Estrogen Receptor B Is a Novel Target in Acute Myeloid Leukemia

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. 2017 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) staining (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 2017 American Association for Cancer Research. Chengdu Biopurify Phytochemicals Ltd. (n ¼ 288) and screened

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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 tor–ligand interactions module in DS. The CDOCKER algorithm Unless otherwise stated, in vitro results are presented as mean 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- fi 0 0 Compound screens against TEX cells have identi ed novel anti- GAGTCTGGTCCTG-3 , reverse ERa:5-TTCGTATCCCACCTTT- – 0 AML targets (4 6) and drugs that have been evaluated in human CATC-3 ). Relative mRNA expression was determined using the clinical trials (17). In our screen, the flavonoid diosmetin was one DDCT fi method as previously described (18), and ef ciency was of two compounds that imparted the greatest reduction in TEX cell calculated using LinRegPCR analysis software (20, 21). Western viability (Fig. 1A; Supplementary Fig. S1A; arrow indicates dios- blotting was performed as previously described (6) and as out- metin; inset: chemical structure). Diosmetin's ability to reduce lined in the Supplementary Methods. growth and proliferation was also shown in a panel of AML cell lines using the MTS assay following a 96-hour incubation period ER reporter assay (Fig. 1B; EC50 values: 3–15 mmol/L). We next assessed diosmetin's A Human Estrogen Receptors Reporter Assay Panel (Indigo effects on functionally defined subsets of primitive human AML Biosciences) was performed according to the manufacturer's pro- and normal hematopoietic cell populations. Treatment of TEX tocol. Briefly, to investigate agonist activity, reporter HeLa cells cells with diosmetin at sublethal doses reduced their ability to were seeded in white 96-well plates and immediately dosed with engraft in the marrow of immune-deficient mice (Fig. 1C; U11 ¼ 1; increasing concentrations of diosmetin. To investigate antagonist P < 0.01). Adding diosmetin into the culture medium had no activity, reporter cells were plated as above, but17-b-estradiol effect on the clonogenic growth of normal hematopoietic cells (E2), a known ERa and ERb agonist, was added at the constant (Fig. 1D; n ¼ 3) but reduced clonogenic growth in a subset of AML submaximal (EC75) concentrations. Following a 24-hour incuba- patient cells (Fig. 1E, n ¼ 8, t11 ¼ 6.5; P < 0.001; Fig. 1F, n ¼ 4; tion period, a luciferase detection reagent was added to the wells Supplementary Tables S1–S2 for patient characteristics). Taken and light emission was quantified using a luminometer. together, diosmetin selectively targets primitive leukemia cells.

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Figure 1. Diosmetin is identified as a novel anti-AML agent. A, A screen of an in-house library identified diosmetin (arrow) as a potent compound capable of reducing TEX cell viability, an AML and surrogate LSC cell line (inset: diosmetin structure). Viability was assessed using the MTS assay. B, Diosmetin's ability to reduce growth and proliferation was tested in a panel of AML cell lines using the MTS assay. Data presented as an average of three independent experiments. C, TEX cells were treated with suboptimal doses of diosmetin (i.e., 10 mmol/L for 24 hours) or a vehicle control, and then live cells were injected via tail vein into NSG mice (n ¼ 6/7 group). After 6 weeks, human AML cells (hCD45þ/CD33þ) in mouse bone marrow were detected by flow cytometry. Data presented as % human cells in individual mouse bone marrow relative to control. D–E, Normal hematopoietic cells (n ¼ 3) or patient-derived AML cells (D: n ¼ 8; E: n ¼ 4) were cultured with diosmetin for 7 to 14 days, and clonogenic growth was assessed by enumerating colonies as described in the Materials and Methods section. Data are presented as % clonogenic growth compared with control SEM, similar to previously described (5). , P < 0.01 and , P < 0.001.

