Metabolic flux analysis and PDX models to understand therapeutic vulnerabilities following inhibition of Ref-1 redox signaling in pancreatic cancer

摘要 胰腺导管腺癌（PDAC）对治疗特别耐药，并且通常表现为 转移性疾病以缺氧、致密基质和代谢重新连接为特征，原始方法和 迫切需要组合策略。我们建议研究抑制氧化还原信号蛋白 和药物组合，其通过冲击肿瘤正在使用的关键途径来选择性地杀死肿瘤， 生存氧化还原因子-1（Ref-1）调节驱动胰腺癌的各种转录因子的活性 细胞增殖和耐药性以及参与细胞代谢的基因。在缺氧条件下， Ref-1显著干扰代谢途径（TCA循环和OXPHOS）和HIF调节基因， 从而减缓胰腺癌共培养球体和异种移植物的生长。第一代Ref-1 抑制剂（APX 3330）完成了I期试验，并显示了32%的缓解率、预测的PK和靶向 没有明显毒性的接触。有6名患者病情稳定，其中4名接受治疗 一段时间（>250天）。基于令人鼓舞的第一阶段数据和详细的结构活性 关系（SAR）计划，我们还确定了处于领先优化的下一代Ref-1抑制剂 阶段，一种筛选对Ref-1抑制敏感的患者的策略，以及 可能与Ref-1抑制协同作用。然而，抗性的适应机制最终会随着 靶向治疗，因此我们也将专注于开发新的组合。我们的假设是 单独靶向Ref-1的氧化还原功能和在机械设计的组合疗法中靶向Ref-1的氧化还原功能将诱导 代谢致死，抑制胰腺癌生长和转移。在目标1中，确定代谢 与Ref-1抑制的结果相关的癌细胞/组织的特征和新肿瘤的预测 代谢靶点以改善Ref-1抑制的功效。我们最近开发的计算预测器 细胞代谢通量将用于研究由于PDAC细胞中Ref-1抑制而引起的代谢变化， 单细胞水平。在目标2中，NMR确定Ref-1和新类似物的直接相互作用、功效、毒性， 和代谢稳定性研究将使我们能够推进候选药物体内研究的最佳候选药物 选择（NIH里程碑4）和IND（研究性新药）提交导致最终的I期试验。 最后，在目标3中，临床前联合治疗中Ref-1的评价将用于克服适应性 阻力为了进一步预测可能被扰动以与Ref-1抑制协同作用的代谢节点， 将使用Aim 1中描述的代谢致死率、细胞代谢通量的计算预测因子。的 将使用体外类器官和小鼠试验研究Ref-1单独和新组合的功效 体内设计总之，为了精确地杀死PDAC，我们将提供一种有效的选择性Ref-1 抑制剂和联合收割机新代谢生物信息学和药物组合，用于增强功效， 对该领域和临床治疗产生重大影响。

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is particularly resistant to therapy and typically presents as metastatic disease. Characterized by hypoxia, dense stroma, and metabolic rewiring, original approaches and combination strategies are desperately needed. We propose to investigate inhibition of a redox signaling protein and drug combinations that selectively kill the tumor by impinging on critical pathways the tumor is using to survive. Redox factor-1 (Ref-1) regulates the activity of various transcription factors that drive pancreatic cancer cell proliferation and drug resistance as well as genes involved in cellular metabolism. Under hypoxia, inhibition of Ref-1 significantly perturbed metabolic pathways (TCA cycle and OXPHOS) and HIF-regulated genes, and thus slowed the growth of pancreatic cancer co-culture spheroids and xenografts. The first-generation Ref-1 inhibitor (APX3330) completed phase I trial and demonstrated 32% response, predicted PK, and target engagement with no significant toxicities. There was disease stabilization in six patients with four on treatment for an extended time (>250 days). Based on encouraging phase I data and a detailed structural-activity relationship (SAR) program, we have also identified next generation Ref-1 inhibitors that are at lead optimization stage, a strategy to screen for patients that have sensitivity to Ref-1 inhibition, and molecular targets that are likely to synergize with Ref-1 inhibition. However, adaptive mechanisms of resistance eventually emerge with targeted therapy, therefore we will also focus on the development of novel combinations. Our hypothesis is that targeting the redox function of Ref-1 alone and in mechanistically designed combination therapies will induce metabolic lethality and inhibit pancreatic cancer growth and metastasis. In Aim 1, identification of metabolic characteristics of cancer cells/tissues that associate with the outcome of Ref-1 inhibition and prediction of new metabolic targets to improve the efficacy of Ref-1 inhibition. Our recently developed computational predictor of cell-wise metabolic flux will be used to study the metabolic changes due to Ref-1 inhibition in PDAC cells at the single cell level. In Aim 2, NMR to establish direct interactions of Ref-1 and the new analogues, efficacy, toxicity, and metabolic stability studies will allow us to advance the top lead candidate(s) for in vivo studies for Candidate Selection (NIH Milestone 4) and IND (Investigational New Drug) submission leading to eventual Phase I trial. Lastly in Aim 3, evaluation of Ref-1 in preclinical combination therapy will be used to overcome adaptive resistance. To further predict metabolic nodes that could be perturbed to synergize with Ref-1 inhibition, creating a metabolic lethality, computational predictor of cell-wise metabolic flux described in Aim1 will be used. The efficacy of Ref-1 alone and in new combinations will be investigated using organoids in vitro and the mouse trial design in vivo. In summary, for a precision approach to kill PDAC, we will deliver a potent and selective Ref-1 inhibitor and combine novel metabolic bioinformatics and drug combinations for enhanced efficacy to have a significant impact on the field and clinical therapeutics.