Monensin induces differentiation and apoptosis in myeloid cell lines

 
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r> M.V. Yusenko, et al. Cancer Letters 479 (2020) 61–70 Fig. 3. Monensin induces differentiation and apoptosis in myeloid cell lines. NB4 and HL60 cells were cultured for 2 days in the presence of the indicated concentrations of monensin. The cells were then stained with against CD11b (A) or CD14 (B) or with annexinV (C) and analyzed by flow cytometry. Columnsindicatethepercentageofpositivecells.Asterisksindicatestatisticalsignificance(*p < 0.05,**p < 0.01,***p < 0.001,Student\'st-test).PanelDshows May-Grünwald-Giemsa staining of the indicated cell lines cultivated for 48 h without or with 100 nM monensin Cell Counting Kit-8 inhibitor. 3.6. Primary murine AML cells are more sensitive to monensin than normal hematopoietic tumor cells frequently expressing oncogenic MYB-NFIB hematopoietic progenitor cells fusion proteins as a result of chromosomal translocations [13]. We in- itially performed viability assays with short-term cultured tumor cells To compare the effect of monensin on primary leukemia cells and derived from two ACC patients (ACC1 and ACC2). These cells were normal hematopoietic progenitor cells, we employed a murine AML highly sensitive to monensin, in contrast to the non-tumorigenic model based on the expression of an MLL-AF9 fusion protein in he- mammary epithelial cell line MCF10A and primary pleomorphic ade- matopoietic progenitor cells [48]. 

Studies with MLL-AF9-transformed noma cells used as controls (Fig. 7A). We also showed that ectopic leukemic cells and primary Kit-positive hematopoietic progenitor cells expression of MYB diminished the inhibitory effect of monensin on the from bone marrow of healthy mice showed that monensin inhibited viability of ACC1 cells, suggesting thatmonensin affectsthe viability of colony formation of the leukemic cells in a concentration-dependent the cells by inhibiting MYB, at least partially (Fig. 7B). Sphere-forming manner (Fig. 6). Importantly, colony formation by the normal hema- assays to assess the effect of monensin on the self-renewing capacity of topoietic progenitors was inhibited significantly less 3xFLAG PEPTIDE inhibitor, consistent with ACCcellsshowedaconcentration-dependentdecreaseinthenumberof the notion that leukemic cells depend more strongly on high levels of spheres, similar to the results of the viability assays (Fig. 7C). Gene MYB activity than normal hematopoietic progenitor cells. expression studies revealed that monensin inhibits the expression of MYB-NFIB, both at the mRNA and protein levels (Fig. 7D), presumably reflecting the transcriptional autoregulation of MYB-NFIB expression 3.7. Monensin inhibits the proliferation of patient-derived ACC cells described before [17]. 

To assess the effect of monensin on the expres- The MYB-inhibitory potential of monensin prompted us to in- sion of target genes regulated by MYB-NFIB, we performed GSEA using the gene expression signature of monensin-treated THP1 cells and the vestigate the effect of monensin on ACC cells. These are non- 65
M.V. Yusenko, et al. Cancer Letters 479 (2020) 61–70 Fig. 4. Effect of ectopic expression of MYB-Δ3on monensin-induced differentiation and apoptosis. NB4 cells infected with a lentivirus encoding a c- terminally truncated MYB (NB4MYBΔ3) or a control lentivirus (NB4control) were treated for 2 days with the indicated concentrations of monensin. Differentiation and apoptosis was then analyzed by staining with against CD11b (A) or CD14 (B) or with annexinV (C). Asterisks indicate statis- tical significance (*p < 0.05, ***p < 0.001, Student\'s t-test). n. s denotes non specific. Panel D shows a Western blot analysis of total cell extracts from NB4MYBΔ3 and NB4control cells, using anti- bodies against MYB and β-actin. Molecular weight markers are indicated to the left. Fig.5. RNAprofilingofmonensin-treatedTHP1cells.THP1cellstreatedfor16hwithoutorwith1μMmonensin.A.Heatmapofgenesdifferentiallyexpressedby alog2-foldchange>1or <−1(padj < 0.05)inthreebiologicalre###http://www.glpbio.com/simage/GA11233-L-NAME-hydrochloride-5.png####plicatesofmonensin-treatedversusuntreatedTHP1cells.B.WesternblotshowingMYBandβ- actinexpressioninmonensin-treatedanduntreatedcells.C.qPCRanalysisofselectedgenesshowingup-ordown-regulationbymonensin.Dataisderivedfromthree biological replicates. Blue bars indicate the log2-fold up- or down-regulation and standard deviation of the indicated genes in monensin-treated versus untreated cells.Greybarsshowthelog2-foldexpressionchangesdeterminedbyRNA-Seq.KnownMYB-regulatedgenesaremarkedwithasterisks.D.Time-dependentdecrease of KIT expression in THP1 cells by monensin. Asterisks indicate statistical significance (***p < 0.001, Student\'s t-test). E-G. GSEA of the monensin-induced gene expression signature and sets of genes activated (E) or repressed (F) by MYB in THP1 cells and up-regulated during ATRA-induced myeloid differentiation (G) o-Phenanthroline. (For interpretation of the references to colour in thisfigure legend, the reader is referred to the Web version of this article.) 66