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TRIB3 Stabilizes High TWIST1 Expression to Promote
Rapid APL Progression and ATRA Resistance
Jian Lin1, 3
, Wu Zhang1, 3,
*, Li-Ting Niu1, Yong-Mei Zhu
1, Xiang-Qin Weng
1, Yan
Sheng1, Jiang Zhu
1 and Jie Xu
1, 2, *
1State Key Laboratory for Medical Genomics, Shanghai Institute of Hematology,
National Research Center for Translational Medicine, Rui-Jin Hospital, Shanghai
Jiao-Tong University School of Medicine and School of Life Sciences and
Biotechnology, Shanghai Jiao-Tong University, 197 Ruijin Er Road, Shanghai
200025, China. 2Translational Medicine Ward, Department of Hematology, Rui-Jin
Hospital, Shanghai 200025, China. 3These authors contributed equally to this work.
*Correspondence: Jie Xu, State Key Laboratory for Medical Genomics and Shanghai
Institute of Hematology Rui-Jin Hospital, affiliated to Shanghai Jiao-Tong University
School of Medicine, Shanghai, 200025, China. E-mail: [email protected]; and
Wu Zhang, State Key Laboratory for Medical Genomics and Shanghai Institute of
Hematology Rui-Jin Hospital, affiliated to Shanghai Jiao-Tong University School of
Medicine, Shanghai, 200025, China. E-mail: [email protected];
Running title: TRIB3 stabilizes TWIST1 to promote APL progression
Key words: Acute promyelocytic leukemia; TWIST1; TRIB3; Early death;
ATRA-resistance
Conflict of interest statement: The authors declare no potential conflicts of interest.
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Abstract
Purpose: The resistance to differentiation therapy and early death caused by fatal
bleeding endangers the health of a significant proportion of APL patients. This study
aims to investigate the molecular mechanisms of ATRA resistance and uncover new
potential therapeutic strategies to block the rapid progression of early death.
Experimental design: The important role of TWIST1 in APL leukemogenesis was
first determined by gain- and loss-of-function assays. We then performed in vivo and
in vitro experiments to explore the interaction of TWIST1 and TRIB3 and develop a
potential peptide-initiated therapeutic opportunity to protect against early death and
induction therapy resistance in patients with APL.
Results: We found that the epithelial-mesenchymal transition (EMT)-inducing
transcription factor TWIST1 is highly expressed in APL cells and is critical for
leukemic cell survival. TWIST1 and TRIB3 were highly co-expressed in APL cells
compared with other subtypes of acute myeloid leukemia (AML) cells. We
subsequently demonstrated that TRIB3 could bind to the WR domain of TWIST1 and
contribute to its stabilization by inhibiting its ubiquitination. TRIB3 depletion
promoting TWIST1 degradation reverses resistance to induction therapy and
improves sensitivity to ATRA. Based on a detailed functional screen of synthetic
peptides, we discovered a peptide analogous to the TWIST1 WR domain that
specifically represses APL cell survival by disrupting the TRIB3/TWIST1 interaction.
Conclusions: Our data not only define the essential role of TWIST1 as an EMT-TF in
APL patients but also suggest that disrupting the TRIB3/TWIST1 interaction reverses
induction therapy resistance and blocks rapid progression of APL early death.
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Translational Relevance
This study was designed to improve the treatment for high-risk APL patients. This
proportion of APL patients continued to suffer from early fatal bleeding and leukemic
extramedullary infiltration, which remains the most important challenge and the
largest obstacle to curing all APL patients. We demonstrate that the unique
EMT-inducing transcription factor TWIST1 is significantly highly expressed in APL
and governs the survival of APL cells. We show that TRIB3 interacts with TWIST1
and stabilizes high TWIST1 expression by repressing TWIST1 ubiquitination. Our
data also suggest that a peptide similar to the WR domain disturbs the
TRIB3/TWIST1 interaction, impairs rapid progression during the early death of APL
and reverses resistance to ATRA therapy. These results reveal the important role of a
specific oncogenic transcriptional factor, TWIST1, in APL leukemogenesis and
suggest a potential peptide-initiated therapeutic opportunity to protect against early
death and induction therapy resistance in patients with APL.
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Introduction
Acute promyelocytic leukemia (APL), which accounts for 10-15% of AML cases, is
characterized by the t(15; 17) chromosomal translocation and is now highly curable
by the combination of granulocytic differentiation induction and the PML-RARα
oncoprotein-targeted agents all-trans retinoic acid (ATRA) and arsenic trioxide (ATO)
(1,2). Despite the striking molecular complete remission (CR) and the very few cases
of relapse associated with ATRA/ATO-based regimens, mortality events typically
result from early fatal bleeding, which remains the most important challenge and the
largest obstacle to curing all APL patients (3). For instance, several studies have
reported that the risk of early hemorrhagic death (HD) reaches an incidence of 10-20%
during the first month of induction (4-6). Importantly, APL patients with a high white
blood cell count face an increased risk of early HD (7). Furthermore, resistance to
ATRA/ATO treatment with PML-RARα mutations still remains a therapeutic
challenge for a significant proportion of APL patients. Thus, we need to better
understand the molecular mechanism of APL pathogenesis and design more effective
therapeutic strategies to block the rapid progression of early death and overcome
resistance.
Oncogenic transcription factors play an important role in the development of
hematological malignancy (8,9). The dysregulation of epithelial-mesenchymal
transition-inducing transcription factors (EMT-TFs), including SNAI1/SNAI2,
ZEB1/ZEB2 and TWIST1/TWIST2, has also been explored within the context of the
aggressive invasion, chemoresistance and poor prognosis of acute myeloid leukemia
(AML) (10-12). TWIST1, a highly conserved basic helix-loop-helix (bHLH) protein,
is a well-characterized EMT-TF that plays a critical role in embryonic development
and cancer metastasis (13,14). Recent studies have shown that TWIST1 is
overexpressed in primary AML samples (15). Our previous study reported that high
expression of TWIST1 in AML contributes to extramedullary infiltration and
promotes leukemic aggressiveness (16). These findings strongly suggest that
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disruption of TWIST1 is involved in leukemogenesis. However, the prognosis of
AML patients with high TWIST1 expression is controversial based on several reports
(15,17). These conflicting reports likely occurred because TWIST1 expression in
AML is heterogeneous (18). TWIST1 expression is higher in APL, an intriguing
AML subtype in which successful clinical tumor differentiation-induction therapy has
been applied and for which the clinical phenotype is inconsistent with the expression
level of TWIST1 (15,18).
