Downregulation of Mir-181a Restores Chemosensitivity to Imatinib Mesylate in Chronic Myeloid Leukemia Cells Resistant to Tyrosine Kinase Inhibitors
Authors: Chorzalska Anna , Kim Javier F , Roder Karim , Olszewski Adam J , Terentyev Dmitry , Dubielecka Patrycja M 
Published: October 2019

Abstract
Abelson Interactor protein 1 (Abi-1) is a negative regulator of Abelson kinase and interacts with its oncogenic form Bcr-Abl1 (Brehme et al., PNAS 2009:106;7414). We have recently shown that Abi-1 also modulates interactions between chronic myeloid leukemia (CML) stem/progenitor cells and their microenvironment, and has a role in acquired drug resistance. Our data indicated that abnormally low levels of Abi-1 in CML cells correlate with increased expression of integrin α4, deregulation of Fak/Akt/Erk signaling, enhanced adhesive properties, quiescence, and resistance to imatinib mesylate (IM, Chorzalska et al., Leukemia 2014:28;2165). We have found miR-181a to be significantly upregulated in IM-resistant CML cells, and miRNA expression profiling identified miR-181a as a potential regulator of Abi-1 expression. Furthermore, a bioinformatics analysis matching the miR-181a seed region to conserved ABI1 sequences using TargetScan 6.2 (Lewis, Burge, & Bartel, Cell 2005;120.15) determined that a 3'-untranslated region of ABI1 (position 131-137) is a target for miR-181a. This suggested that miR-181a may regulate Abi-1 levels in IM-resistant CML cells. We hypothesized that decreasing the levels of miR-181a in those cells would restore the intracellular levels of Abi-1, and consequently reduce the expression of integrin α4, decrease the adhesion and quiescence, and restore their sensitivity to IM.
To test this hypothesis, we designed and constructed a miRNA sponge (Figure 1) using the methodology established by Kluvier et al. (Methods, 2012:58;113). The sponge can endogenously sequester and functionally reduce levels of miR-181a in CML IM-resistant cells (K562-STI-R cell line), thus allowing to measure the effect of miR-181a loss on Abi-1, integrin α4 levels and chemoresistance. K562-STI-R CML cell line was electroporated with pcDNA3-SanDI vector encoding the miR-181a sponge or a control mismatched miRNA sponge.
Using qPCR, we confirmed a 35% percent decrease in miR-181a transcript levels in IM-resistant K562-STI-R CML cell line expressing the design miR-181a sponge, compared with controls (p=0.0015). This was paralleled by a 38% increase in ABI1 mRNA levels (p=0.046) and about 40% increase in Abi-1 protein levels (p=0.03) as evaluated by immunoblotting followed by densitometry, indicating that functionally decreased intracellular levels of miR-181a resulted in increased expression of ABI1. Because we previously demonstrated that down-regulation of ABI1 increases expression of integrin α4 and adhesion of IM-resistant CML cells while decreasing their proliferative potential, we then evaluated ITGA4 transcript levels and expression of integrin α4 in CML cells transfected with the miR-181a sponge. We observed a 20% decrease in ITGA4 transcript and integrin α4 protein levels compared with controls (p=0.051). Phenotypic evaluation of the chemoresistant K562-STI-R cells expressing the miR-181a sponge showed a 36% (p=0.04) increase in the cell number. Decreased number of Annexin V-positive cells suggested that miR-181a sequestration had a modest inhibitory effect on apoptosis. We then evaluated chemosensitivity of the miR-181a sponge-expressing K562-STI-R cells by culturing them in increased concentrations of IM (10, 100 and 200 µM), and measuring apoptosis by Annexin V staining using flow cytometry. We observed 30% (p=0.01), 46% (p=0.023) and 68% (p=0.018) increase in the number of apoptotic cells in miR-181a sponge-expressing cells compared with control cultured at 10, 100 and 200 µM of IM, respectively.
In summary, this study provides the first piece of evidence that miR-181a is an epigenetic regulator of ABI1, and that downregulation of miR-181a by a miRNA sponge can effectively increase the intracellular levels of Abi-1 in IM-resistant CML cells, consequently increasing their sensitivity to Bcr-Abl inhibition. Utilization of targeted miRNA inhibitors may represent a new approach to restore chemosensitivity of IM-resistant CML cells.
Figure 1. Schematic representation of the design miR-181a sponge and the control sponge. miRNA antisense binding sites (MBS) contain central mismatch at position 9-12 of the miRNA sequence (bulge). For the control sponge, a scrambled sequence at positions 2-8 was generated. The two MBS are separated by a short 4-6 nt sequence spacer. Bold flanking letters indicate overhangs compatible with the restriction endonuclease SanDI. Figure 1. Schematic representation of the design miR-181a sponge and the control sponge. miRNA antisense binding sites (MBS) contain central mismatch at position 9-12 of the miRNA sequence (bulge). For the control sponge, a scrambled sequence at positions 2-8 was generated. The two MBS are separated by a short 4-6 nt sequence spacer. Bold flanking letters indicate overhangs compatible with the restriction endonuclease SanDI.

Disclosures
Olszewski: Bristol-Myers Squibb, Inc.: Consultancy; Genentech, Inc.: Research Funding.