Active-Site Tryptophan, the Target of Antineoplastic C-Terminal Binding Protein Inhibitors, Mediates Inhibitor Disruption of CtBP Oligomerization and Transcription Coregulatory Activities.

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.

Combined Targeting of NAD Biosynthesis and the NAD-dependent Transcription Factor C-terminal Binding Protein as a Promising Novel Therapy for Pancreatic Cancer.

Cancer therapies targeting metabolic derangements unique to cancer cells are emerging as a key strategy to address refractory solid tumors such as pancreatic ductal adenocarcinomas (PDAC) that exhibit resistance to extreme nutrient deprivation in the tumor microenvironment. Nicotinamide adenine dinucleotide (NAD) participates in multiple metabolic pathways and nicotinamide phosphoribosyl transferase (NAMPT) is one of the key intracellular enzymes that facilitate the synthesis of NAD. C-terminal binding proteins 1 and 2 (CtBP) are paralogous NAD-dependent oncogenic transcription factors and dehydrogenases that nucleate an epigenetic complex regulating a cohort of genes responsible for cancer proliferation and metastasis. As adequate intracellular NAD is required for CtBP to oligomerize and execute its oncogenic transcriptional coregulatory activities, we hypothesized that NAD depletion would synergize with CtBP inhibition, improving cell inhibitory efficacy. Indeed, depletion of cellular NAD via the NAMPT inhibitor GMX1778 enhanced growth inhibition induced by either RNAi-mediated CtBP1/2 knockdown or the CtBP dehydrogenase inhibitor 4-chlorophenyl-2-hydroxyimino propanoic acid as much as 10-fold in PDAC cells, while untransformed pancreatic ductal cells were unaffected. The growth inhibitory effects of the NAMPT/CtBP inhibitor combination correlated pharmacodynamically with on-target disruption of CtBP1/2 dimerization, CtBP2 interaction with the CoREST epigenetic regulator, and transcriptional activation of the oncogenic target gene TIAM1. Moreover, this same therapeutic combination strongly attenuated growth of PDAC cell line xenografts in immunodeficient mice, with no observable toxicity. Collectively, our data demonstrate that targeting CtBP in combination with NAD depletion represents a promising therapeutic strategy for PDAC. SIGNIFICANCE: Effective precision therapies are lacking in PDAC. We demonstrate that simultaneous inhibition of NAD metabolism and the oncoprotein CtBP is potently effective at blocking growth of both PDAC cells in culture and human PDAC-derived tumors in mice and should be explored further as a potential therapy for patients with PDAC.