In this study, we demonstrate that the EMT-TF TWIST1 is significantly highly
expressed in APL and governs the survival of APL cells. We show that TRIB3
interacts with TWIST1 and stabilizes high TWIST1 expression by repressing
TWIST1 ubiquitination. Our data also suggest that a peptide similar to the WR
domain disturbs the TRIB3/TWIST1 interaction, impairs rapid progression during the
early death of APL and reverses resistance to ATRA therapy. These results contribute
to a better understanding of the molecular mechanism of APL pathogenesis and will
allow for designing more effective therapeutic strategies to block the rapid
progression of early death and overcome resistance.
Materials and Methods
Cells and mice
Leukemic cell lines were cultured in RPMI-1640 medium (Invitrogen, Grand Island,
USA) supplemented with 10% FBS (Invitrogen, Grand Island, USA), and HEK293T
cells were grown in DMEM (Invitrogen, Grand Island, USA) supplemented with 10%
FBS. All cell lines, including NB4-R1 and PR9 cell line, were obtained from the
Shanghai Institute of Hematology as previously described (19-21). NB4-R1, a de
novo ATRA-resistant cell line isolated from parental NB4 cells, was obtained from Dr.
Michel Lanotte (Hospital Saint Louis, Paris, France) (22). HMRP8-PML-RARα
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transgenic mice were generated on an FVB/NJ background using the human MRP8
expression cassette (23). The reproducible APL transplantation model can be
propagated by injecting APL blasts, isolated from hMRP8-PML-RARα transgenic
mice, into the tail vein of syngeneic recipients (24). All transplanted APL cells were
highly purified by flow sorting to exclude the dying cells, and then injected into the
immunodeficient mice or syngeneic recipients. NB4-luc or R1-luc cells were injected
into sub-lethally irradiated eight-week-old NOD/SCID mice through tail veins. All
animal experiments were conducted in accordance with the approved guidelines
provided by the Laboratory Animal Resource Center of Shanghai Jiao-Tong
University School of Medicine.
Patient samples
Primary AML samples were obtained from the bone marrow of diagnosed AML
patients. Leukemic blasts were purified and harvested in the mononuclear layer via
density gradient centrifugation. Human primary AML samples were mainly obtained
from Shanghai Rui-Jin Hospital with written informed consent from each patient and
research ethics board approval in accordance with the Declaration of Helsinki.
Flow cytometry analysis.
Cells were suspended in FACS buffer containing 1% FBS and 0.1% NaN3. The data
were collected on an LSR-Fortessa X20 flow cytometer (BD, Franklin Lakes, USA).
Antibodies were purchased from BD Biosciences (BD, New Jersey, USA), including
anti-CD11b and Annexin V/PI apoptosis flow kit.
Morphological staining.
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Murine PB or BM cells were cytospun onto slides and stained with Wright-Giemsa
staining solution by following manufacture’s manual. The samples were evaluated
under a light microscope (BX61, Olympus).
β-Gal staining.
Senescence-associated b-galactosidase (SA--Gal) staining was detected with the
senescence detection kit (Abcam, Cambridge, USA). The senescent cells were
quantitated from at least six random fields according to the manufacturers’ protocols.
Colony formation unit (CFU) assay.
To evaluate methylcellulose colony-forming unit (CFU) colony numbers in human or
mouse leukemic cells, highly purified sorted cells were plated in duplicate and
cultured in MethoCult medium (Stem Cell Technologies) in 12-plate dishes. On day
11, CFUs were counted from three independent experiments using the manufacturers’
protocols.
shRNA viral vector construction and delivery.
The TWIST1 or TRIB3 shRNA sequence was converted from a pair of previously
reported shRNA oligos (16,25). To generate cells stably expressing TWIST1-shRNA,
TRIB3-shRNA and the negative control-shRNA (NC), the expression cassettes were
transduced into leukemic cells with lentiviral vectors. siRNA oligonucleotides against
TWIST1 were transfected using Lipofectamine 2000 (Invitrogen, Grand Island, USA).
qRT-PCR analysis.
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Total cellular RNA was extracted with RNeasy micro kit or RNeasy mini kit
(QIAGEN, Valencia, CA) by following the manufacture’s manual. For quantitative
real-time RT-PCR, reactions were performed by using SYBR Premix Ex Taq
(Applied Takara Bio Inc.) on an ABI 7500 Real-Time PCR system. The primer
sequences of reference gene GAPDH were previously described (26).
Western blotting, immunohistochemical staining and immunofluorescence.
The resulting cell pellets were lysed with the whole cell lysis buffer in the boiling
water for at least 10 min. The antibodies used included anti--Actin (Sigma, St. Louis,
MO), anti-PML-RAR (Abcam, Cambridge, USA), anti-TRIB3 (Proteintech,
Chicago, USA) and anti-TWIST1 (Santa Cruz Biotechnology, Santa Cruz, CA).
Proteins signals were visualized using the Immobilon Western kit (Millipore, Billerica,
USA). Murine brain sections and spinal cord slices were prepared for HE staining or
human CD45 (Cell signaling, Danvers, USA) immunohistochemical staining. Cells or
frozen tissue samples were fixed in 4% PFA and then permeabilized in 0.2% Triton
X-100. Murine brain sections and spinal cord slices were prepared for murine c-Kit
(Santa Cruz Biotechnology, Santa Cruz, CA) immunohistochemical staining. Optical
sections of the cells were observed under a Leica TCS SP8 confocal microscope
(Leica Microsystems, Wetzlar, Germany).
Treatment of APL mice.
20 days after inoculation of APL cells into syngeneic mice or NOD/SCID mice,
peptidomimetics (10mg/kg, twice) or ATRA (10mg/kg, once) and ATO (10mg/kg,
once) (Sigma-Aldrich, St. Louis, USA) treatment was started by daily intra-peritoneal
injection for 6 to 10 successive days.
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Bioluminescence imaging (BLI) in vivo.
NB4 cells or APL murine blasts carrying a luciferase reporter were transplanted into
mice. The luciferase substrate was injected into living animals before imaging. Then
BLI was performed by according to the manufacturers’ protocols (Xenogen IVIS
Spectrum, PerkinElmer).
coIP assay.
Cell extracts were prepared with lysis buffer (50 mM pH 7.5 Tris, 150 mM NaCl, 0.5%
Triton X-100, 10% glycerol, 2 mM EDTA, 1 mM PMSF, 20 mM, Protease Inhibitor
Cocktail, Phosphatase Inhibitor Cocktail, and 2 mM DTT). Supernatants were then
incubated with preconjugated anti-FLAG M2 (Sigma), anti-MYC (Biotool), anti-HA
(Biotool), anti-GFP (MBL), or anti-IgG (CST) beads at 4 ℃ overnight. The beads
were sequentially washed 5 times with co-IP lysis buffer. The bound proteins were
eluted with 2% SDS lysis buffer and boiled at 100 ℃ for 15 min. The proteins were
analyzed by western blotting according to the standard protocol.
In vivo ubiquitination assays.
