Cell-Based Drug Discovery: Identification and Optimization of Small Molecules that Reduce c-MYC Protein Levels in Cells.

Elevated expression of the c-MYC oncogene is one of the most common abnormalities in human cancers. Unfortunately, efforts to identify pharmacological inhibitors that directly target MYC have not yet yielded a drug-like molecule due to the lack of any known small molecule binding pocket in the protein, which could be exploited to disrupt MYC function. We have recently described a strategy to target MYC indirectly, where a screening effort designed to identify compounds that can rapidly decrease endogenous c-MYC protein levels in a MYC-amplified cell line led to the discovery of a compound series that phenocopies c-MYC knockdown by siRNA. Herein, we describe our medicinal chemistry program that led to the discovery of potent, orally bioavailable c-MYC-reducing compounds. The development of a minimum pharmacophore model based on empirical structure activity relationship as well as the property-based approach used to modulate pharmacokinetics properties will be highlighted.

Leucine-rich repeat-containing protein 59 mediates nuclear import of cancerous inhibitor of PP2A in prostate cancer cells.

Using yeast two-hybrid analysis, we identified several novel protein interactions for the oncoprotein Cancerous Inhibitor of PP2A (CIP2A) and confirmed a subset of these interactions in human cancer cell lines. Analysis of the interaction in prostate carcinoma cells between CIP2A and leucine-rich repeat-containing protein 59 (LRRC59) suggests that CIP2A is translocated into the nucleus at G2/M through its association with LRRC59. Recent work by others has demonstrated that nuclear CIP2A disrupts mitotic checkpoints, which promotes deregulation of the cell cycle and increases cancerous phenotypes. Thus, we provide a novel therapeutic mechanism for inhibiting CIP2A function in cancerous cells via targeting the CIP2A-LRRC59 interaction.

Cancerous inhibitor of protein phosphatase 2A promotes premature chromosome segregation and aneuploidy in prostate cancer cells through association with shugoshin.

Yeast two-hybrid (Y2H) studies have shown that cancerous Inhibitor of protein phosphatase 2A (CIP2A) interacted with several proteins, including leucine-rich repeat-containing protein 59 (LRRC59), suggesting that CIP2A may interact with the chromosome maintenance protein, shugoshin (Sgol1). We previously showed that LRRC59 interacted with CIP2A, which was required for CIP2A nuclear localization. Thus, we predicted that CIP2A and Sgol1 may also interact. Sgol1 is a nuclear protein that regulates chromosome segregation during cell division via protection of cohesin ring proteins. Here, we demonstrated that Sgol1 and the C-terminus of CIP2A interact in prostate carcinoma cell lines in a protein phosphatase 2A (PP2A)-dependent manner. Moreover, we demonstrated that depletion of CIP2A in PC-3 cells decreases premature chromosome segregation, whereas overexpression of CIP2A in an immortalized prostate cell line increases premature chromosome segregation. Importantly, we further showed that CIP2A depletion decreases the incidence of aneuploidy and stabilizes cohesin complex proteins, while overexpression of CIP2A destabilizes Sgol1. Thus, our findings strongly suggest that CIP2A promotes cell cycle progression, premature chromosome segregation, and aneuploidy, possibly through a novel interaction with Sgol1.

PME-1 modulates protein phosphatase 2A activity to promote the malignant phenotype of endometrial cancer cells.

Protein phosphatase 2A (PP2A) negatively regulates tumorigenic signaling pathways, in part, by supporting the function of tumor suppressors like p53. The PP2A methylesterase PME-1 limits the activity of PP2A by demethylating its catalytic subunit. Here, we report the finding that PME-1 overexpression correlates with increased cell proliferation and invasive phenotypes in endometrial adenocarcinoma cells, where it helps maintain activated ERK and Akt by inhibiting PP2A. We obtained evidence that PME-1 could bind and regulate protein phosphatase 4 (PP4), a tumor-promoting protein, but not the related protein phosphatase 6 (PP6). When the PP2A, PP4, or PP6 catalytic subunits were overexpressed, inhibiting PME-1 was sufficient to limit cell proliferation. In clinical specimens of endometrial adenocarcinoma, PME-1 levels were increased and we found that PME-1 overexpression was sufficient to drive tumor growth in a xenograft model of the disease. Our findings identify PME-1 as a modifier of malignant development and suggest its candidacy as a diagnostic marker and as a therapeutic target in endometrial cancer.

