Pevonedistat (PEVO) is a selective inhibitor of neddylation-dependent protein degradation and has been shown to enhance the efficacy of the hypomethylating drug 5-azacytidine (AZA) in leukemia therapy. However, despite promising results, even combination therapy fails due to the emergence of resistance. Our recent data showed that AZA alters redox homeostasis and oxidative modifications of proteins, which further contributed to the selective pressure of AZA. PEVO activates the main antioxidant defense pathway by inhibiting the degradation of nuclear factor erythroid 2-related factor 2 (NRF2), a substrate of ubiquitin ligase Kelch-like ECH-associated protein 1 (KEAP1), and thus also affects cellular redox homeostasis. Redox changes are sensed by protein thiol groups which undergo various oxidative modifications leading to modulation in protein function. Therefore, in the presented work we investigated the impact of PEVO on redox homeostasis and redox proteome with the aim to reveal the role of redox homeostasis in the response of MDS/AML cells to the combined therapy.
We used cell line models of MDS/AML myeloblasts sensitive and resistant to AZA and PEVO developed in our laboratory. We performed quantitative proteomic analysis using TMT10-plex and Orbitrap Fusion Tribrid MS instrument on PEVO-sensitive cells treated with PEVO and PEVO-resistant cells. We identified and quantified 6,886 proteins. PEVO treatment affected the level of 98 proteins, and resistance was associated with changes in levels of 566 proteins. Principal component analysis separated the analyzed groups, with differing levels of NRF2 target proteins contributing most to separating the PEVO-treated sample. Indeed, the activity of NRF2, determined by its accumulation in the nucleus, was significantly increased in the PEVO-treated sample. This was associated with changes in the oxidative state monitored by flow cytometry using fluorescent probes. Interestingly, enhanced activity of NRF2 was detected in untreated PEVO-resistant cells as well. Mass spectrometry-based redox proteomic analysis of reduced and oxidized cysteine thiol groups using iodoTMT isobaric labelling and Orbitrap Fusion Tribrid MS instrument identified 26,265 cysteine-containing peptides from 6,634 proteins and showed that PEVO resistance is accompanied by changes in the redox state of 228 proteins. We identified increased oxidation on cysteines 289 and 290 of Sequestosome-1, previously shown to be important for the autophagic clearance of ubiquitinated proteins.
We have identified prominent modifications in proteins with roles in autophagy, indicating a role in autophagy regulation that could contribute to the adaptive mechanisms leading to the emergence of resistance. Our results indicate that the altered redox state has a significant role in PEVO and AZA resistance.