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

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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.