Stabilization of Notch1 by the Hsp90 Chaperon is Crucial for T Cell

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Stabilization of Notch1 by the Hsp90 Chaperon is Crucial

for T Cell Leukemogenesis

Zhaojing Wang1,2,3*, Yufeng Hu3*, Daibiao Xiao2, Jingchao Wang2, Chuntao Liu3,

Yisheng Xu4, Xiaomeng Shi4, Peng Jiang5, Liang Huang6, Peng Li7, Hudan Liu1,2,

Guoliang Qing1,2#

Authors’ Affiliations: 1Zhongnan Hospital of Wuhan University, Wuhan, P. R. China; 2Medical Research Institute, Wuhan University, Wuhan, P. R. China; 3School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China; 4Protein Facility, Center of Biomedical Analysis, Tsinghua University, Beijing, P. R. China; 5School of Life Sciences, Tsinghua University, Beijing, P. R. China; 6Department of Hematology, Affiliated Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P. R. China; 7South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, P. R. China *These authors contribute equally to this work.

The authors disclose no potential conflicts of interest.

This work was supported by grants from the National Natural Science Foundation of

China (81470332 to HL and 81372205 to GQ).

Running title: Stabilization of Notch1 by the Hsp90 chaperon # Corresponding Author:

Guoliang Qing, Phone: 86-27-68750015, Email: [email protected],

Medical Research Institute, Wuhan University, 185 East Lake Rd,

Wuhan, P. R. China, 430071

Research. on April 13, 2018. © 2017 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 January 31, 2017; DOI: 10.1158/1078-0432.CCR-16-2880

http://clincancerres.aacrjournals.org/

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Abstract

Purpose: Notch1 deregulation is assuming a focal role in T-cell acute lymphoblastic

leukemia (T-ALL). Despite tremendous advances in our understanding of Notch1

transcriptional programs, the mechanisms by which Notch1 stability and turnover are

regulated remain obscure. The goal of the present study is to identify intracellular

Notch1 (ICN1, the activated form of Notch1) binding partner(s) regulating its stability

and activity.

Experimental Design: We employed immunoaffinity purification to identify

ICN1-associating partner and used co-immunoprecipitation to verify the endogenous

protein interaction. Pharmacological or shRNA-mediated inhibition was applied in

loss-of-function assays to assess the role of tentative binding partner(s) in modulating

ICN1 protein stability as well as affecting T-ALL cell expansion in vitro and in vivo.

Mechanistic analysis involved protein degradation and polyubiquitination assays.

Results: We identify the Hsp90 chaperon as a direct ICN1-binding partner essential

for its stabilization and transcriptional activity. T-ALL cells exhibit constitutive

endogenous ICN1-Hsp90 interaction and Hsp90 depletion markedly decreases ICN1

levels. The Hsp90-associated E3 ubiquitin ligase Stub1 mediates the ensuring

proteasome-dependent ICN1 degradation. Administration of 17-AAG or PU-H71, two

distinct Hsp90 inhibitors, depletes ICN1, inhibits T-ALL cell proliferation and triggers

dramatic apoptotic cell death. Systemic treatment with PU-H71 reduces ICN1




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expression and profoundly inhibits murine T-ALL allografts as well as human T-ALL

xenografts.

Conclusions: Our findings demonstrate Hsp90 blockade leads to ICN1 destabilization,

providing an alternative strategy to antagonize oncogenic Notch1 signaling with

Hsp90 selective inhibitors.




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Translational relevance

T cell acute lymphoblastic leukemias (T-ALLs) are aggressive and heterogeneous

hematological tumors resulting from the malignant transformation of T cell

progenitors. The major challenges in the treatments of T-ALLs are dose-limiting

toxicities of chemotherapeutics and drug resistance. Notch1 signaling is one of the

prominent oncogenic pathways in T-ALLs which reciprocally present an ideal

malignancy for studying Notch1-driven cancer. Anti-Notch1 strategy has emerged as

a promising targeted therapy for T-ALL treatments. Yet Notch inhibition by

γ-secretase inhibitor has not achieved impressive therapeutic efficacy, raising the urge

for an alternative approach to Notch1 inhibition. We here demonstrate intracellular

Notch1 protein stability is dependent on active Hsp90 chaperon. Specific Hsp90

inhibitors accelerate Notch1 degradation, leading to T-ALL cell growth inhibition in

vitro as well as in disease models. These findings rationalize Hsp90 antagonists as a

treatment strategy for patients with T-ALL or other Notch1-dependent tumors.




