Key Points

  • A novel RUNX1 mutation that precludes its methylation has been found in a familial AML pedigree.

  • Loss of RUNX1 methylation in HSCs confers resistance to apoptosis, a hallmark of a preleukemic clone.

Abstract

RUNX1 is among the most frequently mutated genes in human leukemia, and the loss or dominant-negative suppression of RUNX1 function is found in myelodysplastic syndrome and acute myeloid leukemia (AML). How posttranslational modifications (PTMs) of RUNX1 affect its in vivo function, however, and whether PTM dysregulation of RUNX1 can cause leukemia are largely unknown. We performed targeted deep sequencing on a family with 3 occurrences of AML and identified a novel RUNX1 mutation, R237K. The mutated R237 residue is a methylation site by protein arginine methyltransferase 1, and loss of methylation reportedly impairs the transcriptional activity of RUNX1 in vitro. To explore the biologic significance of RUNX1 methylation in vivo, we used RUNX1 R233K/R237K double-mutant mice, in which 2 arginine-to-lysine mutations precluded RUNX1 methylation. Genetic ablation of RUNX1 methylation led to loss of quiescence and expansion of hematopoietic stem cells (HSCs), and it changed the genomic and epigenomic signatures of phenotypic HSCs to a poised progenitor state. Furthermore, loss of RUNX1 R233/R237 methylation suppressed endoplasmic reticulum stress–induced unfolded protein response genes, including Atf4, Ddit3, and Gadd34; the radiation-induced p53 downstream genes Bbc3, Pmaip1, and Cdkn1a; and subsequent apoptosis in HSCs. Mechanistically, activating transcription factor 4 was identified as a direct transcriptional target of RUNX1. Collectively, defects in RUNX1 methylation in HSCs confer resistance to apoptosis and survival advantage under stress conditions, a hallmark of a preleukemic clone that may predispose affected individuals to leukemia. Our study will lead to a better understanding of how dysregulation of PTMs can contribute to leukemogenesis.

