Therapeutic Targeting of Tumor-Derived R-Spondin

Published OnlineFirst December 30, 2015; DOI: 10.1158/0008-5472.CAN-15-0561 Cancer Tumor and Stem Cell Biology Research

Therapeutic Targeting of Tumor-Derived R-Spondin Attenuates b-Catenin Signaling and Tumorigenesis in Multiple Cancer Types Cecile Chartier, Janak Raval, Fumiko Axelrod, Chris Bond, Jennifer Cain, Cristina Dee-Hoskins, Shirley Ma, Marcus M. Fischer, Jalpa Shah, Jie Wei, May Ji, Andrew Lam, Michelle Stroud, Wan-Ching Yen, Pete Yeung, Belinda Cancilla, Gilbert O'Young, Min Wang, Ann M. Kapoun, John Lewicki, Timothy Hoey, and Austin Gurney

Abstract

Deregulation of the b-catenin signaling has long been associ- clonal antibody antagonists of RSPO family members and found ated with cancer. Intracellular components of this pathway, that anti-RSPO treatment markedly inhibited tumor growth in including axin, APC, and b-catenin, are frequently mutated in a human patient-derived tumor xenograft models, either as single range of human tumors, but the contribution of speciﬁc extra- agents or in combination with chemotherapy. Furthermore, cellular ligands that promote cancer development through this blocking RSPO signaling reduced the tumorigenicity of cancer signaling axis remains unclear. We conducted a reporter-based cells based on serial transplantation studies. Moreover, gene- screen in a panel of human tumors to identify secreted factors that expression analyses revealed that anti-RSPO treatment in respon- stimulate b-catenin signaling. Through this screen and further sive tumors strongly inhibited b-catenin target genes known to be molecular characterization, we found that R-spondin (RSPO) associated with cancer and normal stem cells. Collectively, our proteins collaborate with Wnt proteins to activate b-catenin. results suggest that the RSPO family is an important stimulator of RSPO family members were expressed in several human tumors b-catenin activity in many human tumors and highlight a new representing multiple malignancies, including ovarian, pancreat- effective approach for therapeutically modulating this fundamen- ic, colon, breast, and lung cancer. We generated speciﬁc mono- tal signaling axis. Cancer Res; 76(3); 1–11. 2015 AACR.

Introduction However, no WNT ligand overexpression has yet been directly associated to the disease in humans (3). b-catenin signaling is essential to developmental processes The R-spondin (RSPO) family of four proteins represents and is deregulated in a wide range of diseases, including cancer another group of secreted factors that enhance b-catenin signal- (1–3). WNT ligands are able to activate b-catenin–dependent ing. RSPO1-4 have a similar structure and harbor two furin signaling by binding to frizzled (FZD) receptor and LRP5/6 domains, which are necessary and sufﬁcient for b-catenin activa- coreceptor complex, inducing the release of unphosphorylated tion, and one thrombospondin type I domain, which promotes b-catenin from a cytosolic destruction complex comprised of non-canonical Wnt signaling (4, 5). Three classes of transmem- axin, APC, CKI, and GSK3 and its translocation to the nucleus brane proteins have been discovered that interact with RSPO: where it associates with the TCF/LEF family of transcription syndecans, Leu-rich repeat-containing G protein-coupled recep- factors and induces canonical gene transcription. Intracellular tors (LGR), and E3 ubiquitin ligases (5–10). Of note, one of these components of the signaling pathway such as APC, axin, and receptors, LGR5, is a robust marker of stem cells (11–18). A b-catenin itself are mutated in a number of human cancers. mechanism of action for RSPO has been elucidated; RSPOs protect frizzled receptors from internalization and degradation and recruit IQGAP1 into the Wnt signaling complex, leading OncoMed Pharmaceuticals Inc., Redwood City, California. to both an increase in b-catenin and Rho GTPase signaling Note: Supplementary data for this article are available at Cancer Research activities (9, 19). The LGR receptors involved in this process are Online (http://cancerres.aacrjournals.org/). members of the superfamily of seven-transmembrane receptors, Current address for C. Bond: Center for Drug Research and Development, suggesting the potential for additional signaling mechanisms Vancouver, BC; current address for J. Shah: ZS Associates, San Mateo, CA; and elicited by RPSO. current address for J. Wei: Rinat Laboratories, Pﬁzer Inc., South San Francisco, Like WNTs, RSPOs have important roles in development and CA. act as powerful stem cell growth factors (20). RSPO1 is used as an Corresponding Author: Cecile Chartier, OncoMed Pharmaceuticals, 800 Che- indispensable agent for in vitro propagation of intestinal stem cells sapeake Drive, Redwood City, CA 94063. Phone: 650-995-8216; Fax: 650-298- and its activity cannot be replaced by exogenous WNT protein 8600; E-mail: [email protected] (21). In vivo, RSPO1 controls the phenotypic sex of humans and doi: 10.1158/0008-5472.CAN-15-0561 mice, RSPO2 is necessary for development of numerous mouse 2015 American Association for Cancer Research. organs, RSPO3 is involved in angiogenesis during placenta