ERs are the predicted molecular target kcal/mol, respectively; Fig. 2C). As a final bioinformatics In primary and validation screens, 5 of the 12 most potent approach, we interrogated publically available AML databases compounds were structurally similar (Fig. 2A). So to investigate (18, 19) to determine whether there is a population of AML the cellular target, a unique structural bioinformatics approach patientswhomayrespondtoanERb-specific small molecule. was used to mine existing protein structural data for known Interestingly, within these databases we identified a subpopu- interactions involving these compounds (Fig. 2A). We first lation of AML patient cells that overexpress ERb while under- screened the PDB for crystallographic or NMR-derived protein expressing ERa, further suggesting a potential clinical utility for structures cobound with diosmetin and these structurally sim- targeting ERb (Supplementary Figs. S2 and S3). ilar compounds. This resulted in an initial set of nine structures, which were subsequently used as templates to find additional Diosmetin is an ERb agonist proteins of similar ligand-binding site composition using the To determine if ERb is diosmetin's molecular target, we first Pocket Similarity Search using Multi-Sketches (PoSSuM) tool. performed ER agonist and antagonist luciferase reporter assays. In This analysis yielded a final list of 92 predicted diosmetin- the agonist assay, ER reporter cells were treated with diosmetin, binding proteins, 52 of which were human. To summarize this and luminescence increased in a dose-dependent manner for ERb list of potential targets, the DAVID tool was used, which reports (Fig. 3A; F5,12 ¼ 162; P < 0.0001) and ERa (Fig. 3B; F5,12 ¼ 245.9; statistically overrepresented functional or protein family cate- P < 0.0001). Given the similarity of the ERa and ERb ligand- gories in gene lists. The analysis predicted nuclear hormone binding domain, dual receptor binding was not surprising. None- receptors, specifically ERs, as a dominant class of diosmetin- theless, diosmetin clearly demonstrated preferential binding binding proteins (Fig. 2B). toward ERb (Fig. 3C; F5,30 ¼ 53.75; P < 0.0001). In the antagonist Molecular docking was next utilized to explore binding inter- assay, reporter cells were coincubated with the ERa and ERb actions of diosmetin in the ligand-binding domain of human ERa ligand, 17b-estradiol (E2), and diosmetin for 24 hours. E2- and ERb receptors (14). The Structure-Based-Design program pre- induced luminescence was not affected demonstrating that dios- dicted diosmetin to bind with greater affinity and stability to metin does not act as an ERa or b antagonist (Fig. 3D and E). ER ERb than ERa (ERa and ERb stability: –22.73 kcal/mol vs. –24.03 agonist activity at 24 hours had no effect on the viability of these kcal/mol, respectively; affinity: –37.94 kcal/mol vs. –39.05 luciferase promoter cells (Supplementary Fig. S4).

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Figure 2. ERb is diosmetin's predicted molecular target. A, A ﬂow chart outlining the structural bioinformatics approach for prediction and analysis of diosmetin- binding proteins from the PDB. B, Initial list of human target proteins identiﬁed in the PDB as cobound with diosmetin-like compounds. C, Molecular docking experiments using the receptor-ligand interactions module (Discovery Studio software) were performed to explore the binding interactions between diosmetin and human ERa and ERb receptors.

