From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Regular Article
MYELOID NEOPLASIA
Interaction of TIF-90 and filamin A in the regulation of rRNA synthesis
in leukemic cells
Le Xuan Truong Nguyen,1 Steven M. Chan,1,2 Tri Duc Ngo,3 Aparna Raval,1 Kyeong Kyu Kim,3 Ravindra Majeti,1,2
and Beverly S. Mitchell1
1
Department of Medicine, Stanford Cancer Institute, and 2Department of Medicine, Institute for Stem Cell Biology and Regenerative Medicine, Stanford
University School of Medicine, Stanford, CA; and 3Department of Molecular Cell Biology, School of Medicine, Sungkyunkwan University, Suwon, Republic of Korea
The transcription initiation factor I (TIF-IA) is an important regulator of the synthesis of
ribosomal RNA (rRNA) through its facilitation of the recruitment of RNA polymerase I (Pol I)
to the ribosomal DNA promoter. Activation of the phosphoinositide 3-kinase (PI3K)/
• Akt/FLNA/TIF-90 signaling
protein kinase B (Akt) pathway, which occurs commonly in acute myelogenous
regulates rRNA synthesis in
acute myelogenous leukemia leukemia, enhances rRNA synthesis through TIF-IA stabilization and phosphorylation.
We have discovered that TIF-IA coexists with a splicing isoform, TIF-90, which is excells.
pressed preferentially in the nucleolus and at higher levels in proliferating and trans• Direct targeting of Akt
formed hematopoietic cells. TIF-90 interacts directly with Pol I to increase rRNA synthesis
has potential therapeutic
as a consequence of Akt activation. Furthermore, TIF-90 binds preferentially to a 90-kDa
applications in acute
cleavage product of the actin binding protein filamin A (FLNA) that inhibits rRNA
myelogenous leukemia
synthesis. Increased expression of TIF-90 overcomes the inhibitory effect of this cleavage
treatment.
product and stimulates rRNA synthesis. Because activated Akt also reduces FLNA
cleavage, these results indicate that activated Akt and TIF-90 function in parallel to
increase rRNA synthesis and, as a consequence, cell proliferation in leukemic cells. These results provide evidence that the direct
targeting of Akt would be an effective therapy in acute leukemias in which Akt is activated. (Blood. 2014;124(4):579-589)
Key Points
Introduction
Alterations in the phosphoinositide 3-kinase (PI3K)/protein kinase
B (Akt) axis are both associated with and causal of malignant transformation in many cancer types including acute myeloid leukemias
(AMLs).1-3 The PI3K/Akt pathway is frequently constitutively
activated in leukemic blasts from AML patients and contributes to
both cell survival and proliferation.4-6 Activation of the target of
rapamycin (TOR) has emerged as a major effector of the PI3K/Akt
pathway’s ability to regulate protein synthesis and as a contributor
to oncogenesis.7-9 In addition to its effects on protein translation, the
mammalian target of rapamycin (mTOR) coordinates the synthesis
of ribosomal proteins and ribosomal RNA (rRNA) to modulate
ribosome biogenesis.10-12 The importance of mTOR activation in
cancer progression has been highlighted by the ability of rapamycin,
a potent and selective inhibitor of mTORC1, to downregulate the
synthesis of rRNA and to inhibit the growth of many tumors.10,13-16
Although there is evidence that activated Akt enhances rRNA
synthesis independent of mTOR,17 the specific mechanisms through
which Akt directly promotes ribosome biogenesis have not yet been
fully elucidated.
The PI3K/Akt/mTOR pathway controls the transcription of rRNA
by polymerase I (Pol I) through transcription initiation factor I
(TIF-IA), the mammalian homolog of yeast Rrn3p.18-20 TIF-IA
interacts with the TATA-binding protein (TBP)-containing factor
TIF-IB/SL1, and both are required to recruit RNA polymerase I (Pol I)
to the ribosomal DNA (rDNA) promoter and generate a productive
transcription initiation complex.21,22 TIF-IA is itself phosphorylated
at multiple sites by a variety of protein kinases with both positive and
negative effects on its ability to initiate transcription at this locus.23-25
It has also been shown that the phosphorylation of TIF-IA by
mTOR enhances rRNA transcription20 and that TIF-IA expression is
essential to maintaining nucleolar architecture and cell viability.26
We have recently shown that Akt regulates TIF-IA activity in part
through the phosphorylation of casein kinase 2 (CK2), enhancing
rRNA synthesis through this mechanism.27 Hence, TIF-IA is a key
intermediate in the regulation of cellular proliferation through the
PI3K/Akt/mTOR signaling network.
We have discovered that TIF-IA coexists with a splicing isoform,
TIF-90, which results from the loss of 30 amino acids encoded
by exon 6 of the gene. TIF-90 is expressed preferentially in the
nucleolus and at higher levels in proliferating and transformed
hematopoietic cells. We therefore interrogated its role as a regulator
of rRNA synthesis. TIF-90 selectively interacts with a 90-kDa
cleavage fragment of the actin binding protein filamin A (FLNA),
which has been shown to have a direct inhibitory effect on rRNA
synthesis.28 Activated Akt inhibits FLNA cleavage, thereby enhancing
rRNA synthesis.29 Based on our studies, we propose a new model
in which TIF-90 functions in concert with Akt to enhance rRNA
synthesis by directly binding with the FLNA cleavage product.
Submitted December 26, 2013; accepted May 2, 2014. Prepublished online as
Blood First Edition paper, May 21, 2014; DOI 10.1182/blood-2013-12-544726.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked “advertisement” in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
There is an Inside Blood Commentary on this article in this issue.
BLOOD, 24 JULY 2014 x VOLUME 124, NUMBER 4
© 2014 by The American Society of Hematology
579
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
580
NGUYEN et al
Methods
Human patient samples
Mononuclear cells were isolated from bone marrow or the peripheral blood
of patients using Stanford Institutional Review Board–approved protocols
by density-gradient centrifugation using Ficoll-Paque Plus (GE Healthcare,
Waukesha, MI). Cells at the interface were removed and washed with
phosphate-buffered saline. Cell pellets were viably frozen at 280°C until use.
All cultures were carried out in defined medium as previously referenced.30
The study was conducted in accordance with the Declaration of Helsinki.
Synthetic siRNA oligonucleotides
The siGENOME SMARTpool for small interfering RNA (siRNA) was purchased from Thermo Scientific (Lafayette, CO). Scrambled control RNA
(SCR) was used as a control. The target sequences for siRNA are shown in the
supplemental Table 1 (available on the Blood Web site).
