NRF2 Signaling Pathway Confers Chemotherapy Resistance In High Risk MDS

ObjectiveMyelodysplastic syndromes(MDS)are a diverse group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and dysplasia of the myeloid cells.Several prognostic scores exist with the International Prognostic Scoring System(IPSS)and its revised vision(IPSS-R)being commonly used to stratify patients into two risk groups,defining lower and higher risk groups.Higher risk MDS patients are followed by an increased risk of developing acute myeloid leukemia(AML)and are correlated with poor clinical outcomes.Treatment strategies were made according to the risk categories of MDS.Cytarabine(Ara-C)is a pyrimidine nucleoside analog that interferes with the synthesis of DNA when the cycle holds in the S phase.Ara-C based therapies have been widely used in the past decades for the management of MDS patients,especially in higher-risk patients.However,the overall response rate of single Ara-C treatment was only 20%?30%.Nuclear factor erythroid 2-related factor 2(NRF2)is a transcription factor that protects cells from oxidative damage.Under electrophilic or oxidative stress,NRF2 is released from its cytosolic inhibitor Kelch-like ECH-associated protein 1(KEAP1),and translocates to the nucleus.It has recently been shown that NRF2 underlies drug resistance in acute myeloid leukemia(AML),chronic myeloid leukemia(CML),and chronic lymphocytic leukemia(CLL).NRF2 binding to antioxidant responsive element(ARE)allows induction of a number of cytoprotective and detoxification genes such as NAD(P)H:quinone oxidoreductase 1(NQO1),heme oxygenase-1(HO-1),and glutamate-cysteine ligase(GCL).Although there are limited studies indicating the mechanisms of NRF2 in drug resistance,the NRF2 target genes,such as HO-1 and GCL,have been reported to facilitate resistance of tumor cells to chemotherapy in AML cells.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.Methods1)To investigate the expression and effects of NRF2 in MDS patients,gene set enrichment analysis(GSEA)was performed using gene-expression profiles.And immunohistochemistry(IHC)were conducted in a large cohort of MDS patients(n=137).Staining results were semi-quantified using an arbitrary score.2)To recapitulate our findings regarding the function of NRF2 in chemo-resistance,we performed in-vitro and in-vivo experiments.All in-vitro experiments were performed both in human MDS-derived cell line SKM-1 and MDS mouse model cell line RUNX1 mutant-transduced MllPTD/WT BM cells(M11PTD/wT/RUNX1-S291fs).We conducted stable NRF2 and DUSP1 shRNA cell lines by lentivirus and confirmed the expression by WB and q-PCR.MDS cells were treated with Ara-C and cell viability was examined by MTS.The cytotoxicity of Ara-C was determined by calculating the half maximal inhibitory concentration(IC50).Apoptosis and cell cycle distribution were detected by flow cytometry.We also established MDS xenograft mouse models through intravenously injecting NRF2 shRNA,DUSP1 shRNA or scramble shRNA SKM-1 cells into NOD/SCID-IL2R?null-SGM3(NSGS)mice.Chromatin immunoprecipitation(ChIP)and q-PCR were performed to validate the relationship between DUSP1 and NRF2.Results1)NRF2 is elevated in higher risk MDS and correlates with inferior overall survivalGSEA in a published MDS cohort(n= 183)revealed that expressions of NRF2 target genes was significantly enriched in higher risk MDS(REAB-1/2)compared to lower-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= 13 7).Consistent with GSEA results,our IHC data indicated that the NRF2 expression levels of BM from higher-risks exceeded lower-risk MDS patients by Revised International Prognostic Scoring System(IPSS-R)(P=0.020).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,391 vs.554 days,P=0.011).2)Re-sensitizing MDS cells to Ara-C treatment in vitro by knockdown of NRF2Pharmacological modulations of NRF2 could regulate chemotherapeutic efficacy of Ara-C in MDS cells.NRF2 downregulation in MDS cell lines,by inhibitor Luteolin,decreases 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 higher concentrations of Ara-C.We also found that NRF2 silenced MDS cell lines with Ara-C treatment tended to be arrested in the S phase.3)NRF2 mediated Ara-C resistance is partly through its direct target gene DUSP1To 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),Ara-C sensitive(IC50<6 ?M Ara-C)and resistant(IC50>80 ?M)AML patient samples(n=10).The data indicated that a group of NRP2 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 ChIP and q-PCR results validated that DUSP1 was a NRF2 direct target gene.DUSP1 inhibitor,BCI,and Ara-C have statistically significant synergistic effects on NRF2 activated SKM-1 cells.Knockdown of DUSP1 by lentivirus shRNA could alos 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 scramble shRNA MDS mice,the tumor weight showed a decreasing trend,but did not show a significant change in the Ara-C treatment group(1.91 g vs.1.53 g with PBS vs.Ara-C treatment group,respectively;P=0.052).Treatment of NRF2 or DUSP1 silencing MDS mice with Ara-C resulted in significantly smaller tumors in the liver(NRF2 silencing MDS mice,2.30 g vs.1.77 g with PBS vs.Ara-C treatment group,respectively,P=0.010;DUSP1 silencing MDS mice,1.84 g vs.1.41 g,respectively,P=0.001).ConclusionOur clinical and experimental results revealed that NRP2 expression levels are elevated in high-risk MDS patients and serve as a statistically significant prognostic variable for OS in patients with MDS.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.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.