Levels of Nrf2 and Keap1 Are Associated with Poor Prognostic Features In Myelodysplastic Syndromes (MDS)

Cellular metabolism and oxidative stress are important in the biology and pathophysiology of hematologic malignancies. Limited studies are available regarding metabolic pathways, including production of reactive oxygen species (ROS), and biology and pathogenesis of MDS. Elevated levels of ROS have been described in other leukemias, linked to increased levels of antioxidant molecules and activation of pathways that protect cells from the oxidative stress. The Nuclear erythroid 2 related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1) - Antioxidant Response Element (ARE) complex is the main signaling pathway involved in oxidative stress. Nrf2 is a transcription factor that induces expression of antioxidant responsive genes like heme oxygenase-1, catalase, glutathione dismutates and superoxide dismutase. With low oxidative stress, Nrf2 is associated with Keap1 in the cytoplasm and is continuously degraded by the E3 ubiquitin ligase complex. Keap1 contains multiple cysteine residues that are modified by oxidation to accelerate Keap1 dissociation from Nrf2, allowing Nrf2 to enter the nucleus and bind to ARE. In MDS, high levels of ROS have been described related to iron overload. We studied expression of Nrf2 and Keap1 in newly diagnosed, untreated patients with MDS to correlate with clinical features, pathogenesis, and clinical outcomes.

24 newly diagnosed pts with MDS and 4 normal donors were evaluated for Nrf2 and Keap1. CD34⁺ cells were sorted from pts and normal donor bone marrow. RNA was isolated using standard protocols and cDNA was obtained by reverse transcriptase using the Invitrogen SuperScript® One-Cycle cDNA Kit. Nrf2 and Keap1 expression were analyzed by qRT-PCR using Applied Biosystems Real-Time PCR 7900 and SYBR® GreenER™ Reagent System from Invitrogen. Patient clinical and clinical-laboratory characteristics, including IPSS, blood counts, bone marrow features, serum ferritin were used for analysis. Patients were followed for time-to-event endpoints, including time to transformation to AML and survival, dated from initial sample collection.

The median pt age was 70 years (range 37–82); 62.5% were male. The median % bone marrow (BM) blast count was 4.5% (range 1–25). Low-risk cytogenetics by metaphase karyotype was noted in 13%, intermediate-risk in 67%, and high-risk in 21%. According to the IPSS scoring system, 25% had high-risk, 17% Int1, 37% Int2, and 21% had low-risk MDS. Bone marrow was hypercellular in 18/24 (75%) pts; 87% for males and 56% for females. The median ANC was 1579/mL (range, 90–17061); hemoglobin was 9.4 gm/dL (range, 6.4–13); and platelet was 63.5/mL (range, 9–1040). The median ferritin level was 859 ng/mL (range, 24–14564); 15/21 (71.4%) had ferritin levels higher than the normal range. The mean relative Nrf2 transcript level was 0.167 (range, .001-.69) vs. 0.004 for normal donors P= 0.147 and the mean relative Keap1 transcript level was 0.014 (range, .003-.05) vs. 0.008 for normal donors P= 0.54. Increased bone marrow (BM) blast was associated with higher Nrf2 transcript level: 7/10 (70%) pts with BM blasts >10% presented higher Nrf2 levels. Increased ferritin level was associated with presence of int-poor cytogenetic features(10/24). The median follow-up time is 11 months (range, 1–43); transformation to AML occurred in 5 pts and overall 18 patients have died. The median survival time is 11 months. There was a trend for shorter survival or Time To Transformation (TTT) associated with higher Nrf2 (median 6 months vs 14 months p=0.328) and higher Keap1 transcript levels (median 6 months vs 14 months p=0.45), although not statistically significant.

CD34+ cells from bone marrow of patients with MDS have higher transcript levels of Nrf2 and Keap1 than normal donor CD34+ cells consistently with data reported in the current licterature for other hematological malignancies, such as CLL and AML. One of the reasons could be the activation and the integrity of Nrf2 signaling in MDS in response to high ROS levels. Among patients with MDS, there was a trend for shorter survival associated with higher Nrf2 and Keap1 transcript levels. These findings suggest that Nrf2/Keap1 signaling may play an important role in the pathophysiology of MDS and merit further investigation and must be confirmed in larger numbers of patients with MDS and normal donors.

No relevant conflicts of interest to declare.