WWP1 Regulates Redox State in Acute Myeloid Leukemia Cells by Modulating TXNIP

Background

Acute myeloid leukemia (AML) is a malignant blood disorder characterized by the abnormal proliferation of immature leukemia cells (leukemic blasts) in the bone marrow. Although significant progress has been made in the treatment of AML in recent years, the long-term survival rate of patients remains low, especially for those with relapsed or refractory disease. Therefore, identifying new therapeutic targets and mechanisms is a critical direction in current AML research.

Redox homeostasis plays a key role in cellular metabolism and survival. The accumulation of reactive oxygen species (ROS) can lead to DNA damage and apoptosis, while antioxidant systems (such as glutathione and the thioredoxin system) maintain normal cellular function by scavenging ROS. Thioredoxin-interacting protein (TXNIP) is an endogenous inhibitor of thioredoxin (TRX). It increases ROS production by inhibiting the reductase activity of TRX, thereby disrupting cellular redox balance. Additionally, TXNIP restricts cell growth and survival by inhibiting glucose uptake and metabolism.

WWP1 is a HECT-type E3 ubiquitin ligase that has been shown to play an oncogenic role in various cancers. However, the mechanisms by which WWP1 functions in AML are not fully understood. This study aims to investigate whether WWP1 regulates TXNIP to influence the redox state and metabolism of AML cells, thereby revealing its oncogenic mechanisms in AML.

Source of the Paper

This paper was led by Professor Francesca Bernassola’s team from the Department of Experimental Medicine at the University of Rome Tor Vergata, Italy. Collaborating institutions include the First Affiliated Hospital of Soochow University in China, the IDI-IRCCS Institute in Rome, Italy, and the IFOM-ETS Institute of Molecular Oncology in Milan, Italy. The paper was published online on October 4, 2024, in the journal Molecular Oncology, with the DOI 10.¹⁰⁰²⁄₁₈₇₈-0261.13722.

Research Process and Results

1. WWP1 Inactivation Leads to Increased ROS Levels in AML Cells

The study first measured the ratio of glutathione (GSH) to oxidized glutathione (GSSG) in WWP1-inactivated AML cells using liquid chromatography-mass spectrometry (LC-MS). The results showed a significant decrease in the GSH/GSSG ratio after WWP1 inactivation, indicating elevated oxidative stress levels in the cells. Further detection of intracellular and mitochondrial ROS levels using fluorescent probes H2DCFDA and MitoSOX revealed a significant increase in ROS levels in AML cells after WWP1 inactivation, particularly mitochondrial ROS. Additionally, the levels of the DNA oxidative damage marker 8-OHdG were significantly elevated, indicating that WWP1 inactivation leads to DNA damage.

2. WWP1 Inactivation Induces DNA Damage via ROS

DNA strand breaks were detected using the comet assay, which showed a significant increase in DNA damage in WWP1-inactivated AML cells. Concurrently, the levels of DNA damage markers γ-H2AX and phosphorylated ATM were also significantly elevated. Treatment with the antioxidant N-acetylcysteine (NAC) significantly reduced DNA damage levels, confirming that WWP1 inactivation induces DNA damage through ROS.

3. TXNIP is a Substrate for WWP1 Ubiquitination

Analysis using the STRING database suggested a potential interaction between WWP1 and TXNIP. Further co-immunoprecipitation experiments confirmed the direct binding of WWP1 to TXNIP. In vivo and in vitro ubiquitination assays demonstrated that WWP1 promotes TXNIP ubiquitination and proteasomal degradation via K48-linked ubiquitin chains. The catalytic mutant of WWP1 (C890A) failed to induce TXNIP ubiquitination, indicating that the catalytic activity of WWP1 is essential for its function.

4. WWP1 Inactivation Leads to TXNIP Stabilization and Functional Activation

After WWP1 inactivation, TXNIP protein levels significantly increased, and its half-life was prolonged. The insulin disulfide reductase assay showed that TRX enzyme activity significantly decreased after WWP1 inactivation, indicating that TXNIP accumulation inhibits the antioxidant function of TRX. Treatment with the TXNIP inhibitor TXNIP-IN-1 or the calcium channel blocker verapamil significantly reduced ROS levels, further confirming the critical role of TXNIP in WWP1 inactivation-induced oxidative stress.

5. WWP1 Inactivation Affects Glucose Metabolism in AML Cells

TXNIP is a negative regulator of glucose metabolism. The study found that after WWP1 inactivation, the mRNA levels of glucose transporters GLUT1 and GLUT4, as well as key glycolytic enzymes LDHA and LDHB, significantly decreased. Seahorse analyzer measurements of glycolytic rates showed that both glycolysis and oxidative phosphorylation levels in AML cells were significantly reduced after WWP1 inactivation, leading to decreased ATP production. Gene silencing of TXNIP partially reversed the inhibitory effects of WWP1 inactivation on glucose uptake and metabolism.

Conclusions and Significance

This study reveals the molecular mechanism by which WWP1 regulates the redox state and glucose metabolism of AML cells through ubiquitin-mediated degradation of TXNIP. Overexpression of WWP1 accelerates TXNIP degradation, enhancing the antioxidant function of TRX and reducing intracellular ROS levels, thereby promoting the survival and proliferation of AML cells. Additionally, WWP1 modulates glucose metabolism through TXNIP, further supporting the growth and survival of AML cells.

This discovery provides a new potential therapeutic target for AML. Inhibiting WWP1 or restoring TXNIP function may enhance the sensitivity of AML cells to chemotherapeutic drugs, thereby improving treatment outcomes. Future research should further explore the role of the WWP1/TXNIP axis in chemotherapy resistance in AML and its potential applications in other cancers.

Research Highlights

1. Novel Molecular Mechanism: First to reveal that WWP1 regulates the redox state and glucose metabolism of AML cells through ubiquitin-mediated degradation of TXNIP.
2. Potential Therapeutic Target: The WWP1/TXNIP axis may serve as a new therapeutic target for AML, particularly for chemotherapy-resistant patients.
3. Comprehensive Experimental Validation: Utilized in vivo and in vitro experiments, ubiquitination analysis, and metabolomics to comprehensively validate the function of WWP1 and its role in AML.