Cell adhesion-induced phosphorylation and inactivation of EZH2 confer drug resistance to acute myeloid leukemia cells.
Cell adhesion-induced phosphorylation and inactivation of EZH2 confer drug resistance to acute myeloid leukemia cells.
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细胞粘附诱导的 EZH2 磷酸化和失活赋予急性髓系白血病细胞耐药性。
DOI：
10.1007/s12185-017-2376-0
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发表时间：
2018
期刊：
影响因子：
2.1
通讯作者：
and Furukawa Y.
中科院分区：
文献类型：
--
作者：
Kikuchi J;Kuroda Y;Koyama D;and Furukawa Y.
在最近一期的《自然医学》杂志上，Göllner等人报道了组蛋白甲基转移酶EZH2的缺失通过抑制EZH2调节的基因，如HOXB7、HOXA9和ABCC1，赋予急性髓性白血病（AML）细胞多药耐药。在他们提出的EZH2失活机制中，cdk1介导的Thr487磷酸化触发E3泛素连接酶TRIM21的募集，从而诱导蛋白酶体依赖性的EZH2降解。在这种情况下，CDK1激活是AML细胞获得耐药性的初始和关键步骤。作者清楚地表明，HSP90稳定CDK1-EZH2复合物，促进EZH2磷酸化；但是，下列问题仍有待澄清。首先，CDK1如何在HSP90稳定后独立于细胞周期变化使EZH2磷酸化，而细胞周期变化是CDK1活性的主要决定因素？其次，耐药AML细胞中的EZH2磷酸化还涉及哪些其他机制？最近，我们发现EZH2在Ser21位点磷酸化，并在多发性骨髓瘤（MM）通过vla -4介导的与骨髓基质细胞（BMSCs）和/或细胞外基质[2]中的纤维连接蛋白相互作用获得耐药过程中失活。Matsunaga等人[[3]]也认为vla4与微量残留AML细胞的耐药有关，这表明vla4也是AML的预后标志物和治疗靶点。根据这些发现，我们假设vla -4介导的与骨髓间充质干细胞和/或纤维连接蛋白的相互作用激活CDK1和/或其他激酶，使AML细胞中的EZH2磷酸化，作为获得多药耐药表型的线索。为了验证这一假设，我们在纤维连接蛋白包被的培养皿中培养AML细胞系HL-60和MV4-11，并证实了对阿霉素（ADM）和阿糖胞嘧啶（Ara-C）的耐药性，IC50值显著增加（图1a）。利用该系统，我们研究了细胞粘附是否诱导AML细胞中EZH2 Ser21和/或Thr487位点磷酸化。如图1b所示，在HL-60和MV4-11细胞获得细胞粘附介导的耐药（CAM-DR）过程中，EZH2的Ser21位点很容易被磷酸化，而Thr487的磷酸化水平是组成型的，不受细胞粘附或药物处理的影响。我们证实，在AML细胞中，与纤维连接蛋白的结合以一种时间依赖性的方式增加了EZH2 Ser21位点的磷酸化水平，而不是Thr487位点。Ser21磷酸化先于或至少与Lys27 （H3K27）组蛋白H3甲基化水平的降低相吻合，这与它在EZH2失活中的作用一致（图1c）。我们之前的研究表明，VLA-4参与激活了PI3K-AKT激酶通路，该通路负责耐药MM细胞[2]中EZH2 Ser21位点的磷酸化。在本研究中，我们研究了该通路是否也在AML细胞的CAM-DR中起关键作用。PI3K-AKT激酶途径抑制剂在临床前研究中对AML细胞发挥细胞毒性，并在I/II期试验中作为单一疗法显示出一定的临床活性(http://www。临床试验。gov标识符NCT01396499和NCT02438761)。在MM细胞[2]中观察到，细胞粘附并没有减轻PI3K和AKT激酶特异性抑制剂LY294002和PF-04691502对HL-60和MV4-11细胞的作用（图1 d）。这些结果表明CAM-DR是通过PI3K/ akt介导的EZH2 Ser21位点磷酸化获得的，并且是Göllner等人描述的EZH2相关耐药的另一种机制。b[1]然而，这种机制的作用程度如何
In a recent issue of Nature Medicine, Göllner et al.[1] reported that loss of the histone methyltransferase EZH2 confers multidrug resistance to acute myeloid leukemia (AML) cells via derepression of EZH2-regulated genes, such as HOXB7, HOXA9 and ABCC1. In their proposed mechanism of EZH2 inactivation, CDK1-mediated Thr487 phosphorylation triggers recruitment of the E3 ubiquitin ligase TRIM21, which induces proteasome-dependent degradation of EZH2. In this scenario, CDK1 activation is the initial and critical step toward the acquisition of drug resistance by AML cells. The authors clearly showed that HSP90 stabilizes the CDK1–EZH2 complex to promote EZH2 phosphorylation; however, the following issues remain to be clarified. First, how does CDK1 phosphorylate EZH2 upon stabilization by HSP90 independently of the cell cycle change, which is a major determinant of CDK1 activity? Second, what other mechanisms are involved in EZH2 phosphorylation in drug-resistant AML cells? Recently, we found that EZH2 is phosphorylated at Ser21 and inactivated during the acquisition of drug resistance by multiple myeloma (MM) via VLA-4-mediated interaction with bone marrow stromal cells (BMSCs) and/or fibronectin in the extracellular matrix [2]. Matsunaga et al.