REFERENCES

REFERENCES
1.
Ito
Y
,
Bae
SC
,
Chuang
LS
.
The RUNX family: developmental regulators in cancer
.
Nat Rev Cancer
.
2015
;
15
(
2
):
81
-
95
.
2.
Sood
R
,
Kamikubo
Y
,
Liu
P
.
Role of RUNX1 in hematological malignancies [published correction appears in Blood. 2018;131(3):373]
.
Blood
.
2017
;
129
(
15
):
2070
-
2082
.
3.
Christen
F
,
Hoyer
K
,
Yoshida
K
, et al
.
Genomic landscape and clonal evolution of acute myeloid leukemia with t(8;21): an international study on 331 patients
.
Blood
.
2019
;
133
(
10
):
1140
-
1151
.
4.
Duployez
N
,
Marceau-Renaut
A
,
Boissel
N
, et al
.
Comprehensive mutational profiling of core binding factor acute myeloid leukemia
.
Blood
.
2016
;
127
(
20
):
2451
-
2459
.
5.
Forbes
SA
,
Beare
D
,
Boutselakis
H
, et al
.
COSMIC: somatic cancer genetics at high-resolution
.
Nucleic Acids Res
.
2017
;
45
(
D1
):
D777
-
D783
.
6.
Koh
CP
,
Wang
CQ
,
Ng
CE
, et al
.
RUNX1 meets MLL: epigenetic regulation of hematopoiesis by two leukemia genes
.
Leukemia
.
2013
;
27
(
9
):
1793
-
1802
.
7.
Metzeler
KH
,
Herold
T
,
Rothenberg-Thurley
M
, et al;
AMLCG Study Group
.
Spectrum and prognostic relevance of driver gene mutations in acute myeloid leukemia
.
Blood
.
2016
;
128
(
5
):
686
-
698
.
8.
Blanc
RS
,
Richard
S
.
Arginine methylation: the coming of age
.
Mol Cell
.
2017
;
65
(
1
):
8
-
24
.
9.
Goyama
S
,
Huang
G
,
Kurokawa
M
,
Mulloy
JC
.
Posttranslational modifications of RUNX1 as potential anticancer targets
.
Oncogene
.
2015
;
34
(
27
):
3483
-
3492
.
10.
Yoshimi
M
,
Goyama
S
,
Kawazu
M
, et al
.
Multiple phosphorylation sites are important for RUNX1 activity in early hematopoiesis and T-cell differentiation
.
Eur J Immunol
.
2012
;
42
(
4
):
1044
-
1050
.
11.
Zhao
X
,
Jankovic
V
,
Gural
A
, et al
.
Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity
.
Genes Dev
.
2008
;
22
(
5
):
640
-
653
.
12.
Tachibana
M
,
Tezuka
C
,
Muroi
S
, et al
.
Phosphorylation of Runx1 at Ser249, Ser266, and Ser276 is dispensable for bone marrow hematopoiesis and thymocyte differentiation
.
Biochem Biophys Res Commun
.
2008
;
368
(
3
):
536
-
542
.
13.
Mizutani
S
,
Yoshida
T
,
Zhao
X
,
Nimer
SD
,
Taniwaki
M
,
Okuda
T
.
Loss of RUNX1/AML1 arginine-methylation impairs peripheral T cell homeostasis
.
Br J Haematol
.
2015
;
170
(
6
):
859
-
873
.
14.
Nakamura-Ishizu
A
,
Matsumura
T
,
Stumpf
PS
, et al
.
Thrombopoietin metabolically primes hematopoietic stem cells to megakaryocyte-lineage differentiation
.
Cell Rep
.
2018
;
25
(
7
):
1772
-
1785.e1776
.
15.
Sherry
ST
,
Ward
MH
,
Kholodov
M
, et al
.
dbSNP: the NCBI database of genetic variation
.
Nucleic Acids Res
.
2001
;
29
(
1
):
308
-
311
.
16.
Abecasis
GR
,
Auton
A
,
Brooks
LD
, et al;
1000 Genomes Project Consortium
.
An integrated map of genetic variation from 1,092 human genomes
.
Nature
.
2012
;
491
(
7422
):
56
-
65
.
17.
Cai
X
,
Gao
L
,
Teng
L
, et al
.
Runx1 deficiency decreases ribosome biogenesis and confers stress resistance to hematopoietic stem and progenitor cells
.
Cell Stem Cell
.
2015
;
17
(
2
):
165
-
177
.
18.
Cabezas-Wallscheid
N
,
Klimmeck
D
,
Hansson
J
, et al
.
Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis
.
Cell Stem Cell
.
2014
;
15
(
4
):
507
-
522
.
19.
Nakamura
Y
,
Arai
F
,
Iwasaki
H
, et al
.
Isolation and characterization of endosteal niche cell populations that regulate hematopoietic stem cells
.
Blood
.
2010
;
116
(
9
):
1422
-
1432
.
20.
Chitteti
BR
,
Kobayashi
M
,
Cheng
Y
, et al
.
CD166 regulates human and murine hematopoietic stem cells and the hematopoietic niche
.
Blood
.
2014
;
124
(
4
):
519
-
529
.
21.
Shoulars
K
,
Noldner
P
,
Troy
JD
, et al
.
Development and validation of a rapid, aldehyde dehydrogenase bright-based cord blood potency assay
.
Blood
.
2016
;
127
(
19
):
2346
-
2354
.
22.
Santaguida
M
,
Schepers
K
,
King
B
, et al
.
JunB protects against myeloid malignancies by limiting hematopoietic stem cell proliferation and differentiation without affecting self-renewal
.
Cancer Cell
.
2009
;
15
(
4
):
341
-
352
.
23.
Liu
Y
,
Elf
SE
,
Miyata
Y
, et al
.
p53 regulates hematopoietic stem cell quiescence
.
Cell Stem Cell
.
2009
;
4
(
1
):
37
-
48
.
24.
Hanahan
D
,
Weinberg
RA
.
Hallmarks of cancer: the next generation
.
Cell
.
2011
;
144
(
5
):
646
-
674
.
25.
Goodell
MA
,
Rando
TA
.
Stem cells and healthy aging
.
Science
.
2015
;
350
(
6265
):
1199
-
1204
.
26.
Koeffler
HP
,
Leong
G
.
Preleukemia: one name, many meanings
.
Leukemia
.
2017
;
31
(
3
):
534
-
542
.
27.
Hsu
JI
,
Dayaram
T
,
Tovy
A
, et al
.