Patient-derived AML cell sensitivity is linked to ERb expression control–treated cells (Fig. 5A). In the absence of doxycycline (i.e., Because diosmetin preferentially bound to ERb, we next deter- no ERb), these cells were insensitive to diosmetin (Fig. 5B); mined whether ERb expression predicted the differential response however, when treated with 100 ng/mL doxycycline (i.e., (i.e., diosmetin sensitive or insensitive) observed in the clono- increased ERb expression), diosmetin imparted toxicity (Fig. genic growth assays (Fig. 1D and E). Interestingly, ERb levels were 5B; F1,8 ¼ 24.44; P < 0.01). Because diosmetin is a flavonoid and elevated in diosmetin-sensitive primary cells (Fig. 4A). In contrast, flavonoids generate ROS (20–22), we confirmed that diosmetin's primary cells (normal and patient-derived) that were insensitive activity is characterized by increases in intracellular ROS, as to diosmetin had no-to-low ERb but elevated ERa mRNA levels measured by DCF-DA and flow cytometry (Supplementary Fig. (Fig. 4B), suggesting that ERb/ERa ratios confer patient-derived S6). Consistent with this observation, ROS increased in cells AML cell sensitivity to diosmetin. In addition, AML cell lines treated with doxycycline and diosmetin but not with diosmetin sensitive to diosmetin (Fig. 1B) express ERb protein, as measured alone (Fig. 5C; t4 ¼ 5.254; P < 0.01). by immunoblotting (Supplementary Fig. S5). To further confirm its functional importance, we generated ERb knockdown OCI-AML2 cells using two independent shRNA vec- ERb is functionally important to diosmetin's activity tors (denoted 16 and 17); knockdown was confirmed by immu- To further define the role of ERb in diosmetin-induced cell noblotting (Fig. 5D). ERb knockdown cells are resistant to dios- death, we utilized doxycycline-inducible flag-tagged ERb U2OS metin-induced death compared with scramble controls (Fig. 5E; cells (U2OS-ERb cells; ref. 7). Treatment with 100 ng/mL doxy- F4,18 ¼ 8.604, P < 0.001). Moreover, consistent with our obser- cycline for 24 hours increased ERb 4-fold compared with vehicle vation that ERb activation is characterized by increased ROS,

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A Agonist - ERb BCAgonist - ERa 4 2,000 2,000 3 1,500 1,500 2 1,000 RLU

RLU 1,000 ER b /ER a

500 1 500

0 0 0 0.0 1.5 12.0 E2 0.0 1.5 3.0 6.0 12.0 E2 0.0 1.5 3.0 6.0 12.0 E2 Diosmetin (mmol/L) Diosmetin (mmol/L) Diosmetin (mmol/L)

a b Antagonist - ER 2,500 Antagonist - ER DE2,500

2,000 2,000

1,500 1,500 RLU RLU 1,000 1,000

500 500

0 0 0.0 1.5 3.0 6.0 12.0 0.0 1.5 3.0 6.0 12.0 Diosmetin (mmol/L) Diosmetin (mmol/L)

Figure 3. Diosmetin is an ER agonist. ERb agonist activity was tested by adding increasing concentrations of diosmetin to HeLa cells containing (A)ERb or (B)ERa under the control of a luciferase reporter. C, Comparison of luminescence at individual diosmetin concentrations highlights diosmetin's preferentially ERb binding. D and E, Diosmetin was tested as an ERb antagonist by adding increasing concentrations of diosmetin in the presence of estradiol (E2) to HeLa cells containing ERa or ERb under the control of a luciferase reporter. Data are presented as relative luciferase intensity (RLU) compared with vehicle control–treated cells. Data presented as an average of three independent experiments. , P < 0.05; , P < 0.01; and , P < 0.001.

diosmetin increased ROS in a dose-dependent manner in trans- mice and after 1 week, intraperitoneal injections of diosmetin duced controls but had little effect in the resistant knockdown cells (50 mg/kg/every other day) were administered for 6 weeks. (Fig. 5F; F4,18 ¼ 5.152; P < 0.01). Together, these data demonstrate Treatment with the ERb agonist had no effect on the presence of þ þ that ERb is functionally important to diosmetin's activity. normal human myeloid cells (hCD45 /CD33 ) in mouse bone marrow compared with vehicle control, as measured by ﬂow ERb targeting imparts anti-AML activity in vivo cytometry (Fig. 6A). Given the cytotoxicity of diosmetin in AML cells, we performed Primary engraftment was next assessed where patient-derived additional experiments in vivo in a functionally deﬁned subset of AML cells were injected into the tail vein of NSG mice and, after 1 primitive human AML and normal hematopoietic cell popula- week, intraperitoneal injections of diosmetin (50 mg/kg/every tions. First, normal HSCs were injected into the tail vein of NSG other day) or vehicle control were administered for 6 weeks.

