Targeting lysosomes with a novel chloroquine derivative induces irreversible lysosomal damage and disrupts autophagosome and lysosome assembly in cancer.

Pancreatic ductal adenocarcinoma (PDAC) exhibits profound therapy resistance driven by lysosome-dependent nutrient recycling, metabolic adaptation, and stress tolerance. Current lysosome targeting agents such as chloroquine (CQ)/hydroxychloroquine (HCQ) show limited efficacy due to transient activity and dose-limiting-toxicities. To overcome these limitations, we developed lysostilbenes, a new class of hybrid small molecules combining the CQ pharmacophore with lysosome-disrupting stilbene analogs. Stilbene pharmacophore is the core structural component of resveratrol. Among the synthesized hybrids, lysostilbene-4 emerged as the lead candidate, demonstrating ~30-40-fold greater cytotoxicity against PDAC cells than parent compounds, while sparing nonmalignant cells. At nanomolar concentrations, lysostilbene-4 induced rapid, irreversible lysosomal membrane permeabilization (LMP), initiating a lysosome mitochondria apoptotic cascade via CTSB (cathepsin B) release, BID cleavage, BAX activation, and caspase-mediated apoptosis. In parallel, it abrogated lysosomal recovery by significantly reducing repair, lysophagy, autophagosome maturation, and uncoupling TFEB-driven transcriptional programs from effective lysosome biogenesis. Reduced TFEB mRNA expression correlated with poor overall-survival and disease-free-survival across multiple cancer patients, with a particularly strong association in pancreatic cancer patients. Using TFEB+/+ and TFEB-/- knockout pancreatic cancer cells we establish that lysostilbene-4 exerts severe cytotoxicity by inducing persistent lysosomal-damage and disrupting autophagosome-lysosome assembly, with vulnerability further amplified in TFEB-deficient cells. This finding underscores TFEB as a key determinant of lysosomal-resilience and a potential predictive biomarker. Importantly, lysostilbene-4 was well tolerated in preclinical mouse-models at supra-therapeutic doses without systemic-toxicity. These findings position lysostilbene-4 as a first-in-class lysosome-targeting therapeutic that enforces sustained lysosomal collapse while compromising adaptive recovery-mechanisms, providing a mechanistically precise and safe strategy against PDAC.Abbreviations: ALG: autophagy-lysosome genes; AMPK: AMP-activated protein kinase; CASM: conjugation of ATG8s to single membranes; CTSB: cathepsin B; LGALS3: galectin 3; LMP: lysosomal membrane permeabilization; LS: lysostilbene; MTOR: mechanistic target of rapamycin kinase; PDAC: pancreatic ductal adenocarcinoma; TCGA: The Cancer Genome Atlas; TFEB: transcription factor EB; ULK1: unc-51 like autophagy activating kinase 1.