Dual Src and MEK Inhibition Decreases Ovarian Cancer Growth and Targets Tumor Initiating Stem-Like Cells Fiona Simpkins1,2,3, Kibeom Jang1,4, Hyunho Yoon1, Karina E

Published OnlineFirst June 29, 2018; DOI: 10.1158/1078-0432.CCR-17-3697

Cancer Therapy: Preclinical Clinical Cancer Research Dual Src and MEK Inhibition Decreases Ovarian Cancer Growth and Targets Tumor Initiating Stem-Like Cells Fiona Simpkins1,2,3, Kibeom Jang1,4, Hyunho Yoon1, Karina E. Hew1,2, Minsoon Kim1,4, Diana J. Azzam1,4, Jun Sun1, Dekuang Zhao1, Tan A. Ince1,5,6, Wenbin Liu7, Wei Guo7, Zhi Wei8, Gao Zhang9, Gordon B. Mills7, and Joyce M. Slingerland1,4,10

Abstract

Purpose: Rational targeted therapies are needed for treat- selumetinib, respectively, showed target kinase inhibition and ment of ovarian cancers. Signaling kinases Src and MAPK are synergistic induction of apoptosis and cell-cycle arrest in vitro, activated in high-grade serous ovarian cancer (HGSOC). and tumor inhibition in xenografts. Gene expression and Here, we tested the frequency of activation of both kinases proteomic analysis conﬁrmed cell-cycle inhibition and in HGSOC and the therapeutic potential of dual kinase autophagy. Dual therapy also potently inhibited tumor- inhibition. initiating cells. Src and MAPK were both activated in tumor- Experimental Design: MEK and Src activation was assayed initiating populations. Combination treatment followed by þ in primary HGSOC from The Cancer Genome Atlas (TGGA). drug washout decreased sphere formation and ALDH1 cells. Effects of dual kinase inhibition were assayed on cell-cycle, In vivo, tumors dissociated after dual therapy showed a marked apoptosis, gene, and proteomic analysis; cancer stem cells; and decrease in ALDH1 staining, sphere formation, and loss of xenografts. tumor-initiating cells upon serial xenografting. Results: Both Src and MAPK are coactivated in 31% of Conclusions: Selumetinib added to saracatinib over- HGSOC, and this associates with worse overall survival on comes EGFR/HER2/ERBB2–mediated bypass activation of multivariate analysis. Frequent dual kinase activation in MEK/MAPK observed with saracatinib alone and targets HGSOC led us to assay the efﬁcacy of combined Src and MEK tumor-initiating ovarian cancer populations, supporting inhibition. Treatment of established lines and primary ovarian further evaluation of combined Src–MEK inhibition in cancer cultures with Src and MEK inhibitors saracatinib and clinical trials. Clin Cancer Res; 1–13. 2018 AACR.

Introduction 1Braman Family Breast Cancer Institute at Sylvester Comprehensive Cancer Center, University of Miami, Miami, Florida. 2Department of Obstetrics & Ovarian cancer is the most lethal gynecologic cancer (1). Gynecology, University of Miami, Miami, Florida. 3Ovarian Cancer Research Despite introduction of targeted therapies, survival has not sig- Center, Division of Gynecology Oncology, Department of Obstetrics & niﬁcantly improved in the last decade (2). Patients with most Gynecology, University of Pennsylvania, Philadelphia, Pennsylvania. 4Depart- advanced ovarian cancers relapse within two years (1), and ment of Biochemistry and Molecular Biology, University of Miami, Miami, tumors become therapy resistant, underscoring the need for new 5 Florida. Department of Pathology and Laboratory Medicine, University of treatment options. High-grade serous ovarian cancer (HGSOC), Miami, Miami, Florida. 6Interdisciplinary Stem Cell Institute, University of Miami, Miami, Florida. 7Department of Systems Biology, The University of the most common and aggressive subtype, is characterized by Texas MD Anderson Cancer Center, Houston, Texas. 8Department of Com- genomic instability and few targetable genetic mutations (3). We puter Science, New Jersey Institute of Technology, Newark, New Jersey. previously showed that Src (4) and the MAPK (5) are frequently 9Wistar Institute, Philadelphia, Pennsylvania. 10Department of Medicine, activated in HGSOC from The Cancer Genome Atlas (TCGA) by University of Miami, Miami, Florida. reverse-phase proteomic analysis (RPPA), identifying these as Note: Supplementary data for this article are available at Clinical Cancer potential therapeutic targets. Research Online (http://clincancerres.aacrjournals.org/). Saracatinib (AZD0530), a potent Src family kinase inhibitor K. Jang, H. Yoon, and K.E. Hew contributed equally to this article. (6), has preclinical antitumor activity in HGSOC (4). Our prior Corresponding Authors: Joyce M. Slingerland, University of Miami Miller School work showed elevated expression of MAPKpT202pY204 is of Medicine, 1501 Northwest 10th Avenue, BRB 708 (C227), Miami, FL 33136. frequent and is an independent prognostic factor for decreased Phone: 305-243-7265; Fax: 305-243-6170; E-mail: [email protected] survival in HGSOC (5). Selumetinib (AZD6244), a noncom- ; and Fiona Simpkins, Division of Gynecology Oncology, Department of OB-GYN, petitive MEK1/2 inhibitor, has clinical activity in low-grade University of Pennsylvania, 3400 Civic Center Boulevard, South Tower, Suite 10- ovarian cancer (7), suppresses serous and clear cell ovarian 176, Philadelphia, PA 19104. Phone: 215-662-3318; Fax: 215-349-5849; E-mail: cancer xenografts (5, 8), and may prove to have clinical utility [email protected]. in HGSOC (5). doi: 10.1158/1078-0432.CCR-17-3697 Stem-like or tumor-initiating cancer cells are emerging as 2018 American Association for Cancer Research. critical mediators of drug resistance (9). The cancer stem cell

