PO.IM01.01 · 免疫学
体内CRISPR筛选揭示Folr1介导的代谢重塑使结直肠癌对PD-1阻断敏感
In vivo CRISPR screening uncovers Folr1-mediated metabolic remodeling that sensitizes colorectal cancer to PD-1 blockade
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：结直肠癌（CRC）是全球癌症相关死亡的主要原因之一。KRAS突变存在于约40-50%的CRC中，其中G12D是最常见的亚型。越来越多的证据表明，KRAS G12D CRC对免疫检查点阻断（ICB）治疗应答不佳，反映出对免疫治疗的内在耐药。为鉴定能够克服这一耐药的关键调控因子，我们在KRAS G12D驱动的CRC模型中开展了体内CRISPR/Cas9遗传筛选。此外，我们研究了导致免疫耐药的潜在机制。
方法：构建了KRAS G12D结直肠癌细胞系MC38K和Caco2K。利用PD-1/PD-L1/MHC-I和ESTIMATE数据设计了一个免疫相关sgRNA文库，并将携带该文库的MC38K细胞在体内抗PD1治疗下进行测试。开展RNA测序和代谢谱分析以阐明免疫相关机制，并使用Seahorse实验验证代谢改变。使用多种CRC模型评估Folr1抑制联合抗PD1治疗，并开发了基于Folr1的多肽疫苗以克服耐药。
结果：KRAS G12D过表达加速肿瘤生长并在MC38K模型中赋予抗PD1耐药。通过体内CRISPR/Cas9筛选，Folr1成为最显著耗竭的基因，在所有与免疫治疗耐药相关的候选基因中排名第一。值得注意的是，Folr1是一个临床可操作的靶点，已有治疗性抗体经FDA批准。对Folr1敲除的MC38K和Caco2K细胞的RNA-seq分析揭示了深刻的代谢重塑，尤其涉及糖酵解。GSEA表明，包括糖酵解、缺氧和mTORC1信号在内的标志性通路被下调，并经qRT-PCR、OCR/ECAR实验和L-乳酸测定验证。在多个体内模型中，Folr1敲除联合抗PD1治疗显著延缓肿瘤生长并延长总生存。此外，基于Folr1的多肽疫苗也显著抑制肿瘤进展。
结论：本研究将Folr1确定为在KRAS G12D驱动的结直肠癌中介导免疫治疗耐药的排名最高的基因。靶向Folr1可重编程肿瘤代谢、增强抗原呈递并恢复对抗PD1治疗的应答性。这些发现确立了Folr1作为一个具有转化意义的治疗靶点，以克服Kras G12D CRC中的免疫耐药。
查看英文原文 English abstract
Introduction: Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide. KRAS mutations are present in approximately 40-50% of CRCs, with G12D being the most prevalent subtype. Accumulating evidence indicates that KRAS G12D CRC responds poorly to immune checkpoint blockade (ICB) therapy, reflecting intrinsic resistance to immunotherapy. To identify key regulators capable of overcoming this resistance, we conducted an in vivo CRISPR/Cas9 genetic screen in a KRAS G12D -driven CRC model. Furthermore, we investigated the underlying mechanisms contributing to immune resistance.
Methods: KRAS G12D colorectal cancer cell lines, MC38K and Caco2K were generated. An immune-related sgRNA library was designed using PD-1/PD-L1/MHC-I and ESTIMATE data, and MC38K cells carrying the library were tested under anti-PD1 treatment in vivo. RNA sequencing and metabolic profiling were performed to elucidate immune-related mechanisms, and Seahorse assays were used to validate metabolic alterations. Multiple CRC models were used to evaluate Folr1 inhibition combined with anti-PD1 therapy, and a Folr1-based peptide vaccine was developed to overcome resistance.
Results: KRAS G12D overexpression accelerated tumor growth and conferred anti-PD1 resistance in MC38K models. Through the in vivo CRISPR/Cas9 screen, Folr1 emerged as the top depleted gene, ranking first among all candidates associated with immunotherapy resistance. Notably, Folr1 is a clinically actionable target with existing therapeutic antibody currently approved by FDA. RNA-seq analysis of Folr1-knockout MC38K and Caco2K cells revealed profound metabolic remodeling, particularly involving glycolysis. GSEA demonstrated that hallmark pathways including glycolysis, hypoxia, and mTORC1 signaling were downregulated, validated by qRT-PCR, OCR/ECAR assays, and L-lactate measurements. In multiple in vivo models, Folr1 knockout combined with anti-PD1 therapy significantly delayed tumor growth and extended overall survival. Furthermore, the Folr1-based peptide vaccine also markedly suppressed tumor progression.
Conclusions: Our study identifies Folr1 as the top-ranked gene mediating immunotherapy resistance in KRAS G12D -driven colorectal cancer. Targeting Folr1 reprograms tumor metabolism, enhances antigen presentation, and restores responsiveness to anti-PD1 therapy. These findings establish Folr1 as a translationally relevant therapeutic target to overcome immune resistance in Kras G12D CRC.
利益披露 Disclosure
B. Jing, None..
J. Overholser, None..
P. Kaumaya, None.