RNA isolation and qRT-PCR
Total cellular RNA was isolated using the RNAeasy Plus mini kit (Qiagen,
Hilden, Germany). Quantitative reverse-transcription polymerase chain
reaction (qRT-PCR) reactions were performed in triplicate with specific
primers (specific primer sequences in supplemental Table 2). qRT-PCR was
carried out on a 7900T Fast real-time PCR system (Applied Biosystems,
Foster, CA). Glyceraldehyde-3-phosphate dehydrogenase RNA was used as
the internal control, and relative gene expression levels were calculated as
DDCt. The results are presented as the fold increase over control.
ChIP assay
Chromatin immunoprecipitation (ChIP) was performed as described by the
manufacturer (Pierce, Rockford, IL). Precleared chromatin was incubated
overnight by rotation with 4 mg of Pol I antibody or IgG antibody as a negative
control. Inmunoprecipitates were resuspended in 50 mL Tris-EDTA buffer.
Inputs and immunoprecipitated DNA samples were quantified by quantitative polymerase chain reaction (qPCR) on a 7900T Fast real-time PCR
system (Applied Biosystems). Primers are listed in supplemental Table 2.
BLOOD, 24 JULY 2014 x VOLUME 124, NUMBER 4
(supplemental Figure 1B). Primers flanking the entire open reading
frame and the region corresponding to the splice variant (supplemental Figure 1B and supplemental Table 2) confirmed the expression of both variants in HeLa and 293T cells (supplemental
Figure 2A). Loss of bp 253-342 results in an in-frame deletion of
30 amino acids from the mature protein. Western blot analysis using
HeLa and 293T lysate demonstrates 2 bands, the smaller of which
corresponds in size to the variant transcript (Figure 1A, top left). We
conclude that the smaller protein represents a splice variant of TIF-IA
that we term TIF-90.
TIF-90 protein is expressed in the K562 leukemic cell line and in
primary AML cells (Figure 1A, top right). To determine whether the
expression of TIF-90 is a function of proliferation and/or transformation, we compared the expression of TIF-IA and TIF-90 in
sorted CD341 cells obtained from normal bone marrow with that
in leukemic mononuclear cells obtained from patients with AML or
a myeloproliferative disease using RT-PCR and qPCR. Although
both isoforms were present in normal hematopoietic precursors,
the expression of both TIF-FL and TIF-90 mRNA was significantly
higher in the leukemic cells (supplemental Figure 2B-D and
Figure 1B, left 2 panels). Western blot analysis of the corresponding
cell lysates demonstrated that TIF-FL and TIF-90 protein expression
levels are also elevated (Figure 1A, bottom). We then compared
the relative levels of TIF-90 expression before and after stimulation
of normal peripheral blood mononuclear cells with phorbol ester
and ionomycin and found a pronounced increase in the expression
of both TIF-90 and TIF-FL as a result of stimulation, as shown
in Figure 1C. Because TIF-IA functions to recruit Pol I to the
preinitiation complex at the rDNA promoter,31-33 we also determined
the extent of Pol I recruitment to rDNA and the levels of pre-rRNA
synthesis in normal and leukemic cells. Leukemic cells demonstrated
significantly higher levels of Pol I binding to rDNA binding and of
59external transcribed spacer (ETS) pre-rRNA synthesis compared
with normal cells (Figure 1B, right 2 panels; supplemental Figure 2E-F).
We therefore hypothesized that the TIF-90 splice variant might play
an important role in the regulation of rRNA synthesis in AML cells.
RNA labeling and analysis
The cells were washed and incubated in phosphate-free Dulbecco’s modified
Eagle medium (Gibco) supplemented with 10% fetal bovine serum for 2 hours
followed by 1-hour labeling with 0.5 mCi [32P] orthophosphate (Perkin
Elmer). Total RNA was extracted with Trizol (Life Technology) following
the manufacturer’s protocol. Equal amounts of RNA (10 mg) were separated
on a 1.2% 4-morpholinepropanesulfonic acid formaldehyde gel. The gel
was dried and visualized by autoradiography.
Statistical analysis
Where indicated, results were compared using the unpaired Student t test
with values obtained from at least 3 independent experiments. P , .05 was
considered significant.
Results
Expression of the TIF-90 splice variant in normal and
leukemic cells
During an investigation of the expression of TIF-IA messenger
RNA (mRNA) in cancer cell lines, we noted the presence of 2 distinct
TIF-IA RNA transcripts (supplemental Figure 1A), suggesting the
presence of a TIF-IA variant. This transcript lacks bp 253-342 of the
TIF-IA mRNA, a region corresponding to exon 6 of the TIF-IA gene
TIF-90 regulates rRNA transcription
TIF-90 colocalizes with Pol I protein at the site of active rRNA
synthesis (Figure 2A and supplemental Figure 3A). Line scans
through 3-dimensional images show that the intensities of TIF-90
and Pol I immunostaining over the rDNA regions occur at similar
locations throughout the nucleus, whereas TIF-FL does not consistently coincide with the location of Pol I (Figure 2A, right). TIF-90
also colocalizes with upstream binding factor (UBF) protein
(supplemental Figure 3B). These data strongly suggest that TIF-90
preferentially binds to Pol I. A structural model of Saccharomyces
cerevisiae Rrn3 indicates that S145 and S185, corresponding to S138
and S199 of human TIF-IA, are essential for interaction with Pol I.34
These amino acids are conserved in TIF-90 (supplemental Figure 3C).
An in vitro GST pull-down assay and coimmunoprecipitation experiments demonstrate that TIF-90 strongly interacts with Pol I,
whereas TIF-FL does so to a lesser extent (Figure 2B).
To determine the relationship between localization of the TIF
proteins and cell growth, cells were grown in medium with 10%
fetal calf serum, serum starved for 12 hours, and then regrown in
the presence of serum. As shown in supplemental Figure 4A, serum
starvation results in the localization of total TIF-IA protein to the
nucleus, whereas the subsequent addition of serum relocalizes TIF-IA
to the nucleolus, as has been previously reported.27,35 These results
suggest that the localization of TIF to the nucleolus is growth dependent.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 24 JULY 2014 x VOLUME 124, NUMBER 4
TIF-90 REGULATES rRNA SYNTHESIS
581
Figure 1. Expression of full-length TIF-IA protein (TIF-FL) and TIF-90 in normal, leukemic, and proliferating hematopoietic cells. (A) Expression of TIF-FL and the
TIF-90 variant in cancer cell lines and primary leukemic cells (AML, n 5 10) (top) and in representative individual normal bone marrow and leukemic cells (bottom) by western
blot. (B) Quantitative expression of (1) TIF-FL mRNA, (2) TIF-90 mRNA, (3) pre-rRNA synthesis, and (4) Pol I occupancy of the rDNA promoter by qPCR in bone marrow
mononuclear cells from normal individuals and from patients with AML. The bar indicates the average value, and the ends of the whiskers represent minimum and maximum
values. Significance was determined using the Student t test. (C) Expression of TIF-FL and TIF-90 transcripts and protein in normal peripheral blood mononuclear cells from 2
individuals before and 22 hours after stimulation with phorbol ester and ionomycin: RT-PCR (left); western blot (right).
We then examined the relative effects of TIF-FL and TIF-90
overexpression on rRNA synthesis in the presence or absence of
endogenous TIF-IA expression. Given the high rate of rRNA turnover
in cells, the levels of pre-rRNA transcript abundance are a valid
approximation of the overall rate of rRNA transcription.16,36,37 We
also used the incorporation of [32P] into newly synthesized RNA as
an adjunctive assay. Analysis of pre-rRNA abundance using both
59ETS qPCR and RNA labeling demonstrated that overexpression
of TIF-90 enhances rRNA synthesis to a greater extent than does
expression of TIF-FL (supplemental Figure 4B), as it also does in
the context of siRNA-induced depletion of endogenous TIF-IA
(supplemental Figure 4B). ChIP analysis revealed that overexpression of TIF-90 also enhances Pol I binding to rDNA
(supplemental Figure 4C), consistent with the increase of pre-rRNA
synthesis. To further determine the relative contributions of TIF-FL
and TIF-90 to pre-rRNA synthesis in primary AML cells, TIF-FL
and TIF-90 were selectively depleted. As shown in Figure 2C-D,
a greater reduction of pre-rRNA synthesis and of Pol I binding to
rDNA results from the selective knockdown of TIF-90 as opposed to
a similar knockdown of TIF-FL. Similarly, depletion of TIF-90 but
not of TIF-FL strongly reduces pre-rRNA synthesis and Pol I binding
to rDNA in both 293T cells (supplemental Figure 4D-E) and K562
cells (supplemental Figure 4F-G).
FLNA interacts with TIF-90 to inhibit rRNA synthesis
The actin binding protein FLNA is a negative regulator of rRNA
synthesis.28 We confirmed that depletion of FLNA enhances
rRNA synthesis and cell proliferation (supplemental Figure 5A-B),
suggesting that FLNA negatively regulates cell proliferation through
inhibition of rRNA synthesis. The induction of the 90-kDa FLNA
cleavage product reduced Pol I binding to rDNA and decreased
pre-rRNA synthesis, as has been shown by Deng et al.28 Activated
Akt, on the other hand, prevents FLNA cleavage by phosphorylating
it at Ser 2152, thereby retaining it in the cytoplasm.29 Supplemental
Figure 5C showed that overexpression of FLNA inhibits TIF-IA
recruitment to rDNA, raising the possibility that FLNA might be an
important intermediate.
To investigate this hypothesis, we first examined the correlation
between TIF-90 and FLNA cleavage with pre-rRNA synthesis and
cell survival in primary AML cells. Indeed, the presence of the FLNA
cleavage product correlates inversely with Pol I binding to rDNA,
pre-rRNA synthesis level, and cell survival in 21 primary AML cells
(Figure 3A-C). Moreover, the endogenous interaction of TIF-IA and
Pol I is reduced in samples with FLNA cleavage (Figure 3D),
whereas knockdown of FLNA in the high FLNA expression group
increased this interaction (Figure 3D). The reduced interaction
between TIF-IA and Pol I in AML cells with high levels of FLNA
cleavage correlates with low levels of rRNA synthesis and shortened cell survival (Figure 3E). Increasing TIF-IA–Pol I binding by
knocking down FLNA in cells with high FLNA cleavage restored
rRNA synthesis and cell survival (Figure 3E). These data suggest that
the expression of FLNA cleavage product inhibits TIF-90 functions
on pre-rRNA synthesis in AML cells.
TIF-IA binds to FLNA in primary AML cells (Figure 4A, left),
and FLNA interacts more strongly with TIF-90 than with TIF-FL
(Figure 4A, right). To determine whether this interaction is direct
or occurs through a Pol I complex intermediate, we used the Pol I–
depleted lysate of FLNA-transfected cells to carry out a binding assay
with GST-tagged TIF-90 protein. Supplemental Figure 5D shows that
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
582
NGUYEN et al
BLOOD, 24 JULY 2014 x VOLUME 124, NUMBER 4
Figure 2. TIF-90 regulates pre-rRNA synthesis. (A) Colocalization of TIF-FL and TIF-90 with Pol I and rDNA using immunostaining and fluorescence in situ hybridization
assays. The 293T cells were transfected with Myc-TIF-FL or Myc-TIF-90 and costained with anti-Myc and anti-Pol I antibodies (left). rDNA was labeled with an rDNA probe as
described in “Materials and methods” (right). Fluorescence intensity was measured along the line through three-dimensional pictures on the left. (B) Interaction of TIF-FL and
TIF-90 with Pol I. Interaction of glutathione S-transferase (GST)-tagged TIF-FL and TIF-90 with Pol I in cell lysate (left). Coimmunoprecipitation of Pol I and Myc-TIF-FL or
Myc-TIF-90 in transfected 293T cells (right). (C and D) Effects of TIF-FL and TIF-90 expression on rRNA synthesis in primary AML cells. Primary AML cell samples (n 5 10)
were combined and transfected with siSCR or siRNAs specific for TIF-FL or TIF-90 for 24 hours. (C) Levels of pre-rRNA expression relative to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) for each sample as determined by qPCR and RNA synthesis using [32P] labeling. (D) Effect of TIF depletion on rDNA promoter occupancy by Pol I.
ChIP assays were performed as described in “Materials and methods” using anti-Pol I antibody. Values represent the mean 6 standard deviation (SD) of triplicate
determinations (n 5 3) (left). The levels of expression of TIF-IA in corresponding samples in panel C are shown by western blot (right).
in the absence of Pol I, GST-TIF-90 still strongly interacts with FLNA,
suggesting a direct interaction. We then mapped the binding domain
of FLNA using sequentially deleted constructs. Only FLNA-wild type
(WT) and the C-terminal (16-24) domain corresponding to the 90-kDa
fragment interacted with TIF-90 (Figure 4B). Deletion of the N-terminal
actin binding domain had no effect on the interaction, and the actin
binding domain alone did not bind to TIF-90 (Figure 4B). The greater
the binding of TIF-90 to FLNA, the less TIF-90 interacts with Pol I,
whereas decreased FLNA expression increases the interaction of TIF90 with Pol I (supplemental Figure 5E). Overexpression of FLNA
strongly inhibits the TIF-90–Pol I interaction and TIF-90–mediated
binding of Pol I to rDNA, as well as cell proliferation (Figure 4C;
supplemental Figure 5F). In addition, with depletion of FLNA,
a reduction in TIF-90 abolishes Pol I binding to rDNA and also
reduces pre-rRNA synthesis and cell proliferation (supplemental
Figure 5G-H). Thus, FLNA negatively regulates rRNA synthesis
predominantly through TIF-90. Furthermore, only the C-terminal
fragment inhibits the interaction of TIF-90 with Pol I and TIF90–mediated enhancement of rRNA synthesis and cell proliferation
(Figure 4D; supplemental Figure 6A). Both FLNA-WT and the
C-terminal (16-24) fragment also inhibit basal levels of pre-rRNA
synthesis and cell proliferation (supplemental Figure 6B). These
data strongly suggest that the C-terminal 90-kDa fragment of FLNA
regulates rRNA synthesis through its interaction with TIF-90. In
summary, the interaction between the C-terminal FLNA fragment and
TIF-90 prevents the recruitment of Pol I to rDNA through TIF-90 and
inhibits both rRNA synthesis and cell proliferation (supplemental
Figure 6C).
