Dysregulation of Transposable Elements and Piwi-Interacting RNAs in Myelodysplastic Neoplasms

Myelodysplastic neoplasms (MDS) are a heterogenous group of malignant hematopoietic stem cell (HSC) disorders characterized by genomic instability, resulting in aberrant differentiation of HSCs, peripheral blood cytopenia, and a tendency toward leukemic transformation. Transposable elements (TEs) are common in the human genome, and their mobilization may represent one of the sources of this instability. TEs are thought to be suppressed, among others, by small noncoding PIWI-interacting RNAs (piRNAs). Although TE and piRNA dysregulation has already been individually described in different hematological malignancies, information on their expression in MDS is still limited. Therefore, our aim was to measure and compare both TE and piRNA expression profiles in parallel in HSCs of different MDS subtypes.

The study was performed on bone marrow CD34⁺ HSCs from 80 MDS patients and 17 healthy donors as controls (CTRs). The MDS diagnoses were established based on the standard WHO 2016 classification criteria, and patients’ prognoses were estimated using the IPSS-R system. For TE expression, RNA-seq libraries were prepared from rRNA-depleted RNA, and after NGS sequencing, TEs were identified and counted by the SalmonTE tool. For piRNA expression, smallRNA-seq libraries were prepared from total RNA, and piRNA reads were mapped to the piRNABank database.

Overall, we identified 687 TEs and 257 piRNAs across the cohort. Although differential expression analysis between MDS patients and CTRs revealed similar expression profiles (13 TEs and 3 piRNAs differentially expressed, FDR < 0.05), there was significant difference between lower risk (LR, IPSS-R ≤ 3.5) and higher risk (HR, IPSS-R ≥ 4) MDS patients (106 TEs and 17 piRNAs dysregulated). Further, we found significantly lower total TE (p = 0.005) and higher total piRNA (p = 0.003) expression in HR-MDS compared to LR-MDS and CTRs, and these two variables negatively correlated (Pearson r = -0.380, p = 0.001). High total piRNA expression also showed a strong association with adverse outcome; median overall survival (OS) of the MDS patients with normal and high piRNA expression reached 64.5 and 18.4 months, and median progression free survival (PFS) was 51.4 and 10.1 months, respectively. In accordance with high piRNA levels, we observed significantly elevated levels of the PIWIL2 gene in HR-MDS patients, and a strong correlation between PIWIL2 and total piRNA levels (Pearson r = 0.624, p < 0.001).

We focused on expression of selected individual molecules: FAM (highly expressed TEs, reduced in LR-MDS), HERV-Fc1 (the most significantly increased TE in MDS compared to CTRs), piR_018780 (highly expressed piRNA, increased in HR-MDS), and PIWIL2. Levels of these molecules correlated with patient outcome; patients with increased piR_018780 and reduced FAM had inferior survival, while lower HERV-Fc1 levels suggested better survival. High level of PIWIL2 was strongly adversely associated with both, OS and PFS, and Cox multivariate analysis proved that a high PIWIL2 level was one of the most significant independent variables predicting survival.

Gene Set Enrichment Analysis showed strong suppression of multiple cellular processes in patients with concurrent low TE and high piRNA expression. These processes included OXPHOS, glycolysis, DNA repair, ROS pathway, G2M cell cycle checkpoint, PI3K/AKT/mTOR signaling, and targets of the MYC transcription factor. Analysis of Biological Processes and GO terms associated with TE and piRNA dysregulation discovered strong suppression of processes connected with immune response: regulation of immune system process, immunoglobulin production, inflammation, and complement activation.

This is the first study to examine dysregulation of TEs and piRNAs in parallel in a large cohort of MDS patients, revealing their potential importance, particularly for the disease progression. It showed significantly reduced TEs and increased piRNA levels in MDS patients with adverse outcome. Dysregulated levels of several TE/piRNA related molecules were significantly associated with shorter patient survival with high PIWIL2 gene expression being an independent prognostic marker. Suppression of key biological processes associated with this dysregulation suggested that MDS cells with low TE and high piRNA levels undergo substantial changes in cellular activity.

Supported by AZV CR (NU20-03-00412) and MH CZ-DRO (UHKT 00023736).