Bioactivation of Napabucasin Triggers Reactive Oxygen Species–Mediated Cancer Cell Death

Abstract

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.

Combined inhibition of Ref‐1 and STAT3 leads to synergistic tumour inhibition in multiple cancers using 3D and in vivo tumour co‐culture models

“…Previous work by Froeling et al established that Napa was bioactivated by NAD(P)H Quinone Oxidoreductase 1 (NQO1), a protein involved in quinone redox cycling, 17 but the role of NQO1 in bioactivation of APX compounds has not been determined. These studies address our hypothesis that a burst of ROS production may be a part of the mechanism of cell death following combination therapy with Napa and Ref‐1 inhibitors.…”

“…16 It can also be bioactivated by NQO1 resulting in ROS production in pancreatic cancer cell lines. 17 Phase III data in colon cancer patients demonstrate that in patients with high levels of p-STAT3 overall survival was greater in Napa-treated patients. 15 Napa is currently being investigated in the clinic for multiple cancer indications where F I G U R E 4 Napabucasin and APX compounds generate ROS in PDAC cells, but only napabucasin is a substrate for NQO1.…”

Phase 1 study of napabucasin, a cancer stemness inhibitor, in patients with advanced solid tumors

“…Napabucasin is a small molecule that has been shown in a recent preclinical study to be bioactivated by NAD(P)H: quinone oxidoreductase-1, and to a lesser extent by cytochrome P450 oxidoreductase, which results in redox cycling and the generation of reactive oxygen species (ROS) [9]. The subsequent increase in ROS levels results in DNA damage and is hypothesized to affect multiple oncogenic cellular pathways, including inhibition of the STAT3 signaling pathway, which has been implicated in cancer stem cell viability [2].…”

Mass balance and pharmacokinetics of an oral dose of 14C‐napabucasin in healthy adult male subjects

“…Napabucasin is an orally administered reactive oxygen species (ROS) generator bioactivated by the intracellular antioxidant NAD(P)H:quinone oxidoreductase 1 (NQO1) 1,2 . Napabucasin exerts its antitumor activity by increasing levels of ROS beyond a cytotoxic threshold, causing cancer cell death 1–3 . Treatment of several cancers with napabucasin alone or in combination with other cancer therapies has been assessed in a number of ongoing clinical trials (congress abstracts, 4–16 and completed clinical trials 17,18 ).…”