Akt enhances rRNA synthesis through TIF-90
We have recently found that activated Akt directly regulates rRNA
synthesis through TIF-IA.27 We therefore asked the role of activated
Akt in regulation of TIF-90 functions. Overexpression of myristoylated Akt (Akt-Myr) significantly enhanced rRNA synthesis with
both TIF proteins, although to a greater extent in TIF-90 cotransfected
cells (supplemental Figure 7A). Akt-Myr also strongly increased
both TIF-90 binding to Pol I and the recruitment of Pol I to rDNA
(supplemental Figure 7B). Although overexpression of TIF-90
alone does not strongly increase cell proliferation, proliferation
is markedly enhanced by the coexpression of Akt-Myr and
TIF-90 (supplemental Figure 7C) and to a lesser extent by the
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 24 JULY 2014 x VOLUME 124, NUMBER 4
TIF-90 REGULATES rRNA SYNTHESIS
583
Figure 3. Relationship of FLNA cleavage and TIF-90 expression to pre-rRNA synthesis in AML cells. (A) Relative expression of phosphorylated Akt (p-Akt) and FLNA in
primary AML cells. Cell lysate (30 mg) from 4 normal bone marrows and 21 AML samples was separated on sodium dodecyl sulfate gels and immunoblotted with the indicated
antibodies (FL, full length; CP, cleavage product). (B and C) Correlation of FLNA cleavage and TIF-90 expression with rRNA synthesis and cell survival. Twenty-one AML
patient samples were divided into 4 groups based on the expression of TIF-90 and presence of the FLNA cleavage product. Measurement of TIF-90 was performed by qPCR
(supplemental Figure 2D), and measurements of FLNA cleavage were carried out using densitometry on western blots (Figure 3A). Samples are divided into low and high
expression relative to the average value for all samples. The value of 59ETS pre-rRNA in supplemental Figure 2E and Pol I recruited to rDNA promoter in supplemental Figure 2F
for each sample was used for graphic representation. (B) Level of pre-rRNA synthesis and RNA labeling with [32P] was performed with the mixture of AML cells from each
group; level of Pol I recruited to rDNA (C, left). The bar indicates the average value and the ends of the whiskers represent minimum and maximum values. Significance was
determined using the Student t test; 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT) assay (C, right). (D and E) Effects of FLNA cleavage on TIF-IA–Pol I
binding, pre-rRNA synthesis, and cell survival in primary AML cells. Fourteen samples from AML patients were divided into low and high FNLA cleavage expression groups
(each group, n 5 7), and the level of FLNA cleavage was measured based on the densitometry results from panel A. (D) TIF-IA protein was immunoprecipitated and
immunoblotted with anti-Pol I antibody (left); effects of knockdown of FLNA on TIF-IA and Pol I binding in primary AML cells (right). A mixture of AML cells (n 5 7) that have
FLNA cleavage levels above the average value was transfected with siSCR or siFLNA for 36 hours. TIF-IA protein was immunoprecipitated and immunoblotted with anti-Pol I
antibody. (E) The 59ETS pre-rRNA was measured by qPCR (left), ChIP assay (middle), and MTT assay (right).
coexpression of Akt-Myr and TIF-FL (supplemental Figure 7C).
Depletion of endogenous Akt or inhibition of Akt activity disrupted
the enhancement of rRNA synthesis by TIF-90 (supplemental
Figure 7D). These results suggest that the coexpression of TIF-90
and p-Akt are important components of the regulation of rRNA
synthesis and cell proliferation. Indeed, treatment of both K562 cells
and AML cells with the Akt inhibitor AZD8055 decreased pre-rRNA
synthesis and cell proliferation (Figure 5A; supplemental Figure 7E).
Depletion of TIF-90 in Akt-transfected 293T cells strongly reduced
pre-rRNA synthesis and cell proliferation, whereas depletion of
TIF-FL had less effect (supplemental Figure 7F-G). Similar
results were obtained in primary AML cells expressing high levels of
p-Akt (Figure 5B).
Akt activation enhances rRNA synthesis by preventing the
cleavage of FLNA
Western blot and densitometry analysis indicated that the expression
of p-Akt correlates inversely with the expression of the FLNA
cleavage product in 21 AML samples (Figures 3A and 5C). We
therefore examined the relationship between p-Akt expression,
FLNA cleavage, and pre-rRNA synthesis. Inhibition of Akt using
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
584
NGUYEN et al
BLOOD, 24 JULY 2014 x VOLUME 124, NUMBER 4
Figure 4. FLNA interacts with TIF-90 and prevents TIF-90 enhancement of rRNA synthesis. (A) Interaction of FLNA and TIF-IA in primary AML cells (left). AML lysate
(500 mg) combined from 10 samples was used for immunoprecipitation experiments. Cell lysate was immunoprecipitated with anti-IgG or anti-FLNA antibody and
immunoblotted with anti-TIF-IA antibody (left). Interaction of TIF-FL and TIF-90 with FLNA (right). The 293T cells were cotransfected with green fluorescent protein (GFP)-FLNA or
vector control, Myc-TIF-FL, or Myc-TIF-90. FLNA protein was immunoprecipitated with anti-GFP antibody and immunoblotted with anti-Myc antibody. (B) Mapping the
interaction domain of FLNA for TIF-90. Schematic structure of FLNA full length and fragments (left); interaction of GST-tagged TIF-90 with FLNA fragments in cell lysate
(right). (C) Effects of FLNA expression on TIF-FL and TIF-90 enhancement of rRNA synthesis. The 293T cells were cotransfected with Myc-tagged TIF-FL or TIF-90 and
vector control or GFP-FLNA. Myc-TIF protein was immunoprecipitated with anti-Myc antibody and immunoblotted with anti-Pol I antibody (left); 59ETS pre-rRNA and RNA
labeling (middle); MTT and colony-forming assay (right). (D) Effects of FLNA full length and fragments on TIF-90 enhancement of rRNA synthesis. The 293T cells were
cotransfected with Myc-TIF-90 and the indicated FLNA constructs. FLNA protein was immunoprecipitated with anti-GFP antibody and immunoblotted with anti-Myc antibody
(left, top row); TIF-90 protein was immunoprecipitated with anti-Myc antibody and blotted for Pol I binding (left, second row); 59ETS pre-rRNA and RNA labeling (middle); MTT
and colony-forming assays (right).
