5’UTR point substitutions and N-terminal truncating mutations of ANKRD26 in acute myeloid leukemia
Marconi et al. Journal of Hematology & Oncology
5'UTR point substitutions and N-terminal truncating mutations of ANKRD26 in acute myeloid leukemia
Caterina Marconi 2 3
Ilaria Canobbio 1 2
Valeria Bozzi 0 2
Tommaso Pippucci 2 3
Giorgia Simonetti 2 6
Federica Melazzini 0 2
Silvia Angori 2 3
Giovanni Martinelli 2 6
Giuseppe Saglio 2 5
Mauro Torti 1 2
Ira Pastan 2 4
Marco Seri 2 3
Alessandro Pecci 0 2
0 Department of Internal Medicine, IRCCS Policlinico San Matteo Foundation and University of Pavia , Pavia , Italy
1 Department of Biology
2 Biotechnology, Laboratories of Biochemistry, University of Pavia , Pavia , Italy
3 Medical Genetics Unit, Department of Medical and Surgical Sciences, University of Bologna , Bologna , Italy
4 Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, MD , USA
5 Department of Clinical and Biological Sciences, San Luigi Hospital, University of Turin , Orbassano, Turin , Italy
6 Department of Experimental, Diagnostic and Specialty Medicine, Institute of Hematology “L. and A. Seràgnoli”, University of Bologna , Bologna , Italy
Thrombocytopenia 2 (THC2) is an inherited disorder caused by monoallelic single nucleotide substitutions in the 5'UTR of the ANKRD26 gene. Patients have thrombocytopenia and increased risk of myeloid malignancies, in particular, acute myeloid leukemia (AML). Given the association of variants in the ANKRD26 5'UTR with myeloid neoplasms, we investigated whether, and to what extent, mutations in this region contribute to apparently sporadic AML. To this end, we studied 250 consecutive, non-familial, adult AML patients and screened the first exon of ANKRD26 including the 5'UTR. We found variants in four patients. One patient had the c.−125T>G substitution in the 5'UTR, while three patients carried two different variants in the 5' end of the ANKRD26 coding region (c.3G>A or c.105C>G). Review of medical history showed that the patient carrying the c.−125T>G was actually affected by typical but unrecognized THC2, highlighting that some apparently sporadic AML cases represent the evolution of a well-characterized familial predisposition disorder. As regards the c.3G>A and the c.105C>G, we found that both variants result in the synthesis of N-terminal truncated ANKRD26 isoforms, which are stable and functional in cells, in particular, have a strong ability to activate the MAPK/ERK signaling pathway. Moreover, investigation of one patient with the c.3G>A showed that mutation was associated with strong ANKRD26 overexpression in vivo, which is the proposed mechanism for predisposition to AML in THC2 patients. These data provide evidence that N-terminal ANKRD26 truncating mutations play a potential pathogenetic role in AML. Recognition of AML patients with germline ANKRD26 pathogenetic variants is mandatory for selection of donors for bone marrow transplantation.
ANKRD26 gene; Acute myeloid leukemia; Inherited predisposition to leukemia; Inherited thrombocytopenia
poorly understood. A recent investigation indicated that
thrombocytopenia of THC2 patients is caused by AN
KRD26 overexpression in megakaryocytes due to defective
downregulation by RUNX1 and FLI1, which, in turn,
derives from impaired binding of these transcription factors
to the mutated 5’UTR .
A growing body of evidence indicates that a significant
proportion of apparently sporadic, adult-onset AML cases
originate from a germline predisposition, which often is
not recognized [5, 6]. Given the association of variants in
the ANKRD26 5’UTR with myeloid neoplasms, we
investigated whether, and to what extent, mutations in this
region contribute to apparently sporadic AML. To this
end, we studied 250 consecutive, non-familial, adult AML
patients and screened the first exon of ANKRD26
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including the 5’UTR. Genomic DNA was obtained from
peripheral blood at the time of diagnosis.
