Although differentiation-inducing agents have significantly improved the management of acute promyelocytic leukemia, no significant progress has been made in the treatment of other acute myeloid leukemias (AML). Numerous proteins involved in tumor development have so-called allosteric disulfide bonds amenable to modifications affecting protein structure and function. We have developed SK053, a small molecule and mechanism-selective inhibitor of enzymes involved in allosteric disulfide bonds formation such as thioredoxin, thioredoxin reductase and protein disulfide isomerase (PDI). The aim of our studies was to evaluate anti-leukemic activity of SK053 in human AML cells.

To validate if SK053 targets PDI, a binding assay and an insulin turbidimetric activity assay were used. Cytostatic/cytotoxic effects in HL60, NB4, KG-1 and MOLM14 cells as well as in primary AML cells were assessed with trypan blue exclusion. Differentiation of AML cells was studied with May-Grünwald-Giemsa staining, nitro blue tetrazolium reduction assay and flow cytometry analysis of CD11b, CD14 and CD15 levels and by RNA sequencing, qRT-PCR and western blotting (WB).

We observed covalent binding of SK053 to PDI and inhibition of its enzymatic activity with IC50 of 10 μM. Since PDI blocks translation of CCAAT enhancer binding protein alpha (CEBPA), a transcription factor crucial for neutrophils maturation, we evaluated the potential of SK053 to induce differentiation and cytostatic/cytotoxic effects in human AML cells. SK053 exerts significant cytostatic/cytotoxic activity in human AML cells (HL60, NB4, KG-1 and MOLM14), and induces differentiation of AML blasts into more mature myeloid cells. Incubation of AML cells with SK053 induced expression of CEBPA and hexokinase 3 mRNA in quantitative RT-PCR and increased amount of CEBPA protein in nuclear fraction measured in WB. Finally, SK053 induces differentiation of primary leukemic cells freshly isolated from AML patients. RNA-seq analysis revealed that incubation of HL60 cells with SK053 down-regulates mRNA for MYC and ID1 oncogenes as well as for histone proteins. Expression of other genes of mature myeloid lineage such as adhesion molecules (collagen 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. The GeneOntology analysis done with the RNAseq results revealed enrichment of gene transcripts regulating myeloid cells differentiation.

SK053 exerts potent anti-leukemic activity and induces differentiation of numerous types of human AML cells. Targeting allosteric disulfide bonds with small molecule inhibitors presents a promising therapeutic strategy in AML.

Citation Format: Dominika Nowis, Justyna Chlebowska, Pawel Gaj, Michal Lazniewski, Malgorzata Firczuk, Karolina Furs, Radoslaw Sadowski, Pawel Leszczynski, Piotr Stawinski, Szymon Klossowski, Ryszard Ostaszewski, Krzysztof Giannopoulos, Rafal Ploski, Dariusz Plewczynski, Jakub Golab. SK053, a small molecule inhibitor of enzymes involved in allosteric disulfide bonds formation, shows potent anti-leukemic effects and induces differentiation of human AML cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5347. doi:10.1158/1538-7445.AM2015-5347