Proteogenomic Characterization Unveils Biomarkers Associated With Chemoresistance in Muscle Invasive Bladder Cancer

Cisplatin-based neoadjuvant chemotherapy (NAC) combined with radical cystectomy has been used as a standard of care for patients with clinically non-metastatic muscle invasive urothelial bladder cancer, but 40-50% patients will benefit from the treatment. Molecular subtypes of muscle invasive bladder cancer (MIBC) provide a possible biomarker roadmap that may help identify patients who may or may not respond to NAC. Recently, by integrating Mass Spectrometry-based proteomics, proteogenomic studies from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) have demonstrated the potential of this approach in prioritizing candidate driver genes and facilitating understanding of the chemotherapy resistance mechanisms. To demonstrate the proteogenomic workflow for investigating the mechanisms underlying chemotherapy resistance in MIBC, we conducted proteogenomic characterization of 46 pre- and 14 post- treatment tumors, including whole exome sequencing (WES), mRNA sequencing, Tandem Mass Tag (TMT)-proteomics, or TMT-phosphoproteomic. These samples were selected from an initial cohort of 143 MIBC tissues and filtered by tumor purity, clinical history, pathological response, and proteomic quality control metrics. The final cohort comprised samples from 53 patients, including 45 with both genomic and proteomic data and 8 with genomic data only.Our multi-omics-based clustering reveals subtypes associated with chemotherapy sensitivity. Protein abundance of a short isoform of ATAD1 and RAF family proteins were identified as biomarkers of chemosensitivity which highlights the importance of proteomics data. Integration of proteomic and phosphoproteomic data reveals Wnt signaling via GSK3B-S9 phosphorylation and the JAK/STAT pathway as potential targets to overcome chemoresistance. Correlations between PD-L1 and TROP-2/NECTIN-4 indicate an additive benefit of combination therapy targeting these proteins. Our data analysis clearly demonstrates that proteogenomic plays an important role in uncovering potential resistance mechanisms that operate only at protein and Post-Translational Modification (PTM) levels. Proteomics data can be accessed through PRIDE with ID PXD060290. RNA-seq and whole-exome sequencing (WES) FASTQ files are archived in the dbGaP database. ]]>
Clinical eligibility criteria were patients with muscle invasive urothelial bladder cancer (MIBC) undergoing cisplatin-based neoadjuvant chemotherapy with either MVAC (methotrexate, vinblastin, doxorubicin, cisplatin) or GemCIS (gemcitabine, cisplatin) and radical cystectomy (RC) or radiation therapy (RT).]]>

基于顺铂的新辅助化疗（neoadjuvant chemotherapy, NAC）联合根治性膀胱切除术，目前已成为临床非转移性肌层浸润性尿路上皮膀胱癌患者的标准治疗方案，但仅40%~50%的患者能从该治疗中获益。肌层浸润性膀胱癌（muscle invasive bladder cancer, MIBC）的分子分型可为筛选可从顺铂新辅助化疗获益的患者提供潜在的生物标志物路线图。

近期，临床蛋白质组肿瘤分析联盟（Clinical Proteomic Tumor Analysis Consortium, CPTAC）开展的蛋白基因组学研究整合了基于质谱的蛋白质组学技术，证实了该方法在优先筛选候选驱动基因、阐明化疗耐药机制方面的应用潜力。

为验证用于探究MIBC化疗耐药机制的蛋白组学工作流程，我们对46份治疗前肿瘤样本及14份治疗后肿瘤样本开展了蛋白基因组学表征，检测内容涵盖全外显子测序（whole exome sequencing, WES）、mRNA测序、串联质谱标签（Tandem Mass Tag, TMT）蛋白质组学及TMT磷酸化蛋白质组学。这些样本初始队列共包含143份MIBC组织，经肿瘤纯度、临床病史、病理应答情况及蛋白质组学质量控制指标筛选后，最终队列纳入53例患者的样本：其中45例同时具备基因组学与蛋白质组学数据，8例仅具备基因组学数据。

基于多组学的聚类分析揭示了与化疗敏感性相关的分子亚型。研究鉴定出ATAD1短亚型蛋白及RAF家族蛋白的表达丰度可作为化疗敏感性的生物标志物，凸显了蛋白质组学数据的重要价值。整合蛋白质组学与磷酸化蛋白质组学数据发现，经GSK3B-S9磷酸化调控的Wnt信号通路与JAK/STAT通路可作为克服化疗耐药的潜在治疗靶点。PD-L1与TROP-2、NECTIN-4的相关性分析提示，靶向这些蛋白的联合治疗可能存在叠加获益。

我们的数据分析清晰证实，蛋白基因组学分析在挖掘仅存在于蛋白质及翻译后修饰（Post-Translational Modification, PTM）层面的潜在耐药机制中发挥关键作用。蛋白质组学数据可通过PRIDE数据库获取，编号为PXD060290。RNA测序及全外显子测序的FASTQ文件存档于dbGaP数据库。

临床入组标准为：接受基于顺铂的新辅助化疗（方案为MVAC：甲氨蝶呤、长春碱、多柔比星、顺铂；或GemCIS：吉西他滨、顺铂）联合根治性膀胱切除术（radical cystectomy, RC）或放射治疗（radiation therapy, RT）的肌层浸润性尿路上皮膀胱癌患者。