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Translational Relevance Materials and Methods Cell culture Ovarian cancers present late and rapidly develop chemo- PEO1R, an estrogen receptor (ER)-positive antiestrogen-resis- therapy resistance. Targeted therapies have yielded modest tant variant of PEO1 was cultured as described in ref. 4. Short gains, with short responses often due to bypass pathway tandem repeat (STR) profiling verified the unique identity of this activation. Thus, strategies to target multiple pivotal signaling line. PEO1R-SIR is a stable Src inhibitor resistant variant, derived nodes, such as Src and MEK, which are both activated in by 12 weeks of continuous exposure to 1 mmol/L saracatinib. OCI- HGSOC, are attractive. We found 31% of HGSOC have acti- E1P was cultured from an ER-positive, primary endometrioid vation of both of these kinases. To subvert therapy resistance, ovarian cancer, OCI-C5x from a primary clear cell, OCI-P5x from a we tested antitumor efficacy of combined Src and MEK inhi- primary serous, and OCI-U1a from a HGSOC in OCMI medium bition. Not only did dual therapy in vivo synergistically inhibit and have been extensively characterized as described previously tumor growth, but residual tumors were markedly depleted for þ (27). All were used below passage twelve. OCMI medium was ALDH1 , sphere-forming, and tumor-initiating stem cells in obtained from Interdisciplinary Stem Cell Institute Live Tissue vivo. Selumetinib added to saracatinib inhibits the EGFR-1 and Culture Service Center (LTCC). Cell lines were authenticated EGFR-2–mediated bypass MEK/MAPK activation observed using ATCC guidelines. Asynchronous cultures were treated with with saracatinib alone and appears to effectively target self- vehicle, 1 mmol/L saracatinib, 200 nmol/L selumetinib, or both renewing ovarian cancer subpopulations. These findings will for 48 hours or longer as indicated for drug assays. All in vitro cell stimulate further in vitro experimentation and support initia- assays described below included at least three biologic replicates tion of clinical trials testing dual Src and MEK inhibitor therapy and triplicate technical repeats. for patients with ovarian cancer. Drugs Saracatinib (AZD0530) is a potent inhibitor of Src family kinases and Abl (6). Saracatinib and selumetinib (AZD6244) from AstraZeneca were dissolved in DMSO. Saracatinib did not (CSC) hypothesis proposes tumors are heterogeneous and exceed 1 mmol/L in vitro to avoid off-target effects (4). Optimal contain a subpopulation of self-renewing cells that give rise selumetinib concentrations were titrated in vitro and in vivo in fl to progeny with reduced proliferation (9, 10). Ascites uid xenografts (5). Dual therapy concentrations were titrated in a from patients with ovarian cancer contains sphere-forming preliminary in vivo experiment (not shown). For xenografts, drugs cells (11) and tumor-initiating cells that are demonstrable in were suspended in sterile 0.5% hydroxypropyl methyl cellulose xenograft models (12). Various surface markers identify ovar- with 0.1% polysorbate (Tween 80). ian cancer stem cell–enriched populations (13–16). Aldehyde þ dehydrogenase activity (ALDH1 )identifies a population Cell-cycle analysis enriched for tumor-initiating cells in both ovarian cancer lines þ Cells were bromodeoxyuridine (BrdU)-labeled, stained with (16–18) and primary tumors (19). ALDH1 cells are increased anti-BrdU antibodies, and propidium iodide (PI) and cell cycle in populations surviving platinum chemotherapy, suggesting assayed as described previously (28). CSCs survive to repopulate after treatment (20). Although 50%–70% of patients with advanced HGSOC achieve a com- Effects of siRNA-mediated Src and MEK knockdown on cell plete clinical response to initial cytoreductive surgery and cycle chemotherapy, 70% will suffer recurrence and ultimately die Three different antisense oligos to either SRC (sc-29228 for of the disease (1). Thus, treatments that target resistant CSCs siSRC), or MEK1 (sc-29396 for siMEK1) and scrambled controls would be useful. from Santa Cruz Biotechnology were transfected at 70% conflu- Targeted therapies have been limited by lack of drug poten- ence. siRNA was added to Lipofectamine 3000 Reagent (Thermo cy, lack of target expression/activation, and by bypass pathway Fisher Scientific) diluted in Opti-MEM Medium (Thermo Fisher activation (21). Bypass activation of MEK and/or AKT limits Scientific) for 15 minutes and then added to cells for 48 hours mTOR/PI3K inhibitor therapy (22, 23). Src inhibition rapidly prior to analysis. mediates MEK/MAPK activation in preclinical breast cancer models (24, 25). Because bypass pathway emergence limits therapeutic targeting of one signaling node, strategies targeting Detection of autophagic vesicles and Annexin V staining more than one pathway may hold promise (26). Here, we Autophagy was evaluated by Cyto-ID Autophagy Detection Kit identify that both pMAPK and pSrc are elevated in nearly one- (ENZO) to selectively label autophagic vacuoles. Annexin V third of HGSOC. Dual Src and MEK blockade potently inhibits staining and flow cytometry used Apoptosis Detection Kit I (BD proliferation and promotes autophagy/apoptosis in vitro and Biosciences) to quantitate the percentage of apoptosis (4). synergistically decreases ovarian cancer xenograft growth. These effects were confirmed by genomic and proteomic anal- Immunoblotting, immunoprecipitation, and kinase assay þ yses. In addition, ALDH1 subpopulation shows Src and Western blot analysis and densitometry were conducted as MAPK activation and dual kinase inhibition targets these described previously (4, 28). Cells were cultured with/without tumor-initiating subpopulations. Finally, tumors dissociated drugs for 48 hours and the same cells were assayed for cell-cycle þ after dual therapy showed a significant depletion of ALDH1 distribution, Western blot analysis, Cyclin E IP blots, and Cyclin and sphere-forming cells and decreased tumor-initiating cells E–associated kinase activity. Cyclin E was precipitated from 300 mg upon serial xenografting. lysate and associated Cyclin E, CDK2, and p27 detected by