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development in mice, and RSPO4 is the gene disrupted in humans received in 2005. It contains the TOP-FLASH reporter system with congenital anonychia (22). In most cases, RSPOs function by comprised of 8 copies of a TCF-binding site (AGATCAAAGG) enhancing Wnt/b-catenin signaling. However, RSPO2 or 3 were upstream of a minimal promoter and firefly luciferase. Cells were also shown to signal through cascades that are separate from the cultured in DMEM, 10% FBS, 500 mg/mL Geneticin (Life Tech- canonical pathway (5, 8, 19, 23–25). nologies) and incubated at 37C, 5% CO2. HEK-293T (CRL- Several lines of evidence suggest a role for RSPOs in cancer 3216) and WNT3A-L cells (CRL-2647) were obtained in 2005 development. The identification of Rspo2 and Rspo3 as sites of from the ATCC and cultured according to the ATCC guidelines. integration for MMTV-induced mammary tumors in mice sup- – ported a role as breast cancer oncogenes (26 28). Ectopic expres- STF reporter assay Rspo2 sion of was shown to increase tumorigenic and invasive Tumor cells were cultured in DMEM, 10% FBS (Life Technol- properties of mouse breast cell lines (25). Genomic rearrange- ogies) for 24 hours at 37 C and 5% CO2. One volume of ments and transcriptional activation in human tumors that result conditioned medium and cells was added on top of a monolayer fi in elevated RSPO expression have been identi ed, suggesting that of HEK-293 STF cells grown in 1 volume of DMEM, 10% FBS. RSPO may be functionally important for tumor development When used, 5 ng/mL of recombinant human RSPO (R&D Sys- – (29 32). However, no direct demonstration of a functional role tems) were added to the reporter cells. WNT3A-conditioned for RSPO in cancer development and maintenance has been medium prepared from WNT3A-L cells was added to the tumor reported to date. cell conditioned medium or purified RSPO at a final concen- Here, we report an analysis of patient-derived xenograft (PDX) tration of 25% v/v. The activity of purified soluble receptors tumor samples for RSPO expression and activity. Functional (10 mg/mL) and mAbs (40 mg/mL) was compared with the RSPO activity was found to be produced by human tumors of control immunoadhesin JAG1-Fc and control antibody LZ1. multiple types. The importance of RSPO in driving the growth of Reporter cells were incubated for 16 hours before luciferase in vivo overexpressing tumors was tested with anti-RSPO mono- activity measurement using Steady-Glo according to the man- clonal antibodies (mAb) directed against various members of the ufacturer's instructions (Promega). family. Efficacy was observed in PDX models established with minimally passaged human tumors of multiple types. Gene- expression analyses revealed that RSPO promoted expression of Real-time RT-PCR RSPO genes associated with the Wnt signaling pathway and normal and For gene-expression analyses, RNA was extracted from cancer stem cells (CSC). These data provide the first demonstra- xenograft tumors using an RNeasy Fibrous Tissue mini kit tion that RSPO family members play a major role in the growth of (Qiagen) according to the manufacturer's instructions. Thirty human cancer and validate RSPO1-3 as therapeutic targets. ng RNA were submitted to a one-step RT-PCR reaction using Applied Biosystems 7900HT instrument and buffer system according to the manufacturer's instructions (Life Technolo- Materials and Methods gies). GUSB was used as endogenous control against which DCts RSPO Xenograft models and tissue processing were calculated and the lowest expressing sample as DDC NOD/SCID mice were purchased from Harlan Laboratories baseline for the t calculations. Relative expressions were and maintained under specific pathogen-free conditions and determined by raising 2 to the power of the negative value of DDC provided with sterile food and water ad libitum. Animals were t for each sample. All gene-expression assays were pur- fi fi housed in a U.S. Department of Agriculture-registered facility in chased from ABI and veri ed for species speci city before accordance with NIH guidelines for the care and use of laboratory screening the OMP PDXs. animals. The mice were allowed to acclimate for several days before the studies. – GUSB: Hs00939627_m1 The establishment of OMP PDX models was described previ- – RSPO1: Hs01045335_mH ously (33). Patient-derived tumors were passaged in mice without – RSPO2: Hs00379983_m1 any intervening cell culture. Tumor cells were stored in liquid – RSPO3: Hs00262176_m1 nitrogen. Experiments for obtaining fresh tumor cells or testing – RSPO4: Hs01382765_m1 antibody therapeutic efficacy were initiated from frozen cell stocks. CR3150, CR1560, CR2506, and CR2513 models were For select target gene-expression analyses, frozen tumor established at Crown BioScience (Beijing, China). tissues were pulverized in liquid nitrogen with a mortar and For in vitro assays and in vivo tumorigenicity assessments, single- pestle. RNA was extracted as above. All RNAs were verified to cell suspensions were obtained from freshly dissociated tumors contain intact ribosomal bands by Bioanalyzer (Agilent). according to a previously described procedure (34). Cells were cDNA template was generated from 50 ng RNA using the Cells then incubated with biotinylated antibodies (a-mouse CD45- Direct One-step qRT-PCR Kit (Life Technologies). PCR was biotin 1:200 dilution and a-mouse H2Kd-biotin 1:100 dilution; performed on ABI 7900HT (Life Technologies) using the rel- BioLegend) on ice for 30 minutes followed by addition of 50 mL ative standard curve method. Gene-expression normalization MagnaBind streptavadin-labeled magnetic beads (Thermo Scien- was performed with GAPDH. Statistical calculations were tific) per 10E6 cells/mL. Mouse cells were removed on a magnetic performed using the t test with Excel software (Microsoft). stand. Primer (forward and reverse) and probe sequences were as follows. TDGF1:50-CCAGAGTGCTGAAGGAATGGA-30, Cell lines 50-CCTCTCTCTTCTATTTGCTTCCTCTT-30,and50-CCTACC- The HEK-293 STF cell line was a kind gift from Dr. Jeremy CAGTCTCCCTGCACACACG-30; ASCL2:50-CCCCTCCCCA- Nathans (Johns Hopkins School of Medicine, Baltimore, MD) CAGCTTCT-30,50-AAATGGATTCTCTGTGCCCTTAGA-30,and

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50-CACCAACACTTGGAGATTTTTCCGGAGG-30; CLDN2:50-CCT- fragments (Crown BioScience PDXs) were injected s.c. into the AAGTCCCCAACCCTCAAC-30,50-GGGCAAAGGGATCCTCTGA- left flankregionof6-to8-week-oldNOD/SCIDmicewitha 30,and50-TGAAACCCCATTCCCTTAAGCCAGGA-30; PTPRO:50- 25-gauge needle. Tumor-bearing animals were randomized GAACTCTGTTGCTGTCTGAGCAA-30,50-GCTCTCACATGTATG- and treatment started when the mean tumor volumes reached CAGGTCAA-30,and50-CGTGGTGCCTAGACTTTGCATTCCTTG- 100 to 150 mm3. Chemotherapeutic agents were given once 30; RPRD1A:50-CACCTGAGGCAGATCATGCA-30,50-ACGGCAC- weekly at various doses ranging from 10 to 100 mg/kg as CATGCTTTTCTCT-30, and 50-CTTTGAGTGCAGTTTGGTCTGAC- specified in the figure legends. Antibodies were dosed once CCCTC-30; LGR5:50-TGGACTCAAGAGACTCAGTAACGTATTAT- weekly or biweekly at a dose ranging from 10 to 25 mg/kg. All 30,50-TAAGCAGAGAAGTAATGTTCCTAACATCA-30,and50-ATT- agents were administered i.p. TAGCTTGGTTTTAGCTGTGTTCTCTCTGGATAACC-30; ZNRF3:50- For the tumorigenicity study, single tumor cell suspensions CCATGTCTTATGTTGAGAGTGTGACA-30,50-TGCAAATATG- prepared as described from control and treated tumors were TAAAATCTGTGTGCAA-30, and 50-TGGAATAATCATTGAAAATG- countedanddilutedto50cellsin100mL 50% Matrigel in FACS ACTAACACAAGACCCTGTAA-30; AXIN2:50-CCTTTCCTCCACA- buffer. Ten NOD/SCID mice were injected s.c. per group. CACCTTCA-30,50-TGTATAGTACAAGTAACAATGGCAAACAG-30, Tumor growth was monitored for 59 days without any further and 50-ATGTACAGATTAACTTAACACAAAAACCCGAACATCAA- treatment. A-30; GAPDH:50-CCACCCATGGCAAATTCC-30,50-TGGGATTTC- 0 0 CATTGATGACAAG-3 ,and5-TGGCACCGTCAAGGCTGAGAA- Microarray analysis 0 CG-3 . RNA was isolated and microarray analyses were conducted as detailed in Supplementary Data. Data are available through Antibodies GEO database, accession GSE73906. Anti-RSPO antibodies were generated by immunizing mice with purified recombinant human RSPO1, RSPO2, or RSPO3 (R&D Systems) followed by hybridoma generation and char- Results acterization. The antibodies LZ1, an anti-lysozyme monoclonal Identification of RSPO as a tumor-derived b-catenin generated by panning a phage display library obtained from stimulating activity MorphoSys (Martinsried/Planegg) and 1B711, a murine mono- To identify possible b-catenin–stimulating activities pro- clonal directed against dinitrophenol (anti-hapten) and duced by human tumor cells, a functional screen was devel- obtained from the ATCC were used as negative controls in the oped. Tumor cells freshly obtained from minimally passaged in vitro and in vivo experiments, respectively (35). Anti-RPSO3 PDXs (referred to as OMP-xxxx tumors hereafter, where xxxx mAb #2 was isolated by a mammalian cell antibody display refers to our internal nomenclature for that tumor PDX line) technique, MAbTrap, in which antibody sequences derived were cocultured with an HEK-293 cell-based reporter cell from the immunized mice were transiently expressed and then line containing a TCF-luciferase reporter (STF). This reporter retained on the cell surface by interaction with a membrane- cell line responds to WNT and RSPO signals through b-catenin– anchored CH2-CH3 antibody fragment. FACS was used to driven transcription. Forty eight OMP tumors (Supplementary isolate cells expressing the antibody of interest and correspond- Table S1) were screened in this assay, of which, three (6.25%) ing cDNA isolated. induced reporter activity more than 2-fold relative to control The binding of anti-RSPO antibodies to RSPO was assessed (Fig. 1A and Supplementary Table S2). OMP-LU2, OMP-LU25, by flow cytometry. First, HEK-293T cells were transiently trans- and OMP-OV38 induced luciferase activity 6.7-, 4.7-, and 3.1- fected with cDNA expression vectors for RSPO extracellular fold over medium alone, respectively. In this survey, WNT3A domainfusedtohumanCD4transmembrane domain fused was also tested, alone as a positive control and added to to GFP using Fugene 6 (Promega). Transfected cells were then the coculture assay. Interestingly, the three tumors that were incubated with 20 mg/mL antibody and washed with PBS. positive for b-catenin signaling activity displayed further ele- Antibody binding was detected with a PE-conjugated anti- vated activation upon addition of WNT3A (Fig. 1A). OMP-LU2, mouse Fc antibody and light signals measured for PE and GFP, OMP-LU25, and OMP-OV38 boosted WNT3A activity 4.3-, 9.6-, using a Canto II instrument (BD Biosciences). and 1.6-fold over WNT3A alone. Such potentiation of WNT To assess ability of anti-RSPO antibodies to block interac- has been noted as a hallmark of RSPO activity, suggesting that tion with LGR, binding studies were conducted. RSPO pro- RSPO ligands might be responsible for the activation of the teinswereexpressedasFcfusionproteinscontainingRSPO b-catenin reporter in the assay (4). Of note, additional OMP N-terminal furin domains and human IgG1 Fc. HEK-293T tumors were shown to score in the assay only in the presence of cells were transiently transfected with cDNA expression vector WNT3A, possibly reflecting lower levels of the b-catenin acti- encoding the N-terminal extracellular domain of human LGR vating factor(s). OMP-B37, OMP-B39, and OMP-LU102 fused to human CD4 transmembrane domain fused to GFP. increased WNT3A activity 2.7-, 9-, and 1.8-fold, respectively After 24 hours, cells were incubated with RSPO–Fc fusion (Fig. 1A and Supplementary Table S2). To examine the tumor- proteins and anti-RSPO antibodies at 10 mg/mL. RSPO bind- derived activity further, FZD8-Fc and LGR5-Fc decoy receptors ing was detected by flow cytometry with a PE-conjugated anti- were tested in the assay. FZD8-Fc acts as an inhibitor of Wnt human Fc antibody and light signals measured for PE and signaling by binding WNT ligands, whereas LGR5-Fc binds to GFP. RSPO and antagonizes RSPO potentiation of Wnt signaling (Fig. 1B and C). In conditions in which FZD8-Fc abolished In vivo efficacy experiments WNT3A-induced reporter activity, it partially inhibited the About 50,000 freshly thawed tumor cells re-suspended in activity induced by the tumor cells alone and dramatically 100 mL 50% Matrigel in FACS buffer (OMP PDXs) or tumor decreased the activity induced by the tumor cells in presence

