MK-1775与帕比司他（Panobinostat）协同杀伤急性髓系白血病细胞的机制研究

发布时间：2018-07-05 16:22

本文选题：急性髓系白血病 + WEE1　；参考：《吉林大学》2015年博士论文

【摘要】：急性髓系白血病（acute myeloid leukemia, AML）是最常见的急性白血病，其治疗现状仍不乐观，成人和儿童的存活率分别只有25%和65%。因此，急需找到新药来治疗这种致命性的疾病。 AML的标准治疗方法是应用阿糖胞苷（cytosine arabinoside, Ara-C）和蒽环类抗生素如柔红霉素（daunorubicin, DNR）的联合化疗，这些药物可以在进行分裂的细胞中引起DNA损伤。然而，DNA损伤会通过激活细胞周期检验点而使细胞周期停滞，为细胞进行DNA修复提供时间并促进细胞存活。因此，细胞周期检验点的活化可能是AML治疗失败的潜在原因。检验点激酶1（checkpoint kinase1, CHK1）是个在DNA复制起始、复制叉稳定、同源重组修复、细胞周期进程和S以及G2/M细胞周期检验点中都起重要作用的蛋白激酶。在应答DNA损伤或复制压力时，CHK1会磷酸化CDC25磷酸酶，从而阻止CDK1/CDK2的活化，进而阻止细胞周期进程，这有利于细胞进行DNA修复而存活。另个重要的细胞周期检验点激酶是WEE1，它的主要功能是磷酸化CDK1和CDK2的第15位酪氨酸残基（Y15）而使它们失去活性，从而阻止细胞周期进程。因此，靶向这些细胞周期检验点激酶能干扰DNA损伤的修复，允许不可修复的DNA损伤累积并最终导致细胞的死亡。组蛋白去乙酰化酶（histone deacetylases, HDACs）是类能重塑染色质并且通过表观遗传学调节基因表达的关键酶类。与大多数抗癌药物样，临床前的研究表明，组蛋白去乙酰化酶抑制剂（histone deacetylase inhibitors, HDACIs）能够诱导肿瘤细胞的周期阻滞、分化和死亡，而对正常细胞的影响较小。然而，HDACIs在临床上的单药作用效果比较有限，所以现在有很多临床实验正在研究HDACIs与其它药物联合的抗肿瘤效果（NCT01242774、NCT01742793、NCT02061449和NCT02145715, clinicaltrials.gov）。早前我们课题组证明了帕比司他（panobinostat，目前最强的广谱HDACI，最近刚获得美国食品与药品管理局的批准用于治疗多发性骨髓瘤）能够下调AML细胞中的CHK1表达水平。据此，我们推测在AML细胞中用帕比司他联合WEE1抑制剂MK-1775会有协同抗AML作用。为了检验上述推断，我们首先检测了MK-1775（第个WEE1的高效选择性抑制剂）在AML细胞株及AML患者临床样本中的抗AML活性。我们通过体外MTT实验发现，无论是初诊或复发的AML临床样本都对MK-1775有相近的敏感性。而且，Ara-C耐药的HL-60细胞（HL-60/Ara-C）与母本HL-60细胞相比对MK-1775更加敏感，这为应用MK-1775治疗复发AML提供了进步的支持。有意思的是，AML临床样本中凡是带有t（15；17）染色体异位的要比不带t（15；17）染色体异位的对MK-1775更加敏感，并有显著性差异。无论在AML细胞株还是临床样本中，MK-1775都诱导了细胞死亡的发生，并伴随有CDK1和CDK2的第15位酪氨酸残基磷酸化水平的下降及DNA损伤的增加。时间进行曲线实验的结果表明，MK-1775处理细胞后能增加CHK1的磷酸化水平（导致CHK1的活化）和DNA损伤，而且这种增加是依赖于CDK活力的，因为CDK抑制剂Roscovitine能逆转这种现象，说明MK-1775通过活化CDK来杀伤AML细胞。随后我们证明，无论是在AML细胞株中还是在AML临床样本中，MK-1775与CHK1抑制剂LY2603618都有协同抗AML作用。接下来，我们检测了帕比司他在AML细胞株及临床样本中的活性及其与MK-1775联合抗AML的作用。我们发现，帕比司他无论是在初诊还是复发AML临床样本中都有几乎相同的药物活性。另外我们还发现，帕比司他与MK-1775的确能够协同引起AML细胞增殖阻滞和细胞死亡。帕比司他单独或与MK-1775联合都能够减少WEE1的蛋白表达水平，同时下调CHK1信号通路。通过慢病毒shRNA沉默CHK1，能显著增强AML细胞株对MK-1775的敏感性。与CHK1相似，沉默WEE1也显著增强了MK-1775和帕比司他引起的细胞死亡。综上所述，我们的研究结果证明了CHK1对MK-1775抗AML活性起关键作用，并指明CHK1活化是导致AML细胞对MK-1775耐药的个可能机制。另外，我们的研究强烈支持应用MK-1775治疗初诊和复发AML患者，，尤其是带有（t15；17）染色体异位的患者。最后，我们的结果证明帕比司他与MK-1775具有协同抗AML作用，这种作用至少部分是通过下调CHK1和/或WEE1来实现的。这为临床开发帕比司他与MK-1775对AML的联合疗法提供了理论和实验依据。
[Abstract]:Acute myeloid leukemia (AML) is the most common acute leukemia. The status of treatment is still not optimistic. The survival rate of adults and children is only 25% and 65%., respectively. Therefore, new drugs are urgently needed to treat this fatal disease.
The standard treatment for AML is the combination of cytosine arabinoside (Ara-C) and anthracycline, such as daunorubicin (daunorubicin, DNR), which can cause DNA damage in split cells. However, DNA damage causes cell cycle stagnation by activating cell cycle checkpoints and D for cells. NA repair provides time and promotes cell survival. Therefore, activation of cell cycle checkpoints may be a potential cause of failure in AML treatment.
Test point kinase 1 (checkpoint kinase1, CHK1) is a protein kinase that plays an important role in the initiation of DNA replication, replicating fork stability, homologous recombination repair, cell cycle process and S and G2/M cell cycle checkpoints. In response to DNA damage or replication pressure, CHK1 phosphorylates CDC25 phosphatase, thereby preventing CDK1/CDK2 from activation, and thus preventing the activation of CDK1/CDK2. The cell cycle process is prevented, which helps the cells to survive DNA repair. Another important cell cycle test point kinase is WEE1. Its main function is to phosphorylate the fifteenth tyrosine residues (Y15) of CDK1 and CDK2, which cause them to lose their activity and thus prevent the cell cycle process. Therefore, these cell cycle test point kinase can be targeted. Repair of DNA damage is allowed to accumulate and ultimately result in death of DNA cells.
