“Two-hit” model for lymphatic malformations. - PROJECT SUMMARY Complex lymphatic anomalies (CLAs) are rare diseases with variable clinical manifestations caused by the abnormal development of lymphatic vessels. CLAs include central conducting lymphatic anomaly (CCLA), generalized lymphatic anomaly (GLA), Gorham-Stout disease (GSD), and kaposiform lymphangiomatosis (KLA). CLA patients exhibit a variety of phenotypes, such as tortuous dilated lymphatic vessels, multifocal lymphatic malformations (LMs), and ectopic lymphatic vessels in bone. Unfortunately, existing treatments for CLAs have significant side effects and are not effective in all patients. Therefore, there is an urgent need to identify new, safer treatments for CLAs. We and others recently identified somatic activating mutations in KRAS in CLA patients. To investigate the effect of hyperactive KRAS signaling on the development of lymphatic vessels, we generated a novel mouse model that expresses an active form of KRAS (KRASG12D) in lymphatic endothelial cells (LECs). We found that KRASG12D induces the formation of LMs in newborn mice but not in adult mice. These results suggest that growing lymphatic vessels in newborn mice are more sensitive to the pathologic effects of KRASG12D than stable lymphatic vessels in adult mice. Based on our preliminary data, we hypothesize that the formation of LMs requires two signals - one triggered by a genetic mutation in LECs and another supplied by a lymphangiogenic factor in the microenvironment. VEGF-C is a growth factor that stimulates lymphangiogenesis during development and in pathological settings. In aim one, we will test whether VEGF-C in the microenvironment renders lymphatic vessels sensitive to the pathogenic effects of KRASG12D. In aim two, we will determine whether directly targeting KRASG12D reverses LMs. Completing these aims will establish that CLAs are developmental disorders requiring a genetic mutation and a permissive lymphangiogenic environment for their development. Thus, like tumors, oncogene-induced LMs require “two hits” for their formation. Our findings will also significantly impact a broad spectrum of vascular and lymphatic malformations by identifying new therapeutic targets and near-clinic drugs that could be repurposed for these disabling, disfiguring, and life- threatening diseases. Importantly, KRAS mutation-specific inhibitors are predicted to have fewer side effects than traditional targeted therapies, a feature that could improve patient compliance with treatment and overall outcomes. We will also provide preclinical evidence for a new imaging modality that could rapidly move to the clinic to image lymphatic function in CLA patients non-invasively.
