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PIWIL4 ACTS AS A PIRNA BINDING, EPIGENETICALLY ACTIVE AND GROWTH REGULATORY PROTEIN IN HUMAN ACUTE MYELOID LEUKEMIA
Author(s): ,
Shiva Bamezai
Affiliations:
University of Ulm,Institute for Experimental Cancer Research,Ulm,Germany
,
Medhanie Mulaw
Affiliations:
University of Ulm,Institute for Experimental Cancer Research,Ulm,Germany
,
Naidu Vegi
Affiliations:
University of Ulm,Institute for Experimental Cancer Research,Ulm,Germany
,
Michaela Feuring-Buske
Affiliations:
University of Ulm,Institute for Experimental Cancer Research,Ulm,Germany
,
Fengbiao Zhou
Affiliations:
Department of Medicine IV,Hematology and Oncology, University Hospital of Halle (Saale),Halle (Saale),Germany
,
Christian Rohde
Affiliations:
Department of Medicine IV,Hematology and Oncology, University Hospital of Halle (Saale),Halle (Saale),Germany
,
Alexei Aravin
Affiliations:
Division of Biology and Biological Engineering,California Institute of Technology, Pasadena,United States
,
Jessica Hoell
Affiliations:
Department of Pediatric Oncology, Hematology and Clinical Immunology,Heinrich-Heine-University,Düsseldorf,Germany
,
Andreas Kloetgen
Affiliations:
Department of Pediatric Oncology, Hematology and Clinical Immunology,Heinrich-Heine-University,Düsseldorf,Germany
,
Arndt Borkhardt
Affiliations:
Department of Pediatric Oncology, Hematology and Clinical Immunology,Heinrich-Heine-University,Düsseldorf,Germany
,
Christoph Plass
Affiliations:
Division of Epigenomics and Cancer Risk Factors,German Cancer Research Center (DKFZ),Heidelberg,Germany
,
Carsten Müller-Tidow
Affiliations:
Department of Medicine IV,Hematology and Oncology, University Hospital of Halle (Saale),Halle (Saale),Germany
,
Vijay P.S. Rawat
Affiliations:
University of Ulm,Institute for Experimental Cancer Research,Ulm,Germany
Christian Buske
Affiliations:
University of Ulm,Institute for Experimental Cancer Research,Ulm,Germany
(Abstract release date: 05/18/17) EHA Library. Bamezai S. 06/24/17; 181716; S429
Shiva Bamezai
Shiva Bamezai
Contributions
Abstract

Abstract: S429

Type: Oral Presentation

Presentation during EHA22: On Saturday, June 24, 2017 from 12:15 - 12:30

Location: Hall E

Background
Piwi proteins are critically important for maintaining the self-renewing stem cell population in lower organisms through epigenetic silencing of transposable elements via DNA methylation and H3K9me3 marks, in close interaction with a novel class of non-coding RNA called piwi interacting RNA (piRNA).

Aims
There are neither precise data on the function of Piwi proteins in human acute myeloid leukemia (AML), nor are there reports on expression of piRNAs in this disease. We employed functional techniques and NGS to understand the role of human PIWI-like protein, PIWIL4 and its associated piRNA in AML.

 

Methods
We assessed the expression of human PIWIL genes in AML and healthy bone marrow cells using qRT-PCR. Murine stem progenitors were transduced with AML specific oncogenes to evaluate the effect on Piwil4 expression. shRNA mediated knockdown (KD) of PIWIL4 was performed on AML cell lines, AML patient bone marrow (BM) cells and healthy cord blood CD34+ stem progenitors and the impact on growth was determined using in vitro and in vivo assays. Western blot, ChIP-seq for H3K9me3 and RNA-seq were performed to assess the impact of PIWIL4 KD on the epigenetic landscape and transcriptome of the AML cell line THP-1. IP for PIWIL4 followed by LC-MS was performed to determine the binding partners of PIWIL4. PAR-CLIP and microarray were performed to identify piRNAs that physically bind to PIWIL4 and to test the impact of PIWIL4 KD on piRNA expression.

Results
Among the family of human PIWIL genes, PIWIL4 showed the highest expression level and was ubiquitously expressed in healthy hematopoietic stem/progenitors, mature lymphoid and myeloid cells. Importantly, PIWIL4 was aberrantly higher expressed in more than 89% of the AML patients (n=68; p< 0.0001) compared to normal CD34+ BM and total BM cells (n=3). Overexpression of AML specific oncogenes in murine stem progenitors, within 96h post-transduction, induced a 6 to 8 fold increase in Piwil4 expression compared to GFP control (n=3, p< 0.0001). Knockdown (KD) of PIWIL4 in AML cell lines significantly impaired proliferation and clonogenic growth in vitro (n=3; p< 0.001) and delayed onset of leukemia in NSG mice (n=8; p< 0.0001). PIWIL4 KD in primary AML patient BM cells lead to 5-fold decrease in clonogenicity (n=3, p<0.001), but had no impact on clonogenicity of healthy stem progenitors in vitro (n=4). Western blot and ChIP-seq (n=2, MACS1.4, p<0.01, FDR<0.01) in THP-1 cell line revealed a marked global reduction in repressive H3K9me3 marks upon PIWIL4 KD. Over 500 promoter and 600 gene body associated loci exhibited loss of H3K9me3 marks. RNA-seq analyses revealed over 4000 differentially expressed genes upon PIWIL4 depletion. 30% of the loci that lost H3K9me3 marks at promoters and gene body were differentially expressed in RNA-seq (fold>0.05, adj. p<0.01). These genes belonged to pathways associated with RNA metabolism, transcription and cell death. Moreover, these genes were enriched for binding sites of SETDB1, an H3K9me3 establishing histone methyltransferase (ENRICHR, p<0.01, FDR<0.01). Notably, using IP/LC-MS, PIWIL4 was found to associate with SETDB1 in 293T cells. 560 unique piRNAs were found to physically bind to PIWIL4 and 981 unique piRNAs were differentially expressed upon PIWIL4 depletion in THP-1 cells.

