SK053, an Inhibitor of Enzymes Involved in Allosteric Disulfide Bonds Formation, Targets Expression of Histone Genes and Induces Differentiation of Human AML Cell

Targeting epigenetic modifiers, such as histone deacetylases, bromodomain and extraterminal (BET) domains or methyltransferases as well as the use of differentiation-inducing agents (all-trans retinoic acid and arsenic trioxide) are rising hopes for development of effective therapeutic strategies for acute myeloid leukemia (AML). However, despite significant advances the cure rates for patients aged sixty or more are still very low. Targeting allosteric disulfide bonds becomes a novel approach to cancer treatment. Several disulfide bonds involved in cancer development and progression have already been identified as potentially druggable targets (Hogg P, Nat Rev Cancer 2013). Our interest in this field focuses on proteins with thioredoxin fold such as protein disulfide isomerase (PDI). We have recently developed SK053, a small molecule inhibitor of thioredoxin/thioredoxin reductase system, that exerts anti-tumor effects both in vitro and in murine tumor models (Klossowski S et al., J Med Chem 2012). Our studies revealed that SK053 is not a target-specific, but mechanism-selective inhibitor of allosteric disulfide bonds formation that also blocks the enzymatic activity of PDI. Biotinylated form of SK053 precipitates both TRX as well as PDI from human acute myeloid leukemia cells. Mass spectrometry analysis as well as molecular docking simulations show that SK053 covalently binds to cysteine C397 localized in the fourth (catalytic) PDI domain. SK053 reduces proliferation and induces monocytic and granulocytic differentiation in various types of human AML cells (HL60, MV4-11, Fig. 1A). Accordingly, it up-regulates expression of selected differentiation-related genes and increases expression of cell membrane differentiation markers (CD11b, CD14 and CD15). SK053 induces differentiation of primary leukemic blasts (Fig. 1B). Our ongoing studies show that silencing of PDI, TXN or PRDX alone using lentiviral shRNA systems does not result in differentiation of HL60 cells (Fig. 1C). RNA-seq analysis revealed that incubation of HL60 cells with SK053 down-regulates mRNA for MYC and ID1 oncogenes, which are involved in the regulation of blast proliferation. Moreover, the transcripts for histone proteins: HIST1H3J, HIST1H2AB, HIST1H1B, HIST1H3H, HIST1H2AH were strongly down-regulated. Drugs that disrupt histone modifiers are in clinical trials with promising results but very little is known about direct targeting of histone proteins. Expression of other genes important for development of myeloid lineage such as adhesion molecules (collagene type XV, fibronectin I, MAC-1), hydrolytic enzymes (carboxypeptidase, proteinase 3, CA12 anhydrase, ADAM19 metalloprotease), proteoglycan 2 (core of eosinophilic granules) and PGLYRP3 (peptidoglycan recognition protein 3) was significantly up-regulated.