Long Non-Coding Transcript in LEF1 Locus Is a Novel Regulator of Cell Cycle and Apoptotic Pathways in Myeloid Leukemic Cells

Myelodysplastic syndrome and acute myeloid leukemia are hematologic disorders characterized by inefficient differentiation and aberrant control of cell growth and apoptosis. In early MDS, blasts, unable to differentiate, exhibit exacerbated proliferation and apoptosis. However, MDS progression and AML transformation are associated with a progressive resistance to apoptosis. The molecular mechanisms that control these cellular decisions are not fully understood. Long non-coding RNAs (lncRNA) have gained great attention in recent years as they have been implicated in a wide variety of biological functions. Here we focus on a long non-coding transcript encoded in the LEF1 locus as a novel regulator of apoptosis and proliferation signaling in hematologic disease.

Our previous work using microarray technology identified a long non-coding RNA, antisense to LEF1 gene, which expression was reduced in myelodysplastic syndrome patients. We mapped the sequence to a previously characterized transcript, LEF1-AS1. This transcript is highly conserved and has been recently associated to B-cell differentiation. We observed a modulation of LEF1-AS1 during the first stages of in-vitro erythroid differentiation from CD34+ HSC to erythroblast (from collection day 6 to day 8 after addition of erythropoietin, n=6, 78% mean reduction, P<0.0001).

Due to reported reduction on LEF1-AS1 expression in MDS and low endogenous expression of LEF1-AS1 in myeloid cells lines, we decided to overexpress the transcript in myeloid cell lines HL60 and K562 to study the function of the lncRNA. Stably transfected cells were obtained by DMRIE-C mediated transfection using pcDNA vector containing full length lef1-AS1 or empty pcDNA vector. Selection was carried out by addition of geneticin to the culture medium.

Unexpectedly, after stable over-expression of LEF1-AS1, we could not detect any alteration of LEF1 expression in the mRNA or protein level in both myeloid cell lines (HL60 and K562). Despite of the lack of regulatory effect upon LEF1, we observed a significant inhibition in cell proliferation evaluated Carboxyfluorescein succinimidyl ester (CFSE) labelling (p<0.05).

To investigate the antiproliferative effects of LEF1-AS1, we analyzed the cell cycle phase distribution of the stably transfected cell HL60 by flow cytometry using propidium iodide DNA staining. LEF1-AS1 over-expression resulted in a significant increase in the proportion of S-phase cells (p=0.01) and no difference in G2/M, suggesting intra S-phase imprisonment.

Additionally, we evaluated the effect of LEF1-AS1 over-expression upon apoptosis in stably transfected HL60 using annexin/Propidium Iodide Apoptosis flow cytometry assay. We observed no difference in the apoptosis level between LEF1-AS1-overexpressing cells and control cells in basal culture conditions; however, LEF1-AS1 over-expression inhibited significantly serum deprivation-induced apoptosis (reduction of 50%, p<0.05).

We screened several apoptosis and proliferation regulators by RT-PCR and western blotting and detected a strong increase in anti-apoptotic BCL2 and CDKN1A/p21 in HL60 cells over-expressing LEF1-AS1. This increase was observed in cells in exponential growth and starvation. Interestingly, we did not observe this antiapoptotic effect in K562, a cell line that do not express BCL2 endogenously.

To confirm that the phenotypical effects of lef1-AS over -expression were not mediated by LEF1 function, we also over-expressed lef1-AS1 in Hela, a cell line lacking endogenous expression of LEF1. Empty vector and lef1-AS1-Hela stably-transfected-cells had no detectable expression of LEF1; however, a similar inhibition of cell proliferation and S-phase accumulation were observed.

To further investigate the mechanism behind these phenotypical effects we carried out a RNA-pulldown experiment coupled with mass spectrometry to identify the proteins that bind to LEF1-AS1. Our preliminary results identified an apoptosis regulator protein in association to this transcript and we are currently working to unravel the implications of this interaction.

Remarkably, BCL2 is simultaneously antiapoptotic and anti-proliferative and S-phase arrest has been associated to p21 up-regulation. Our results depict LEF1-AS1 as a fine-tuner of proliferation and survival decisions, acting, at least partially, through modulation of BCL2 and CDKN1A/p21.