For the in vivo ubiquitination assays, 293T cells were transiently transfected with
plasmids for HA-tagged ubiquitin, Flag-PML-RARα, GFP-TWIST1, Myc-TRIB3 and
other indicated constructs. 24 hours after transfection, cells were treated with 10 μM
MG-132 or DMSO for 24 hours, and then washed with PBS and collected by
centrifugation. Cells were lysed in coIP lysis buffer. The lysate was subjected to co-IP
or IP using anti-HA-conjugated agarose beads for overnight at 4 °C. The samples
were loaded, separated by SDS-PAGE and immunoblotted with the indicated
antibodies as described above.
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Statistical Analysis.
Data are presented as arithmetic means ± SEM. Kaplan-Meier survival analysis,
student’s t tests or tests were used to calculate P values where appropriate. P < 0.05
was considered to be significant.
Results
High TWIST1 expression in patients with APL
To evaluate the expression levels of EMT-TFs in AML, we analyzed microarray data
for different cytogenetic codes from 199 AML samples from the E-MTAB-3444
database (Fig. S1A). Detailed quantitative assessment showed that TWIST1
expression, but not TWIST2, SNAI1/2 and ZEB1/2 expression, was significantly
higher in t (15;17) APL samples than in other cytogenetic types of AML, including t
(8;21), inv (16), MLL-(11q23), t (9;11) and inv (3)/t (3;3) (Fig. 1A and Fig. S1B).
We also examined EMT-TF mRNA expression using the RNA-seq database of The
Cancer Genome Atlas (TCGA) and verified the higher quantity of TWIST1 mRNA in
the M3 subtype of AML according to FAB classification (Fig. 1B and Fig. S1C). To
further confirm the aberrant high expression of TWIST1 in APL, we quantified the
mRNA and protein levels of APL blasts from primary AML patients (Fig. 1C, 1D,
Fig. S1D and Supplementary Table 1). Indeed, higher levels of the TWIST1 mRNA
and protein were found in APL samples than in non-APL samples. Consistent with
the data from human leukemic cells, higher TWIST1 expression was found in
leukemic cells from PML-RARα transgenic mice than in blasts from other AML
mouse models (Fig. 1E). In a series of AML cell lines, the typical APL cell line NB4
presented higher TWIST1 expression than the other cell lines (Fig. S1E). To assess
whether TWIST1 expression was correlated with the expression of the PML-RARα
oncoprotein, we performed ZnSO4-induced PML-RARα expression in PR9 cells. As
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expected, ZnSO4-induced PML-RARα expression upregulated the expression of
TWIST1 in a time-dependent manner, although TWIST1 and PML-RARα were not
completely colocalized (Fig. 1F). Consistent with this finding, TWIST1 was also
correlated with PML-RARα expression in NB4 cells, with partial colocalization (Fig.
S1F). Thus, these results demonstrate that TWIST1, as an EMT-TF, is highly
expressed in APL cells.
High TWIST1 expression promotes APL progression
To assess whether high TWIST1 expression plays an important role in APL
progression, we introduced negative control (NC)-short hairpin RNA (shRNA) or
sh-TWIST1-1/2 into two types of APL cells (NB4 cells and APL murine blasts) and
found that sh-TWIST1-1 caused significant suppression of TWIST1 expression in
both types of cells (Fig. S2A). We observed that sh-TWIST1-1 decreased
PML-RARα expression and induced NB4 cell apoptosis and differentiation but not
senescence (Fig. 2A, 2B and Fig. S2B). The efficacy and specificity of TWIST1
shRNAs were confirmed by rescue via TWIST1 overexpression (Fig. S2C).
Consistent with the findings obtained in the NB4 cell line, sh-TWIST1-1 expression
induced apoptosis and differentiation in leukemic blasts of APL transgenic murine,
which were generated on an FVB/NJ background using the human MRP8 expression
cassette (Fig. S2D-F). To evaluate the role of TWIST1 in APL progression in vivo,
we inoculated TWIST1-knockdown NB4 cells into sublethally irradiated NOD/SCID
mice by tail vein injection. At 3 weeks after transplantation, TWIST1 knockdown
significantly suppressed APL cell invasion and prolonged the survival of recipient
mice (Fig. 2C and D). Similarly, APL transgenic murine blasts expressing
sh-TWIST1-1 generated fewer leukemic cells and had a much longer survival rate
than the NC mice (Fig. S2G-I). Interestingly, in contrast to the NC-shRNA groups, in
which the mice developed a spinning in a circle syndrome or even paralysis, we did
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not observe significant central nervous system (CNS) infiltration of APL cells in the
TWIST1-knockdown groups, indicating that TWIST1 as an EMT-TF may contribute
to APL extramedullary infiltration (Fig. 2C, 2E and Fig. S2G, S2J). As reported
previously, the serious situation associated with APL relapse and poor prognosis
largely predominated in the CNS infiltration (27-29), we showed that downregulation
of TWIST1 in APL cells prevented CNS invasion in mice, though we also observed
skin and eye involvement in some NC-shRNA cases of our APL mouse models (Fig.
S2K). We also examined the effect of TWIST1 depletion on blasts from primary APL
patients and confirmed the importance of TWIST1 in maintaining APL cell survival
(Fig. 2F, 2G and Fig. S2L). Collectively, these data suggest that TWIST1 plays an
important role in APL cell survival and is critical for APL disease progression.
TRIB3 interacts with TWIST1 in APL cells
As mentioned previously, TWIST1 correlated with the expression of the PML-RARα
oncoprotein but was not completely colocalized with PML-RARα. We then
investigated whether there was evidence linking the molecular mechanisms of
PML-RARα-driven APL to an indirect influence of TWIST1. Notably, the activity of
TWIST1 as an EMT-TF seemed to be maintained through a TRIB3/p62-dependent
interaction (30,31). The Tribbles proteins (TRIB1, TRIB2 and TRIB3) have been
shown to play critical roles in leukemogenesis via different mechanisms (32-38).
TRIB3 has been reported to suppress the degradation of PML-RARα through
interacting with PML-RARα and PML (39). Given the importance of TWIST1 and
TRIB3 in APL pathogenesis, we assessed whether TWIST1 and TRIB3 were highly
coexpressed in APL cells compared with other subtypes of AML. We analyzed the
profiling data from the E-MTAB-3444 and TCGA databases and verified high TRIB3
expression in previously studied APL samples (Fig. S3A and B). As expected,
TWIST1 expression was significantly correlated with the expression of TRIB3 in
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APL patient samples (Fig. 3A). Consistent with this finding, TWIST1 and TRIB3
were also highly coexpressed in NB4 cells or ZnSO4-induced PR9 cells (Fig. 3B and
Fig. S3C). As high expression of TWIST1 and TRIB3 in APL cells, we suspected that
TWIST1 might interact with TRIB3 to regulate PML-RARα function. We found that
TWIST1 coimmunoprecipitated with TRIB3 in NB4 cells and APL murine blasts (Fig.
