Abstract 2722: An added twist: drug-mediated pan-inhibition of KDM catalysis causes selective reduction of mRNAs that encode a small subset of KDM enzymes

Abstract Background: The class of 2-oxoglutarate-requiring mono-iron dioxygenases (MIDOs) that demethylate specific lysine, histidine, or arginine residues of histones is receiving attention for its role in human diseases, in particular cancer (10.1016/j.ejmech.2022.114143). The steric events during MIDO catalysis were resolved at subatomic scale in 1982 by two students at Philipps University, Marburg/Germany (HAG mechanism [10.1016/0022-5193(82)90320-4, 10.1016/0022-5193(82)90320-4]). They presented a classification of all conceivable inhibitors and rapidly reduced their seminal approach to practice, establishing pyridine dicarboxylates and oxalylglycines as MIDO inhibitors, now in global use. In 1995, Cornell researchers employed HAG metrics to identify, among FDA-approved drugs, deferiprone (DFP) as MIDO inhibitor, enabling control of key protein families in vitro and in vivo (e.g. US-6080766-A). In 2019, DEF was identified as pan-selective inhibitor of histone lysine demethylating MIDOs (KDMs [10.1038/s41598-019-39214-1]). KDMs have, beyond their enzymatic activity, critical non-enzymatic functions in the physical assembly of the transcriptional machinery (10.3390/ijms25136900). We noted that DFP not only inhibits key MIDOs at clinically relevant serum concentrations, but also decreases selectively their encoding mRNAs, a precondition for reducing their biosynthesis. Hypothesis: DEF not only inhibits KDM catalysis pan-selectively, but also reduces the biosynthesis of particular KDMs via knockdown of their mRNAs. Methods: Cell culture of ARK1; repeated, timed (12/24/48 hrs) RNA-Seq using clinical concentrations of DFP (and deferoxamine [DFOX] to test for iron depletion); flow cytometry for MIDO-dependent proteins (collagens, collectins, fibrillins) to confirm biologically effective intracellular / intracompartmental concentrations of DFP. Results: mRNAs encoding 15 histone lysine demethylating MIDOs were detected. 11 mRNAs for KDMs were expressed irrespective of time point monitored, duration of DFOX (15 µM) or DFP (100 and 150 µM) exposure, or DFP concentration. These non-reactive mRNAs were: KDM2A, 3B, 4A, 4C, 4D, 5A-C, 6A, 7A, 7C. The expression of KBM2B, KBM3A, KBM4B, and KBM6B increased at least 1.8 fold between 12 and 48 hrs. (FDR p <0.00000) in controls, with marginal effect of DFOX. By contrast, DFP uniformly abolished this rise of expression, causing 2-fold reductions of the four mRNAs relative to controls at 48 hrs. The slopes of the dose-response curve changed markedly, e.g. from +3 in control to below -0.5 for KBM2B, KBM3A, and KBM4B on DFP. Remarkably, at 12 hrs. DFP increased their mRNAs distinctly above controls, e.g. doubled KDM3A expression. The disruptive DFP effects were fully evident at 100 µM. Conclusions: Drug inhibition of KDM catalysis can reduce KDM expression, thus also affecting non-enzymatic KDM functions. Citation Format: Hartmut Martin Hanauske-Abel, 1 Mainul Hoque, 1 Seema Husain, 1 Sukhwinder Singh, 1 Axel R. Hanauske, 2 Patricia Soteropoulos1. An added twist: drug-mediated pan-inhibition of KDM catalysis causes selective reduction of mRNAs that encode a small subset of KDM enzymes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2722.