title: Targeting of BMI-1 with PTC-209 shows potent anti-myeloma activity and impairs the tumour microenvironment.
abstract: BACKGROUND
  The polycomb complex protein BMI-1 (BMI-1) is a putative oncogene reported to be overexpressed in multiple myeloma (MM). Silencing of BMI-1 was shown to impair the growth and survival of MM cells. However, therapeutic agents specifically targeting BMI-1 were not available so far. Here, we investigated PTC-209, a novel small molecule inhibitor of BMI-1, for its activity in MM.
  
  
  METHODS
  BMI-1 expression was analysed in human MM cell lines and primary MM cells by using publically available gene expression profiling (GEP) data. The anti-MM activity of PTC-209 was investigated by viability testing, cell cycle analysis, annexin V and 7-AAD staining, quantification of cleaved poly(ADP-ribose) polymerase (PARP), JC-1 as well as colony formation assays. Deregulation of central myeloma growth and survival genes was studied by quantitative PCR and flow cytometry, respectively. In addition, the impact of PTC-209 on in vitro osteoclast, osteoblast and tube formation was analysed.
  
  
  RESULTS
  We confirmed overexpression of BMI-1 in MM patients by using publically available GEP datasets. Of note, BMI-1 expression was further increased at relapse which translated into significantly shorter overall survival in relapsed/refractory patients treated with bortezomib or dexamethasone. Treatment with PTC-209 significantly decreased viable cell numbers in human MM cell lines, induced a G1 cell cycle arrest, promoted apoptosis and demonstrated synergistic activity with pomalidomide and carfilzomib. The anti-MM activity of PTC-209 was accompanied by a significant decrease of cyclin D1 (CCND1) and v-myc avian myelocytomatosis viral oncogene homolog (MYC) expression as well as upregulation of cyclin-dependent kinase inhibitor 1A (CDKN1A) and cyclin-dependent kinase inhibitor 1B (CDKN1B). We also observed upregulation of NOXA (up to 3.6 ± 1.2-fold induction, P = 0.009) and subsequent downregulation of myeloid cell leukemia 1 (MCL-1) protein levels, which likely mediates the apoptotic effects of PTC-209. Importantly, the anti-MM activity was upheld in the presence of stromal support or myeloma growth factors insulin-like growth factor 1 (IGF-1) and interleukin 6 (IL-6). In the MM microenvironment, PTC-209 impaired tube formation, impaired osteoclast development and decreased osteoblast formation in a dose-dependent manner (P < 0.01 at 1 μM, respectively). The latter might be attributed to an induction of DKK1 and was reversed by concurrent anti-DKK1 antibody treatment.
  
  
  CONCLUSIONS
  We confirmed overexpression of BMI-1 in MM highlighting its role as an attractive drug target and reveal therapeutic targeting of BMI-1 by PTC-209 as a promising novel therapeutic intervention for MM.
authors.affiliation: Wilhelminen Cancer Research Institute, Department of Medicine I, Wilhelminenspital, Montleartstraße 37, 1160, Vienna, Austria. arnold.bolomsky@extern.wienkav.at.
authors.forename: Arnold
authors.initials: A
authors.lastname: Bolomsky
authors.affiliation: Center for Medical Statistics, Informatics and Intelligent Systems, Medical University of Vienna, Vienna, Austria. karin.schlangen@meduniwien.ac.at.
authors.forename: Karin
authors.initials: K
authors.lastname: Schlangen
authors.affiliation: Center for Medical Statistics, Informatics and Intelligent Systems, Medical University of Vienna, Vienna, Austria. wolfgang.schreiner@meduniwien.ac.at.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Schreiner
authors.affiliation: Wilhelminen Cancer Research Institute, Department of Medicine I, Wilhelminenspital, Montleartstraße 37, 1160, Vienna, Austria. niklas.zojer@wienkav.at.
authors.forename: Niklas
authors.initials: N
authors.lastname: Zojer
authors.affiliation: Wilhelminen Cancer Research Institute, Department of Medicine I, Wilhelminenspital, Montleartstraße 37, 1160, Vienna, Austria. heinz.ludwig@wienkav.at.
authors.forename: Heinz
authors.initials: H
authors.lastname: Ludwig
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D004357:Drug Synergism; D020869:Gene Expression Profiling / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006574:Heterocyclic Compounds, 2-Ring / Q000494:pharmacology / Q000627:therapeutic use*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D006801:Humans; D009101:Multiple Myeloma / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects / Q000235:genetics; D009842:Oligopeptides / Q000494:pharmacology; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics; D063150:Polycomb Repressive Complex 1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013792:Thalidomide / Q000031:analogs & derivatives / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology / Q000627:therapeutic use*; D059016:Tumor Microenvironment / Q000187:drug effects* / Q000235:genetics
chemical_list: C069077:BMI1 protein, human; D006574:Heterocyclic Compounds, 2-Ring; D009842:Oligopeptides; C586999:PTC-209; D013844:Thiazoles; D013792:Thalidomide; C524865:carfilzomib; D003907:Dexamethasone; C467566:pomalidomide; D063150:Polycomb Repressive Complex 1