We found three different variants in four patients,
whose clinical features are reported in Additional file 1:
Table S1. One patient carried the c.−125T>G
substitution in the 5’UTR that was previously reported as
responsible for THC2 . Review of personal and family
history disclosed that this subject had thrombocytopenia
since childhood, and one sister and her son had
independently received the diagnosis of THC2 due to the
same mutation. We could confirm that the c.−125T>G
had a germinal origin (Additional file 1: Table S1).
Therefore, this AML case represented the evolution of a
typical but unrecognized THC2.
Two patients carried the c.3G>A variant of ANKRD26
that is predicted to cause the loss of the physiologic start
codon (p.Met1?). In both patients, we could analyze the
DNA from different tissues (urinary epithelium, saliva,
and blood collected in complete remission), which
demonstrated the germinal origin of the variant. Finally, one
patient had the c.105C>G substitution resulting in the
generation of a stop codon at position 35 (p.Tyr35*). We
thus investigated the effects of these two variants in the
5’ end of the ANKRD26 coding region.
In patient 2 carrying the c.3G>A, we could obtain RNA
from the whole blood collected at the time of diagnosis.
We found that ANKRD26 mRNA expression was strongly
increased (about ninefold changes) in the patient
compared with healthy controls (Fig. 1), similarly to what was
observed with megakaryocytes and hematopoietic
progenitors of THC2 patients .
Both the c.3G>A and the c.105C>G are predicted to
cause either the complete loss of the ANKRD26 protein or
the synthesis of a shorter isoform starting from an ATG
Fig. 1 ANKRD26 is strongly overexpressed in the peripheral blood of one
AML patient carrying the c.3G>A variant. Real-time PCR on cDNA from
the whole blood showed a significant increase in ANKRD26 expression in
patient 2 with respect to three healthy controls. Data reported
represent the mean of the three independent experiments and
are expressed as mean ± S.E.M. Statistical analysis was performed
by Mann–Whitney non-parametric test
downstream the physiologic start codon. To investigate
this aspect, we cloned the 5’UTR and either the wild-type
(WT) or the mutant ANKRD26 coding sequences in a 3’
FLAG-tagged vector. After transfection in HeLa cells,
ANKRD26 expression was assessed by immunoblotting
using the three different antibodies: an anti-FLAG against
the C-terminal tag, the JA3 antibody against the
Nterminus of human ANKRD26 (residues 1–218), and the
SDI antibody recognizing an ANKRD26 internal epitope
(residues 289–388) (Additional file 1: Methods).
Transfection of WT as well as the mutant constructs resulted in the
expression of proteins recognized by the anti-FLAG
antibody (Fig. 2a). The WT protein migrated at a molecular
mass of about 200 kDa, the predicted mass of full-length
ANKRD26, whereas both mutants migrated at a slightly
lower mass. Interestingly, the WT ANKRD26 was
recognized by all the three antibodies, while the mutant proteins
were detected by the anti-FLAG and the SDI, but not the
JA3 antibody against the N-terminus (Fig. 2a). These
results indicate that the c.3G>A and the c.105C>G have a
very similar effect, resulting in the expression of a slightly
shorter protein compared to WT ANKRD26, with a
truncated N-terminus and a preserved downstream sequence.
This picture is consistent with the translation starting from
a downstream ATG and then proceeding with a correct
and complete reading (Additional file 1: Table S2).
To investigate the stability of the mutant proteins in
cells, we blocked the protein synthesis by adding
cycloheximide to HeLa cultures 24 h after transfection and
measured the kinetics of the subsequent reduction of
the amounts of transfected proteins. These experiments
showed that both variants presented a similar stability as
the WT protein (Additional file 1: Figure S1).
We then investigated whether these mutant proteins
maintain their function. The best known functional activity
of ANKRD26 is the modulation of different kinase
signaling pathways [4, 7], especially the MAPK/ERK pathway.
