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P. Lin et al.
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. Over the last decades, Ara-C-based therapies have been widely used to manage MDS patients, especially those at higher risk.4 However, the overall response rate of single Ara-C treatment was only approximately 20-30%.5,6 Clinically, MDS patients who remained unresponsive to the routine treatment of Ara-C (100mg/m2) were defined as Ara-C-resistant MDS patients.
Nuclear factor erythroid 2-related factor 2 (NRF2, also called NFE2L2) is a transcription factor that protects cells from oxidative damage.7,8 Under oxidative stress, NRF2 is released from its cytosolic inhibitor Kelch-like ECH-asso- ciated protein 1 (KEAP1) and translocates to the nucleus.9 It has recently been shown that NRF2 underlies drug resistance in acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CLL).10-12 NRF2 binding to antioxidant respon- sive element (ARE) allows induction of a number of cyto- protective and detoxification genes, such as NAD(P)H: quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), and glutamate-cysteine ligase (GCL).7,13,14 Few studies have shown the mechanisms of NRF2 in drug resistance. NRF2 target genes, such as HO-1, have been reported to facilitate resistance of tumor cells to chemotherapy in AML cells.15,16
Here, we aimed to correlate NRF2 expression and its clinical outcome in a large cohort of MDS patients (n=137). We also performed in vitro and in vivo experiments to validate our findings regarding the function of NRF2 in chemo-resistance in MDS. We found that NRF2 expres- sions were elevated in higher risk MDS and correlated with inferior clinical outcomes. High levels of NRF2 reduced MDS cell sensitivity to Ara-C treatment partly through its direct target gene DUSP1.
Methods
Immunohistochemistry
Immunohistochemistry (IHC) was performed on 4 μm thick bone marrow (BM) sections. BM samples were stained for NRF2 expression (dilution 1:200; Abcam, UK) or DUSP1 expression (dilution 1:100; Abcam, UK). Samples were incubated using pri- mary antibody for 30 minutes (min) at 37°C. Secondary antibody (dilution 1:50; Dako, Denmark) was applied for 15 min. Binding was visualized by the horseradish peroxidase (HRP)/3,3’- diaminobenzidine (DAB) kit (ZSGB-BIO, China). Staining results were semi-quantified using an arbitrary score as follows: No stain- ing, 0; Pale yellow, 1; Tan, 2; Brown, 3; Nuclear staining in 0-25% of cells, 0; Nuclear staining in 25-50% of cells, 1; Nuclear staining in 50-75% of cells, 3; Nuclear staining in 75-100% of cells, 4. The stain color score multiplied by the nuclear staining proportion score is the final IHC score.
In vitro cytotoxicity assay
Myelodysplastic syndrome cell lines (5×105/mL) and primary
MDS cells (1×106/mL) were seeded in 96-well flat bottom plates and treated with increasing concentrations of Ara-C. Cell prolifer- ation was determined using the MTS proliferation assay. 20 ml of MTS (Promega, USA) was added to 100 ml of cell suspension, and cells were further incubated in 5% CO2 for 3-4 hours at 37 °C. The plates were then analyzed on an enzyme immunoassay plate reader at 490 nm. The half inhibitory concentration (IC50) values
of Ara-C were calculated by Prism Graphpad software. All exper- iments were performed in triplicate.
Mice models
NOD/SCID-IL2Rγnull-SGM3 (NSGS) mice were bred and main- tained in Cincinnati Children’s Hospital Medical Center (CCHMC).17 Mice were randomized into six groups. NRF2 shRNA SKM-1 (transfected with shRNA targeting NRF2), DUSP1 shRNA SKM-1 (transfected with shRNA targeting DUSP1), and scramble shRNA SKM-1 were resuspended in 300 ml phosphate buffer saline (PBS) and then injected intravenously into the non-irradiat- ed mice (1 million cells per mouse). Ten days after cell inoculation (Day 0), the mice received 50 milligram/ kilogram (mg/kg) of Ara- C or PBS once a day for five consecutive days (Day 10-15). Ara-C and PBS were injected intraperitoneally. All experiments were per- formed in accordance with protocols approved by the Institutional Review Board of CCHMC.
Statistical analysis
Data were analyzed using SPSS 16.0 and GraphPad Prism 6. Statistical analyses were performed using Student t-test or one-/two-way ANOVA with multiple comparisons correction. P<0.05 was considered statistically significant.
Results
NRF2 is elevated in higher risk MDS and correlates with inferior overall survival
To explore NRF2 expression and its clinical outcome in MDS, we performed immunohistochemistry (IHC) on 137 MDS patients and 17 controls (Figure 1A). IHC staining results showed that NRF2 was over-expressed in the BM biopsies from MDS patients (P<0.01) (Figure 1B). The NRF2 levels of intermediate-, high-, and very high-risk IPSS-R patients exceeded that of low-risk IPSS-R patients (P=0.004) (Online Supplementary Table S1). To validate the expression of NRF2 with its downstream target signature in MDS, we analyzed published gene expression profiles of CD34+ BM cells purified from samples obtained from 183 MDS patients and 17 healthy controls.18 We per- formed gene set enrichment analysis (GSEA) to explore the downstream targets’ signature of NRF2 in this cohort.19 Although the expression of NRF2 is not signifi- cantly activated in MDS patients compared to the healthy controls (P=0.225) (Online Supplementary Figure S1A), NRF2 expression is significantly enriched in higher risk MDS patients (MDS-EB-1/2) when compared to those with lower risk MDS (MDS-SLD/MDS-RS) (P=0.020) (Figure 1C). Leading edge genes are shown in Online Supplementary Appendix Lists 1 and 2. It is worth noting that MDS patients with higher NRF2 expression 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) (Figure 1D). We fur- ther performed CD34 and NRF2 double staining by immunofluorescence with MDS patient BM aspiration samples (Online Supplementary Figure S1B). CD34 and NRF2 double staining in particular cells demonstrated that NRF2 was also expressed at protein levels in CD34+ cells (Online Supplementary Figure S1C). High NRF2 expression levels were closely associated with higher risk according to the 2016 WHO subtype (P=0.022), IPSS cytogenetics (P=0.001), and IPSS categories (P=0.001). There were no significant differences in other clinical features between
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