a PI3K/mTOR inhibitor (LY294002) but not of mTORC1 (rapamycin)
or mitogen-activated protein kinase (PD98059) induced FLNA
cleavage (supplemental Figure 8A, right). The induction of the
FLNA cleavage product reduced Pol I binding to rDNA and
decreased pre-rRNA synthesis, as has been shown by Deng et al28
(supplemental Figure 8A, left and middle). In primary AML cells, the
presence of the cleavage fragment is indicative of decreased Pol I
binding to rDNA and a decrease in both pre-rRNA synthesis and cell
survival (Figure 5C-D). To confirm the effect of Akt activation on
these interactions, we first treated K562 cells with AZD8055, an
inhibitor of mTORC1 and 2. Figure 6E confirms that Akt is inhibited
by this compound, and supplemental Figure 10A shows that the inhibition occurs after 1 hour of exposure and that rRNA synthesis
continues to decrease over 24 hours. Supplemental Figure 8B
demonstrates that AZD8055 induces FLNA cleavage in a dosedependent manner. The decrease in pre-rRNA synthesis and Pol I
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 24 JULY 2014 x VOLUME 124, NUMBER 4
TIF-90 REGULATES rRNA SYNTHESIS
585
Figure 5. Akt regulates rRNA synthesis through TIF-90 and FLNA cleavage. (A) Effects of Akt inhibition on rRNA synthesis and cell proliferation in AML cells. K562 and
a mixture of primary AML cells (n 5 10) were treated with AZD8055 for 3 hours (left). The 59ETS pre-rRNA and RNA labeling were performed as shown. Values for qPCR
represent the mean 6 SD of triplicate determinations (n 5 3). Western blot demonstrating Akt inhibition is shown in supplemental Figure 7E. K562 and a mixture of primary
AML cells (n 5 10) were treated with AZD8055, and cell survival and proliferation were determined by MTT assay and western blot using the proliferation markers proliferating
cell nuclear antigen (PCNA) and antigen Ki-67 (Ki67) (right). (B) Effects of TIF-90 depletion on rRNA synthesis, promoter occupancy, and cell proliferation in primary AML
cells. A mixture of AML cells (n 5 7) with p-Akt expression levels above the mean value (as determined by densitometry on western blot; Figure 3A) was transfected with
siSCR or siTIF-90 for 24 hours. The 59ETS pre-rRNA (left), ChIP assay (middle), and MTT assay and western blot (right). (C and D) Correlation of p-Akt with cleavage of
FLNA, pre-rRNA synthesis, and cell survival in primary AML cells. Twenty-one samples from AML patients were divided into low and high p-Akt expression groups, and the
level of FLNA cleavage was measured based on the densitometry results from Figure 3A. (C) Correlation of p-Akt with FLNA cleavage. (D) Correlation of p-Akt with pre-rRNA
synthesis (left); correlation of p-Akt and Pol I binding to rDNA (middle); MTT assay with a mixture of AML cells (low p-Akt, n 5 9; high p-Akt, n 5 12) (right). (E) Effects of
AZD8055 on FLNA cleavage in the presence of Akt or Akt-Myr. K562 cells were transfected with the indicated Akt constructs or treated with AZD8055 or no drug. Western
blots were performed as indicated (left); 59ETS pre-rRNA was measured by qPCR (middle) and MTT and colony-forming assay (right).
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
586
NGUYEN et al
BLOOD, 24 JULY 2014 x VOLUME 124, NUMBER 4
Figure 6. Effects of TIF-90 expression on FLNA C-terminal (FLNA-C) (16-24) regulated rRNA synthesis. (A) Correlation of FLNA cleavage and TIF-90 expression with
rRNA synthesis. AML samples with high levels of FLNA cleavage as determined by western blot were divided into low and high TIF-90 expression groups. The level of rRNA
synthesis (left) and the recruitment of Pol I to the rDNA promoter (right) were determined for each set of samples. (B) Effects of TIF-90 depletion and FLNA cleavage on rRNA
synthesis and cell survival in primary AML cells. AML samples with high levels of both FLNA cleavage and TIF-90 (n 5 5) were transfected with siSCR or siTIF-90 (20 nM).
The 59ETS pre-rRNA (left); ChIP assay and western blot (middle); MTT assay (right). (C and D) Effects of TIF-90 overexpression on the FLNA-C (16-24)–mediated decrease
in rRNA synthesis, Pol I binding to rDNA, and cell proliferation. K562 cells were transfected with vector control of FLNA-C (16-24) before transfection with vector control or
Myc-TIF-90. (C) The 59ETS pre-rRNA was measured by qPCR (left); ChIP assay with anti-Pol I antibody (right). Values for qPCR represent the mean 6 SD of triplicate
determinations. (D) MTT and colony-forming assay (left); western blot (right). (E and F) Effects of TIF-90 overexpression on AZD8055-mediated decrease in rRNA synthesis,
Pol I binding to rDNA, and cell proliferation. K562 cells were treated with AZD8055 before transfection with vector control or Myc-TIF-90. (E) Western blot with indicated
antibodies. (F) ChIP assay with anti-Pol I antibody (left); 59ETS pre-rRNA was measured by qPCR (middle); MTT and colony-forming assay (right).
promoter occupancy that occurs with AZD8055 treatment correlates
with the induction of FLNA cleavage (supplemental Figure 8C).
AZD8055 treatment also inhibits the proliferation of K562 cells as
well as their ability to form colonies (supplemental Figure 8D-E),
whereas overexpression of Akt-Myr both prevents FLNA cleavage
and enhances pre-rRNA synthesis and cell proliferation (Figure 5E).
AZD8055 also reverses the effect of Akt-WT in preventing FLNA
cleavage but has less effect on Akt-Myr (Figure 5E). Treatment with
2 more specific Akt inhibitors (MK-2206 and Akt inhibitor viii) also
induces FLNA cleavage (supplemental Figure 8F) and decreases
pre-RNA synthesis and cell proliferation in K562 and AML cells
(supplemental Figure 8G-H). These data support the conclusion that
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 24 JULY 2014 x VOLUME 124, NUMBER 4
activation of Akt inhibits FLNA cleavage and thereby enhances prerRNA synthesis and cell proliferation.
