AXL介导ROS1突变肺癌产生克唑替尼耐药及其分子机制研究

Although patients with lung cancer whose tumors are positive for ROS1 fusion have benefited from crizotinib, the long-term overall survival and progression-free survival of these patients are still limited due to the acquisition of drug resistance. No optimal therapeutic regimens are yet available to overcome drug resistance in that the underlying mechanisms of resistance to crizotinib in patients with ROS1 fusion-positive lung cancer are still unclear. In our previous studies, we discovered that inflammasomes regulated by AXL and NLRP3 mediate the acquired resistance to crizotinib in ROS1 fusion-positive lung cancer cells. Based on these findings, we hypothesize that the acquired drug resistance is mediated by bypass of activation of AXL. On the other hand that activated AXL up-regulates NLRP3 via NF-κB signaling, and activated NLRP3 inflammasomes reduce ROS1 fusion protein which resulted in resistance to crizotinib for the loss of target . In this proposal, we will uncover the regulatory mechanisms of acquired resistance mediated by the AXL-NLRP3-ROS1 signaling axis in vitro and in vivo. Moreover, we will investigate the therapeutic effects of the combination of crizotinib plus the inhibition of AXL or NLRP3 with shRNA-mediated depletion or small molecule inhibitors in crizotinib-resistant lung cancer murine model. We expect that the proposed studies will provide new insights into molecular mechanisms that underlie therapy resistance to crizotinib and identify rationale-based combination therapies to overcome resistance to crizotinib for patients with ROS1 fusion-positive lung cancers.

克唑替尼治疗ROS1突变型肺癌在临床上取得了显著效果，但获得性耐药仍是一个普遍且严峻的问题，因其分子机制并不完全清楚，目前尚无有效解决办法。我们前期研究首次发现AXL和NLRP3炎症小体参与调控ROS1突变型肺癌产生克唑替尼耐药，初步探索了其机制，并提出假说：活化的AXL一方面通过旁路激活导致耐药；另一方面通过NF-κB信号通路上调NLRP3，激活的NLRP3炎症小体下调ROS1蛋白，促使克唑替尼失去作用靶点导致ROS1突变型肺癌产生耐药。本项目拟在细胞水平干预AXL、NLRP3并深入研究转录前水平调控ROS1的具体分子机制，阐明AXL-NLRP3-ROS1信号轴在耐药中的作用，同时在小鼠模型中应用AXL或NLRP3抑制剂治疗ROS1突变耐药型肺癌，探索治疗肺癌耐药的新靶点。相关原创性发现及深入研究将为临床治疗克唑替尼耐药型肺癌提供新靶点及理论依据。

本项目针对克唑替尼治疗ROS1融合性非小细胞肺癌获得性耐药产生且分子机制尚不清晰这一难点问题，通过研究证实 GAS6/AXL是调控非小细胞肺癌对多种TKI形成耐药的共同关键因子，并首次在ROS1 融合型非小细胞肺癌克唑替尼耐药予以证实；AXL及其配体GAS6通路激活诱导耐药产生，阻断AXL及配体GAS6可逆转耐药。机制研究揭示，NLRP3通过其相互作用蛋白UPF1 介导的非选择性mRNA降解下调ROS1 mRNA，从而抑制克唑替尼药物的靶点蛋白表达，促进耐药形成。同时，N-糖基化修饰通过调控AXL蛋白膜定位及稳定性，影响AXL信号通路活性；GLT8D1是AXL 糖基化修饰的关键糖基转移酶，敲除GLT8D1可抑制AXL糖基化修饰，促进AXL蛋白通过溶酶体途径降解。进一步体外机体内实验研究证实敲除及抑制GLT8D1可逆转AXL介导的TKI获得性耐药，GLT8D1可作为ROS1 融合型非小细胞肺癌克唑替尼耐药后的有效治疗靶点。本项目研究为临床治疗克唑替尼耐药型肺癌提供新靶点及理论依据。