The effect of CTBP dehydrogenase inhibitors MTOB and 4-Cl-HIPP in J774.1 cells

C-terminal binding proteins (CTBPs) are conserved transcriptional repressors involved in cancer and inflammation. Uniquely amongst transcriptional coregulators, CTBPs possess a functional dehydrogenase domain. Since multiple malignancies display elevated CTBP levels, CTBP inhibitors targeting this dehydrogenase domain have been developed. While the importance of CTBPs dehydrogenase function for transcriptional regulation remains unclear, multiple studies have relied CTBP inhibitors such as MTOB and 4-Cl-HIPP. In vitro studies have confirmed binding of these compounds to CTBP’s active site, however evidence for specificity is currently lacking. To address this, we treated wildtype or Ctbp1, 2 double knockout J774.1 cells with MTOB or 4-Cl-HIPP and performed RNAseq. We observed that both inhibitors elicit distinct transcriptional changes indicating non-overlapping modes of action. Moreover, the majority of changes induced by either inhibitor are observed in knockout cells indicative of off-target effects. We hypothesize that those CTBP dehydrogenase inhibitors might lack specificity to CTBPs. To investigate whether commonly used inhibitors of C-terminal binding proteins (CTBPs) MTOB (4-methylthio-2-oxybutyrate) and 4-Cl-HIPP (3-(4-Chlorophenyl)-2-(hydroxyimino)propanoic acid) specifically affect CTBPs function, wildtype and Ctbp1-/-, Ctbp2-/- (Ctbp1/2dKO) cells of the macrophage cell line J774.1 were treated with either inhibitor or a vehicle control (n=3 for each combination of genotype and treatment). Following RNA extraction, library preparation and RNA sequencing we assess transcription rates altered between wildtype and knockout and compare alterations by genotype to effects elicited by the inhibitors versus vehicle control. To define specificity we then investigate whether inhibitor effects observed in the wildtype are present in CTBP1/CTBP2 knockout cells.