Targeting of BMI-1 with PTC-209 shows potent anti-myeloma activity and impairs the tumour microenvironment

Abstract

BackgroundThe 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.MethodsBMI-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.ResultsWe 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0247-4) contains supplementary material, which is available to authorized users.

Bmi1 – A Path to Targeting Cancer Stem Cells

“…More importantly, diminished Bmi1 levels reduced the self-renewal capacity of CSCs, which in turn correlated with lowered tumourigenic potential in vitro and in vivo. In keeping with the original observations, cells treated with a Bmi1 inhibitor became more apoptotic 77,[81][82][83] and underwent cell cycle arrest at G 0 phase. 81,82 Collectively, these studies have provided a strong rationale for including Bmi1-targeted therapy in the treatment strategies for patients presenting with malignancies displaying elevated Bmi1 expression.…”

“…Mechanistic insight into kinases involved in post-translational modification of Bmi1 came from the yeast two-hybrid interaction assay, which identified MAPKAP kinase 3pK as a regulator of Bmi1 chromatin association, among other PcG proteins. 80 In recent years, small molecule inhibitors developed by PTC Therapeutics ( South Plainfield, NJ, US), were designed to promote phosphorylation of Bmi1, and have been tested in colorectal carcinomas, 81 lung adenocarcinomas, 82 multiple myeloma (MM), 83 prostate cancer 77 and medulloblastoma (unpublished data). In all cases, small molecule inhibitor resulted in decreased Bmi1 protein levels and reduced activity of the PRC1 complex.…”

Epigenetic‐Based Mechanisms of Osteoblast Suppression in Multiple Myeloma Bone Disease

“…Further, BMI1 shRNA was effective in reducing tumor growth of MM xenografts in mice . Anti‐MM activity of PTC209 was significant even in the presence of MM growth factors insulin‐like growth factor 1 (IGF1) and IL6 as well as in cocultures with BMSCs . In the same study, PTC209 impaired OB formation in a dose‐dependent manner.…”

“…BMI1 inhibition, using a small molecule inhibitor, PTC209, induced apoptosis in MM cell lines and primary MM cells in vitro . PTC209 exhibited synergistic and additive anti‐MM effects when combined with pomalidomide and carfilzomib, as well as EZH2 and BET‐targeting epigenetic inhibitors . Further, BMI1 shRNA was effective in reducing tumor growth of MM xenografts in mice .…”

Update on the role and therapeutic potential of polycomb repressive complexes in (biliary tract) cancer

“…Bolomsky et al found that BMI1 was overexpressed in multiple myeloma (MM) patients and associated with short overall survival [10]. PTC-209 reduced BMI1 levels and number of viable MM cells in vitro.…”