Genetic Activation of NRF2 By KEAP1 Inhibition Induces Fetal Hemoglobin Expression and Triggers Anti-Oxidant Stress Response in Erythroid Cells
Authors: Gupta Dipti , Lessard Samuel , Moore Nancy , Duan Jicheng , Nakamura Yukio , Yang Feng-Chun , Hicks Alexandra , Light David R. , Krishnamoorthy Sriram 
Published: February 2020

Nuclear factor (erythroid derived-2)-like 2 (NRF2), a basic leucine zipper transcription factor, is sequestered in the cell cytosol by Keap1, a kelch domain protein. Under steady state, this interaction results in ubiquitination and proteasomal degradation of the NRF2 protein. Modification of critical cysteine residues leads to conformational changes in KEAP1, resulting in nuclear translocation of newly synthesized NRF2 and modulation of downstream gene expression. NRF2 activation by compounds that modify KEAP1 such as dimethyl fumarate has been shown to induce γ-globin in erythroid cell systems. Moreover, NRF2 alleviates oxidative stress associated with SCD by activating antioxidant enzymes including catalase, glutathione peroxidase and superoxide dismutase (SOD), that scavenge free radicals (Chirico and Pialoux 2012, Belcher, Chen et al. 2017, Krishnamoorthy, Pace et al. 2017).
In the present work, we employed CRISPR Cas9 mediated knockout (KO) of KEAP1 in HUDEP2 cells and CD34+ derived erythroid cells to study its impact on downstream gene and protein expression relevant to sickle cell disease such as fetal hemoglobin (HbF) induction and anti-oxidant stress responses. Genetic KO of KEAP1 by six different gRNAs resulted in populations with indel levels from 30 to 95%, as determined by a mutation specific digital droplet PCR (ddPCR) and next-generation sequencing (NGS). LC/MS confirmed KEAP1 knockdown at the protein level. Consequently, NRF2 protein level in the nuclear extract was elevated in KEAP1 KO cells as demonstrated by nuclear NRF2 bound to antioxidant response element (ARE) in a DNA-binding assay. qPCR analysis revealed a robust and significant induction of the NRF2 dependent enzyme, NAD(P)H quinone dehydrogenase 1 (NQO1) and γ-globin. Gene expression levels of NQO1 and γ-globin inductions correlated significantly with the indel percentages; cell populations with ~80% indels showed about 20-fold induction in NQO1, and about 10-fold induction in γ-globin gene expression levels. Upon differentiation for 7 days in culture, KEAP1 KO cells showed up to 4-fold induction in γ-globin protein levels measured by flow cytometry and HPLC. Up-regulation of HbF and NQO1 protein were confirmed using LC/MS analysis. Additionally, KEAP1 KO clonal populations continued to show robust induction of NQO1 and HbF at gene and protein expression levels. To elucidate the mechanism of γ-globin induction, chromatin immunoprecipitation (ChIP) analysis in the KEAP1 KO cells demonstrated enrichment of NRF2 recruitment in the ARE sequences of the promoter regions of NQO1 and γ-globin genes.
Next, KEAP1 KO was carried out in bone marrow derived CD34+ cells using a CRISPR Cas9 RNP system. In agreement with our observations in HUDEP2 cells, knockdown of KEAP1 in CD34+ cells resulted in induction of the expression of γ-globin, NQO1 and HO1. Additionally, treatment with CDDO-Me, an NRF2 activator compound, resulted in a 4-fold induction in γ-globin mRNA levels in primary SCD patient derived CD34+ cells (by ddPCR), a 2.5-fold induction in HbF (by flow cytometry), and a 2.5-fold up-regulation in both Aγ and Gγ globin levels (by ultra-performance liquid chromatography). When CDDO-Me treated cells were subjected to ex-vivo hypoxia challenge, we observed a dose dependent inhibition in sickling with a maximal decrease of 55% at 250 nM, as evaluated by IDEAS imaging flow cytometry.
We further evaluated the antioxidant response with NRF2 activation in these cell systems. KEAP1 KO in HUDEP2 cells evaluated using RNA-sequencing showed enrichment of genes that participate in various protective mechanisms including NRF2 mediated oxidative stress response and glutathione redox reaction as analyzed by ingenuity pathway analysis (IPA). We subjected KEAP1 KO CD34+ cells to 4% hypoxia for 2 hours and observed a reduction in total reactive oxygen species levels as measured by flow cytometry compared to the control cells. These findings suggest that Nrf2 activation in erythroid cells offer a protective phenotype by improving the oxidative stress defense mechanisms, a well established pathophysiological manifestation of SCD.
In conclusion, KEAP1 inhibition in erythroid cell lineage and subsequent NRF2 activation leads to HbF induction and improvement in the antioxidant stress enzymes. Overall these data demonstrate a multi-faceted potential benefit of NRF2 activation in sickle cell disease.

Disclosures
Gupta: Sanofi: Employment. Lessard:Sanofi: Employment. Moore:Sanofi: Employment. Duan:Sanofi: Employment. Hicks:Sanofi: Employment. Light:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment.