Proteogenomics analysis to identify acquired resistance-specific alterations in melanoma PDXs on MAPKi therapy [RNA-seq]

Therapeutic approaches to treat melanoma include small molecule drugs that target activating protein mutations in pro-growth signaling pathways like the MAPK pathway. While beneficial to the approximately 50% of patients with activating BRAFV600 mutation, mono- and combination therapy with MAPK inhibitors is ultimately associated with acquired resistance. To better characterize the mechanisms of MAPK inhibitor resistance in melanoma, we utilize patient-derived xenografts and apply proteogenomic approaches leveraging genomic, transcriptomic, and proteomic technologies that permit the identification of resistance-specific alterations and therapeutic vulnerabilities. A specific challenge for proteogenomic applications comes at the level of data curation to enable multi-omics data integration. Here, we present a proteogenomic approach that uses custom curated databases to identify unique resistance-specific alternations in melanoma PDX models of acquired MAPK inhibitor resistance. We demonstrate this approach with a NRASQ61L melanoma PDX model from which resistant tumors were developed following treatment with a MEK inhibitor. Our multi-omics strategy addresses current challenges in bioinformatics by leveraging development of custom curated proteogenomics databases derived from individual resistant melanoma that evolves following MEK inhibitor treatment and is scalable to comprehensively characterize acquired MAPK inhibitor resistance across patient-specific models and genomic subtypes of melanoma. A patient-derived xenograft (PDX) of an NRASQ61L patient melanoma tumor was established as non-dissociated tumor fragments in NOD scid gamma (NSG) mice and serially passaged without dissociation, in vitro growth cycles, or adjunctive in vivo growth supplements such as Matrigel. To derive the acquired-resistant NRASQ61L tumors, we implanted one tumor fragment (passage #2) per NSG mouse. A cohort of tumor-bearing mice with similar tumor volumes were selected for experimentation. When the tumors reached ~500 mm3, a mouse was treated with the vehicle-chow (V), while four mice were treated with trametinib-embedded chow (R1,2,4,5) to achieve 5 mg/kg/day dosing. All trametinib-treated tumors displayed tumor regression at this chosen dosage. Tumor tissues for NRASQ61L vehicle (V) and four treated samples (R1,2,4,5) were harvested at end-points and prepared for whole exome sequencing (WES) and bulk RNA-sequencing (RNA-seq).

黑色素瘤的治疗手段包括靶向促增殖信号通路（如丝裂原活化蛋白激酶通路（mitogen-activated protein kinase pathway, MAPK））中激活型蛋白突变的小分子药物。尽管该疗法对约50%携带BRAFV600激活突变的患者有益，但MAPK抑制剂单药及联合治疗最终仍会引发获得性耐药。为更深入解析黑色素瘤中MAPK抑制剂耐药的机制，本研究采用患者来源异种移植瘤（patient-derived xenografts, PDX）模型，并运用蛋白基因组学（proteogenomic）方法，整合基因组学、转录组学与蛋白质组学技术，以鉴定耐药特异性改变及治疗脆弱靶点。蛋白基因组学应用的一大核心挑战在于如何完成多组学数据整合所需的数据整理工作。在此，我们提出一种基于定制整理数据库的蛋白基因组学方法，用于在获得性MAPK抑制剂耐药的黑色素瘤PDX模型中鉴定独特的耐药特异性改变。我们利用NRASQ61L型黑色素瘤PDX模型验证了该方法：经丝裂原活化蛋白激酶激酶（mitogen-activated protein kinase kinase, MEK）抑制剂处理后，该模型可诱导产生耐药肿瘤。我们的多组学策略通过开发源自MEK抑制剂治疗后演化的耐药黑色素瘤样本的定制整理蛋白基因组数据库，解决了生物信息学领域当前的诸多挑战，且该策略可扩展应用，以全面解析不同患者特异性模型及黑色素瘤基因组亚型中的获得性MAPK抑制剂耐药机制。我们在非肥胖糖尿病重症联合免疫缺陷（NOD scid gamma, NSG）小鼠中构建了NRASQ61L型黑色素瘤患者来源异种移植瘤（PDX）：采用未解离的肿瘤碎片进行接种，且无需解离、体外培养周期或基质胶（Matrigel）等辅助体内生长补充剂即可进行连续传代。为获得获得性耐药的NRASQ61L型肿瘤，我们向每只NSG小鼠接种第2代肿瘤碎片。选取肿瘤体积均一的荷瘤小鼠队列开展实验。当肿瘤体积达到约500 mm³时，1只小鼠给予载体饲料（V组），4只小鼠给予曲美替尼（trametinib）包埋饲料（R1、R2、R4、R5组），给药剂量为5 mg/kg/天。该给药剂量下，所有曲美替尼处理组的肿瘤均出现消退。在实验终点收集NRASQ61L型载体对照组（V组）及4个处理组（R1、R2、R4、R5组）的肿瘤组织，用于全外显子测序（whole exome sequencing, WES）及批量RNA测序（bulk RNA-sequencing, RNA-seq）。