Targeting of BMI-1 with PTC-209 shows potent anti-myeloma activity and impairs the tumour microenvironment.
Targeting of BMI-1 with PTC-209 shows potent anti-myeloma activity and impairs the tumour microenvironment.
复制标题
DOI：
10.1186/s13045-016-0247-4
复制
发表时间：
2016-03-02
影响因子：
28.5
通讯作者：
Ludwig H
中科院分区：
文献类型：
--
作者：
Bolomsky A;Schlangen K;Schreiner W;Zojer N;Ludwig H
多梳复合蛋白BMI-1（BMI-1）是一种推定的癌基因，据报道在多发性骨髓瘤（MM）中过表达。BMI-1的沉默显示损害MM细胞的生长和存活。然而，特异性靶向BMI-1的治疗剂迄今不可用。在这里，我们研究了PTC-209，一种新的BMI-1的小分子抑制剂，其在MM中的活性。BMI-1的表达在人MM细胞系和原代MM细胞中进行了分析，通过使用实验室可用的基因表达谱（GEP）数据。PTC-209的抗MM活性通过活力测试、细胞周期分析、膜联蛋白V和7-AAD染色、切割的聚（ADP-核糖）聚合酶（PARP）、JC-1的定量以及集落形成测定来研究。通过定量PCR和流式细胞术分别研究了中央骨髓瘤生长和存活基因的失调。此外，分析了PTC-209对体外破骨细胞、成骨细胞和管形成的影响。我们通过使用可获得的GEP数据集证实了MM患者中BMI-1的过表达。值得注意的是，BMI-1表达在复发时进一步增加，这导致用硼替佐米或地塞米松治疗的复发/难治性患者的总生存期显著缩短。用PTC-209处理显著降低人MM细胞系中的活细胞数，诱导G1细胞周期停滞，促进细胞凋亡，并显示与泊马度胺和卡非佐米的协同活性。PTC-209的抗MM活性伴随着细胞周期蛋白D1（CCND 1）和v-myc禽骨髓细胞瘤病病毒癌基因同源物（MYC）表达的显着降低，以及细胞周期蛋白依赖性激酶抑制剂1A（CDKN 1A）和细胞周期蛋白依赖性激酶抑制剂1B（CDKN 1B）的上调。我们还观察到NOXA的上调（高达3.6 ± 1.2倍诱导，P = 0.009）和随后髓样细胞白血病1（MCL-1）蛋白水平的下调，这可能介导PTC-209的凋亡作用。重要的是，在基质支持物或骨髓瘤生长因子胰岛素样生长因子1（IGF-1）和白细胞介素6（IL-6）存在下，抗MM活性得以维持。在MM微环境中，PTC-209以剂量依赖性方式损害管形成、损害破骨细胞发育和成骨细胞形成（分别为1 μM时P < 0.01）。后者可能归因于DKK 1的诱导，并通过同时抗DKK 1抗体治疗逆转。我们证实了BMI-1在MM中的过表达，突出了其作为有吸引力的药物靶点的作用，并揭示了PTC-209对BMI-1的治疗靶向作用，作为MM的一种有前途的新型治疗干预。本文的在线版本（doi：10.1186/s13045-016-0247-4）包含补充材料，可供授权用户使用。
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. 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. 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. 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. The online version of this article (doi:10.1186/s13045-016-0247-4) contains supplementary material, which is available to authorized users.