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Preclinical characterization of a potent and selective inhibitor of the histone demethylase KDM1A for MLL leukemia.
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2013
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Histone ModificationsMixed-phenotype Acute LeukemiaEpigenetic ChangeE13543 BackgroundCell DifferentiationEpigeneticsTumor BiologyMyeloid NeoplasiaHematological MalignancyMll LeukemiaStem CellsRadiation OncologyCancer ResearchHistone Demethylase Kdm1aGene ExpressionEpigenetic RegulationCell BiologyChromatin FunctionBiomolecular EngineeringChromatinChromatin StructureChromatin RemodelingNatural SciencesSelective InhibitorCancer GenomicsMedicineDrug Discovery
e13543 Background: The lysine-specific demethylase KDM1A catalyzes the removal of methyl groups from mono and dimethylated H3K4/K9, changes that have inductive or inhibitory effects on gene expression. KDM1A is a key regulator of leukemia stem cell (LSC) potential and is required for MLL-AF9 oncogenic transformation. Inhibition of KDM1A expression or activity overcomes the differentiation block in AML. Mixed lineage leukemia (MLL) is a form of AML that is especially sensitive to inhibition of KDM1A. Methods: Gene expression was analyzed using microarrays, cell differentiation was assessed by FACS, viability and apoptosis were assessed by MTT assay and PI staining, me 2 H3K4 levels were quantified by qPCR and ChIPseq. Results: We used computational models based on reported X-ray structures fine-tuned with the SAR of a first series of molecules to design and synthesize >800 KDM1A inhibitors. ORY-1001 is an enantiomerically pure KDM1A inhibitor (IC50 <20nM) with high selectivity against related FAD dependent aminoxidases (MAO-A/B, IL4I1, KDM1B >100uM, SMOX 7uM). ORY-1001 does not inhibit non-related histone modifiers, and is clean in a CEREP diversity panel. Treatment of THP-1 (MLL-AF9) cells with ORY-1001, results in a time/dose dependent me 2 H3K4 accumulation at KDM1A target genes and concomitant induction of differentiation markers (EC50 me 2 H3K4 and FACS CD11b <1nM). Microarray analysis of treated THP-1 cells reveals differentiation towards a monocyte-like phenotype. ORY-1001 induces apoptosis in THP-1 and inhibits proliferation and colony formation of MV(4;11) (MLL-AF4) cells (EC50 <1nM). Daily oral administration of doses <0.020mg/kg leads to significantly reduced tumor growth in rodent MV(4;11) xenografts. In vivo and in vitro IND/IMPD enabling studies have shown that ORY-1001 presents excellent oral bioavailability, target exposure and activity in vivo. The compound is stable in hepatocytes (Clint<0.6 mL/min/g liver, @1uM) and does not exhibit CYP (IC 50 >100uM) or hERG (%inhib.<2%, @10uM) inhibition. No off-target activity has been detected in 28d rat toxicology studies. Conclusions: A potent and selective KDM1A inhibitor is described with in vitro and vivo anti-leukemic efficacy.