The High NRF2 Expression Confers Chemotherapy Resistance through Upregulated DUSP1 in Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are a diverse group of clonal marrow stem cell disorders characterized by ineffective hematopoiesis and dysplasia of the cellular elements. Although low-dose cytarabine (Ara-c) has been considered as one of the frontline therapies for high-risk MDS, the overall response rate of single Ara-c treatment was only 30%~40%. Nuclear factor erythroid 2-related factor 2 (NRF2), a basic leucine zipper transcription factor, has been reported to play a pivotal role in chemical detoxification. It is not yet known whether NRF2 has utility as a prognostic biomarker, or whether an elevated NRF2 level is associated with Ara-c resistance in MDS patients.

To investigate the expression and effects of NRF2 in MDS patients, gene set enrichment analysis (GSEA) and immunohistochemistry (IHC) were performed. GSEA in a published MDS cohort (n=183) revealed that expressions of NRF2 target genes was significantly enriched in high risk MDS (REAB-1/2) compared to low-risks (RARS and RA). We further detected NRF2 expression levels using IHC staining in the bone marrow (BM) biopsies from a cohort of MDS patients (n=111). Consistent with GSEA results, our IHC data indicated that the NRF2 expression levels of BM from high-risks exceeded low-risk MDS patients by Revised International Prognosis Scoring System (IPSS-R). Importantly, MDS patients with higher NRF2 levels (IHC scores, 4~6) displayed worse overall survival (OS) than patients with lower NRF2 levels (IHC scores, 0~3) (median, 15.7 vs. 22.7 months, P=0.007). Subgroup analysis showed that higher NRF2 levels correlated with poor OS only in chemotherapy group (P=0.04) but not in decitabine (P=0.38) or supportive care group (P=0.19).

To recapitulate our findings regarding the function of NRF2 in chemo-resistance, we performed in-vitro and in-vivo experiments. All experiments were performed both in human MDS-derived cell line SKM-1 and MDS mouse model cell line RUNX1 mutant-transduced Mll^PTD/WT BM cells (Mll^PTD/WT/RUNX1-S291fs). The cytotoxicity of Ara-c was determined by calculating the half maximal inhibitory concentration (IC50). NRF2 downregulation in MDS cell lines, by inhibitor luteolin, increases IC50 of Ara-c. On the contrary, upregulation of NRF2, mediated by agonist sulforaphane, induces resistance of MDS cells to Ara-C. To better define how suppression of NRF2 sensitized MDS cells to Ara-c, we used lentivirus-mediated shRNA for knockdown of NRF2. Knockdown of NRF2 markedly enhanced early and late apoptosis in MDS cell lines treated with Ara-c. We also established xenograft mouse models through intravenously injecting NRF2 shRNA or scramble shRNA SKM-1 cells into NOD/SCID-IL2Rγ^null-SGM3 (NSGS) mice. Knockdown of NRF2 significantly sensitized tumors to Ara-c in our SKM-1 transplanted mouse model. To further investigate the mechanisms involved in NRF2-mediated Ara-c resistance, we analyzed published gene-expression profiles of a large cohort of MDS patients (n=183) and Ara-C sensitive (IC50 <6 μM AraC) and resistant (IC50 >80 μM) AML patient samples (n=10). The data indicated that a group of NRF2 target genes might be responsible for Ara-C resistence. Interestedly, DUSP1 was one of the genes upregulated in both high-risk MDS patients and Ara-c resistant AML patients. Our chromatin immunoprecipitation (ChIP) and qPCR results validated that DUSP1 was a NRF2 direct target gene. Importantly, downregulation of DUSP1 by inhibitor or lentivirus shRNA could abrogate Ara-c resistance in NRF2 elevated MDS cell lines, indicating that NRF2 confers Ara-c resistance partly through its downstream target gene DUSP1 in MDS cells.

In conclusion, our clinical and experimental results reveal that 1) NRF2 expression levels are elevated in high-risk MDS patients and serve as a statistically significant prognostic variable for OS in patients with MDS, especially for MDS patients who have received chemotherapy. 2) Pharmacological inhibition of NRF2 re-sensitizes MDS cells to Ara-c treatment while activation of NRF2 by agonist resulted in the reduced sensitivity to Ara-c. 3) NRF2 mediates Ara-c resistance through its direct target gene DUSP1 . Taken together, our findings suggest that silencing NRF2 sensitizes MDS cells to Ara-c treatment; targeting NRF2 in combination with conventional chemotherapy could pave the way for high risk MDS therapy.