NRF2 Signaling Promotes Survival and Drug Resistance Of Acute Myeloid Leukaemia Through Induction Of Mir-125b-1

We have previously shown that Nrf2, an important regulator of several biologic processes involved in the progression of cancer, has abnormal constitutive expression in AML. As post-transcriptional regulation by micro RNAs (miRNAs) affects the malignant phenotype of a number of diseases, in this study, we aimed to identify and characterise NRF2 regulated miRNAs in AML.

Initially a qRT-PCR miRNA array was utilised to investigate miRNA expression in response to NRF2 lentiviral mediated knockdown in the AML cell line THP-1. To verify these results analysis of miRNA identified in the array were performed in response to transfection of siRNA against the NRF2 inhibitors Keap1 and Bach1. Subsequently we performed bioinformatic analysis to identify putative NRF2 binding sites upstream of the miRNA coding regions. From this we identified miR-125b as a putative NRF2 regulatory miRNA.

There are two miR125b paralogs coding for the same mature sequence in humans, miR-125b-1 on chromosome 11 in a cluster containing let-7a-2 and miR-100 and miR-125b-2 on chromosome 21 in a cluster with miR-99a and let-7c. To identify which paralog is upregulated by NRF2 qRT-PCR of the miRNA in each cluster was performed and referenced to the immature miR125b sequences which do differ between each paralog. Results show that NRF2 regulates miR-125b-1, but does not regulate miR-125b-2 or the other miRNAs within the miR125b-1 or the miR125b-2 clusters, implying that mir-125b-1 contains an alternative promoter which is regulated by NRF2.

To locate the NRF2 promoter site in miR125b-1 electrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChiP) were carried out which confirmed NRF2 binding to the putative ARE site 946bp upstream of the 5’ of the miR-125b-1. To confirm NRF2 capacity to transcriptionally regulate miR-125b-1, we cloned the miR-125b-1 promoter into the PGL4 luciferase vector (PGL4/p125b) and found that NRF2 knockdown significantly inhibited PGL4/p125b luciferase activity compared to control knockdown.

Finally to identify the functional relevance of miR-125b-1 in a broad range of AMLs, miR-125b-1 mimic and antagomiRs were transfected into 3 AML cell lines and 11 primary AML samples. Results show a 10-25% increase in annexin V staining in the miR-125b-1 antagomiR experiments compared with the mimic or controls. Moreover miR-125b antagomiR increased the cytotoxicity of conventional AML therapy (daunorubicin and cytosine arabinoside). These results confirm the cytoprotective role of miR-125b-1 in AML.

In summary we find that NRF2 regulation of miR-125b-1 acts as a novel survival pathway in human AML.