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Introduction

Notch1 is a highly conserved transmembrane receptor that elicits signaling

transduction required for cell proliferation, survival and differentiation. Normally

transmembrane ligands in adjacent cells stimulate Notch1 receptor by inducing

step-wise intramembrane proteolysis to produce a transcriptional effector ICN1,

which specifically turns on target gene expression (1). Aberrantly activated Notch1

signaling has been implicated in a variety of tumors, including T-ALL, a malignant

disorder of thymocyte progenitors (2). The central role of Notch1 in T cell

transformation was realized upon the identification of activating mutations in the

NOTCH1 gene present in about 60% of T-ALL cases (3). Once obtained these

activating mutations, Notch1 signaling is potentiated by either eliciting

ligand-independent activation or prolonging ICN1 half-life. These genetic lesions

remarkably enhance ICN1 transcriptional programs and the expression of downstream

genes that promote leukemogenesis such as c-MYC (4-7). Tremendous efforts have

been paid in identifying downstream targets regulated by Notch1 signaling, whereas

less attention is gained to understand the upstream mechanisms sustaining aberrant

Notch1 activities, particularly those involved in Notch1 stabilization.

Heat shock protein 90 (Hsp90) is an abundant, highly conserved molecular

chaperone crucial for correct folding and maturation of a variety of cellular proteins

that regulates cell survival, proliferation and apoptosis. The increased expression of

Hsp90 that is observed in many tumor types reflects the efforts of malignant cells to

maintain homeostasis in a hostile environment as well as tolerate alterations from




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numerous genetic lesions which often result in aberrant accumulations of

oncoproteins. This dependence on Hsp90 appears to be a vulnerability of cancer cells

and has led to the development of drugs aimed at depleting the molecular chaperon

and degrading cancer proteome, leading to loss of tumor cell viability (8). Several

Hsp90 inhibitors are currently being tested in both preclinical models and clinical

settings (9). Many have shown promises in solid tumors (10-12) as well as

hematological malignancies (13-15). Inhibition of Hsp90 leads to degradation of its

client proteins through the ubiquitin-dependent proteasome pathway (16).

Downregulation of many Hsp90 substrates is in large part dependent on Stub1 activity,

the U-box ubiquitin E3 ligase that interacts with the Hsp90 chaperon complex to favor

substrate degradation (17).

Current treatment guidelines for patients with T-ALL include only conventional

chemotherapy and overall prognosis of T-ALL remains unsatisfied (18). Anti-Notch

targeted therapy by γ-secretase inhibitors, which inhibit Notch proteolytic processing

and subsequent activation, has been launched for a long time. Unfortunately, clinical

application of these inhibitors has been hampered owing to their limited efficacies and

considerable side-effects (19). Thus, attempts to seek alternative approaches to

Notch1 inhibition become imperative for effective T-ALL therapies.

In the current study, we identify Hsp90 as a novel ICN1 binding partner crucial

for its stabilization and oncogenic function. Inhibition of Hsp90 leads to

Stub1-mediated ICN1 ployubiquitination and subsequent degradation by the 26S




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proteasome. Two different categories of Hsp90 inhibitors, geldanamycin analogues

and purine analogues, exhibit marked cytotoxicities against Notch1-dependent T-ALL

cells as well as murine models. These findings suggest Hsp90 inhibition as a potential

alternative approach and also a new horizon of anti-Notch1 strategy to benefit patients

with T-ALL and other Notch1-addicted malignancies.