Activated Akt promotes rRNA synthesis by preventing the
interaction of TIF-90 with the FLNA cleavage product
Overexpression of constitutively activated Akt-Myr prevents the
inhibition of the TIF-90–Pol I interaction by the FLNA 16-24
fragment and increases Pol I recruitment to rDNA, rRNA synthesis,
and proliferation (supplemental Figure 9A). Co-overexpression of
Akt and TIF-90 in the presence of FLNA 1-15 enhances pre-rRNA
synthesis and cell proliferation, confirming that only the C-terminal
fragment is required for the inhibitory effect (supplemental
Figure 9A). Similar effects of Akt on FLNA proteins and endogenous
TIF-IA were observed in K562 cells (supplemental Figure 9B-C).
Overexpression of both Akt-WT and Akt-Myr enhances TIF-90
binding to Pol I, Pol I–rDNA recruitment, rRNA synthesis, and
proliferation, while preventing FLNA cleavage (supplemental
Figure 9D). Although treatment with AZD8055 disrupted the
downstream effects of Akt-WT, it had only minimal effects on
Akt-Myr (supplemental Figure 9D).
Higher levels of TIF-90 expression are associated with increased
pre-rRNA synthesis in primary AML cells (Figure 6A), whereas
decreased TIF-90 expression is associated with a decrease in
pre-rRNA synthesis and cell survival (Figure 6B). We therefore
asked whether enhanced expression of TIF-90 reverses the
FLNA-mediated repression of rRNA synthesis. Figure 6C-D shows
that overexpression of TIF-90 partially increases Pol I recruitment
to rDNA, rRNA synthesis, and cell proliferation, each of which is
inhibited by FLNA 16-24. Although overexpression of TIF-90 does
not affect the induction of FLNA cleavage by Akt inhibitors, TIF-90
expression does promote Pol I–rDNA binding and increase rRNA
synthesis and proliferation in the presence of Akt inhibition
(Figure 6E-F; supplemental Figure 9E). These data indicate that
higher levels of TIF-90 expression enhance rRNA synthesis by
reversing the inhibitory effects of FLNA 16-24.
Finally, we examined the effects of AZD8055 treatment on 10
AML primary samples. AZD8055 decreased the phosphorylation of
Akt and increased FLNA cleavage (Figure 7A), while inhibiting
pre-rRNA synthesis and cell survival (Figure 7B-C; supplemental
Figure 10B). Similar results were observed with MK-2206 and
Akti-viii inhibitors (supplemental Figure 10C-D). In summary, these
results support the conclusion that the p-Akt/FLNA/TIF-90 signaling
pathway is important in maintaining the viability and growth potential
of AML cells.
Discussion
We have found that TIF-90, a splice variant of TIF-IA, is coexpressed
with TIF-IA and is increased in expression at both the mRNA and
protein levels in proliferating cells and in the cells of patients with
acute leukemia. Exogenously expressed TIF-90 localizes within both
the nucleus and the nucleolus, whereas expression of TIF-FL is
predominantly nuclear. In contrast, when actively growing cells are
stained for endogenous TIF with an antibody that recognizes both
forms, TIF is located predominantly in the nucleolus, shifting to the
nucleus in quiescent cells that have been deprived of serum. To
further validate the role of TIF-90, we demonstrated that exogenous
expression leads to a greater increase in overall levels of 59ETS
pre-rRNA than does expression of TIF-FL in both the presence and
TIF-90 REGULATES rRNA SYNTHESIS
587
absence of endogenous TIF expression. Moreover, specific depletion
of endogenous TIF-90 in primary AML cells results in a significant
decrease in pre-rRNA synthesis and proliferation. From these results,
it is reasonable to conclude that TIF-90 plays a major role in the
regulation of rRNA synthesis in AML cells.
In order to define more specifically the upstream signaling
pathways that affect the activity of TIF-90, we examined the role of
Akt activation. Akt has been shown to regulate ribosome biogenesis
at multiple levels and to interact with both mTORC1 and c-Myc
to stimulate 59ETS pre-rRNA transcription.17 Although a number
of studies had attested to the role of the mTOR pathway as the
major link between nutrient availability, cell growth, and rRNA
synthesis,16,20,38,39 recent work by ourselves and others has revealed
that inhibition of mTORC1 by rapamycin does not ablate rRNA
synthesis, whereas inhibition of Akt results in a much more
pronounced decrease in 59ETS pre-RNA levels.17 Our data support
a central role for activated Akt in markedly enhancing the effects
of TIF-90 on pre-rRNA synthesis. This evidence is based on the
following observations: coexpression of p-Akt and TIF-90 has
a greater effect on rRNA synthesis than does the coexpression of
p-Akt and TIF-FL; the level of p-Akt correlates with TIF-90
expression and the level of pre-rRNA synthesis in primary AML
cells; and selective TIF-90 depletion inhibits rRNA synthesis in
AML cells expressing high levels of p-Akt.
FLNA plays a major intermediary role in this pathway. It
interacts with more than 45 functionally diverse proteins including
nuclear proteins and serves as a scaffold in a number of signaling
networks.40,41 Studies had previously shown that Akt activation
induces FLNA cleavage to generate a 90-kDa C-terminal product.29
These data suggested to us that TIF-90 might interact directly with
FLNA. We therefore determined that FLNA interacts with TIF-90
through its C-terminal domain and that the expression of TIF-90
reverses the inhibition of rRNA synthesis that results from FLNA
cleavage, while depletion of endogenous FLNA enhances the
TIF-90–Pol I interaction. These results support 2 additional
pathways involving TIF-90 that regulate rRNA synthesis: one in
which FLNA-C suppresses rRNA synthesis when TIF-90 expression is limiting and a second pathway through which activated Akt
increases rRNA synthesis by both increasing TIF-90 levels and
inhibiting the cleavage of FLNA to FLNA-C (Figure 7D). This
conclusion is borne out in both in vitro experiments in cell lines
and in vivo correlative observations on primary AML cells
(Figure 6A). TIF-90, and not TIF-FL, is thus the key intermediate
in this pathway.