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B 10,000 JAG1.Fc A FZD8.Fc 7,500 No Wnt3A+JAG1.Fc Control 60 5,000 Tumor 25,00

Wnt3A Luciferase activity (RLU) 0 40 Tumor+Wnt3A 0.0001 0.01 1 100 C µg/mL fusion protein 20,000 80,000 RSPO1 RSPO2

15,000 60,000 20 10,000 40,000

5,000 20,000 Fold luciferase induction

0 Luciferase activity (RLU) 0 0 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 40,000 15,000 RSPO3 RSPO4 LU2 PN7 LU25 30,000 LU102 OV19 OV38 10,000 20,000

Tumor 5,000 10,000 Luciferase activity (RLU) Luciferase activity (RLU)0 Luciferase activity (RLU) 0 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 LGR5.Fc (µg/mL) LGR5.Fc (µg/mL) D LU2 LU25 LU102 OV38 40,000 125,000 15,000 Control 250,000

100,000 Tumor 30,000 200,000 10,000 Wnt3A 150,000 75,000 20,000 100,000 50,000 Tumor+Wnt3A 5,000 10,000 50,000 25,000 Relative luciferase Relative luciferase Relative luciferase Relative luciferase

0 0 0 0

FZD8.FcLGR5.Fc FZD8.FcLGR5.Fc FZD8.FcLGR5.Fc FZD8.FcLGR5.Fc Control.Fc Control.Fc Control.Fc Control.Fc

Figure 1. Detection of OMP tumor sample-derived b-catenin activation. The ability of human tumor-derived soluble factors to activate b-catenin was assessed in the STF assay. A, fold inductions are shown for a subset of the 48 OMP tumor samples tested. They represent the ratio of tumor (red bars), WNT3A (green bars), and tumor þ WNT3A (purple bars)-induced luciferase activity relative to the medium alone. B, FZD8.Fc inhibitory activity was demonstrated in the STF reporter assay. STF cells were exposed to WNT3A or non-WNT3A (solid triangle) conditioned medium supplemented with the indicated concentrations of FZD8.Fc (hollow dots). Mouse JAG1-Fc (solid dots) was used as negative control. Results are displayed as luciferase activity levels (RLU, relative light units) as a function of the test agent's concentration (mg/mL). C, serial dilutions of LGR5.Fc were used to inhibit the induction of luciferase by RSPO1 (squares), RSPO2 (circles), RSPO3 (triangles), or RSPO4 (diamonds) added to WNT3A-conditioned medium. Results are shown as the measured RLUs as a function of the decoy protein concentration. D, both FZD8.Fc and LGR5.Fc inhibited the luciferase reporter activity (RLUs) of STF cells stimulated with tumor cell cultures supplemented (purple bars) or not (red bars) with WNT3A. WNT3A activity was only inhibited by FZD8.Fc (green bars).

of exogenously added WNT3A. Conversely, although LGR5-Fc family member was highly expressed within a tumor as deter- remained mostly ineffective at inhibiting WNT3A alone, it mined by quantitative real-time polymerase chain reaction efficiently decreased the activity of the tumor cells alone and (qPCR; Fig. 2 and Supplementary Table S2). Thus, OMP-LU2, supplemented with WNT3A. LGR5-Fc reduced the activity of OMP-LU25 and OMP-OV38 expressed high levels of RSPO2 the cocultures containing added WNT3A to a level comparable or RSPO3. Additional RSPO mRNA-positive tumors, for exam- with that observed with WNT3A alone (Fig. 1D). These results ple, OMP-OV19 and OMP-PN7 (Fig. 1A and Supplementary suggest that the activation of the b-catenin reporter observed in Table S2), had not been identified by the in vitro functional the tumor cell culture assay reflects the presence of RSPO activity assay, maybe due to the low sensitivity of the assay. It has potentiating low levels of endogenous WNT ligands. The mod- been reported that RSPO2 and RSPO3 genes are targets of est inhibition of the tumor-induced activation by FZD8-Fc rearrangements in colon cancer and that resulting fusions in- suggests the presence of a distinct (WNT or non-WNT) ligand crease gene expression (29, 31). On the basis of these reports, not bound by FZD8-Fc. we performed a PCR-based screen and also mRNAseq analy- Interestingly, the tumors previously found to exhibit sis. These analyses did not find fusions in either RSPO2 b-catenin activity possessed markedly elevated levels of par- or RSPO3 in our tumor bank, including RSPO2-high OMP- ticular RSPO family members. Generally, only a single RPSO C28, suggesting that other mechanisms are involved in the

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100,000 250,000 RSPO1 RSPO2 80,000 200,000 LU2 60,000 OV19 150,000 RQ RQ 40,000 100,000