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immunoblotting (28). Cyclin E–Cdk2 precipitates were assayed Analysis of activated Src and MAPK in HGSOC data from TCGA/ for Histone H1 activity as described previously (28). TCPA data The Cancer Genome Atlas/The Cancer Proteome Atlas (TCGA/ Sphere formation and Aldefluor assays TCPA) presents RPPA of 178 proteins and phospho-proteins from For sphere assays, cells were cultured with or without saraca- over 400 untreated new HGSOCs (30) at http://cancergenome. tinib (1 mmol/L) or selumetinib (200 nmol/L) or both for 48 nih.gov/cancersselected/ovarian. Publicly available RPPA data for hours followed by 48-hour drug washout prior to seeding as 405 primary HGSOCs was analyzed for levels of activated Src and single-cell suspensions in limiting dilutions (500, 1,000, and MAPK. Kaplan–Meier curves were prepared to display overall 2,000 cells) on ultra-low attachment plates (Corning) in Mam- survival dichotomized by tumors showing both pT202pY204- mocult media (20). Spheres > 75 mm diameter were counted after MAPK (pMAPK) and pY416Src (pSrc) above the median versus all 14–21 days. Xenograft tumors from each experimental group others in 405 HGSOC tumors. HGSOC survival distributions were dissociated, pooled, and plated in triplicate sphere assays. were compared using the log-rank test. For multivariate analysis, Drug-treated cells were also assayed for ALDH1 activity by high pSrc/high pMAPK was evaluated together with race, age at Aldefluor kit (StemCell Technologies; ref. 20). Aldefluor-positive pathologic diagnosis (as a continuous variable), year of diagnosis, and -negative cells were sorted by flow cytometry (18) for Western no macroscopic residual disease, residual disease 11–20 mm, blot analysis. residual disease > 20 mm residual disease, tumor grade, clinical stage, disease subtype using Cox regression in 338 cancers with Tumor xenografts complete data. Animal experiments were approved by the Institutional Animal Care and Use Committee. Estradiol pellets 0.36 mg/90 days Gene array and RPPA (Innovative Research) were implanted subcutaneously into 5- RPPA and Illumina gene expression analyses were carried out week-old female NOD/SCID mice because they improve tumor- on PEO1R with and without drug treatments for 48 hours in vitro igenicity in vivo (Charles River; ref. 4). PEO1R cells (106 in 100 mL from three biologic repeat samples. The complete method for Matrigel) were injected in the mammary fat pad as described in RPPA is described at https://www.mdanderson.org/research/ ref. 19. The mammary fat pad was chosen because ovarian CSCs research-resources/core-facilities/functional-proteomics-rppa- are most reliably xenografted in the mammary fad pad compared core.html. with other traditional sites (peritoneal cavity and ovarian bursa) Gene expression analysis of these samples followed MIAME in NSG mice and recapitulated the heterogeneity of primary guidelines of the Microarray Gene Expression Data Society as serous ovarian cancer, as assessed by histology, surface immuno- described in ref. 3. The raw data of gene expression microarrays phenotype, and expression of p53, WT1, and CK7 (19). When generated from Illumina Chips were normalized, background- tumors reached 70 mm3, mice were divided into four groups (n ¼ corrected, and summarized using the R package "lumi." Probes 10/group): (i) untreated (E2 only); (ii) selumetinib [oral gavage, 5 below background level (detection P < 0.01) were excluded and mg/kg/day (5)]; (iii) saracatinib [oral gavage, 20 mg/kg/day (4)]; differential expression was identified with Bayes-adjusted vari- (iv) both drugs together. Animals were weighed and tumors ance analysis using the Bioconductor Limma package. To reduce measured using calipers and volumes were calculated twice week- false positives, unexpressed probes were removed. The R package ly using (long-side short-side2)/2. Mice were sacrificed when "limma" was employed for gene differential expression analysis, control tumors reached approximately 1,000 mm3 for tumor followed by multiple test correction by the Benjamini–Hochberg harvesting, dissociation, and serial transplantation. procedure. Genes with adjusted P < 0.05 and fold change > 2 were claimed as significantly differentially expressed. These data are IHC of xenografts available in NCBI GEO with accession number GSE112371. Xenografts were immunostained after antigen retrieval by boil- The same differential expression analysis method was ing in sodium citrate (10 mmol/L, pH 6.0) for 45 minutes, applied to RPPA data. RPPA data processing used SuperCurve followed by primary antibody treatment for Ki67 (Ki-67PAb, (SuperCurve Package. R package version 1.4.1.2011) as described 1:500 Abcam) and ALDH1 (ALDH1 mAb, 1:200, Cell Signaling previously (30, 31). Bar plots for relative RPPA for different Technology), and scored as described previously (5, 29). Four to treatments groups were generated using R package "ggplot2", five high-power fields (60) from at least three tumors/treatment using P < 0.05 to define significant RPPA proteins. Corresponding group were chosen and 80–100 cells/field counted for nuclear "logFC" values of selected proteins were magnified 100X and Ki67 or for ALDH1 staining. The mean score represents % positive applied as y-axis to present the log2-fold change between treat- cells from at least 400 cells counted. Cleaved caspase-3 was ment and control groups. detected by staining with cleaved caspase-3 mAb (1:100, Cell Signaling Technology) and 546 AlexaFluor (1:200, Invitrogen) Statistical analysis of in vitro and in vivo data and direct immunofluorescence as described previously (4). For all analyses comparing more than two conditions, results were analyzed by ANOVA. One-way or two-way ANOVA assessed Ex vivo tumor dissociation, limiting dilution sphere, and tumor difference among means. For 2 2 factorial experiments, inter- initiation assays actions were tested by two-way ANOVA followed by Tukey To quantitate drug effects on stem-like cells, primary tumors honesty significance test, with P 0.05 indicating a significant were dissociated and resuspended for sphere assays or to quan- difference. Effects on cell cycle and viability were analyzed using titate tumor-initiating cells in vivo in a second set of recipient mice. the median-effect method of Chou and Talalay (32). Post hoc For tumor-initiating cell assays, 5,000 or 20,000 cells were analysis by Dunnett multiple comparisons test was used to injected in 100 mL Matrigel into the mammary fad pad of estro- calculate differences between individual groups after the ANOVA, gen-supplemented NOD-SCID mice (n ¼ 6/group). for example, between dual therapy and control. Student t test was

