Transcriptional regulation of amino acid metabolism by KDM2B, in the context of ncPRC1.1 and in concert with MYC and ATF4
IntroductionKDM2B encodes a JmjC domain-containing histone lysine demethylase, which functions as an oncogene in several types of tumors, including TNBC. This study was initiated to address the cancer relevance of the results of our earlier work, which had shown that overexpression of KDM2B renders mouse embryonic fibroblasts (MEFs) resistant to oxidative stress by regulating antioxidant mechanisms. MethodsWe mainly employed a multi-omics strategy consisting of RNA-Seq, quantitative TMT proteomics, Mass-spectrometry-based global metabolomics, ATAC-Seq and ChIP-seq, to explore the role of KDM2B in the resistance to oxidative stress and intermediary metabolism. These data and data from existing patient datasets were analyzed using bioinformatic tools, including exon-intron-split analysis (EISA), FLUFF and clustering analyses. The main genetic strategy we employed was gene silencing with shRNAs. ROS were measured by flow cytometry, following staining with CellROX and various metabolites were measured with biochemical assays, using commercially available kits. Gene expression was monitored with qRT-PCR and immunoblotting, as indicated. ResultsThe knockdown of KDM2B in basal-like breast cancer cell lines lowers the levels of GSH and sensitizes the cells to ROS inducers, GSH targeting molecules, and DUB inhibitors. To address the mechanism of GSH regulation, we knocked down KDM2B in MDA-MB-231 cells and we examined the effects of the knockdown, using a multi-omics strategy. The results showed that KDM2B, functioning in the context of ncPRC1.1, regulates a network of epigenetic and transcription factors, which control a host of metabolic enzymes, including those involved in the SGOC, glutamate, and GSH metabolism. They also showed that KDM2B enhances the chromatin accessibility and expression of MYC and ATF4, and that it binds in concert with MYC and ATF4, the promoters of a large number of transcriptionally active genes, including many, encoding metabolic enzymes. Additionally, MYC and ATF4 binding sites were enriched in genes whose accessibility depends on KDM2B, and analysis of a cohort of TNBCs expressing high or low levels of KDM2B, but similar levels of MYC and ATF4 identified a subset of MYC targets, whose expression correlates with the expression of KDM2B. Further analyses of basal-like TNBCs in the same cohort, revealed that tumors expressing high levels of all three regulators exhibit a distinct metabolic signature that carries a poor prognosis. ConclusionsThe present study links KDM2B, ATF4, and MYC in a transcriptional network that regulates the expression of multiple metabolic enzymes, including those that control the interconnected SGOC, glutamate, and GSH metabolic pathways. The co-occupancy of the promoters of many transcriptionally active genes, by all three factors, the enrichment of MYC binding sites in genes whose chromatin accessibility depends on KDM2B, and the correlation of the levels of KDM2B with the expression of a subset of MYC target genes in tumors that express similar levels of MYC, suggest that KDM2B regulates both the expression and the transcriptional activity of MYC. Importantly, the concerted expression of all three factors also defines a distinct metabolic subset of TNBCs with poor prognosis. Overall, this study identifies novel mechanisms of SGOC regulation, suggests novel KDM2B-dependent metabolic vulnerabilities in TNBC, and provides new insights into the role of KDM2B in the epigenetic regulation of transcription. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=122 HEIGHT=200 SRC="FIGDIR/small/548031v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1bac894org.highwire.dtl.DTLVardef@1c6977eorg.highwire.dtl.DTLVardef@103d73corg.highwire.dtl.DTLVardef@127b1ff_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsThe knockdown of KDM2B in basal-like breast cancer cell lines lowers the levels of GSH and sensitizes the cells to ROS inducers, GSH targeting molecules, and DUB inhibitors. KDM2B regulates intermediary metabolism by targeting the expression of a host of metabolic enzymes, including those in the SGOC, glutamate, and GSH metabolism. KDM2B enhances chromatin accessibility of MYC and ATF4 and promotes their expression. MYC and ATF4 binding sites are enriched in the promoters of genes whose accessibility depends on KDM2B. KDM2B functioning in the context of ncPRC1.1, binds the promoters of transcriptionally active genes, including those encoding KDM2B-regulated metabolic enzymes, in concert with MYC and ATF4. Basal-like TNBCs expressing high levels of all three regulators, exhibit a distinct metabolic signature that is associated with poor prognosis.