PO.ET03.05 · 实验与分子治疗
EGFR突变NSCLC中通过新型keratin 17+药物耐受持留细胞群体的多谱系耐药演化
Multi-lineage evolution of drug resistance via a novel keratin 17+ drug tolerant persister population in EGFR -mutant NSCLC
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：EGFR突变肺癌最初对一线靶向治疗有反应。然而，耐药不可避免地通过多种机制产生。耐药肿瘤普遍源自在初始治疗中存活的药物耐受持留细胞（DTPC）。尽管有这一认识，EGFR突变DTPC的生物学特性尚未得到充分理解。本研究旨在探究促成治疗耐药演化的DTPC表型。
方法：通过用>IC90浓度的奥希替尼处理EGFR突变细胞系14天来生成DTPC模型。14天后，用10X 3'单细胞RNA测序（scRNAseq）对细胞进行分析。使用Seurat整合数据、归一化数据、聚类细胞并进行差异表达分析。使用UCell对细胞的生物学通路活性进行评分。使用scanpy分析来自Gardner等ERPMT小鼠模型的scRNAseq数据。使用IRB批准的方案在MD Anderson癌症中心收集接受治疗的肺癌患者的人体样本。用10X 5' scRNAseq对样本进行分析。使用scanpy整合数据、归一化数据并聚类细胞。使用Palantir进行轨迹分析。
结果：对细胞系配对进行了比较DTPC和对照细胞的差异表达（DE）分析。利用DE结果，我们生成了共识基因特征，捕获在DTPC模型中普遍上调或下调的基因。对这些基因的审查发现，KRT17在奥希替尼DTPC中高度上调。我们发现，与KRT17-DTPC相比，KRT17+DTPC上调上皮-间质转化（EMT）、干性和异常basaloid特征。亚聚类显示，KRT17 DTPC是异质性的，标记非重叠的EMT、增殖和MET表达群体。为验证我们的体外发现，我们分析了来自多个体内来源的scRNAseq数据集。在GEMM模型中，Krt17+细胞在突变EGFR受抑制后填充最小残留病灶（MRD）。Krt17+细胞是一个干样群体，在残留肿瘤细胞失去肺泡上皮特性、发生神经内分泌转化之前出现。最后，我们通过研究人类患者临床标本证实了发现的临床相关性。与未接受治疗的样本相比，KRT17+细胞在奥希替尼治疗的MRD样本中显著富集。轨迹分析表明，KRT17表达表征了先于通过多种已知机制（包括MET扩增和组织学转化（HT））产生耐药的细胞群体。
结论：我们的数据表明，在初始治疗中存活的EGFR突变细胞汇聚为一种新型KRT17+DTPC状态，该状态作为一个多能祖细胞群体，能够利用多种机制抵抗治疗，包括HT。
查看英文原文 English abstract
Background: EGFR mutant lung cancers initially respond to frontline targeted therapy. However, resistance inevitably develops through diverse mechanisms. Resistant tumors universally emerge from drug tolerant persister cells (DTPCs) that survive initial treatment. Despite this knowledge, the biology of EGFR mutant DTPCs is not well understood. This study was conducted to interrogate DTPC phenotypes that enable evolution of therapy resistance.
Methods: DTPC models were generated by treating EGFR mutant cell lines with >IC90 concentrations of osimertinib for 14 days. After 14 days, cells were profiled with 10X 3' single cell RNA sequencing (scRNAseq). Seurat was used to integrate data, normalize data, cluster cells, and conduct differential expression analyses. UCell was used to score cells for activity of biology pathways. scanpy was used to analyze scRNAseq data from the Gardner et al. ERPMT mouse model. Human patient samples were collected from lung cancer patients treated at MD Anderson Cancer Center using IRB approved protocols. Samples were profiled with 10X 5' scRNAseq. scanpy was used to integrate data, normalize data, and cluster cells. Palantir was used to perform trajectory analyses.
Results: Differential expression (DE) analysis comparing DTPCs and control cells were conducted for cell line pairs. Using DE results, we generated consensus gene signatures capturing genes universally upregulated or downregulated across DTPC models. Review of these genes identified that KRT17 was highly upregulated in osimertinib DTPCs. We found KRT17 + DTPCs upregulate epithelial-to-mesenchymal (EMT), stemness, and aberrant basaloid signatures compared to KRT17 - DTPCs. Subclustering showed that KRT17 DTPCs are heterogeneous and label non-overlapping EMT, proliferative, and MET expressing populations. To validate our in vitro findings, we analyzed scRNAseq datasets from multiple in vivo sources. In GEMM models, Krt17 + cells populate minimal residual disease (MRD) following suppression of mutant EGFR. Krt17 + cells were a stem-like population that emerged as residual tumor cells lost alveolar epithelial identity prior to neuroendocrine transformation. Lastly, we confirmed the clinical relevance of our findings by investigating human patient clinical specimens. Compared to treatment naïve samples, KRT17 + cells were significantly enriched in osimertinib-treated MRD samples. Trajectory analyses demonstrated that KRT17 expression characterized cell populations that preceded development of resistance through several known mechanisms, including MET amplification and histologic transformation (HT).