Figure 4. ERb expression confers patient-derived AML cell sensitivity to diosmetin. ERa and ERb gene expressions were measured by qPCR data in primary AML samples sensitive (A; n ¼ 5) and insensitive (B; n ¼ 6) to diosmetin. Data presented as relative mRNA expression normalized to GAPDH. (Normal hematopoietic cells denoted as N; primary insensitive AML samples denoted as IN.)

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Figure 5. ERb is diosmetin's molecular target. A, U2OS-ERb human osteosarcoma cells, with ERb under a doxycycline-inducible promoter, were incubated in the presence or absence of doxycycline (100 ng/mL) for 72 hours. Expression of ERb was measured in the presence of increasing doxycycline concentrations (100–500 ng/mL) using Western blotting. Experiments were performed three times, and representative blots are shown. B, Viability of U2OS-ERb cells was measured in the presence or absence of diosmetin with or without doxycycline (100 ng/mL) for 72 hours using the ANN/PI assay. Data presented as an average of three independent experiments and as percent viable (ANN/PI) relative to zero controls (C) ROS was measured in U2OS-ERb cells treated with diosmetin (24 hours at 10 mmol/L) or a vehicle control in the presence of doxycycline (100 ng/mL), using DCF-DA staining as outlined in the Materials and Methods section. D, ERb (gene: ESR2) gene silencing was achieved by lentiviral-mediated transduction and conﬁrmed by immunoblotting. Densitometry calculated as outlined in the Materials and Methods section. E, Viability of ERb knockdown cells was tested with increasing concentrations of diosmetin by the ANN/PI assay. Data presented as an average of three independent experiments. F, ROS was measured in knockdown cells using DCF-DA staining, as outlined in the Materials and Methods section. Data presented as an average of three independent experiments. , P < 0.05; , P < 0.01; and , P < 0.001.

Human myeloid cells were then measured in mouse bone marrow. flavonoid was capable of inducing selective toxicity in leukemia Treatment with diosmetin significantly reduced LSC primary and LSCs with no effect on normal cells in vitro and in vivo.We engraftment compared with vehicle control–treated mice (Fig. subsequently demonstrated that selective AML toxicity was relat- 6B; U ¼ 21; P < 0.05). In secondary transplant assays, an equal ed to ERb targeting, highlighting this ER subtype as a novel anti- number of viable leukemia cells from control or diosmetin-treated AML target. mice were injected into the tail vein of NSG mice (n ¼ 5/group) and, ER isoforms, ERa and ERb, are encoded by genes at different after 6 weeks, engraftment was assessed in mouse bone marrow. chromosomal locations and perform different cellular roles (23). Human cells isolated from mice treated with the diosmetin had While ERa activation results in cell proliferation, ERb targeting significantly reduced LSC secondary engraftment compared with results in cell senescence or death (24, 25). ERb activation induced vehicle control–treated mice (Fig. 6C; U ¼ 2; P < 0.05). Analysis of death in prostate (25), colon (26), and breast (26, 27) cancer complete blood counts, bilirubin, serological markers (i.e., alkaline tissue, and loss of ERb in mice results in prostate hyperplasia and phosphatase or creatine kinase, which are markers of kidney func- myeloproliferative disease (e.g., increased myeloid cell numbers tion or liver function, respectively), and mouse body weights in bone marrow, blood, spleen, and lymph nodes) resembling showed no difference between control and diosmetin treatment chronic myeloid leukemia (CML; ref. 28, 29). In this study, ERb groups (Fig. 6C–H). Together, this demonstrates the selective activ- targeting with diosmetin-induced selective toxicity that was ity of the ERb agonist diosmetin against primitive leukemia cells. dependent on AML cell ERb expression. Patient-derived AML cells with elevated ERb mRNA were diosmetin sensitive, whereas insensitive cells had low-to-nondetectable ERb levels. Moreover, Discussion ERb knockdown conferred resistance, whereas overexpression, A high-throughput screen identified diosmetin as a novel anti- using doxycycline-inducible cells, resulted in enhanced cell death AML compound, and validation studies determined that this following ERb targeting with diosmetin. Indeed, ERb ligand

