PO.ET03.04 · 实验与分子治疗
利用全基因组 CRISPR 筛选鉴定克服横纹肌肉瘤化疗耐药的合成致死靶点
Using genome wide CRISPR screening to identify synthetic lethal targets for overcoming chemoresistance in rhabdomyosarcoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
横纹肌肉瘤（RMS）是最常见的儿童软组织肉瘤，其化疗耐药仍是改善生存结局的主要障碍，尤其是在高危腺泡状 RMS（ARMS）中——ARMS 约占所有 RMS 病例的三分之一，因其强烈的转移倾向而临床上更具侵袭性，复发后生存率低于 24%。耐药是治疗失败的主要驱动因素，导致近 80-90% 的癌症相关死亡。尽管伊立替康仍是 RMS 中最有效的化疗药物之一，但伊立替康相关化疗耐药的出现凸显了迫切需要开发合理的联合疗法，以针对 RMS 细胞的遗传稳健性和功能冗余性。为克服生存结局的停滞和内在耐药，我们进行了全基因组成簇规律间隔短回文重复序列（CRISPR）敲除筛选，以开发和鉴定专门针对克服 ARMS 细胞伊立替康耐药的新型治疗弱点和有效联合策略。我们的研究首次鉴定出 47 个候选预后基因，其缺失可能使 RMS 细胞对伊立替康治疗敏感。除若干可成药靶点外，通路富集分析揭示 DNA 损伤修复通路是 RMS 细胞伊立替康耐药中下调最显著的通路，提示靶向该通路可与伊立替康诱导合成致死相互作用。在排名靠前的命中基因中，我们验证了 FGFR4 和 PARP1 是耐药的关键调节因子，siRNA 介导的这些基因沉默可使 RMS 细胞对伊立替康治疗敏感。最重要的是，FGFR4 作为一种基因依赖性在 ARMS 中显著富集。此外，使用现有小分子抑制剂对这些基因进行药理学抑制，无论作为单药还是联合使用，均显著增强了伊立替康敏感性并进一步降低细胞活力，且具有良好的治疗窗口，支持其临床转化潜力。我们目前正在研究这些药物组合在 RMS 模型中的机制基础和体内疗效。本研究为开发个体化和靶向治疗策略、改善 RMS 患者结局提供了一个有前景的框架。
查看英文原文 English abstract
Chemotherapy resistance in Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, remains a major obstacle to improve survival outcomes, particularly in high-risk alveolar RMS (ARMS), which accounts for approximately one-third of all RMS cases and is clinically more aggressive due to its strong propensity for metastasis, with survival rates fall below 24% after relapse. Drug resistance is a primary driver of treatment failure, contributing to nearly 80-90% of cancer-related mortality. Although irinotecan remains one of the most effective chemotherapeutic agents in RMS, the emergence of irinotecan-related chemoresistance highlights the urgent need to develop rational combination therapies to target the genetic robustness and functional redundancy of RMS cells. To overcome the stagnation in survival outcomes and intrinsic drug resistance, a genome wide Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Knockout screening was performed to develop and identify novel therapeutic vulnerabilities and effective combination strategies specifically tailored to overcome irinotecan resistance in ARMS cells. For the first time, our study identified 47 candidate prognostic genes whose deletion potentially sensitizes RMS cells to irinotecan treatment. Along with several druggable targets, pathway enrichment analysis revealed that the DNA damage repair pathway is the most significantly downregulated in irinotecan-resistance in RMS cells, suggesting that targeting this pathway could induce a synthetic lethal interaction with irinotecan. Among the top hits, we validated FGFR4 and PARP1 as key modulators of resistance, and siRNA-mediated silencing of these genes sensitizes RMS cells to irinotecan treatment. Most importantly, FGFR4 was significantly enriched as a gene dependency in ARMS. In addition, pharmacological inhibition of these genes using available small-molecule inhibitors markedly enhanced irinotecan sensitivity and further reduced cell viability, both as single agents and in combination, with a favorable therapeutic window, supporting their potential for clinical translation. We are currently investigating the mechanistic basis and in vivo efficacy of these drug combinations in RMS models. This study provides a promising framework for developing personalized and targeted therapeutic strategies to improve outcomes for RMS patients.
利益披露 Disclosure
A. Mazhar, None..
Q. Wang, None..
S. Gadde, None..
D. Ravindraraja, None..
B. Cheung, None..
G. Marshall, None.