK9 mutant cells. Our immunofluorescence studies show the activated form of the NEK9 protein localizes to the base of the cilium, a new discovery in NEK9 biology. We have previously shown this activation signal to be increased across the NEK9 mutations. Together, these preliminary data support ciliary dysfunction to be a result of NEK9 mutations. To study the involvement of NEK9 and cilia in comedogenesis in vivo, we have generated a transgenic mouse using a tamoxifen-dependent, basal keratinocyte specific promoter (K14-CreERT) to drive transcription of the NEK9 mutation most commonly reported in NC. We will time transgene induction with follicular morphogenesis onset and perform immunofluorescence and advanced microscopy methods to interrogate defects in canonical follicular development. We will continue to obtain NC and EIC tissue from our institution and perform WES to identify new candidate variants that may contribute to comedogenesis. We will generate stratified skin equivalent models to continually test the newly identified mutations for ciliary phenotypes. Lastly, we will use the known biology of NEK9 to systematically test downstream effectors for possible mechanisms of ciliary dysfunction. This training proposal represents an integrated scientific approach and new learning experiences that harness techniques in cell biology, computational genetics, and pathology to yield novel insights into the mechanisms of comedogenesis.
