Here we report a RNA-seq study that elucidates the specificity of two substrate competitive inhibitors of C-terminal binding proteins (CTBP) 1 and 2.

CTBPs are paralogous transcriptional co-regulators that harbour a d-isomer specific 2-hydroxyacid dehydrogenase domain (D2-HDH) and their overexpression is associated with cancer (Boyd, Subramanian et al., 1993, Dcona, Morris et al., 2017, Schaeper, Boyd et al., 1995). Additionally, CTBPs are mediators of inflammation (Li, Zhang et al., 2020, Li, Zhang et al., 2022, Saijo, Collier et al., 2011, Shen, Kapfhamer et al., 2017). Therefore, inhibition of CTBP function may provide clinical tools to target malignancies or chronic inflammatory diseases.

The D2-HDH domain of CTBPs reduces or oxidises yet unknown substrates using the co-enzyme NAD+/NADH. 4-methylthio-2-oxobutanoate (MTOB) as a substrate analogue that binds the substrate-binding pocket of CTBPs in a CTBP-specific way (Hilbert, Grossman et al., 2014) , thereby antagonizing transcriptional regulation by CTBPs (Straza, Paliwal et al., 2010) . Using MTOB as a lead structure, 1 ^st generation CTBP inhibitors such 2-(hydroxyimino)-3-phenylpropanoic acid and its 2- and 4-chloro analogues were developed (Korwar, Morris et al., 2016) .

To test the specificity of the two CTBP inhibitors MTOB and 4-Cl-HIPP (3-(4-Chlorophenyl)-2-(hydroxyimino) propanoic acid), we generated a Ctbp1/2 double knockout (dKO) in the macrophage cell line J774.1 ( Fig. 1A ). Wildtype and Ctbp1/2 dKO cells were treated with either DMSO as vehicle control, 1.5 mM MTOB or 250 µM 4-Cl-HIPP for 4 h to investigate direct drug effects on transcription. The concentrations of both inhibitors were chosen in accordance with published dose response curves (Achouri, Nöel et al., 2007, Dcona, Chougoni et al., 2023, Dcona, Damle et al., 2019, Hilbert, Morris et al., 2015, Straza, Paliwal et al., 2010). RNA was collected and drug effects assessed by RNA-seq.

Principal component analysis of the RNA-seq results shows a clear separation of double knockout and wildtype cells, explaining 46.8% of variance. Of the inhibitors, MTOB has the strongest influence on gene expression, driving 19.4% of gene expression variation. 4-Cl-HIPP, on the other hand effects only 3.9% of variation. Surprisingly, the inhibitor effects were observed in wildtype and Ctbp1/2 dKO cells on this global view, indicating off-target effects of both inhibitors ( Fig. 1B ).

A more detailed analysis of gene expression in response to either MTOB or 4-Cl-HIPP revealed 290 and 35 differentially expressed genes in wildtype cells, respectively ( Fig. 1C ). A comparison of inhibitor effects in wildtype to the effect in Ctbp1/2 dKO cells showed that both inhibitors also effect gene expression even when CTBPs 1 and 2 are not expressed further confirming their off-target effects. For MTOB, 93 of the 290 regulated genes were CTBP-dependent ( Fig. 1C top right), whereas for 4-Cl-HIPP 17 of the 35 genes showed CTBP dependency ( Fig. 1C right bottom). Among the CTBP-dependent 4-Cl-HIPP effected genes “regulation of translation elongation” enriched significantly by overrepresentation analysis for gene ontologies of biological process. MTOB, on the other hand, regulates “defense response to viruses”, “innate immune response signaling” and “type I interferon signaling” ( Fig. 1D ). Those results indicate that 4-Cl-HIPP and MTOB do regulate different gene sets, even among their CTBP-dependent target genes hinting towards a different mode of action. To further elucidate the similarities or differences of both inhibitors, we specifically looked at CTBP-dependently regulated genes for both inhibitors (17 for 4-Cl-HIPP, 93 for MTOB) by blotting the z-scaled variance stabilized counts as heatmap and performed clustering analysis by Euclidian distance. Again, MTOB had the stronger effect of the two inhibitors as it contributed 93 genes to the gene set. Among the CTBP-dependent MTOB target genes we identified genes mimicking the Ctbp1/2 double knockout effect and genes that do not mimic the effect of Ctbp1/2 loss-of-function ( Fig. 1E ). The CTBP-dependent MTOB effects that are not mimicked by Ctbp1/2 loss-of-function may depend on its dehydrogenase activity or oligomerization state.

In summary, the two CTBP inhibitors MTOB and 4-Cl-HIPP have distinct modes of actions of which only a subset is mimicked by Ctbp1/2 loss-of-function. More importantly, both inhibitors show effects in Ctbp1/2 double knockout cells indicative for off-target effects. We could not identify specific pathways enriched among the off-target genes, but other D2-HDH-containing dehydrogenases may potentially be targeted by those inhibitors. One potential candidate is phosphoglycerate dehydrogenase (PHGDH). Its loss was recently associated with an M2 to M1 phenotype switch in tumour-associated macrophages (Cai, Li et al., 2024) .

