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J. Chlebowska-Tuz et al.
SK053 increased the levels of CCAAT enhancer-binding protein a and upregulated mRNA levels for differentiation-associated genes. Finally, SK053 decreased the survival of blasts and increased the per- centage of cells expressing the maturation-associated CD11b marker in primary cells isolated from bone marrow or peripheral blood of patients with acute myeloid leukemia. Collectively, these results provide a proof-of-concept that protein disulfide isomerase inhibition has potential as a therapeutic strategy for the treatment of acute myeloid leukemia and for the development of small-molecule inhibitors of protein disulfide isomerase.
Introduction
Acute myeloid leukemia (AML), the most prevalent acute leukemia among adults, is a malignancy of myeloid lineage cells characterized by the inhibition of cell differentiation leading to accumulation of abnormal white blood cells.1 The use of differentiation-inducing agents, such as all-trans retinoic acid and arsenic triox- ide, for the treatment of acute promyelocytic leukemia has brought remarkable therapeutic effects.2,3 However, not all patients with acute promyelocytic leukemia ben- efit from differentiation treatment and there has been no such significant progress in the treatment of other types of AML.4 The development of new therapeutic agents exerting anti-leukemic effects by targeting unique cellular mechanisms of differentiation is still, therefore, a pressing need of clinical importance.5 It is particularly desirable to develop differentiation-promot- ing compounds that induce terminal differentiation of leukemic cells leading to cell cycle arrest followed by cell death, and obviate overt cytotoxicity.
A critical transcription factor involved in the develop- ment and differentiation of myeloid lineage cells is CCAAT enhancer-binding protein a (C/EBPa). In C/EBPa-deficient mice granulocyte differentiation is blocked,6 and C/EBPa expression in bipotential myeloid progenitors is sufficient to induce granulocytic development.7 Dysregulation of C/EBPa activity is fre- quently observed in AML patients. Lack of, aberrant or suboptimal C/EBPa activity can result from genomic mutations in the CEBPA gene,8 transcriptional suppres- sion originating from promoter hypermethylation, or functional inactivation by phosphorylation.9 A transla- tional block that occurs in cells experiencing endoplas- mic reticulum stress has also been reported as a mech- anism leading to C/EBPa downregulation at the mRNA level.10 Various mechanisms such as loss of Ca2+ home- ostasis, inhibition of disulfide bond formation, oxida- tive stress, or hypoxia, lead to endoplasmic reticulum stress, which triggers the unfolded protein response. The role of the unfolded protein response is to restore protein homeostasis and normal endoplasmic reticulum function. Accordingly, this response has been reported to be upregulated in a significant percentage of patients with AML and to be associated with a more favorable course of the disease.10
We have previously developed SK053, a pep- tidomimetic inhibitor of thioredoxin that exerts cytosta- tic/cytotoxic effects and endoplasmic reticulum stress- mediated apoptosis in tumor cells.11 Conspicuously, we have observed that AML cells incubated with SK053 undergo growth arrest followed by differentiation into more mature myeloid stages and cell death. We, there- fore, employed RNA sequencing and a biotin affinity
probe-labeling approach to identify the molecular mechanism of the differentiation-promoting effects of SK053, revealing protein disulfide isomerase (PDI) as a druggable target for AML treatment.
Methods
A detailed description of the methods used can be found in the Online Supplementary Data file.
Culture conditions for the acute myeloid leukemia cell lines
NB4, MOLM14, HS-5 and HL-60 cells were cultured in RPMI 1640 (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone), 100 mg/mL streptomycin and 100 U/mL penicillin (P/S, Sigma-Aldrich). KG1 cells were cultured in IMDM (Gibco) supplemented with 20% heat-inactivated fetal bovine serum (Hyclone) and P/S. HeLa cells were cultured in DMEM (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone) and P/S. All cells were cultured at 37°C in a fully humidified atmosphere of 5% CO2.
Acute myeloid leukemia cell differentiation assays
Myeloid differentiation was assessed by staining cytospun cells with May-Grünwald-Giemsa as well as in flow cytometry by examining the surface expression of CD11b. A conventional semi-quantitative microscopic nitroblue tetrazolium (NBT; Sigma-Aldrich) assay was used to determine the production of superoxide anion upon stimulation with phorbol myristate acetate in differentiated AML cells. A modified quantitative NBT assay was established and implemented by dissolving the blue formazan particles from equal cell numbers in sodium hydroxide, followed by measurement of absorbance at 620 nm using an ASYS UVM 340 microplate reader (Biochrom, Cambridge, UK).
RNA extraction and quantitative real-time polymerase chain reaction
Total RNA was extracted from HL-60 cells using an RNA purification kit (EurX) and relative gene expression was quanti- fied using a LightCycler II 480 Real-Time PCR System (Roche, Basel, Switzerland), LightCycler Probes Master and hydrolysis probes [Universal Probe Library (UPL), Roche] according to the manufacturer's recommendations.
Westernblot
HL-60 cells were washed twice with ice-cold phosphate- buffered saline, lysed in RIPA buffer (50 mmol/L Tris base, 150 mmol/L NaCl, 1% NP-40, 0.25% sodium deoxycholate, and 1 mmol/L EDTA) or cytoplasmic/nuclear fractions of proteins were extracted using an NE-PER Kit (Pierce). Immunoblotting was done via standard procedures using the antibodies accord- ing to the manufacturer’s instructions.
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