Date of Award
Doctor of Philosophy (PhD)
Cell & Molecular Biology
Edward M. Behrens
Langerhans Cell Histiocytosis (LCH) is an inflammatory myeloid neoplasia characterized by abnormal cells, called “LCH cells,” closely related to conventional dendritic cells (DCs). LCH cells universally exhibit hyperactive-ERK signaling due to gain-of-function mutations in the Ras-ERK pathway, commonly BRAFV600E. The pathologic features of LCH cells and the mechanisms by which hyperactive-ERK drives them remains largely unknown, and the need for more effective, safer, and curative therapies are needed. We aimed to better understand the DC-specific consequences of harboring BRAFV600E. Since DCs rely on ERK signaling to produce inflammatory molecules in response to environmental cues, we hypothesized that BRAFV600E-DCs have enhanced inflammatory response to pathogenic stimuli. To address this, we used a transgenic mouse model of LCH which expresses BRAF-V600E in DCs (BRAF-V600Efl/+:CD11c-Cre) and used them to generate mixed-bone marrow chimeras to titrate the number of mutated DCs present. We observed increased LPS-induced circulating cytokines in the transgenic mice, and an inflammatory phenotype that was dependent on the number of LCH cells reconstituted in the chimeras. Additionally, we generated bone-marrow derived DCs (BMDCs) and specifically investigated TLR4-induced TNFα production with or without pharmacologic BRAFV600E inhibition. We observed a reversible increase in secreted TNFα and a partially reversible increase in TNFα protein per-cell, despite a decrease in TLR4 signaling and Tnfa transcripts compared to controls. This prompted us to examine ERK-driven, post-transcriptional mechanisms that contribute to TNFα production and secretion using biochemical and cellular assays. We identified a reversible increase in TACE activation, the enzyme required for TNFα secretion, and most strikingly, a largely irreversible increase in translation efficiency of transcripts, including TNFα, and a reversible increase in the rate of ribosomal elongation. A high throughput exploration of the translatome revealed an enrichment of LPS-response transcripts. These data suggest that LCH cells are hyper-responsive to pathogenic cues which may contribute to LCH pathogenesis. Specifically, these data demonstrate that BRAFV600E exploits multiple post-transcriptional mechanisms in DCs to amplify inflammatory responses. Together, this research advances our understanding of DC biology and LCH pathobiology, lays the groundwork for future studies to further investigate the mechanisms and consequences of both the hyper-responsiveness and altered translatome of LCH cells, and offers novel molecular targets for potential therapeutics.
Minichino, Danielle E., "Novel Mechanisms Of Braf-V600e Regulation Of Inflammation In Dendritic Cells Of Langerhans Cell Histiocytosis" (2022). Publicly Accessible Penn Dissertations. 5477.