Identification of monensin as a potent MYB inhibitor. A. Schematic illus- tration of the HEK-MYB-Luc

 M.V. Yusenko, et al. Cancer Letters 479 (2020) 61–70 Fig. 1. Identification of monensin as a potent MYB inhibitor. A. Schematic illus- tration of the HEK-MYB-Luc reporter cell line. The carry a stably integrated MYB-inducible luciferase reporter gene, an expression vector for human MYB-2KR under the control of a modified CMV pro- moter with Tet-operator sites positioned close to the transcriptional start site and an expression vector for the Tet-repressor. The HEK-Luc cell line carries a constitutively activeluciferasereportergenedrivenbythe CMV promoter. B. HEK-MYB-Luc and HEK- Luccellsgrownfor12hinpresenceof1μg/ ml doxycyclin and the indicated con- centrations of monensin were analyzed for luciferase activity. 

 

Columns show the mean fluorescence and viability with standard deviations, normalized to cells cultured without monensin. Asterisks indicate sta- tistical significance compared to the un- treated control (**p < 0.01, ***p < 0.001, Student\'s t-test). C. HEK- MYB-Luc cells treated with the indicated concentrations of monensin and with or without 1 μg/ml doxycyclin were analyzed by western blotting for MYB and β-actin expression. D. HEK-MYB-Luc cells treated as indicated in B were analyzed for cell viability. MYB protein levels were not decreased in the presence of salinomycin significantly less in the presence of the proteasome inhibitor MG132 but slightly increased by nigericin. (Fig. 2D). Thus, monensin targets MYB by two distinct mechanisms, Salinomycinhaspreviouslyattractedinterestasananti-tumoragent namelybyinhibitingitsactivityandinducingitsdegradation,atleastin that inhibits the survival of breast cancer stem cells [38]. In these cells, AML cells. Interestingly, there was no decrease of MYB expression in salinomycin triggers a process referred to as ferroptosis [39] in which HEKMYBLuccellswhentreatedwithmonensinfortwodays(Fig.2B).A lipid peroxides are generated that ultimately exert the cytotoxic effect possibleexplanationforthisdiscrepancyofMYBstabilitybetweenAML [40].WethereforewonderedwhethertheinhibitionofMYBactivityby and HEKMYBLuc cells is suggested by work that has implicated the F- monensin was caused by a mechanism involving lipid peroxides or, box protein Fbw7 as a substrate-specific adapter of the SCF-Fbw7 E3 moregenerally,reactiveoxygenspecies(ROS).Toaddressthis,weused ubiquitin ligase complex in the control of MYB degradation [42,43]. different scavengers of ROS including N-acetyl cysteine, α-tocopherol, The adenovirus E1A protein was shown to interact with the SCF-Fbw7 and Na-ascorbate, as well as the ferroptosis inhibitor ferrostatin-1. complex and to inhibit its activity [44]. 

 

Since the HEKMYBLuc cells Suppl. Fig.4 shows that these compounds did not interfere with the expressE1A,thismightexplainwhyMYBisnotdegradedinthesecells. inhibition of MYB activity by monensin, suggesting that###http://www.glpbio.com/simage/GA11233-L-NAME-hydrochloride-3.png#### the inhibitory MYBactivityiscrucialformaintaining myeloidleukemiacells inan mechanism is not related to ferropotosis and the generation of ROS. undifferentiated state and for preventing apoptosis [4]. We therefore analyzed if monensin induces apoptosis or differentiation of myeloid leukemia cells Sotrastaurin (AEB071). Fig. 3 shows that the expression of the differentiation 3.3. Treatment of AML cell lines with monensin leads to degradation of markers CD11b and CD14 in NB4 and HL60 was increased in a MYB and induction of differentiation and apoptosis concentration-dependent manner by monensin. Monensin-induced dif- ferentiation was also observed by May-Grünwald-Giemsa staining of Toexploretheeffectofmonensinonmyeloidleukemiacellswefirst several AML cell lines, which displayed changes in the nuclear/cyto- performed viability assays on a panel of different human myeloid leu- plasmic ratio and in the size and morphology of the nuclei Chloramphenicol. Some cells kemia cell lines. Whereas all cell lines tested, including HEKMYBLuc showed also increased vesicle formation (Fig. 3D). Besides differentia- cells, were sensitive to monensin when treated for 48 h, some AML cell tion, monensin also induced cell death in a dose-dependent manner, as lines exhibited EC values for loss of viability clearly below 1 μM 50 demonstrated by annexin V expression (Fig. 3). Similar results were (Fig.2A).Theseresultsareinaccordancewithanearlierstudy,showing obtained for salinomycin and nigericin (suppl. Fig.5). that human AML cell lines are highly sensitive to low concentrations of monensin [41]. 

 

To investigate the effect of monensin on MYB expres- sion weperformedWesternblotting ofAMLcelllinestreatedfor2days 3.4. EctopicexpressionofaC-terminallytruncatedversionofMYBpartially with monensin. Interestingly, in all cases we observed a strong con- suppresses the inhibitory activity of monensin centration-dependent decrease in the amount of MYB protein (Fig. 2B). This decrease occurred gradually upon monensin treatment because it To investigate whether the induction of differentiation and apop- was less prominent after a shorter treatment time Cell Counting Kit-8 stability. Northern blot ana- tosis by monensin was caused by inhibition of MYB or by an unrelated lysis of MYB mRNA from monensin-treated or untreated NB4 cells was mechanism, we asked whether ectopic expression of an activated, C- terminally truncated version of MYB counteracts the effects of mon- onlyslightlydecreasedaftertwodaysoftreatment(Fig.2C),suggesting that the strong decrease of MYB in the presence of monensin was ensin [45]. Ectopic expression of truncated rather than full-length MYB mainly due to degradation of the protein. We confirmed this by also allowed us to monitor its expression without an additional epitope showing that monensin treatment decreased MYB protein levels tag. We employed lentivirus-infected NB4 cells expressing a C- 63