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Our earlier studies had shown that overexpression of the JmjC domain histone demethylase KDM2B renders mouse embryo fibroblasts (MEFs) resistant to oxidative stress due to the role of KDM2B in the regulation of antioxidant mechanisms. Here we present evidence that the knockdown of KDM2B in basal-like breast cancer cell lines results in a decrease of Glutathione (GSH) levels, a secondary increase of intracellular ROS levels and in enhanced sensitivity to deubiquitinase inhibitors. The expression of the Glutamate-Cystine antiporter, the Glutamate-Cysteine Ligase GCLC/GCLM and the Glutathione Peroxidase GPX4, all of which regulate GSH abundance was not affected. To address the mechanism of the GSH regulation we carried out RNA-Seq, quantitative proteomics and metabolomics analyses in shKDM2B- and empty vector-transduced MDA-MB-231 cells. The results showed that the KD of KDM2B causes major shifts in metabolism and that one of the metabolic pathways whose activity depends on KDM2B is the SGOC pathway, which has a major role in GSH biosynthesis. Experiments in cultured cells confirmed the importance of KDM2B in the regulation of this pathway and they also showed that the inhibition of the pathway via the KD of KDM2B is partly responsible for the shKDM2B-induced inhibition of cell proliferation in culture and in xenograft experiments in NSG mice. More important, the transcriptomic signature of the SGOC pathway correlates with the expression of KDM2B in basal like mammary adenocarcinomas in the TCGA database. The genes encoding the majority of the enzymes in the SGOC pathway are known to be regulated by MYC and ATF4 and our data show that both MYC and ATF4 are under the regulatory control of KDM2B. ATAC-Seq and ChIP-Seq experiments in shKDM2B and control MDA-MB-231 cells also showed that KDM2B binds the promoter region of not only MYC and ATF4, but also of the genes encoding the SGOC enzymes and that it regulates chromatin accessibility and the abundance of H3K4me3/H3K27Ac active histone marks in these promoters. Overall, our data indicate that KDM2B regulates the SGOC pathway by targeting MYC and ATF4 and by making the promoters of the genes encoding the SGOC enzymes accessible to these regulators. Overall, our data provide new evidence on SGOC regulation, and identify novel KDM2B-dependent metabolic vulnerabilities in basal like breast cancer. Citation Format: Evangelia Chavdoula, Vollter Anastas, Allesandro La Ferlita, Julian Aldana, Giuseppe Carota, Mariarita Spampinato, Sameer Parashar, Ilaria Cosentini, Burak Soysal, Giovanni Nigita, Michael Freitas, Philip Tsichlis. KDM2B regulates Serine-Glycine-One Carbon (SGOC) metabolism by targeting the SGOC enzyme genes via a combination of direct and indirect epigenetic mechanisms. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6038.

Protein post-translational modification (PTM) is crucial to modulate protein interactions and activity in various biological processes. Emerging evidence has revealed PTM patterns participate in the pathology onset and progression of various diseases. Current PTM identification relies mainly on mass spectrometry-based approaches that limit the assessment to the entire protein population in question. Here we report a label-free method for the detection of the single peptide with only one amino acid modification via electronic fingerprinting using reengineered durable channel of phi29 DNA packaging motor, which bears the deletion of 25-amino acids (AA) at the C-terminus or 17-AA at the internal loop of the channel. The mutant channels were used to detect propionylation modification via single-molecule fingerprinting in either the traditional patch-clamp or the portable MinION™ platform of Oxford Nanopore Technologies. Up to 2000 channels are available in the MinION™ Flow Cells. The current signatures and dwell time of individual channels were identified. Peptides with only one propionylation were differentiated. Excitingly, identification of single or multiple modifications on the MinION™ system was achieved. The successful application of PTM differentiation on the MinION™ system represents a significant advance towards developing a label-free and high-throughput detection platform utilizing nanopores for clinical diagnosis based on PTM.

Epigenetic and metabolic alterations in cancer cells are intertwined. The concentration of metabolites can influence the activity of chromatin modifiers, which in turn can act as metabolic sensors that translate changes in cellular metabolism to transcriptional reprogramming. In the present study, we investigated the role of histone demethylase KDM2B in the metabolic reprogramming of the triple-negative breast cancer (TNBC), in which KDM2B is selectively expressed at high levels. Knockdown of KDM2B in TNBC cell lines reduced their proliferation rate and tumor growth in vivo. Transcriptomic, proteomic, and metabolomic profiling demonstrated that the Serine-Glycine pathway and One Carbon metabolism (SGOC) and other amino acid biosynthetic and catabolic processes are downregulated by the knockdown of KDM2B. Additionally, we see reduction of metabolites produced via these pathways (purines, pyrimidines, formate, glutathione and NADPH). Importantly, the expression of the enzymes involved in the SGOC metabolic pathway (e.g. PHGDH, PSAT1, PSPH, SHMT2, MTHFD1L, MTHFD2 and DHFR) depends on c-MYC, NRF2, and ATF4 which our data show that they are under the positive regulatory control of KDM2B. The epistatic relationship between these factors, with the expression of the enzymes of the SGOC pathway and the effects of the KDM2B knockdown on chromatin occupancy and accessibility of the promoters of these factors is in progress and will be presented. Analysis of TCGA data showed positive and statistically significant correlations between KDM2B and the SGOC gene signature in TNBC patients. In addition, the metabolic pathway signature that distinguishes control and shKDM2B-transduced cells corresponds to the metabolic signature of a subset of TNBCs, which have been reported to carry poor prognosis. The present study highlights the role of the epigenetic factor KDM2B as an upstream regulator of the metabolic reprogramming of TNBC. Citation Format: Evangelia Chavdoula, Vollter Anastas, Alessandro La Ferlita, Julian Aldana, Anuvrat Sircar, Michael A. Freitas, Lalit Sehgal, Philip N. Tsichlis. The epigenetic factor KDM2B alters the serine-glycine synthesis pathway and the one-carbon metabolism (SGOC) in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3019.

Breast cancer (BC) is a highly heterogeneous disease associated with metabolic reprogramming. The shifts in the metabolome caused by BC still lack data from Latin populations of Hispanic origin. In this pilot study, metabolomic and lipidomic approaches were performed to establish a plasma metabolic fingerprint of Colombian Hispanic women with BC. Data from 1H-NMR, GC-MS and LC-MS were combined and compared. Statistics showed discrimination between breast cancer and healthy subjects on all analytical platforms. The differentiating metabolites were involved in glycerolipid, glycerophospholipid, amino acid and fatty acid metabolism. This study demonstrates the usefulness of multiplatform approaches in metabolic/lipid fingerprinting studies to broaden the outlook of possible shifts in metabolism. Our findings propose relevant plasma metabolites that could contribute to a better understanding of underlying metabolic shifts driven by BC in women of Colombian Hispanic origin. Particularly, the understanding of the up-regulation of long chain fatty acyl carnitines and the down-regulation of cyclic phosphatidic acid (cPA). In addition, the mapped metabolic signatures in breast cancer were similar but not identical to those reported for non-Hispanic women, despite racial differences.