3C and Fig. S3D), and this finding was confirmed in HEK293T cells by coexpressing
various tagged plasmids or performing glutathione S-transferase (GST) pull-down
(Fig. 3D and E). Additionally, using a coimmunostaining assay, we observed that
TWIST1 was strongly colocalized with TRIB3 (Fig. 3F, 3G and Fig. S3E). Taking
into account the previously confirmed the interaction of TRIB3 and PML-RARα (39),
we further investigated the relationship among these three proteins. We showed the
co-localization of TWIST1, TRIB3 and PML-RARα in both NB4 and
plasmid-coexpressing HEK293T cells (Fig. S3F). According to Flag-PML-RARα
immunoprecipitation of APL murine blasts and plasmid-coexpressing HEK293T cells,
TRIB3, but not TWIST1, bound directly to PML-RARα (Fig. S3G and H). These
results suggest that TWIST1 interacts with TRIB3 to indirectly modulate
PML-RARα.
TRIB3 protects TWIST1 from ubiquitination and stabilizes high TWIST1
expression
Human TWIST1 is an 18-kDa protein that contains 202 amino acids, which is much
smaller than the PML-RARα fusion oncoprotein. To determine whether TWIST1
protein has a short half-life compared to PML-RARα oncoprotein, we performed the
cycloheximide (CHX) chase assay with APL cells. Using the translational inhibitor
CHX, we observed that APL cells accumulated endogenous TWIST1 and TRIB3 but
not the corresponding mRNA after treatment with the proteasome inhibitor MG-132.
The CHX chase assay indicated that TWIST1 was degraded more rapidly than
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PML-RARα (Fig. 3H, Fig. S3I and J). In vivo ubiquitination assays also showed
increased levels of ubiquitinated TWIST1 after MG132 treatment (Fig. S3K and L).
To check the possibility that TWIST1 is a direct transcriptional target of PML-RARα,
we screened the identified putative PML-RARα oncoprotein-binding sites provided
by Wang et al.(20), and we didn’t find the binding characteristic of TWIST1 as a
direct transcription target of PML-RARα. These data demonstrate that APL cells
degrade endogenous TWIST1 in a proteasome-dependent manner. Interestingly, we
observed the half-life of TWIST1 is longer than half-life of TRIB3 (Fig. 3H).
Considering that TWIST1 interacted with TRIB3 in APL cells, we hypothesized that
TRIB3 might inhibit ubiquitination and degradation of TWIST1 in a
TRIB3-dependent manner. TRIB3 knockdown decreased the protein level of
endogenous TWIST1 and shortened the protein half-life in NB4 cells, which was
reversed by MG-132 treatment (Fig. 3I, Fig. S3M and S3N). In vivo ubiquitination
assays also showed increased levels of ubiquitinated TWIST1 after TRIB3
knockdown (Fig. 3J). Together, these data indicate that TRIB3 stabilizes high
TWIST1 expression in APL cells through preventing ubiquitination.
TRIB3 inhibition promotes TWIST1 degradation and reverses resistance to
ATRA therapy
Although APL has been highly curable with ATRA-based differentiation therapy, a
fraction of patients still relapse and become resistant to ATRA (40,41). We found that
ATRA treatment decreased the protein levels of TWIST1 and TRIB3 in
ATRA-sensitive NB4 cells but not in ATRA-resistant NB4-R1 (R1) cells (Fig. 4A).
This finding led us to consider whether TRIB3 cooperated with TWIST1 to contribute
to ATRA resistance. To test this hypothesis, we introduced NC-shRNA or
sh-TRIB3-1/2 into R1 cells and observed significant suppression of TRIB3 expression
with sh-TRIB3-2 (Fig. S4A). Furthermore, sh-TRIB3-2 slightly decreased TWIST1
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protein levels and promoted R1 cell differentiation but not cell apoptosis (Fig.
S4B-D). Silencing of TRIB3 also reduced TWIST1 expression in response to ATRA
and greatly reversed ATRA resistance in R1 cells (Fig. 4B, 4C and Fig. S4E). In vivo
ubiquitination assays revealed increased levels of ubiquitinated TWIST1 after TRIB3
knockdown in response to ATRA treatment (Fig. 4D). Moreover, TWIST1
overexpression rescued TWIST1 protein levels and impaired the differentiation
induced by TRIB3 knockdown (Fig. 4E, 4F and Fig. S4F-J). To evaluate the role of
TRIB3/TWIST1 in APL ATRA resistance in vivo, we inoculated TRIB3-knockdown
and/or TWIST1-overexpressing R1 cells into sublethally irradiated NOD/SCID mice
by tail vein injection. At 25 days after transplantation with 6-day ATRA treatment,
TRIB3 knockdown significantly reversed ATRA resistance in R1 cells and prolonged
the survival of recipient mice (Fig. 4G and H). Similarly, TWIST1 overexpression
rescued the suppression of R1 cells and shortened the survival of recipient mice
caused by TRIB3 knockdown in vivo (Fig. 4G and H). Interestingly, compared with
the NC-shRNA groups, reduced CNS infiltration of R1 cells was observed in the
TRIB3-knockdown groups, and modest CNS infiltration of R1 cells was observed in
the TWIST1-overexpressing group, indicating that TWIST1 may contribute to APL
extramedullary infiltration (Fig. 4I). These results show that loss of TRIB3 promotes
TWIST1 degradation and reverses resistance to ATRA therapy.
The WR domain of TWIST1 is required for TWIST1/TRIB3 binding and
TWIST1 stabilization in APL
Because the TRIB3/TWIST1 interaction is involved in the protein stability and
functional maintenance of the TWIST1 protein, we further analyzed the binding
interface residues in detail. We constructed mutants of TWIST1 and TRIB3 deleted
for various domains (Fig. S5A and B). We found that both the bHLH domain and the
WR domain of TWIST1 interacted with the C terminus of TRIB3, although the bHLH
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domain only weakly contributes to this interaction (Fig. 5A). We have thus unveiled
the critical binding domains of TWIST1 with TRIB3. As observed before, the binding
of TRIB3 and TWIST1 stabilized high TWIST1 expression in APL cells (Fig. 3J and
Fig. S3K and L). Considering that TRIB3 interacts mostly with the WR domain of
TWIST1, we hypothesized that the TRIB3-C terminus: TWIST1-WR domain
interaction might stabilize high TWIST1 expression. As expected, HEK293T IP
assays showed that TRIB3 was able to inhibit the ubiquitination of TWIST1, and this
inhibition mainly occurred between the C-terminus of TRIB3 and the WR domain of
TWIST1 (Fig. 5B, Fig. S5C and D).
