High dose acetaminophen with n-acetylcysteine rescue as a novel STAT3 inhibitor with anti-cancer stem cell properties

高剂量对乙酰氨基酚与 n-乙酰半胱氨酸救援作为具有抗癌干细胞特性的新型 STAT3 抑制剂

基本信息

批准号：
10368472
负责人：
Alexander Neuwelt
金额：
--
依托单位：
VA VETERANS ADMINISTRATION HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-10-01 至 2026-09-30
项目状态：
未结题

项目摘要

High dose acetaminophen (AAP) with delayed n-acetylcysteine (NAC) rescue has shown promise in early phase clinical trials, inducing disease shrinkage in 8/14 and an objective response in 3/14 assessable patients with diverse tumor types. However, the mechanism of anti-tumor activity of high dose AAP is not well understood. It was previously felt that high dose AAP has anti-tumor activity via similar processes as its mechanism of hepatotoxicity, namely free radical injury and glutathione depletion. However, pre-clinical and clinical evidence suggests that glutathione is not depleted within the tumor in response to high-dose AAP treatment. This suggests an alternate mechanism of anti-tumor activity of high dose AAP. In this proposal, we outline preliminary data suggesting that: 1.) High dose AAP has anti-cancer stem cell (CSC) activity and 2.) High dose AAP functions as a novel inhibitor of STAT3 phosphorylation. STAT3 is a protein that has previously been shown to play an important role in the maintenance and proliferation of a viable CSC population. We hypothesize that the anti-CSC activity in non-small cell lung cancer (NSCLC) of high dose AAP is via a STAT3 dependent mechanism. In specific aim #1, sub-aim A, we propose to evaluate the mechanism of STAT3 inhibition by AAP. We have obtained preliminary data suggesting that AAP binds STAT3 in the SH2 domain via a strong hydrogen bond formed at Lys626. We aim to validate these preliminary findings using orthogonal approaches such as microscale thermopheresis (MST). These studies will be performed in collaboration with co-mentor Umesh Desai, PhD, head of Medicinal Chemistry at Virginia Commonwealth University (VCU). In sub-aim B, we evaluate the mechanism of the anti-CSC activity of high dose AAP. We will generate constructs of human NSCLC with altered STAT3 proteins: empty vector (STAT3 KO), STAT3 K626K (WT) and STAT3 K626A (predicted to have decreased AAP-binding). We will determine the sensitivity of STAT3-altered constructs to the anti-CSC effects of AAP or AAP + NAC. If human NSCLC cells with STAT3 K626A have decreased responsiveness to the anti-CSC effects of high dose AAP, this would suggest that the anti-CSC effects of AAP are mediated by direct binding to STAT3. In specific aim #2, we will evaluate the mechanisms of synergy between chemotherapy (in NSCLC without targetable driver mutation) and targeted therapy (in NSCLC with EGFR or ALK driver alteration) with AAP, both in vitro and in vivo. We will investigate whether AAP-enhances chemotherapy/targeted therapy anti-tumor activity via a a STAT3 dependent mechanism. In this CDA application, Dr. Neuwelt proposes to work with an outstanding group of established scientists whom have the experience and expertise necessary to mentor Dr. Neuwelt on the proposed project. Dr. Neuwelt’s primary mentor will be Dr. Patel, a senior VA-merit funded researcher with expertise in cancer stem cell biology. Dr. Larner, a co-mentor on the project, discovered the role of STAT3 in mitochondrial function and has dedicated much of his career to understanding STAT3 physiology. If awarded a CDA, Dr. Neuwelt will enroll in relevant classes at local Universities, attend scientific conferences, take care of VA-based patients, and grow his research lab, all with the goal of becoming an independent VA-based physician scientist.

高剂量对乙酰氨基酚 (AAP) 与延迟的 n-乙酰半胱氨酸 (NAC) 救援在早期已显示出希望阶段临床试验，在 8/14 中诱导疾病缩小，并在 3/14 可评估患者中产生客观反应然而，高剂量AAP的抗肿瘤活性机制尚不清楚。此前人们认为高剂量 AAP 通过与其相似的过程具有抗肿瘤活性。肝毒性的机制，即自由基损伤和谷胱甘肽耗竭，然而，临床前和临床证据表明，高剂量 AAP 不会导致肿瘤内的谷胱甘肽耗尽这表明了高剂量 AAP 的抗肿瘤活性的另一种机制。概述初步数据表明：1.) 高剂量 AAP 具有抗癌干细胞 (CSC) 活性，2.) 高剂量 AAP 作为 STAT3 磷酸化的新型抑制剂发挥作用，STAT3 是一种具有以下功能的蛋白质。先前已被证明在可行的 CSC 的维持和增殖中发挥重要作用我们研究了高剂量 AAP 对非小细胞肺癌 (NSCLC) 的抗 CSC 活性。是通过 STAT3 依赖机制实现的。在具体目标 #1 的子目标 A 中，我们建议评估该机制。我们已经获得了初步数据，表明 AAP 在 SH2 中结合 STAT3。我们的目标是使用 Lys626 上形成的强氢键来验证这些初步发现。正交方法，例如微尺度热交换（MST），这些研究将在与联合导师 Umesh Desai 博士、弗吉尼亚联邦大学药物化学负责人合作大学（VCU）。在子目标 B 中，我们评估了高剂量 AAP We 的抗 CSC 活性的机制。将生成具有改变的 STAT3 蛋白的人类 NSCLC 构建体：空载体 (STAT3 KO)、STAT3 K626K (WT) 和 STAT3 K626A（预计 AAP 结合减少）我们将确定敏感性。 STAT3 改变的构建体对 AAP 或 AAP + NAC 的抗 CSC 作用（如果具有 STAT3 的人 NSCLC 细胞）。 K626A 对高剂量 AAP 的抗 CSC 作用的反应性降低，这表明 AAP 的抗 CSC 作用是通过直接结合 STAT3 介导的。在具体目标 #2 中，我们将评估化疗（在没有靶向驱动突变的非小细胞肺癌）和靶向化疗之间的协同机制我们将在体外和体内研究 AAP 疗法（针对具有 EGFR 或 ALK 驱动基因改变的 NSCLC）。 AAP 是否通过 STAT3 依赖性增强化疗/靶向治疗的抗肿瘤活性在这个 CDA 申请中，Neuwelt 博士建议与一个杰出的既定团队合作。拥有指导 Neuwelt 博士完成拟议项目所需经验和专业知识的科学家。 Neuwelt 博士的主要导师是 Patel 博士，他是一位 VA 优秀资助的高级研究员，具有癌症方面的专业知识该项目的共同导师 Larner 博士发现了 STAT3 在线粒体中的作用。如果获得 CDA，博士将其职业生涯的大部分时间致力于理解 STAT3 生理学。 Neuwelt 将在当地大学注册相关课程、参加科学会议、照顾 VA 的患者，并扩大他的研究实验室，所有这些都是为了成为一名独立的退伍军人管理局医生科学家。