Identifying and disabling new pathways for macromolecular recycling in pancreatic cancer

PROJECT SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is an incurable malignancy with an overall median survival of 11 months post-diagnosis. PDAC develops in an oxygen-poor, nutrient-deprived microenvironment. Consequently, PDAC cells and tumors are highly dependent on enhanced lysosome biogenesis and activity to facilitate degradation, clearance and recycling of macromolecular substrates delivered by increased rates of autophagy and micropinocytosis. Recent studies show some beneficial effects of combination therapy with general lysosomal inhibition (hydroxychloroquine) in prolonging PDAC patient survival. However, to effectively target the lysosome in this cancer we must first identify key PDAC-specific lysosomal factors that enable pro-survival catabolic activities unique to this tumor and its growth pattern, and which could be targeted with precision medicine approaches. Our labs implemented a systematic proteomic- based analysis of lysosomes immunopurified from PDAC versus non-PDAC 2D cultured cells and in vivo PDAC tumors to generate a comprehensive map of PDAC-specific lysosomal proteins at primary as well as secondary organ sites. These studies highlighted two lysosomal hydrolases as essential for PDAC growth. Phospholipase B domain containing 1 (PLBD1), a putative hydrolase of unknown function, is upregulated in PDAC tumor specimens, and its ablation leads to massive morphological and functional disruption of PDAC lysosomes, suppressing tumor growth under all paradigms. A second PDAC-specific hydrolase was Legumain (LGMN), which was enriched in lysosomes of PDAC cells growing in the liver, a major secondary colonization site but not the pancreas. Accordingly, depleting LGMN suppresses liver colonization by PDAC cells without affecting their growth in the pancreas, pointing to LGMN as the first organ site-specific lysosomal factor for PDAC growth. Building on these foundational discoveries, we will: 1- explore PLBD1’s role as an aminopeptidase that removes branched-chain amino acids (BCAAs) from substrates, affecting PDAC cell metabolism. We will use mass spectrometry to identify PLBD1-dependent substrates and investigate how their breakdown supports PDAC growth. 2-dissect how the LGMN enable metastatic growth of PDAC in liver, particularly how breakdown of liver-specific macromolecular substrates by LGMN fuel PDAC metabolism as well as the mechanisms that promote LGMN expression in this organ. 3- develop small-molecule inhibitors for PLBD1 and LGMN via both covalent and non-covalent chemistry to evaluate their potential as therapeutic targets in PDAC. Together, these studies will uncover key lysosomal proteins supporting PDAC growth and suggest new therapeutic strategies targeting lysosomal functions.