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used for comparisons of two conditions only. Tumor-initiating stem cell frequency was calculated using the LCalc software http:// www.stemcell.com/tutorials/lcsetup.exe (StemCell Technologies). To test for drug synergy in in vitro assays, the coefficient of drug interaction (CDI) was calculated as CDI ¼ (2 drug combination divided by the control)/(drug 1 divided by the control)(drug 2 divided by the control). CDI < 1 indicates synergistic drug interaction; CDI ¼ 1 indicates additivity; and CDI > 1 indicates antagonism. This method was also used to evaluate differences between final tumor volumes between groups. In vivo tumor growth curves were compared using an ANOVA for multiple comparisons tests with GraphPad PRISM (GraphPad Software) to determine differences in the growth curve slopes of dual-treated tumors compared with other groups. The combination ratio method was used to analyze for drug synergy on xenograft growth (24, 33). Fractional tumor volume (FTV) was defined as mean-final tumor volume of drug treated animals divided by mean-final tumor volume of untreated controls (only estradiol). The combination ratio compared the FTV expected if there were no synergy, with the observed FTV. The combination ratio was calculated as (FTV of saracatinib FTV of selumetinib)/observed FTV of combination. Observed and Figure 1. expected FTV are described as: Effects of pMAPK and pSrc on HGSOC survival. A, Kaplan–Meier curve Expected FTV ¼ (mean FTV of saracatinib) (mean FTV of shows worse HGSOC survival with both intratumor pMAPK and pSrc above selumetinib) median on RPPA (n ¼ 405; logrank P ¼ 0.004). B, Multivariate analysis shows Observed FTV ¼ final tumor volume combined therapy/final elevation of both pMAPK and pSrc above median, greater age, lack of tumor volume estradiol alone macroscopic disease, and any residual disease are independent prognostic factors. Combination ratio ¼ Expected FTV/Observed FTV. A combination ratio > 1 indicates synergy; a ratio < 1 indicates a less than additive effect. modest effects of each monotherapy (mean % S phase ¼ 24.6% for SI, and 20.65% for MI vs. control of 45.9%), both drugs together caused synergistic cell-cycle inhibition (mean % S- Results phase ¼ 4.25%). A coefficient of drug interaction or CDI < 1 High expression of both pMAPK and pSrc is associated with indicates synergy: CDI ¼ 0.38 for decrease in mean percent decreased HGSOC survival S-phase cells, %S; P ¼ 0.0224, for dual therapy versus TCGA shows 18% of HGSOCs have activating Ras/Raf/MEK/ control Fig. 2A. Drug effects were verified by siRNA-mediated MAPK pathway mutations (5). Src activates the Ras/Raf/MEK/ kinase knockdown. While loss of each kinase alone caused partial MAPK pathway. Because ovarian cancers frequently express G1 arrest, dual knockdown of Src and MEK together synergistically activated pSrc (4) and pMAPK (5), we determined the extent decreased the %S compared with each siRNA alone (CDI ¼ 0.77, P to which both kinases are activated together in the RPPA dataset < 0.0001 for dual knockdown vs. control, Supplementary Fig. S1A of 405 HGSOCs in TCGA. pSrc (pY416) was detected in 74% and S1B). After 48 hours, saracatinib decreased pY416-Src (pSrc). (n ¼ 300/405) and pMAPK (pT202Y204) was detected in 76% Although selumetinib decreased pT202pY204-MAPK (pMAPK), (n ¼ 310/405) of these tumors, respectively. In nearly one-third it increased pSrc (Fig. 2B). In addition, MEK inhibitor alone (31% or 126/405) of HGSOCs, both pMAPK and pSrc levels increased pS473-AKT (pAKT), but saracatinib alone and dual were above the median, and these patients had a shorter inhibitor treatment both decreased pAKT (see also densitometry; survival (median 40.4 months) compared with all others Supplementary Fig. S1C). The increased pMEK observed after (median survival ¼ 48.9 months; n ¼ 405; P ¼ 0.004, Fig. selumetinib reflects drug action to lock MEK1/2 into a confor- 1A). Multivariate analysis showed elevation of both pMAPK mation that enables substrate/ATP binding but disrupts catalysis, and pSrc independently predicts poor overall survival (HR ¼ yielding an inactive conformation that reacts with anti-pMEK 1.43; 95% CI, 1.05-1.95; P ¼ 0.024; Fig. 1B). antibody (34). To determine whether p27 was mediating cell-cycle arrest, Src and MEK inhibitors synergistically induce cell-cycle arrest in cyclin E-CDK2–bound p27 and associated kinases activities were ovarian cancer cell lines and primary cultures assayed. While Cyclin E and CDK2 levels were unchanged, the Because Src and MAPK are frequently both activated in HGSOC, levels of CDK inhibitor, p27 (Fig. 2C), and Cyclin E-bound p27 we tested the therapeutic potential of inhibition of both kinases. detected by immunoprecipitation/Western blot analyis (Fig. 2D) The HGSOC line, PEO1R, was treated with saracatinib (Src both increased modestly with each drug, and increased most inhibitor; SI, 1 mmol/L), selumetinib (MEK inhibitor; MI, 200 significantly with combination therapy. Densitometric quantita- nmol/L), or both for 48 hours, followed by cell-cycle analysis (Fig. tion of mean p27 levels and of p27 bound to cyclin E are graphed 2). Saracatinib causes partial G1 arrest at 1 mmol/L with little effect on the right for data in Fig. 2C and D. Very little p21 is expressed in at lower doses (4). Similarly, selumetinib caused only partial G1 these cells (not shown). Cyclin E precipitates were tested for arrest, with no greater effect above 200 nmol/L (5). Despite kinase activity in the same lysates recovered for Fig. 2B and C.

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Figure 2. Effects of dual Src and MEK inhibition on signaling and cell cycle. A–E, PEO1R controls (C) were treated with saracatinib (1 mmol/L, Src inhibitor, SI), selumetinib (200 nmol/L, MEK inhibitor, MI), orboth for 48 hours. The same lysateswere used in B–E. Graphs show mean SEM; signiﬁcant differencesbetween multiple comparisons versus control were calculated by ANOVA with post hoc comparisons between treated groups and control. A, Cell-cycle distribution; z, P ¼ 0.01 for both drugs versus control, CDI ¼ 0.38 (See also Supplementary Fig. S1). B and C, Western blot analysis shows indicated proteins (densitometry in Supplementary Fig. S1C). p27 densitometry in C, right, , P ¼ 0.0008; , P ¼ 0.0004; z, P ¼ 0.00002. D, Cyclin E IP/Western blotted for Cyclin E, p27, and Cdk2. Cyclin E-bound p27 densitometry: , P ¼ 0.0005; , P ¼ 0.0007; z, P ¼ 0.00001. E, Cyclin E-Cdk2 activity graphed as % max; , P ¼ 0.03; , P ¼ 0.006; z, P ¼ 0.000004; CDI ¼ 0.77. F–I, For OCI-C5x and OCI-P5x, controls (C) were treated as in A. F and H, Cell-cycle distribution. Differences in %S for OCI-C5x: , P ¼ 0.01; , P ¼ 0.03; z, P ¼ 0.0002; CDI ¼ 0.60; for OCI-P5x: , P ¼ 0.02; , P ¼ 0.1; z, P ¼ 0.0067; CDI ¼ 0.52. Western blots for OCI-C5x (G) and OCI-P5x (I). Densitometry in Supplementary Fig. S2.