Assembly of the SLIP1-SLBP complex on histone mRNA requires heterodimerization and sequential binding of SLBP followed by SLIP1.

The SLIP1-SLBP complex activates translation of replication-dependent histone mRNAs. In this report, we describe how the activity of the SLIP1-SLBP complex is modulated by phosphorylation and oligomerization. Biophysical characterization of the free proteins shows that whereas SLIP1 is a homodimer that does not bind RNA, human SLBP is an intrinsically disordered protein that is phosphorylated at 23 Ser/Thr sites when expressed in a eukaryotic expression system such as baculovirus. The bacterially expressed unphosphorylated SLIP1-SLBP complex forms a 2:2 high-affinity (K(D) < 0.9 nM) heterotetramer that is also incapable of binding histone mRNA. In contrast, phosphorylated SLBP from baculovirus has a weak affinity (K(D) ~3 µM) for SLIP1. Sequential binding of phosphorylated SLBP to the histone mRNA stem-loop motif followed by association with SLIP1 is required to form an "active" ternary complex. Phosphorylation of SLBP at Thr171 promotes dissociation of the heterotetramer to the SLIP1-SLBP heterodimer. Using alanine scanning mutagenesis, we demonstrate that the binding site on SLIP1 for SLBP lies close to the dimer interface. A single-point mutant near the SLIP1 homodimer interface abolished interaction with SLBP in vitro and reduced the abundance of histone mRNA in vivo. On the basis of these biophysical studies, we propose that oligomerization and SLBP phosphorylation may regulate the SLBP-SLIP1 complex in vivo. SLIP1 may act to sequester SLBP in vivo, protecting it from proteolytic degradation as an inactive heterotetramer, or alternatively, formation of the SLIP1-SLBP heterotetramer may facilitate removal of SLBP from the histone mRNA prior to histone mRNA degradation.

Ets1 and Elk1 transcription factors regulate cancerous inhibitor of protein phosphatase 2A expression in cervical and endometrial carcinoma cells.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) has been identified as a proto-oncogene that is overexpressed in various types of human cancers. CIP2A acts by inhibiting protein phosphatase 2A-dependent destabilization of c-Myc, resulting in increased cell proliferation. Here, we have characterized the proximal promoter region of the human CIP2A gene in cervical, endometrial and liver carcinoma cells. The 5' flanking minimal proximal promoter of the CIP2A gene consists of putative binding sites for Ets1 and Elk1 in forward and reverse orientations. Here, we show that Ets1 and Elk1 binding is essential for CIP2A basal expression in several urogenital cancer cell lines. Interestingly, both Ets1 and Elk1 are required together for CIP2A expression, as siRNA knockdown of Ets1 and Elk1 together decreased CIP2A gene transcription, whereas knockdown of Ets1 or Elk1 alone had no effect. Moreover, ectopic expression of Ets1 and Elk1 together increased CIP2A expression. To gain physiological significance of the Ets1 and Elk1 regulation we observed, a panel of matched human cervical carcinoma samples was analyzed for the expression of CIP2A and Ets1 and/or Elk1. We found a direct correlation between the levels of CIP2A and the levels of Ets1 and Elk1. Our results suggest that the binding of Ets1 and Elk1 together to the proximal CIP2A promoter is absolutely required for CIP2A expression in cervical, endometrial and liver carcinoma cell lines. Thus, different factors regulate CIP2A expression in a cell-type specific manner. As previous work has shown a requirement for only Ets1 in prostate and gastric carcinomas, our results now indicate that CIP2A regulation is more complex than previously determined.