M.V. Yusenko, et al. Cancer Letters 479 (2020) 61–70 demonstrated for monensin

 M.V. Yusenko, et al. Cancer Letters 479 (2020) 61–70 demonstrated for monensin, strongly interfere with the proliferative unclear if, and how, changes of the intracellular ion homeostasis cause potential of AML and ACC cells. Our data showed that monensin in- inhibition of MYB activity. Monensin, salinomycin, and nigericin have hibits MYB activity without affecting MYB expression (Fig. 1). Simi- different preferences for monovalent ions, with monensin and nigericin larly,treatmentofTHP1leukemiacellsfor16hwithmonensinaffected showing at least 10-fold preferences for Na + over K+, while salino- the expression of a significant number of MYB-regulated genes without mycin prefers K+ over Na + ions [49], making it difficult to relate the decreasingMYBexpression(Fig.5).InhibitionofMYBactivitywasfast, inhibition of MYB by all three compounds to their ion selectivity pro- as illustrated by the effect of monensin on the expression of the KIT file. Our data also exclude formation of ROS and the process of fer- gene, which showed inhibitory effects already after 2–4 h of monensin roptosis, which is triggered by salinomycin in breast cancer stem cells treatment (Fig. 5D). In AML cells, MYB expression was strongly de- and involves the generation of lipid-ROS [40] in the inhibition of MYB. creased upon prolonged treatment with monensin, presumably as a Using microscale thermophoresis [58] or DARTS (target identification result of proteasomal degradation (Fig. 2). This could simply be a usingdrugaffinityresponsivetargetstability)assays[59]wealsofailed consequence of the induction of myeloid differentiation, which oc- to detect direct binding of monensin to MYB (unpublished data). Thus, curred concurrently with monensin treatment and is known to be ac- the mechanismbywhichmonensin affects MYBactivity and expression companied by a decrease of MYB expression [4], or could represent a remains to be elucidated in future studies. distinct activity of monensin. In any case, down-regulation of MYB In summary, we have shown for the first time that a single com- expression further contributes to the efficacy of monensin as a MYB pound with significant MYB-inhibitory activity is active at nanomolar inhibitor. Besides inducing differentiation, monensin interfered with concentration against from two different malignancies dependent the viability of myeloid leukemia cell lines and induced apoptosis. Al- on deregulated MYB expression. We cannot completely exclude that though monensin has a variety of activities in eukaryotic cells [49–51], MYB-independent activities of monensin contribute to its inhibitory ectopic expression of an activated version of MYB counteracted the effectsonAMLandACCcells.However,ectopic expressionofactivated effectsofmonensin.Thiswasmostsignificantincaseoftheinductionof MYB clearly reversed some of these effects. Furthermore, gene expres- differentiation (Fig. 4), indicating that the activity of monensin is sion profiling has shown a significant overlap of genes affects by mediated, at least to a significant part, by inhibition of MYB. The fact monensin treatment and MYB silencing in two different tumor types 3xFLAG molecular weight. thatMYBinhibitionplaysamajorroleintheanti-proliferativeeffectsof These observations support the notion that the effects induced by monensin is also in line with the observation that primary leukemic monensin are mediated to a significant part by inhibition of MYB. cells derived from a mouse model of AML are more sensitive to mon- Further studies to unravel the inhibitory mechanism of monensin and ensin than normal hematopoietic progenitor cells (Fig. 6). Thus, mon- related polyether compounds, to identify structural features that are ensin acts in a manner reflecting the higher MYB-dependence of the responsible for their inhibitory activity and to explore their ther###http://www.glpbio.com/simage/GA11366-H-D-Leu-Thr-Arg-pNA-acetate-salt-1.png####apeutic leukemic cells compared to normal hematopoietic progenitor cells potential for MYB-dependent malignancies will therefore be highly in- [11,12],whichisaprerequisiteforpotentialtherapeuticapplicationsof teresting. monensin in AML. Interestingly, a previous study has shown that monensin treatment reduces the size of tumors formed by murine Availability of data and materials WEHI-3BD leukemia cells in an in vivo mouse model [41], further supporting the therapeutic potential of monensin. Alldataarepresentedinthisarticleandthesupplementarydatafile. The MYB-inhibitory activity of monensin is strongly corroborated In. by the observation that monensin also interferes potently with the RNA-Seq, data are available at GEO under accession number viability and non-adherent growth of human ACC cells, in contrast to GSE130657. non-tumorigenic breast cells and primary pleomorphic adenoma cells. This is a highly significantfinding as it confirms preferential targeting Funding of malignant versus non-malignant cells (Fig. 6), also in epithelial cell types cck-8 price. 

GSEA showed that monensin down-regulates the expression of a This work was supported by the Else Kröner-Fresenius-Stiftung, the significant number of genes activated by MYB-NFIB in ACC cells. We Deutsche Krebshilfe, the Adenoid Cystic Carcinoma Research also found that increased MYB expression in ACC cells reduces their Foundation (ACCRF), the Swedish Cancer Society, the Swedish sensitivity towards monensin. Taken together, these findings suggest Childhood Cancer Fund, and the Swedish state under the agreement thattheanti-proliferativeactivityofmonensininACCcellsismediated, between the government and the county councils AT13387, the ALF-agreement at least in part, by inhibition of the MYB-NFIB fusion. Similar to the (ALFGBG-721711). down-regulation of MYB in AML cells, monensin caused a decrease of MYB-NFIB expression in ACC cells, however, the underlying mechan- Author contributions isms appear to be different. While MYB mRNA was only slightly sup- pressedinAMLcells,MYB-NFIBmRNAwasstronglydown-regulatedby M.Y., M.K.A., A.T., L.A.G., and A.J. designed and performed ex- monensin in ACC cells. Most likely, this is due to the reported auto- periments and analyzed data; M.-F.A.P., and J.-H.M. designed experi- regulation of MYB-NFIB in ACC cells as a consequence of the chromo- ments; J.P.v.K. helped design screening experiments. G.S. and K.-H.K. somal translocation in these cells [17].

 

 The observation that monensin conceived the study, designed experiments, analyzed data and wrote interferes with the autoregulatory loop of MYB-NFIB in ACC cells also the manuscript. All authors reviewed the results and approved thefinal provides further evidence for its ability to target MYB-NFIB activity. version of the manuscript. The efficacy of monensin as an anti-tumor agent has been demon- strated in several studies before, using models of pancreatic, colon and Declaration of competing interest prostate cancer. In these studies monensin was shown to induce oxi- dative stress and to deregulate avariety of signalling pathways, suchas The authors declare that they do not have any conflict of interest to EGFR, Wnt and androgen signalling [52–55]. Monensin and the related disclose. compounds salinomycin and nigericin are members of the large group of polyether  that have diverse biological activities. They act as ionophores to mediate the transport of monovalent K+ and Acknowledgements Na + ions over biological membranes and thereby disturb the in- tracellular ion homeostasis and the function of intracellular organelles, We thank B. Berkenfeld and P. Pieloch for technical assistance and such as mitochondria and the Golgi apparatus [56,57]. It is currently the Core Facility Genomics at the Medical Faculty of the University of 68