Discovery of small molecules that induce lysosomal cell death in cancer cells using a phenotypic screening platform

The lysosomal cell death, (LCD), pathway is a caspase-independent, cell death pathway that has been suggested as a possible target for cancer therapy; making the development of sensitive and specific high-throughput (HT) assays to identify LCD-inducers highly desirable. Here, we report a two-step HT screening platform to reliably identify LCD-inducers. First we identify compounds that kill through a non-apoptotic pathway. A multiplexed phenotypic image-based assay using a Gal-3 reporter was then used to further classify hits as LCD-inducers. The identification of permeablized lysosomes in our phenotypic assay is not affected by changes in the lysosomal pH, thus resolving an important limitation in currently used methods. We have validated our platform in a screen by identifying 24 LCD-inducers, some of them previously known to be LCD-inducers. Although most LCD-inducers were cationic amphiphilic drugs, (CADs) our data also gave new insights into the biology of LCD suggesting that lysosomal accumulation and ASM inhibition are not sufficient or necessary for the induction of LCD. Accordingly, we have identified a non-CAD LCD-inducer, which is of great interest in the field. Overall our results demonstrate a robust rapid HT platform to identify novel LCD-inducers that will also be very useful for gaining deeper insights in to the role of role of lysosomal membrane permeabilization in drug-induced toxicity.