多靶点干预：CUDC-907协同ABT-199抑制AML的分子机制

The 5-year survival rate for acute myeloid leukemia (AML) in adults is a dismal 20-30%, highlighting the need for new therapeutic approaches for treating this deadly disease. The anti-apoptotic Bcl-2 family proteins have been an area of great interest as drug targets for the treatment of various malignancies, including leukemia. Although the Bcl-2 selective inhibitor ABT-199 shows promising anti-leukemic activity in AML cell line models and primary patient samples, intrinsic drug resistance remains a concern. One mechanism of ABT-199 resistance that has been identified in AML cell lines is the binding of Mcl-1 to pro-apoptotic Bim, which is released during ABT-199 treatment. This interaction results in the stabilization of Mcl-1, inhibition of Bim, and ultimately the inhibition of cell death/apoptosis. These results demonstrate that Mcl-1 is critical for the intrinsic resistance to ABT-199 in AML cells. In addition, recent studies indicate that the DNA replication stress response could be another intrinsic mechanism of resistance to ABT-199 in AML cells. Therefore, to overcome ABT-199-resistance, combining ABT-199 with a drug that can decrease/inhibit Mcl-1, up-regulate Bim, and abolish the DNA replication stress response would improve ABT-199 activity in resistant AML cells. The PI3K/Akt and MEK/ERK signaling pathways enhance Mcl-1 stability but decrease Bim stability, while CHK1 and Wee1 play critical roles in the DNA replication stress response. The recently developed histone deacetylase (HDAC) and PI3K dual inhibitor, CUDC-907, simultaneously inhibits PI3K/Akt and MEK/ERK signaling pathways, suggesting that it can down-regulate Mcl-1 and up-regulate Bim. Further, as a HDAC inhibitor, it might also down-regulate CHK1 and Wee1 in AML cells as indicated by our most recent studies. Therefore, we hypothesize that combing CUDC-907 with ABT-199 would overcome the intrinsic resistance to ABT-199 through down-regulation of Mcl-1, up-regulation of Bim, and abolishment of the DNA replication stress response, leading to synergistic anti-leukemic activities against AML. Our preliminary studies strongly support this hypothesis. We found that CUDC-907 indeed caused increased Bim and decreased Mcl-1 protein levels in AML cell lines treated with various concentrations of CUDC-907. This was accompanied by concentration-dependent induction of apoptosis in both AML cell lines and primary patient samples. Further, CUDC-907, in combination with ABT-199, resulted in synergistic induction of apoptosis in AML cell lines and primary patient samples. In this study, we will use AML cell lines and primary patient samples to thoroughly investigate the molecular mechanism by which CUDC-907 synergistically enhances the anti-leukemic activity of ABT-199. The results will form a solid foundation for the clinical evaluation of CUDC-907 combined with ABT-199 for treating AML.

我们的前期研究发现，部分AML细胞系及临床样本对Bcl-2选择性抑制剂ABT-199产生内源性耐药。Mcl-1和DNA复制压力应答可能是这一内源性耐药的潜在机制。PI3K和ERK可调控Mcl-1表达，CHK1和Wee1可调控DNA复制压力应答。由于CUDC-907可抑制PI3K和ERK活性，并下调CHK1和wee1表达。因此猜测CUDC-907一方面靶向PI3K和ERK，导致Mcl-1下调，从而逆转Mcl-1介导的ABT-199耐药；另一方面下调CHK1和Wee1，消除DNA复制压力应答，阻断其介导的另一个ABT-199耐药机制。因此，将二者联合将产生协同抗AML活性。本项目拟利用体外AML模型，阐明CUDC-907增加ABT-199活性的分子机制，证实CUDC-907与ABT-199对AML的协同抑瘤活性，为联合应用CUDC-907和ABT-199治疗AML提供理论基础及实验依据。

Bcl-2抑制剂ABT-199 (Venetoclax)和PI3K/HDAC双抑制剂CUDC-907 (Fimepinostat)是治疗AML的潜力新药。本项目使用这两种药物多靶点干预AML细胞中促进细胞增殖和存活的信号传导通路，明确了CUDC-907单独或联合ABT-199的抗AML活性及相应的分子机制。课题组在2017到2018的两年间，按既定计划顺利完成所有研究内容，取得如下结果：1）明确CUDC-907具有良好的体内、体外抗AML活性；2）证明上调Bim、下调Mcl-1、Wee1、CHK1、RRM1和c-Myc、诱导DNA损伤和复制压力是CUDC-907抗AML的重要分子机制；3）明确CUDC-907和ABT-199联合应用具有出色的体外协同抗AML活性，并取得初步的体内抗AML活性结果；4）证明CUDC-907与ABT-199通过相互克服AML细胞对凋亡的耐受及协作诱导DNA损伤和DNA复制压力来协同杀伤AML细胞。在项目进行期间（2018年11月），本研究涉及的主要药物ABT-199已经被美国FDA批准与地西他滨、阿扎胞苷或低剂量阿糖胞苷联合使用来治疗75岁及以上或者无法耐受常规化疗的AML患者。本项目的研究成果为CUDC-907和ABT-199在AML临床治疗中的联合应用提供了实验与理论依据。