Knockdown Of The Piwi - Like Protein 4 (PIWIL4) Delays Leukemic Growth and Is Associated With Gross Changes In The Global Histone Methylation Marks In Human MLL - Rearranged AML

Abstract

Piwi proteins belong to a class of proteins which were shown to be critically involved in the maintenance of the self-renewal property of stem cells in lower organisms. Furthermore, it was shown that they preserve genomic integrity through epigenetic silencing of transposable elements via CpG methylation and repressive histone modifications such as H3K9me3 in close interaction with a novel class of non-coding RNA called piRNA. So far there are neither precise data on the function of Piwi proteins in human acute myeloid leukemia, nor are there reports on expression of piRNAs in this disease. In a first step we tested PIWIL gene expression levels in normal human hematopoietic cells and leukemic patient samples by qRT-PCR. Among the family of human PIWI genes, PIWIL4 showed the highest expression level and was ubiquitously expressed in normal hematopoietic stem/progenitors, mature lymphoid and myeloid cells. Importantly, PIWIL4 showed aberrantly high expression in more than 72% of the AML patients (n=68; p< 0.0001) compared to normal CD34+ bone marrow (BM) and total BM cells (n=3). Notably, in nine of the ten MLL-AF9 rearranged AML patients, PIWIL4 was 64-fold higher expressed compared to normal CD34+ BM (p<0.0001) and 8-fold higher compared to inv(16), PML-RARa or cytogenetically normal AML patients (p<0.0001). To further validate this finding we analysed gene expression data performed on CD34+ human cord blood cells transduced with MLL-AF9 (n=9) vs AML-ETO (n=6) vs MYH11 (n=3): of note, PIWIL4 showed a 6 fold increase in expression in the MLL-AF9 transduced cells compared to the other experimental arms. Stable knockdown of PIWIL4 in the MLL rearranged AML cell lines MV4-11 (MLL-AF4) and THP-1 (MLL-AF9) significantly impaired growth in vitro (n=3) reducing proliferation and clonogenic growth by 83%/93% and 91%/93%, respectively. In addition, depletion of PIWIL4 delayed onset of leukemia in NSG mice transplanted with MV4-11/ THP-1 cells transduced with shPIWIL4 compared to the scrambled control (shRNA: AML onset 48/62d after transplantation vs. 30/30 days in the scrambled control; n=4/8 per arm; p< 0.0001/p<0.001). ChIP-seq analysis revealed that depletion of PIWIL4 in the THP1 cell line results in a marked global reduction in repressive H3K9me3 marks and in an increase in activating H3K4me3 marks as compared to cells transduced with the scrambled control. RNA-seq analyses revealed over 2500 differentially expressed genes upon PIWIL4 depletion with 60% of the genes being upregulated compared to the scrambled control (p<0.05). Among them genes involved in cell cycle such as RB1, P21, TGFB1 as well as epigenetic modifiers such as SETDB1, HDAC1,2 and demethylating enzyme TDG were differentially expressed. RB1 and EED, a protein necessary for PRC2 complex function, displayed an increase in expression and loss of H3K9me3 modifications on their promoters upon knockdown of PIWIL4. To prove piRNA expression in human AML and to test any association between PIWIL4 expression and piRNA signatures, microarray analyses covering 23,677 piRNAs was performed on the MLL-AF9 rearranged THP-1 cell line, of which 14193 piRNAs showed expression levels higher than 4 (arbitrary log2 scale). PIWIL4 knockdown induced differential expression of 981 piRNAs (p≤0.01, fold change ≥2), of which 527 were downregulated and 454 upregulated. Thus, collectively, we could show for the first time that PIWIL4 expression is deregulated in human AML, affects leukemic growth, shapes epigenetic marks and impacts piRNA expression in this disease. Disclosures: No relevant conflicts of interest to declare.

Transposable elements and Piwi‑interacting RNAs in hemato‑oncology with a focus on myelodysplastic syndrome (Review)

“…This depletion was found to be associated with a global reduction in the levels of repressive H3K9me3 signatures and an increase in the levels activating H3K4me3 signatures (143). In addition, it was demonstrated that Piwil4 depletion altered the piRNA expression profile in AML cells with rearranged MLL (143). The role of Piwi has also been previously studied in chronic myelogenous leukemia (CML) (144).…”

“…Depletion of Piwil4 in mixed-lineage leukemia (MLL) rearranged AML cell lines was found to impair cell proliferation and clonogenic growth whilst delaying the onset of leukemia in mice (143). This depletion was found to be associated with a global reduction in the levels of repressive H3K9me3 signatures and an increase in the levels activating H3K4me3 signatures (143). In addition, it was demonstrated that Piwil4 depletion altered the piRNA expression profile in AML cells with rearranged MLL (143).…”

“…To date, the function of the piRNA/Piwi axis in leukemia has only been partially explored, with the majority of the emphasis on Piwi proteins rather than piRNAs. Bamezai et al (143) showed that Piwil4 showed aberrantly high expression in >72% patients with AML. Depletion of Piwil4 in mixed-lineage leukemia (MLL) rearranged AML cell lines was found to impair cell proliferation and clonogenic growth whilst delaying the onset of leukemia in mice (143).…”

“…Bamezai et al (143) showed that Piwil4 showed aberrantly high expression in >72% patients with AML. Depletion of Piwil4 in mixed-lineage leukemia (MLL) rearranged AML cell lines was found to impair cell proliferation and clonogenic growth whilst delaying the onset of leukemia in mice (143). This depletion was found to be associated with a global reduction in the levels of repressive H3K9me3 signatures and an increase in the levels activating H3K4me3 signatures (143).…”

Genome-Wide Tiling Array Analysis of HPV-Induced Warts Reveals Aberrant Methylation of Protein-Coding and Non-Coding Regions

“…Furthermore, various DM protein-coding genes in warts have been previously associated with cancers other than squamous cell carcinoma. Encoding an argonaute family protein, the piwi-like protein 4 (PIWIL4) gene was reported to be highly expressed in breast cancer cells, and its knockdown was found to lessen leukemic growth [46,47]. Moreover, increased expression of the protein tyrosine phosphatase (PTPRA) gene and promoter methylation of the calcium-binding protein 39-like (CAB39L) gene were associated with gastric cancer [48,49].…”