[3] also implicated VLA-4 in drug resistance in minimal residual AML cells, which suggests that VLA-4 is also a prognostic marker and therapeutic target of AML. From these findings, we hypothesized that VLA-4-mediated interaction withBMSCs and/or fibronectin activates CDK1 and/or other kinases to phosphorylate EZH2 in AML cells as a cue to acquire the multidrug-resistant phenotype. To test this hypothesis, we cultured AML cell lines HL-60 and MV4-11 in fibronectin-coated dishes and confirmed the development of resistance to doxorubicin (ADM) and cytosine arabinoside (Ara-C), as evidenced by a significant increase in the IC50 values (Fig. 1 a). Using this system, we investigated whether cell adhesion induces EZH2 phosphorylation at Ser21 and/or Thr487 in AML cells. As shown in Fig. 1 b, EZH2 was readily phosphorylated at Ser21 in HL-60 and MV4-11 cells during the acquisition of cell adhesion-mediated drug resistance (CAM-DR), whereas the level of Thr487 phosphorylation was constitutive and unaffected by cell adhesion or drug treatment. We verified that the attachment to fibronectin increased the phosphorylation level of EZH2 at Ser21, but not Thr487, in AML cells in a time-dependent manner. Ser21 phosphorylation preceded, or at least coincided, with a decrease in the methylation level of histone H3 at Lys27 (H3K27), consistent with its role in EZH2 inactivation (Fig. 1 c). We previously showed that VLA-4 engagement activates the PI3K–AKT kinase pathway, which is responsible for EZH2 phosphorylation at Ser21 in drug-resistant MM cells [2]. In the present study, we investigated whether this pathway also plays a pivotal role in CAM-DR of AML cells. Inhibitors of the PI3K–AKT kinase pathway exert cytotoxicity against AML cells in preclinical studies and show some clinical activity in phase I/II trials as monotherapies (http://www. clinicaltrials. gov identifier NCT01396499 and NCT02438761). As observed in MM cells [2], cell adhesion did not mitigate the effects of specific inhibitors for PI3K and AKT kinase, LY294002 and PF-04691502, on HL-60 and MV4-11 cells (Fig. 1 d). These results suggest that CAM-DR is acquired via PI3K/AKT-mediated EZH2 phosphorylation at Ser21 and operates as an additional mechanism in the EZH2-related drug resistance described by Göllner et al.[1] However, the extent to which this mechanism contributes