To directly examine the functional effect of interface residues critical for binding
between TRIB3 and TWIST1, we generated four synthetic peptides containing the
interface residues and various lysine sites from the TWIST1 C terminus (Fig, 5C,
upper panel). We fused these peptides to a cationic cell-penetrating peptide (42). The
peptides were designed in the orientation containing K residues or the WR domain
and were termed peptides 1, 2 and 3. We also designed an inactive peptide (termed
peptide con) as a negative control. To determine the ability of peptidomimetics to
dissociate the TRIB3/TWIST1 complex in APL cells, we purified the
TRIB3/TWIST1 complex by IP using anti-TWIST1 antibodies in the presence of 1
µM peptidomimetics and determined its composition by western blotting. Compared
to peptide con treatment, competition with peptide 3 led to significant dissociation of
the cellular TRIB3/TWIST1 complex (Fig. 5C, bottom panel). We then treated NB4
and R1 cells with peptidomimetics and observed that peptide 3, but not the peptide
con, peptide 1 or peptide 2, induced significant differentiation and apoptosis in APL
cells rather than other leukemic cells or normal hematopoietic cells (Fig. 5D, Fig.
S5E and 5F). Consistent with the significant changes in cell surface CD11b and
annexin V, we observed a significant decrease in the protein abundance of
TRIB3/TWIST1 and a robust increase in intracellular caspase-3 and PARP cleavage
after peptide 3 treatment for only 8 hours (Fig. S5G). Moreover, in vivo
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ubiquitination assays showed increased levels of ubiquitinated TWIST1 with peptide
3 treatment (Fig. S5H). Thus, peptide 3 is a specific peptide mimetic inhibitor of
TRIB3/TWIST1 complex assembly in APL cells and induces apoptosis and
differentiation of APL cells in vitro.
WR domain peptidomimetic inhibition of the TRIB3/TWIST1 interaction
impairs rapid progression during the early death of APL.
Despite the striking long-term leukemia-free survival rate after the ATRA/ATO-based
regimen, the progression of APL, including early death (ED) and differentiation
therapy resistance, still affects the health of a significant proportion of APL patients
(3,7). To mimic the progressive clinical pattern of APL ED, we began to treat APL
mouse models with ATRA/ATO or peptidomimetics at 20 days post transplantation
(Fig. S6A). We noted that APL mice had very high peripheral blasts at this stage and
died within five days if not treated, which was very similar to the clinical pattern of
APL ED (Fig. S6B). We transplanted NB4 cells into NOD/SCID mice, and at 20 days
posttransplantation, we treated the engrafted mice with ATRA/ATO, peptide 3 or
control peptide for 30 days. Peptide 3 significantly delayed leukemia progression,
extended survival and reduced CNS infiltration of APL cells (Fig. 6A-C and Fig.
S6C). Consistent with the results obtained in APL cell-engrafted NOD/SCID mice,
APL transgenic mice treated with peptide 3 exhibited significantly delayed disease
latency and death compared to the mice in the control or ARTA/ATO group (Fig. 6D
and E). We also noted that peptide 3, the WR domain peptide mimetic, inhibited APL
cell proliferation and impeded CNS infiltration in vivo (Fig. S6D and S6E). We then
examined whether peptide 3 interfered with TWIST1: TRIB3 interaction to reverse
resistance to ATRA treatment. As expected, the use of peptide 3 in vitro greatly
impaired ATRA resistance in R1 cells (Fig. S6F). To evaluate the role of
peptidomimetics in APL ATRA resistance in vivo, we inoculated R1 cells into
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sublethally irradiated NOD/SCID mice by tail vein injection. At 20 days after
transplantation, the mice were treated with ATRA and peptidomimetics, and then
monitored for response to ATRA treatment. Consistent with the use of peptide 3 in
vitro, the WR domain peptidomimetic significantly impeded leukemia growth and
sensitivity to ATRA and prolonged the survival of R1 cell recipient mice (Fig. 6F
and 6G). We further confirmed the therapeutic effect of peptide 3 in preventing rapid
progression of primary APL ED patient blasts in vitro. We screened five samples of
ED from the collected APL bone marrow specimens and found that they all involved
cases of high WBC and peripheral blast counts (Fig. S6G). We treated leukemia cells
with peptide 3 in vitro and confirmed that it can promote leukemia cell differentiation
and apoptosis in a short time compared with ATRA/ATO or peptide con (Fig. 6H and
Fig. S6H). Therefore, the WR domain peptidomimetic can rapidly promote APL cell
differentiation and apoptosis, and can prevent early death of APL and reverse
induction therapy resistance (Fig. 6I).
Discussion
Although the importance of EMT-TFs, including the TWIST, SNAIL, and ZEB
families, has been well documented in the tumorigenesis of epithelial cancers, the role
of EMT-TFs in hematological malignancies is still unknown. In this study, we found
that EMT-TF TWIST1 is highly expressed in APL patients and is critical for leukemic
cell survival. In light of a recent report indicating that the stress protein TRIB3
inhibits the degradation of PML-RARα and promotes APL progression, we found that
TWIST1 and TRIB3 were highly coexpressed in APL cells compared with other
subtypes of AML. We subsequently demonstrated that TRIB3 could strongly bind to
the WR domains of TWIST1 to stabilize TWIST1 by inhibiting its ubiquitination.
The success of clinical APL studies has led to the current highly curative of APL
therapy. With differentiation therapy, over 80% of patients with APL achieve
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19
long-term leukemia-free survival. However, a proportion of APL patients suffer from
early fatal bleeding and leukemic extramedullary infiltration, and the underlying
mechanisms of this difference are largely unknown (27-29). Based on a detailed
analysis and a functional screening of synthetic peptides, we discovered a peptide
analogous to the TWIST1 WR domain that rapidly and specifically represses APL
cell survival by disrupting the TRIB3/TWIST1 interaction. Targeting rapid TWIST1
degradation could protect against early death in APL and improved sensitivity to
ATRA. Furthermore, our data also showed that TWIST1 governed CNS infiltration
during progression in NB4-xenograft mice and APL transplantable mice.
Cell-penetrating peptides may competitively inhibit the TWIST1/TRIB3 interaction
and repress CNS progression by initiating APL cell differentiation and apoptosis. Our
previous study also reported that high expression of TWIST1 in AML contributes to
extramedullary infiltration and promotes leukemic aggressiveness (16). A pioneering
study using the MLL-AF9 mouse model revealed that the EMT-inducer ZEB1
contributed to leukemic blast invasion and was associated with poor survival in AML
patients. These observations suggest that EMT-TFs not only play important roles in
solid tumors but also promote leukemic progression and aggressive extramedullary
infiltration. Thus, EMT-TFs may be novel therapeutic targets for disease progression
in patients with relapsed and refractory AML.
TWIST1 contains two highly conserved and functionally different domains: the
bHLH domain for DNA binding and the WR domain for heterodimer formation (43).
