中药单体改构药CP5322促进急性髓系白血病细胞凋亡及其机制研究

Acute myeloid leukemia (AML) is a most common malignancy with high heterogeneity, but the traditional chemotherapeutic regimen showed less than 30% overall survival of 5 years. Compounds with low toxicity and high effect are on urgent for clinical trail. We firstly screened out the modified Chinese herbal monomer CP5322, which possesses the potential of both HDACi and apigenin. In vitro, CP5322 significantly induced the apoptosis of AML cells at nanomolar scale, which was more efficient than the typical chemotherapeutic drug cytarabine (Ara-C) and the HDACs inhibitor Chidamide. Furthermore, CP5322 was low toxicity to normal cells. In vivo, CP5322 suppressed tumor growth in tumor-bearing mice, cleared away the AML cells in leukemia mouse model, inhibited the liver metastasis, and prolonged the survival of AML mice. It’s worth noting that CP5322 was low toxicity to the organs and bone marrow in normal mice. Our previous study showed that t(8;21) AML cells were more sensitive to CP52322 than the AML1-ETO (AE) negative cells. Both HDACs and PI3K/AKT，related to AE fusion protein，were inhibited by CP5322, which suggests that CP5322 may affect the stability of AE fusion protein. Thus, our project is aim to explore the mechanism of t(8;21) AML cells apoptosis induced by CP5322, and the effect of CP5322 on the t(8;21) AML mice, which will provide new idea and theoretic basis for a potential application of CP5322 in clinical treatment against t(8;21) AML.

急性髓系白血病(AML)是较为常见的高异质性恶性肿瘤，但传统化疗药的5年总生存率不到30%，高效低毒药物为临床亟需。我们首次筛选出的中药单体改构药CP5322，具备芹菜素和HDACi的双重功效，能在纳摩尔级剂量诱导AML细胞凋亡，其抑制率优于Ara-C和Chidamide，且细胞毒性低。同时，CP5322可抑制荷瘤鼠的肿瘤生长，清除白血病模型鼠的AML细胞，抑制其肝转移，延长生存期，且无损重要器官。由AML1-ETO (AE)融合蛋白导致的t(8;21)型AML细胞对CP5322尤其敏感。我们前期研究发现，与AE融合蛋白相关的HDACs和PI3K/AKT均被抑制，推测CP5322可能与AE的稳定性密切相关。本课题旨在进一步探究CP5322促t(8;21)型AML细胞凋亡的作用机制，及其对t(8;21)型AML小鼠的治疗作用，进而为t(8;21)型AML的治疗提供新的思路和理论基础。

长期以来，表观调节异常一直被认为是急性髓系白血病（AML）发病的关键驱动因素。然而，靶向AML表观遗传改变的诸如组蛋白去乙酰化酶抑制剂（HDACi）等表观遗传药物仅表现出有限的临床疗效，且机制不明确。幸运的是，我们筛选出了一种名为Apigenin-Vorinostat-Conjugate（AVC，原名为CP5322）的新型表观遗传药，它为我们提供了处理异质性恶性肿瘤的可能性。AVC对HDAC的抑制作用表现在HDAC的表达、酶活性和组蛋白乙酰化水平上。我们通过AML小鼠模型的细胞活力测定和肿瘤进展检测其对AML的疗效，发现细胞凋亡是导致细胞死亡的主要方式。通过RNA测序证明了该机制，并通过蛋白质组分析器阵列试剂盒，实时PCR，蛋白质印迹和挽救实验进行了验证。我们发现AVC有效地抑制了AML细胞的发生，而对正常细胞几乎无损伤。Kasumi-1细胞对AVC（IC50= 0.024μM）的敏感性至少比伏立诺他（IC50 = 0.513μM）和Ara-C（IC50 = 0.4366μM）高20倍。此外，它可以有效地逆转AML小鼠模型中的肿瘤发生，同时保持关键器官的安全，证明了AML治疗的可行性和良好的安全性。机制上，AVC可以对接到c-Kit的活性口袋，阻隔c-Kit的磷酸化及其下游的增殖信号PI3K / Akt途径，并最终抑制白血病发生的进展。这也许可以解释为什么 AVC"偏向"于 c-Kit 标记的 t（8;21） AML。总的来说，这些临床前数据表明，利用类黄酮-HDACi-偶联物作为下一代表观遗传学药物用于针对AML的临床治疗，具有很大的潜力。