Purpose: Napabucasin (2-acetylfuro-1,4-naphthoquinone or BBI-608) is a small molecule currently being clinically evaluated in various cancer types. It has mostly been recognized for its ability to inhibit STAT3 signaling. However, based on its chemical structure, we hypothesized that napabucasin is a substrate for intracellular oxidoreductases and therefore may exert its anticancer effect through redox cycling, resulting in reactive oxygen species (ROS) production and cell death. Experimental Design: Binding of napabucasin to NAD(P) H:quinone oxidoreductase-1 (NQO1), and other oxidoreductases, was measured. Pancreatic cancer cell lines were treated with napabucasin, and cell survival, ROS generation, DNA damage, transcriptomic changes, and alterations in STAT3 activation were assayed in vitro and in vivo. Genetic knockout or pharmacologic inhibition with dicoumarol was used to evaluate the dependency on NQO1. Results: Napabucasin was found to bind with high affinity to NQO1 and to a lesser degree to cytochrome P450 oxidoreductase (POR). Treatment resulted in marked induction of ROS and DNA damage with an NQO1-and ROS-dependent decrease in STAT3 phosphorylation. Differential cytotoxic effects were observed, where NQO1-expressing cells generating cytotoxic levels of ROS at low napabucasin concentrations were more sensitive. Cells with low or no baseline NQO1 expression also produced ROS in response to napabucasin, albeit to a lesser extent, through the one-electron reductase POR. Conclusions: Napabucasin is bioactivated by NQO1, and to a lesser degree by POR, resulting in futile redox cycling and ROS generation. The increased ROS levels result in DNA damage and multiple intracellular changes, one of which is a reduction in STAT3 phosphorylation.

Napabucasin is an NAD(P)H:quinone oxidoreductase 1 (NQO1)bioactivatable small molecule hypothesized to affect multiple oncogenic pathways. In a prespecified, retrospective analysis of the napabucasin phase III CO.23 study, overall survival was longer for napabucasin versus placebo in patients expressing phosphorylated STAT3 (pSTAT3) in tumor cells and cells of the tumor microenvironment (TME). We hypothesized that a connection may exist between NQO1 expression in cancer cells and pSTAT3 in tumor cells and the TME. In 3D spheroid cocultures of cancer cells and cancer-associated fibroblasts, the antitumor activity of napabucasin was NQO1 dependent. The levels of cytokines such as IL6, CXCL10, and GM-CSF were higher in NQO1-positive versus NQO1deleted cocultures. These differentially secreted cytokines promoted STAT3 phosphorylation in tumor cells and the TME. NQO1-expressing, napabucasin-sensitive tumor cells can modify tumor cells and the TME to promote STAT3 phosphorylation, suggesting that pSTAT3 may be used to identify a subpopulation of patients who would likely respond to napabucasin. Implications: pSTAT3 is a potential biomarker for patient response to the anticancer drug napabucasin.

Paxillin is an adaptor protein associated with focal adhesion complex, and is activated by tyrosine phosphorylation through focal adhesion kinase (FAK) and Src kinase. Recent studies reveal that serine phosphorylation of paxillin by JNK and p38 MAPK is essential for cell migration or neurite extension, but their cellular targets remain unclear. In this study, we examined the requirement of paxillin phosphorylation by p38 MAPK or JNK in T-cell motility and activation using paxillin mutants at the respective phosphorylation sites, Ser85, and Ser178. (S85A)-paxillin, (S178A)-paxillin, or (S85A/S178A)-paxillin inhibited the motility of NIH/3T3 fibroblasts, but did not interfere with T-cell migration and integrin-mediated T-cell adhesion. In contrast, activation of T cells was effectively suppressed by (S85A/S178A)-paxillin. Transgenic (S85A/S178A)-paxillin expression inhibited T-cell proliferation and reduced the production of IL-2, IFN-γ, and IL-4. In searching for signals modulated by (S85A/S178A)-paxillin, we found that NFAT activation was specifically blocked by (S85A/S178A)-paxillin. This could be partly attributed to diminished stromal interaction molecule 1 (STIM1) expression and attenuated TCR-induced Ca 2+influx. Our results demonstrate that dual phosphorylation of paxillin by JNK and p38 MAPK is essential for T-cell activation and suggest that NFAT is a functional target of the JNK/p38 phosphorylated paxillin.

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