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Effective Inhibition of Bone Morphogenetic Protein Function By Published OnlineFirst September 8, 2015; DOI: 10.1158/1535-7163.MCT-14-0956 Small Molecule Therapeutics Molecular Cancer Therapeutics Effective Inhibition of Bone Morphogenetic Protein Function by Highly Specific Llama-Derived Antibodies Silvia Calpe1, Koen Wagner2, Mohamed El Khattabi3, Lucy Rutten3, Cheryl Zimberlin4, Edward Dolk3, C. Theo Verrips3, Jan Paul Medema4, Hergen Spits2, and Kausilia K. Krishnadath1,5 Abstract Bone morphogenetic proteins (BMP) have important but signaling. Epitope binning and docking modeling have shed distinct roles in tissue homeostasis and disease, including light into the basis for their BMP specificity. As opposed to the carcinogenesis and tumor progression. A large number of BMP wide structural reach of natural inhibitors, these small mole- inhibitors are available to study BMP function; however, as culestargetthegroovesandpocketsofBMPsinvolvedin most of these antagonists are promiscuous, evaluating specific receptor binding. In organoid experiments, specific inhibition effectsofindividualBMPsisnotfeasible.Becausetheonco- of BMP4 does not affect the activation of normal stem cells. genic role of the different BMPs varies for each neoplasm, Furthermore, in vitro inhibition of cancer-derived BMP4 non- highly selective BMP inhibitors are required. Here, we describe canonical signals results in an increase of chemosensitivity the generation of three types of llama-derived heavy chain in a colorectal cancer cell line. Therefore, because of their variable domains (VHH) that selectively bind to either BMP4, high specificity and low off-target effects, these VHHs could þ to BMP2 and 4, or to BMP2, 4, 5, and 6. These generated VHHs represent a therapeutic alternative for BMP4 malignancies. have high affinity to their targets and are able to inhibit BMP Mol Cancer Ther; 14(11); 2527–40. Ó2015 AACR. Introduction ical intracellular effectors (3, 4). Translocation to the nucleus of these transcription factors initiates transcription of BMP target Bone morphogenetic proteins (BMP) are growth factors that genes (5, 6). belong to the TGFb superfamily. They consist of around 20 BMPs are emerging as attractive therapeutic targets due to members, classified into distinct subfamilies, depending on their multiple roles in a variety of diseases, in particular cancer. their sequence homology and functionality. BMP2, BMP4 and For many years, the BMP field has been dogged by the assump- BMP5, BMP6, BMP7 form two subgroups that have been tion that many BMPs are functionally interchangeable. How- extensively studied (1). They mediate their function by binding ever, there is increasing evidence indicating the disparate to two molecules of type 1 (BMPR1 ,BMPR1 , or ACVR1) and a b actions of BMPs (7). For instance, whereas BMP4 and BMP2 two molecules of type 2 (BMPR2, ACVRL1, ActRII, or ActRII ) B seem to facilitate metastasis and invasion in several cancers, serine/threonine kinase receptors (2). Depending on the pres- BMP6 and BMP7 present a suppressive role in metastatic breast ence or absence of preformed BMPR complexes, the signaling is cancer and melanomas (8). Most intriguing is the role of BMP4, mediated via the SMAD family of transcription factors follow- which presents opposing roles depending on the neoplasm ingadefined canonical pathway or by less defined noncanon- (7, 9). In certain malignancies, such as glioblastoma (10) and myeloma (11), high levels of BMP4 are associated with less malignant features while in breast (12), ovarian (13), lung 1Center for Experimental and Molecular Medicine, Academic Medical (14), and prostate (15), BMP4 is involved in epithelial–mes- Center, Amsterdam, the Netherlands. 2AIMM Therapeutics, Academic enchymal transition (EMT) and metastatic behavior. In gastro- 3 Medical Center, Amsterdam, the Netherlands. QVQ BV, Utrecht, the intestinal cancers such as pancreas (16), colon (17), and gastric Netherlands. 4Laboratory for Experimental Oncology and Radiobiol- ogy, Center for Experimental Molecular Medicine, Academic Medical cancer (18), BMP4 has been associated with tumor progression Center, University of Amsterdam, Amsterdam, the Netherlands. and chemotherapy resistance. Therefore, to resolve and inhibit 5 Department of Gastroenterology and Hepatology, Academic Medical the individual action of BMP4 in tumors, the generation of Center, Amsterdam, the Netherlands. potent and specific BMP4 inhibitors is warranted. Note: Supplementary data for this article are available at Molecular Cancer Targeting and regulating BMP signaling at the extracellular Therapeutics Online (http://mct.aacrjournals.org/). receptor level is clinically highly attractive (19). Neutralization Corresponding Author: Kausilia