Brownfield, Douglas G

Abstract CCAAT/enhancer-binding protein alpha (C/EBPα) is a critical lineage-defining transcription factor and tumor suppressor. Despite its clinical significance in conditions like acute myeloid leukemia and hepatocellular carcinoma (HCC), C/EBPα has remained undruggable due to its intrinsically disordered regions and lack of small-molecule binding pockets. In this study, we describe the development of a novel Oligonucleotide-based PROteolysis-TArgeting Chimera (Oligo-PROTAC or O'PROTAC) strategy designed to achieve targeted degradation of endogenous C/EBPα. We designed and synthesized a 10-nucleotide double-stranded DNA decoy derived from a C/EBPα-binding motif and conjugated it via variable linkers to the E3 ligase ligands pomalidomide or VH032. A total of six candidates were synthesized and evaluated in 3T3-J2 and Huh7 cell lines. We demonstrate that these O'PROTACs successfully localize to the nucleus and achieve dose-dependent degradation of C/EBPα, with the most potent candidates exhibiting sub-micromolar DC50 via lipofection delivery. Notably, this degradation occurred without inducing significant cytotoxicity. We observe a transcriptional shift consistent with loss of C/EBPα, including downregulation of cooperative factors involved in liver lineage specification and their downstream target genes. This work establishes O'PROTACs as a viable modality for targeting C/EBPα, offering a novel strategy for mechanistic investigation and future therapeutic targeting.

Abstract Metabolic vulnerabilities in cancer have been targeted primarily to suppress tumor growth, but less is known about the metabolic requirements for tumor cell invasion. Here we report that lipid catabolism by cytosolic and lysosomal lipases supports pancreatic cancer cell invasion through both overlapping and distinct functional and metabolic mechanisms. Lysosomal acid lipase (LAL)-dependent lipid droplet catabolism promotes invadopodia formation and stabilization, enabling extracellular matrix degradation. In addition to modulating cellular energetics, lipidomics revealed that lipid droplet catabolism regulates cholesterol and membrane phospholipid levels. Using spatially resolved biosensors and cholesterol imaging, we found that lysosomal lipid catabolism occurs at invadopodia and sustains local ATP and membrane cholesterol. These findings identify spatially organized lipid catabolism as a mechanism that couples local energetics and membrane remodeling during the earliest steps of pancreatic cancer cell invasion.

Summary Physical forces shape cell behavior, yet how they integrate with signaling to control fate and disease remains unclear. The alveolar epithelium is patterned by FGF signaling and mechanical stretch, but how these cues specify AT1 and AT2 cells is poorly understood. Here we show that cell membrane tension (CMT) is a conserved regulator of epithelial fate in mouse and human lungs. CMT drops before differentiation and is spatially patterned, defining where bipotent progenitors acquire AT1 or AT2 identity. Lower CMT enhances FGFR2 endocytosis and ERK signaling to drive AT2 differentiation and permits architectural remodeling that enables stretch-mediated YAP/TAZ nuclear entry for AT1 maturation. β-catenin elevates CMT cell-intrinsically independent of its role in canonical WNT transcription, while embedding, osmotic compression, or fibroblast wrapping elevate CMT extrinsically. Combined intrinsic and extrinsic tension traps alveolar epithelial cells in a KRT8⁺ transitional state seen in fibrotic lungs. Membrane tension thus integrates physical and molecular cues linking morphogenesis to fibrosis. Graphical Abstract In Brief We identify a conserved drop in cell membrane tension (CMT) that gates epithelial fate transitions across mouse and human lungs. In bipotent progenitors, reduced CMT promotes Alveolar Type 2 (AT2) fate via FGFR2 endocytosis and ERK signaling, as well as Alveolar Type 1 (AT1) fate by enabling architectural remodeling required for YAP/TAZ nuclear entry. We further show that intrinsic β-catenin and extrinsic confinement cues, including mesenchymal contact, converge to elevate CMT, restricting differentiation and—in adult AT2s—inducing a KRT8⁺ transitional state associated with fibrosis. Highlights Cell membrane tension drops before differentiation and is required for AT1 and AT2 fate acquisition. Reduced tension enhances FGFR2 endocytosis and ERK signaling to drive AT2 specification. Reduced tension permits architectural remodeling and YAP/TAZ nuclear entry required for AT1 maturation. Intrinsic β-catenin and extrinsic confinement elevate CMT to restrict differentiation and together strongly induce a KRT8⁺ transitional state associated with fibrosis.