Recently, the WR domain was also reported to exhibit transactivation activity and
interact with RUNX2, SOX9, p53, RELA and p62 (31,44-47). Notably, through a
p62-dependent interaction, the WR domain is necessary for the proteolytic activity of
TWIST1 (31). TRIB3 inhibited autophagic substrate clearance by interacting with p62
and led to UPS-dependent accumulation of EMT-TFs, such as TWIST1, ultimately
promoting tumor growth and metastasis (30). These results strongly implicated that
TRIB3 was highly implicated in TWIST1 degradation. Here, we provided clear
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20
evidence that the binding of TRIB3 to the WR domain inhibited TWIST1
ubiquitination and stabilized high TWIST1 expression to promote APL progression
and ATRA resistance. Intriguingly, despite the absence of lysine sites in the WR
domain, the TWIST1 WR domain was required for TWIST1 ubiquitylation, which
appears to be caused by recruitment of potential ubiquitin E3 ligases.
APL is commonly driven by the t (15;17) chromosomal translocation, which yields
the PML-RARα fusion oncoprotein as a transcriptional repressor. ATRA- and/or
ATO-triggered PML-RARα proteolysis are required for the elimination of APL cells.
Recent studies have shown that PML-RARα has a half-life of over 8 hours and
requires approximately 12 hours to be partially degraded in response to ATRA and/or
ATO (39,48,49). If this is the case, a significant number of APL patients who
experience rapid progression and a high-risk state, including high white blood cell
(WBC) counts, fatal bleeding and severe infection, would not rapidly repress APL
cell survival via ATRA- and/or ATO-initiated PML-RARα degradation. Similarly, the
proposal that ATRA- and/or ATO-triggered PML-RARα degradation in vitro and in
vivo exerts any effect has been controversial. Therefore, we hypothesized that
PML-RARα induces a gain-of-function transcriptional activation to upregulate
modulators of APL pathogenesis. We provided clear evidence that high TWIST1
expression promotes APL progression and that TWIST1 proteolysis initiated by a
novel peptide might help reshape the therapeutic design for rapid APL eradication.
Although a previous study indicated that TRIB3 suppresses the degradation of
PML-RARα by interacting with PML-RARα and PML, it is difficult to determine
how TRIB3 instability might protect the half-life of PML-RARα. Our results strongly
suggest that the binding of TRIB3 to TWIST1 inhibits TWIST1 ubiquitination and
stabilizes high TWIST1 expression to promote APL progression and ATRA
resistance. This characteristic is a very effective target for preventing APL early death
and ATRA resistance.
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21
Acknowledgements
We thank Dr. Chun-Lin Shen for her assistance with NOD/SCID in vivo
bioluminescence imaging (BLI). This study was founded by the National Natural
Science Foundation of China (81800099, 81400106, 81430002, 81770206), the
Shanghai Rising-Star Program (17QA1402200, 19QA1407800), the Shanghai
Excellent Youth Medical Talents Training Program (2018YQ09), and National
Science and Technology Major Project (2018ZX09101001). Dr. Wu Zhang gratefully
acknowledges to be supported by Global Scholar-in-Training Award (GSITA) from
AACR.
Author contributions
J.X. and W.Z. conceived the project and designed the study. J.L., W.Z. and L-T.N.
performed the experiments, interpreted the data and prepared the manuscript. Y-M.Z.,
X-Q.W., and Y.S. provided assistance with clinical data analysis and technical
support. J.Z. contributed to the funding support and revised the manuscript during the
revision. J.L. and W.Z. wrote the manuscript. J.X. supervised the entire project.
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Figure legends
Figure 1: High TWIST1 expression in APL.
(A) Relative TWIST1 mRNA expression levels in 199 human AML cases with six
different AML cytogenetic aberrations from the gene expression microarray of the
E-MTAB-3444 database, including t (15;17) (n = 26), t (8;21) (n = 46), inv (16) (n =
48), MLL (11q23) (n = 43), t (9;11) (n = 21), and inv (3)/t (3;3) (n = 15) (see also
Supplementary Figure 1A, **P < 0.01).
(B) In silico analysis of TWIST1 mRNA expression levels in different FAB subtypes
of AML patients (n = 173), including M0 (n = 16), M1 (n = 44), M2 (n = 38), M3 (n =
16), M4 (n = 34), M5 (n = 18), M6 (n = 2), M7 (n = 3), and not classified (NC, n = 2).
The raw RNA-seq data were obtained from the TCGA database (*P < 0.05, **P <
0.01, N/A: not applicable).
(C) Quantitative real-time PCR (qRT-PCR) assay of the mRNA expression levels of
TWIST1 in primary blasts from newly diagnosed APL M3 patients (n = 22) versus
CD34+ bone marrow (BM) cells from healthy donors (n = 6) and primary blasts from
other AML subtypes, including M1 (n = 7), M2 (n = 14), M4 (n = 23), M5 (n = 18),
and M6 (n = 6) (Data represent the mean ± SEM of three assays, *P < 0.05, **P <
0.01).
(D) The semi-quantitative analysis of western blot data showing TWIST1 and
PML-RARα expression in primary leukemic blasts obtained from newly diagnosed
APL M3 patients (n =12) versus CD34+ bone marrow (BM) cells from healthy donors
(n = 4) and other AML subtypes, including M1 (n = 7), M2 (n = 12), M4 (n = 14), M5
(n = 12), and M6 (n = 5). Data represent the mean ± SEM of three assays, *P < 0.05,
**P < 0.01.
(E) qRT-PCR analysis of TWIST1 mRNA expression in blasts from different AML
mouse models after bone marrow transplantation (BMT) of murine hematopoietic
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stem/progenitor cells (HSPCs) freshly transduced with the indicated oncogenic fusion
genes (top panel). The data represent the mean ± SEM of three assays. ***P < 0.001.
In these murine leukemic blasts, the protein levels of TWIST1 and PML-RARα were
detected by western blotting. Three independent western blotting replicates were
performed (bottom panel).
(F) PR9 cells were incubated with 200 μM ZnSO4 for the indicated times, and the cell
lysates were blotted with an anti-TWIST1 or anti-PML-RARα antibody (top panel).
The data represent immunoblots of three independent assays. Immunofluorescence
microscopic inspection of the expression of TWIST1 or PML-RARα in control and
ZnSO4-induced PR9 cells for indicated time. Representative images were obtained in
six random fields from three independent biological replicates. Scale bar, 2 μm.
Figure 2: High TWIST1 expression promotes APL progression.
(A) Lentiviruses carrying sh-Negative Control (NC) or sh-TWIST1-1 were used to
transduce NB4 cells. Western blot showing the protein levels of TWIST1,
PML-RARα, p21, p-p53, cleaved PARP and cleaved caspase-3 in the transduced cells.