AML is associated with poor long-term survival, and new
therapeutic approaches are urgently needed. The activation of the
PI3K/Akt pathway is relatively common in AML leukemic cells and
is associated with a poorer outcome.5,6 Recent research has shown
that the compound AZD8055, an inhibitor of both Akt and mTORC1,
may have more efficacy in inhibiting the growth of leukemic cells
in vitro and in vivo than the mTORC1-specific inhibitor rapamycin
and its analogs.42-45 AZD8055 decreases cell proliferation and cell
cycle progression and induces autophagy, resulting in a significant
decrease of AML cell survival without affecting the fate of normal
CD341 hematopoietic progenitors. Treatment with AZD8055 also
markedly represses the growth of AML xenografts in mouse models
at well-tolerated doses.43 We now show that AZD8055 inhibits the
effect of activated Akt by inducing the cleavage of FLNA, thereby
repressing rRNA synthesis in AML cells. Consistent with its inhibition of activated Akt, AZD8055 also significantly decreases
AML cell proliferation and colony formation. These results, together
with our previous findings, provide a comprehensive overview of
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
588
BLOOD, 24 JULY 2014 x VOLUME 124, NUMBER 4
NGUYEN et al
Figure 7. Akt inhibition induces FLNA cleavage and a decrease in rRNA synthesis and cell proliferation in AML cells. (A) Effects of AZD8055 on p-Akt expression and
FLNA cleavage in primary AML cells. Ten AML patient samples were treated with AZD8055 (20 nM) or vehicle for 3 hours, and 30 mg lysate of protein was used for western
blot and immunoblotted with the indicated antibodies. (B and C) Effects of AZD8055 on Pol I binding to rDNA, pre-rRNA synthesis, and cell survival in AML primary cells. Six
AML samples were treated as in panel A. (B) ChIP assay was performed with anti-Pol I antibody. (C) The 59ETS pre-rRNA was measured by qPCR (top); RNA was labeled
with 32P (bottom). Values for qPCR represent the mean 6 SD of triplicate determinations. (D) Schematic model of the regulation of rRNA synthesis by activated Akt as
mediated through both the inhibition of FLNA cleavage and TIF-90 activity.
the mechanisms by which activated Akt directly enhances rRNA
synthesis (Figure 7D). These mechanisms provide support for the
direct targeting of Akt as a therapeutic strategy in AML.
Authorship
Acknowledgments
Contribution: L.X.T.N. and B.S.M. designed the research; L.X.T.N.,
S.M.C., T.D.N., and A.R. performed the research; L.X.T.N., K.K.K.,
R.M., and B.S.M. analyzed the data; and L.X.T.N. and B.S.M. wrote
the manuscript.
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
Correspondence: Beverly S. Mitchell, Lorry Lokey Building,
265 Campus Dr, Suite G2167, Stanford, CA 94305-5456; e-mail:
[email protected].
The authors thank Dr Fumihiko Nakamura for FLNA constructs.
This work was supported by a translational research grant and
by a SCOR award from the Leukemia and Lymphoma Society.
References
1. Martelli AM, Tazzari PL, Evangelisti C, et al.
Targeting the phosphatidylinositol 3-kinase/Akt/
mammalian target of rapamycin module for acute
myelogenous leukemia therapy: from bench to
bedside. Curr Med Chem. 2007;14(19):2009-2023.
2. Polak R, Buitenhuis M. The PI3K/PKB signaling
module as key regulator of hematopoiesis:
implications for therapeutic strategies in leukemia.
Blood. 2012;119(4):911-923.
3. Sujobert P, Bardet V, Cornillet-Lefebvre P, et al.
Essential role for the p110delta isoform in
phosphoinositide 3-kinase activation and cell
proliferation in acute myeloid leukemia. Blood.
2005;106(3):1063-1066.
4. Min YH, Eom JI, Cheong JW, et al. Constitutive
phosphorylation of Akt/PKB protein in acute
myeloid leukemia: its significance as a prognostic
variable. Leukemia. 2003;17(5):995-997.
5. Xu Q, Simpson SE, Scialla TJ, Bagg A, Carroll M.
Survival of acute myeloid leukemia cells requires PI3
kinase activation. Blood. 2003;102(3):972-980.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
BLOOD, 24 JULY 2014 x VOLUME 124, NUMBER 4
6. Martelli AM, Nyåkern M, Tabellini G, et al.
Phosphoinositide 3-kinase/Akt signaling pathway
and its therapeutical implications for human acute
myeloid leukemia. Leukemia. 2006;20(6):
911-928.
7. Sabatini DM, Erdjument-Bromage H, Lui M,
Tempst P, Snyder SH. RAFT1: a mammalian
protein that binds to FKBP12 in a rapamycindependent fashion and is homologous to yeast
TORs. Cell. 1994;78(1):35-43.
8. Jacinto E, Hall MN. Tor signalling in bugs, brain
and brawn. Nat Rev Mol Cell Biol. 2003;4(2):
117-126.
9. Guertin DA, Sabatini DM. An expanding role for
mTOR in cancer. Trends Mol Med. 2005;11(8):
353-361.
10. Hannan KM, Brandenburger Y, Jenkins A, et al.
mTOR-dependent regulation of ribosomal gene
transcription requires S6K1 and is mediated by
phosphorylation of the carboxy-terminal activation
domain of the nucleolar transcription factor UBF.
Mol Cell Biol. 2003;23(23):8862-8877.
TIF-90 REGULATES rRNA SYNTHESIS
TIF-IA links rRNA synthesis to nutrient availability.
Genes Dev. 2004;18(4):423-434.
21. Miller G, Panov KI, Friedrich JK, Trinkle-Mulcahy
L, Lamond AI, Zomerdijk JC. hRRN3 is essential
in the SL1-mediated recruitment of RNA
Polymerase I to rRNA gene promoters. EMBO J.
2001;20(6):1373-1382.
22. Yuan X, Zhao J, Zentgraf H, Hoffmann-Rohrer U,
Grummt I. Multiple interactions between RNA
polymerase I, TIF-IA and TAF(I) subunits regulate
preinitiation complex assembly at the ribosomal
gene promoter. EMBO Rep. 2002;3(11):
1082-1087.
23. Zhao J, Yuan X, Frödin M, Grummt I.
ERK-dependent phosphorylation of the
transcription initiation factor TIF-IA is required for
RNA polymerase I transcription and cell growth.
Mol Cell. 2003;11(2):405-413.
24. Hoppe S, Bierhoff H, Cado I, et al. AMP-activated
protein kinase adapts rRNA synthesis to cellular
energy supply. Proc Natl Acad Sci USA. 2009;
106(42):17781-17786.
11. Mayer C, Grummt I. Ribosome biogenesis and
cell growth: mTOR coordinates transcription by all
three classes of nuclear RNA polymerases.
Oncogene. 2006;25(48):6384-6391.
25. Mayer C, Bierhoff H, Grummt I. The nucleolus as
a stress sensor: JNK2 inactivates the transcription
factor TIF-IA and down-regulates rRNA synthesis.
Genes Dev. 2005;19(8):933-941.
12. Wang X, Proud CG. The mTOR pathway in the
control of protein synthesis. Physiology
(Bethesda). 2006;21:362-369.
26. Yuan X, Zhou Y, Casanova E, et al. Genetic
inactivation of the transcription factor TIF-IA leads
to nucleolar disruption, cell cycle arrest, and
p53-mediated apoptosis. Mol Cell. 2005;19(1):
77-87.