20,000 50,000 C28 PN7 0 0 Breast Colon Lung Mel Ov Panc Breast Colon Lung Mel Ov Panc

20,000 15,000 RSPO3 RSPO4 LU25 15,000 LU102 10,000 10,000 RQ RQ 5,000 5,000 OV38

0 0 Breast Colon Lung Mel Ov Panc Breast Colon Lung Mel Ov Panc

Figure 2. RSPO gene-expression proﬁle across multiple different tumor types. RSPO1, 2, 3,and4 gene expression in breast, colon, lung, melanoma, ovary, and pancreas tumor models was assessed by real time RT-PCR and expressed as a relative quantity using the lowest positive sample as baseline and GUSB as endogenous reference. Each purple bar represents one patient-derived tumor sample.

overexpression of RSPO in these tumors. RSPO3-high human RSPO antagonists inhibit the growth of human tumor CRC tumors possessing the reported PTPRK–RSPO3 gene xenografts fusion were identified at an external service provider (Sup- The impact of inhibiting RSPO signaling on tumor growth plementary Fig. S1). was assessed using PDXs in mice. Tumor-bearing mice were To investigate the functional consequence of inhibiting the treated with antibodies to individual RSPO family members. activity of specific RSPOs, mAbs were generated that bind RSPO1, Anti-RSPO treatment produced significant inhibition of tumor RSPO2, or RSPO3 (Fig. 3A) and block the ability of RSPO to growth in several types of human tumors (Fig. 5A). Growth of potentiate WNT stimulation of the b-catenin luciferase reporter OMP-OV19, an ovarian tumor noted to express RSPO1,was through blocking binding to LGR (Fig. 3B and C). All antibodies inhibited by antibody to RSPO1. OMP-C28 and OMP-PN7, a were shown to be specific for their targeted RSPO and not to cross- colon and a pancreatic tumor, respectively, noted to express react with the other RSPOs. Anti-RSPO antibodies were tested for RSPO2, were inhibited by antibody to RSPO2. Anti-RSPO3 impact on the tumor-induced b-catenin activity observed in the displayed significant antitumor activity in several RSPO3- luciferase reporter screen. Antibody to RSPO2 blocked the activity high tumors, including non–smallcelllungcancer(NSCLC) produced by OMP-LU2 tumor cells and antibody to RSPO3 models OMP-LU25 and OMP-LU102, colorectal cancer models blocked the activity observed in response to OMP-LU25, OMP- CR3150, CR2513, and CR2506, and ovarian cancer OMP- LU102, and OMP-OV38 tumor cells (Fig. 4). In each case, the OV38. Colon tumor CR3150 is noteworthy as a tumor posses- antibodies inhibited the reporter activity back to control levels. sing a RSPO3 chromosomal translocation identical to those None of the three antibodies affected the reporter activity when previously reported (29). Thus, anti-RSPO treatments showed non–b-catenin–inducing tumor cells such as OMP-PN25 and single-agent therapeutic activity in most RSPO-high models. OMP-M6 were used in the assay (Supplementary Fig. S2). Overall, Combination with chemotherapy, including taxane treatment the results parallel the relative RSPO gene-expression pattern in NSCLC and ovarian models, gemcitabine treatment in pan- (Fig. 2, Supplementary Table S2), strongly implicating the RSPO creatic tumor, and irinotecan in colorectal cancer models, proteins as the inducers of the observed b-catenin signaling resulted in further significant inhibition of tumor growth activity produced by these human tumor cells. In particular, the beyond the impact of chemotherapy alone (Fig. 5A). The above data revealed RSPO2 or RSPO3 as the principal factors mechanism of combination activity with paclitaxel was inves- responsible for the b-catenin activity derived from several of our tigated in OMP-OV38. Anti-RSPO3 treatment combined with patient-derived tumors, making potential therapeutic targets of paclitaxel was found to increase mitotic cell death (Supple- RSPO2 and RSPO3. mentary Fig. S3).

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a-RSPO1 Control RSPO1 A C -+++

An-RSPO1

An-RSPO2 RSPO1 binding LGR5 LGR5 LGR5 LGR5

An-RSPO3 #1 a-RSPO2 Control RSPO2 -+++

Anbody binding An-RSPO3 #2

No Ab RSPO2 binding LGR5 LGR5 LGR5 LGR5 RSPO1 RSPO2 RSPO3 RSPO4

a-RSPO3 #1 Control RSPO3 B -+++ 15,000 5,000 h-RSPO1 h-RSPO2 m-RSPO1 4,000 m-RSPO2 10,000 3,000

2,000 5,000 1,000 RSPO3 binding Relave luciferase Relave luciferase 0 0 WNT3A - + ++ ++ WNT3A - ++ + + + RSPO1 - - +++ + RSPO2 -- + + + + LGR5 LGR5 LGR5 LGR5 a-RSPO1 mAb - --- ++ a-RSPO2 mAb - --- ++

a-RSPO3 #2 Control RSPO3

100,000 -+++ 150,000 h-RSPO3 7,500 m-RSPO3 100,000 5,000

2,500 50,000 Relave luciferase 0 Relave luciferase 0 RSPO3 binding WNT3A - +++ WNT3A - ++++ + h-RSPO3 - - + + RSPO3 - - + + + + a-RSPO3 mAb1 --- + a-RSPO3 mAb2 -- -- ++ LGR5 LGR5 LGR5 LGR5

Figure 3. Characterization of anti-RSPO1, RSPO2, and RSPO3 mAbs. RSPO-binding properties of the anti-RSPO mAbs were assessed by flow cytometry. The ability of anti-RSPO antibodies to inhibit RSPO signaling function was monitored in the cell-based b-catenin reporter assay. A, RSPO1, 2, 3, or 4 was displayed at the surface of HEK-293 cells by means of a transiently transfected RSPO1, 2, 3, or 4.CD4TM.GFP fusion cDNA expression vector that tethers RSPO to the cell surface via a transmembrane anchor. Binding of the RSPO moieties by the anti-RSPO1, anti-RSPO2, and two anti-RSPO3 mAbs was detected using a PE-conjugated secondary antibody. The PE (y axis) and GFP/FITC (x axis) signals are plotted for each individual cell analyzed. The relative increase of both signals translates specific antibody binding to the antigen. B, anti-RSPO antibodies' inhibitory activity was confirmed in the STF reporter assay. STF cells were exposed to WNT3A conditioned medium supplemented with RSPO1, RSPO2, or RSPO3. Anti-RSPO antibodies were added. Results are displayed as luciferase activity levels (RLU, relative light units). C, LGR5 was displayed at the surface of HEK-293T cells by means of a transiently transfected LGR5.CD4TM.GFP fusion cDNA expression vector that expresses the N-terminal extracellular domain of LGR5 tethered to the cell surface via a transmembrane anchor. Ability of anti-RSPO mAb to block the binding of the indicated RSPO was assessed by incubating cells with RSPO-Fc fusion protein and mAb as indicated and followed by detection of bound RSPO protein with PE-conjugated anti-human Fc secondary antibody. Specific RSPO-LGR5 binding is indicated by detection of cells within the inset box of the flow-cytometry plot.

