PO.ET03.05 · 实验与分子治疗
Neuregulin-1（NRG1）导向疗法在NRG1融合阳性癌症中诱导碱性磷酸酶胎盘型（ALPP）表达：临床前数据与治疗前景
Neuregulin-1(NRG1 ) -directed therapies induce alkaline phosphatase placental (ALPP) expression in NRG1 -fusion positive cancers: Preclinical data and therapeutic perspective
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：NRG1融合阳性（NRG1+）癌症定义了实体瘤的一种新的分子亚型。NRG1嵌合蛋白通过ERRB3/ERRB2二聚化进行信号传导，激活PI3K/AKT和MAPK信号通路，促进促肿瘤特性。针对NRG1+癌症的治疗依赖于双特异性抗ERBB2/ERBB3抗体如zenocutuzumab（zeno）或ERBB2抑制剂如afatinib（afa）。这些疗法的临床疗效有限，提示NRG1/ERBB3通路的生物学尚未被完全理解。我们的目的是了解NRG1+癌症对NRG1导向疗法的耐药机制。
材料与方法：我们通过使用MDA-MB-175细胞连续暴露于浓度递增的afa，建立了afa耐药模型。它们在存在256 nM afatinib的情况下生长。afa敏感性通过Prestoblue试验评估。MDA-MB-175细胞携带内源性PPP6R3-TENM4-NRG1融合，对afa、zeno及其他NRG1导向疗法敏感。对afa初治和afa耐药的MDA-MB-175的Bulk RNA测序数据进行了无监督分析和比较分析。这些模型的特征分析通过定量RT-PCR和western blot完成。
结果：生成了五个afa耐药MDA-MB-175模型，与敏感细胞相比IC50升高10至100倍。RNA-seq数据的无监督分析显示，afa耐药（n=5）和afa初治（n=5）的MDA-MB-175形成两个不同的聚类。ALPP是afa耐药模型中差异表达最显著的十个基因之一（log2FoldChange = 8.1，p = 2.46.10-20），并且是唯一一个在afa初治中确认无表达、而在afa耐药MDA-MB-175中在RNA和蛋白水平均有表达的基因。在afa耐药模型中，无afa培养2-8周后ALPP表达消失。在afa初治和afa耐药的MDA-MB-175中，暴露于afa一到两天后观察到ALPP表达升高。对afa耐药模型中NRG1-ERBB3通路的研究显示phospho-ERBB3和phospho-ERK降低，而总ERBB3和ERK无变化。
结论：afa在NRG1+ MDA-MB-175细胞中诱导了ALPP的早期表达，且afa耐药与高ALPP表达以及ERBB3和ERK磷酸化降低相关。目前正在使用ALPP KO和过表达的NRG1+模型（包括NRG1+工程化临床前模型和来自暴露于NRG1导向疗法患者的NRG1+患者来源模型）验证ALPP是否为相关靶点。zeno和zongertinib对临床前模型中ALPP表达的影响，以及ALPP在一队列NRG1+非小细胞肺癌和胰腺导管腺癌中的表达研究正在进行中。总体而言，ALPP可能代表NRG1+癌症中一个有吸引力的靶点，或可预防对NRG1导向疗法的耐药。
查看英文原文 English abstract
Background: NRG1- fusion positive ( NRG1 +) cancers define a new molecular subtype of solid tumors. NRG1 chimeric proteins signal through ERRB3/ERRB2 dimerization, activating PI3K/AKT and MAPK signalling pathways, promoting pro-tumoral properties. NRG1 + cancers targeting relies on bispecific anti-ERBB2/ERBB3 antibodies such as zenocutuzumab (zeno) or ERBB2 inhibitors such as afatinib (afa). These therapies have limited clinical efficacy, suggesting that the biology of the NRG1/ERBB3 pathway is not totally understood. Our objective is to understand the mechanisms of resistance to NRG1-directed therapies in NRG1 + cancers.
Material and Methods: We have generated afa-resistant models by continuous exposure to increasing concentrations of afa, using MDA-MB-175 cells. They grew in presence of 256 nM afatinib. Afa-sensitivity was assessed by Prestoblue test. MDA-MB-175 cells exhibit an endogenous PPP6R3-TENM4-NRG1 fusion and are sensitive to afa, zeno and other NRG1-directed therapy. Bulk RNA sequencing data from afa-naive and afa-resistant MDA-MB-175 were subjected to unsupervised and comparative analysis. Characterization of these models was completed by quantitative RT-PCR and western blotting.
Results: Five afa-resistant MDA-MB-175 models were generated, showing 10-to-100-fold increase in IC50 as compared to sensitive cells. RNA-seq data unsupervised analysis showed that afa-resistant (n=5) and afa-naive (n=5) MDA-MB-175 formed two distinct clusters. ALPP was one of the ten most differentially expressed genes in afa-resistant models (log2FoldChange = 8.1, p = 2.46.10 -20 ) and the only one with a confirmed lack of expression in afa-naive and expression in afa-resistant MDA-MB-175 at the RNA and protein levels. In afa-resistant models, ALPP expression disappeared after 2-8 weeks when cultured without afa. In afa-naive and afa-resistant MDA-MB-175, an increase of ALPP expression was observed after one or two days of afa exposure. Investigation of NRG1-ERBB3 pathway in afa-resistant models showed a decrease of phospho-ERBB3 and phospho-ERK with no variation of total ERBB3 and ERK.
Conclusion: Afa induced early ALPP expression in NRG1 + MDA-MB-175 cells, and afa-resistance was associated with high ALPP expression and a decrease of ERBB3 and ERK phosphorylation. Validation of ALPP as a relevant target is ongoing using ALPP KO and overexpressing NRG1+ models, including NRG1+ engineered preclinical models and NRG1+ patient-derived models from patients exposed to NRG1-directed therapies. Zeno and zongertinib impact on ALPP expression in preclinical models as well as ALPP expression in a cohort of NRG1 + non-small cell lung cancer and pancreatic ductal adenocarcinoma is ongoing. Overall, ALPP may represent an attractive target in NRG1+ cancers that may prevent resistance to NRG1-directed therapies.
利益披露 Disclosure
M. Barre, None..
C. Thiollier-Schmitt, None..
M. Issenmann, None..
C. Cortay, None..
E. Cros-Perrial, None..
S. Ortiz-Cuaran, None..
N. Gadot, None..
E. Voilin, None..
A. Ferrari, None..
E. Cumunel, None..
C. Dumontet, None..
L. Jordheim, None.
M. Duruisseaux,
Pfizer g., Board of Directors, non-salaried role), ), Other, Remuneration for participation in scientific meetings.
Merus ).
Takeda g., Board of Directors, non-salaried role), ).
Guardant g., Board of Directors, non-salaried role), ), Other, Remuneration for participation in scientific meetings.
Eli Lilly ).
Boehringer Ingelheim g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings.
Astra Zeneca g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings.
Roche g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings.
BMS g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings.
Abbvie g., Board of Directors, non-salaried role).
MSD g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings.
Novartis g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings.
GSK g., Board of Directors, non-salaried role).
Sanofi g., Board of Directors, non-salaried role).
Amgen g., Board of Directors, non-salaried role), Other, Remuneration for participation in scientific meetings.
Regeneron g., Board of Directors, non-salaried role).
Revolution medecine g., Board of Directors, non-salaried role).
Novocure g., Board of Directors, non-salaried role).