Histone deacetylase (histone deacetylases, HDACs) is the key enzyme that can remould chromatin and regulate gene expression through epigenetics. With most anticancer drugs, pre clinical studies have shown that the histone deacetylase inhibitor (histone deacetylase inhibitors, HDACIs) can induce the cycle of tumor cells. Block, differentiation, and death have little effect on normal cells. However, the effect of HDACIs on clinical monotherapy is limited, so many clinical trials are now studying the antitumor effects of the combination of HDACIs and other drugs (NCT01242774, NCT01742793, NCT02061449 and NCT02145715, clinicaltrials.gov). Earlier our group It has been shown that panobinostat, the strongest broad-spectrum HDACI, recently approved by the US Food and Drug Administration for the treatment of multiple myeloma, can reduce the level of CHK1 expression in AML cells. Accordingly, we speculate that the use of the WEE1 inhibitor, MK-1775 in AML cells, has a synergistic anti AML effect.
To test the above inference, we first detected the anti AML activity of MK-1775 (the highly selective inhibitor of WEE1) in the AML cell line and the clinical samples of the AML patients. We found that the initial and recurrent AML clinical samples were sensitive to MK-1775 in both the AML cell line and the AML patient's clinical samples. Moreover, Ara-C resistant HL-60 cells (HL-60) /Ara-C) is more sensitive to MK-1775 than maternal HL-60 cells, which provides progressive support for the application of MK-1775 to relapse AML. It is interesting that all AML clinical samples with t (15; 17) chromosomes are more sensitive to MK-1775 than without t (15; 17) chromosome heterotopic, and have significant differences in AML cell lines. In clinical samples, MK-1775 induced the occurrence of cell death, accompanied by a decrease in the phosphorylation level of fifteenth tyrosine residues with CDK1 and CDK2 and the increase of DNA damage. The results of the time curve experiment showed that MK-1775 treated cells could increase the phosphorylation level of CHK1 (the activation of CHK1) and DNA damage, and this increase Addition is dependent on CDK activity, because the CDK inhibitor Roscovitine can reverse this phenomenon, indicating that MK-1775 can kill AML cells by activating CDK. Then we demonstrate that both MK-1775 and CHK1 inhibitor LY2603618 both in AML cell lines and in AML clinical samples have synergistic resistance to AML.
Next, we examined the activity of pariselt in AML cell lines and clinical samples and the role of MK-1775 in combination with AML. We found that Parkinson has almost the same drug activity in both primary and relapse AML clinical samples. In addition, we also found that Paris and MK-1775 do cooperate with MK-1775 to cause AML finer. Cell proliferation block and cell death. Both alone or in combination with MK-1775 can reduce the protein expression level of WEE1 and reduce the CHK1 signaling pathway. The silence of CHK1 through the lentivirus shRNA can significantly enhance the sensitivity of the AML cell line to MK-1775. Similar to CHK1, the silence of WEE1 also significantly enhanced the cells caused by MK-1775 and the passages. Death.
To sum up, our results show that CHK1 plays a key role in the anti AML activity of MK-1775 and indicates that CHK1 activation is a possible mechanism that causes AML cell resistance to MK-1775. Furthermore, our study strongly supports the application of MK-1775 in the treatment of patients with primary and recurrent AML, especially those with (T15; 17) chromosome ectopia. Finally, we The results showed that the synergistic anti AML effect of MK-1775 was partly achieved by downregulation of CHK1 and / or WEE1, which provided a theoretical and experimental basis for the clinical development of the combined therapy of MK-1775 and MK-1775 for AML.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R733.71

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