High-Dose Acetaminophen with Concurrent CYP2E1 Inhibition Has Profound Anticancer Activity without Liver Toxicity.

Acetaminophen (AAP) is metabolized by a variety of pathways such as sulfation, glucuronidation, and fatty acid amide hydrolase-mediated conversion to the active analgesic metabolite AM404. CYP2E1-mediated metabolism to the hepatotoxic reactive metabolite NAPQI (N-acetyl-p-benzoquinone imine) is a minor metabolic pathway that has not been linked to AAP therapeutic benefits yet clearly leads to AAP liver toxicity. N-acetylcysteine (NAC) (an antioxidant) and fomepizole (a CYP2E1 inhibitor) are clinically used for the treatment of AAP toxicity. Mice treated with AAP in combination with fomepizole (plus or minus NAC) were assessed for liver toxicity by histology and serum chemistry. The anticancer activity of AAP with NAC and fomepizole rescue was assessed in vitro and in vivo. Fomepizole with or without NAC completely prevented AAP-induced liver toxicity. In vivo, high-dose AAP with NAC/fomepizole rescue had profound antitumor activity against commonly used 4T1 breast tumor and lewis lung carcinoma lung tumor models, and no liver toxicity was detected. The antitumor efficacy was reduced in immune-compromised NOD-scid IL2Rgammanull mice, suggesting an immune-mediated mechanism of action. In conclusion, using fomepizole-based rescue, we were able to treat mice with 100-fold higher than standard dosing of AAP (650 mg/kg) without any detected liver toxicity and substantial antitumor activity. SIGNIFICANCE STATEMENT: High-dose acetaminophen can be given concurrently with CYP2E1 inhibition to allow for safe dose escalation to levels needed for anticancer activity without detected evidence of toxicity.

High-Dose Acetaminophen with N-acetylcysteine Rescue Inhibits M2 Polarization of Tumor-Associated Macrophages.

High-dose acetaminophen (AAP) with N-acetylcysteine (NAC) rescue is among the few treatments that has shown activity in phase I trials without achieving dose-limiting toxicity that has not progressed to evaluation in later line studies. While the anti-tumor effects of AAP/NAC appear not to be mediated by glutathione depletion and free radical injury, the mechanism of anti-tumor effects of AAP/NAC has not been definitively characterized. In vitro, the effects of AAP/NAC were evaluated on bone marrow derived macrophages. Effects of AAP on IL-4/STAT6 (M2) or IFN/LPS/STAT1 (M1) signaling and downstream gene and protein expression were studied. NAC reversed the AAP toxicity in the normal liver but did not reverse AAP cytotoxicity against tumor cells in vitro. AAP/NAC selectively inhibited IL-4-induced STAT6 phosphorylation but not IFN/LPS-induced STAT1 phosphorylation. Downstream, AAP/NAC inhibited IL-4 induction of M2-associated genes and proteins but did not inhibit the IFN/LPS induction of M1-associated genes and proteins. In vivo, AAP/NAC inhibited tumor growth in EF43.fgf4 and 4T1 triple-negative breast tumors. Flow cytometry of tumor-associated macrophages revealed that AAP/NAC selectively inhibited M2 polarization. The anti-tumor activity of high-dose AAP/NAC is lost in macrophage-depleted mouse syngeneic tumor models, suggesting a macrophage-dependent mechanism of action. In conclusion, our study is the first to show that high-dose AAP/NAC has profound effects on the tumor immune microenvironment that facilitates immune-mediated inhibition of tumor growth.

Antagonism between Prdm16 and Smad4 specifies the trajectory and progression of pancreatic cancer.

The transcription factor Prdm16 functions as a potent suppressor of transforming growth factor-beta (TGF-ß) signaling, whose inactivation is deemed essential to the progression of pancreatic ductal adenocarcinoma (PDAC). Using the KrasG12D-based mouse model of human PDAC, we surprisingly found that ablating Prdm16 did not block but instead accelerated PDAC formation and progression, suggesting that Prdm16 might function as a tumor suppressor in this malignancy. Subsequent genetic experiments showed that ablating Prdm16 along with Smad4 resulted in a shift from a well-differentiated and confined neoplasm to a highly aggressive and metastatic disease, which was associated with a striking deviation in the trajectory of the premalignant lesions. Mechanistically, we found that Smad4 interacted with and recruited Prdm16 to repress its own expression, therefore pinpointing a model in which Prdm16 functions downstream of Smad4 to constrain the PDAC malignant phenotype. Collectively, these findings unveil an unprecedented antagonistic interaction between the tumor suppressors Smad4 and Prdm16 that functions to restrict PDAC progression and metastasis.