Estrogen Receptor B Is a Novel Target in Acute Myeloid Leukemia
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Published OnlineFirst August 23, 2017; DOI: 10.1158/1535-7163.MCT-17-0292 Companion Diagnostic, Pharmacogenomic, and Cancer Biomarkers Molecular Cancer Therapeutics Estrogen Receptor b Is a Novel Target in Acute Myeloid Leukemia Sarah-Grace Rota1,2, Alessia Roma1,2, Iulia Dude2, Christina Ma2, Robert Stevens3, Janet MacEachern3, Joanna Graczyk3, Shaundrei Mabriel G. Espiritu4, Praveen N. Rao2, Mark D. Minden5, Elena Kreinin2, David A. Hess6, Andrew C. Doxey4, and Paul A. Spagnuolo1,2 Abstract Acute myeloid leukemia (AML) is a devastating disease char- patient-derived AML cells with high levels of ERb were sensitive, acterized by poor patient outcome and suboptimal chemother- whereas cells with low ERb were insensitive to diosmetin. Knock- apeutics. Here, a high-throughput screen identified diosmetin, a down of ERb confirmed resistance, whereas overexpression citrus flavonoid, with anti-AML activity. Diosmetin imparted enhanced sensitivity to diosmetin, which was demonstrated to selective toxicity against leukemia and leukemia stem cells in vitro be mediated by reactive oxygen species signaling. In summary, and in vivo with no effect on normal hematopoietic stem cells. these studies highlight targeting of ERb with diosmetin as a Mechanistically, we demonstrated that diosmetin targets estrogen potential novel therapeutic strategy for the treatment of AML. receptor (ER) b.ERb expression conferred cell sensitivity, as Mol Cancer Ther; 16(11); 2618–26. Ó2017 AACR. Introduction Materials and Methods Acute myeloid leukemia (AML) is an aggressive hemato- Cell culture logic malignancy with poor patient outcome characterized Cell lines from the ATCC were cultured in 5% CO2 at 37 C. clinically by an accumulation of immature myeloid cells in They were obtained in 2012, and no authentication was per- the bone marrow (1). Current induction therapy fails to formed thereafter. Additional details can be found in the Sup- achieve long-term remission, and the 5-year-survival rates for plementary Materials. U2OS human osteosarcoma cells with adult patients (i.e., >60yearsold)islessthan10%(2).New epitope (FLAG)-tagged ERb under the doxycycline promoter therapeutics to improve AML patient outcomes are clearly (hereafter denoted U2OS-ERb) were grown in RPMI and were needed. generated by Monroe and colleagues (7). Primary human AML To identify novel anti-AML drugs, we created a unique library samples were provided by Drs. Mark Minden (Princess Margaret of food-derived bioactive compounds and screened this library Cancer Center), Joanna Graczyk, Janet MacEachern, and Robert against TEX leukemia cells, an AML and surrogate leukemia Stevens (Grand River Cancer Centre) and were cultured in IMDM, stem cell (LSC) line (3) used successfully in high-throughput and supplemented with 20% FCS and antibiotics. These cells were screens (4–6). Through this approach, we identified diosmetin isolated from the peripheral blood of consenting AML patients as a novel drug with anti-AML activity. Mechanistically, we who had at least 80% malignant cells among the mononuclear show that diosmetin binds to estrogen receptor (ER) b and cells. Normal hematopoietic stem cells (HSC) were purchased demonstrate that ERb is functionally important to diosmetin's from Stem Cell Technologies or provided by Dr. David Hess activity. (Western University). The collection and use of human tissue for this study was approved by Institutional Ethics Review boards (University Health Network, Toronto, ON, Canada, Western University, and University of Waterloo, Waterloo, Ontario). 1Department of Food Science, University of Guelph, Guelph, Ontario, Canada. Cell growth and viability 2School of Pharmacy, University of Waterloo, Kitchener, Ontario, Canada. Cell growth and viability was measured using the 3Grand River Cancer Centre, Kitchener, Ontario, Canada. 4Department of Biol- 5 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- ogy, University of Waterloo, Waterloo, Ontario, Canada. Princess Margaret (4-sulfophenyl)-2H-tetrazolium inner salt (MTS) reduction assay Cancer Center, Ontario Cancer Institute, Toronto, Ontario, Canada. 