Abstract The site- and methylation state-specific modification of lysine residues on histone proteins is a dynamic epigenetic modification that plays a key role in chromatin structure and gene regulation. In particular, methylation of histone H3 at lysine residue 4 (H3K4) is implicated in activation of transcription, whereas lysine methylation of histone H3 at residue 27 (H3K27) is correlated with repression of transcription. It is now well established that the dynamic interplay between histone methyltransferases and demethylases provides an important layer in tuning transcriptional responses and programs and collectively, these chromatin modifications are fundamentally involved in proliferation, stem cell self renewal and differentiation. The H3K4 methylation state found at transcriptional start sites is the result of the interplay between SET domain containing methyltransferases such as mixed lineage leukemia (MLL) and histone demethylase members of the JARID1 (KDM5) family of 2-oxoglutarate and Fe2+ dependent oxygenases (2OG oxygenases). On the other hand, the methyltransferase enhancer of zeste homolog 2 (EZH2) catalyzes the S-adenosylmethionine dependent trimethylation of H3K27, which then recruits the polycomb repressive complex (PRC) leading to gene silencing, whereas the 2-OG dependent oxygenases UTX (KDM6A) and JmjD3 (KDM6B) catalyse demethylation of methylated H3K27 residues. Both types of demethylases, namely KDM5B and KDM6A are implicated in myeloma as shown in multiple genetic analyses. Using combined screening and structure-guided approaches potent and selective chemotypes for human KDM5 and KDM6 histone demethylases were identified and used as tools to interrogate histone methylation biology and anti-proliferative responses in multiple myeloma. Whereas the amino-alkylated -pyridine carboxylate derivative KDOAM25 is a cell permeable KDM5 selective inhibitor with in vitro IC50 values < 100 nM, the molecule GSK-J4, based on a pyridine-pyrimidine scaffold has been previously characterized as cell-permeable KDM6 inhibitor with an IC50 below 100 nM and 10-fold selectivity over KDM5 demethylases. Inhibition of KDM5 enzymes by KDOAM25 led to an expected increase of global H3K4me3 levels and enrichment of cell cycle regulator genes resulting in a concomitant cell cycle arrest in a subset of myeloma cell lines. On the other hand, treatment with GSK-J4 led, in patient-derived co-culture systems and all multiple myeloma lines tested, to an apoptotic response, via inhibition of histone demethylases JARID1B, UTX and JmjD3. In conclusion, treatment with the inhibitor GSK-J4 in myeloma systems counteracts Myc-driven metabolic dependencies and impairs glutamine utilisation in a unique way that leads to selected amino acid depletion, eliciting the integrated stress response (ISR) in part via GCN2-dependent phosphorylation of eIF2α, ATF4 activation and induction of pro-apoptotic genes. Citation Format: Udo Oppermann. Inhibiting histone demethylases to target metabolic dependencies in multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr SY27-01.