本文選題：急性髓系白血病 + WEE1�。� 參考：《吉林大學(xué)》2015年博士論文

【摘要】：急性髓系白血�。╝cute myeloid leukemia, AML）是最常見的急性白血病，其治療現(xiàn)狀仍不樂觀，成人和兒童的存活率分別只有25%和65%。因此，急需找到新藥來治療這種致命性的疾病。 AML的標(biāo)準(zhǔn)治療方法是應(yīng)用阿糖胞苷（cytosine arabinoside, Ara-C）和蒽環(huán)類抗生素如柔紅霉素（daunorubicin, DNR）的聯(lián)合化療，這些藥物可以在進(jìn)行分裂的細(xì)胞中引起DNA損傷。然而，DNA損傷會(huì)通過激活細(xì)胞周期檢驗(yàn)點(diǎn)而使細(xì)胞周期停滯，為細(xì)胞進(jìn)行DNA修復(fù)提供時(shí)間并促進(jìn)細(xì)胞存活。因此，細(xì)胞周期檢驗(yàn)點(diǎn)的活化可能是AML治療失敗的潛在原因。 檢驗(yàn)點(diǎn)激酶1（checkpoint kinase1, CHK1）是個(gè)在DNA復(fù)制起始、復(fù)制叉穩(wěn)定、同源重組修復(fù)、細(xì)胞周期進(jìn)程和S以及G2/M細(xì)胞周期檢驗(yàn)點(diǎn)中都起重要作用的蛋白激酶。在應(yīng)答DNA損傷或復(fù)制壓力時(shí)，CHK1會(huì)磷酸化CDC25磷酸酶，從而阻止CDK1/CDK2的活化，進(jìn)而阻止細(xì)胞周期進(jìn)程，這有利于細(xì)胞進(jìn)行DNA修復(fù)而存活。另個(gè)重要的細(xì)胞周期檢驗(yàn)點(diǎn)激酶是WEE1，它的主要功能是磷酸化CDK1和CDK2的第15位酪氨酸殘基（Y15）而使它們失去活性，從而阻止細(xì)胞周期進(jìn)程。因此，靶向這些細(xì)胞周期檢驗(yàn)點(diǎn)激酶能干擾DNA損傷的修復(fù)，允許不可修復(fù)的DNA損傷累積并最終導(dǎo)致細(xì)胞的死亡。 組蛋白去乙酰化酶（histone deacetylases, HDACs）是類能重塑染色質(zhì)并且通過表觀遺傳學(xué)調(diào)節(jié)基因表達(dá)的關(guān)鍵酶類。與大多數(shù)抗癌藥物樣，臨床前的研究表明，組蛋白去乙酰化酶抑制劑（histone deacetylase inhibitors, HDACIs）能夠誘導(dǎo)腫瘤細(xì)胞的周期阻滯、分化和死亡，而對(duì)正常細(xì)胞的影響較小。然而，HDACIs在臨床上的單藥作用效果比較有限，所以現(xiàn)在有很多臨床實(shí)驗(yàn)正在研究HDACIs與其它藥物聯(lián)合的抗腫瘤效果（NCT01242774、NCT01742793、NCT02061449和NCT02145715, clinicaltrials.gov）。早前我們課題組證明了帕比司他（panobinostat，目前最強(qiáng)的廣譜HDACI，最近剛獲得美國食品與藥品管理局的批準(zhǔn)用于治療多發(fā)性骨髓瘤）能夠下調(diào)AML細(xì)胞中的CHK1表達(dá)水平。據(jù)此，我們推測(cè)在AML細(xì)胞中用帕比司他聯(lián)合WEE1抑制劑MK-1775會(huì)有協(xié)同抗AML作用。 為了檢驗(yàn)上述推斷，我們首先檢測(cè)了MK-1775（第個(gè)WEE1的高效選擇性抑制劑）在AML細(xì)胞株及AML患者臨床樣本中的抗AML活性。我們通過體外MTT實(shí)驗(yàn)發(fā)現(xiàn)，無論是初診或復(fù)發(fā)的AML臨床樣本都對(duì)MK-1775有相近的敏感性。而且，Ara-C耐藥的HL-60細(xì)胞（HL-60/Ara-C）與母本HL-60細(xì)胞相比對(duì)MK-1775更加敏感，這為應(yīng)用MK-1775治療復(fù)發(fā)AML提供了進(jìn)步的支持。有意思的是，AML臨床樣本中凡是帶有t（15；17）染色體異位的要比不帶t（15；17）染色體異位的對(duì)MK-1775更加敏感，并有顯著性差異。無論在AML細(xì)胞株還是臨床樣本中，MK-1775都誘導(dǎo)了細(xì)胞死亡的發(fā)生，并伴隨有CDK1和CDK2的第15位酪氨酸殘基磷酸化水平的下降及DNA損傷的增加。時(shí)間進(jìn)行曲線實(shí)驗(yàn)的結(jié)果表明，MK-1775處理細(xì)胞后能增加CHK1的磷酸化水平（導(dǎo)致CHK1的活化）和DNA損傷，而且這種增加是依賴于CDK活力的，因?yàn)镃DK抑制劑Roscovitine能逆轉(zhuǎn)這種現(xiàn)象，說明MK-1775通過活化CDK來殺傷AML細(xì)胞。隨后我們證明，無論是在AML細(xì)胞株中還是在AML臨床樣本中，MK-1775與CHK1抑制劑LY2603618都有協(xié)同抗AML作用。 接下來，我們檢測(cè)了帕比司他在AML細(xì)胞株及臨床樣本中的活性及其與MK-1775聯(lián)合抗AML的作用。我們發(fā)現(xiàn)，帕比司他無論是在初診還是復(fù)發(fā)AML臨床樣本中都有幾乎相同的藥物活性。另外我們還發(fā)現(xiàn)，帕比司他與MK-1775的確能夠協(xié)同引起AML細(xì)胞增殖阻滯和細(xì)胞死亡。帕比司他單獨(dú)或與MK-1775聯(lián)合都能夠減少WEE1的蛋白表達(dá)水平，同時(shí)下調(diào)CHK1信號(hào)通路。通過慢病毒shRNA沉默CHK1，能顯著增強(qiáng)AML細(xì)胞株對(duì)MK-1775的敏感性。與CHK1相似，沉默WEE1也顯著增強(qiáng)了MK-1775和帕比司他引起的細(xì)胞死亡。 綜上所述，我們的研究結(jié)果證明了CHK1對(duì)MK-1775抗AML活性起關(guān)鍵作用，并指明CHK1活化是導(dǎo)致AML細(xì)胞對(duì)MK-1775耐藥的個(gè)可能機(jī)制。另外，我們的研究強(qiáng)烈支持應(yīng)用MK-1775治療初診和復(fù)發(fā)AML患者，，尤其是帶有（t15；17）染色體異位的患者。最后，我們的結(jié)果證明帕比司他與MK-1775具有協(xié)同抗AML作用，這種作用至少部分是通過下調(diào)CHK1和/或WEE1來實(shí)現(xiàn)的。這為臨床開發(fā)帕比司他與MK-1775對(duì)AML的聯(lián)合療法提供了理論和實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R733.71

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 姚宏亮;楊竹林;李永國;;胃良惡性病變組織中CHK1和PLK1的表達(dá)及意義[J];中南大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2010年10期

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