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ral stem cell fate by modifying ROS signaling to activate NRF2-dependent pathways.32,33 Future studies may help to elucidate the possible mechanisms involved in high NRF2 expression levels in higher-risk MDS patients. Importantly, our survival analysis indicated that MDS patients with higher NRF2 levels in BM cells correlated with worse OS than patients with lower NRF2 levels (P=0.011) (Figure 1D). The discrepancy between our results and the previously published data may be related to the methods, cells studied (mononuclear cells of PB vs. BM), or sample size (31 vs. 137). This needs to be further investigated in larger independent cohorts.
Ara-C is widely used as a treatment approach in higher- risk MDS patients; however, single Ara-C treatment has limited therapeutic effect. Drug resistance is the major cause of treatment failure. High NRF2 proteins in human primary AML cells have been shown to be driven by NF- κB and knockdown of NRF2 reduced colony formation of AML cells in response to treatment of Ara-C and daunoru- bine.10 However, there are no research reports on the role of NRF2 in mediating drug resistance in MDS. The SKM- 1 cell line was established from a Japanese male patient in 1985 who was initially diagnosed as higher-risk MDS (MDS-EB-2).34 It has been reported that SKM-1 cells had a higher IC50 of Ara-C than other myeloid leukemia cell lines, indicating that SKM-1 cells were more Ara-C resist- ant.35,36 The incidences of MLL-PTD and RUNX1 muta- tions showed an increase in higher-risk MDS compared to lower-risk MDS.37 Thus, SKM-1 cells and MDS mouse model cell line RUNX1 mutant-transduced MllPTD/WT BM cells (MllPTD/WT/RUNX1-S291fs) were used in our study. BM mononuclear cells from Ara-C-sensitive and Ara-C-resis- tant MDS patients were also studied. Our results indicated that NRF2-mediated drug resistance in MDS was similar to other conditions.10,38
Luteolin is a potential NRF2 inhibitor that can promote the degradation of NRF2 mRNA. Our results revealed that NRF2 downregulation in primary MDS cells, by inhibitor Luteolin, decreased IC50 of Ara-C. As primary MDS cells were mostly composed of non-transformed cells, we also validated our results in human and mouse MDS cell lines. Downregulation of NRF2 by Luteolin could also enhance the chemotherapeutic efficacy of Ara-C in MDS cell lines. SFN is a well-known NRF2 ago- nist. SFN is an isothiocyanate that forms a KEAP1– Sulforaphane thionoacyl adduct to stabilize NRF2.39 We found that upregulation of NRF2, mediated by agonist SFN, induced resistance of MDS cells to Ara-C. Previous reports in AML indicated the protective role of NRF2 against apoptosis.15 NRF2 regulates homologs miR-125B1 and miR-29B1 to repress the apoptosis induced by the front-line AML chemotherapy agent daunorubicin.40 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 apoptosis and triggered S-phrase arrest in MDS cell lines treated with Ara-C. Taken together, NRF2 levels regulate MDS cells’ sensitivity to Ara-C therapy.
There is increasing evidence to suggest that NRF2 tar- get genes, such as HO-1, NQO1, and multidrug resist- ance-associated protein (MRP), are involved in cytopro- tection and detoxification, thus providing drug resistance in anti-cancer therapy.12,41,42 To determine the mecha- nisms of NRF2-mediated Ara-C resistance in MDS, we performed GSEA analysis on published data and then
discovered a list of the genes (n=37) up-regulated in both high-risk MDS patients and Ara-C-resistant AML patients. One of the genes on the list is DUSP1 (also known as MKP1), which regulates mitogen-activated protein kinase (MAPKinase) by dephosphorylation of threonine and tyrosine residues.43 DUSP1 may play an important role in the cellular response to environmental oxidative stress and agents that damage DNA.44 However, little is known about the relationship between DUSP1 and NRF2 or the effect of DUSP1 on chemo-resis- tance in MDS. Our ChIP q-PCR and q-PCR data indicat- ed DUSP1 was an NRF2 direct target gene. Our IHC and immunoblotting data showed that DUSP1 expressions were elevated in higher-risk or Ara-C-resistant MDS. Given the small number of MDS patients studied, future validation with larger cohorts is needed.
Interestingly, downregulation of DUSP1 by inhibitor or lentivirus shRNA could abrogate Ara-C resistance in NRF2-elevated MDS cells. There is growing evidence to demonstrate that NRF2 activation by antioxidant inter- ventions increased cancer cell migration and induced tumor metastasis by decreasing ROS levels.45,46 It has been suggested that NRF2 improved sensitivity of AML cells to chemotherapy by compromising the ability of the AML cell to scavenge the ROS.38 Our results suggest- ed that ROS signaling pathways may play limited roles in NRF2-mediated Ara-C resistance in MDS cells. We identified a larger cohort of the genes (n=331) up-regulat- ed in both high-risk MDS patients and Ara-C-resistant AML patients. We found significant enrichment of the up-regulated genes in 12 KEGG pathways pertaining to cell signaling (e.g. MAPK and JAK-STAT signaling), immune responses (e.g. chemokine signaling and lyso- some signaling), and cell death (e.g. apoptosis and FoxO signaling). It has been reported that alterations of SETD2 (encoding the histone 3 lysine 36 trimethyltransferase) and EZH2 (catalyzing the trimethylation of lysine 27 of histone H3) also led to resistance to DNA damaging- chemotherapy such as Ara-C in leukemia via different mechanisms.47,48 Our current study indicated that NRF2 conferred Ara-C resistance partly through DUSP1 in MDS. Other signaling pathways identified in this study warrant further investigation in the future.
Our data showed that silencing NRF2 or DUSP1 signif- icantly sensitized tumors to Ara-C by measuring tumor size in the livers of our SKM-1-transplanted mouse mod- els. SKM-1 is a cell line carrying a large number of muta- tions, affecting ASXL1, BCORL1, EZH2, SF1, STAG2, TET2, TP53, and WT1, which are close to the character- istics of high-risk MDS.49 Thus, SKM-1 cell lines were used in this study. To better establish stable NRF2 or DUSP1 knockdown MDS mouse models, SKM-1 cells were transfected and then injected into NSGS mice. SKM-1 cells mainly generated tumors in the livers but not in BM or peripheral blood of NSGS mice. Although not the ideal model, our in vivo results indicated that silencing NRF2 or DUSP1 increased the sensitivity of SKM-1 cells to Ara-C treatment. Future MDS patient- derived xenograft models are needed to validate our find- ings.50
In conclusion, our clinical and experimental results revealed that NRF2 expression levels are elevated in high-risk MDS patients and serve as a statistically signif- icant prognostic variable for OS in MDS patients. Pharmacological inhibition of NRF2 re-sensitizes MDS
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haematologica | 2019; 104(3)