Not all RSPO-high tumors were sensitive to RSPO inhibition. cell suspension and 50 cells were injected into recipient mice for a For example, the NSCLC OMP-LU2, despite high levels of RPSO2 second (treatment free) round of propagation. The ability of this mRNA, and colorectal cancer CR1560, despite high levels of low cell number to re-grow a tumor provided a direct measure of RSPO3 mRNA, were not significantly inhibited by anti-RSPO2 their CSC content. In such a setting, anti-RSPO3 significantly and anti-RSPO3, respectively, whether alone or combined with reduced the tumorigenicity of OMP-LU25 tumor cells both as a chemotherapy (Fig. 5A). Analysis of LGR4, 5, and 6 gene expres- single agent and in combination with paclitaxel (Fig. 5B). sions revealed no correlation between receptor expression and Although all 10 control mice and 8 of 9 paclitaxel group mice resistance to antibody treatment (Supplementary Fig. S4). Tumors developed tumors, the anti-RSPO3 and combination-treated that did not express RSPO were unresponsive to antibody treat- tumor cells engrafted poorly (7:10 and 6:10, respectively) and ment (Supplementary Fig. S5). grew slowly. Similar reductions in tumorigenicity after anti-RSPO The responsive NSCLC model OMP-LU25 was used to assess treatment were observed in other tumors, including recently the impact of RSPO3 inhibition on CSCs, also known as tumor obtained pancreatic and lung tumors that were not included in initiating cells. For this, tumors previously treated with antibody our original panel (Supplementary Fig. S6). In addition, levels of and/or chemotherapy were harvested and processed to a single- CD44, a well-known colon CSC marker, were significantly

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LU2 LU25 20,000 150,000

15,000 Figure 4. 100,000 Inhibition of tumor-derived b-catenin 10,000 activity by the anti-RSPO antibodies. The 50,000 use of speciﬁc anti-RSPO antibodies in the 5,000 tumor-STF cell coculture reporter assay

ﬁ Relative luciferase identi ed RSPO ligands as the tumor- Relative luciferase 0 0 derived factors responsible for inducing b-catenin transcriptional activity. OMP-LU2, -LU25, -LU102, and -OV38 cells LU102 OV38 were tested for their ability to induce 80,000 2,000 luciferase activity of a TCF-responding element in absence (1st series) and 60,000 1,500 presence (2nd series) of WNT3A when combined with a control mAb (red bars), 40,000 1,000 anti-RSPO1 (tan bars), anti-RSPO2 (purple 20,000 bars), or anti-RSPO3 mAb1 (blue bars) 500 antibody. Effect of the anti-RSPO

Relative luciferase ND ND ND 0 Relative luciferase 0 antibodies could not be assessed (ND) on OV38 in absence of WNT3A. Relative luciferase activity (RLU) was plotted for -WNT3A +WNT3A -WNT3A +WNT3A each condition next to a no tumor cell control (black bars). No tumor + control mAb Tumor + control mAb Tumor + anti-RSPO1 Tumor + anti-RSPO2 Tumor + anti-RSPO3 reduced in anti–RSPO3-treated tumors (Supplementary Fig. S7; not be associated with growth inhibition of non-RSPO/Wnt- ref. 33). These results show that RSPO3 is important to maintain driven tumors. CSCs in multiple tumor types. To further study the mechanism of action of RSPO3, microarray analysis was performed on anti–RSPO3-treated colon tumors. RSPO blockade inhibits stem cell signaling pathways in Findings were consistent with the qPCR results (Fig. 6) and also tumors revealed new insights into the RSPO3 mechanism of action RSPO1 functions as a potent regulator of stem cell growth in (Supplementary Table S3). In the three responsive models, mod- colon (21). The expression of a panel of genes associated with ulation of Wnt signaling was confirmed by the downregulation of stem cell and/or RSPO signaling was, thus, monitored in anti– numerous Wnt genes that included CLDN2, EPHB2, CD44, LRP4, RSPO3-treated colorectal cancer xenografts. Control and anti- ASCL2, AXIN2, LGR5, PTPRO, TDGF1, and ZNRF3. In addition, body-treated tumor RNAs from the in vivo efficacy experiments effects on Notch signaling were revealed by the downregulation of conducted with CR3150, CR2506, CR2513, and CR1560 were genes such as NOTCH1 and PSEN1. Both Wnt and Notch path- tested for the expression of ASCL2, AXIN2, CLDN2, LGR5, ways are associated with CSCs (43–45). Consistently, numerous PTPRO, RPRD1A, TDGF1,andZNRF3 genes, using qPCR. Strik- stem cell genes were inhibited by anti-RSPO3 in the three respon- ingly, 7 of the 8 genes were downregulated in response to anti- ders. Interestingly, differentiation genes, including BAMBI, RSPO3 in all three sensitive colorectal cancer models (Fig. 6). CADM1, POU2F2, and RGS2 were upregulated. Most of the genes The downregulation was highly significant in at least two of the described above were not significantly modulated by anti-RSPO3 models for these genes. For example, ASCL2 expression could in the nonresponsive CR1560. Together, these results show that no longer be detected in any of the anti–RSPO3-treated CR2506 RSPO3 blockade inhibited CSC signaling pathways in colorectal and CR2513 samples; LGR5 was downregulated 7.24- and cancer, and suggest that the tumor cells may adopt a more 5.75-fold in treated CR2506 and CR3150 tumors, respectively, differentiated cell fate. Microarray data for anti-RSPO3 plus and TDGF1 mRNA levels were reduced in all three models, up irinotecan-treated CR2506 further support this mechanism by to 234-fold. All 7 downregulated genes are reported canonical showing that most Wnt and stem cell genes targeted by anti- Wntsignalingtargetgenes(36,37).Severalofthesegenes, RSPO3 remained modulated upon addition of chemotherapeu- ASCL2, LGR5,andTDGF1, have also been directly associated tics, with some of them responding to a greater extent to the with CSCs in various tumor types, supporting a role for the combination than to anti-RSPO3 alone (Supplementary Table RSPO pathway in CSC biology and consistent with the in vivo S3). Overall, the gene-expression data support the inhibition of tumorigenicity results presented herein (38–40). Downregula- self-renewal pathways as the main mechanism of anti-RSPO3's tion of Wnt and stem cell genes by anti-RSPO treatment could antitumor activity. be involved in the sensitization of tumor cells to chemotherapeutic agents (41, 42). Two of the 8 studied genes, AXIN2 and ZNRF3, were downregulated by anti-RSPO3 in the nonrespond- Discussion ing CR1560 model. The two genes are the only Wnt target genes Because the initial discovery of WNT1 as a gene product of the panel that were highly expressed in this model, possibly capable of promoting breast cancer thirty years ago, there reflecting a lower state of Wnt signaling activation. Thus, iso- has been a recognition that this signaling pathway may con- lated Wnt targets could be regulated by anti-RSPO3 therapy and tribute to human cancer (46). This recognition has been greatly

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) 1,000 3 Ovary 2,000 Colon 1,500 Lung 1,500 Pancreas OMP-OV19 OMP-C28 OMP-LU2 OMP-PN7 800 1,500 1,000 1,000 600 1,000 400 500 500 500 200

Avg. tumor vol (mm Avg. 0 0 0 0 0 5 10 15 20 25 30 35 01020 30 01020 30 0510 15 20 Days post treatment Days post treatment Days post treatment Days post treatment

) 1,500 2,000 2,000 3 Colon 1,500 Colon Colon Colon CR3150 CR2513 CR2506 CR1560 1,500 1,500 1,000 1,000 1,000 1,000 500 500 500 500

Avg. tumor vol (mm Avg. 0 0 0 0 010203040 50 02040 010203040 50 010203040 Days post treatment Days post treatment Days post treatment Days post treatment

) B 3 2,000 Lung 3,000 Lung 1,500 Ovary

) 2,500

OMP-LU25 OMP-LU102 OMP-OV38 3 1,500 2,000 2,000 1,000 1,000 1,500 1,000 500 1,000 500 500

Avg. tumor vol (mm Avg. 0 0 0 05 1510 20 01020 30 0 5 10 15 20 3025 volume (mm Tumor 0 Days post treatment Days post treatment Days post treatment