Three independent western blotting replicates were performed.
(B) The flow cytometric scatter plots present differentiated cells (CD11b+, upper
panel) and apoptotic cells (annexin V+, bottom panel) in NB4 cells with or without
TWIST1 mRNA knockdown. Column diagram showing the percentage of CD11b+
cells and annexin V+
cells in transduced NB4 cells. The values are presented as the
mean ± SEM (n = 6 per group). ***P < 0.001.
(C) A representative bioluminescence image of mice transplanted with NB4-luc cells
stably transduced with NC or sh-TWIST1-1. Quantitative luciferase bioluminescence
was monitored at week 3-post xenografting. Representative BLI images and
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quantitation data were from six independent experiments; n = 6 for each group. **P <
0.01.
(D) Kaplan-Meier analysis shows the survival rates of mice receiving 2×106 NB4-luc
cells stably expressing a nontargeting NC or an shRNA targeting TWIST1
(sh-TWIST1-1) (n = 6 for each model).
(E) Hematoxylin and eosin (HE) staining of brain biopsies and spinal cord biopsies
collected from mice transplanted with NB4-luc cells stably transduced with NC or
sh-TWIST1-1 at day 20 post xenografting (left panel). Representative hCD45+
immunohistochemical staining of the murine brain and spinal cord (right panel).
Carmine arrows indicate the CNS-infiltrating NB4-luc cells in transplanted mice.
Black triangles and squares denote the cerebral parenchyma and spinal cavities,
respectively. Dashed lines indicate the meninges. Images are representative of six
independent experiments. Scale bar, 100 μm.
(F) TWIST1, PML-RARα, cleaved PARP, cleaved caspase-3 and p21 expression in
blasts from three APL patients that were transduced with a nontargeting siRNA (NC)
or a siRNA targeting TWIST1 (si-TWIST1) for 48 hours in vitro. Three independent
western blotting replicates were performed.
(G) Representative scatter plots of differentiated cells (CD11b+, upper panel) and
apoptotic cells (annexin V+, bottom panel) in blasts from three APL patients with or
without TWIST1 mRNA knockdown. The quantitative measurement presents the
percentages of CD11b+ cells and annexin V
+ cells in transduced APL patients’ blasts.
Three independent assays were performed for each group. The values are presented as
the mean ± SEM (n = 6 per group). **P < 0.01, ***P < 0.001.
Figure 3: TRIB3 interacts with TWIST1 in APL cells.
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(A) A correlation analysis of the relative TWIST1 and TRIB3 mRNA expression in
TCGA AML patients with either APL (n = 16) or non-APL disease (n = 157).
(B) Total lysates of the indicated AML cell lines were extracted, and TWIST1 and
TRIB3 protein levels were detected by western blotting. TWIST1 and TRIB3 protein
level were highly correlated in AML cell lines. The data are representative
immunoblots of three independent assays.
(C) NB4 cell extracts were subjected to IP with immunoglobulin G (IgG) and
anti-TWIST1 or anti-TRIB3 Abs and blotted with the indicated Abs. The interaction
of TWIST1 and TRIB3 was evaluated by Co-IP in NB4 cells. The data are
representative immunoblots of three independent assays.
(D) HEK293T cells were cotransfected with TWIST1-Myc and TRIB3-Flag
expression plasmids. Cell extracts were subjected to IP with anti-Myc or anti-Flag
Abs and blotted with the indicated Abs. Ectopically expressed TWIST1 and TRIB3
interact in HEK293T cells. The data are representative immunoblots of three
independent assays.
(E) Retrieved proteins were evaluated by western blotting. The GST-only protein was
used as the negative control. In vitro interaction of TWIST1 and TRIB3 was detected
with a GST pull-down assay. The data are representative immunoblots of three
independent assays.
(F-G) Colocalization of TWIST1 and TRIB3 was detected in NB4 and HEK293T
cells with immunostaining. For (F), anti-TWIST1 and anti-TRIB3 Abs were used for
immunostaining. For (G), TWIST1-Myc and TRIB3-Flag were ectopically expressed
in HEK293T cells, and anti-Myc or anti-Flag Abs were used. Images are
representative of at least six random fields. Scale bar, 2 μm.
(H) NB4 cells were incubated with CHX (10 μg/mL) or CHX plus MG132 (10 μM)
for the indicated times. TWIST1, TRIB3 and PML-RARα protein level were detected
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30
by immunoblotting (left panel). The semi-quantitative analysis of TWIST1 and
TRIB3 protein expression in NB4 cells subjected to the indicated treatment were
shown (right panel). The data represent the mean ± SEM of three assays.
(I) The statistics and quantification of relative TWIST1 expression level were
performed by densitometry of protein expression levels presented relative to GAPDH
in the same lane, and were compared with the western blotting assay of 0-min control
groups (see also Supplementary Figure 3N). The data represent the mean ± SEM of
three assays.
(J) The effect of TRIB3 depletion on TWIST1 ubiquitination in vivo. The cell extracts
of Control (NC) or TRIB3-silenced (sh-TRIB3-1) NB4 treated with MG132 (10 μM)
in vitro for 12h were subjected to IP with immunoglobulin G (IgG) and anti-TWIST1
Abs and blotted with an ubiquitin Ab. The data represent immunoblots of three
independent assays.
Figure 4: TRIB3 inhibition promotes TWIST1 degradation and reverses
resistance to ATRA therapy.
(A) TWIST1, TRIB3 and PML-RARα expression levels in NB4 and R1 cells treated
with ATRA (1 µM) and harvested at the indicated times. Three independent western
blotting replicates were performed.
(B) TWIST1, TRIB3 and PML-RARα protein expression in transduced R1 cells after
treatment with ATRA (1 µM) for the indicated times. R1 cells stably transduced with
a nontargeting shRNA (NC) or an shRNA targeting TRIB3 (sh-TRIB3-2). The
measurements of protein levels were obtained from three independent western
blotting replicates.
(C) The flow cytometric scatter plots present differentiated cells (CD11b+) in R1 cells
with or without TRIB3 mRNA knockdown after treatment with ATRA (1 µM) for the
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31
indicated times (top panel). Column diagram showing the percentage of CD11b+ cells
in transduced R1 cells (bottom panel). The values are presented as the mean ± SEM
(n=6 per group). ***P < 0.001
(D) Control (NC) or TRIB3-silenced (sh-TRIB3-2) R1 cells after treatment with
ATRA (1 µM) for 24 hours were subjected to IP with immunoglobulin G (IgG) and
anti-TWIST1 Abs and blotted with an ubiquitin Ab. The data represent immunoblots
of three independent assays.
(E) R1 cells were stably transduced with NC or an shRNA targeting TRIB3 mRNA
(sh-TRIB3-2), followed by transduction with a lentiviral vector carrying a control or
TWIST1 construct. In these cells, after treatment with ATRA (1 µM) for the indicated
times, the expression levels of TWIST1 and TRIB3 were detected by western blotting.
