Sam68 Allows Selective Targeting of Human Cancer Stem Cells.

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/ß-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/ß-catenin signaling within CSCs. Disruption of CBP-ß-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.

Reversible lineage-specific priming of human embryonic stem cells can be exploited to optimize the yield of differentiated cells.

The clinical use of human embryonic stem cells (hESCs) requires efficient cellular expansion that must be paired with an ability to generate specialized progeny through differentiation. Self-renewal and differentiation are deemed inherent hallmarks of hESCs and a growing body of evidence suggests that initial culture conditions dictate these two aspects of hESC behavior. Here, we reveal that defined culture conditions using commercial mTeSR1 media augment the expansion of hESCs and enhance their capacity for neural differentiation at the expense of hematopoietic lineage competency without affecting pluripotency. This culture-induced modification was shown to be reversible, as culture in mouse embryonic fibroblast-conditioned media (MEF-CM) in subsequent passages allowed mTeSR1-expanded hESCs to re-establish hematopoietic differentiation potential. Optimal yield of hematopoietic cells can be achieved by expansion in mTeSR1 followed by a recovery period in MEF-CM. Furthermore, the lineage propensity to hematopoietic and neural cell types could be predicted via analysis of surrogate markers expressed by hESCs cultured in mTeSR1 versus MEF-CM, thereby circumventing laborious in vitro differentiation assays. Our study reveals that hESCs exist in a range of functional states and balance expansion with differentiation potential, which can be modulated by culture conditions in a predictive and quantitative manner.

Human embryonic stem cell-derived hematopoietic cells maintain core epigenetic machinery of the polycomb group/Trithorax Group complexes distinctly from functional adult hematopoietic stem cells.

Hematopoietic cells derived from human embryonic stem cells (hESCs) have a number of potential utilities, including the modeling of hematological disorders in vitro, whereas the use for cell replacement therapies has proved to be a loftier goal. This is due to the failure of differentiated hematopoietic cells, derived from human pluripotent stem cells (hPSCs), to functionally recapitulate the in vivo properties of bona fide adult hematopoietic stem/progenitor cells (HSPCs). To better understand the limitations of differentiation programming at the molecular level, we have utilized differential gene expression analysis of highly purified cells that are enriched for hematopoietic repopulating activity across embryonic, fetal, and adult human samples, including in vivo explants of human HSPCs 8-weeks post-transplantation. We reveal that hESC-derived hematopoietic progenitor cells (eHPCs) fail to express critical transcription factors which are known to govern self-renewal and myeloid/lymphoid development and instead retain the expression of Polycomb Group (PcG) and Trithorax Group (TrxG) factors which are more prevalent in embryonic cell types that include EZH1 and ASH1L, respectively. These molecular profiles indicate that the differential expression of the core epigenetic machinery comprising PcGs/TrxGs in eHPCs may serve as previously unexplored molecular targets that direct hematopoietic differentiation of PSCs toward functional HSPCs in humans.

Identification of drugs including a dopamine receptor antagonist that selectively target cancer stem cells.

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.

Kaiso regulates Znf131-mediated transcriptional activation.

Kaiso is a dual-specificity POZ-ZF transcription factor that regulates gene expression by binding to sequence-specific Kaiso binding sites (KBS) or methyl-CpG dinucleotide pairs. Kaiso was first identified as a binding partner for the epithelial cell adhesion regulator p120(ctn). The p120(ctn)/Kaiso interaction is reminiscent of the beta-catenin/TCF interaction and several studies have suggested that Kaiso is a negative regulator of the Wnt/beta-catenin TCF signaling pathway. To gain further insight into Kaiso's function, we performed a yeast two-hybrid screen using the Kaiso POZ domain as bait. This screen identified the POZ-ZF protein, Znf131, as a Kaiso-specific binding partner. GST pull-down assays confirmed that the interaction is mediated via the POZ domain of each protein, and co-immunoprecipitation experiments further supported an in vivo Kaiso-Znf131 interaction. Using a Cyclic Amplification and Selection of Targets (CAST) approach, we identified the 12-base pair DNA palindrome sequence GTCGCR-(X)(n)-YGCGAC as a potential Znf131 binding element (ZBE). In vitro studies using electrophoretic mobility shift assay (EMSA) demonstrated that Znf131 binds the ZBE via its zinc finger domain. Znf131 DNA-binding specificity was confirmed using competition assays and ZBE mutational analyses. An artificial promoter-reporter construct containing four tandem copies of the ZBE was constructed and used to assess Znf131 transcriptional properties. We observed dose-dependent transcriptional activation of this artificial promoter-reporter by Znf131 in both epithelial and fibroblast cells, suggesting that Znf131 is a transcriptional activator. Kaiso overexpression significantly decreased the Znf131-mediated transcriptional activation, and interestingly, co-expression of the Kaiso-specific interaction partner p120(ctn) relieved Kaiso's inhibition of Znf131-mediated transcriptional activation. These findings indicate that Znf131 is a transcriptional activator, a less common function of POZ-ZF proteins, that is negatively regulated by its heterodimerization partner Kaiso.

Nuclear trafficking of the POZ-ZF protein Znf131.

Znf131 is a member of the BTB/POZ family of transcription factors with roles in development and carcinogenesis. Like many members of this protein family, Znf131 displays robust nuclear localization in cultured cells, but the mechanism(s) of Znf131 nuclear trafficking is unknown. Here, we report the mechanism of Znf131 nuclear localization. Visual inspection of the Znf131 amino acid sequence revealed three basic regions (BR-1, -2 and -3) with the potential to serve as nuclear localization signals (NLS). Of the three basic regions, only BR-1 functioned independently to efficiently target heterologous beta-gal-GFP fusion proteins to HeLa cell nuclei. However, a Znf131 truncation mutant containing BR-2 and BR-3 efficiently targeted heterologous beta-gal-GFP fusion proteins to HeLa cell nuclei. Mutational analysis of full-length GFP-tagged Znf131 revealed that loss of any one BR alone did not prevent Znf131 nuclear localization. This apparent redundancy in NLS activity was due to the fact that intact BR-1 or BR-2 alone could target full-length Znf131 to nuclei. Consequently, simultaneous mutation of BR-1 and BR-2 abolished full-length Znf131 nuclear localization. Therefore, BR-1 and BR-2 are functional NLSs for Znf131 and as such are designated NLS-1 and NLS-2. Finally, wild type Znf131, and not a Znf131 NLS-defective mutant (NLS-1m/NLS-2m) interacted preferentially with the nuclear import receptor Importin-alpha3 in vitro.