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)

Aravin, A.

Bamezai, S.

Borkhardt, A.

Buske, C.

Feuring-Buske, M.

Hoell, J.

Kloetgen, A.

Mulaw, M.

Müller-Tidow, C.

Plass, C.

Rawat, V. P.

Rohde, C.

Vegi, N.

Zhou, F.

Caltech Authors - Main

Background: Acute myeloid leukemia (AML) is characterized by rapid growth
and block in differentiation of myeloid progenitors. The AML blast is defined by
having “open” chromatin. We hypothesized that alterations of chromatin com-
paction may promote AML. Reversing those changes could represent a novel
therapeutic approach.
Aims: Gain of chr21q22 is the most common focal amplification in complex
karyotype AML. HMGN1 is a chromatin-regulatory protein on 21q22 known to
affect lymphoid development, and our preliminary data suggested that HMGN1
could directly mediate a myeloid differentiation block. Since HMGN1 is known
to decompact chromatin and alter histone marks, our goal was to define and
therapeutically target the mechanisms by which HMGN1 overexpression dis-
rupts myeloid differentiation and promotes clonal dominance.
Methods: We immortalized bone marrow progenitors from wild-type (WT) or
OE-HMGN1 mice (transgenic overexpressing HMGN1) with an estrogen
receptor-HoxB8 fusion protein. Using exogenous estrogen to control nuclear
translocation of HoxB8, we analyzed synchronized myeloid differentiation by
flow cytometry, RNAseq, and TMT proteomic analysis. We performed MINT-
ChIP-seq (MNase Indexed T7-chromatin IP) to measure the histone marks
H3K27ac, H3K27me3, H3K4me3 and total Histone H3. We also measured
histone marks in hematopoietic stem and progenitor subpopulations in vivo.
We performed competitive bone marrow transplantation with CD45.1 WT and
CD45.2 OE-HMGN1 donors and measured the relative contribution to
hematopoiesis over time.
Results: Synchronized differentiation in WT cells progressed over 6 days from
myeloid progenitors to mature neutrophils and monocytes, analyzed by cell
surface markers, morphology, and gene and protein expression. OE-HMGN1
cells proliferated faster and remained as undifferentiated myeloblasts (84%
Cd11b+Gr1+ in WT vs 4% in OE-HMGN1, p=0.002; Fig A). Gene set enrich-
ment analysis revealed more similarity to undifferentiated hematopoiesis and
leukemia signatures in OE-HMGN1 cells. MINT-ChIP indicated higher global
and locus-specific levels of H3K27ac in OE-HMGN1 cells (Fig B, upper panel),
consistent with an increase in gene transcription, confirmed by RNA-seq. We
found a specific increase in HoxA cluster expression in OE-HMGN1 cells, high-
est at HoxA7 and HoxA9, genes known to be important in AML pathogenesis.
In agreement with gene expression, among the most differentially measured
histone peaks genome-wide were higher H3K27ac at HoxA gene promoters at
all differentiation time points analyzed (Fig B, lower panel). Competitive trans-
plantation demonstrated an advantage to OE-HMGN1 stem and progenitor
cells. The clonal dominance of OE-HMGN1 over WT cells extended to all pop-
ulations analyzed (long- and short-term HSCs, multipotent progenitors, CMP,
GMP and MEP; Fig C) and to mature lineages (myeloid, B and T cells). MINT-
ChIP in LK and LKS stem and progenitor cells revealed an increase in H3K27ac
peaks at cell cycle and leukemia-related genes in the context of OE-HMGN1.
H3K27 acetylation is catalyzed by the CBP/p300 histone acetyltransferase
(HAT), suggesting that HAT inhibition could target leukemias with HMGN1 over-
expression. Indeed, treatment of myeloid progenitors with the CBP/p300
inhibitor C646 rescued the differentiation block in OE-HMGN1 cells (93%
CD11b+Gr1+ in WT vs 80% in OE-HMGN1, p=NS).
Figure 1.
Summary/Conclusions: Our study suggests that HMGN1 overexpression
blocks myeloid differentiation and promotes proliferation in hematopoietic pro-
genitors via increased H3K27 acetylation. Targeting epigenetic changes down-
stream of HMGN1 or interfering with HMGN1 itself may represent a novel ther-
apeutic strategy in AML.
S429
PIWIL4 ACTS AS A PIRNA BINDING, EPIGENETICALLY ACTIVE AND
GROWTH REGULATORY PROTEIN IN HUMAN ACUTE MYELOID
LEUKEMIA
S. Bamezai1,*, M. Mulaw1, N. Vegi1, M. Feuring-Buske1, F. Zhou2, C. Rohde2,
A. Aravin3, J. Hoell4, A. Kloetgen4, A. Borkhardt4, C. Plass5, C. Müller-Tidow2,
V.P. Rawat1, C. Buske1
1University of Ulm, Institute for Experimental Cancer Research, Ulm, 2Depart-
ment of Medicine IV, Hematology and Oncology, University Hospital of Halle
(Saale), Halle (Saale), Germany, 3Division of Biology and Biological Engineer-
ing, California Institute of Technology, Pasadena, United States, 4Department
of Pediatric Oncology, Hematology and Clinical Immunology, Heinrich-Heine-
University, Düsseldorf, 5Division of Epigenomics and Cancer Risk Factors,
German Cancer Research Center (DKFZ), Heidelberg, Germany
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 land-
scape 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). Overex-
pression 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 asso-
ciated 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.
Summary/Conclusions: 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.
S430
METTL3 CONTROLS TRANSLATION OF TARGET MRNAS BY N6
METHYLATION OF ADENOSINE RESIDUES IN THEIR CODING SEQUENCE
AND CONSTITUTES A NOVEL THERAPEUTIC VULNERABILITY OF ACUTE
MYELOID LEUKAEMIA
K. Tzelepis1,*, I. Barbieri2, L. Pandolfini2, J. Shi3, S.C. Robson2, V. Migliori2,
A.J. Bannister2, N. Han2, E. De Braekeleer4, H. Ponstingl5, C.R. Vakoc6,
G.S. Vassiliou1, T. Kouzarides2
1Haematological Cancer Genetics, Wellcome Trust Sanger Institute, 2Gurdon
Institute, Cambridge, United Kingdom, 3Perelman School of Medicine, Univer-
sity of Pennsylvania, Philadelphia, United States, 4Wellcome Trust Sanger Inti-
tute, 5Wellcome Trust Sanger Institute, Cambridge, United Kingdom, 6Cold
Spring Harbor Laboratory, Cold Spring Harbor, New York, United States
Background: Acute myeloid leukaemia (AML) patient survival remains below
30% and there have been no major new anti-AML therapies for decades.
154 | haematologica | 2017; 102(s2)
22nd Congress of the European Hematology Association


PIWIL4 Acts as a piRNA Binding, Epigenetically Active and Growth Regulatory Protein in Human Acute Myeloid Leukemia

https://authors.library.caltech.edu/79106/1/Bamezai_2017p154.pdf

PIWIL4 Acts as a piRNA Binding, Epigenetically Active and Growth Regulatory Protein in Human Acute Myeloid Leukemia

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