Three independent western blotting replicates were performed.
(F) Column diagram showing the percentage of CD11b+ cells in transduced R1 cells.
The values are presented as the mean ± SEM (n=6 per group). **P < 0.01, ***P <
0.001.
(G) A representative bioluminescence image of mice transplanted with R1-luc cells
stably transduced with NC, sh-TRIB3-2 or sh-TRIB3-2-TWIST1 (sh-TRIB3-2,
followed by transduction with a lentiviral vector carrying a control or TWIST1
construct). Quantitative luciferase bioluminescence was monitored at day 25 post
xenografting after treatment with ATRA (10 mg/kg). Representative BLI images and
quantitation data were from six independent experiments; n = 6 for each group. **P <
0.01, ***P < 0.001.
(H) Kaplan-Meier analysis shows the survival rates of mice receiving 2×106 R1-luc
cells stably expressing a nontargeting NC, an shRNA targeting TRIB3 (sh-TRIB3-2)
or sh-TRIB3-2-TWIST1 after 6-day treatment with ATRA (10 mg/kg, once daily) (n
= 6 for each model).
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(I) HE staining of brain biopsies collected from mice transplanted with R1-luc cells
stably transduced with NC, sh-TRIB3 or sh-TRIB3-2-TWIST1 at day 25 post
xenografting after treatment with ATRA (10 mg/kg) (left panel). Representative
hCD45+ immunohistochemical staining of the murine brain (right panel). Carmine
arrows indicate the CNS-infiltrating R1-luc cells in transplanted mice. Images are
representative of at least six random fields. Scale bar, 100 μm.
Figure 5: The WR domain of TWIST1 is required for the binding of
TWIST1/TRIB3 complex and TWIST1 stabilization.
(A) Mapping of TWIST1 regions involved in C-terminal binding to TRIB3. Upper:
Diagram of TWIST1 deletion mutants. Bottom: HEK293T cells were cotransfected
with the indicated TWIST1 and TRIB3-C-Myc constructs. The cell extracts were
subjected to IP with an anti-Myc Ab. The data are representative immunoblots of
three independent assays.
(B) HEK293T cells were cotransfected with the indicated TWIST1-△ bHLH,
TRIB3-C and HA-Ub constructs. Cell extracts were subjected to IP with an anti-GFP
Ab and blotted with an HA Ab. The data represent immunoblots of three independent
assays.
(C) Schematic illustration showing that different peptides target relevant amino acid
sequences, which include the key lysine sites and the WR domain of the TWIST1
protein. Mapping aa106-aa202 sequences of human TWIST1 protein regions involved
in C-terminal peptide binding (upper). Three peptides containing common sequences
of penetrating peptides were designed to competitively inhibit the binding of TWIST1
and TRIB3 (middle). The cell extracts of NB4 or R1 treated with different peptides (1
µM) for 12 hours were subjected to IP with immunoglobulin G (IgG) and
anti-TWIST1 Abs and blotted with the indicated Abs (bottom left). Quantitative and
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33
statistic analysis of TRIB3/TWIST1 gray values from IP (bottom right). The data are
representative immunoblots of three independent assays.
(D) Representative scatter plots of differentiated cells (CD11b+, upper panel) and
apoptotic cells (annexin V+, bottom panel) in NB4 or R1 cells treated with different
peptides (1 µM) for 12 hours (upper). The quantitative measurements present the
percentages of CD11b+ cells and annexin V
+ cells in NB4 or R1 cells treated with
different peptides (bottom). Three independent assays were performed for each group.
The values are presented as the mean ± SEM (n=6 per group). ***P < 0.001.
Figure 6: WR domain peptidomimetic inhibition of the TRIB3/TWIST1
interaction impairs rapid progression during the early death of APL.
(A) A representative bioluminescence image of mice transplanted with NB4-luc cells
after peptide con (10 mg/kg twice daily), ATRA (10 mg/kg once daily)/ATO (4
mg/kg once daily) or peptide 3 (10 mg/kg twice daily) treatment in vivo.
Representative BLI images were from six independent experiments.
(B) Quantitative luciferase bioluminescence was monitored at day 25-post
xenografting related to (A). (n = 6 for each group, ***P < 0.001)
(C) Kaplan-Meier analysis shows the survival rates of mice receiving 2×106 NB4-luc
cells after peptide 3 or ATO/ATRA treatment in vivo (n = 6 for each model). The
black arrow indicates that administration begins at day 20.
(D) A representative bioluminescence image of mice transplanted with APL murine
-luc cells after peptide con (10 mg/kg twice daily), ATRA (10 mg/kg once
daily)/ATO (4 mg/kg once daily) or peptide 3 (10 mg/kg twice daily) treatment in
vivo. Quantitative luciferase bioluminescence was monitored at day 23-post
xenografting. The values are presented as the mean ± SEM (n=6 per group). ***P <
0.001.
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34
(E) Kaplan-Meier analysis shows the survival rates of mice receiving 1×106
APL
murine-luc cells after peptide con, peptide 3 or ATO/ATRA treatment in vivo (n = 6
for each model). The black arrow indicates that administration begins at day 20.
(F) A representative bioluminescence image of mice transplanted with APL-luc cells
after peptide control (10 mg/kg twice daily)/ATRA (10 mg/kg once daily) or peptide
3 (10 mg/kg twice daily)/ATRA treatment (10 mg/kg once daily) in vivo. Quantitative
luciferase bioluminescence was monitored at day 25-post xenografting.
Representative BLI images and quantitation data were from six independent
experiments; n = 6 for each group. Scale bar, 1 cm. ***P < 0.001.
(G) Kaplan-Meier analysis shows the survival rates of mice receiving APL-luc cells
after peptide control/ATRA or peptide 3/ATRA treatment in vivo (n = 6 for each
model). The black arrow indicates that administration begins at day 20.
(H) Representative scatter plots of differentiated cells (CD11b+) and apoptotic cells
(annexin V+) in blasts from five APL early death patients with peptide con (2 µM),
peptide 3 (2 µM) or ATRA (1 µM)/ATO (1 µM) treatment in vitro for 8 hours. The
quantitative measurements present the percentages of CD11b+ cells and annexin V
+
cells in cells treated with peptide con, peptide 3 or ATRA/ATO. Four independent
assays were performed for each group. The values are presented as the mean ± SEM.
(I) An illustration of TRIB3 stabilizing high TWIST1 expression to promote APL
rapid progression and ATRA resistance.
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Published OnlineFirst June 24, 2019.Clin Cancer Res Jian Lin, Wu Zhang, Li-Ting Niu, et al. APL Progression and ATRA ResistanceTRIB3 Stabilizes High TWIST1 Expression to Promote Rapid
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