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Figure 6. Diosmetin targets leukemia stem cells in vivo. A, Normal CD34þ cells from cord blood were injected via tail vein into NSG mice (n ¼ 5/group). After 1 week, mice were treated intraperitoneally with 50 mg/kg/every other day with diosmetin. After 6 weeks, normal human myeloid cells (hCD45þ/CD33þ) in mouse bone marrow were detected by flow cytometry. B, Patient-derived AML cells were injected via tail vein into NSG mice (n ¼ 10/group). After 1 week, mice were treated intraperitoneally with 50 mg/kg/every other day with diosmetin. After 6 weeks, human AML cells (hCD45þ/CD33þ) in mouse bone marrow were detected by flow cytometry. C, Secondary engraftments were performed, as detailed in the Materials and Methods section, Human AML cells (hCD45þ/CD33þ) in mouse bone marrow were detected by flow cytometry after 6 weeks. For all experiments, data were normalized to vehicle-treated controls and presented as percent engraftment. D–I, Compared with control-treated animals, diosmetin-treated mice had no changes in body weights, white blood cells, red blood cells, bilirubin levels,or serum levels of alkaline phosphatase (marker or kidney function) or creatine kinase (marker of liver damage). , P < 0.05.

activation or transfection of ER-negative cell lines with ERb results characteristic associated with improved patient outcome. In one in reduced proliferation, induction of apoptosis, and/or inhibi- study, ERa was specifically tested; however, no differentiation tion of tumor formation in prostate (30), colon (31), lymphoma was made between ERa and ERb in the other two studies (38–40). (32, 33), and breast (26, 27) cancer models. Thus, ERb targeting ERb methylation (i.e., decreased ERb activity) is observed in colon exerts antiproliferative effects and is demonstrated here, for the or breast tumors (41, 42). It is important to note; however, that first time, as a novel anti-AML/LSC target. while receptor expression is critical, other factors influence the High ratios of ERb/ERa conferred AML cell sensitivity. Inter- cellular response to ER interactions with estrogen hormones or rogation of publically available AML patient genetic databases selective estrogen receptor modulators (SERMs; e.g., tamoxifen, identified an AML patient subpopulation with this phenotype raloxifene; ref. 43). Indeed, tamoxifen is an ER antagonist in confirming the potential prognostic significance of ERb and breast, but a weak ER agonist in bone tissue, whereas raloxifene clinical utility of ERb drug targeting. Similarly, elevated ERb levels is a strong agonist in bone but a weak agonist in breast tissue (44). were observed in CLL patients compared with normal samples Although future studies are needed to further define the role of ERs (34). Low ERb/ERa ratios were observed in breast tumors com- in myeloid cells and leukemia pathogenesis, our study points to pared with matched normal adjacent tissue (35), and the presence the direct antitumorigenic and prognostic potential of ERb in of ERb (ratio, 1.0–1.5) resulted in favorable patient response to AML. chemotherapy (36). Downregulation and degradation of ERb by Although a wide spectrum of ERb agonists are under clinical Pescadillo (PES1) resulted in lower ERb/ERa ratios and breast development (AUS-131, fosfestrol, KB-9520, sulfestrol; refs. 42 cancer progression in mice; elevated ERa and PES1 coupled with and 45), none are FDA approved or have been tested clinically downregulated ERb was also found in breast cancer patients (37). in AML. Diosmetin is widely available and when provided Moreover, ER hypermethylation in AML is a common clinical orally as diosmin at 10 mg/kg (the aglycone prodrug form),

