An intestinal stem cell niche in Apc mutated neoplasia targetable by CtBP inhibition

Abstract

C-terminal binding protein 2 (CtBP2) drives intestinal polyposis in the Apc min mouse model of human Familial Adenomatous Polyposis. As CtBP2 is targetable by an inhibitor of its dehydrogenase domain, understanding CtBP2’s role in adenoma formation is necessary to optimize CtBP-targeted therapies in Apc mutated human neoplasia. Tumor initiating cell (TIC) populations were substantially decreased in Apc min Ctbp2+/- intestinal epithelia. Moreover, normally nuclear Ctbp2 was mislocalized to the cytoplasm of intestinal crypt stem cells in Ctbp2+/- mice, both Apc min and wildtype, correlating with low/absent CD133 expression in those cells, and possibly explaining the lower burden of polyps in Apc min Ctbp2+/- mice. The CtBP inhibitor 4-chloro-hydroxyimino phenylpyruvate (4-Cl-HIPP) also robustly downregulated TIC populations and significantly decreased intestinal polyposis in Apc min mice. We have therefore demonstrated a critical link between polyposis, intestinal TIC’s and Ctbp2 gene dosage or activity, supporting continued efforts targeting CtBP in the treatment or prevention of Apc mutated neoplasia.

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

“…5). For this purpose, we transfected expression plasmids encoding wt, G189A, W324F, and W324G-mutated CtBP2 cDNA into HCT116 cells homozygously deleted for CtBP2 using CRISPR techniques (Chawla et al, 2018). Intact transfected cells were then incubated with DSG at 37°C for 30 minutes, and total cellular protein was immunoblotted for CtBP2.…”

“…Cells [wild-type (wt) or CRISPR] were maintained according to the American Type Culture Collection's recommendation. HCT116; p53-/- (Straza et al, 2010), HCT116;CtBP2-/- (Chawla et al, 2018), and MDA-MB-231 cells were maintained in RPMI 1640 or Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) FBS and penicillin-streptomycin, unless specified. Cells were maintained in a humidified incubator equilibrated with 5% CO 2 at 37°C.…”

“…In light of this evidence, CtBP could be considered a potential therapeutic target in cancer. Our group has developed CtBP inhibitors targeting the dehydrogenase domain that are able to modulate CtBP target genes, induce selective cytotoxicity in cancer cells, and attenuate polyposis in the Apc min/1 mouse model of human familial adenomatous polyposis (Straza et al, 2010;Korwar et al, 2016;Sumner et al, 2017;Chawla et al, 2018). Chemically, CtBP dehydrogenase-domain inhibitors were designed to assimilate the structural attributes of 2-keto-4methylthiobutyrate (MTOB), an a-ketoacid substrate of CtBP that acts as inhibitor of its dehydrogenase activity at higher concentration ( Fig.…”

“…In light of this evidence, it is imperative to understand the physiologic role for conserved W318/324 in CtBP1/2 activities, as this residue is the key affinity target of the parent substrate competitive inhibitor HIPP that has already exhibited promising preclinical antineoplastic activity (Korwar et al, 2016;Sumner et al, 2017;Chawla et al, 2018). To better understand the biologic effect of W318/324targeted inhibitors on CtBP function, we have used sitedirected mutagenesis, wherein W324 was mutated to simulate the conditions of inhibitor interaction/inhibition of W324 biochemical activity.…”

CtBP determines ovarian cancer cell fate through repression of death receptors

“…Relative to a potential role for CtBP as a key oncogenic driver or dependency, our previous studies have shown that CtBP2 is a dependency for APC mutated neoplasia in the Min mouse intestinal polyposis model of human Familial Adenomatous Polyposis 23 . We further demonstrated that CtBP2 haploinsufficiency reduced tumor initiating cell (TIC) abundance in APC min/+ intestines, suggesting the oncogenic role of CtBP2 in intestinal neoplasia relates to its promotion of TIC activities 24 . These findings were more recently mirrored by similar findings in a mouse model of human pancreatic adenocarcinoma (PDAC), where CtBP2 deficiency slowed tumor growth, abrogated metastases, and severely attenuated expression of TIC markers 25 .…”