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Cyclin E-CDK2 activity was most strongly inhibited by both drugs Dual Src and MEK inhibition induces autophagy and apoptosis (P < 0.000004 for dual therapy compared with control, CDI ¼ in ovarian cancer cells 0.77; Fig. 2E). As little as a 2–3-fold rise in Cyclin E–bound p27 PEO1R, OCI-C5x, and OCI-P5x cells were treated with saraca- can fully inhibit cyclin E-Cdk2. Thus, p27 is a mediator of drug tinib (1 mmol/L), selumetinib (200 nmol/L) or both, or with response in vitro. paclitaxel as positive control for 48 hours and evaluated for Most available ovarian cancer lines are heavily passaged, apoptosis. Cleaved PARP-1 levels showed little or no change derived from metastatic tumors, and do not always reflect the with each monotherapy, but rose significantly with dual Src and biologic phenotype of the primary cancer (27). Drug responses MEK inhibition in all models (Fig. 3A–C, densitometry; Supple- were thus validated in early passage, primary ovarian cancer mentary Fig. S4A). Similarly, Annexin V-positive cells increased by cultures (27). In the endometrioid primary culture, OCI-E1P, 2.5- to 4-fold with dual therapy in all models, with more modest saracatinib (1 mmol/L), or selumetinib (200 nmol/L) for 48 hours effects with each monotherapy (Fig. 3D–F). each modestly decreased the %S (P ¼ 0.0002 for saracatinib and In autophagic cells, microtubule-associated protein light chain P ¼ 0.005 for selumetinib), while dual therapy synergistically 3-I (LC3-I) is cleaved to LC3-II (35). LC3-II was variably increased decreased %S (P < 0.00001, CDI ¼ 0.86; Supplementary Fig. with either kinase inhibitor but increased most significantly with S1D). Dual Src and MEK inhibitor action was also evaluated in dual kinase inhibition (Fig. 3G–I, densitometry; Supplementary primary cultures from an ER-negative clear cell ovarian cancer Fig. S4B). To further evaluate autophagy, drug-treated cells were (OCI-C5x) and from a HGSOC (OCI-P5x) using saracatinib (1 labeled with Cyto-ID-Green autophagy dye, assayed by flow mmol/L), selumetinib (200 nmol/L), or both for 48 hours. Dual cytometry (36). Autophagic cells increased most significantly therapy caused synergistic cell-cycle inhibition in OCI-C5x, with with combination treatment (PEO1R P < 0.00006; OCI-C5x CDI ¼ 0.6 for mean %S (P ¼ 0.0002; Fig. 2F) and in OCI-P5x with P < 0.0052; OCI-P5x P < 0.0029 for dual therapy vs. CDI ¼ 0.52 (P ¼ 0.0067; Fig. 2H). pSrc and pMAPK were control; Fig. 3J–L). Autophagy is initially a survival mechanism, decreased by their respective inhibitors, confirming drug targeting but leads to cell death under prolonged stress. Combination (Fig. 2G and I). p27 increased most with dual therapy in OCI-C5x treatment appears to activate autophagy, ultimately leading to and OCI-P5x (Fig. 2G and I; Supplementary Fig. S2A and S2B). apoptosis. Thus, for the both the established PEO1R HGSOC line and in primary cultures of HGSOC, endometrioid, and clear cell cancer GSA and RPPA confirm dual therapy inhibits cell cycle and origin, dual therapy caused greater cell-cycle inhibition than upregulates autophagy either monotherapy. Gene expression analysis and proteomic analysis by RPPA evaluated drug effects over 48 hours in PEO1R. Gene set enrich- Emergence of saracatinib resistance is associated with EGFR ment analysis (GSEA) showed the top 10 genes from the top gene and HER2/ERBB2 activation and is overcome by combined Src sets (cell cycle, DNA replication, and Aurora kinase) were all and MEK inhibition decreased by dual therapy compared with untreated controls. Despite promising preclinical antitumor activity, resistance GSEA plots show effects on cell cycle, DNA replication, Aurora, to saracatinib monotherapy emerges rapidly in vitro and in vivo and autophagy genes of combination therapy versus control, with (4, 24, 25). To evaluate potential signaling pathway activation normalized enrichment scores (NES) shown (Fig. 4A; Supple- with emergence of acquired saracatinib resistance, PEO1R were mentary Fig. S5). Autophagy was the top upregulated gene set, cultured in the continuous presence of saracatinib (drug confirming observations in Fig. 3. renewed every 3 days) and cell lysates were recovered weekly RPPA showed the 20 proteins most decreased by dual treatment (at weeks 0, 1, 2, 3, and 4). Lysates were then evaluated for compared with monotherapy all promote cell cycle/proliferation phosphorylated and total Src and MAPK. Within 2 weeks, (cyclin B1, FOXM1, NDRG1, CDK1, pRb, CHK1, YB1 TTF1, saracatinib no longer decreased pSrc, and pMAPK increased MDM2) or MAPK/and PI3K signaling (pS6, p70S6K, MAPK, by week 3 (Supplementary Fig. S3A). Notably, treatment of Src 4EBp1, PRAS40; Fig. 4B). The top 20 upregulated proteins includ- inhibitor-resistant PEO1R cells (PEO1R-SIR), with saracatinib ed apoptosis mediators (caspase 7, Bim, FOX03A), cell-cycle (1 mmol/L) together with selumetinib (200 nmol/L) synergis- inhibitors (p27, p21), and TSC-1, which opposes the PI3K/mTOR tically inhibited cell cycle progression within 48 hours (P < pathway (Fig. 4B and C). The similarity between triplicate samples 0.00001 for % S-phase cells after dual therapy compared with in each group is displayed in Fig. 4C. control, CDI ¼ 0.52), with little effect after each monotherapy þ (Supplementary Fig. S3B). Src and MEK inhibitors target ALDH1 and sphere-forming Signaling kinase activation was compared with/without drug cells þ treatment by RPPA in PEO1R and PEO1R-SIR. RPPA showed ALDH1 and ALDH1 cells were sorted by flow cytometry. In þ pY1068-EGFR was 1.65-fold higher (P ¼ 0.009) and pY1248- PEO1R, ALDH1 cells had more sphere-forming and colony- þ HER2/ERBB2 was 1.3-fold higher (P ¼ 0.05) in PEO1R-SIR than forming cells than ALDH1 cells (Fig. 5A and B). ALDH1 cells parental PEO1R while total kinase levels were similar. Western expressed higher pMAPK and pSrc than ALDH1 cells in PEO1R blot analysis confirmed EGFR, HER2/ERBB2, and showed Raf and in two primary cultures (Fig. 5C–E; densitometry in Supple- activation in PEO1R-SIR compared with PEO1R, while total mentary Fig. S6A–S6C). kinase levels were unchanged. Selumetinib rapidly inhibited Each drug alone decreased ALDH1 activity moderately, but þ MAPK, despite persistent activation of these upstream kinases both together synergistically decreased the % ALDH1 cells in all (Supplementary Fig. S3C and S3D). Thus, prolonged saracatinib models tested (P ¼ 0.0048 for dual therapy vs. control for PEO1R, exposure mediates bypass activation of EGFR, HER2/ERBB2, and CDI ¼ 0.96; P ¼ 0.000001 for OCI-C5x, CDI ¼ 0.52; P ¼ 0.00002 Raf/MEK. Addition of selumetinib circumvents the MAPK acti- for OCI-P5x, CDI ¼ 0.57; Fig. 5F–H; and P ¼ 0.0008 for OCI-U1a, vation that arises after prolonged saracatinib exposure. CDI ¼ 0.98; Supplementary Fig. S6D).

