Background: The polycomb complex protein BMI-1 (BMI-1) is a putative oncogene reported to be overexpressed\nin multiple myeloma (MM). Silencing of BMI-1 was shown to impair the growth and survival of MM cells. However,\ntherapeutic agents specifically targeting BMI-1 were not available so far. Here, we investigated PTC-209, a novel\nsmall molecule inhibitor of BMI-1, for its activity in MM.\nMethods: BMI-1 expression was analysed in human MM cell lines and primary MM cells by using publically\navailable gene expression profiling (GEP) data. The anti-MM activity of PTC-209 was investigated by viability testing,\ncell cycle analysis, annexin V and 7-AAD staining, quantification of cleaved poly(ADP-ribose) polymerase (PARP), JC-1\nas well as colony formation assays. Deregulation of central myeloma growth and survival genes was studied by\nquantitative PCR and flow cytometry, respectively. In addition, the impact of PTC-209 on in vitro osteoclast,\nosteoblast and tube formation was analysed.\nResults: We confirmed overexpression of BMI-1 in MM patients by using publically available GEP datasets. Of note,\nBMI-1 expression was further increased at relapse which translated into significantly shorter overall survival in\nrelapsed/refractory patients treated with bortezomib or dexamethasone.\nTreatment with PTC-209 significantly decreased viable cell numbers in human MM cell lines, induced a G1 cell cycle\narrest, promoted apoptosis and demonstrated synergistic activity with pomalidomide and carfilzomib. The anti-MM\nactivity of PTC-209 was accompanied by a significant decrease of cyclin D1 (CCND1) and v-myc avian\nmyelocytomatosis viral oncogene homolog (MYC) expression as well as upregulation of cyclin-dependent kinase\ninhibitor 1A (CDKN1A) and cyclin-dependent kinase inhibitor 1B (CDKN1B). We also observed upregulation of NOXA\n(up to 3.6 �± 1.2-fold induction, P = 0.009) and subsequent downregulation of myeloid cell leukemia 1 (MCL-1)\nprotein levels, which likely mediates the apoptotic effects of PTC-209. Importantly, the anti-MM activity was upheld\nin the presence of stromal support or myeloma growth factors insulin-like growth factor 1 (IGF-1) and interleukin 6\n(IL-6).\nIn the MM microenvironment, PTC-209 impaired tube formation, impaired osteoclast development and decreased\nosteoblast formation in a dose-dependent manner (P < 0.01 at 1 �¼M, respectively). The latter might be attributed to\nan induction of DKK1 and was reversed by concurrent anti-DKK1 antibody treatment.\nConclusions: We confirmed overexpression of BMI-1 in MM highlighting its role as an attractive drug target and\nreveal therapeutic targeting of BMI-1 by PTC-209 as a promising novel therapeutic intervention for MM.
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