Abstract
The deregulation of lineage control programs is often associated with the progression of haematological malignancies. The molecular regulators of lineage choices in the context of tyrosine kinase inhibitor (TKI) resistance remain poorly understood in chronic myeloid leukemia (CML). To find a potential molecular regulator contributing to lineage distribution and TKI resistance, we undertook an RNA-sequencing approach for identifying microRNAs (miRNAs). Following an unbiased screen, elevated miRNA182-5p levels were detected in Bcr-Abl-inhibited K562 cells (CML blast crisis cell line) and in a panel of CML patients. Earlier, miRNA182-5p upregulation was reported in several solid tumours and haematological malignancies. We undertook a strategy involving transient modulation and CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats)-mediated knockout of the MIR182 locus in CML cells. The lineage contribution was assessed by methylcellulose colony formation assay. The transient modulation of miRNA182-5p revealed a biased phenotype. Strikingly, Δ182 cells (homozygous deletion of MIR182 locus) produced a marked shift in lineage distribution. The phenotype was rescued by ectopic expression of miRNA182-5p in Δ182 cells. A bioinformatic analysis and Hes1 modulation data suggested that Hes1 could be a putative target of miRNA182-5p. A reciprocal relationship between miRNA182-5p and Hes1 was seen in the context of TK inhibition. In conclusion, we reveal a key role for miRNA182-5p in restricting the myeloid development of leukemic cells. We propose that the Δ182 cell line will be valuable in designing experiments for next-generation pharmacological interventions.
Highlights
The pathways that regulate haematopoietic differentiation are well understood and have served as paradigms in developmental biology.[1]
High miRNA182-5p expression is associated with tyrosine kinase inhibitor (TKI) resistance in Chronic myeloid leukemia (CML) cells
To assess the miRNAs that were modulated in the context of resistance to imatinib, Illumina sequencing was performed on RNA extracted from imatinibtreated K562 cells
Summary
The pathways that regulate haematopoietic differentiation are well understood and have served as paradigms in developmental biology.[1] With the discovery of microRNAs (miRNAs), there has been an interest in analysing the role of these molecules in haematopoiesis and related disease states.[1,2,3,4]. Examples of such miRNAs are miRNA223, miRNA486, miRNA144 and miRNA451.6,7 in the context of hematopoietic development and malignancies, a miRNA of particular interest is miRNA182-5p.5–9. Over the past decade or so, a considerable body of literature has documented the striking clinical remissions that Gleevec and next-generation tyrosine kinase inhibitor (TKI) have achieved.[21,22,23] there is a small but significant set of CML patients who develop resistance to TKIs in the absence of specific identifiable tyrosine kinase mutations.[24,25] The mechanisms associated with this class of relapse are currently poorly understood
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