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J. Chlebowska-Tuz et al.
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Figure 3. SK053 inhibits enzymatic activity of protein disulfide isomerase. (A) A turbidimetric assay of recombinant human PDI-mediated insulin disulfide reduction (mean ± SD of 6 experiments). (B) Determination of the IC50 value for SK053 with SigmaPlot software. (C) Fluorescence lifetime imaging (FLIM) of HeLa cells trans- fected with plasmid encoding an endoplasmic reticulum-tuned fluorescent redox-responsive probe (roGFPiE) tracking the activity of the PDI in cells; 100 mM dithio- threitol (DTT), a reducing agent, was used as a positive control. A representative result of a series of experiments is presented.
sion of which has been shown to correlate inversely with C/EBPa activity, increased self-renewal of leukemic cells as well as a block in blast differentiation.16 shRNA-mediat- ed knock-down of PDI (P4HB) expression significantly suppressed the proliferation of HL-60 cells (Figure 4D,E). Lentiviral transduction of HL-60, MOLM14 and KG1 cells with another shRNA sequence confirmed that PDI knock- down is associated with significant growth suppression (Figure 4F,G) and upregulates C/EBPa protein (Online Supplementary Figure S7A). PDI knock-down also increased the percentage of CD11b+ HL-60 cells as compared with nontargeting controls, but failed to do so in MOLM14 and KG1 cells (Figure 4H). Unexpectedly, SK053 strongly sup- pressed the growth and induced differentiation of HL-60 cells transduced with two independent shRNA sequences targeting CEBPA (Online Supplementary Figure S7B-D), indi- cating that the presence of this differentiation-associated transcription factor is not necessary for the antileukemic effects of SK053.
Protein disulfide isomerase inhibition targets leukemia-initiating cells and is effective in primary acute myeloid leukemia cells
AML is characterized by a small population of self- renewing leukemic stem cells referred to as leukemia-ini- tiating cells that give rise to a large population of imma- ture leukemic blasts.17 Since leukemia-initiating cells are intrinsically resistant to chemotherapeutics and AML fre- quently relapses due to incomplete elimination of leukemia-initiating cells18 we sought to investigate the effects of SK053 on the survival of CD123+CD34+CD38-
leukemia-initiating cell-like cells within the KG1 AML cell line. We observed a concentration-dependent decrease in the percentage of these cells when incubated with SK053 (Figure 5A,B). Moreover, pretreatment of KG1 cells with SK053 decreased the clonogenic potential of these cells in a colony formation assay in vitro (Figure 5C,D). Moreover, SK053 exerted antileukemic effects in primary AML blasts isolated from bone marrow or peripheral blood of AML patients. We observed a decrease in blast survival in all samples from six patients investigated, accompanied by an increased percentage of cells expressing CD11b marker in five out of the six sam- ples (Figure 6).
Discussion
In this study we demonstrate that SK053 induces differ- entiation of AML cells. SK053 has been developed as a thioredoxin inhibitor and was reported to induce endo- plasmic reticulum stress and apoptosis of tumor cells.11 Thus, it was to some extent unexpected to observe that SK053 induces cytostatic/cytotoxic effects against HL-60 cells (Figure 1A,B), while thioredoxin knock-down with shRNA turned out to be largely ineffective in suppressing the growth of these cells (Online Supplementary Figure S6C). This observation prompted us to look for an addi- tional cellular target for SK053. Using biotinylated SK053 as a bait in a biotin affinity probe-labeling approach we observed that SK053 binds to PDI (Figure 2, Online Supplementary Figure S9). Molecular docking confirmed
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haematologica | 2018; 103(11)