C-terminal binding proteins (CtBP1/2) are oncogenic transcriptional coregulators and dehydrogenases often overexpressed in multiple solid tumors, including breast, colon, and ovarian cancer, and associated with poor survival. CtBPs act by repressing expression of genes responsible for apoptosis (e.g., PUMA, BIK) and metastasis-associated epithelial-mesenchymal transition (e.g., CDH1), and by activating expression of genes that promote migratory and invasive properties of cancer cells (e.g., TIAM1) and genes responsible for enhanced drug resistance (e.g., MDR1). CtBP's transcriptional functions are also critically dependent on oligomerization and nucleation of transcriptional complexes. Recently, we have developed a family of CtBP dehydrogenase inhibitors, based on the parent 2-hydroxyimino-3-phenylpropanoic acid (HIPP), that specifically disrupt cancer cell viability, abrogate CtBP's transcriptional function, and block polyp formation in a mouse model of intestinal polyposis that depends on CtBP's oncogenic functions. Crystallographic analysis revealed that HIPP interacts with CtBP1/2 at a conserved active site tryptophan (W318/324; CtBP1/2) that is unique among eukaryotic D2-dehydrogenases. To better understand the mechanism of action of HIPP-class inhibitors, we investigated the contribution of W324 to CtBP2's biochemical and physiologic activities utilizing mutational analysis. Indeed, W324 was necessary for CtBP2 self-association, as shown by analytical ultracentrifugation and in vivo cross-linking. Additionally, W324 supported CtBP's association with the transcriptional corepressor CoREST, and was critical for CtBP2 induction of cell motility. Notably, the HIPP derivative 4-chloro-HIPP biochemically and biologically phenocopied mutational inactivation of CtBP2 W324. Our data support further optimization of W318/W324-interacting CtBP dehydrogenase inhibitors that are emerging as a novel class of cancer cell-specific therapeutic.

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C-terminal Binding Proteins (CtBP) 1 and 2 constitute a family of oncogenic transcriptional co-regulators overexpressed in tumor tissues that are associated with worse prognostic outcome and aggressive tumor characteristics in multiple cancer types. Specifically, CtBP has been found to repress expression of genes responsible for apoptosis and EMT (eg. BIK and CDH1) and promote expression of genes that partake in the migration of cancer cells and those that are responsible for enhanced drug resistance (eg. TIAM1 and MDR1). CtBP2 is also critically required for colon cancer stem cell self-renewal. CtBP is unique among transcription co-regulators in harboring a conserved D-isomer specific 2-hydroxyacid dehydrogenase (D2DH) domain, which reduces an alpha-keto acid substrate to an alpha-hydroxy acid in the presence of NADH. The presence of NADH also facilities oligomerization of CtBP, leading to assembly of higher order complexes of CtBP with both DNA binding transcription factors and histone modifying enzymes that then leads to modulation target genes. We have identified hydroxyimino-3-phenylpropanoic acid and its 4-chloro derivative (HIPP; 4-Cl-HIPP) as potent substrate competitive inhibitors of the CtBP dehydrogenase (IC50’s=240nM, 180 nM), which also disrupt CtBP oligomerization, promoter localization, and transcriptional regulation. Co-crystallization of HIPP and CtBP1/2 indicated a strong π-π interaction between the HIPP phenyl ring and the indolyl ring in tryptophan W318/324 of CtBP1/2, indicating that this tryptophan is critical to CtBP interaction with HIPP inhibitors, dehydrogenase function, and quaternary structure. Of note W318/324, though conserved in CtBP1/2, is unique among D2DH, suggesting that a better understanding of W318/324 role and function in CtBP structure and function is critical to optimizing design of inhibitors that targeting this evolutionary unique residue among dehydrogenases. To further elucidate the mechanism of action in catalysis and oligomerization, as well as functional importance for transcription of CtBP2 W324, we analyzed enzyme kinetics, oligomerization, transcriptional co-regulatory activity and cell migration in a series of CtBP2 W324 mutants overexpressed in breast and colon cancer cell lines with concomitant knockdown (siRNA) or knockout (CRISPR/Cas9) of CtBP2. Our data demonstrates that mutation of W324 abrogated dehydrogenase activity, oligomerization, transactivation of the validated CtBP target gene TIAM1 and induction of migration. In summary, our findings suggest that the W324 residue is critical for CtBP2’s function and its unique conservation in CtBP1/2 vs. other dehydrogenases will allow the development of high specificity CtBP W318/324 inhibitors to limit potential toxicity due to off target inhibition of related metabolic dehydrogenases. Citation Format: Martin M. Dcona, Benjamin L. Morris, Priyadarshan K. Damle, Zaid Nawaz, Francisco Zarate Perez, Michael J. Dennis, Sahib J. Singh, William E. Royer, Keith C. Ellis, Steven R. Grossman. Tryptophan 318/324, the target of C-terminal binding protein (CtBP) inhibitors, plays a critical role in CtBP enzymatic activity, oligomerization and transcriptional coregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3204. doi:10.1158/1538-7445.AM2017-3204

C-terminal binding proteins (CtBP) 1 and 2 are transcriptional coregulators that are upregulated in several cancers, including a majority of studied breast, colorectal, and ovarian tumor samples. CtBPs drive many cellular oncogenic properties, including migration, invasion, proliferation, and survival, in part through repression of tumor suppressor genes, such as E-cadherin, PTEN, BRCA1, and p16. CtBPs encode an intrinsic dehydrogenase activity regulated by both intracellular NADH concentration and the putative substrate 4-methylthio-2-oxobutyric acid (MTOB). NADH binding induces CtBP dimerization, which regulates the recruitment of transcriptional regulatory complexes. High levels of MTOB appear to inhibit CtBP dehydrogenase function and induce cytotoxicity among cancer cells in a CtBP-dependent manner. However, the function of the substrate-binding domain has yet to be examined with regard to CtBP's oncogenic activity. To this end, we have created several point mutations in the substrate-binding pocket of CtBP and have determined key residues for CtBP's enzymatic activity using an in vitro dehydrogenase assay. We have found that a conserved tryptophan in the catalytic domain, and specifically its aromatic moiety, is imperative for enzyme activity. This tryptophan is unique to CtBP family proteins and distinguishes the CtBP substrate-binding domain from that of all other families of dehydrogenases. Additionally, we found arginine and histidine residues lining the substrate pocket that are necessary for CtBP dehydrogenase function. Knowledge of these residues allows the directed synthesis of drugs with increased potency and higher CtBP specificity. Moreover, to investigate the importance of the catalytic domain on CtBP's oncogenic potential, we have transfected human cancer cell lines with several CtBP substrate-binding domain mutants and observed the effects on cellular processes, including growth, survival, migration, and recruitment of key components of the transcriptional repressor complex. From this work we have investigated the utility of CtBP, and specifically the CtBP substrate-binding domain, as a target for cancer therapeutics. We also provide preliminary insights into the function of this domain in cellular models of cancer. Citation Format: Benjamin L. Morris, Priyadarshan Damle, Zaid Nawaz, Steven R. Grossman. Evaluation of critical residues in the C-terminal binding protein (CtBP) dehydrogenase domain contributing to substrate binding, catalysis, and oncogenic activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2199. doi:10.1158/1538-7445.AM2015-2199