Organoids derived from human pluripotent stem cells are a potentially powerful tool for high-throughput screening (HTS), but the complexity of organoid cultures poses a significant challenge for miniaturization and automation. Here, we present a fully automated, HTS-compatible platform for enhanced differentiation and phenotyping of human kidney organoids. The entire 21-day protocol, from plating to differentiation to analysis, can be performed automatically by liquid-handling robots, or alternatively by manual pipetting. High-content imaging analysis reveals both dose-dependent and threshold effects during organoid differentiation. Immunofluorescence and single-cell RNA sequencing identify previously undetected parietal, interstitial, and partially differentiated compartments within organoids and define conditions that greatly expand the vascular endothelium. Chemical modulation of toxicity and disease phenotypes can be quantified for safety and efficacy prediction. Screening in gene-edited organoids in this system reveals an unexpected role for myosin in polycystic kidney disease. Organoids in HTS formats thus establish an attractive platform for multidimensional phenotypic screening.

We have isolated cDNAs for four human genes (DPDE1 through DPDE4) closely related to the dnc learning and memory locus ofDrosophila melanogaster. The deduced amino acid sequences of the Drosophila and human proteins have considerable homology, extending beyond the putative catalytic region to include two novel, highly conserved, upstream conserved regions (UCR1 and UCR2). The upstream conserved regions are located in the amino-terminal regions of the proteins and appear to be unique to these genes. Polymerase chain reaction analysis suggested that these genes encoded the only homologs of dnc in the human genome. Three of the four genes were expressed in Saccharomyces cerevisiae and shown to encode cyclic AMP-specific phosphodiesterases. The products of the expressed genes displayed the pattern of sensitivity to inhibitors expected for members of the type IV, cyclic AMP-specific class of phosphodiesterases. Each of the four genes demonstrated a distinctive pattern of expression in RNA from human cell lines.Regulation of cyclic AMP (cAMP) is important in central nervous system (CNS) function in both invertebrates and mammals. Studies of two different invertebrates in particular have demonstrated a role for cAMP in learning and memory. InAplysia snails, long-term facilitation of neurons in the gill retraction reflex occurs with learning. These neuronal changes are associated with alterations in activity of several components of the cAMP signalling pathway, particularly adenylyl cyclase and a cAMP-dependent protein kinase (32). In Drosophila melanogaster, numerous mutations in genes affecting learning have been isolated, including dunce (dnc), which encodes a cAMP-specific phosphodiesterase (PDE), and rutabaga (rut), which encodes a calcium-calmodulinstimulated adenylyl cyclase (10,16,28). Electrophysiologic studies of neurons from dnc-and rut-flies have shown alterations in synaptic plasticity, suggesting that the functional changes occurring in learning in wild-type flies are similar to those seen in Aplysia snails (40). These observations suggest that modulation of the cAMP pathway can influence learning and memory in organisms widely separated in evolution.Evidence for a functional role of dunce-like PDEs in the mammalian CNS initially came from the development of specific inhibitors. Inhibitors of cAMP-specific PDEs, including the drug rolipram, have clinical activity as antidepressants (11). The initial biochemical analysis of mammalian CNS PDEs, and of these inhibitors, was limited to testing in partially purified preparations, which can contain multiple PDE isoforms (1). More recently, cDNAs for homologs of dnc have been cloned from rats (5,7,9,(36)(37)(38) and shown to be rolipram sensitive (14,37,38

Cancers that appear pathologically similar often respond differently to the same drug regimens. Methods to better match patients to drugs are in high demand. We demonstrate a promising approach to identify robust molecular markers for targeted treatment of acute myeloid leukemia (AML) by introducing: data from 30 AML patients including genome-wide gene expression profiles and in vitro sensitivity to 160 chemotherapy drugs, a computational method to identify reliable gene expression markers for drug sensitivity by incorporating multi-omic prior information relevant to each gene’s potential to drive cancer. We show that our method outperforms several state-of-the-art approaches in identifying molecular markers replicated in validation data and predicting drug sensitivity accurately. Finally, we identify SMARCA4 as a marker and driver of sensitivity to topoisomerase II inhibitors, mitoxantrone, and etoposide, in AML by showing that cell lines transduced to have high SMARCA4 expression reveal dramatically increased sensitivity to these agents.

Glioblastoma Multiforme (GBM) continues to have a poor patient prognosis despite optimal standard of care. Glioma stem cells (GSCs) have been implicated as the presumed cause of tumor recurrence and resistance to therapy. With this in mind, we screened a diverse chemical library of 2,000 compounds to identify therapeutic agents that inhibit GSC proliferation and therefore have the potential to extend patient survival. High-throughput screens (HTS) identified 78 compounds that repeatedly inhibited cellular proliferation, of which 47 are clinically approved for other indications and 31 are experimental drugs. Several compounds (such as digitoxin, deguelin, patulin and phenethyl caffeate) exhibited high cytotoxicity, with half maximal inhibitory concentrations (IC50) in the low nanomolar range. In particular, the FDA approved drug for the treatment of alcoholism, disulfiram (DSF), was significantly potent across multiple patient samples (IC50 of 31.1 nM). The activity of DSF was potentiated by copper (Cu), which markedly increased GSC death. DSF–Cu inhibited the chymotrypsin-like proteasomal activity in cultured GSCs, consistent with inactivation of the ubiquitin-proteasome pathway and the subsequent induction of tumor cell death. Given that DSF is a relatively non-toxic drug that can penetrate the blood-brain barrier, we suggest that DSF should be tested (as either a monotherapy or as an adjuvant) in pre-clinical models of human GBM. Data also support targeting of the ubiquitin-proteasome pathway as a therapeutic approach in the treatment of GBM.