| Summary: | Acute Myeloid Leukaemia refers to the excess proliferation of myeloid progenitor cells. Whilst the 5-year survival rate is 40% in younger patients, this falls to 5% in patients over 65 and resistance to front-line chemotherapy agents remain a problem. We previously identified NRF2, a regulator of anti-oxidant genes, to be constitutively activated in AML and this correlated with resistance to chemotherapy. Recent studies have also suggested NRF2 also plays a more oncogenic role. To further understand NRF2's role in both chemotherapy resistance and oncogenesis we looked at its ability to regulate miRNA in AML. Using a miRNA array, we identified several miRNAs, including miR-125b and miR-29b, whose expression correlated with that of NRF2 in both cell lines and AML patient samples. Both miRNAs exist as paralogs, in that they contain the same miRNA sequence but exist in different genomic locations and we used qPCR to identify miR-125b1 and miR-29b1 as paralogs regulated by NRF2. Using a reporter assay we confirmed the activity of putative NRF2-ARE binding sites in both miRNA promoters. To understand the function of both in AML we manipulated their expression using miRNA 'mimics' or antagomIRs. Individual manipulation of either miRNA resulted in a slight increase in apoptosis. However, the miRNAs appeared to act synergistically as when expressed simultaneously a significant increase in apoptosis was seen both in cell lines and patient blasts. Manipulation of both miRNA also resulted in the increased sensitivity of AML cells to chemotherapy agents. BAK1, STAT3 and AKT2 were shown to be targets of both miRNAs providing a novel mechanism by which NRF2 expression can affect AML cells. To further study the role of NRF2 in AML we used CRISPR-Cas9 to generate NRF2-deficient cells. CRISPR guide RNA were designed to target NRF2 Exons 1, 2 and genome editing validated in HEK293T cells. Leukaemic cell lines (K562 and THP-1) were virally transduced with guides targeting Exon 4 of NRF2. Once editing was verified clones were derived by growing selected cells in Methycellulose-containing medium. Putative clones were initially screened using MG-132 (to stabilise NRF2) and further confirmed by treatment with the NRF2 inducers CDDO-Me and Sulforaphane. Verified clones were characterised by Sanger Sequencing. In addition to CRISPR-Cas9 we also validated a number of other gene-editing methodologies including CRISPR-Cpf-1 and TALENs. Overall these methodologies represent powerful tools to further characterise the role of NRF2 in AML.
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