Control mAb Anti-RSPO1 Anti-RSPO2 Anti-RSPO3 Paclitaxel Anti-RSPO3 Control mAb Chemo Chemo + anti-RSPO1 Chemo + anti-RSPO2 Chemo + anti-RSPO3

Paclitaxel + anti-RSPO3

Figure 5. Inhibition of RSPOhigh tumor growth in vivo using speciﬁc anti-RSPO antibodies. Ovarian (OMP-OV19 and -OV38), pancreatic (OMP-PN7), lung (OMP-LU2, -LU25, and -LU102), and colorectal (OMP-C28, CR3150, CR2513, CR2506, and CR1560) xenograft models were treated with anti-RSPO1 (green symbols), anti-RSPO2 (blue symbols), or anti-RSPO3 mAb2 (red symbols) antibody alone (up-pointing triangles) or in combination with chemo (down-pointing triangles) to assess the role of RSPO in driving the in vivo growth of these models. Anti-RSPO1 was used at 10 mg/kg once weekly; anti-RSPO2 was used at 10 (OMP-PN7) or 15 (OMP-LU2) mg/kg once weekly or 25 mg/kg every other week (OMP-C28); anti-RSPO3 mAb2 was used at 25 mg/kg once weekly. The chemo agents and regimens were as follows: 15 mg/kg taxol once weekly for OMP-OV19, -OV38, and –LU25, 10 mg/kg taxol once weekly for OMP-LU2, 100 mg/kggemcitabineonceweeklyforOMP-PN7,and10mg/kgirinotecanonceweeklyforOMP-C28,CR3150,CR2513,CR2506,andCR1560.A,tumorvolumes were measured weekly, averaged for each treatment group (n ¼ 10) and graphed as a function of time to obtain growth curves relative to the control antibody (open black circles) and chemo (black circles) groups. Tumor models were deemed responsive when P values calculated using a two-way ANOVA analysis and Bonferroni post-tests were <0.05 (), <0.01 (), or <0.001 () at termination. Antibody alone was compared with control antibody and chemo combination was compared with control antibody þ chemo agent as depicted by black vertical lines on the graphs. B, the tumorigenicity of anti–RSPO3- treated OMP-LU25 tumors was assessed upon passaging low cell doses in na€ve NOD/SCID mice. All 10 individual tumor volumes were plotted for each group after 100% take rate was observed in the control group.

strengthened by the understanding that intracellular compo- expressed due to retroviral MMTV integration, there has not nents of the signaling cascade, APC, axin, and b-catenin, are been clear evidence that WNT1 is frequently overexpressed mutated in approximately 90% of human colon tumors (47). in human tumors. To address this question of which factor Mutations in Wnt pathway components have also now been might be responsible for driving b-catenin signaling in human observed in a broad range of human tumors (3). Recently, tumors, we surveyed a series of human tumors for production therapeutic agents have been developed that target the Wnt of b-catenin signaling activity using a functional reporter pathway, and these agents show efficacy in a range of preclinical assay. This effort has identified RSPO family members as impor- tumor models (43, 48). OMP-18R5 (vantictumab), an antibody tant stimulators of b-catenin signaling activity in a number that functions by blocking WNT binding to 5 of the 10 human of human tumors. Patient-derived lung and ovarian tumor frizzled receptors, demonstrates activity in inhibiting the growth cells were shown to induce b-catenin activation in an in vitro of a range of human tumor xenograft models, indicating that cell-based assay and the activity was directly dependent on extracellular Wnt pathway signals play an important role in the RSPO2 or RSPO3 expression as demonstrated using specific growth of many human tumors. Despite this progress, a major blocking anti-RSPO antibodies. Interestingly, although RSPO1- unanswered question remains the specific contribution of var- or RSPO4-high tumors were identified, none of them displayed ious members of the large family of WNT ligands in human any detectable activity in the STF coculture assay. This could tumors, and other signaling proteins that might contribute to reflect the assay detection limit or/and the induction of differ- b-catenin activation, particularly in tumors that do not possess ential b-catenin activation levels by different RSPO proteins. known mutations impacting intracellular signaling compo- Subsequently, the growth of several of the RSPO-producing nents. For instance, despite the demonstration that murine tumors was shown to be driven by the RSPO activity when WNT1 can drive the formation of breast tumors when over- implanted in NOD/SCID mice. Using anti-RSPO antibodies

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RSPO Signaling in Cancer

ASCL2 AXIN2 CLDN2 LGR5 0.3 0.25 4 0.8

0.20 3 0.6 0.2 0.15 2 0.4 0.10 0.1 1 0.2 0.05 Gene expression (/GAPDH) Gene expression (/GAPDH) Gene expression (/GAPDH) 0.0 Gene expression (/GAPDH) 0.00 0 0.0 CR3150 CR2506 CR2513 CR1560 CR3150 CR2506 CR2513 CR1560 CR3150 CR2506 CR2513 CR1560 CR3150 CR2506 CR2513 CR1560

PTPRO RPRD1A TDGF1 ZNRF3 1.0 1.5 0.6 0.4

0.8 0.3 1.0 0.4 0.6 0.2 0.4 0.5 0.2 0.1 0.2 Gene expression (/GAPDH) Gene expression (/GAPDH) Gene expression (/GAPDH) Gene expression (/GAPDH) 0.0 0.0 0.0 0.0 CR3150 CR2506 CR2513 CR1560 CR3150 CR2506 CR2513 CR1560 CR3150 CR2506 CR2513 CR1560 CR3150 CR2506 CR2513 CR1560