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Figure 3. Effects of Src and MEK inhibition on apoptosis and autophagy in OVCA cells. A–F, Untreated controls (C) of the indicated lines were treated with paclitaxel (100 nmol/L), saracatinib (1 mmol/L, SI), selumetinib (200 nmol/L, MI), or both SI and MI (both) for 48 hours. Graphs show mean SEM; signiﬁcant differences between multiple comparisons versus control were calculated by ANOVA with post hoc comparisons between treated groups and control. A–C, Western analysis shows PARP and cleaved PARP drug. See Supplementary Fig. S4A for densitometry. D–F, Quantitation of apoptotic cells by ﬂow cytometry for Annexin V. For PEO1R: , P ¼ 0.00008; , P ¼ 0.003; z, P ¼ 0.002; #, P ¼ 0.00002; for OCI-C5x: , P ¼ 0.0009; , P ¼ 0.0006; #, P ¼ 0.00001; for OCI-P5x: , P ¼ 0.00055; , P ¼ 0.023; #, P ¼ 0.0026. G–I, Western blot analysis for LC3B-1 and LC3B-II after treatment with either SI, MI, or both. For densitometry, see Supplementary Fig. S4B. J–L, Autophagic lysosomes detected as in Materials and Methods. Differences for PEO1R: , P ¼ 0.002; , P ¼ 0.0003; z, P ¼ 0.00006; for OCI-C5x: , P ¼ 0.016; z, P ¼ 0.0029; for OCI-P5x: z, P ¼ 0.0052.

Drug effects on sphere formation were evaluated. After Combination treatment decreases tumor growth and targets drug treatment for 48 hours, followed by a 48-hour drug tumor-initiating stem cells in vivo more effectively than either washout, all groups had similar cell-cycle profiles, confirming monotherapy similar proliferation in the bulk population prior to sphere Drug effects were next investigated in vivo. Saracatinib and seeding (Supplementary Fig. S6E–S6H). Combination treat- selumetinib each alone modestly decreased PEO1R xenograft ment decreased sphere-forming cell abundance more than tumor growth, but both together not only suppressed tumor either drug alone in PEO1R, OCI-C5x, OCI-P5x, and OCI- growth, but caused a modest tumor regression. Growth curve þ U1a (Fig. 5I–K; Supplementary Fig. S6I). Thus, ALDH1 popu- analysis revealed significant differences in the slope of dual treated lations in both PEO1R and in primary cultures derived from tumor versus all other groups (control vs. both; P ¼ 0.0001, SI vs. aggressive high-grade papillary serous and clear cell ovarian both; P ¼ 0.0142, MI vs. both; P ¼ 0.0027). Tests for synergy using cancers are Src and MAPK driven, and dual therapy with the combination ratio (24, 33) showed a combination ratio > 1.4 þ saracatinib and selumetinib targets the ALDH1 and sphere- at all time points after week one, indicating a synergistic drug forming populations. effect (Fig. 6A). Between week 1.5 and the end of the experiment,

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Figure 4. Gene expression and RPPA analysis of control and drug-treated PEO1R cells. A–C PEO1R was treated with saracatinib (SI, 1 mmol/L), selumetinib (MI, 200 nmol/L), or both for 48 hours, then gene and protein expression were evaluated by Illumina microarray and RPPA. A, GSEA plots of cell cycle, DNA replication, Aurora, and autophagy after combination therapy versus control with normalized enrichment score (NES). See also Supplementary Fig. S5. B, Clustering analysis of normalized RPPA data shows the top 20 proteins down- or upregulated by treatment. LogFC

represents the log2 fold change between treatment and controls. C, Heatmap representation of selected triplicate repeat RPPA data from controls or drug-treated cells shows inter-sample variability.

mean tumor volume increased by nearly 5-fold in untreated apoptosis could fully account for the marked decrease in xenograft controls, and by 2.05 and 2.8 fold respectively in saracatinib- growth following dual therapy. Attenuation of the stem cell and selumetinib-treated tumors, while the mean volume of dual population was notable in surviving tumor cells. Notably the þ treated tumors showed a modest decrease (P ¼ 0.00006 for dual ALDH1 -stained stem-like cells (29) decreased significantly with therapy vs. control, with a CDI ¼ 0.42 indicating drug synergy for each drug alone, and most notably with dual therapy (Fig. 6E; final tumor volume change; Fig. 6B). Supplementary Fig. S7). In vivo, while tumor cell proliferation decreased up to 40% To further evaluate drug effects on ovarian cancer stem-like cells of dual treated tumor cells remained Ki67 positive (Fig. 6C). in vivo, treated xenografts and control tumors were excised, dis- Apoptosis, detected as cleaved caspase on immunofluorescence sociated, and cells were either seeded for sphere formation or (IF), increased modestly from 6% in untreated controls to 16% in transplanted in limiting dilutions into a second series of recipient dual drug-treated tumors (Fig. 6D). Tumor necrosis was similar in mice to assay tumor-initiating stem cell abundance. Sphere for- all tumors. Thus, in vivo, neither the antiproliferative effect, nor mation was most significantly decreased in cells surviving after

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Figure 5. Stem cell effects of Src and MEK inhibition. A and B, Sphere (, P ¼ 0.0005; A) and soft agar colony formation (, P ¼ 0.0029; B) in ALDH1þ and ALDH1 PEO1R cells. Differences by Student t test. C–E, Western blot analysis of ALDH1þ and ALDH1 populations. Densitometry in Supplementary Fig. S6A–S6C. F–H, Mean % ALDH1þ cells SEM in indicated lines after 48 hours of saracatinib (1 mmol/L, SI), selumetinib (200 nmol/L, MI), or both. For PEO1R: , P ¼ 0.045; , P ¼ 0.106; z, P ¼ 0.0048 (F). For OCI-C5x: , P ¼ 0.0002; , P ¼ 0.0003; z, P ¼ 0.000001 (G). For OCI-P5x: , P ¼ 0.003; , P ¼ 0.0029; z, P ¼ 0.00002 (H). See also Supplementary Fig. S6D for OCI-U1a. I–K, PEO1R, OCI-C5x, and OCI-P5x were treated with SI, MI, or both or mock treated for 48 hours, followed by 48- hour drug washout and then 2,000, 1,000, or 500 cells seeded in biologic triplicate sphere assays. Graphs show mean SEM. For PEO1R 2,000 cells: , P ¼ 0.0006; , P ¼ 0.0001; z, P ¼ 0.00002; 1,000 cells: , P ¼ 0.01; , P ¼ 0.00005; z, P ¼ 0.00002; 500 cells: , P ¼ 0.3; , P ¼ 0.003; z, P ¼ 0.0005 (I). For OCI-C5 2,000 cells: , P ¼ 0.0004; , P ¼ 0.0008; z, P ¼ 0.00005; 1,000 cells: , P ¼ 0.0009; , P ¼ 0.0003; z, P ¼ 0.00007; 500 cells: , P ¼ 0.0006; , P ¼ 0.001; z, P ¼ 0.0003 (J). For OCI-P5 2,000 cells: , P ¼ 0.0003; , P ¼ 0.0006; z, P ¼ 0.00008; 1,000 cells: , P ¼ 0.007; , P ¼ 0.008; z, P ¼ 0.002; 500 cells: , P ¼ 0.009; , P ¼ 0.001; z, P ¼ 0.00043 (K). For OCI-U1a sphere data, see Supplementary Fig. S6E. See also Supplementary Fig. S6F–S6I for cell- cycle proﬁles at the time of seeding.