K. Krishnadath, Academic Medical Center, of BMP by several natural antagonists and chemical inhibitors Meibergdreef 9, Amsterdam 1105AZ, the Netherlands. Phone: 31-0- has already been proven successful for the suppression of 205666703; Fax: 31-0-206917033; E-mail: [email protected] metastasis in prostate (15), lung (14), as well as breast cancer doi: 10.1158/1535-7163.MCT-14-0956 (20). Albeit significant progress has been made in generating Ó2015 American Association for Cancer Research. highly specificandlesstoxicsmall-moleculeinhibitorsthat www.aacrjournals.org 2527 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst September 8, 2015; DOI: 10.1158/1535-7163.MCT-14-0956 Calpe et al. target BMP type I receptors (21–23), these inhibitors are with 100 ng/mL of BMP2, BMP4, BMP5, BMP6, or BMP7 (R&D still nonselective. Therefore, the clinical use of current BMP Systems) for 4 hours with or without VHHs or Noggin at the inhibitors is questionable (24). For instance, in vivo treatment indicated concentrations. C2C12 mouse myoblasts (stably trans- with the natural antagonist Coco or the chemical inhibitor fected with a reporter plasmid for BMP activity) were a kind LDN-193189 results in reactivation of breast cancer (25) or gift of Dr. L. Zilberberg and Dr. D. Rifkin (ref. 30; New York colon tumor burden (26) in mice with a particular mutatio- University, School of Medicine, New York, NY). CL-11 and nal phenotype, respectively. Therefore, these off-target effects Colo-678 were obtained from the COSMIC project at the Sanger could lead to undesirable side effects if used in the clinic. Institute, UK. The rest of the cell lines were obtained from ATCC. Llama-derived antibodies are small proteins of about 15 kDa All cell lines were obtained more than 6 months ago, and no that, as opposed to conventional antibodies, their antigen-bind- authentication has been performed by the authors. ing fragment is formed by just the variable domain of the heavy chain, hence their denomination as VHHs (27). Because of their Western blot unique structure, VHHs can bind specifically and with high Protein isolation and SDS-PAGE separation were performed as affinity to their antigens, often due to their ability to bind to described previously (31). Briefly, EPC2-hTERT and organoids hidden epitopes within grooves or cavities. This peculiar charac- were lysed with 100 mL of M-PER buffer (Sigma-Aldrich), whereas teristic was leveraged to design specific and highly effective the mouse organoids were lysed at a ratio of 1:20 with T-PER inhibitors of BMP4. In the present study, we describe the char- buffer (Sigma-Aldrich). Cell debris were pelleted and supernatant acterization of five anti-BMP4 llama–derived VHHs. Specificity to was collected. For protein detection, the following antibodies different BMPs allowed their classification into 3 groups: VHHs were used: antiphospho-SMAD1/5/8 at 1:500; p-p38 (T180/ specific for BMP4; BMP2, 4; and BMP2, 4, 5, 6. We demonstrate Y182) at 1:500, pAkt (ser473) at 1:1,000, total Akt at 1:500, total that their binding affinities are greater than those published for p38 at 1:500 (Cell Signaling Technology); anti-b-actin at 1:1,000, Noggin, a natural BMP antagonist, and for conventional anti- anti-SMAD4 at 1:500, anti-pERK (Y204) at 1:1,000 (Santa Cruz); BMP4 antibodies. We show that the VHHs effectively inhibit BMP anti-GAPDH at 1:500 (Millipore); anti-BMP7 MAB3541 and anti- downstream signaling and transcriptional activation of BMP BMP6 AF507 (R&D Systems) at 1:1,000; and anti-BMP5 ab10858 target genes. Our epitope and docking experiments, as well as (Abcam), anti-BMP2 500-P195 (PeproTech), and anti-BMP4 mutagenesis analyses, provide structural evidence for their MAB757 (R&D Systems) all at 1:500. affinity and specificity. These studies show that each VHH binds to a different molecular interface within the BMP molecule and, BMP activity luciferase reporter assay subsequently, unveil the binding regions that shape BMP spec- C2C12 cells were plated in 96-well plates at 5 Â 103 cells per ificity. Finally, we show that our VHHs inhibit endogenous BMP well, and cells were allowed to attach overnight. DMEM (100 mL) function as they can replace Noggin in ex vivo organoid cultures with 0.1% BSA was added in each well. Cells were treated in þ as well as increase in vitro chemosensitivity of BMP4 colorectal triplicate with BMPs, VHHs, or controls at the indicated concen- cancer cells. trations for 16 hours. Wells with unstimulated cells or no cells were added as controls. Luciferase activity was measured by adding 100 mL of luciferase substrate solution from the Bright- Materials and Methods Glo Luciferase Assay System (Promega Benelux). After 3 minutes Generation of llama-derived anti-BMP4 VHHs of incubation, luciferase activity
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