Control antibody Anti-RSPO3

Figure 6. Downregulation of select Wnt target genes in anti–RSPO3-treated colorectal cancer xenografts. ASCL2, AXIN2, CLDN2, LGR5, PTPRO, RPRD1A, TDGF1,and ZNRF3 genes were analyzed for expression levels in colorectal cancer xenografts CR3150, CR2506, CR2513, and CR1560 treated with control antibody (small plaid pattern bars) or anti-RSPO3 (large plaid pattern bars). Only high quality RNA samples were included in the analysis, resulting in the following sample sizes: n ¼ 15 for control antibody in CR3150 and CR2506, n ¼ 14 for control antibody in CR2513 and CR1560, n ¼ 3 for anti-RSPO3 in CR3150, n ¼ 10 for anti-RSPO3 in CR2506, n ¼ 8foranti-RSPO3inCR2513,andn ¼ 9 for anti-RSPO3 in CR1560. Results were expressed as relative quantity over GAPDH endogenous control gene. P values were calculated using a t test with Welch correction and anti–RSPO3-induced decrease considered significant when below ,0.05,,0.01,or,0.001. alone and in combination with standard of care chemothera- which the antibody would inhibit tumor growth by decreasing peutic agents, we were able to significantly decrease the growth CSC frequency, a most desirable outcome for long-term cancer rate of RSPO3-positive lung and ovarian tumors that were therapies. CSCs are defined as the most tumorigenic subset identified in our cell-based screen. In addition, RSPO-expres- of malignant cells that supports tumor growth by conferring sing tumors with no detectable ex vivo b-catenin–activating stem cell properties, including self-renewal and multipotency properties, were also shown to respond to antibody-mediated to tumor cells (49). The major impact of RSPO on stem cells is anti-RSPO treatment. Anti-RSPO1 was shown to inhibit the in thought to be due to RSPO potentiation of the Wnt/b-catenin vivo growth of an RSPO1-high ovarian tumor and anti-RSPO2 signaling and thus, one hypothesis is that RSPO may have that of RSPO2-high colon and pancreas tumors. This suggests limited signaling activity in absence of WNT ligands (4, 50). that the reporter assay used to monitor tumor cell–derived In this model, tumors may achieve enhanced b-catenin signal- RSPO activity is not sensitive enough to allow for detecting low ing by elevated RSPO, which functions to potentiate low levels of functional RSPO or that a b-catenin–independent levels of WNT that are present in the tumor microenvironment, function of RSPO is at play in these models. Our results also rather than by substantial upregulation of the expression of suggest that gene-expression analysis may be sufficient to WNT family members. Thus, it appears that, in many tumors, a identify tumors that respond to anti-RSPO therapy. According- synergistic potentiation of b-catenin function is mediated by ly, three xenograft models in which no RSPO gene expression tumor-derived RSPO and blocking RSPO represents a most wasdetectedfailedtorespondtoantibody-mediatedRSPO efficient therapeutic approach in these tumors. inhibition. Testing additional models will be required to further defining minimal RSPO gene-expression levels associated with anti-RSPO treatment sensitivity. Finally, the identification Disclosure of Potential Conflicts of Interest fl of an anti-RSPO2 antibody-resistant lung tumor and an anti– No potential con icts of interest were disclosed. RSPO3-resistant colon tumor despite respective high RSPO2 and RSPO3 expression indicates that not all instances of high Authors' Contributions RSPO expression may reflect tumor dependence upon this Conception and design: C. Chartier, C. Bond, J. Cain, M.M. Fischer, W.-C. Yen, signaling axis. Of note, RSPO4 was not tested as a therapeutic A.M. Kapoun, J. Lewicki, T. Hoey, A. Gurney target in this work. However, its relative prevalence across the Development of methodology: C. Chartier, F. Axelrod, C. Bond, M.M. Fischer, OMP tumor bank supports evaluating RSPO4 in future studies. B. Cancilla Overall, the comprehensive set of in vivo efficacy data presented Acquisition of data (provided animals, acquired and managed patients, here demonstrates the therapeutic value of RSPO blockade to provided facilities, etc.): C. Chartier, J. Raval, F. Axelrod, C. Bond, M.M. Fischer, treat RSPO-expressing tumors. J. Shah, J. Wei, M. Ji, A. Lam, M. Stroud, W.-C. Yen, P. Yeung, B. Cancilla – Analysis and interpretation of data (e.g., statistical analysis, biostatistics, In anti RSPO3-sensitive colorectal cancer models, the sig- computational analysis): C. Chartier, J. Raval, C. Bond, J. Cain, S. Ma, nificant downregulation of target genes associated with both M.M. Fischer, J. Shah, J. Wei, M. Ji, W.-C. Yen, B. Cancilla, G. O'Young, normal stem cells and CSCs supports a mechanism of action by M. Wang, A.M. Kapoun, T. Hoey, A. Gurney

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Writing, review, and/or revision of the manuscript: C. Chartier, J. Cain, ence for their assistance. Research funding was provided by OncoMed M.M. Fischer, W.-C. Yen, A.M. Kapoun, J. Lewicki, T. Hoey, A. Gurney Pharmaceuticals. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Chartier, S. Ma, G. O'Young, T. Hoey Study supervision: C. Chartier, C. Bond, P. Yeung, T. Hoey Grant Support Other (as the lead in the in vivo experiment): C. Dee-Hoskins Research funding was provided by OncoMed Pharmaceuticals. The costs of publication of this article were defrayed in part by the payment of Acknowledgments page charges. This article must therefore be hereby marked advertisement in The authors thank many people at OncoMed Pharmaceuticals for their accordance with 18 U.S.C. Section 1734 solely to indicate this fact. contributions to this work, including Peter Stathis, Ian Scott, Esohe Idu- sogie, Jim Evans, Xiaomei Song, Diane Pardi, and Michael Mulkerrin. Received February 25, 2015; revised October 12, 2015; accepted November 5, The authors thank Zhun Wang, Jie Cai, and Henry Li from Crown Biosci- 2015; published OnlineFirst December 30, 2015.

