Summary/Abstract Endothelial cells (ECs) are key components of both blood and lymphatic vessels, and aberrant EC proliferation due to abnormalities in various signaling pathways contributes to a wide range of vascular anomalies including vascular malformation and tumors. Lymphatic malformation (LM) and lymphangiosarcoma (LAS) are vascular anomalies originating from lymphatic ECs. While LM mostly remains as a benign disease, a fraction of LM patients progress to the highly aggressive and deadly LAS. Although LM (e.g. chronic lymphedema in breast cancer patients) has been recognized as a risk factor for LAS, very little is known about underlying mechanisms regulating the progression of LM to LAS. The long-term goal of the proposed studies is to understand the molecular and cellular mechanisms of LM progression to LAS in order to develop new strategies for effective prevention and treatment of this deadly disease. In prior funding periods, we developed a mouse model with inducible EC-specific deletion of Tsc1 tumor suppressor gene (Tsc1iΔEC mice), which recapitulates salient features of human LAS. Using a new tumor cell line derived from Tsc1iΔEC mice, we further showed that autophagy blockade by knockout or knockdown of different autophagy genes inhibited vascular tumor cell proliferation in vitro and tumorigenicity in vivo. Transcriptional profiling and additional mechanistic analysis suggested a role for the autophagy-dependent expression of osteopontin and its potential autocrine stimulation of Jak/Stat3 signaling in promoting tumor cell proliferation and tumorigenicity. More importantly, by generating EC-specific double conditional knockout of an essential autophagy gene Fip200 in Tsc1iΔEC model, we found that autophagy inhibition, while not affecting the initial LM development, blocked LM progression to LAS. We also prepared a new Tsc1/Fip200-4A double conditional knockin mouse for further analysis of the mechanisms of regulation of LM progression to LAS by FIP200-mediated autophagy in vivo. In addition, we performed a genome- wide CRISPR-Cas9 screen and identified several genes with tumor suppressive functions for vascular tumor growth in vivo, including Rasa1 encoding a negative regulator of Ras signaling, suggesting potential synergy between Ras/MAPK and mTORC1 signaling in LM development and progression to LAS. Based on these strong supporting data and using our unique mouse and cell models, we propose to 1) determine the mechanisms of autophagy regulation of vascular tumor cell proliferation and tumorigenicity; 2) examine targeting of autophagy and its downstream pathways to block LM progression to LAS; and 3) explore the potential synergy between Ras and mTORC1 signaling in the development and progression of vascular tumors. Together, these studies will significantly advance our understanding of the molecular and cellular mechanisms of LM progression to LAS, that may contribute to the future design of effective prevention and novel ther...