Connectivity Map-based discovery of parbendazole reveals targetable human osteogenic pathway Andrea M. Bruma, Jeroen van de Peppela, Cindy S. van der Leijea, Marijke Schreuders-Koedama, Marco Eijkenb, Bram C. J. van der Eerdena, and Johannes P. T. M. van Leeuwena,1 aDepartment of Internal Medicine, Erasmus MC, 3015 CN Rotterdam, The Netherlands; and bArcarios BV, 3015 CN Rotterdam, The Netherlands Edited by John T. Potts, Massachusetts General Hospital, Charlestown, MA, and approved September 4, 2015 (received for review January 26, 2015) Osteoporosis is a common skeletal disorder characterized by low molecules to a user’s selected gene signature of the phenotype bone mass leading to increased bone fragility and fracture suscepti- of interest using a pattern-matching algorithm with a high level bility. In this study, we have identified pathways that stimulate of resolution and specificity. The screening results in a list of differentiation of bone forming osteoblasts from human mesenchy- compounds with a highly correlating gene expression pattern to mal stromal cells (hMSCs). Gene expression profiling was performed that of the phenotype of interest, which has the potential to aid in hMSCs differentiated toward osteoblasts (at 6 h). Significantly in finding a novel treatment for a disease or to identify novel regulated genes were analyzed in silico, and the Connectivity Map pathways or genes involved in a complex biological process. To (CMap) was used to identify candidate bone stimulatory com- date, the CMap has been successfully used to identify compounds pounds. The signature of parbendazole matches the expression and combination therapies that show promise in the treatment of changes observed for osteogenic hMSCs. Parbendazole stimulates osteoarthritic pain (5), adenocarcinoma (6), kidney disease (7), osteoblast differentiation as indicated by increased alkaline phospha- gliomas (8), and NK cell neoplasms (9). tase activity, mineralization, and up-regulation of bone marker genes Our aim was to identify previously unidentified anabolic ALPL SPP1 (alkaline phosphatase/ , osteopontin/ , and bone sialoprotein therapeutic targets by genomic, proteomic, and bioinformatic IBSP II/ ) in a subset of the hMSC population resistant to the apoptotic dissection of human mesenchymal stromal cell (hMSC)-derived effects of parbendazole. These osteogenic effects are independent of osteoblasts. Therefore, we used the CMap to identify compounds glucocorticoids because parbendazole does not up-regulate gluco- with a matching gene expression profile to human mesenchymal corticoid receptor (GR) target genes and is not inhibited by the GR stem cells undergoing osteogenic differentiation. By following antagonist mifepristone. Parbendazole causes profound cytoskel- this approach, we aimed to not only discover novel compounds etal changes including degradation of microtubules and increased that stimulate osteogenic differentiation, but also novel pro- focal adhesions. Stabilization of microtubules by pretreatment with cesses underlying this process. Taxol inhibits osteoblast differentiation. Parbendazole up-regulates bone morphogenetic protein 2 (BMP-2) gene expression and activity. Results Cotreatment with the BMP-2 antagonist DMH1 limits, but does Parbendazole Has the Strongest Correlating Gene Signature to hMSCs not block, parbendazole-induced mineralization. Using the CMap Undergoing Osteoblast Differentiation. Using a pattern-matching we have identified a previously unidentified lineage-specific, bone anabolic compound, parbendazole, which induces osteo- algorithm, the CMap links compounds with disease or physio- genic differentiation through a combination of cytoskeletal changes logical phenotypes by measuring similarities in gene expression and increased BMP-2 activity. (3). To identify compounds that may exert bone anabolic effects due to their ability to stimulate genes regulated during osteoblast Connectivity Map | osteoblast | mesenchymal stem cell | cytoskeleton | osteoporosis Significance steoporosis is a common and devastating bone disease char- Osteoporosis, a disease characterized by increased bone fra- Oacterized by reduced bone mass and increased fragility and gility and fracture risk, affects over 20% of the ever-growing fracture risk. It has been estimated that an osteoporotic fracture elderly population. It is important to further our knowledge of occurs once every 8 s worldwide (1), and direct healthcare costs in bone cell biology so we can develop new bone anabolic Europe alone are at least V31.7 billion annually (2). Osteoporosis treatments. By combining genomic and bioinformatic tools means porous bones and occurs when bone remodeling is disrupted. against the backdrop of osteogenic differentiating