PPM1D mutations drive clonal hematopoiesis in response to cytotoxic chemotherapy
.
Cell Stem Cell
.
2018
;
23
(
5
):
700
-
713.e6
.
28.
Matsumura
T
,
Nakamura-Ishizu
A
,
Takaoka
K
, et al
.
TUBB1 dysfunction in inherited thrombocytopenia causes genome instability
.
Br J Haematol
.
2019
;
185
(
5
):
888
-
902
.
29.
Hetz
C
,
Papa
FR
.
The unfolded protein response and cell fate control
.
Mol Cell
.
2018
;
69
(
2
):
169
-
181
.
30.
Beck
D
,
Thoms
JA
,
Perera
D
, et al
.
Genome-wide analysis of transcriptional regulators in human HSPCs reveals a densely interconnected network of coding and noncoding genes
.
Blood
.
2013
;
122
(
14
):
e12
-
e22
.
31.
Wu
JQ
,
Seay
M
,
Schulz
VP
, et al
.
Tcf7 is an important regulator of the switch of self-renewal and differentiation in a multipotential hematopoietic cell line
.
PLoS Genet
.
2012
;
8
(
3
):
e1002565
.
32.
Kohlmann
A
,
Grossmann
V
,
Klein
HU
, et al
.
Next-generation sequencing technology reveals a characteristic pattern of molecular mutations in 72.8% of chronic myelomonocytic leukemia by detecting frequent alterations in TET2, CBL, RAS, and RUNX1
.
J Clin Oncol
.
2010
;
28
(
24
):
3858
-
3865
.
33.
McNerney
ME
,
Brown
CD
,
Peterson
AL
, et al
.
The spectrum of somatic mutations in high-risk acute myeloid leukaemia with -7/del(7q)
.
Br J Haematol
.
2014
;
166
(
4
):
550
-
556
.
34.
Luo
X
,
Feurstein
S
,
Mohan
S
, et al
.
ClinGen Myeloid Malignancy Variant Curation Expert Panel recommendations for germline RUNX1 variants
.
Blood Adv
.
2019
;
3
(
20
):
2962
-
2979
.
35.
Goyama
S
,
Yamaguchi
Y
,
Imai
Y
, et al
.
The transcriptionally active form of AML1 is required for hematopoietic rescue of the AML1-deficient embryonic para-aortic splanchnopleural (P-Sp) region
.
Blood
.
2004
;
104
(
12
):
3558
-
3564
.
36.
Lutterbach
B
,
Westendorf
JJ
,
Linggi
B
,
Isaac
S
,
Seto
E
,
Hiebert
SW
.
A mechanism of repression by acute myeloid leukemia-1, the target of multiple chromosomal translocations in acute leukemia
.
J Biol Chem
.
2000
;
275
(
1
):
651
-
656
.
37.
Ichikawa
M
,
Asai
T
,
Saito
T
, et al
.
AML-1 is required for megakaryocytic maturation and lymphocytic differentiation, but not for maintenance of hematopoietic stem cells in adult hematopoiesis [published correction appears in Nat Med. 2005;11(1):102]
.
Nat Med
.
2004
;
10
(
3
):
299
-
304
.
38.
Cai
X
,
Gaudet
JJ
,
Mangan
JK
, et al
.
Runx1 loss minimally impacts long-term hematopoietic stem cells
.
PLoS One
.
2011
;
6
(
12
):
e28430
.
39.
Jacob
B
,
Osato
M
,
Yamashita
N
, et al
.
Stem cell exhaustion due to Runx1 deficiency is prevented by Evi5 activation in leukemogenesis
.
Blood
.
2010
;
115
(
8
):
1610
-
1620
.
40.
Growney
JD
,
Shigematsu
H
,
Li
Z
, et al
.
Loss of Runx1 perturbs adult hematopoiesis and is associated with a myeloproliferative phenotype
.
Blood
.
2005
;
106
(
2
):
494
-
504
.
41.
Chen
MJ
,
Yokomizo
T
,
Zeigler
BM
,
Dzierzak
E
,
Speck
NA
.
Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter
.
Nature
.
2009
;
457
(
7231
):
887
-
891
.
42.
van Galen
P
,
Kreso
A
,
Mbong
N
, et al
.
The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress
.
Nature
.
2014
;
510
(
7504
):
268
-
272
.
43.
Tsuru
A
,
Imai
Y
,
Saito
M
,
Kohno
K
.
Novel mechanism of enhancing IRE1α-XBP1 signalling via the PERK-ATF4 pathway
.
Sci Rep
.
2016
;
6
(
1
):
24217
.
44.
Han
J
,
Back
SH
,
Hur
J
, et al
.
ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death
.
Nat Cell Biol
.
2013
;
15
(
5
):
481
-
490
.
45.
Teske
BF
,
Wek
SA
,
Bunpo
P
, et al
.
The eIF2 kinase PERK and the integrated stress response facilitate activation of ATF6 during endoplasmic reticulum stress
.
Mol Biol Cell
.
2011
;
22
(
22
):
4390
-
4405
.
46.
Rücker
FG
,
Schlenk
RF
,
Bullinger
L
, et al
.
TP53 alterations in acute myeloid leukemia with complex karyotype correlate with specific copy number alterations, monosomal karyotype, and dismal outcome
.
Blood
.
2012
;
119
(
9
):
2114
-
2121
.
47.
Sashida
G
,
Harada
H
,
Matsui
H
, et al
.
Ezh2 loss promotes development of myelodysplastic syndrome but attenuates its predisposition to leukaemic transformation
.
Nat Commun
.
2014
;
5
(
1
):
4177
.
48.
Booth
CAG
,
Barkas
N
,
Neo
WH
, et al
.
Ezh2 and Runx1 mutations collaborate to initiate lympho-myeloid leukemia in early thymic progenitors
.
Cancer Cell
.
2018
;
33
(
2
):
274
-
291.e8
.
49.
An
N
,
Khan
S
,
Imgruet
MK
, et al
.
Gene dosage effect of CUX1 in a murine model disrupts HSC homeostasis and controls the severity and mortality of MDS
.
Blood
.
2018
;
131
(
24
):
2682
-
2697
.
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