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ERb in AML

diosmetin reached a Cmax of 126 mmol/L within 2 hours with a Authors' Contributions half-life of 26–48 hours (46). In addition, administration of Conception and design: S.-G. Rota, P.A. Spagnuolo 500 mg of daflon, a common product used for chronic venous Development of methodology: S.-G. Rota, A. Roma, D.A. Hess, A.C. Doxey, insufficiency that contains 450 mg of diosmin, results in 0.17– P.A. Spagnuolo Acquisition of data (provided animals, acquired and managed patients, 1.32 mmol/L diosmetin plasma concentrations (47, 48) with provided facilities, etc.): I. Dude, C. Ma, R. Stevens, J. MacEachern, P.N. Rao, minimal reported toxicities (49, 50). Our study provides a clear P.A. Spagnuolo rationale for preclinical testing and/or potential clinical eval- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, uation of ER-based therapies for AML, specifically focusing on computational analysis): S.-G. Rota, A. Roma, I. Dude, C. Ma, S.M.G. Espiritu, potent ERb agonists such as diosmetin. Similar to our anti-AML P.N. Rao, P.A. Spagnuolo in vivo observations, treatment with ERb agonists has shown Writing, review, and/or revision of the manuscript: S.-G. Rota, A. Roma, D.A. Hess, A.C. Doxey, P.A. Spagnuolo antitumor effects in lymphoma (33), prostate (30), and breast Administrative, technical, or material support (i.e., reporting or organizing (51, 52) cancer models. Tamoxifen also enhanced cytarabine's data, constructing databases): S.-G. Rota, A. Roma, C. Ma, M.D. Minden, antitumor activity in AML xenografts (53). However, tamoxifen E. Kreinin, D.A. Hess, P.A. Spagnuolo decreased bone marrow cell numbers and increased myeloid Study supervision: P.A. Spagnuolo progenitor apoptosis in healthy mice (53), which was convinc- ingly shown to be ERa mediated (i.e., effects not shown in Acknowledgments ERb / mice). This emphasizes the need for caution when We are grateful to Drs. John E. Dick and David G. Monroe for their generous b applying SERM therapies, as these agents exert tissue- and gift of TEX cells and doxycycline-inducible ER cells, respectively; Rose Hurren, a b Jean Flanagan, Martin Ryan, and Nancy Gibson for assisting with the engraft- context-dependent activities and both ER and ER are found ment assays; and the practitioners at the Cancer Centres of Grand River and – on hematopoietic progenitors (54 56). Hence, clinical evalu- Princess Margaret for their assistance in procuring primary patient samples. ation, specifically within the hematopoietic compartment, of potent and selective ERb agonistsisrequiredtotranslatethese Grant Support observations into patient benefits.Nonetheless,ourwork This work was supported by grants to P.A. Spagnuolo by the Stem demonstrates a potential clinical benefitofERb agonists in Cell Network, Leukemia and Lymphoma Society of Canada, University of AML therapy, particularly in a subpopulation of patients who Waterloo, Ontario Research Fund, and the Canadian Foundation of Inno- b a vation; to Rao PPN by the Natural Sciences and Engineering Research present with favorably high ER /ER ratios. Council of Canada (NSERC) and the Early Researcher Award; and to In conclusion, ERb expression conferred AML cell sensitivity to A.C. Doxey by NSERC. the ERb agonist, diosmetin, highlighting diosmetin as a novel The costs of publication of this article were defrayed in part by the payment of drug and ERb as a potential novel anti-AML target. page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Disclosure of Potential Conﬂicts of Interest Received April 5, 2017; revised July 11, 2017; accepted August 16, 2017; No potential conﬂicts of interest were disclosed. published OnlineFirst August 23, 2017.

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2626 Mol Cancer Ther; 16(11) November 2017 Molecular Cancer Therapeutics

Estrogen Receptor β Is a Novel Target in Acute Myeloid Leukemia

Sarah-Grace Rota, Alessia Roma, Iulia Dude, et al.

Mol Cancer Ther 2017;16:2618-2626. Published OnlineFirst August 23, 2017.

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