RPPA profiling of ES8 Ewing sarcoma xenografts after Endoglin-targeting antibody-drug conjugate treatments

Endoglin (EDG) is a cell surface protein with an important role in the establishment of neo-angiogenesis and vasculogenic mimicry. EDG is part of the transforming growth factor-β (TGF-β) family, acting as an important co-receptor. EDG is shed from the cell surface into the extracellular compartment by matrix metalloproteinase 14 (MMP14), in its soluble form (sEDG). Both transmembrane and soluble forms of EDG exert important signaling functions in the development of new blood vessels and tumour progression. To better understand the role of EDG in Ewing sarcoma (ES), a deadly neoplasm of late childhood and adolescence, we test the efficacy of OMTX703, an endoglin-targeting antibody-drug conjugate in ES8 xenograft. Having determined an optimal dose for OMTX703, an additional experiment was conducted to assess the mechanism(s) of OMTX703 action and its potential mechanism(s) of resistance following a 2-week exposure to OMTX703 at 0, 10, 30, and 60 mg/kg; 246 proteins were assessed by reverse-phase protein array (RPPA). Analysis of variance (ANOVA), Pearson’s correlation as distance metric and Ward’s linkage as the clustering method using a false discovery rate (FDR) of 0.01, identified 60 proteins that discriminated between treatment groups (Matrix#1-Normalized Values). To investigate the proteomic changes associated with the heightened clinical activity of the 60 mg/kg dose, a secondary analysis was performed, which grouped the 10 mg/kg OMTX703 samples and the 10 mg/kg OMTX003 ones with the placebo-treated samples (Matrix#2-Normalized Values). Using a FDR of 0.0001, an absolute log2 fold change of 1.5, Pearson’s correlation as distance metric and Ward’s linkage as the clustering method, 22 proteins were discriminately identified between the 3 treatment groups (Matrix#2-Normalized Values). Notably, a protein regulator of altered metabolism (RPS6) was exclusively upregulated following OMTX703 (60mg/kg), and a second metabolism biomarker (LDHA) was down-expressed in the 30 and 60 mg/kg-treated groups. Conversely, BRD4 was one of about a dozen proteins that were preferentially down-regulated in samples treated only by 60 mg/kg. An RPPA analysis of ES8 xenografts treated with OMTX003 (n=8, a nacked mAb anti endoglin at 10 mg/kg), OMTX703 (an endoglin-targeting antibody-drug conjugate with cytolisin at 10, 30 and 60 mg/kg (n=8 each)) or control vehicle (n=7) were performed simultaneously using the same array. Lysates were processed, spotted onto nitrocellulose-coated FAST slides, probed with 300 validated primary antibodies, and detected using earlier methods. MicroVigene software program (VigeneTech) was used for automated spot identification, background correction, and individual spot-intensity determination. Expression data was normalized for possible unequal protein loading, taking into account the signal intensity for each sample for all antibodies tested. Raw log2 intensity values were normalized for global protein expression by median centering across 246 antibodies tested. Principal component analysis was used to check for a batch effect and feature-by-feature two-sample t-tests were used to assess differences between treatment and control groups. We also used feature-by-feature one-way analysis of variance (ANOVA) followed by the Tukey test to perform pair comparisons for all groups. Beta-uniform mixture models were used to fit the resulting p value distributions to adjust for multiple comparisons. The cutoff p values and number of significant proteins were computed for several different false discovery rates (FDRs).

内皮糖蛋白（Endoglin, EDG）是一种细胞表面蛋白，在新生血管生成（neo-angiogenesis）与血管生成拟态（vasculogenic mimicry）的构建过程中发挥关键作用。EDG属于转化生长因子-β（transforming growth factor-β, TGF-β）家族成员，作为重要的共受体参与调控相关生物学过程。基质金属蛋白酶14（matrix metalloproteinase 14, MMP14）可将EDG从细胞表面剪切脱落至细胞外间隙，形成可溶性形式（soluble form, sEDG）。EDG的跨膜型与可溶性亚型均在新生血管发育及肿瘤进展过程中承担重要的信号传导功能。为深入阐明EDG在尤文肉瘤（Ewing sarcoma, ES）——一种高发于儿童及青少年的致死性恶性肿瘤——中的生物学功能，我们评估了靶向内皮糖蛋白的抗体偶联药物（antibody-drug conjugate）OMTX703在ES8异种移植模型中的抗肿瘤疗效。在确定OMTX703的最优给药剂量后，我们开展了补充实验以探究OMTX703的作用机制，以及以0、10、30、60 mg/kg剂量连续给药2周后产生耐药性的潜在通路；本实验采用反相蛋白阵列（reverse-phase protein array, RPPA）对246种蛋白进行检测分析。采用方差分析（analysis of variance, ANOVA）、以皮尔逊相关系数（Pearson’s correlation）作为距离度量指标、沃德联结法（Ward’s linkage）作为聚类方法，并设定错误发现率（false discovery rate, FDR）为0.01，最终筛选出60种可区分不同给药组的差异蛋白（矩阵1-标准化值，Matrix#1-Normalized Values）。为探究60 mg/kg剂量组临床活性增强相关的蛋白质组学变化，我们开展了二次分析：将10 mg/kg OMTX703样本、10 mg/kg OMTX003样本与安慰剂处理样本进行合并分组（矩阵2-标准化值，Matrix#2-Normalized Values）。采用FDR=0.0001、绝对log2倍数变化≥1.5、皮尔逊相关系数作为距离度量、沃德联结法作为聚类方法，最终在3个给药组间筛选出22种差异蛋白（矩阵2-标准化值，Matrix#2-Normalized Values）。值得注意的是，代谢调控蛋白RPS6仅在OMTX703（60 mg/kg）组中显著上调，而另一代谢生物标志物LDHA则在30 mg/kg及60 mg/kg给药组中表达下调。与之相反，BRD4是仅在60 mg/kg给药组中优先下调的十余种蛋白之一。本研究同时采用同一蛋白阵列平台，对经以下试剂处理的ES8异种移植瘤开展RPPA分析：OMTX003（n=8，10 mg/kg剂量下的靶向内皮糖蛋白裸单克隆抗体）、OMTX703（10、30、60 mg/kg剂量下的携带溶胞素（cytolisin）的靶向内皮糖蛋白抗体偶联药物，每组n=8），以及对照溶媒（n=7）。样品裂解液经处理后点样于硝酸纤维素包被的FAST玻片，采用300种经过验证的一抗进行免疫孵育，通过既往报道的实验方法完成信号检测。采用MicroVigene软件（VigeneTech公司开发）实现斑点自动识别、背景校正及单斑点信号强度定量。考虑到不同样本间蛋白上样量可能存在不均一性，我们针对所有检测抗体的信号强度对表达数据进行标准化校正，以消除上样偏差。原始log2强度值通过对246种检测抗体的信号进行中位数中心化处理，以实现整体蛋白表达水平的标准化。采用主成分分析（principal component analysis）检验批次效应，采用逐特征两样本t检验评估给药组与对照组间的表达差异。我们同时采用逐特征单因素方差分析（ANOVA）结合Tukey检验，完成所有组间的两两比较。采用β-均匀混合模型（beta-uniform mixture models）拟合所得p值分布，以校正多重比较带来的假阳性误差。针对多种不同的错误发现率（FDR）阈值，我们计算了临界p值及显著差异蛋白的数量。