Abstract
Autophagy is an evolutionarily conserved process by which cellular structures may be degraded for ongoing biogenesis. Following nutrient deprivation, autophagy functions critically to replenish metabolic precursors by recycling nonessential structures. Recent evidence has identified the induction of autophagy following growth factor withdrawal in preclinical models of cancer. As the autophagic response in these studies requires abrogation of apoptotic effectors, autophagy emerges as a relevant cell survival mechanism in human cancer. While growth factor depletion is an uncommon approach in cancer therapy, small molecule inhibition of dominant growth factor pathways is a central focus of targeted chemotherapeutic development. Therefore, we predicted that small molecule inhibition of growth factor pathways will prompt an autophagic response in human cancers, and that additional inhibition of autophagy will result in a synergistic and tumor-selective cytotoxicity. As there are few known inhibitors of autophagy, we developed a forward chemical genetic discovery approach utilizing high-throughput, high-content, epifluorescent microscopy. A human glioblastoma cell line stably expressing an EGFP-LC3 fusion protein was cultured at low confluency in 384-well plate format. Four hours following 100 nL pin-transfer of an arrayed small molecule library, cells were fixed and nuclei were counter-stained with Hoechst. Images were acquired and analyzed using automated microscopy and scripting software (MetaXPress; Molecular Devices, Sunnyvale, CA). The designed algorithm proved capable of quantitative cell-scoring for the presence of characteristic, fluorescent, LC3-positive autophagic punctae, as well as discrete annotation of vesicle size permitting the differentiation of likely autophagy inducers from inhibitors of autophagosome-lysosome fusion. Statistical rendering of four phenotypic measurements for the selection of assay positives was performed in the ChemBank analysis environment (NCI-Initiative for Chemical Genetics, Cambridge, MA). With an interest in discovering immediate candidates for clinical investigation, we profiled a 3000-member small molecule library of off-patent pharmaceutical products and bioactive compounds with known pharmacology. Six ligands were discovered which demonstrated a phenotype characteristic of late inhibition of autophagosomal maturation. Each is an established pharmaceutical product. At least three of these molecules have previous annotation as inhibitors of vesicle function in the medical literature. With an interest in profiling these ligands for synergy with growth factor pathway inhibitors in cancer, we established a novel platform for assessing synergistic cytotoxicity in high-throughput format. Data collected from these experiments confirm the synergistic antineoplastic activity of known and novel inhibitors of autophagy when combined with receptor tyrosine kinase inhibition in model systems of hematologic and epithelial tumors. These experiments serve as the scientific basis for clinical studies planned in lung cancer, breast cancer and multiple myeloma. Additionally, these data identify a panel of small molecule inducers of autophagy of plausible utility in protein aggregation disorders such as Huntington’s, Parkinson’s and Prion Diseases.
Organoids in image-based phenotypic chemical screens
