NOTCH4ΔL12_16 sensitizes lung adenocarcinomas to EGFR-TKIs through transcriptional down-regulation of HES1

Resistance to epidermal growth factor tyrosine kinase inhibitors (EGFR-TKI) remains one of the major challenges in lung adenocarcinoma (LUAD) therapy. Here, we found an increased frequency of the L12_16 amino acid deletion mutation in the signal peptide region of NOTCH4 (NOTCH4ΔL12_16) in EGFR-TKI-sensitive patients. Functionally, exogenous induction of NOTCH4ΔL12_16 in EGFR-TKI -resistant LUAD cells sensitized them to EGFR-TKIs. This process was mainly mediated by the reduction of the intracellular domain of NOTCH4 (NICD4) caused by the NOTCH4ΔL12_16 mutation, which resulted in a lower localization of NOTCH4 in the plasma membrane. Mechanistically, NICD4 could transcriptionally upregulate the expression of HES1 by competitively binding to the gene promoter relative to p-STAT3. Because p-STAT3 can downregulate the expression of HES1 in EGFR-TKI-resistant LUAD cells, the reduction of NICD4 induced by NOTCH4ΔL12_16 mutation, leads to a decrease in HES1. Moreover, inhibition of the NOTCH4-HES1 pathway using inhibitors and siRNAs abolished the resistance of EGFR-TKI. Overall, we report that the NOTCH4ΔL12_16 mutation sensitizes LUAD patients to EGFR-TKIs through transcriptional down-regulation of HES1 and that targeted blockade of this signaling cohort could reverse EGFR-TKI -resistance in LUAD, providing a potential approach to overcome resistance to EGFR-TKI -therapy.

表皮生长因子酪氨酸激酶抑制剂（epidermal growth factor tyrosine kinase inhibitors, EGFR-TKI）耐药仍是肺腺癌（lung adenocarcinoma, LUAD）治疗面临的核心挑战之一。本研究发现，在EGFR-TKI敏感型肺腺癌患者体内，NOTCH4信号肽区域的L12_16位氨基酸缺失突变（NOTCH4ΔL12_16）的检出频率显著升高。功能实验显示，在EGFR-TKI耐药的肺腺癌细胞中外源诱导NOTCH4ΔL12_16表达，可使细胞恢复对EGFR-TKI的敏感性。该过程主要由NOTCH4ΔL12_16突变引发的NOTCH4胞内结构域（intracellular domain of NOTCH4, NICD4）水平降低所介导，后者会减少NOTCH4在细胞膜上的定位。机制研究表明，NICD4可通过与p-STAT3竞争性结合基因启动子，转录上调HES1的表达；而在EGFR-TKI耐药的肺腺癌细胞中，p-STAT3可下调HES1的表达，因此NOTCH4ΔL12_16突变诱导的NICD4水平降低，会导致HES1表达量下降。此外，利用抑制剂与小干扰RNA（small interfering RNA, siRNA）阻断NOTCH4-HES1通路，可完全消除EGFR-TKI耐药表型。综上，本研究证实NOTCH4ΔL12_16突变通过下调HES1的转录水平，使肺腺癌患者对EGFR-TKI产生敏感性；靶向阻断该信号轴可逆转肺腺癌的EGFR-TKI耐药，为克服EGFR-TKI治疗耐药提供了潜在的干预策略。