References 1. Wang J, Sinha T, Wynshaw-Boris A. Wnt signaling in mammalian devel- 19. Carmon KS, Gong X, Yi J, Thomas A, Liu Q. RSPO-LGR4 functions via opment: lessons from mouse genetics. Cold Spring Harb Perspect Biol IQGAP1 to potentiate Wnt signaling. Proc Natl Acad Sci U S A 2014;111: 2012;4:pii: a007963. E1221–9. 2. Clevers H, Nusse R. Wnt/b-catenin signaling and disease. Cell 2012; 20. Schuijers J, Clevers H. Adult mammalian stem cells: the role of Wnt, Lgr5, 149:1192–205. and R-spondins. EMBO J 2012;31:2685–96. 3. Polakis P. Wnt signaling in cancer. Cold Spring Harb Perspect Biol 2012;4: 21. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et al. pii: a008052. Single Lgr5 stem cells build crypt-villus structures in vitro without a 4. Kazanskaya O, Glinka A, del Barco Barrantes I, Stannek P, Niehrs C, mesenchymal niche. Nature 2009;459:262–5. Wu W. R-Spondin2 is a secreted activator of Wnt/beta-catenin 22. Jin YR, Yoon JK. The R-spondin family of proteins: emerging regulators of signaling and is required for Xenopus myogenesis. Dev Cell 2004;7: WNT signaling. Int J Biochem Cell Biol 2012;44:2278–87. 525–34. 23. Friedman MS, Oyserman SM, Hankenson KD. Wnt11 promotes osteoblast 5. Ohkawara B, Glinka A, Niehrs C. Rspo3 binds syndecan 4 and induces maturation and mineralization through R-spondin 2. J Biol Chem 2009; Wnt/PCP signaling via clathrin-mediated endocytosis to promote morpho- 284:14117–25. genesis. Dev Cell 2011;20:303–14. 24. Mulvaney JF, Yatteau A, Sun WW, Jacques B, Takubo K, Suda T, et al. 6. Carmon KS, Gong X, Lin Q, Thomas A, Liu Q. R-spondins function as Secreted factor R-Spondin 2 is involved in refinement of patterning of ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta- the mammalian cochlea. Dev Dyn 2013;242:179–88. catenin signaling. Proc Natl Acad Sci U S A 2011;108:11452–7. 25. Klauzinska M, Baljinnyam B, Raafat A, Rodriguez-Canales J, Strizzi L, Greer 7. de Lau W, Barker N, Low TY, Koo BK, Li VS, Teunissen H, et al. Lgr5 YE, et al. Rspo2/Int7 regulates invasiveness and tumorigenic properties of homologues associate with Wnt receptors and mediate R-spondin signal- mammary epithelial cells. J Cell Physiol 2012;227:1960–71. ling. Nature 2011;476:293–7. 26. Lowther W, Wiley K, Smith GH, Callahan R. A new common integration 8. Glinka A, Dolde C, Kirsch N, Huang YL, Kazanskaya O, Ingelfinger D, et al. site, Int7, for the mouse mammary tumor virus in mouse mammary tumors LGR4 and LGR5 are R-spondin receptors mediating Wnt/beta-catenin and identifies a gene whose product has furin-like and thrombospondin-like Wnt/PCP signalling. EMBO Rep 2011;12:1055–61. sequences. J Virol 2005;79:10093–6. 9. Hao HX, Xie Y, Zhang Y, Charlat O, Oster E, Avello M, et al. ZNRF3 27. Theodorou V, Kimm MA, Boer M, Wessels L, Theelen W, Jonkers J, promotes Wnt receptor turnover in an R-spondin-sensitive manner. et al. MMTV insertional mutagenesis identifies genes, gene families Nature 2012;485:195–200. and pathways involved in mammary cancer. Nat Genet 2007;39: 10. Koo BK, Spit M, Jordens I, Low TY, Stange DE, van de Wetering M, et al. 759–69. Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis 28. Callahan R, Mudunur U, Bargo S, Raafat A, McCurdy D, Boulanger C, et al. of Wnt receptors. Nature 2012;488:665–9. Genes affected by mouse mammary tumor virus (MMTV) proviral inser- 11. Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, et al. tions in mouse mammary tumors are deregulated or mutated in primary Identification of stem cells in small intestine and colon by marker gene human mammary tumors. Oncotarget 2012;3:1320–34. Lgr5. Nature 2007;449:1003–7. 29. Seshagiri S, Stawiski EW, Durinck S, Modrusan Z, Storm EE, Conboy CB, 12. Jaks V, Barker N, Kasper M, van Es JH, Snippert HJ, Clevers H, et al. Lgr5 et al. Recurrent R-spondin fusions in colon cancer. Nature 2012;488: marks cycling, yet long-lived, hair follicle stem cells. Nat Genet 2008;40: 660–4. 1291–9. 30. Watson AL, Rahrmann EP, Moriarity BS, Choi K, Conboy CB, Greeley AD, 13. Barker N, Huch M, Kujala P, van de Wetering M, Snippert HJ, van Es JH, et al. Canonical Wnt/beta-catenin signaling drives human schwann cell et al. Lgr5(þve) stem cells drive self-renewal in the stomach and build long- transformation, progression, and tumor maintenance. Cancer Discov lived gastric units in vitro. Cell Stem Cell 2010;6:25–36. 2013;3:674–89. 14. Brzeszczynska J, Ramaesh K, Dhillon B, Ross JA. Molecular profile of organ 31. Shinmura K, Kahyo T, Kato H, Igarashi H, Matsuura S, Nakamura S, et al. culture-stored corneal epithelium: LGR5 is a potential new phenotypic RSPO fusion transcripts in colorectal cancer in Japanese population. marker of residual human corneal limbal epithelial stem cells. Int J Mol Mol Biol Rep 2014;41:5375–84. Med 2012;29:871–6. 32. Gong X, Yi J, Carmon KS, Crumbley CA, Xiong W, Thomas A, et al. Aberrant 15. de Visser KE, Ciampricotti M, Michalak EM, Tan DW, Speksnijder EN, Hau RSPO3-LGR4 signaling in Keap1-deficient lung adenocarcinomas pro- CS, et al. Developmental stage-specific contribution of LGR5(þ) cells to motes tumor aggressiveness. Oncogene 2015;34:4692–701. basal and luminal epithelial lineages in the postnatal mammary gland. 33. Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, Cho RW, et al. Phenotypic J Pathol 2012;228:300–9. characterization of human colorectal cancer stem cells. Proc Natl Acad Sci 16. Plaks V, Brenot A, Lawson DA, Linnemann JR, Van Kappel EC, Wong KC, U S A 2007;104:10158–63. et al. Lgr5-expressing cells are sufficient and necessary for postnatal 34. Dylla SJ, Beviglia L, Park IK, Chartier C, Raval J, Ngan L, et al. Colorectal mammary gland organogenesis. Cell Rep 2013;3:70–8. cancer stem cells are enriched in xenogeneic tumors following chemother- 17. Huch M, Dorrell C, Boj SF, van Es JH, Li VS, van de Wetering M, et al. In vitro apy. PLoS ONE 2008;3:e2428. þ expansion of single Lgr5 liver stem cells induced by Wnt-driven regen- 35. Rothe C, Urlinger S, Lohning C, Prassler J, Stark Y, Jager U, et al. The human eration. Nature 2013;494:247–50. combinatorial antibody library HuCAL GOLD combines diversification of 18. Yee KK, Li Y, Redding KM, Iwatsuki K, Margolskee RF, Jiang P. Lgr5-EGFP all six CDRs according to the natural immune system with a novel display marks taste bud stem/progenitor cells in posterior tongue. Stem Cells method for efficient selection of high-affinity antibodies. J Mol Biol 2008; 2013;31:992–1000. 376:1182–200.

OF10 Cancer Res; 76(3) February 1, 2016 Cancer Research

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36. Van der Flier LG, Sabates-Bellver J, Oving I, Haegebarth A, De Palo M, Anti 43. Gurney A, Axelrod F, Bond CJ, Cain J, Chartier C, Donigan L, et al. Wnt M, et al. The Intestinal Wnt/TCF Signature. Gastroenterology 2007;132: pathway inhibition via the targeting of Frizzled receptors results in 628–32. decreased growth and tumorigenicity of human tumors. Proc Natl Acad 37. Kim M, Kim H, Jho EH. Identiﬁcation of ptpro as a novel target gene of Wnt Sci U S A 2012;109:11717–22. signaling and its potential role as a receptor for Wnt. FEBS Lett 2010; 44. Hoey T, Yen WC, Axelrod F, Basi J, Donigian L, Dylla S, et al. DLL4 blockade 584:3923–8. inhibits tumor growth and reduces tumor-initiating cell frequency. 38. Zhu R, Yang Y, Tian Y, Bai J, Zhang X, Li X, et al. Ascl2 knockdown results in Cell Stem Cell 2009;5:168–77. tumor growth arrest by miRNA-302b-related inhibition of colon cancer 45. Yen WC, Fischer MM, Axelrod F, Bond C, Cain J, Cancilla B, et al. Targeting progenitor cells. PLoS ONE 2012;7:e32170. Notch signaling with a Notch2/Notch3 antagonist (tarextumab) inhibits 39. Barker N, Ridgway RA, van Es JH, van de Wetering M, Begthel H, van den tumor growth and decreases tumor-initiating cell frequency. Clin Cancer Born M, et al. Crypt stem cells as the cells-of-origin of intestinal cancer. Res 2015;21:2084–95. Nature 2009;457:608–11. 46. Nusse R. Wnt signaling. Cold Spring Harb Perspect Biol 2012;4: 40. da Silva-Diz V, Sole-Sanchez S, Valdes-Gutierrez A, Urpi M, Riba-Artes D, a011163. Penin RM, et al. Progeny of Lgr5-expressing hair follicle stem cell con- 47. Schepers A, Clevers H. Wnt signaling, stem cells, and cancer of the tributes to papillomavirus-induced tumor development in epidermis. gastrointestinal tract. Cold Spring Harb Perspect Biol 2012;4:a007989. Oncogene 2013;32:3732–43. 48. Baarsma HA, Konigshoff M, Gosens R. The WNT signaling pathway from 41. Steg AD, Bevis KS, Katre AA, Ziebarth A, Dobbin ZC, Alvarez RD, et al. Stem ligand secretion to gene transcription: molecular mechanisms and phar- cell pathways contribute to clinical chemoresistance in ovarian cancer. Clin macological targets. Pharmacol Ther 2013;138:66–83. Cancer Res 2012;18:869–81. 49. Clevers H. The cancer stem cell: premises, promises, and challenges. 42. Liu YS, Hsu HC, Tseng KC, Chen HC, Chen SJ. Lgr5 promotes cancer Nat Med 2011;17:313–9. stemness and confers chemoresistance through ABCB1 in colorectal cancer. 50. Krausova M, Korinek V. Wnt signaling in adult intestinal stem cells and Biomed Pharmacother 2013;67:791–9. cancer. Cell Signal 2014;26:570–9.

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Therapeutic Targeting of Tumor-Derived R-Spondin Attenuates β-Catenin Signaling and Tumorigenesis in Multiple Cancer Types

Cecile Chartier, Janak Raval, Fumiko Axelrod, et al.

Cancer Res Published OnlineFirst December 30, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-0561

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