þ combination treatment (P ¼ 0.0002 for dual therapy vs. controls, sing ALDH1 , and decreased abundance of both sphere-forming CDI ¼ 0.71; Fig. 6F). Tumor-initiating cells were assayed by cells and tumor-initiating stem cells in vivo on serial assays. injection of 5,000 or 20,000 dissociated tumor cells/group into each of 6 recipient mice. Tumors formed from dissociated, dual therapy–treated xenograft cells showed little growth compared Discussion with those arising from the same number of monotherapy-treated Recent targeted monotherapies directed against various signal- or control tumor cells (Fig. 6G). Tumor cell populations persisting ing kinases have shown activity in HGSOC, but limited response after combination therapy in vivo contained fewer tumor-initiat- duration (37). Because targeting single pathways is often limited ing cells and formed tumors with greater latency compared with by bypass pathway activation (38), targeting multiple signaling monotherapy (shown for 5,000 cell group, Fig. 6H). Tumor- nodes might yield greater, and more durable responses. Most initiating stem cell (TISC) frequency decreased from 1/3,053 solid tumors appear to be initiated by a stem cell–like subpop- untreated tumor cells to 1/15,898 cells surviving dual therapy ulation that resists therapy and mediates recurrence (29, 39). In an (P ¼ 0.018, Fig. 6I). Thus, dual MEK and Src inhibitor treated effort to subvert drug resistance, we tested whether combined use primary tumors showed a signiﬁcant attenuation of cells expres- of two rationally targeted therapies might have greater antitumor

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Figure 6. Synergistic effects of saracatinib and selumetinib on tumor volumes and tumor initiating cells in vivo. A, Mean volume SEM is graphed for PEO1R xenograft controls (C) and groups treated with saracatinib (SI), selumetinib (MI), and both. B, Mean fold tumor volume change SEM between week 1.5 and harvest. , P ¼ 0.0006; , P ¼ 0.007; z, P ¼ 0.00006; for indicated groups versus control; CDI ¼ 0.42. Graphs show mean SEM; signiﬁcant differences between multiple comparisons versus control were calculated by ANOVA with post hoc comparisons between treated groups and control. C and D, Tumors from A above were assayed by IHC or IF and mean % positive cells graphed as mean SEM; signiﬁcant differences between multiple comparisons versus control were calculated by ANOVA with post hoc comparisons between treated groups and control. IHC Ki67 , P ¼ 0.99; , P ¼ 0.05; z, P < 0.0001 (C), Caspase 3 IF , P ¼ 0.99; , P ¼ 0.54; z, P ¼ 0.004 (D); and ALDH1 IHC , P ¼ 0.32; , P ¼ 0.01; z, P < 0.0001 (E; photomicrographs in Supplementary Fig. S7). F, PEO1R xenografts from A above were dissociated and 5,000 pooled tumor cells were seeded in triplicated sphere assays. , P ¼ 0.004; , P ¼ 0.1734; z, P ¼ 0.0002; CDI ¼ 0.71. G–I, Dissociated tumor cells from A above were pooled for each group and implanted into NOD/SCID mice in a limiting dilution tumor-initiating assay as described in Materials and Methods. Mean tumor volume/time is graphed in G. Mean % tumor-free mice/time is graphed for the 5,000 cell groups in H; tumor-initiating stem cell (TISC) frequency from limiting dilution cell injections, P ¼ 0.018 for dual therapy versus control shown in I.

