Dissecting lysosomal metabolic dependencies in pancreatic cancer through functional genomics

BACKGROUND:Lysosomal nutrient recycling and acquisition pathways employed at an elevated capacity in pancreatic cancer cells in response to the harsh metabolic microenvironment in which they subsist. We have others have previously studied autophagy or lysosomal inhibition strategies and identified the metabolic adaptations they effect on cancer cells. These studies have thus suggested that the lysosome plays a role in regulating iron, lipids, nucleotides, and amino acids, among other nutrients. However, these studies have differed in their experimental approaches, pharmacological agent, and molecular targets, and it is unclear whether these multiple conclusions are broadly generalizable across all autophagy or lysosome inhibition strategies. METHODS AND RESULTS:To address these issues, here we perform parallel metabolism-focused CRISPR activation and knockout screens using 8 different autophagy or lysosome inhibitors in pancreatic cancer cell lines. From these, we determined that iron homeostasis is primarily affected by agents that affect lysosomal pH, such as bafilomycin A1 and Ammonium, but not by other inhibitors of lysosomal function such as apilimod, ESK981, or chloroquine, or by ULK1 inhibitors ULK101, SBI-0206965, or DCC-3116. Pairing the CRISPR screen data with transcriptomics, we further identified that lipid homeostasis was affected by all lysosome inhibitors but not ULK inhibitors. These multi-omics approaches specifically highlighted Farnesyl Diphosphate Synthase (FDPS), a gene involved in cholesterol synthesis as a prominent metabolic vulnerability revealed upon lysosome inhibition, whether with chloroquine, bafilomycin A1, or apilimod. DISCUSSION:Our study demonstrates a clear metabolic distinction between inhibiting autophagy compared to inhibiting the lysosome. Our data further highlight that disturbances in lipid metabolism are a general effect of lysosome inhibition, while disruptions in iron homeostasis may primarily be due to disturbances in lysosomal pH.