LEF1-AS1, long non-coding RNA, inhibits proliferation in myeloid malignancy.

LEF1 antisense RNA 1 (LEF1-AS1) is an antisense long non-coding RNA encoded in the lymphoid enhancer-binding factor 1 (LEF1) locus. LEF1-AS1 is a conserved transcript dysregulated in hematopoiesis. This study aimed to functionally characterize the role of this transcript in myeloid malignancy and explore a possible regulatory effect of LEF1-AS1 upon LEF1. We show that LEF1-AS1 is highly expressed in normal hematopoietic stem cells but barely detectable in myeloid malignant cell lines. Additionally, bone marrow cells from myelodysplastic syndrome (n=12) and acute myeloid malignancy patients (n=28) expressed significantly reduced levels of LEF1-AS1 compared to healthy controls (n=15). Artificial LEF1-AS1 over-expression inhibited proliferation in HL60 and led to an upregulation of tumor suppressors p21 and p27, and reduced ERK1/2 activation. Unexpectedly, no underlying modulation of LEF1 was detected. Ectopic expression of LEF1-AS1 also inhibited proliferation in HELA, a cell line lacking endogenous expression of LEF1, supporting a LEF1-independent mechanism. Additionally, transient over-expression of LEF1-AS1 in AML patient cells also led to reduced proliferation and colony formation capacity. We used a mass spectrometry-based proteomics approach. Proteomic quantification identified the modulation of an important metabolic regulator, Fumarase, and concomitant accumulation of the metabolite fumarate.

ited proliferation in HL60 and led to an upregulation of tumor suppressors p21 and p27, and reduced ERK1/2 activation. Unexpectedly, no underlying modulation of LEF1 was detected. Ectopic expression of LEF1-AS1 also inhibited proliferation in HELA, a cell line lacking endogenous expression of LEF1, supporting a LEF1-independent mechanism. Additionally, transient over-expression of LEF1-AS1 in AML patient cells also led to reduced proliferation and colony formation capacity. We used a mass spectrometry-based proteomics approach. Proteomic quantification identified the modulation of an important metabolic regulator, Fumarase, and concomitant accumulation of the metabolite fumarate.

K E Y W O R D S
LEF1-AS1, long non-coding RNA, acute myeloid malignancy unspliced transcript and ultimately on the regulation of LEF1. 6 Based on this evidence and as LEF1 coding gene has an established role in myeloid malignancy, 7,8 we suspected a cis-regulatory mechanism between the antisense non-coding transcript and LEF1 in the haematopoietic system. To investigate the role of LEF1-AS1 in the regulation of LEF1 and myeloid malignancy pathogenesis, we used a stable transfection approach to overexpress the transcript in myeloid cell line HL60. Stably transfected cells were obtained by DMRIE(1,2dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide) -C-mediated transfection using pcDNA vector containing full length LEF1-AS1 or empty pcDNA vector, and 3 weeks of geneticin selection. This method was adapted from Grinstein et al 9 and details are presented in Supplementary information. Unexpectedly, we could not detect any significant alteration of LEF1 expression after overexpression of LEF1-AS1 ( Figure 1A). Despite the lack of effect upon LEF1, we observed that LEF1-AS1 overexpression led to inhibition of proliferation as shown by two different proliferation-specific assays.
In line with this reduction in proliferation, an increased expression of tumour suppressors CDKN1A (p21) and CDKN1B (p27) was detected in the mRNA and protein levels ( Figure 1B; Supplementary).  Figure 1E,F), and no effect upon LEF1 expression (no detection by RT-PCR in control and LEF1-AS1 cells).
Lacking an obvious candidate mediator of this anti-proliferative function of LEF1-AS1, we resorted to a mass spectrometry-based proteomics approach to characterize the function of LEF1-AS1.
Whole-cell lysates were processed and analysed by Q-tof mass spectrometry. A total of over 500 proteins were identified (see complete list in a supplementary file) and 16 were differentially expressed (P-value cut-off of 0.05, see Table S2). We were able to validate the most relevant results by western blot (Figure 2A). Histone 3 (H3), a marker of proliferation, 10,11 was reduced, in line with our functional results. Talin is a protein involved in cell-to-cell and cellto-substrate adhesion and migration and it has a role in metastasis of several cancers, but its contribution to leukaemogenesis is not clear. Talin up-regulation was also observed in Hela and may play a role in LEF1-AS1 function in other tissues (Supplementary). We also validated the increased protein expression of RAB7A, a small GTPase that regulates exocytosis/endocytosis-mediated protein/ RNA trafficking. 12 Remarkably, the activation of RAB7A is associated with the increased endocytic degradation of epidermal growth factor receptor, 13  We observed that LEF1-AS1 is lost in myeloid malignant cells.
Expression of LEF1-AS1 was shown to be reduced in haematopoietic stem cells from myelodysplastic syndrome patients, 3 we observed the same pattern in total bone marrow cells. MDS is a haematologic disorder characterized by blood cytopenia and increased risk of developing AML. 3 This loss of expression is also observed in AML patients' bone marrows suggesting this suppression may be an important step in malignization and disease progression.
As we demonstrated, the artificial re-expression of LEF1-AS1 reduces proliferation of myeloid cell line HL60, non-haematopoietic F I G U R E 2 A, Validation of proteins modulated by LEF1-AS1 detected by mass spectrometry using western blot, statistical analysis (paired t test) was performed using the relative optical densities of the bands of three independent experiments (see bands used in the analysis in Supplementary).