qPCR analysis of the effects of monensin on theexpressionofselectedgenesinACC1cellssupportedthisconclusion (Fig cck8 ic50. 7F).

 
M.V. Yusenko, et al. Cancer Letters 479 (2020) 61–70 set of genes down regulated by MYB-NFIB knockdown in ACC cells [23] MF498. We observed a significant overlap between genes suppressed by monensin in THP1 and genes activated byMYB-NFIB in ACCcells, indicating that monensin targets MYB-NFIB-dependent gene transcrip- tion in ACC cells (Fig. 7E). qPCR analysis of the effects of monensin on theexpressionofselectedgenesinACC1cellssupportedthisconclusion (Fig cck8 ic50. 7F). 4. Discussion MYB has emerged as a candidate drug target for human malig- nancies whose development depends on deregulated MYB expression, such as AML and ACC. Although targeting transcription factors has Fig. 6. Effect of monensin on clonal growth of primary murine AML cells traditionally been considered difficult, recent work from our and other and normal hematopoietic progenitors. Kit positive cells from bone marrow of healthy mice and murine AML cells carrying the MLL-AF9 on cofusion groups has demonstrated that small molecule targeting of MYB is fea- (generated via retroviral transduction of murine Kit positive HPCs with MLL- sibleandhastherapeuticpotential[20,21,23–27].Thishassetthestage AF9 and subsequent injection into syngeneic mice) were subjected to colony for identification and characterization of molecular scaffolds for de- formation assays in the absence or presence of 50 or 100 nM monensin.

 Equal velopment of new MYB inhibitors. numbers of cells were plated with DMSO (black bars) or with monensin (grey We have employed a MYB reporter cell line [24] to screen the bars). Columns show the relative colony number normalized to the DMSO Prestwick Chemical Library of 1280 approved, off-patent drugs for control. Asterisks indicate statistical significance (*p < 0.05, ***p < 0.001, novel MYB inhibitors. We found that the polyether ionophores mon- Student\'s t-test). ensin, salinomycin, and nigericin potently inhibit MYB activity and, as Fig. 7. Effects of monensin on the proliferation of ACC cells. A. Cell viability assay of ACC cells from two patients (ACC1 and ACC2), primary pleomorphic adenoma cells (PA), and non-tumorigenic mammary epithelial cells (MCF10A) treated with monensin at the indicated concentrations for 72 h. B. ACC1 cells transduced with a retroviral expression vector encoding MYB or an empty control vector were treated for 72 h with monensin and analyzed for cell viability. 

 

MYB mRNAexpressionlevelsintransducedcells(quantifiedbyqPCR)areshownontheright.C.Sphere-formingassayofACC1cellstreatedwithdifferentconcentrations of monensin. D. Effects of monensin on MYB-NFIB mRNA and protein levels in ACC1 cells. Cells were treated with the compound for 72 h. E. GSEA of the gene expressionsignatureofmonensin-treatedTHP1cellsandthesetofgenesdown-regulatedafterknockdownofMYB-NFIBinACCcells(fromref.22).G.qPCRanalyses confirming down-regulation of selected MYB target genes by monensin in ACC1 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Student\'s t-test). 67
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