PO.MCB01.01 · 分子与细胞生物学
新型治疗：CDK11抑制在肾脏横纹肌样瘤中的应用
Novel treatment: CDK11 inhibition in rhabdoid tumor of the kidney
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
肾脏横纹肌样瘤(RTK)是一种侵袭性的儿科恶性肿瘤，尽管进行了广泛治疗，其预后仍极差。其高治疗耐药性和疾病复发需要寻找新的治疗策略。RTK以SMARCB1基因缺失为特征，导致细胞周期、细胞增殖和RNA剪接方面的下游功能障碍。已知周期蛋白依赖性激酶(CDK)调控这些过程，已有几种CDK抑制剂被用于癌症治疗。CDK11通过剪接因子SF3B1调控细胞周期和RNA剪接的已知功能，以及其在多种癌症中的过表达，使其成为一个潜在的治疗靶点。在本研究中，我们评估了CDK11抑制在RTK中的治疗潜力。基于Therapeutically Applicable Research To Generate Effective Treatments(TARGET)-RT数据库，RTK组织(n=63)的CDK11表达显著高于正常肾组织(n=6)。我们还证实在两个RTK细胞系(G401和JMU-RTK-2)中的CDK11蛋白表达高于人胚胎肾细胞(HEK293)(p分别为0.05、0.13)。一种CDK11抑制剂OTS964表现出显著的剂量依赖性细胞毒性，在G401中的IC50为33.1 nM，在JMU-RTK-2中的IC50为19.3 nM。接下来，我们用25 mg/kg的OTS964或载体对照处理移植了JMU-RTK-2肿瘤的裸鼠。OTS964单药治疗显著延长了生存期(中位生存期：46.5天对37.0天)并抑制了肿瘤进展(p=0.002)，且无临床毒性迹象。最后，我们研究了OTS964的作用机制。在IC80为80 nM(G401)和30 nM(JMU-RTK-2)时，OTS964处理显著增加了cleaved PARP、cleaved caspase-3和p53的表达。在8小时(G401)和12小时(JMU-RTK-2)时显示出G2/M周期阻滞。已知OTS964可抑制剪接因子SF3B1(剪接体的核心组分)的磷酸化，从而调控RNA剪接。我们通过已知的SF3B1依赖性转录本DNAJB1的未剪接转录本积累增加，证实了SF3B1抑制。总之，我们的数据证明了CDK11抑制在RTK中的治疗疗效。使用OTS964抑制CDK11抑制了RTK细胞生长并在小鼠异种移植模型中延长了生存期。OTS964通过caspase-3激活诱导凋亡，伴随G2/M细胞周期阻滞、p53上调，以及通过SF3B1抑制导致的RNA剪接破坏。在未来的研究中，我们计划研究CDK11抑制与现有治疗的协同组合。
查看英文原文 English abstract
Rhabdoid tumor of the kidney (RTK) is an aggressive pediatric malignancy with extremely poor outcomes despite extensive treatment. Its high treatment resistance and disease relapses necessitates a search for new therapeutic strategies. RTK is marked by the loss of SMARCB1 gene, leading to downstream dysfunctions in cell cycle, cell proliferation, and RNA splicing. Cyclin dependent kinases (CDKs) are known to regulate these processes, with several CDK inhibitors already being used in cancer treatment. CDK11's known function in regulating cell cycle and RNA splicing via splicing factor SF3B1 and its overexpression in various cancers makes it a potential therapeutic target. In this study, we evaluated the therapeutic potential of CDK11 inhibition in RTK. Based on the Therapeutically Applicable Research To Generate Effective Treatments (TARGET)-RT database, RTK tissues (n = 63) have significantly higher CDK11 expression compared to normal kidney tissues (n = 6). We also confirmed higher CDK11 protein expression in two RTK cell lines (G401 and JMU-RTK-2) than in human embryonic kidney cells (HEK293) (p = 0.05, 0.13, respectively). A CDK11 inhibitor, OTS964, showed significant dose-dependent cytotoxicity with IC50 of 33.1 nM in G401 and 19.3 nM in JMU-RTK-2. Next, we treated nude mice engrafted with JMU-RTK-2 tumors with 25 mg/kg of OTS964 or vehicle control. OTS964 monotherapy significantly prolonged survival (median survival: 46.5 days vs 37.0 days) and suppressed tumor progression (p = 0.002) without clinical signs of toxicity. Finally, we investigated OTS964's mechanisms of action. OTS964 treatment at IC80 of 80 nM (G401) and 30 nM (JMU-RTK-2) significantly increased expression of cleaved PARP, cleaved caspase-3, and p53. G2/M cycle arrest was demonstrated at 8 hours (G401) and 12 hours (JMU-RTK-2). OTS964 is known to inhibit phosphorylation of the splicing factor SF3B1, a core component of the spliceosome, thereby regulating RNA splicing. We confirmed SF3B1 inhibition by increased accumulation of the unspliced transcript DNAJB1, a known SF3B1-dependent transcript. In conclusion, our data demonstrated the therapeutic efficacy of CDK11 inhibition in RTK. CDK11 inhibition using OTS964 suppressed RTK cell growth and prolonged survival in mouse xenograft model. OTS964 induced apoptosis via caspase-3 activation with G2/M cell cycle arrest, p53 upregulation, and RNA splicing disruption via SF3B1 inhibition. In future studies, we plan to investigate synergistic combination of CDK11 inhibition with existing therapies.
利益披露 Disclosure
K. Lam, None..
Y. Murakami, None..
S. Fukui, None..
E. Helmke, None..
N. Satake, None.