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

大剂量对乙酰氨基酚(AAP)延迟N-乙酰半胱氨酸(NAC)抢救在早期显示出希望 阶段临床试验，导致8/14的疾病缩小和3/14可评估的患者的客观反应 肿瘤类型多种多样。然而，大剂量AAP的抗肿瘤作用机制尚不清楚。 明白了。以前人们认为，高剂量的AAP具有抗肿瘤活性，其过程与其类似 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，对大剂量AAP的反应，肿瘤内的谷胱甘肽没有被耗尽。 治疗。这提示了大剂量AAP抗肿瘤作用的另一种机制。在这项提案中，我们 概述初步数据表明：1.)高剂量AAP具有抗肿瘤干细胞(CSC)活性和2。 大剂量AAP作为一种新的STAT3磷酸化抑制剂发挥作用。STAT3是一种具有 以前被证明在维持和扩散可行的CSC方面发挥着重要作用 人口。我们推测，大剂量AAP对非小细胞肺癌(NSCLC)的抗CSC活性 是通过STAT3依赖的机制。在具体目标#1，分目标A中，我们建议评价该机制 AAP对STAT3的抑制作用。我们已经获得了初步数据，表明AAP与SH2中的STAT3结合 结构域通过在Lys626形成的强氢键来实现。我们的目标是用以下方法验证这些初步发现 微尺度热分离(MST)等正交法。这些研究将在 与共同导师、弗吉尼亚联邦药物化学系主任Uesh Desai博士合作 大学(VCU)。在次目标B中，我们评估了大剂量AAP抗CSC活性的机制。我们 将产生带有改变的STAT3蛋白的人NSCLC的构建：空载体(STAT3 KO)，STAT3 K626K(WT)和STAT3 K626A(预测已降低AAP结合)。我们将确定敏感度 STAT3改变的结构对AAP或AAP+NAC的抗CSC效应的影响。如果携带STAT3的人非小细胞肺癌细胞 K626A降低了对大剂量AAP抗CSC作用的反应性，这可能提示 AAP的抗CSC作用是通过与STAT3直接结合介导的。在具体目标2中，我们将评估 非小细胞肺癌化疗(无靶向驱动基因突变)与靶向协同作用机制的研究 体外和体内AAP治疗(EGFR或ALK驱动因素改变的非小细胞肺癌)。我们会调查的 AAP是否通过a-STAT3依赖增强化疗/靶向治疗抗肿瘤活性 机制。在这份CDA申请中，纽韦尔特博士建议与一群杰出的 拥有指导纽韦尔特博士所需经验和专业知识的科学家。 纽韦尔特博士的主要导师将是帕特尔博士，他是退伍军人事务部资助的高级研究员，具有癌症方面的专业知识 干细胞生物学。拉纳博士是该项目的共同导师，他发现了STAT3在线粒体中的作用 他的大部分职业生涯都致力于了解STAT3生理学。如果被授予CDA，Dr。 纽韦尔特将参加当地大学的相关课程，参加科学会议，照顾基于退伍军人管理局的 患者，并发展他的研究实验室，所有这些都是为了成为一名独立的退伍军人事务部医生 科学家。

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.