Targeting antioxidant enzymes enhances the therapeutic efficacy of the BCL-X_L inhibitor ABT-263 in KRAS-mutant colorectal cancers

Highlights

• The BCL-X_L targeting agent, ABT-263 increases intracellular reactive oxygen species levels.

• Pharmacological inhibition of antioxidant enzymes enhances ABT-263-mediated apoptosis.

• 2-methoxyestradiol exhibits synergism with ABT-263 in KRAS-mutant colorectal cells and patient-derived xenografts.

• Combination treatment of ABT-263 and 2-methoxyestradiol inhibits mTOR-dependent translation of MCL-1 protein.

Abstract

Cancer chemotherapeutic drugs exert cytotoxic effects by modulating intracellular reactive oxygen species (ROS) levels. However, whether ROS modulates the efficacy of targeted therapeutics remains poorly understood. Previously, we reported that upregulation of the anti-apoptotic protein, BCL-X_L, by KRAS activating mutations was a potential target for KRAS-mutant colorectal cancer (CRC) treatment. Here, we demonstrated that the BCL-X_L targeting agent, ABT-263, increased intracellular ROS levels and targeting antioxidant pathways augmented the therapeutic efficacy of this BH3 mimetic. ABT-263 induced expression of genes associated with ROS response and increased intracellular ROS levels by enhancing mitochondrial superoxide generation. The superoxide dismutase inhibitor, 2-methoxyestradiol (2-ME), exhibited synergism with ABT-263 in KRAS-mutant CRC cell lines. This synergistic effect was attributed to the inhibition of mTOR-dependent translation of the anti-apoptotic MCL-1 protein via caspase 3-mediated cleavage of AKT and S6K. In addition, combination treatment of ABT-263 and 2-ME demonstrated a synergistic effect in in vivo patient-derived xenografts harboring KRAS mutations. Our data suggest a novel role for ROS in BH3 mimetic-based targeted therapy and provide a novel strategy for treatment of CRC patients with KRAS mutations.

Introduction

Reactive oxygen species (ROS) are highly reactive oxygen-containing molecules, such as superoxide, hydrogen peroxide, and hydroxyl radicals [1]. Precise control of intracellular ROS levels is required to regulate cellular signal transduction and survival. Uncontrolled ROS cause irreversible damage to cellular components and can result in cell death [2]. Cancer cells characteristically contain high levels of ROS and consequently display high levels of antioxidants [2]. Traditional cytotoxic chemotherapy drugs used in cancer treatment generally increase ROS production and overwhelm the intracellular reduction capacity, resulting in the induction of cell death via apoptosis and necrosis [1]. Several molecularly targeted agents that specifically manipulate dysregulated target proteins or pathways have been recently developed and clinically applied; however, studies investigating the influence of ROS in targeted cancer treatment have been limited.

Colorectal cancer (CRC) is the third most prevalent cancer and the second most common cause of cancer-related deaths worldwide [3]. Mutations in the KRAS gene are detected in approximately 40% of CRC patients [4]; patients harboring KRAS mutations exhibit poor prognoses compared with patients with the wild-type KRAS gene [5,6]. Moreover, patients with KRAS mutant tumors are usually resistant to anti-epidermal growth factor receptor (EGFR) therapies, such as panitumumab and cetuximab [7]. Previous studies found that patients with KRAS mutations exhibit higher expression levels of the anti-apoptotic protein BCL-X_L and suggested BCL-X_L as a therapeutic target in CRC patients with KRAS mutations [[8], [9], [10]]. ABT-263 (navitoclax) is a B-cell lymphoma-2 homology 3 (BH3) mimetic that inhibits the function of anti-apoptotic BCL-2 proteins. Monotherapy using ABT-263 usually results in therapeutic resistance; therefore, additional strategies for ABT-263-based combination therapies is in urgent need of development.

In this study, we investigated the effect of the targeted agent ABT-263 on intracellular ROS status in CRC cells. ABT-263 increased cellular ROS levels, particularly of mitochondrial superoxide. Inhibition of antioxidant defense enzymes, especially superoxide dismutase (SOD), augmented the therapeutic effect of ABT-263 in vitro and in vivo in patient-derived xenograft (PDX) models. These data suggest a novel role for ROS in mediating the effects of the targeted therapeutic ABT-263 and provide an additional strategy for combination therapeutics in CRC patients with KRAS mutations.