Materials and Methods

Cell lines and reagents

SIL-ALL, HPB-ALL, DND41 and CUTTL1 cells were kindly provided by

Warren Pear (University of Pennsylvania; April, 2011). 293T, MOLT-4, JURKAT and

CCRF-CEM cells were purchased from American Type Culture Collection (June,

2012). All cell lines were maintained as described (3), authenticated using the variable

number of tandem repeats (VNTR) PCR assay, cultured for fewer than 6 months after

resuscitation and tested for mycoplasma contamination every 3 months using

MycoAlert (Lonza).

pcDNA3-Flag-ICN1 was obtained from Warren Pear (University of

Pennsylvania). pcDNA3-HA-Hsp90β was a gift from William Sessa (Addgene

plasmid # 22487) (20), and the Myc-tagged Stub1 constructs (wild-type or mutants)

were provided by Bin Li (Institut Pasteur of Shanghai, Chinese Academy of

Sciences)(21). For the experiments in which Hsp90β (or Stub1) was silenced in

T-ALL cells, shRNA sequences were obtained from the collection of the RNAi

Consortium (22) and cloned into a lentiviral vector PLKO.1. For ICN1 (or Stub1)




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overexpression in T-ALL cells, the protein coding sequence was cloned into a

lentiviral vector pCDH. Primers used for shRNAs and molecular cloning are listed in

Supplementary Table S1. Chemicals and antibodies used in the study are listed in

Supplementary Table S2.

Identification of ICN1 interacting partners

293T cells stably expressing Flag-tagged ICN1 were lysed for 30 minutes and

subjected to centrifugation at 12,000 g for 15 minutes. The resulting supernatant was

incubated with antibody against Flag epitope (Sigma) at 4 °C for 4 hours, followed by

addition of protein G-agarose (Roche) (4 °C overnight). Immunoprecipitated proteins

were washed and eluted, followed by SDS-PAGE and Commassie blue staining (23).

Gel bands were excised and digested with trypsin overnight. The resulting peptides

were analyzed by the Protein Facility, Center of Biomedical Analysis at Tsinghua

University (Beijing, China).

Chromatin immunoprecipitation (ChIP)

ChIP was performed as described (24, 25). Briefly, SIL-ALL cells were fixed

with 1% paraformaldehyde at room temperature for 10 minutes. Precleared chromatin

was immunoprecipitated with antibody against cleaved Notch1 (Val1744) for 16 h and

then salmon sperm DNA saturated protein G agarose (Millipore) for 1 hour at 4 °C.

Eluted DNA was quantified by CFX Connect Real-Time PCR System (BioRad) using

specific primers (Supplementary Table S1).




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Mice

Animal procedures were approved by the Animal Experimentations Ethics

Committee of Guangzhou Institutes of Biomedicine and Health, Chinese Academy of

Science.

Human T-ALL xenograft: 2.5 × 106 CUTLL1 cells were tail vein injected into 6-8

weeks NOD-SCID IL-2 receptor gamma-deficient (NSI) mice (Guangzhou Institutes

of Biomedicine and Health, Chinese Academy of Science) that had been subjected to

100cGy whole-body irradiation (Precision X-ray). PU-H71 (75 mg/kg, thrice weekly)

or vehicle (10 mmol/L PBS) was intraperitoneally injected into NSI mice two days

after engraftment. T-ALL development in vivo was monitored by periodic blood

drawings and flow cytometry analysis of human CD8 and CD45. After four weeks of

treatment, mice were sacrificed and assessed for disease progression.

Murine T-ALL transplant: Murine T-ALL cells driven by KrasG12D/Notch1L1601P

mutations were described by Chiang et al (4). 2.5 × 106 murine T-ALL cells were

injected into 6-8 weeks half-lethally irradiated (450cGy) C57BL/6 mice (Beijing HFK

Bioscience). Five days post injection, mice were intraperitoneally injected with

PU-H71 (50 mg/kg, thrice weekly) or vehicle (10 mmol/L PBS). After three weeks of

treatment, mice were sacrificed and assessed for leukemia progression by flow

cytometry analysis of GFP+ as well as CD4+CD8+ cells in the bone marrow and

spleen.




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Statistics

Significance analysis between groups were performed using Student's t-test, with

P < 0.05 considered significant.

