TRIB3 Stabilizes High TWIST1 Expression to Promote Rapid ... · TRIB3 Stabilizes High TWIST1 Expression to Promote Rapid APL Progression and ATRA Resistance Jian Lin1, 3, Wu ... Shanghai

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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.

Research. on May 29, 2020. © 2019 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 24, 2019; DOI: 10.1158/1078-0432.CCR-19-0510

mailto:[email protected]


http://clincancerres.aacrjournals.org/

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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




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




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.




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




27

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




28

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.




29

(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




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




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).




32

(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




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.




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.






















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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TRIB3 Stabilizes High TWIST1 Expression to Promote Rapid ... · TRIB3 Stabilizes High TWIST1 Expression to Promote Rapid APL Progression and ATRA Resistance Jian Lin1, 3, Wu ... Shanghai