PO.ET09.05 · 实验与分子治疗
联合SUMO抑制与伊立替康增强胰腺癌细胞毒性
Combining SUMO inhibition and irinotecan increases pancreatic cancer cytotoxicity
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：胰腺癌（PDAC）对化疗的反应为短暂性，这促使人们需要改进的治疗选择。伊立替康（IRI）是PDAC一线和二线治疗的标准治疗药物。SUMO化是拓扑异构酶1（Top-1）上的一种翻译后修饰，可调节其DNA结合。我们假设将IRI与Subasumstat（SST——一种SUMOi药物）联用将在降低Top-1效力方面产生协同作用，从而增强凋亡。
方法：人单细胞RNA测序（scRNASeq）：使用人胰腺癌单细胞图谱识别FOLFIRINOX后表达SUMO2/3的癌细胞。体外协同作用：用IRI和/或SST（1nM-500nM，24小时）处理KPC46、FC1245、FC1245-吉西他滨耐药的小鼠PDAC细胞；用cell titer glo评估活力；使用Combenefit生成协同作用图。蛋白质印迹（WB）：用SST和/或IRI（100nM）处理KPC46和FC1245细胞24小时；检测Top-1（染色质蛋白组分）、gamma-H2AX和切割的caspase-3以验证凋亡。生存实验：NSG小鼠接受原位KPC46细胞注射——腹腔注射SST（15mg/kg q48h）和/或IRI（12.5mg/kg 每周）；用超声评估肿瘤体积（TV）。人PDAC培养：将患者肿瘤切片在200nM SST和/或IRI中孵育5天；对EPCAM和PanCK PDAC细胞标志物进行流式细胞术（FC）检测。
结果：scRNASeq显示与相邻正常组织相比，SUMO2/3在PDAC原发肿瘤细胞中高表达，且表达SUMO2/3的癌细胞在FOLFIRINOX治疗后持续存在。SST+IRI之间显著的体外协同作用低至50nM即可发生。WB显示SST+IRI组的染色质结合Top-1、全细胞γ-H2AX和切割的caspase-3增加最为明显（条带强度：IRI:0.1，SST:0.15，SST+IRI:0.52；p=0.009）。SST+IRI处理在体内的协同细胞毒性使生存显著改善（IRI:16，SST:17，SST+IRI=24天；p=0.008）并使TV减小（治疗2周后；平均TV：IRI=358mm3，SST[所有小鼠死亡]，SST+IRI=132mm3；p=0.001）。在人PDAC切片中，SST+IRI处理使PANCK+和EPCAM+细胞的减少最为明显（IRI:24.4%，SST:15.5%，SST+IRI:9.5%）。
结论：SST+IRI构成了一种有前景的PDAC联合治疗方案，在小鼠和人模型中均观察到协同的PDAC细胞毒性。在临床上，SST可作为FOLFIRINOX的辅助手段，或SST+IRI可作为一种二线替代治疗，并有可能纳入RAS抑制剂。
查看英文原文 English abstract
Introduction: Pancreatic cancer (PDAC) has a transient response to chemotherapy, motivating the need for improved treatment options. Irinotecan (IRI) is a standard of care agent for both 1 st and 2 nd line PDAC therapy. SUMOylation is a post translational modification on topoisomerase 1 (Top-1) that modulates its DNA binding. We hypothesized that combining IRI with Subasumstat (SST-a SUMOi agent) would synergize in reducing Top-1 efficacy, leading to augmented apoptosis.
Methods: Human single cell RNA sequencing (scRNASeq) : using the Human Pancreatic Cancer Single-Cell Atlas to identify SUMO2/3 expressing cancer cells after FOLFIRNINOX. In Vitro Synergy : KPC46, FC1245, FC1245-Gem Resistant murine PDAC cells treated with IRI and/or SST (1nM-500nM, 24hrs); viability assessed with cell titer glo; synergy plots generated using Combenefit. Western Blots (WB) : KPC46 and FC1245 cells treated with SST and/or IRI (100nM) for 24 hours; probed for Top-1 (chromatin protein fraction), gamma-H2AX, and cleaved caspase-3 to validate apoptosis. Survival Experiments : NSG mice underwent orthotopic KPC46 cell injections-IP injections with SST (15mg/kg q48h) and/or IRI (12.5mg/kg qwkly); tumor volume (TV) assessed with ultrasound. Human PDAC Cultures : patient tumor slices incubated in 200nM SST and/or IRI for 5 days; flow Cytometry (FC) conducted for EPCAM and PanCK PDAC cell markers.
Results: scRNASeq showed SUMO2/3 is highly expressed PDAC primary tumor cells as compared to adjacent normal tissue and that SUMO2/3 expressing cancer cells persist after FOLFIRINOX treatment. Significant in vitro synergy between SST+IRI occurred as low as 50nM. WB showed the greatest increase in chromatin bound Top-1, total cell ϒ- H2AX, and cleaved caspase 3 (Band Intensity: IRI: 0.1, SST:0.15, SST+IRI: 0.52; p=0.009) occurred for SST+IRI. Synergistic cytotoxicity of SST+IRI treatment in vivo resulted in significantly improved survival (IRI:16, SST:17, SST+IRI=24 days; p=0.008) and reduced TV (after 2 wk-treatment; AvgTV: IRI=358mm3, SST [all mice dead], SST+IRI=132mm3; p=0.001). In human PDAC slices, SST+IRI treatment yielded the greatest decrease in PANCK+ and EPCAM+ cells (IRI:24.4%, SST:15.5%, SST+IRI:9.5%).
Conclusion: SST+IRI constitutes a promising combination treatment for PDAC with synergistic PDAC cytotoxicity observed in both murine and human models. Clinically, SST could serve as an adjunct to FOLFIRINOX or SST+IRI could be an alternative 2nd line treatment with potential to include a RAS inhibitor.
利益披露 Disclosure
A. Courelli, None..
L. Ren, None..
H. Tiriac, None..
Y. Chen, None..
A. Lowy, None.