Selective targeting of cancer stem cells (CSCs) offers promise for a new generation of therapeutics. However, assays for both human CSCs and normal stem cells that are amenable to robust biological screens are limited. Using a discovery platform that reveals differences between neoplastic and normal human pluripotent stem cells (hPSC), we identify small molecules from libraries of known compounds that induce differentiation to overcome neoplastic self-renewal. Surprisingly, thioridazine, an antipsychotic drug, selectively targets the neoplastic cells, and impairs human somatic CSCs capable of in vivo leukemic disease initiation while having no effect on normal blood SCs. The drug antagonizes dopamine receptors that are expressed on CSCs and on breast cancer cells as well. These results suggest that dopamine receptors may serve as a biomarker for diverse malignancies, demonstrate the utility of using neoplastic hPSCs for identifying CSC-targeting drugs, and provide support for the use of differentiation as a therapeutic strategy.

Human embryonic stem cells (hESC) have potential applications as tools for drug screening to identify small molecule regulators of self-renewal or differentiation. Elucidating the mechanisms governing lineage commitment in hESC will allow for efficient derivation of specified cell types for clinical use. Recognizing the early steps in loss of pluripotency is key to achieving both goals of drug screening and derivation of therapeutically relevant cell types. Here we report the use of a real time cell cycle fluorescent reporter for the first time in hESC that indicates onset of differentiation in a lineage unbiased manner. Pluripotent hESC possess a short cell cycle length, due primarily to a truncated G1 phase. G1 lengthens concomitant with differentiation. Stable hESC lines expressing the live cell cycle reporter exhibit fluorescence only during G1. Due to the short length of pluripotent G1 phase, G1 fluorescence is only weakly and transiently detected, however it is quickly increased to easily detectable levels upon onset of differentiation. We hypothesize that lengthened G1 phase can be used as an indicator of differentiation status of individual human embryonic stem cells. Cells with lengthened G1 are typically negative for pluripotency markers OCT4, Tra-1-60 and SSEA-3 following differentiation. Differentiated cells with lengthened G1 also demonstrate increased levels of lineage-specific differentiation markers at both the protein and mRNA level. Automated image analysis of hESC indicates this mutually exclusive relationship between lengthened G1 and pluripotency exists both on the cellular level and in colonies as a whole. Here we have shown that lengthened G1 indicates both loss of pluripotency and gain of lineage markers.

Targeting of human cancer stem cells (CSCs) requires the identification of vulnerabilities unique to CSCs versus healthy resident stem cells (SCs). Unfortunately, dysregulated pathways that support transformed CSCs, such as Wnt/β-catenin signaling, are also critical regulators of healthy SCs. Using the ICG-001 and CWP family of small molecules, we reveal Sam68 as a previously unappreciated modulator of Wnt/β-catenin signaling within CSCs. Disruption of CBP-β-catenin interaction via ICG-001/CWP induces the formation of a Sam68-CBP complex in CSCs that alters Wnt signaling toward apoptosis and differentiation induction. Our study identifies Sam68 as a regulator of human CSC vulnerability.

Spatial localization of primitive leukemic cells is restricted to niches shared with their normal counterparts, and their ability to retain occupancy of these niches is rivaled by normal HSPCs.