ANKRD26 regulates ERK phosphorylation in mouse
embryonic fibroblasts . Hyperactivation of ERK in human
megakaryocytes is the mechanism of thrombocytopenia in
THC2 and increased ERK signaling at the level of the
myeloid progenitors could contribute to predisposition to
myeloid malignancies . In HeLa cells, transfection of
either WT or mutant ANKRD26 (but not of the empty
vector) resulted in a marked phosphorylation of ERK, while
it had no effects on some other signal transduction kinases
such as AKT or p38MAPK (Fig. 2a, b). The efficiency of
exogenous ANKRD26 in phosphorylating ERK was
measured as the p-ERK/ERK ratio weighted for FLAG: this
value was 2.7- to 3.3-fold higher for the mutants compared
with WT ANKRD26 (Fig. 2c). We concluded that the
Ntruncated ANKRD26 variants do maintain the ability to
activate the ERK pathway of the WT protein and could be
even more potent ERK activators than the WT ANKRD26.
Fig. 2 The c.3G>A and c.105C>G variant result in the synthesis of N-terminal truncated proteins that maintain the ability to phosphorylate ERK.
ANKRD26-FLAG wild-type (WT) or mutant (c.3G>A or c.105C>G) constructs, or the empty vector, were transfected into HeLa cells. A further control was
performed by avoiding DNA loading during transfection (no DNA). a Cells were lysed 48 h after transfection and an aliquot of 20 μg of protein was
analyzed by immunoblotting. Transfection of both mutant constructs resulted in bands running at a slightly lower molecular mass compared to the
WT band, which were recognized by the anti-FLAG and the SDI antibodies, but not by the JA3 antibody. Moreover, transfection of the WT as well as
the mutant proteins (but not of the empty vector) induced the phosphorylation of ERK. Tubulin was used as loading control. b Transfection of WT or
mutant ANKRD26 had no effects on phosphorylation of signaling kinases AKT or p38-MAPK. A lysate of platelets stimulated by 10 μM TRAP was used
as positive control (ctrl+). c The ability of transfected ANKRD26-FLAG in phosphorylating ERK was measured as the P-ERK/ERK ratio weighted for the
amount of FLAG, as determined by densitometric analysis of the respective bands. This value was significantly higher for both mutants compared to
WT ANKRD26 (***P < 0.001). Data reported represent the mean of three independent experiments and are reported as mean ± S.E.M. Statistical analysis
was performed by Student t test
Interestingly, the c.3G>A and the c.105C>G variants
were not present in an in-house cohort of 510
consecutive control individuals of the same geographic origin
(Additional file 1: Methods) and resulted in a
significantly higher frequency in our cohort of AML patients
in comparison to the non-The Cancer Genome Atlas
subset of the Exome Aggregation Consortium
(exac.broadinstitute.org), with p values of 0.012 and 0.032 for
the c.3G>A and c.105C>G, respectively.
In summary, the analysis of a large case series showed
that variants in the ANKRD26 5’UTR are infrequent among
non-familial AML patients. However, some apparently
sporadic, adult-onset AML cases represent the evolution of
an unrecognized THC2. Identification of these cases is
imperative especially in patients who are candidates for
hematopoietic stem cell (HSC) transplantation from a
family donor, in order to avoid the use of HSC from a donor
affected by the same inherited disorder. In fact, several
reports indicate that the use of HSC from donors with
germline mutations predisposing to hematological
malignancies resulted in the development of donor-derived
leukemia in the recipient and/or poor transplant
engraftment [5, 8–10]. Of note, the sister of patient 1 with THC2
developed chronic myelomonocytic leukemia 2 years after
the onset of AML in the proband.
