Activation of Autophagy Via the SQSTM1-KEAP1-NRF2 Axis Induced By Pevonedistat Provides a Temporary Readjustment of MDS/AML Cells Leading to the Development of Resistance

Introduction: Myelodysplastic syndromes (MDS) are difficult-to-treat myeloid malignancies characterized by dysplasia, peripheral cytopenia, and progression to acute myeloid leukemia (AML). The standard treatment for high-risk MDS is inhibition of DNA methyltransferase with 5-azacytidine (AZA). Pevonedistat (PEVO) is a small molecule inhibitor of the activating enzyme NEDD8 that downregulates Cullin ring ligases (CRLs), which interferes with the trafficking and degradation of proteins in the proteasome and leads to the accumulation of CRL substrates. One of the CRL substrates is Nuclear factor erythroid 2-related factor 2 (NRF2) which represents a first line of antioxidant defense and has been previously implicated in drug resistance. The combination of AZA with PEVO was compared to AZA arm in patients with MDS in phase 2, multicenter, global, randomized, controlled, open-label PANTHER trial (NCT02610777, L. Ades 2022). AZA+PEVO arm was as safe as AZA alone and provided benefits in the OS and ORR among patients with higher-risk MDS. However, the primary endpoint, EFS, was not met, which may also implicate the emergence of therapeutic resistance. In the present study, we investigated the role of the NRF2 antioxidant pathway in the development of resistance to PEVO.

Methods: We derived PEVO-resistant (PEVO-R) clone from AZA-resistant cells (PEVO-S) developed in our lab (Minarik et al., 2022) by sequential treatment of the human OCI-M2 MDS/AML cell line with PEVO and analyzed them using whole exome sequencing, transcriptomic and proteomic analyses. We applied a quantitative mass spectrometry-based proteomic approach to identify protein targets of NRF2-driven redox changes in PEVO-S and PEVO-R clones. Autophagy was determined using flow cytometry and immunodetection.

Results: DNA sequencing revealed mutation of the PEVO target such as Ubiquitin activating enzyme 3 UBA3^Leu227Valat the site of interaction with NEDD8, which resulted in loss of response to PEVO. The transcriptomic analysis identified 14,484 protein-coding genes, of which 6,097 were significantly differentially expressed in PEVO-treated PEVO-S compared to untreated control. Resistance to PEVO displayed 2,241 differentially expressed genes in comparison to PEVO-S. We identified 148 out of 353 (42%) overexpressed NRF2-targets in PEVO-treated PEVO-S in contrast to 48 genes in PEVO-R. Neither NRF2 nor its upstream regulator Kelch-like associated protein 1 (KEAP1) were differentially expressed, instead PEVO induced expression of sequestosome-1 (SQSTM1), providing a positive feedback loop for NRF2 activation.

We also found higher accumulation of NRF2 in the nucleus of PEVO-R cells in comparison to PEVO-S cells. The proteome of PEVO-R cells was significantly more oxidized compared to PEVO-S. Out of 6,367 identified and quantified cysteine peptides the PEVO-R cells displayed 275 significantly more oxidized cysteines compared to PEVO-S. Moreover, PEVO-R cells had significantly oxidized CYS²⁸⁹ and CYS²⁹⁰ of SQSTM1. The S-acylation of those cysteines was linked to autophagy promotion. Indeed PEVO-R cells had markedly active autophagy and were sensitive to autophagy inhibitors in comparison to PEVO-S.

Conclusions: Our data suggest that PEVO induces accumulation of SQSTM1, thereby activating autophagy and NRF2, and placing cells in a transient state that protects them from oxidative damage associated with PEVO administration. This allows mutagenesis to occur which results in complete loss of PEVO responsiveness possibly via mutating UBA. Thus, the SQSTM1-KEAP1-NRF2 pathway appears to be a major checkpoint during the treatment of MDS with PEVO and could stay behind the loss of therapeutic efficacy in MDS patients.

Grants: AZV (NU21-08-00312), GAUK (273223), GAČR (24-10353S), EXCELES (LX22NPO5102).