Gu Siyu, Hou Yue, Dovat Katarina, Dovat Sinisa, Song Chunhua, Ge Zheng
Department of Hematology, Zhongda Hospital, School of Medicine, Southeast University, Institute of Hematology Southeast University, 87 Dingjiaqiao Street, Nanjing, 210009, China.
Hershey Medical Center, Pennsylvania State University Medical College, Hershey, 17033, USA.
Exp Hematol Oncol. 2023 Feb 27;12(1):23. doi: 10.1186/s40164-023-00383-5.
More effective targeted therapy and new combination regimens are needed for Acute myeloid leukemia (AML), owing to the unsatisfactory long-term prognosis of the disease. Here, we investigated the synergistic effect and the mechanism of a histone deacetylase inhibitor, Chidamide in combination with Cladribine, a purine nucleoside antimetabolite analog in the disease.
Cell counting kit-8 assays and Chou-Talalay's combination index were used to examine the synergistic effect of Chidamide and Cladribine on AML cell lines (U937, THP-1, and MV4-11) and primary AML cells. PI and Annexin-V/PI assays were used to detect the cell cycle effect and apoptosis effect, respectively. Global transcriptome analysis, RT-qPCR, c-MYC Knockdown, western blotting, co-immunoprecipitation, and chromatin immunoprecipitation assays were employed to explore the molecule mechanisms.
The combination of Chidamide with Cladribine showed a significant increase in cell proliferation arrest, the G0/G1 phase arrest, and apoptosis compared to the single drug control in AML cell lines along with upregulated p21 expression and downregulated CDK2/Cyclin E2 complex, and elevated cleaved caspase-9, caspase-3, and PARP. The combination significantly suppresses the c-MYC expression in AML cells, and c-MYC knockdown significantly increased the sensitivity of U937 cells to the combination compared to single drug control. Moreover, we observed HDAC2 interacts with c-Myc in AML cells, and we further identified that c-Myc binds to the promoter region of RCC1 that also could be suppressed by the combination through c-Myc-dependent. Consistently, a positive correlation of RCC1 with c-MYC was observed in the AML patient cohort. Also, RCC1 and HDAC2 high expression are associated with poor survival in AML patients. Finally, we also observed the combination significantly suppresses cell growth and induces the apoptosis of primary cells in AML patients with AML1-ETO fusion, c-KIT mutation, MLL-AF6 fusion, FLT3-ITD mutation, and in a CMML-BP patient with complex karyotype.
Our results demonstrated the synergistic effect of Chidamide with Cladribine on cell growth arrest, cell cycle arrest, and apoptosis in AML and primary cells with genetic defects by targeting HDAC2/c-Myc/RCC1 signaling in AML. Our data provide experimental evidence for the undergoing clinical trial (Clinical Trial ID: NCT05330364) of Chidamide plus Cladribine as a new potential regimen in AML.
由于急性髓系白血病（AML）的长期预后不尽人意，因此需要更有效的靶向治疗和新的联合治疗方案。在此，我们研究了组蛋白去乙酰化酶抑制剂西达本胺与嘌呤核苷抗代谢物类似物克拉屈滨联合使用在该疾病中的协同作用及机制。
采用细胞计数试剂盒 - 8法和Chou - Talalay联合指数法检测西达本胺和克拉屈滨对AML细胞系（U937、THP - 1和MV4 - 11）及原发性AML细胞的协同作用。分别用PI和Annexin - V/PI法检测细胞周期效应和凋亡效应。采用全转录组分析、RT - qPCR、c - MYC敲低、蛋白质免疫印迹、免疫共沉淀和染色质免疫沉淀试验来探索分子机制。
与单药对照组相比，西达本胺与克拉屈滨联合使用使AML细胞系中的细胞增殖停滞、G0/G1期停滞和凋亡显著增加，同时p21表达上调，CDK2/Cyclin E2复合物下调，裂解的caspase - 9、caspase - 3和PARP升高。该联合用药显著抑制AML细胞中的c - MYC表达，与单药对照组相比，c - MYC敲低显著增加了U937细胞对联合用药的敏感性。此外，我们观察到HDAC2在AML细胞中与c - Myc相互作用，并且我们进一步确定c - Myc与RCC1的启动子区域结合，该区域也可通过c - Myc依赖性被联合用药抑制。一致地，在AML患者队列中观察到RCC1与c - MYC呈正相关。此外，RCC1和HDAC2高表达与AML患者的不良生存相关。最后，我们还观察到该联合用药显著抑制了伴有AML1 - ETO融合、c - KIT突变、MLL - AF6融合、FLT
3 - ITD突变的AML患者以及一名具有复杂核型的CMML - BP患者的原代细胞生长并诱导其凋亡。
我们的结果证明了西达本胺与克拉屈滨在AML及具有遗传缺陷的原代细胞中对细胞生长停滞、细胞周期停滞和凋亡具有协同作用，其作用机制是通过靶向AML中的HDAC2/c - Myc/RCC1信号通路。我们的数据为正在进行的西达本胺加克拉屈滨作为AML新潜在治疗方案的临床试验（临床试验编号：NCT05330
364）提供了实验证据。