6Robarts Research Institute, University of Western Ontario, London, Ontario, Canada. (Promega) or Annexin V and propidium iodide (ANN/PI) stain- ing (Biovision), according to the manufacturer's protocol and as Note: Supplementary data for this article are available at Molecular Cancer previously described (6, 8). Colony formation assays were per- Therapeutics Online (http://mct.aacrjournals.org/). formed, as previously described (9) and as outlined in the Corresponding Author: Paul A. Spagnuolo, University of Guelph, 50 Stone Road, Supplementary Methods. Guelph, ON N1G 2W1, Canada. Phone: 519-824-4120; Fax: 519-824-6631; E-mail: [email protected] Chemical screen and reactive oxygen species measurements doi: 10.1158/1535-7163.MCT-17-0292 Food-derived bioactive molecules were obtained from Ó2017 American Association for Cancer Research. Chengdu Biopurify Phytochemicals Ltd. (n ¼ 288) and screened 2618 Mol Cancer Ther; 16(11) November 2017 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst August 23, 2017; DOI: 10.1158/1535-7163.MCT-17-0292 ERb in AML as outlined in the Supplementary Methods. Reactive oxygen RNAi knockdown of ERb species (ROS) measurements were conducted as detailed in the Lentiviral infections were performed as described (9). Briefly, Supplementary Methods (10). OCI-AML2 cells (5  106) were resuspended and incubated overnight at 37C in media (6.5 mL) containing protamine sulfate m Target prediction using structural bioinformatics and (5 g/mL) and 1 mL of virus cocktail (which contains a puromycin molecular docking antibiotic resistance gene and the shRNA sequence). Next, Starting with diosmetin, we searched the Protein Data Bank the virus was removed by centrifugation, and the cells were (PDB) for protein structures capable of binding diosmetin-like washed and resuspended in fresh media containing puromycin m ligands. We identified an initial set of 9 structures cobound with (1 g/mL). Following puromycin selection, live cells were plated four diosmetin-like ligands: biochanin A (QSO), genistein for viability assays. The shRNA coding sequences (Sigma Chem- b 0 (GEN), kaempherol (KMP), and luteolin (LU2). This set was ical) were: ER (TRCN0000003328) shRNA clone 16 5 -CCGGG- expanded to include proteins with similar binding site composi- ATGCTTTGGTTTGGGTGATTCTCGAGAATCACCCAAACCAAAG- 0 b 0 tions using the PoSSuM (Pocket Similarity Search using Multi- CATCTTTTT-3 , (TRCN0000003327) ER shRNA clone 17 5 - CCGGCCTTAATTCTCCTTCCTCCTACTCGAGTAGGAGGAAGG- Sketches) tool with default parameters (11, 12). This search 0 yielded a final set of 92 proteins with predicted diosmetin-binding AGAATTAAGGTTTTT-3 ; and a TRC scramble control. potential. The Database for Annotation, Visualization and Inte- grated Discovery (DAVID) tool (13) was then used to summarize In vivo models and identify overrepresented functions common to this group of NOD/SCID gamma mice (NSG; Jackson Laboratory or Univer- potential target proteins. sity of Western Ontario) were used for xenograft and engraftment The molecular docking experiments were conducted using the assays as previously described (5, 6, 16) and detailed in the computational software Discovery Studio (DS), Structure-Based- Supplementary Methods. For engraftment experiments, human þ þ Design program from BIOVIA/Accelrys Inc., as previously myeloid cells (hCD45 /CD33 ) were detected in femoral bone fl described (14, 15). Brie y, diosmetin was built using the small marrow by flow cytometry following a 6-week engraftment peri- a  molecules module in DS. The crystal structures of ER (pdb id: 1 od. All animal studies were carried out according to the regula- b  7R) and ER (pdb id: 1 7J) with genistein bound was obtained tions of the Canadian Council on Animal Care and with the from PDB. The protein was prepared using the macromolecules approval of the University of Waterloo, Animal Care Committee. module in DS. Ligand-binding site was defined by selecting a 12 Å radius sphere using genistein crystal structure after which it was deleted. Then molecular docking was performed using the recep- Statistical analysis Æ tor–ligand interactions module in DS. The CDOCKER algorithm Unless otherwise stated, in vitro results are presented as mean Æ was used to find the most appropriate binding modes of dios- SD, whereas in vivo results are presented as mean SEM. Data metin using CHARMm force field. The docked poses obtained were analyzed with GraphPad Prism 4.0 (GraphPad Software) were ranked based on the CDOCKER energies and CDOCKER using one-way ANOVA with Tukey post hoc analysis for between- interactions energies in kcal/mol. group comparisons or standard Student t tests where appropriate and Mann–Whitney t tests were used for animal experiments. P 0.05 was accepted as being statistically significant. mRNA and protein detection qPCR was performed as previously described (9) in triplicate using an ABI 7900 Sequence Detection System (Applied Biosys- Results tems) with 5 ng of RNA equivalent cDNA, SYBR Green PCR Master A screen for novel anti-AML compounds identifies diosmetin fi mix (Applied Biosystems), and 300 nmol/L of ER-speci c primers To identify novel anti-AML compounds, we screened our (forward ERb:50-TGCTCAATTCCAGTATGTACC-30, reverse ERb: 0 0 0 unique in-house library against TEX cells using the MTS assay. 5 -ATGAGGTGAGTGTTTGAGAG-3 , forward ERa:5-CATTATG-