Here we report corin, a synthetic hybrid agent derived from the class I HDAC inhibitor (entinostat) and an LSD1 inhibitor (tranylcypromine analog). Enzymologic analysis reveals that corin potently targets the CoREST complex and shows more sustained inhibition of CoREST complex HDAC activity compared with entinostat. Cell-based experiments demonstrate that corin exhibits a superior anti-proliferative profile against several melanoma lines and cutaneous squamous cell carcinoma lines compared to its parent monofunctional inhibitors but is less toxic to melanocytes and keratinocytes. CoREST knockdown, gene expression, and ChIP studies suggest that corin’s favorable pharmacologic effects may rely on an intact CoREST complex. Corin was also effective in slowing tumor growth in a melanoma mouse xenograft model. These studies highlight the promise of a new class of two-pronged hybrid agents that may show preferential targeting of particular epigenetic regulatory complexes and offer unique therapeutic opportunities.

dMammalian genomes harbor three CoREST genes. rcor1 encodes CoREST (CoREST1), and the paralogues rcor2 and rcor3 encode CoREST2 and CoREST3, respectively. Here, we describe specific properties of transcriptional complexes formed by CoREST proteins with the histone demethylase LSD1/KDM1A and histone deacetylases 1 and 2 (HDAC1/2) and the finding that all three CoRESTs are expressed in the adult rat brain. CoRESTs interact equally strongly with LSD1/KDM1A. Structural analysis shows that the overall conformation of CoREST3 is similar to that of CoREST1 complexed with LSD1/KDM1A. Nonetheless, transcriptional repressive capacity of CoREST3 is lower than that of CoREST1, which correlates with the observation that CoREST3 leads to a reduced LSD1/KDM1A catalytic efficiency. Also, CoREST2 shows a lower transcriptional repression than CoREST1, which is resistant to HDAC inhibitors. CoREST2 displays lower interaction with HDAC1/2, which is barely present in LSD1/KDM1A-CoREST2 complexes. A nonconserved leucine in the first SANT domain of CoREST2 severely weakens its association with HDAC1/2. Furthermore, CoREST2 mutants with increased HDAC1/2 interaction and those without HDAC1/2 interaction exhibit equivalent transcriptional repression capacities, indicating that CoREST2 represses in an HDAC-independent manner. In conclusion, differences among CoREST proteins are instrumental in the modulation of protein-protein interactions and catalytic activities of LSD1/KDM1A-CoREST-HDAC complexes, fine-tuning gene expression regulation.

Nurr1 is a transcription factor essential for the development of ventral dopaminergic neurons. In search for regulatory mechanisms of Nurr1 function, we identified the SUMO (small ubiquitin-like modifier)-E3 ubiquitinprotein isopeptide ligase, PIAS␥, as an interaction partner of Nurr1. Overexpressed PIAS␥ and Nurr1 co-localize in the nuclei of transfected cells, and their interaction is demonstrated through co-immunoprecipitation and glutathione S-transferase pulldown assays. Co-expression of PIAS␥ with Nurr1 results in a potent repression of Nurr1-dependent transcriptional activation of an artificial NGFI-B response element (NBRE) reporter as well as of a reporter driven by the native tyrosine hydroxylase promoter. We identified two consensus sumoylation sites in Nurr1. The substitution of lysine 91 by arginine in one SUMO site enhanced the transcriptional activity of Nurr1, whereas the substitution of lysine 577 by arginine in the second SUMO site decreased transcriptional activity of Nurr1. Interestingly, PIAS␥-induced repression of Nurr1 activity does not require the two sumoylation sites, because each mutant is repressed as efficiently as the wild type Nurr1. In addition, the mutations do not alter Nurr1 nuclear localization. Finally, we provide evidence that Nurr1 and PIAS␥ co-exist in several nuclei of the rodent central nervous system by demonstrating the co-expression of Nurr1 protein and PIAS␥ mRNA in the same cells. In conclusion, our studies identified PIAS␥ as a transcriptional co-regulator of Nurr1 and suggest that this interaction may have a physiological role in regulating the expression of Nurr1 target genes.

Genesis of midbrain dopamine (DA) neurons depends on Nurr1, a nuclear receptor expressed during development and adulthood in these neurons. Nurr1 is required for the expression of genes of dopaminergic phenotype such as tyrosine hydroxylase and DA transporter. The expression of the tyrosine kinase receptor RET also depends on Nurr1 during development. However, it is unknown whether RET expression is regulated by Nurr1 during adulthood, and the mechanism by which Nurr1 regulates RET expression. Using an adeno-associated vector-delivered anti-Nurr1 ribozyme, we knocked-down Nurr1 expression unilaterally in the substantia nigra (SN) of adult rats. Animals injected with the ribozyme displayed a 57.3% decrease in Nurr1 mRNA in the SN accompanied by decreased DA extracellular levels in the striatum. RET mRNA in the injected SN and RET protein in the ipsilateral striatum decreased 76.9% and 47%, respectively. Tyrosine hydroxylase and DA transporter mRNA did not change in Nurr1 knocked-down SN. Nurr1 induced the transcription of the human RET promoter in cell type and concentration-dependent manner. Nurr1 induction of RET promoter is independent of NBRE elements. These results show that the expression of RET in rat adult SN is regulated by Nurr1 and suggest that RET is a transcriptional target of this nuclear receptor.