Conclusions: Our data demonstrate that EGFR-mutant cells surviving initial treatment converge onto a novel KRT17 + DTPC state which serves as a multipotent progenitor population capable of leveraging diverse mechanisms to resist therapy, including HT.
利益披露 Disclosure
B. B. Morris, None.
M. B. Nilsson,
Spectrum Pharmaceuticals Other, MN receives licensing fees and royalties from spectrum pharmaceuticals.
E. Earlie, None..
E. E. Gardner, None..
H. Chen, None..
S. G. Trevino, None..
A. J. Halliday, None..
Y. Xi, None..
J. Wang, None.
N. Vokes,
AstraZeneca ), Other, Consultant/Advisory Board.
Guardant Other, Consulting/Advisory Board; Honoraria.
Bristol Myers Squibb ), Other, Trial support.
Catalyst Pharmaceuticals Other, Consulting/Advisory Board.
ImmunityBio Other, Consulting/Advisory Board.
Sanofi ), Other, Trial support.
Mirati ).
Circulogene ), Other, Funding.
Pfizer Other, Consulting/Advisory Board.
Summit Therapeutics Other, Consulting/Advisory Board; Trial support.
OncoHost Other, Consulting/Advisory Board; Trial support.
Regeneron Other, Trial support; travel reimbursement.
Amgen Other, Trial support.
IDEAYA Other, Trial support.
EMD serono Other, Trial support.
Dava Onc Other, Honoraria.
Cardinal Health Other, Honoraria.
MJH Other, Honoraria.
Medlive Other, Honoraria.
HMP Global Other, Honoraria.
D. Gibbons,
Menarini Ricerche Other, Scientific Advisory Board.
Onconova Other, Scientific Advisory Board.
Aktis Oncology Other, Scientific Advisory Board.
Eli Lilly ), Other, Scientific Advisory Board.
Ideology Health Other, Honoraria.
NGM Biopharmaceuticals ).
Boehringer Ingelheim ).
Mirati ).
Bristol Myers Squibb ).
J. Zhang,
Merck ).
Johnson and Johnson ), Other, Consulting.
Norvartis ), Other, Consulting.
Summit Therapeutics ).
Helius ).
AstraZeneca Other, Consulting.
GenePlus Other, Consulting.
OrigMed Other, Consulting.
Innovent Other, Consulting.
Varian Other, Consulting.
Catalyst Therapeutics Other, Consulting.
A. M. Laughney, None.
Y. Y. Elamin,
AstraZeneca ), Other, Advisory Board.
Eli Lilly Other, Advisory Board.
Norvartis Other, Advisory Board.
Sanofi Other, Advisory Board.
Takeda ), Other, Advisory Board.
Bristol Myers Squibb Other, Advisory Board.
Nuvation ), Other, Advisory Board.
Mirati Other, Advisory Board.
Merus ), Other, Advisory Board.
Tahioo ), Other, Advisory Board.
BluePrint Medicines Other, Advisory Board.
Catalyst Pharmaceuticals Other, Advisory Board.
Johnson & Johnson Other, Advisory Board.
Kestrel Therapeutics Other, Advisory Board.
Telligene ).
Ellipses ).
X. Le,
ArriVent ), Other, Consulting.
Bayer AG ), Other, Consulting.
Boehringer Ingelheim ), Other, Consulting.
Eli Lilly ), Other, Consulting.
EMD Serono ), Other, Consulting.
Janssen ), Other, Consulting.
Dizal ), Other, Consulting.
Regeneron ), Other, Consulting.
Takeda ).
Teligene ), Other, Consulting.
ThermoFisher ).
AstraZeneca Other, Consulting.
AbbVie Other, Consulting.
Abion Other, Consulting.
Allist Other, Consulting.
Bristol Myers Squibb Other, Consulting.
Daiichi-Sankyo Other, Consulting.
BioNTech Other, Consulting.
Roche/Genentech Other, Consulting.
Taiho Other, Consulting.
J. V. Heymach,
AstraZeneca ), Other, Advisory Board.
Boehringer Ingelheim ), Other, Advisory Board.
Bristol Myers Squibb ), Other, Advisory Board.
Mirati ), Other, Advisory Board.
Takeda ), Other, Advisory Board.
Taiho ).
Tenaci-T Therapeutics Other, Business ownership.
Spectrum Pharmaceuticals Other, Licensing/royalties.
Clinical Care Targeted Communications Other, Speaking event.
Physicians Education Resource Other, Speaking event.
Prime Education Other, Speaking event.
Bayer Other, Advisory Board.
Eli Lilly Other, Advisory Board.
Janssen Pharmaceuticals Other, Advisory Board.
Jazz Pharmaceuticals Other, Advisory Board.
OncoHost Other, Advisory Board.
BioNTech AG Other, Advisory Board.
Curio Science Other, Advisory Board.
DAVA Oncology Other, Advisory Board.
EMD Serono Other, Advisory Board.