PO.ET02.12 · 实验与分子治疗
靶向AURKA和TRIP13利用有丝分裂脆弱性并增强Rb缺陷型免疫健全肺癌模型中的抗肿瘤免疫
Targeting AURKA and TRIP13 exploits mitotic vulnerability and enhances antitumor immunity in Rb-deficient immunocompetent lung cancer models
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
Rb缺失是多种癌症中的常见事件，并产生可被治疗性利用的脆弱性。我们此前报道，HPV阳性、Rb缺陷型癌症依赖两种有丝分裂检查点调控因子——Aurora激酶A(AURKA)和TRIP13——以实现正常的有丝分裂退出。这两种蛋白的双重抑制诱导有丝分裂灾难和细胞死亡。为确定这种依赖性是否延伸至HPV驱动的肿瘤之外并影响肿瘤免疫微环境，我们在Rb缺陷型肺癌模型中评估了AURKA和TRIP13抑制的联合作用。为测试该组合，我们使用了TRIP13抑制剂DCZ0415和AURKA抑制剂alisertib。我们首先评估了DCZ0415抑制TRIP13的能力。与先前报道一致，CETSA和亲和层析实验证实DCZ0415与TRIP13结合。用10μM DCZ0415处理癌细胞可在下拉实验中减少MAD2与CDC20的结合，表明DCZ0415选择性地干扰TRIP13介导的MAD2转化。TRIP13的ATP酶活性基本不受影响，提示一种不依赖激酶的抑制机制。经CellTiter-Glo实验测定，DCZ0415在小鼠非小细胞肺癌(NSCLC)细胞系中的IC₅₀值为：344SQ为20μM，344P为16μM。在凋亡实验(Annexin V)中，DCZ0415单独未诱导可测量的细胞死亡，而alisertib(200 nM)在72小时后导致约25%的凋亡。相比之下，联合治疗产生了显著更高的凋亡。在344SQ细胞中，联合治疗诱导约40%的细胞死亡，而DCZ0415单用为4.6%，alisertib单用为25%。在344P细胞中观察到类似结果，联合治疗诱导约55%的凋亡。我们接下来在一个PD1耐药、Rb缺陷型免疫健全小鼠模型中评估了该组合。与任一单药相比，联合治疗使肿瘤生长显著减少，至第14天已出现显著差异。对肿瘤浸润免疫细胞(TILs)的初步免疫分析揭示了若干组合特异性效应。树突状细胞丰度增加，提示肿瘤内抗原呈递增强。该组合还降低了肿瘤中髓源性抑制细胞(MDSC)的水平，表明肿瘤免疫微环境向抑制性较低的方向转变。此外，TIL分析显示总体T细胞浸润增加，CD4⁺和CD8⁺ T细胞群体相对于单药均升高。T细胞的多功能性也得到增强，表现为GZMB⁺和IFNγ⁺效应T细胞的频率更高。总之，这些发现将AURKA和TRIP13联合抑制确定为治疗Rb缺陷型癌症的一种有前景的治疗方法，并提供了其在NSCLC中疗效和免疫调节潜力的首个体内证据。
查看英文原文 English abstract
Rb loss is a common event in multiple cancers and creates vulnerabilities that can be therapeutically exploited. We previously reported that HPV-positive, Rb-deficient cancers depend on two mitotic checkpoint regulators, Aurora kinase A (AURKA) and TRIP13, for proper mitotic exit. Dual inhibition of these proteins induces mitotic catastrophe and cell death. To determine whether this dependency extends beyond HPV-driven tumors and affects the tumor immune microenvironment, we evaluated the combination of AURKA and TRIP13 inhibition in Rb-deficient lung cancer models. To test the combination, we used the TRIP13 inhibitor DCZ0415 and the AURKA inhibitor alisertib. We first assessed the ability of DCZ0415 to inhibit TRIP13. Consistent with previous reports, DCZ0415 binds to TRIP13 as confirmed by CETSA and affinity chromatography assays. Treatment of cancer cells with 10µM DCZ0415 reduced MAD2 association with CDC20 in pull-down assays, indicating that DCZ0415 selectively interferes with TRIP13-mediated MAD2 conversion. TRIP13 ATPase activity was largely unaffected, suggesting a kinase-independent inhibitory mechanism. The IC₅₀ values for DCZ0415 in murine non-small cell lung cancer (NSCLC) cell lines were 20μM for 344SQ and 16μM for 344P, as determined by CellTiter-Glo assays. In apoptosis assays (Annexin V), DCZ0415 alone did not induce measurable cell death, while alisertib (200 nM) resulted in ~25% apoptosis after 72 hours. In contrast, the combination treatment produced significantly greater apoptosis. In 344SQ cells, the combination induced ~40% cell death, compared with 4.6% for DCZ0415 and 25% for alisertib alone. Similar findings were observed in 344P cells, with combination treatment inducing ~55% apoptosis. We next evaluated the combination in a PD1-resistant, Rb-deficient immunocompetent mouse model. The combination produced a marked reduction in tumor growth compared with either agent, with significant differences apparent by day 14. Preliminary immunoprofiling of tumor-infiltrating immune cells (TILs) revealed several combination-specific effects. Dendritic cell abundance increased, suggesting enhanced antigen presentation within the tumor. The combination also reduced myeloid-derived suppressor cell (MDSC) levels in the tumor, indicating a shift toward a less suppressive tumor immune microenvironment. Additionally, TIL analysis showed increased overall T-cell infiltration, with both CD4⁺ and CD8⁺ T-cell populations elevated relative to single agents. T-cell polyfunctionality was also enhanced, as indicated by higher frequencies of GZMB⁺ and IFNgamma⁺ effector T cells. Together, these findings identify combined AURKA and TRIP13 inhibition as a promising therapeutic approach for Rb-deficient cancers and provide the first in vivo evidence of its efficacy and immune-modulating potential in NSCLC.
利益披露 Disclosure
S. Ghosh, None..
J. L. Anderson, None..
A. G. Sikora, None.