Control cells were incubated without mon- MYB inhibition is feasible and has therapeutic potential

M.V. Yusenko, et al. Cancer Letters 479 (2020) 61–70 I B) genes, leading to disruption of the 3′-end of MYB mRNA, loss of Isolation of polyadenylated RNA and Northern blotting was performed MYB negative regulatory elements, and overexpression of oncogenic as described before [34]. Blots were hybridized sequentially to radi- MYB-NFIB fusion proteins [13]. MYB may also be activated by copy olabeled probes specific for MYB and ribosomal protein S17 mRNAs. numbergainorjuxtapositionofenhancerelementsfromNFIBandother Radioactive bands were quantified with a phosphor image analyzer. fusion partner genes [16,17]. Overall, thesefindings have greatly stimulated the interest in MYB 2.4. Flow cytometry as a target for drug development [1,3,18]. Although transcription fac- torsaretraditionally considereddifficulttotarget,initialapproaches to Approximately one million were cultured for 2 days in RPMI inhibit MYB activity or expression by small molecule or peptido-mi- 1640 medium containing 10% fetal calf serum and the desired con- metic inhibitors have already proven successful and have shown that centration of monensin. Control cells were incubated without mon- MYB inhibition is feasible and has therapeutic potential [3,19–27]. ensin. The cells were then stained with anti-human CD11b-FITC (clone Despite the success of these initial pre-clinical studies, it is not clear ICR F44, BioLegend) or CD14-FITC antibody (clone 63D3, BioLegend) whether they can be used in the clinic. None of the compounds have so and analyzed by a flow cytometer. 

Side scatter and forward scatter far been tested in clinical trials and it is possible that they may have profileswereusedtoexcludedebrisandnon-viablecells.Toanalyzethe unwanted side effects. Therefore, it is important to expand the collec- fraction of dead cells, the cells were double-stained with FITC-annexin- tion of potential MYB inhibitors. The aim of this study was to identify V (BioLegend, Biozol, Eching, Germany) and propidium iodide (PI) to novel inhibitory compounds and, thereby, to expand the available determine the percentage of apoptotic/necrotic cells. molecular scaffolds as starting points for further development of MYB Additional methods are described in supplementary information. inhibitors. Here,weemployedtheMYBreportercelllineHEK-MYB-Luc[24]to 3. Results screen a library of 1280 approved small molecule drugs for compounds that inhibit MYB activity. Our data show that the polyether ionophore 3.1. Monensin suppresses MYB activity in a cell-based screening system monensin A (henceforth referred to as monensin) is a potent MYB in- hibitor. Characterization of its effects on AML and ACC cells suggests We have screened the Prestwick Chemical Library®, a collection of that monensin and related compounds might be promising candidates 1280 off-patent approved drugs, for the ability to inhibit luciferase for further development as novel MYB inhibitors. activity in the MYB reporter cell line HEK-Myb-Luc. This library has been used frequently to repurpose approved drugs for new applications 2. Materials and methods [see references 35,36,37]. HEK-Myb-Luc cells harbor a doxycycline- inducible expression system for an activated version of human MYB 2.1. Cells (referred to as MYB-2KR) and a MYB-inducible luciferase reporter gene [24](Fig. 1A). MYB-2 KR is a sumoylation-deficient MYB mutant that HEK-MYB-Luc and HEK-Luc reporter cells have been described has a higher transactivation potential than wild type MYB [28,29]. previously [24]. HEK-MYB-Luc cells express a doxycycline-inducible Initialscreeningofthelibraryat5μMconcentration(seescatterplotin version of human MYB (MYB-2KR) which drives the ex###http://www.glpbio.com/simage/GA11233-L-NAME-hydrochloride-2.png####pression of the suppl.Fig.1),testingcandidate compounds atlowerconcentrations and MYB-inducibleluciferasereportergenepGL4-5xMRE(GG)-Myc[28,29]. elimination of cytotoxic compounds or compounds that inhibit luci- HEK-Luc cells carry a stably integrated luciferase gene under the con- feraseitself,identifiedmonensin,apolyetherionophoreantibioticfrom trol of the CMV. HL60, NB4, KG1,OCI-AML3, MV4-11, THP-1, Kasumi- Streptomyces cinnamonensis, as a novel inhibitor of MYB activity. In 1 and U937 are established human myeloid leukemia lines. Murine addition, the several DNA damaging agents, including etoposide, to- MLL-AF9 transformed AML cells were obtained as described before potecan, and camptothecin were identified as potential MYB inhibitors [21]. Murine hematopoietic Kit-positive progenitor cells were isolated (unpublisheddata).Thesecompoundswerenotstudiedfurtherbecause from the femur of C57/BL6 wildtype mice and enriched on the basis of our previous work had already established a connection between MYB Kit expression by immunomagnetic beads (Miltenyi Biotec, Bergisch and DNA damage signaling [24]. Most of the compounds that showed Gladbach, Germany) and Kit antibodies. Colony formation assays with strong inhibition in theprimaryscreen werealso excludedfromfurther murine AML cells and normal HPC were performed as previously de- studybecausetheywerehighlycytotoxicorinhibitedluciferaseactivity scribed [21,30]. Patient-derived, MYB-NFIB positive ACC cells (ACC1 itself. and ACC2) and pleomorphic adenoma (PA) cells were isolated and Monensin inhibited MYB activity in HEK-MYB-Luc cells in a con- cultivated in vitro as described [13 AS1517499,31]. MCF10A is a nontumorigenic centration-dependent manner with an EC concentration of slightly 50 mammary gland epithelial cell line that was obtained from ATCC and less than 1μM(Fig. 1B). 

By contrast, luciferase activity in the HEK-Luc cultured as previously described [32]. cellsthatconstitutivelyexpressluciferasefromastablyintegratedCMV promoter-based reporter plasmid was virtually not affected by mon- 2.2. Chemicals ensin. Monensin also had no significant effect on the MYB expression level or the viability of the reporter cell line after 12 h treatment The Prestwick Chemical Library® was obtained from Prestwick (Fig. 1C and D). Since the screening cell line employed the activated Chemical (Illkirch Cell Counting Kit-8 solubility, France). Doxycyclin, monensin A (purity 90–95%), MYB-mutant 2 KR, we also compared the inhibitory effect of monensin salinomycin (purity ≥98%), nigericin (purity ≥98%), N-acetyl-cy- on the activity of MYB-2KR and wild type MYB. Both forms of MYB steine, α-tocopherol, sodium ascorbate, ferrostatin-1 and MG132 were were inhibited similarly (suppl. Fig.2). obtained from Sigma-Aldrich (München, Germany), dissolved in ethanol or DMSO and stored as 10 mM stock solutions at−70 °C. 3.2. The MYB-inhibitory activity of monensin is shared by salinomycin and nigericin 2 3x FLAG for sale.

3. Protein and RNA analysis To investigate whether other polyether ionophores have similar Total cell extracts were prepared by boiling the cells in SDS-sample inhibitory activity we tested salinomycin and nigericin for their ability bufferorpreparedusingRIPAlysisbuffer(MerckMillipore,Darmstadt, to inhibit MYB activity. Experiments with HEK-Myb-Luc and HEK-Luc Germany), followed by SDS polyacrylamide gel electrophoresis and cells(suppl.Fig.3)showedthatbothcompoundsinhibitMYBactivityin Western blotting with antibodies against MYB (5E11 [33], or EP769Y, a concentration-dependent manner similar to monensin, while the lu- Abcam) or β-actin (Sigma-Aldrich, AC-15, and Abcam, ab8226). ciferase activity of the HEK-Luc cells was not significantly affected.