Mechanistic study of the Cullin3-CRL3^KEAP1-NRF2 neddylation axis in chemotherapy resistance of Acute Myeloid Leukemia stem cells

Abstract

Abstract Cullin3-CRL3^KEAP1-NRF2 neddylation axis is a core mechanism of chemotherapy resistance in leukemia stem cells (LSCs), and targeting NAE can overcome existing treatment bottlenecks. Background: The recurrence of acute myeloid leukemia (AML) is rooted in the inherent and acquired resistance of LSCs to chemotherapy, yet the mechanisms by which LSCs sense and adapt to chemotherapy-induced oxidative stress remain unclear. Although neddylation has been shown to regulate cell fate in various solid tumors, its role and molecular details in LSC resistance are still unknown. Methods: This study utilized CD34⁺CD38⁻ LSCs isolated from the bone marrow of newly diagnosed or relapsed AML patients and the THP-1 cell line to systematically analyze the function of the Cullin3-CRL3^KEAP1-NRF2 neddylation axis in Ara-C resistance. The specific strategies included: ① Establishing an Ara-C gradient resistance model and intervening with the NAE inhibitor MLN4924 to assess LSC survival, ROS levels, and mitochondrial membrane potential; ② Employing high-sensitivity NEDD8 mass spectrometry and Co-IP to identify the neddylation sites of Cullin3 and its interaction dynamics with KEAP1 post-chemotherapy; ③ Constructing a KEAP1-NRF2 dual fluorescence reporter system to track NRF2 nuclear translocation in real-time under oxidative stress; ④ Using CRISPR-Cas9 to mediate a Cullin3-KR (K712R) mutation to block its neddylation and verify the integrity of the axis on downstream expression of ALDH1A1, ABCB1, and BCL-2; ⑤ Utilizing flow cytometry to detect LSC apoptosis and ROS probes to monitor redox balance; ⑥ Evaluating the in vivo synergistic efficacy and survival benefit of MLN4924 combined with Ara-C in NSG mouse transplantation models. Results: Chemotherapy-induced oxidative stress triggered significant neddylation of the 712th lysine of Cullin3 within 4 hours, enhancing the E3 ligase activity of CRL3^KEAP1, leading to the ubiquitination and degradation of KEAP1, thereby relieving its inhibition on NRF2. Following NRF2 nuclear translocation, transcription of ALDH1A1, ABCB1, and BCL-2 was initiated, endowing LSCs with enhanced aldehyde dehydrogenase activity, increased drug efflux capacity, and anti-apoptotic advantages. Notably, MLN4924 or the Cullin3-KR mutation completely blocked this axis, significantly reducing intracellular NRF2 activity and decreasing the proportion of ALDH1A1⁺/ABCB1⁺ LSCs, resulting in an increase in Ara-C-induced apoptosis rates from 28% to 71%. In the NSG mouse model, the median survival of the MLN4924 and Ara-C combination treatment group extended to 82 days, significantly benefiting compared to the monotherapy groups (Ara-C 37 days, MLN4924 41 days, p<0.001), with a 2.8 log reduction in human LSC burden in the bone marrow. Conclusion: This study reveals for the first time that the chemotherapy-induced Cullin3-CRL3^KEAP1-NRF2 neddylation axis is a critical switch for LSC adaptation to oxidative stress and resistance. Targeting NAE with MLN4924 can precisely block this axis, providing a novel combination therapy strategy for eradicating LSCs and preventing AML relapse.