Generation of Ctbp1 and 2 double knockout cells

To assemble a functional gRNA, 2 µl Alt-R CRISPR-Cas9 tracrRNA labelled with ATTO 550 and 2 µl crRNA (IDT, 100 µM) were mixed in 20 µl duplex buffer, heated to 95°C for 5 min and slowly cooled back to room temperature within two hours. 0.7 µl annealed oligonucleotides (50 µM) were mixed with 0.5 µl Alt-R Cas9 (IDT) in 1.8 µl PBS and incubated for 20 min at room temperature to assemble RNP complexes. 500,000 cells of the cell line J774.1 were washed with PBS and resuspended in 12 µl electroporation buffer R (Thermo Fisher Scientific). 4 µl of electroporation enhancer (15 µM IDT), 8 µl of electroporation buffer R and 12 µl cell suspension were added to the assembled RNP complexes. The solution was mixed with a 10 µl electroporation tip, the filled tip was placed in the Neon electroporation device (Thermo Fisher Scientific) in 5 mL buffer E and subjected to 3x 1400 V for 10 ms each. Then cells were seeded in 6-well plates pre-filled with full growth medium. Next day, cells were suspended in FACS buffer (5 mM EDTA, 2% FBS, PBS), pipetted through a cell strainer to obtain single cell suspension and subjected to FACS (BD Aria II). To enrich for successfully transfected cells ATTO 550 positive cells were sorted in a container filled with growth medium. Cells were diluted to 2 cells/ 100 µl and 100 µl aliquots were seeded on 96-well plates for single cell outgrowth. During this time, cells were monitored closely to ensure monoclonal origin. After reaching confluence, clonal colonies were propagated and subjected to genotyping by PCR. Selected clones were confirmed as either wildtype or knockout by sequencing of PCR products and western blotting.

Inhibitor treatment of J774.1

J774.1 wildtype or Ctbp1/2 double knockout cells were seeded at 250,000 cells/ well of a 24-well plate in 1 mL full growth medium (DMEM high glucose, 10 % FBS, 1 % Penicillin/ Streptomycin) and incubated at 37 °C and 5 % CO ₂ overnight. Next day, cells were treated with 1.5 mM MTOB (2 M in PBS, Sigma Aldrich Cat. No.: K6000), 250 µM 4-Cl-HIPP (500 mM in DMSO, gifted by W.E.Royer) or a vehicle control (PBS/ DMSO) for 4 h.

Western blot

Cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris (pH 7.4), 1% Nonidet P-40, 0.5% sodium-deoxycholate, 0.1% sodium-dodecylsulfate). 6x Laemmli-buffer (375 mM Tris-HCl (pH 6.8), 6% SDS, 4.8 % glycerol, 9% 2-mercaptoethanol and 0.03% bromphenolblue) was added to a final concentration of 1X and protein lysates were boiled at for 10 min 95 °C. Proteins were separated by size using a 8% polyacrylamide gel in running buffer (25 mM Tris, 192 mM glycine, 0.1 % SDS) and transferred to a PVDF membrane in transfer buffer (25 mM Tris, 192 mM glycine, 20 % ethanol) using semi-dry transfer. Membranes containing protein lysates were blocked for 1 h in 5 % BSA in TBS-T (150 mM NaCl, 10 mM Tris , 0.1 % Tween-20) and incubated with primary antibody overnight at 4°C. The primary antibody was removed and membranes were washed thrice with TBS-T before incubation with HRP-coupled secondary antibodies for 1 h at room temperature. Membranes were washed again thrice with TBS-T before HRP substrate was added. Chemiluminescence was detected using the Sapphire Azure Bioscanner.

RNA-seq

Total RNA was extracted from treated cells using the ReliaPrep RNA Miniprep System (Promega Cat. No.:Z6012) according to the manufacturer’s instructions. The RNA quality was determined on an Agilent 2100 Bioanalyzer with the RNA 6000 Nano kit, following the manufacturer’s instructions. Library preparation and rRNA depletion were conducted using the TruSeq unstranded mRNA Library Prep kit (Illumina) starting with 1 µg of RNA for each biological triplicate. The samples were sequenced on the Illumina NovaSeq6000 with 2x100bp paired-end.

Data analysis

NGS data quality was assessed with FastQC (RRID: SCR 014583, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).

Gene-level quantification was performed with Salmon version 1.9.0 (RRID: SCR_017036 (Patro, Duggal et al., 2017) ). Settings were: -libType A, -gcBias, -biasSpeedSamp 5 using the mm39 (M28, GRCm39) reference transcriptome provided by Gencode (Frankish, Diekhans et al., 2021) . Gene count normalization and differential expression analysis were performed with DESeq2 version 1.44.0 (RRID: SCR_015687 (Love, Huber et al., 2014) ) after import of gene-level estimates with “tximport” version 1.32.0 (RRID: SCR_016752 (Soneson, Love et al., 2015) ) in R (RRID: SCR_001905, R version 4.1.1 (Team, 2018) ).

For gene annotation, Ensembl gene IDs were mapped to symbols using the Bioconductor package “AnnotationHub” version 3.12.0 (RRID: SCR_024227 (Morgan & Shepherd, 2024) ). Genes with expression higher than a 10 ^th percentile, fold change of 1.5, and Benjamini-Hochberg-adjusted p value < 0.05 were called significantly changed. Plots were generated with “ggplot2” version 3.5.1 (RRID: SCR_014601 (Wickham, 2016) ) or “pheatmap” version 1.0.12 (RRID: SCR_016418; https://github.com/raivokolde/pheatmap ) packages and GO enrichment performed with “clusterProfiler” version 4.12.6 (RRID: SCR 016884 (Yu, Wang et al., 2012) ).

Data availability

RNA-seq data is available at GSE281668.

For interactive data exploration: https://franzig.shinyapps.io/ctbpinhibitors/.

Table 1. CrRNA for CRISPR clones

Table 2. Antibodies for Western blot

Table 3. Primer sequences

Table 4. Software