新型CRM1抑制剂KPT-330协同增强ABT-199抗AML活性及机制研究

An increasing body of evidence supports that chemotherapy-resistant acute myeloid leukemia stem cells are responsible for disease recurrence. Therefore, there is an urgent need of new therapeutic regimens which can eliminate both bulk leukemia cells and stem cells to improve the survival rate of AML patients. The Bcl-2-selective inhibitor ABT-199 and CRM1-selective inhibitor KPT-330 exhibit promising efficacy against both bulk leukemia cells and stem cells in AML. Given that KPT-330 has potential effect on downregulation of Mcl-1 which can sequestrate Bim causing intrinsic resistance to ABT-199, we postulate that combining KPT-330 with ABT-199 could result in synergistic anti-AML effect. Our preliminary studies demonstrate that KPT-330 downregulates Mcl-1, releases Bim from Mcl-1, and synergistically enhances ABT-199-induced apoptosis in AML cell lines and primary patient samples. Importantly, colony forming assays showed that compared to single drug treatments, the ABT-199 and KPT-330 combination caused significant decrease of AML-CFUs (colony forming units) in primary AML patient samples. These results demonstrate that KPT-330 can enhance the anti-AML activity of ABT-199 against both bulk AML cells and progenitor cells. However, the effect of ABT-199 and KPT-330 combination on AML stem cells and the molecular mechanism by which KPT-330 suppresses Mcl-1 expression remain unknown. CRM1 cargo protein E2F1 and IκB are both involved in the regulation of Mcl-1 expression. E2F1 binds to the Mcl-1 promoter and represses Mcl-1 gene transcription, while IκB negatively regulates Mcl-1 expression through inhibiting NF-κB which binds to the Mcl-1 promoter and promotes Mcl-1 gene transcription. Based on the results from our preliminary studies and literature, we hypothesize that KPT-330 enhances ABT-199-induced apoptosis by promoting nuclear retention of E2F1 and IκB through inhibition of CRM1, resulting in decreased expression of Mcl-1, leading to synergistic anti-AML activity aganist both bulk leukemia cells and stem cells in AML. In this project, we will use AML cell lines, primary patient samples and in vivo AML animal models to investigate the anti-AML activity and the underlying molecular mechanisms of the combination treatment with ABT-199 and KPT-330. The results of this study will form a solid foundation for the clinical evaluation of ABT-199 combined with KPT-330 for treating AML.

高复发率是治疗急性髓系白血病（AML）所面临的最大困境。化疗难以清除的AML干细胞是疾病复发的根源。Bcl-2抑制剂ABT-199可同时靶向AML主体细胞和干细胞，但Mcl-1限制其抗AML活性。我们的前期研究表明，CRM1抑制剂KPT-330可通过下调Mcl-1增加ABT-199的抗AML活性，但KPT-330调控Mcl-1的机制尚不明确。本项目拟利用AML细胞株、患者临床样本以及动物模型，考察ABT-199与KPT-330联合应用对AML主体细胞和干细胞的抑制活性，阐明KPT-330通过诱导E2F1和IκB核定位，抑制Mcl-1表达，从而增强ABT-199抑制AML活性的分子机制。本项目将为该新型AML治疗方案的临床转化奠定坚实的理论基础及实验依据。

急性髓系白血病（AML）是我国成人急性白血病中最常见的一种类型。目前AML的常规治疗方案是以阿糖胞苷、柔红霉素、去甲基化试剂以及维奈克拉（ABT-199/Venetoclax）为基础的化疗。多数患者治疗后可达到完全缓解，但复发率极高，且一旦复发便对化疗药物丧失敏感性并伴随有其他并发症。成年AML患者的五年生存率仅为26%。核转运蛋白1（XPO1/CRM1在多种实体肿瘤和血液系统恶性肿瘤中过表达，且与患者的预后不良相关。KPT-330（Selinexor）和KPT-8602（Eltanexor）是近年来新开发的口服CRM1选择性抑制剂，能够特异性靶向癌细胞，极具临床应用潜力。其中，KPT-330已被美国食品和药品管理局（FDA）批准上市，联合地塞米松用于复发性或难治性多发性骨髓瘤的治疗。在本研究中，我们证明CRM1抑制剂能够下调CHK1、WEE1、RAD51、RRM2和c-Myc的mRNA以及蛋白水平，诱导DNA损伤和细胞凋亡。其中，下调c-Myc是抑制CRM1诱导DNA损伤的起始因素，而下调CHK1、WEE1、RAD51和RRM2则在诱导DNA损伤的后期起作用。进一步研究结果表明，Venetoclax能够通过抑制DNA损伤修复，使CRM1抑制剂所造成的DNA损伤不断积累，最终导致更多的细胞凋亡。本研究进一步完善了CRM1抑制剂与Venetoclax协同抗AML的分子机制，为二者联合治疗AML提供了更加充足的理论基础和实验依据。