Dynamic BH3 Profiling with Patient Derived Organoids of Esophageal Cancer and Mesothelioma Enable Precision-Based Targeting of the Mitochondrial Apoptotic Pathway

摘要/摘要 食道癌和恶性胸膜间皮瘤很难治疗，因为它们通常有过度的包膜。 反流和石棉的反复侮辱导致了3000个体细胞突变。识别一种突变或致病途径 肿瘤细胞通过新的突变和旁路途径绕过靶点。因为这个原因， 以线粒体通路为靶点是一种显著改进的方法，因为它们 致癌驱动蛋白下游和通路突变。 最近，我们发现癌前病变的Barrett‘s食道细胞慢性暴露于胆盐诱导 通过一种机制进行恶性转化，这种机制被称为‘少数MOMP(线粒体外膜 渗透性)‘。MOMP受B细胞淋巴瘤-2(Bcl2)家族蛋白的调节，这些蛋白分为 与BH3(BH3)结构域相互作用的促和抗凋亡蛋白。少数民族MOMP部分 将凋亡机制的内在途径激活到不足以导致细胞死亡的水平；相反，它 促进基因组不稳定、细胞转化和肿瘤发生。我们注意到在《少数民族MOMP》中， 巴雷特细胞通过上调抗凋亡蛋白Mcl-1来抵抗细胞凋亡。当我们锁定目标时 MCL-1、少数MOMP从亚致死的线粒体激活转向坦率的凋亡与肿瘤细胞 死了。动态BH3图谱(DBP)提供了一种检测哪些抗凋亡蛋白负责的方法 每种肿瘤的抗药性。通过测定相关的抗凋亡蛋白，一类化合物BH3 模仿，瞄准那些特定的蛋白质。最近，一个生化“工具箱”利用DBP来识别合适的 BH3在过表达bcl2蛋白的小鼠细胞中的模拟。患者衍生的有机化合物(PDO)可以 总结肿瘤对治疗的反应。如果一个在PDO中使用DBP的综合生化工具包 识别能够直接从患者肿瘤中抵抗的蛋白质，然后基于精确的靶向 Bcl2蛋白为克服难治性癌症提供了一种治疗策略。 我们的目标是破坏线粒体平衡，线粒体平衡促进癌症发生，但通过直接抑制细胞凋亡 以抗药性蛋白为靶标。我们将建立一个生化工具包来预测治疗 利用DBP-PDO模型对食道癌和间皮瘤的反应。以这些蛋白质为靶点将 通过阻断代偿性抗凋亡蛋白，使少数人的MOMP向凋亡方向转变。我们的假设是 DBP-PDO模型在肿瘤活检后7天内是一种临床可操作的生物检测方法。这股海流 研究将允许我们通过以下方式完全绕过难以处理的旁路机制和癌细胞的突变 靶向下游的线粒体抗性机制。这一新战略有望改变少数族裔 MOMP导致坦率的凋亡，从而使这些细胞容易受到标准治疗的影响。

SUMMARY / ABSTRACT Esophageal cancer and malignant pleural mesothelioma are difficult to treat because they typically harbor over 3000 somatic mutations from the repeated insults of reflux and asbestos. Identifying one mutation or pathway to target is circumvented by the tumor cell through new mutations and bypass pathways. For this reason, targeting the mitochondrial pathways represents a substantially improved approach because they are downstream of oncogenic driver proteins and pathway mutations. Recently, we have shown that chronic exposure of pre-neoplastic, Barrett’s esophageal cells to bile salt induced malignant transformation through a mechanism termed, ‘Minority MOMP (mitochondrial outer membrane permeabilization)’. MOMP is regulated by the B-cell lymphoma-2 (Bcl-2) family of proteins that are divided into pro- and anti-apoptotic proteins that interact at Bcl-2 homology-3 (BH3) domains. Minority MOMP partially activates the intrinsic pathway of the apoptotic machinery to a level not sufficient to result in cell death; rather, it promotes genomic instability, cellular transformation, and tumorigenesis. We noted that in ‘Minority MOMP’, Barrett’s cells resisted apoptosis through the upregulation of the anti-apoptotic protein, Mcl-1. When we targeted Mcl-1, Minority MOMP shifted from the sub-lethal mitochondrial activation to frank apoptosis and tumor cells died. Dynamic BH3 profiling (DBP) provides an assay to measure which anti-apoptotic proteins are responsible for the resistance for each tumor. By determining the relevant anti-apoptotic protein, a class of compounds, BH3 mimetics, target those specific protein. Recently, a biochemical ‘toolkit’ utilized DBP to identify the appropriate BH3 mimetic in murine cells that overexpressed bcl-2 proteins. Patient-derived organoids (PDO) can recapitulate tumor response to therapy. If a comprehensive biochemical toolkit utilizing DBP in PDOs identifies proteins that enable resistance directly from patient tumors, then precision-based targeting of the Bcl-2 proteins provides a therapeutic strategy to overcome treatment-refractory cancers. Our goal is to disrupt the mitochondrial balance that enables carcinogenesis but blocks apoptosis by directly targeting the proteins responsible for resistance. We will establish a biochemical toolkit to predict treatment response in esophageal cancer and mesothelioma by utilizing a DBP-PDO model. Targeting these proteins will shift Minority MOMP toward apoptosis by blocking the compensatory anti-apoptotic proteins. Our hypothesis is that the DBP-PDO model is a clinically actionable bioassay within seven days from tumor biopsy. This current research will allow us to circumvent intractable bypass mechanisms and mutations of cancer cells altogether by targeting the downstream mitochondrial resistance mechanism. This novel strategy is expected to shift Minority MOMP toward frank apoptosis and thus render these cells vulnerable to standard therapies.