Conclusion
Thus, collectively, we could show for the first time that PIWIL4 expression is deregulated in human AML and acts as a piRNA binding, epigenetically active growth regulatory protein in human AML.

Session topic: 3. Acute myeloid leukemia - Biology

Abstract: S429

Type: Oral Presentation

Presentation during EHA22: On Saturday, June 24, 2017 from 12:15 - 12:30

Location: Hall E

Background
Piwi proteins are critically important for maintaining the self-renewing stem cell population in lower organisms through epigenetic silencing of transposable elements via DNA methylation and H3K9me3 marks, in close interaction with a novel class of non-coding RNA called piwi interacting RNA (piRNA).

Aims
There are neither precise data on the function of Piwi proteins in human acute myeloid leukemia (AML), nor are there reports on expression of piRNAs in this disease. We employed functional techniques and NGS to understand the role of human PIWI-like protein, PIWIL4 and its associated piRNA in AML.

 

Methods
We assessed the expression of human PIWIL genes in AML and healthy bone marrow cells using qRT-PCR. Murine stem progenitors were transduced with AML specific oncogenes to evaluate the effect on Piwil4 expression. shRNA mediated knockdown (KD) of PIWIL4 was performed on AML cell lines, AML patient bone marrow (BM) cells and healthy cord blood CD34+ stem progenitors and the impact on growth was determined using in vitro and in vivo assays. Western blot, ChIP-seq for H3K9me3 and RNA-seq were performed to assess the impact of PIWIL4 KD on the epigenetic landscape and transcriptome of the AML cell line THP-1. IP for PIWIL4 followed by LC-MS was performed to determine the binding partners of PIWIL4. PAR-CLIP and microarray were performed to identify piRNAs that physically bind to PIWIL4 and to test the impact of PIWIL4 KD on piRNA expression.

Results
Among the family of human PIWIL genes, PIWIL4 showed the highest expression level and was ubiquitously expressed in healthy hematopoietic stem/progenitors, mature lymphoid and myeloid cells. Importantly, PIWIL4 was aberrantly higher expressed in more than 89% of the AML patients (n=68; p< 0.0001) compared to normal CD34+ BM and total BM cells (n=3). Overexpression of AML specific oncogenes in murine stem progenitors, within 96h post-transduction, induced a 6 to 8 fold increase in Piwil4 expression compared to GFP control (n=3, p< 0.0001). Knockdown (KD) of PIWIL4 in AML cell lines significantly impaired proliferation and clonogenic growth in vitro (n=3; p< 0.001) and delayed onset of leukemia in NSG mice (n=8; p< 0.0001). PIWIL4 KD in primary AML patient BM cells lead to 5-fold decrease in clonogenicity (n=3, p<0.001), but had no impact on clonogenicity of healthy stem progenitors in vitro (n=4). Western blot and ChIP-seq (n=2, MACS1.4, p<0.01, FDR<0.01) in THP-1 cell line revealed a marked global reduction in repressive H3K9me3 marks upon PIWIL4 KD. Over 500 promoter and 600 gene body associated loci exhibited loss of H3K9me3 marks. RNA-seq analyses revealed over 4000 differentially expressed genes upon PIWIL4 depletion. 30% of the loci that lost H3K9me3 marks at promoters and gene body were differentially expressed in RNA-seq (fold>0.05, adj. p<0.01). These genes belonged to pathways associated with RNA metabolism, transcription and cell death. Moreover, these genes were enriched for binding sites of SETDB1, an H3K9me3 establishing histone methyltransferase (ENRICHR, p<0.01, FDR<0.01). Notably, using IP/LC-MS, PIWIL4 was found to associate with SETDB1 in 293T cells. 560 unique piRNAs were found to physically bind to PIWIL4 and 981 unique piRNAs were differentially expressed upon PIWIL4 depletion in THP-1 cells.

Conclusion
Thus, collectively, we could show for the first time that PIWIL4 expression is deregulated in human AML and acts as a piRNA binding, epigenetically active growth regulatory protein in human AML.

Session topic: 3. Acute myeloid leukemia - Biology

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