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efficacy, and potentially target the ovarian cancer–initiating vitro and synergistically reduced lung xenograft growth (47). population. While Src is frequently activated in HGSOC, saracatinib is a TCGA analyses show frequent genetic aberrations that activate pan-Src family inhibitor, and thus effects on other Src family the Ras/Raf/MEK/MAPK pathway in HGSOCs (3). In addition, kinases could contribute to results observed. proteomic RPPA has shown that HGSOCs with high pMAPK have There is strong evidence that a population with ovarian CSC- a worse overall survival compared with tumors without MAPK like properties in vitro and in vivo is selected for by chemotherapy activation (5). MEK inhibitors, such as selumetinib, have activity with platinum agents (17, 49). Ovarian cancer stem-like cells in low-grade serous ovarian cancers but have not been fully express surface markers CD133, CD44, CD24, CD117/cKit, and þ explored in HGSOC (7). MEK/MAPK pathway gene alterations demonstrate ALDH1 activity (50–53). In addition, CD133 cells correlate with MEK inhibitor drug response in low-grade serous with ALDH1 activity show higher sphere and tumor-initiating cell þ cancers (40). abundance (17). Chemotherapy enriches for ALDH1 surviving We previously showed that most HGSOCs in the TCGA/TCPA cells (29, 49) and cancers recurring after chemotherapy showed þ þ þ dataset express activated pSrc (4), a major regulator of prolifer- increased ALDH1 , CD44 , and CD133 cells compared with ation and metastasis (41). Despite the frequent activation of Src, matched primaries (49). Preclinical strategies to target ovarian the Src family kinase inhibitor, dasatinib has shown limited tumor-initiating cells include targeting CSC marker–positive cells activity as a single agent for recurrent HGSOC, with only 21% with siRNA (29, 54), antibodies, or inhibitors to CD44 or CD117/ of patients progression-free after 6 months (42). Another Src cKit (52, 55). Other studies have utilized CSC-targeted dendritic family kinase inhibitor, saracatinib, evaluated in combination cell vaccination (50–53), and epigenetic drugs to target CSCs with chemotherapy for recurrent platinum-resistant ovarian can- (56). Several reports also predict successful CSC targeting by cer, showed no added clinical benefit (43). Interestingly, saraca- Notch (57) and Wnt (55) inhibitors. tinib monotherapy rapidly gives rise to MEK/MAPK bypass path- Here, we show potential for dual Src and MEK inhibition to way activation, and drug resistance in breast cancer xenografts (24, therapeutically target ovarian cancer initiating cells. Src is a key þ 25). Because Src (4) and MAPK (5) are both activated in HGSOC, mediator of malignant stem cell self-renewal (58). The ALDH1 we tested the potential therapeutic efficacy of combined Src/MEK populations showed higher pSrc and pMAPK than ALDH1 cells. blockade Moreover, saracatinib and selumetinib synergistically reduced þ Here we show that saracatinib and selumetinib synergistically ALDH1 cells and sphere formation in vitro in both PEO1R and induced cell cycle arrest in vitro in HGSOC, and in high-grade in primary cultures. After prolonged MEK and Src inhibitor endometrioid and clear cell ovarian carcinoma primary cultures. exposure in vivo, apoptosis and proliferation were only modestly þ Autophagy is initially a survival mechanism, but under prolonged changed. However dual therapy decreased the ALDH1 popula- stress, leads to cell death (44). Dual therapy upregulated both tion, and dramatically reduced sphere-forming and tumor-initi- autophagy and apoptosis in all the models. Combination therapy ating cells in xenograft tumors dissociated after in vivo treatment, may initially activate autophagy but ultimately lead to apoptosis. suggesting Src and MEK inhibition target the tumor-initiating GSEA confirmed these findings. The top 20 gene sets downregu- population. While ovarian cancers comprise multiple histologic lated by dual versus monotherapies govern cell-cycle progression subtypes, characterized by different molecular profiles that war- and DNA replication, while autophagy drivers were upregulated. rant different treatment strategies (59), current studies have GSEA changes were validated further by RPPA. potential application not only to serous, but also to other ovarian Resistance to saracatinib monotherapy emerged rapidly in cancer subtypes. Although in vivo studies were limited to the PEO1 PEO1R, with loss of Src inhibition and activation of putative cell line, in vitro data in primary HGSOC and clear cell ovarian bypass mechanisms via EGFR, HER2/ERBB2, Raf, and MAPK over cancer cultures predict potential generalizability to other ovarian four weeks in culture. Treatment of saracatinib-resistant cells with cancer subtypes. selumetinib potently inhibited MAPK despite upregulation of Characterization of pathways required for CSC survival and upstream MEK activators by saracatinib. Saracatinib resistance self-renewal may yield novel more effective strategies to overcome also developed within weeks in vivo, in PEO1R xenografts, as in drug resistance. Treatment with saracatinib and selumetinib other models (24, 25). Addition of selumetinib to saracatinib together modestly decreases proliferation and induces cell death, overcame resistance to each monotherapy, yielding synergistic but most critically appears to target CSCs. Both orally available antitumor activity in vivo. Thus, dual treatment might circumvent drugs were well-tolerated in phase I/II studies. Application of acquisition of saracatinib resistance. While the MEK inhibitor, saracatinib/selumetinib for ovarian cancer treatment will require selumetinib, rapidly activated AKT, saracatinib inhibited this phase I trials of dual therapy to ensure tolerability, bioavailability, kinase and prevented selumetinib-mediated AKT activation. and to develop clinical biomarkers of target inhibition. Current Thus, selumetinib-induced bypass AKT activation (38, 45) and data support further clinical evaluation of dual MEK and Src saracatinib-induced bypass EGFR/MEK activation are both sub- inhibitor combinations. verted by dual Src and MEK blockade in our ovarian cancer models. Disclosure of Potential Conflicts of Interest Recent preclinical data also support combined use of Src and G.B. Mills has ownership interests (including patents) at Catena Pharma- MEK inhibitors in other malignancies such as breast (24, 46) and ceuticals, ImmunoMet, Myriad Genetics, PTV Ventures, and Spindletop Ven- lung cancer (47), and melanoma (48). Dual MEK and Src inhi- tures; reports receiving speakers bureau honoraria from Allostery, AstraZeneca, fi bition induced apoptosis and decreased breast cancer metastasis ISIS Pharmaceuticals, Lilly, MedImmune, Novartis, P zer, Symphogen, and Tarveda; is a consultant/advisory board member for Adventist Health, Astra- (46). In melanoma, although MEK inhibition decreased cell Zeneca, Allostery, Catena Pharmaceuticals, Critical Outcome Technologies, ISIS proliferation, it increased invasion, and dual Src/MEK blockade Pharmaceuticals, ImmunoMet, MedImmune, Lilly, Novartis, Precision Medi- impaired this (48). Combined treatment with saracatinib and the cine, Provista Diagnostics, Signalchem Lifesciences, Symphogen, Takeda/Mil- MEK inhibitor, PD0325901, reversed EMT, decreased invasion in lenium Pharmaceuticals, Tarveda, and Tau Therapeutics; and reports receiving

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commercial research grants from Adelson Medical Research Foundation, Astra- Writing, review, and/or revision of the manuscript: F. Simpkins, K. Jang, Zeneca, Breast Cancer Research Foundation, Critical Outcome Technologies, H. Yoon, K.E. Hew, Z. Wei, G. Zhang, G.B. Mills, J.M. Slingerland Illumina, Karus, Komen Research Foundation, NanoString, Pﬁzer, and Takeda/ Administrative, technical, or material support (i.e., reporting or organizing Millenium Pharmaceuticals. No potential conﬂicts of interest were disclosed by data, constructing databases): F. Simpkins, J.M. Slingerland the other authors. Study supervision: F. Simpkins, J.M. Slingerland

Authors' Contributions Acknowledgments Conception and design: F. Simpkins, K.E. Hew, D.J. Azzam, G.B. Mills, Saracatinib and selumetinib were provided by AstraZeneca. This work was J.M. Slingerland funded by NCI K08 CA151892-01 award (to F. Simpkins) and by a grant from Development of methodology: F. Simpkins, K. Jang, H. Yoon, K.E. Hew, the Breast Cancer Research Foundation (to J. Slingerland). D.J. Azzam, D. Zhao, Z. Wei Acquisition of data (provided animals, acquired and managed patients, The costs of publication of this article were defrayed in part by the payment of provided facilities, etc.): F. Simpkins, K. Jang, H. Yoon, M. Kim, D.J. Azzam, page charges. This article must therefore be hereby marked advertisement in J. Sun, T.A. Ince, G.B. Mills, J.M. Slingerland accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): F. Simpkins, K. Jang, H. Yoon, K.E. Hew, M. Kim, Received December 15, 2017; revised April 6, 2018; accepted June 28, 2018; W. Liu, W. Guo, Z. Wei, G. Zhang, G.B. Mills, J.M. Slingerland published ﬁrst June 29, 2018.

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www.aacrjournals.org Clin Cancer Res; 2018 OF13

Dual Src and MEK Inhibition Decreases Ovarian Cancer Growth and Targets Tumor Initiating Stem-Like Cells

Fiona Simpkins, Kibeom Jang, Hyunho Yoon, et al.

Clin Cancer Res Published OnlineFirst June 29, 2018.

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