human Bone remodeling is a balancing act between removal of old bone mesenchymal stromal cells, we have identified a previously and formation of new bone, which is achieved by two distinct cells, unidentified bone anabolic compound that induces osteoblast the osteoclast and osteoblast, respectively. When uncoupling of differentiation in a subset of the hMSC population through these two processes takes place, bone resorption can overtake bone cytoskeletal changes and increased bone morphogenetic pro- formation, resulting in osteoporosis. Most osteoporosis treatments, tein 2 activity. Through this novel approach we identified an such as bisphosphonates, reduce bone resorption and result in important mechanism of lineage allocation and demonstrated modest increases in bone density; however, these treatments do the significance of cytoskeletal organization in osteogenic not result in a true bone anabolic effect, so patients do not regain differentiation, providing us with a novel mechanism for bone formation to target for new osteoporosis treatments. bone that has been lost at time of diagnosis. An ideal treatment CELL BIOLOGY would stimulate bone formation as well, to help repair the damage Author contributions: A.M.B., J.v.d.P., M.E., B.C.J.v.d.E., and J.P.T.M.v.L. designed re- already done to the bone microarchitecture and strength; with this search; A.M.B., J.v.d.P., C.S.v.d.L., M.S.-K., and B.C.J.v.d.E. performed research; A.M.B., in mind, our goal was to search for previously unidentified J.v.d.P., M.S.-K., B.C.J.v.d.E., and J.P.T.M.v.L. analyzed data; and A.M.B., B.C.J.v.d.E., and molecules and/or mechanisms that stimulate human osteoblast J.P.T.M.v.L. wrote the paper. differentiation and bone formation. The authors declare no conflict of interest. The connectivity map (CMap) is a web-based tool that allows This article is a PNAS Direct Submission. for screening of compounds against a genome-wide disease or 1To whom correspondence should be addressed. Email: [email protected]. physiological gene signature (3, 4). This screening is achieved This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. by comparing microarray data from more than 1,300 small 1073/pnas.1501597112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1501597112 PNAS | October 13, 2015 | vol. 112 | no. 41 | 12711–12716 Downloaded by guest on September 28, 2021 Table 1. CMap permuted results showing compounds with FACS analysis to look at apoptosis and proliferation. Parbendazole significant positive correlation to osteogenic hMSCs gene increased apoptosis at day 5 and 8 of culture compared with con- signature trol-treated hMSCs (44.8–58.5 vs. 15.8–17.9%, respectively; Fig. A Rank Compound name Cell line Mean CMap score nPvalue 2 ). We also found that at days 5 and 8 of culture, parbendazole significantly increased proliferation compared with control- 1 Parbendazole PC3 0.855 2 <0.00001 treatment (24.7–26.1 vs. 9.1–10.4% Ki67+, respectively; Fig. 2B). 2 Dexamethasone PC3 0.913 2 <0.00001 To verify the overall viability of hMSCs treated with parbenda- 3 Fludrocortisone PC3 0.768 2 0.00002 zole, we performed a PrestoBlue assay, a metabolism-based as- 4 Halcinonide PC3 0.788 2 0.00002 say as a readout for cell viability. Parbendazole treatment led to 5 Fludroxycortide PC3 0.775 2 0.00002 a decrease in cell viability compared with both controls and dex- 6 Flumetasone PC3 0.854 2 0.00002 treated hMSCs starting at day 4 of culture (Fig. 2C). These 7 Flunisolide PC3 0.718 2 0.00002 findings reveal that parbendazole is capable of increasing both 8 Fluocinonide PC3 0.72 2 0.00002 proliferation and apoptosis in hMSCs, and because apoptosis (i.e., Top matching compounds from the Connectivity Map based on the increased cell death and decreased cell survival) exceeds prolif- average of the replicates of a single compound for each cell line. Shading eration conditions, the total accumulation of cells is reduced in indicates a glucocorticoid. Top matching compound and only nonglucocorti- parbendazole-treated cells. coid, parbendazole, in white. Rank is based on the P value calculated from the CMap scores all of the replicates of a single compound in a single cell Parbendazole Induces Osteoblast Differentiation Independent of line. Score is based on the relative strength of a given signature in an in- Glucocorticoid Receptor Signaling. To determine if parbendazole stance from the total set of instances calculated upon execution of a query. induces osteogenic differentiation through direct GR–mediated stimulation of osteoblast marker genes, similar to dex, we per- formed quantitative gene expression analyses for known GR differentiation, we performed a CMap analysis in which we searched target genes following parbendazole treatment.