Results

Identification of Hsp90 as a novel ICN1-binding protein

To identify novel partners of ICN1, we generated 293T cells expressing

Flag-tagged ICN1. Flag-bound immunoprecipitates from these cells were resolved by

SDS-PAGE and visualized by silver staining (Fig. 1A). Potential specific bands were

excised and subjected to liquid chromatography tandem mass spectrometry

(LC-MS/MS) analysis, which led to the identification of Hsp90 as a protein associated

with ICN1 (Fig. 1B and Supplementary Table S3).

To validate the mass spectrometry analysis, we enforced the expression of

Flag-ICN1 and HA-Hsp90 in 293T cells for reciprocal immunoprecipitation and

confirmed associations between both proteins (Fig. 1C). Moreover,

co-immunoprecipitation using SIL-ALL and CUTLL1 cell lysates validated the

specific interaction between endogenous ICN1 and Hsp90 in T-ALL cells (Fig. 1D).

To define the precise region(s) for Hsp90-ICN1 interaction, we expressed full-length

HA-tagged Hsp90 in combination with respective Flag-tagged fragments of ICN1 in

293T cells (Fig. 1E). The N-terminal region of ICN1 (amino acids 1761-2155)

containing the RAM and ANK domains did not interact with Hsp90, whereas the

C-terminal region (amino acids 2156-2555) interacted as strongly as the full-length




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protein (Fig. 1E, Lanes 1-4). As the C-terminal region of ICN1 contains the TAD

domain, the PEST domain and a linker peptide (amino acids 2425-2442; for simplicity,

we named it H) between these two domains, we expressed different mutants of these

interacting modules with Hsp90 and determined their contribution to the association

between the two proteins. Interestingly, deletion of either the TAD domain (H-PEST,

2425-2555) or the PEST domain (TAD-H, 2215-2442) alone did not impair the

interaction with Hsp90 (Fig. 1E, Lanes 6 and 8). Yet lack of the linker peptide H

(TAD, 2215-2424 or PEST, 2443-2555) resulted in complete loss of protein

interaction (Fig. 1E, Lanes 5 and 7). To evaluate the importance of the peptide H in

mediating ICN1-Hsp90 interaction, we synthesized a Flag-tagged peptide H as well as

a control peptide (amino acids 1861-1888 of ICN1) and examined their interaction

with Hsp90. As expected, the Flag-tagged peptide H but not the control peptide

efficiently pulled down Hsp90 from lysates of 293T cells expressing HA-tagged

Hsp90 (Fig. 1E, Lanes 9 and 10). Altogether, these results provide strong evidence

supporting that Hsp90 is a direct ICN1 binding partner, and amino acids 2425-2442

within ICN1 are responsible for the interaction between ICN1 and Hsp90.

Hsp90 inhibition depletes ICN1 and impairs Notch1 transcriptional activities

To assess potential roles of Hsp90 in regulating Notch1, we treated multiple

T-ALL cell lines (SIL-ALL, CUTLL1, HPB-ALL, MOLT-4 and JURKAT) with

17-AAG, a well-characterized Hsp90 inhibitor (26). As shown in Fig. 2A,

administration of 17-AAG dose-dependently depleted ICN1 expression in all the cell




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lines examined (Fig. 2A). Similar results were obtained when PU-H71, a purine

analog Hsp90 inhibitor (27), was applied in CUTLL1, HPB-ALL and JURKAT cells.

Again, PU-H71 induced downregulation of ICN1 protein in a time-dependent manner

in these cells (Fig. 2B). The effect of Hsp90 blockade on ICN1 decrease was not only

restricted to Notch1 mutant cells (SIL-ALL, CUTLL1, HPB-ALL and MOLT-4), but

also applied to JURKAT cells which express wild-type Notch1. To exclude potential

drug-associated non-specific effects, we infected HPB-ALL or CUTLL1 cells with

lentiviruses expressing Hsp90β specific shRNAs (#1, #2 or #3) or a control shRNA.

