Down-Regulation of Microrna 223 and 148a Mediates Resistance of Human Acute Myeloid Leukemia Cells to Pan-Histone Deacetylase (HDAC) Inhibitors.
Authors: Chen Jianguang , Kandaswamy Veena , Fiskus Warren , Wang Yongchao , Rao Rekha , Balusu Ramesh , Buckley Kathleen M. , Atajada Peter , Bhalla Kapil N. 
Published: October 2019

Abstract
Abstract 2401
Poster Board II-378
MicroRNA (miR) alterations are highly involved in the pathogenesis of leukemia. However, the role of miRs in de novo or acquired resistance of cancer cells to therapeutic agents has not been fully elucidated. Recently, we reported the isolation and characterization of HL-60/LR cells, derived from human acute myeloid leukemia HL-60 cells, that are resistant to pan-histone deacetylase (HDAC) inhibitors (HDIs), including vorinostat and panobinostat (Blood. 2008; 112: 2896). To explore the role of miRs in acquiring resistance to HDIs, we performed a miR microarray analysis of the parental HL-60 and HL-60/LR cells. Compared to HL-60 cells, expression of thirteen microRNAs were discovered to be significantly increased (> 4-fold) and fourteen miRs were markedly down-regulated (> 4-fold) in HL-60/LR cells. Alterations in the expression of three of the most promising upregulated (miR-21, miR-126 and miR-146a) and down-regulated (miR-223, miR-148a and miR-342) miRs were confirmed by Q-PCR in HL-60/LR cells. The expression of miR-223, miR-148a and miR-342 was also significant lower in the relatively HDI-resistant K562 cells as compared to HDI-sensitive U937 and HL-60 cells. Conversely, miR-126 and miR-146a expressions were higher in K562 cells compared to U937 and HL-60 cells. Short term (24 hours) treatment with panobinostat (10 to 50 nM) did not alter the expression of miR-223 or miR-148a expression in HL-60 cells. As compared to treatment with either agent alone, co-treatment with the histone methyl transferase EZH2 antagonist 3-deazaneplanocin (DZnep, 1.0 uM) and DNA methyl transferase inhibitor decitabine (2.0 uM) induced miR-223 and miR-148a levels and mediated apoptosis of HL-60/LR cells, suggesting that an epigenetic silencing mechanism(s) may be involved in the down-modulation of miR-223 and miR-148a in HL-60/LR cells. To determine whether the alterations in the miR levels were mechanistically involved in conferring resistance to HDIs, we engineered through retroviral transduction stable ectopic expressions of miR-223, miR-148a and miR-342 into HL-60/LR cells and miR-21 and miR-146a into HL-60 cells. Ectopic expression of miR-223 and miR-148a significantly increased the sensitivity of HL-60/LR cells to panobinostat and vorinostat. In contrast, re-expression of miR-342 had an insignificant effect on HDI sensitivity. Increased expression of miR-21 and miR-146a did not confer resistance to the HDIs in HL-60 and U937 cells. Next, by Western analyses, we compared the expression levels of several of the predicted target proteins of miR-223 and miR-148a, (as predicted by the computer programs TargetScan and picTAR), in HL-60 versus HL-60/LR cells, as well as in the HL-60/LR cells with stable ectopic expression of miR-223 and miR-148a. Several candidate proteins including GRP94, Ribosomal protein S6 kinase MSK1, MEF2C and DNMT1 showed higher level of expression in HL-60/LR versus HL-60 and were down-regulated in miR-223 or miR-148a transduced HL-60/LR cells, suggesting that these proteins may confer resistance against HDI. Parenthetically, miR-223 was shown to be a myeloid-specific miR which negatively regulates progenitor proliferation and granulocyte differentiation and activation. miR-223 mutant mice have an expanded granulocytic compartment resulting from an increase in the number of granulocyte progenitors. In summary, our observations indicate that high miR-223 and miR-148a levels may be predictive biomarkers for sensitivity to HDIs in human AML cells. Additionally, induction of miR-223 and miR-148a by EZH2 antagonist may increase sensitivity and overcome resistance to HDIs in human AML cells. Targeting the levels and/or activity of the miR-223 and miR-148a target proteins may also be an effective strategy in enhancing the activity of HDI based combination therapy against AML.

Disclosures:
Atajada: Novartis: Employment. Bhalla:Merck: Honoraria; Novartis: Honoraria, Research Funding.