Moreover, we observed that mutations in the ANKRD26
coding sequence resulting in the truncation of the protein
N-terminus also have a regulatory effect, causing ANKRD26
overexpression and thus playing a potential role in AML. In
fact, ANKRD26 overexpression is the proposed
pathogenetic mechanism for both thrombocytopenia and
predisposition to AML in THC2 patients . Since none of the
patients 2–4 presented thrombocytopenia before AML, we
suggest that, unlike THC2 mutations, the coding variants
described here induce ANKRD26 overexpression though a
mechanism independent of RUNX1/FLI1 interaction with
the 5’UTR of the gene and possibly due to increased mRNA
stability. In this way, the transcription factors are still able
to bind the 5’UTR and downregulate ANKRD26 in
megakaryocytes, thus avoiding thrombocytopenia. Whatever the
mechanisms of ANKRD26 upregulation, we showed
that these N-truncated isoforms are stable in cells and
have a strong ability to activate the MAPK/ERK
pathway. Although further investigation is required, the
present data strongly suggest that N-terminal
truncating mutations of ANKRD26 have a potential
pathogenetic role in apparently sporadic AML. Since our
investigation was restricted to non-familial AML cases,
prevalence of ANKRD26 pathogenetic variants in AML
could be greater than we found.
Additional file 1: Table S1. Main clinical and laboratory features of the
AML patients with ANKRD26 mutations identified by the present investigation.
Table S2. Prediction of translation start codon presence in the ANKRD26
coding sequence and relative protein size. Figure S1. The stability of
ANKRD26 mutant proteins is similar to that of the WT counterpart. HeLa cells
were transfected by ANKRD26-FLAG WT or mutant constructs and cultured in a
12-well plate. Protein synthesis was blocked 24 h after transfection by addition
to the cell culture of cycloheximide 100 mM diluted in DMSO. Control
conditions were carried out by adding the same amount of DMSO alone. Cells were
then lysed just before the addition of cycloheximide or DMSO (time 0) and 8,
24, and 48 h after the addition of cycloheximide or DMSO and analyzed by
immunoblotting with anti-FLAG and anti-tubulin antibodies. The histograms
show the amount of proteins expressed as FLAG/tubulin ratio and referred to
time 0 of each condition. After the addition of cycloheximide, WT ANKRD26
expression decreased to about 60% at 8 h, to 45% at 24 h, and to 20% at
48 h. The expression was significantly lower after cycloheximide treatment
compared with DMSO alone at each time point, indicating that protein
synthesis was efficiently blocked by cycloheximide (***P < 0.001; **P < 0.01; *P
< 0.05). Overall, mutant and WT proteins showed a similar kinetic of reduction
after cycloheximide treatment. Data reported represent the mean of three
independent experiments and are reported as mean ± S.E.M. Statistical analysis
was performed by Student t test. Methods. (DOCX 200 kb)
This work was supported in part by grants from the Telethon Foundation,
Italy (GGP10089), the Cariplo Foundation, Italy (2012–0529, 2010–0807), the
Italian Ministry of Health (RF-2010-2310098), and the Intramural Research
Program of the NIH, National Cancer Institute, Center for Cancer Research.
Availability of data and materials
All data generated and analyzed during the current study are included in the
submitted article and its supplementary information file. Materials as well as
additional information are available from the corresponding author on
CM, MS, and AP designed the research, interpreted the data, and wrote the
manuscript. GSi, GM, and GSa designed the research, enrolled the patients,
and interpreted the data. MT and IP designed the research and interpreted
the data. IC, VB, TP, FM, and SA performed the experiments and interpreted
the data. All the authors critically revised the manuscript and approved the
Ethics approval and consent to participate
The study was approved by the Institutional Review Board of the Institute of
Hematology “L. and A. Seràgnoli”, University of Bologna, Bologna, Italy, and the
Institutional Review Board of the San Luigi Hospital, University of Turin, Orbassano,
Turin, Italy—the two institutions that enrolled the patients. All patients provided
written informed consent in accordance with the Declaration of Helsinki.
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