Influence of monensin on viability and MYB expression of human AML cell lines

 M.V. Yusenko, et al. Cancer Letters 479 (2020) 61–70 Fig. 2. Influence of monensin on viability and MYB expression of human AML cell lines. A. Cells were cultured for 48 h in the presence of the indicated concentrations of monensin, followed by an MTS assay. Data is presented as percentage of remaining viable relative to cells cultured without monensin. B. Different AML cell lines and HEKMYBLuc cells were treated for 24 or 48 h with the indicated concentrations of monensin. Total cell extracts were then analyzed by western blotting for MYB and β-actin expression. C. Northern blot analysis of MYB mRNA expression in NB4 cells treated for 2 days with 0.3 or 1 μM monensin. Ribosomal protein S17 mRNA was used as a loading control. The numbers refer to the amounts of MYB mRNA relative to untreated NB4 cells. D. NB4 cells were treatedfor12hwithdifferentamountsofmonensinintheabsenceorpresence of10μMMG132. Totalcellextracts werethenanalyzed bywesternblottingforMYB and β-actin expression LY3009120. terminally truncated MYB (MYB-Δ3) and compared them to NB4 cells independent replicate samples of monensin-treated and control cells infected with a control lentivirus. Fig. 

 

4A shows that the presence of revealed a large number of genes whose expression was up- (1688 MYB-Δ3 significantly diminished the ability of monensin to induce genes) or down-regulated (722 genes) at least two-fold by monensin differentiation,asdemonstratedbythenumberofCD11b-positivecells. (Fig.5A).We selected several up- and down-regulated genes and tested Similarly, using CD14 as a differentiation marker, MYB-Δ3 suppressed their expression by qRT-PCR, confirming the changes in expression the differentiation induced by monensin (Fig 3xFLAG PEPTIDE inhibitor. 4B). The induction of induced by monensin (Fig. 5C). These changes were detected within a apoptosis by monensin was only slightly reduced by MYB-Δ3(Fig. 4C). few hours of treatment with monensin (Fig. 5D), as exemplified for the These experiments suggested that the effect of monensin on the dif- MYB-regulated KIT gene [47]. We then performed gene set enrichment ferentiation of leukemic cells was mediated to a large extent by the analysis (GSEA) with the published gene sets of MY###http://www.glpbio.com/simage/GA11233-L-NAME-hydrochloride-4.png####B-activated and inhibition of MYB, whereasthe cytotoxic effects appearto be less MYB- -repressed genes [46]. GSEA determines whether pre-defined sets of dependent. Analysis of MYB protein levels showed that the expression genes (e.g. MYB-activated or -repressed) show significant, coordinated oftheendogenousfull-lengthMYBwasstronglyinhibitedbymonensin, differences between two biological states, in our case, between mon- as demonstrated before (Fig. 2B), whereas MYB-Δ3 continued to be ensin-treated and control cells. Thus, the GSEA plot in Fig. 5E shows expressed in the presence of monensin (Fig. 4D). 

We have not in- that genes previously identified as activated by MYB were significantly vestigated the reasons for the persistent expression of MYB-Δ3 in the enriched in monensin-repressed genes [46]. We also detected a sig- presence of monensin. It is possible that the C-terminal part of MYB is nificant correlation between genes repressed by MYB [46] and genes required for its degradation Prostaglandin J2, but this remains to be explored. activated by monensin treatment (Fig. 5F). Thus, these findings con- firmed the MYB-inhibitory activity of monensin on a transcriptome- wide scale. Additional GSEA analyses revealed that monensin induces 3.5. Monensin interferes with the activation of MYB-target genes in AML differentiation of THP1 as indicated by the correlation between cells monensin-upregulated genes and genes upregulated by tretinoin (all- trans-retinoic-acid), an inducer of differentiation of myeloid cells To better understand how monensin exerts its inhibitory effects on (Fig. 5G). We also detected significant overlaps between genes down- leukemic cells, we performed transcriptome profiling of THP1 cells regulated by monensin and E2F targets and G2/M checkpoint genes treated for 16hwith 1μM monensinand untreated cells as control. We (suppl. Fig.6), suggesting that monensin also interferes with cell cycle usedTHP1cellsbecausepreviousstudieshadalreadyidentifiedup-and progression. down-regulated genes after silencing of MYB in these cells [46]. Wes- tern blotting showed that the amount of MYB was not significantly affectedafter16hofmonensin-treatment(Fig.5B).RNA-seqanalysisof 64