PROJECT SUMMARY. Progression to metastatic disease from Non-Small Cell Lung Cancer (NSCLC) is a significant cause of mortality. Central nervous system (CNS) metastases, which carry poor prognosis and limited treatment options, can form intraparenchymally (IP) within the tissue of the brain or within the cerebrospinal fluid (CSF) filled spaces between the leptomeninges. The latter, termed leptomeningeal disease (LMD), is difficult to diagnose and treat, and the pathophysiological mechanisms underlying progression to LMD are poorly understood. 60% of patients with LMD have past or concurrent IP metastases, but the mechanisms promoting a switch to LMD invasion remain unknown. Interestingly, patients with EGFR-mutant NSCLC are more likely to progress to LMD, particularly at resistance to tyrosine kinase inhibitors (TKIs), the standard of care for these patients. While third-generation TKIs such as Osimertinib show excellent brain penetrance, resistant CNS disease, including LMD, remains a pressing clinical problem. The primary aims of this project are to identify mechanisms underlying progression to and persistence of LMD, and determine what factors favor this progression in cases of TKI-resistant EGFR-mutant disease. I hypothesize that in a subset of IP cases, progression to LMD occurs through invasion of parenchymal cells through the perivascular spaces in the brain, and that this progression is promoted by mechanisms that synergistically foster TKI resistance. I have validated multiple NSCLC murine models of comorbid IP and LMD following intra-arterial injection, including an EGFR- mutant model that emerges at late-stage TKI resistance as well as a syngeneic model. In Aim 1, I will utilize spatiotemporal barcoding of these lines to chart the anatomical routes cells traverse to reach the leptomeningeal space. In particular, I will determine whether LMD metastases descend from parenchymal metastases, or enter the CSF from systemic circulation across the blood-CSF-barrier. I will then assess how pathways downstream of mechanosensing by β1-integrin, previously shown by our laboratory to promote CNS TKI resistance, may modulate the ability of EGFR-mutant cells to complete this journey at resistance. Then, in Aim 2, I will investigate the mechanistic role of the protein Tissue Inhibitor of Metalloproteinases 1 (TIMP-1), identified in our biorepository samples as upregulated in the CSF of patients with LMD. TIMP-1 can signal through β1-Integrin and CD63 to promote anchorage-independent survival, mirroring a phenotype observed in our LMD model lines in vitro. Given the role of β1-integrin in this pathway, I will investigate whether stromal TIMP-1 levels play a dual role in EGFR-mutant LMD by promoting cell survival while also promoting signaling underlying TKI resistance. As a graduate student in Dr. Don Nguyen’s laboratory in the Pathology department at Yale University, I have the support of a diverse array of translational and clinical researchers as my...