13. Neshat MS, Mellinghoff IK, Tran C, et al.
Enhanced sensitivity of PTEN-deficient tumors to
inhibition of FRAP/mTOR. Proc Natl Acad Sci
USA. 2001;98(18):10314-10319.
14. Sawyers CL. Will mTOR inhibitors make it as
cancer drugs? Cancer Cell. 2003;4(5):343-348.
15. Mahajan PB. Modulation of transcription of rRNA
genes by rapamycin. Int J Immunopharmacol.
1994;16(9):711-721.
16. James MJ, Zomerdijk JC. Phosphatidylinositol
3-kinase and mTOR signaling pathways regulate
RNA polymerase I transcription in response to
IGF-1 and nutrients. J Biol Chem. 2004;279(10):
8911-8918.
17. Chan JC, Hannan KM, Riddell K, et al. AKT
promotes rRNA synthesis and cooperates with
c-MYC to stimulate ribosome biogenesis in
cancer. Sci Signal. 2011;4(188):ra56.
18. Schnapp A, Pfleiderer C, Rosenbauer H, Grummt
I. A growth-dependent transcription initiation
factor (TIF-IA) interacting with RNA polymerase I
regulates mouse ribosomal RNA synthesis.
EMBO J. 1990;9(9):2857-2863.
19. Bodem J, Dobreva G, Hoffmann-Rohrer U, et al.
TIF-IA, the factor mediating growth-dependent
control of ribosomal RNA synthesis, is the
mammalian homolog of yeast Rrn3p. EMBO Rep.
2000;1(2):171-175.
20. Mayer C, Zhao J, Yuan X, Grummt I. mTORdependent activation of the transcription factor
27. Nguyen XT, Mitchell BS. Akt activation enhances
ribosomal RNA synthesis through casein kinase II
and TIF-IA. Proc Natl Acad Sci USA. 2013;
110(51):20681-20686.
28. Deng W, Lopez-Camacho C, Tang JY, et al.
Cytoskeletal protein filamin A is a nucleolar
protein that suppresses ribosomal RNA gene
transcription. Proc Natl Acad Sci USA. 2012;
109(5):1524-1529.
29. Wang Y, Kreisberg JI, Bedolla RG, Mikhailova M,
deVere White RW, Ghosh PM. A 90 kDa fragment
of filamin A promotes Casodex-induced growth
inhibition in Casodex-resistant androgen receptor
positive C4-2 prostate cancer cells. Oncogene.
2007;26(41):6061-6070.
30. Xu Q, Thompson JE, Carroll M. mTOR regulates
cell survival after etoposide treatment in primary
AML cells. Blood. 2005;106(13):4261-4268.
31. Nomura M. Ribosomal RNA genes, RNA
polymerases, nucleolar structures, and synthesis
of rRNA in the yeast Saccharomyces cerevisiae.
Cold Spring Harb Symp Quant Biol. 2001;66:
555-565.
32. Grummt I. Life on a planet of its own: regulation of
RNA polymerase I transcription in the nucleolus.
Genes Dev. 2003;17(14):1691-1702.
33. Drygin D, Rice WG, Grummt I. The RNA
polymerase I transcription machinery: an
589
emerging target for the treatment of cancer. Annu
Rev Pharmacol Toxicol. 2010;50:131-156.
34. Blattner C, Jennebach S, Herzog F, et al.
Molecular basis of Rrn3-regulated RNA
polymerase I initiation and cell growth. Genes
Dev. 2011;25(19):2093-2105.
35. Szymański J, Mayer C, Hoffmann-Rohrer U,
Kalla C, Grummt I, Weiss M. Dynamic subcellular
partitioning of the nucleolar transcription factor
TIF-IA under ribotoxic stress. Biochim Biophys
Acta. 2009;1793(7):1191-1198.
36. Allo SN, McDermott PJ, Carl LL, Morgan HE.
Phorbol ester stimulation of protein kinase C
activity and ribosomal DNA transcription. Role in
hypertrophic growth of cultured cardiomyocytes.
J Biol Chem. 1991;266(32):22003-22009.
37. Stefanovsky VY, Pelletier G, Hannan R, GagnonKugler T, Rothblum LI, Moss T. An immediate
response of ribosomal transcription to growth
factor stimulation in mammals is mediated by
ERK phosphorylation of UBF. Mol Cell. 2001;8(5):
1063-1073.
38. Grewal SS, Evans JR, Edgar BA. Drosophila
TIF-IA is required for ribosome synthesis and cell
growth and is regulated by the TOR pathway.
J Cell Biol. 2007;179(6):1105-1113.
39. Li H, Tsang CK, Watkins M, Bertram PG, Zheng
XF. Nutrient regulates Tor1 nuclear localization
and association with rDNA promoter. Nature.
2006;442(7106):1058-1061.
40. Feng Y, Walsh CA. The many faces of filamin:
a versatile molecular scaffold for cell motility and
signalling. Nat Cell Biol. 2004;6(11):1034-1038.
41. Popowicz GM, Schleicher M, Noegel AA, Holak
TA. Filamins: promiscuous organizers of the
cytoskeleton. Trends Biochem Sci. 2006;31(7):
411-419.
42. Tamburini J, Green AS, Bardet V, et al.
Protein synthesis is resistant to rapamycin and
constitutes a promising therapeutic target in acute
myeloid leukemia. Blood. 2009;114(8):1618-1627.
43. Willems L, Chapuis N, Puissant A, et al. The dual
mTORC1 and mTORC2 inhibitor AZD8055 has
anti-tumor activity in acute myeloid leukemia.
Leukemia. 2012;26(6):1195-1202.
44. Sini P, James D, Chresta C, Guichard S.
Simultaneous inhibition of mTORC1 and
mTORC2 by mTOR kinase inhibitor AZD8055
induces autophagy and cell death in cancer cells.
Autophagy. 2010;6(4):553-554.
45. Chresta CM, Davies BR, Hickson I, et al.
AZD8055 is a potent, selective, and orally
bioavailable ATP-competitive mammalian target
of rapamycin kinase inhibitor with in vitro and in
vivo antitumor activity. Cancer Res. 2010;70(1):
288-298.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2014 124: 579-589
doi:10.1182/blood-2013-12-544726 originally published
online May 21, 2014
Interaction of TIF-90 and filamin A in the regulation of rRNA synthesis in
leukemic cells
Le Xuan Truong Nguyen, Steven M. Chan, Tri Duc Ngo, Aparna Raval, Kyeong Kyu Kim, Ravindra
Majeti and Beverly S. Mitchell
Updated information and services can be found at:
http://www.bloodjournal.org/content/124/4/579.full.html
Articles on similar topics can be found in the following Blood collections
Myeloid Neoplasia (1689 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.