Page 105 - 2018_11-Haematologica-web

Protein disulfide isomerase as a target in leukemia
Isolation of primary acute myeloid leukemia blasts and evaluation of the anti-leukemic effects of SK053
The study was approved by the Institutional Review Board of the Medical University of Lublin (approval n. KE-0254/71/2010) and the Medical University of Warsaw (approval n. KB 182/2011) and was conducted according to the Declaration of Helsinki. Each patient signed written informed consent to the procedures. Leukemic cells were obtained from the bone mar- row of patients with AML and isolated by 1.077 g/mL Histopaque (Sigma-Aldrich) density gradient centrifugation. Cells were seeded in IMDM culture medium, supplemented with 10% heat-inactivated fetal bovine serum at a density 5×105 cells/mL and incubated for 72 h with SK053. Cytotoxic effects, growth and differentiation upon incubation with SK053 were evaluated with methods described above. 7-AAD staining was used to discriminate dead cells. Additionally, anti-CD33 antibody (BD Biosciences, cat. n. 551378) was used to identify myeloid cells among all peripheral blood mononuclear cells. The patients’ basic characteristics are shown in Online Supplementary Table S11.
Statistical analysis
Data were analyzed using Microsoft Excel 2010 and GraphPad Prism 6.0 for Windows (GraphPad Software Inc., La Jolla, CA, USA) software. The results were analyzed for signif- icance using a two-tailed Student t-test or one-way ANOVA with a Dunnett post-hoc test. Statistical significance was defined as P values <0.05.
Results
SK053 induces differentiation of acute myeloid leukemia cells
HL-60 acute promyelocytic leukemia cells were incu- bated for 24 to 120 h with increasing concentrations of SK053 and cell growth as well as cytotoxic effects were determined by counting viable cells and flow cytometry. SK053 inhibited cell growth in a time- and concentra- tion-dependent manner (Figure 1A). We observed similar cytotoxic effects of SK053 against HL-60 cells grown in cell culture medium alone or on the top of bone marrow stromal cells (HS5) mimicking the interaction between AML blasts and the bone marrow niche (Figure 1B). HL- 60 cells incubated with SK053 also showed features of more mature myelopoietic stages with a decreased nucleus/cytoplasm ratio and a greater reduction of NBT (Figure 1C), corresponding to NADPH oxidase activity. An NBT reduction assay (Figure 1D) as well as increased surface levels of CD11b measured in flow cytometry (Figure 1E) confirmed the differentiation-promoting effects of SK053. Quantitative real-time polymerase chain reaction analysis revealed that incubation of HL-60 cells with SK053 resulted in increased levels of tran- scripts for differentiation-associated genes, such as dif- ferentiation-driving transcription factors (CEBPB, CEBPE), early markers of neutrophil differentiation (HK3) and genes expressed by mature granulocytes (CSF3R encoding G-CSFR, CSF2R encoding GM-CSFR, ELANE and MPO) (Figure 1F). Cytostatic/cytotoxic and differentiation-promoting effects of SK053 were also observed in NB4, another acute promyelocytic leukemia cell line, as well as in non-acute promyelocytic leukemia AML cell lines, such as MOLM14 and KG1 (Online Supplementary Figure S1).
SK053 induces gene expression profile associated with stress response and differentiation
To get further insight into the mechanisms of SK053 activity in AML cells, we sequenced and analyzed tran- scriptomes of controls and SK053-treated HL-60 cells using next-generation sequencing. Analysis of transcrip- tomes of controls and HL-60 cells incubated with SK053 for 48 h (Online Supplementary Figure S2) revealed differen- tial expression of genes (Online Supplementary Figure S3A, Online Supplementary Table S1) that, in gene ontology analysis, clustered into two major groups: (i) biological processes (Online Supplementary Figure S3B, Online Supplementary Table S2) involving the stress response (e.g. genes encoding calreticulin or proteasome subunits), reg- ulation of apoptosis (e.g. genes encoding Bcl-2-related protein, CFLAR or TNFRSF10B) and myeloid cell differen- tiation (CBFB, JAK2 or PIR) and (ii) molecule function (Online Supplementary Figure S3C, Online Supplementary Table S3) associated with changes in oxidoreductases activity [e.g. NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, ATP synthase or cytochrome c]. Furthermore, a 5-day incubation of HL-60 cells with SK053 induced the expression of genes regulating myeloid cell differentiation (JUN, CSF1, ID2) and cell pro- liferation (BCL6, PNMT) (Online Supplementary Figure S4A-C, Online Supplementary Tables S4-S6). Immunoblotting of the lysates of HL-60 cells confirmed the results of the next-generation sequencing analysis showing that SK053 induces endoplasmic reticulum stress and the unfolded protein response (Online Supplementary Figure S5).
SK053 binds to and inhibits protein disulfide isomerase
We then used lentiviral particles encoding short hairpin RNA (shRNA) to target thioredoxin in HL-60 cells. Intriguingly, thioredoxin knock-down with shRNA failed to inhibit the growth of HL-60 cells (Online Supplementary Figure S6A). Subsequently, we used two other shRNA sequences targeting thioredoxin (Online Supplementary Figure S6B) and assessed the effects of thioredoxin knock- down in HL-60 and MOLM14 cells. While both hairpins effectively suppressed the growth of MOLM14 cells, only one shRNA slightly inhibited and the other had no effect on the growth of HL-60 cells (Online Supplementary Figure S6C,D). These observations indicate that inhibition of thioredoxin might not be responsible for the differentia- tion-inducing effects of SK053 in HL-60 cells. Therefore, using a biotin affinity probe-labeling approach followed by mass spectrometry, we sought to identify other poten- tial intracellular SK053 targets in these cells. Incubation of HL-60 cells for 4 h with SK-BIO, an active, biotinylated SK053 analog (see Online Supplementary Figure S8 for the general structures of compound used herein), followed by pulldown with avidin-coated beads and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), revealed a prominent band of ~50-70 kDa molecular weight in silver-stained gels (Figure 2A). An inactive, biotinylated analog of SK053 that lacks the electrophilic double bond (referred to as SK-IN, Online Supplementary Figure S8) failed to precipitate any protein. The ~50-70 kDa protein was identified in mass spectrometry to be a protein disulfide isomerase (PDI), a product of the P4HB gene (Online Supplementary Figure S9A-D, Online Supplementary Table S7). SDS-PAGE followed by the
haematologica | 2018; 103(11)
1845