Deregulated Expression of Long Noncoding RNAs H19, LEF1-AS1, TCL6, and WT1-AS1 Predicts Poor Outcome of Patients with Myelodysplastic Syndromes
Authors: Dostalova Merkerova Michaela , Loudova Nikoleta , Szikszai Katarina , Krejcik Zdenek , Klema Jiri , Pecherkova Pavla , Hrustincova Andrea , Vesela Jitka , Cermak Jaroslav , Jonasova Anna , Belickova Monika 
Published: October 2019

Abstract
INTRODUCTION: Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell (HSC) disorders characterized by dysplastic and ineffective blood cell production and a tendency for transformation to acute myeloid leukemia (AML). Because of the heterogeneity of MDS, the development of additional molecular tools able to refine the Revised International Prognosis Scoring System (IPSS-R), predict outcome and monitor the response to treatment would be highly beneficial.
Long noncoding RNAs (lncRNAs) are protein-noncoding RNAs that play multiple roles in hematopoietic differentiation. They contribute to the pathogenesis of hematologic malignancies, representing a new class of potential biomarkers and therapeutic targets.
AIMS: In this study, we aimed to identify lncRNAs with deregulated expression in MDS that could serve as novel potential molecular markers of poor outcome.
METHODS: The discovery cohort included 54 MDS patients, 14 AML patients with myelodysplasia-related changes (AML-MRC), and 9 controls. Agilent microarrays were used to profile lncRNAs expression in CD34+ bone marrow (BM) cells. Data analyses were focused on the identification of lncRNAs with altered levels between MDS patients (i) with (very) low vs. (very) high risk IPSS-R, and (ii) with progression-free survival (PFS) shorter vs. longer than 2 years. RT-qPCR was applied to validate expression levels of selected deregulated lncRNAs (H19, TCL6, LEF1-AS1, and WT1-AS) and levels of their protein-coding counterparts (LEF1 and WT1) in an independent validation cohort (79 MDS and 14 AML-MRC patients, and 13 controls). Further, we examined whether the expression of H19, TCL6, LEF1, and WT1 changed in patients treated with azacitidine (AZA). For this purpose, we retrieved data from our previous microarray analysis (GEO GSE77750) performed on a cohort of 19 MDS, 3 chronic myelomonocytic leukemia (CMML), 10 AML-MRC patients and 10 controls.
RESULTS: In the MDS patients with higher risk IPSS-R, 23 lncRNAs were downregulated (e.g., CXADRP3, LEF1-AS1, PCED1B-AS1, SLC8A-AS1, and TCL6) and 35 lncRNAs were upregulated (e.g., BCAR4, FAM225A, FARP1-AS1, H19, and RBPMS-AS1) vs. lower risk IPSS-R (|logFC| ≥ 1, p ≤ 0.01). Furthermore, we identified significant upregulation of e.g. H19, SATB2-AS1, and WT1-AS in patients with PFS < 2 years vs. those with longer survival.
For subsequent study, we selected H19, LEF1-AS1, TCL6, and WT1-AS lncRNAs based on their known association with hematopoiesis. In the validation cohort, we proved that downregulation of LEF1-AS1 and TCL6 and upregulation of H19 and WT1-AS were associated with poor PFS and OS (p < 0.01, Figure 1). Further, we correlated lncRNA expression levels with multiple clinical parameters (sex, age, BM blasts, hemoglobin, neutrophils, platelets, and IPSS-R-based cytogenetics) and found out that only level of H19 was independent from all these variables whereas expression of other lncRNAs was associated with BM blast count. Multivariate analysis confirmed that H19 level, BM blasts, and platelets remained independent factors for OS and PFS in MDS (p < 0.05). Importantly, WT1-AS level, platelets, and hemoglobin were identified as independent variables for survival of MDS patients with BM blast counts < 5 % (p < 0.05). Additionally, we measured levels of appropriate protein-coding counterparts of LEF1-AS1 (i.e. LEF1) and WT1-AS (i.e. WT1) and found strong positive correlation between these pairs (p < 0.001), indicating their co-transcriptional regulation.
Because expression of the studied lncRNAs significantly varied between lower and higher risk MDS patients, we further investigated impact of AZA treatment on the expression of H19, TCL6, LEF1, and WT1 in higher risk disease. However, we detected neither any difference in their expression between responders and nonresponders nor any significant return to normal expression levels caused by AZA therapy.
CONLUSIONS: Our data proved that particular lncRNAs are specifically deregulated in higher risk MDS. Altered transcript levels of these lncRNAs may serve as independent markers of poor survival, even in the patients with low BM blast counts, providing additional information about prognosis of MDS patients beyond the currently used scoring system.
The work was supported by grants 17-31398A, 16-33617A, 16-33485A, and 00023736 from the Ministry of Health of the Czech Republic.

Disclosures
No relevant conflicts of interest to declare.