PO.ET02.09 · 实验与分子治疗
利用 AVITI24™ 多组学空间生物学平台剖析过氧化物还原酶 3（PRX3）药理学抑制和基因敲除的效应
Profiling the effects of Peroxiredoxin 3 (PRX3) pharmacological inhibition and genetic deletion with the AVITI24™ multi-omic spatial biology platform
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摘要 Abstract
中文摘要
靶向肿瘤细胞的代谢脆弱性（包括肿瘤细胞逃逸氧化应激）是肿瘤学中一种有吸引力的治疗策略。硫链丝菌素（Thiostrepton，TS）是 RSO-021 的活性药物成分，是一种处于临床阶段的过氧化物还原酶 3（PRX3）共价抑制剂，目前正处于治疗胸膜间皮瘤的 2 期试验中（NCT05278975）。PRX3 表达支持肿瘤细胞逃逸氧化应激，而通过 CRISPR/Cas9 进行基因敲除或用 TS 对 PRX3 进行药理学靶向可抑制间皮瘤肿瘤生长。为进一步剖析细胞对 PRX3 敲除和 TS 抑制的反应，我们利用 AVITI24™ 多组学空间生物学平台，在单次运行中对数十万个细胞进行分析。使用 Teton™ CytoProfiling 检测，我们分析了高维形态学、靶向转录组数据和多重蛋白质组数据，从而能够将分子标志物与通路水平的读数直接关联。差异表达通过两种互补方法进行评估。单细胞分析使用条件间的 Wilcoxon 秩和检验，而伪批量（pseudobulk）分析将重复孔聚合为伪批量组并使用 DESeq 进行差异表达检验。使用 log₂FC 阈值 0.1 鉴定每种条件的标志基因，并比较两种方法的结果以评估一致性。在 H-MESO-1 人间皮瘤细胞系中敲除 PRX3 在 RNA 水平（如 CDK1 和 Kif11）和蛋白质水平（如 phospho-p38、phospho-AKT1、phospho-CDK1）降低了细胞周期和增殖基因的表达。TS 处理 H-MESO-1 细胞增加了与丝裂原活化蛋白激酶（MAPK）信号（MAPK10、MAPK11）、应激反应（HSP70）和凋亡（FASLG）相关基因的转录本和蛋白质水平。TS 处理降低了与线粒体功能、细胞迁移和生长相关基因的表达。TS 处理未在 PRX3 敲除细胞中引起与野生型处理细胞相同的基因表达效应，MAPK 信号是 PRX3 敲除和 TS 处理之间一个共同上调的特征（如 MAPK13 和 MAP3K5）。总之，AVITI24™ 多组学空间生物学平台提供了一个稳健的工作流程，支持剖析对间皮瘤肿瘤细胞中 PRX3 的基因和药理学靶向，进一步支持 PRX3 作为一种新颖且可干预的癌症治疗靶点。
查看英文原文 English abstract
Targeting metabolic vulnerabilities of tumor cells, including tumor cell escape from oxidative stress, is an attractive therapeutic approach in oncology. Thiostrepton (TS), the active pharmaceutical ingredient of RSO-021, is a clinical stage peroxiredoxin 3 (PRX3) covalent inhibitor currently in phase 2 testing for the treatment of pleural mesothelioma (NCT05278975). PRX3 expression supports tumor cell escape from oxidative stress and genetic deletion via CRISPR/Cas9 or pharmacological targeting of PRX3 with TS inhibits mesothelioma tumor growth. To further profile cellular responses to PRX3 deletion and inhibition with TS we utilized the AVITI24™ multi-omic spatial biology platform to profile hundreds of thousands of cells in a single run. Using the Teton™ CytoProfiling assay, we analyzed high-dimensional morphology, targeted transcriptomic data, and multiplexed proteomic data, enabling direct linkage of molecular markers to pathway-level readouts. Differential expression was evaluated using two complementary approaches. Single-cell analyses used a Wilcoxon rank-sum test between conditions, while pseudobulk analyses aggregated replicate wells into pseudo-bulk groups and used DESeq for differential expression testing. Marker genes for each condition were identified using a log₂FC threshold of 0.1, and results from both methods were compared to assess concordance. Deletion of PRX3 in the H-MESO-1 human mesothelioma cell line reduced expression of cell cycle and proliferation genes at the RNA level (ie. CDK1 and Kif11) and protein level (ie. phospho-p38, phospho-AKT1, phospho-CDK1). TS treatment of H-MESO-1 cells increased transcript and protein levels of genes related to Mitogen-activated protein kinase (MAPK) signaling (MAPK10, MAPK11), stress response (HSP70), and apoptosis (FASLG). TS treatment reduced expression of genes related to mitochondrial function, cell migration, and growth. TS treatment did not elicit the same effects on gene expression in PRX3 deleted cells as wild type treated cells with MAPK signaling being one common increased feature across PRX3 deletion and TS treatment (ie. MAPK13 and MAP3K5). Together the AVITI24™ multi-omic spatial biology platform provides a robust workflow that supported profiling genetic and pharmacological targeting of PRX3 in mesothelioma tumor cells, further supporting PRX3 as a novel and actionable therapeutic target in cancer.
利益披露 Disclosure
V. Gibson,
RS Oncology Other, Research presented in this abstract was supported by a Research Contract between RS Oncology and The University of Vermont.
S. Lymer,
Element Biosciences Employment.
T. Wolf,
Element Biosciences Employment.
B. Cunniff,
RS Oncology Independent Contractor, Stock, ), Travel.