Hsp90β shRNA #2 and #3, which exhibited a profound silencing effect, markedly

downregulated ICN1 expression in both T-ALL cell lines (Fig. 2C), validating that

Hsp90 is required for aberrant ICN1 accumulation in this context.

The implication of Hsp90 in ICN1 regulation prompted us to investigate whether

its inhibition affects Notch1 transcriptional activities. We analyzed five representative

Notch1 targets in CUTLL1 cells by quantitative PCR. Gapdh and Actin, two

constitutively expressed genes not regulated by Notch1, were included as negative

controls. Compared with the mock treatment, administration of PU-H71 significantly

decreased the transcription of representative Notch1 targets (Fig. 2D). In support of

this notion, chromatin immunoprecipitation (ChIP) revealed that administration of

PU-H71 in SIL-ALL cells significantly decreased the occupancy of ICN1 on the Hes1

promoter (Fig. 2E). The Actin promoter was used as a negative control. These

findings argue that Hsp90 plays a crucial role in mediation of transcriptional

activation by oncogenic Notch1 in T-ALL cells.




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Hsp90 inactivation causes proteasome-mediated ICN1 degradation

We postulate that stabilization of ICN1 might be a result of protection from

degradation by physical association with the multi-component Hsp90 chaperone

complex. To test this and further delineate the kinetics of ICN1 degradation, we

performed protein stability assays in two T-ALL cell lines (CUTLL1 and SIL-ALL)

that express high levels of ICN1 and are extremely sensitive to γ-secretase inhibitor

treatment. In the γ-secretase inhibitor DAPT pre-treated CUTLL1 cells,

administration of 17-AAG markedly accelerated the turnover of ICN1 protein (Fig.

3A, left panel). Time-course analysis revealed that pharmacological inhibition of

Hsp90 significantly shortened the half-life of endogenous ICN1 from 13.8 hours to

7.4 hours (Fig. 3A, right panel). Similar results were obtained in SIL-ALL (Fig. 3B)

as well as T6E, a murine T-ALL cell line (data not shown). Moreover, Hsp90

inhibition also significantly destabilized ectopically expressed ICN1 upon

cyclohemixide (CHX) pre-treatment to block protein synthesis; time-course analysis

revealed that it significantly shortened the half-life of exogenous ICN1 from 16.3

hours to 8.5 hours (Fig. 3C). Addition of the 26S proteasome inhibitor (MG132)

completely blocked ICN1 destabilization in response to 17-AAG in multiple T-ALL

cell lines (Fig. 3D), further corroborating that Hsp90 regulates ICN1 expression

predominantly by preventing proteasomal degradation. In support of this notion, we

observed that 17-AAG treatment significantly enhanced ICN1 polyubiquitination in

293T cells (Fig. 3E, Lane 2-3). Collectively, these results suggest that acceleration of

ICN1 degradation upon Hsp90 inactivation is strictly dependent on the




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ubiquitin-proteasome pathway.

The ubiquitin E3 ligase Stub1 negatively regulates ICN1

The observation that enhanced ICN1 polyubiquitylation upon 17-AAG treatment

prompted us to identify the E3 ligase that mediates ICN1 degradation. In this regard,

the Hsp90-associated ubiquitin E3 ligase Stub1 (Stip1 homology and U-box

containing protein 1, also called CHIP) is recruited to induce proteasomal degradation

of misfolded or aggregated molecules (17). We depleted Stub1 by a specific shRNA

and then subjected these cells to 17-AAG treatment. In comparison to the mock

treatment, Stub1 depletion largely prevented 17-AAG-induced ICN1 degradation in

HPB-ALL and MOLT-4 cells (Fig. 4A). Consistent with prior findings that Stub1

reduces chaperone efficiency and induces substrate degradation (17, 28, 29), ectopic

expression of Stub1 markedly diminished endogenous ICN1 expression in 293T and

multiple T-ALL cells even when Hsp90 inhibitors were absent (Fig. 4B). Exogenous

Stub1 expression markedly accelerated Flag-tagged ICN1 degradation upon 17-AAG

treatment of 293T cells (Fig. 4C); time-course analysis revealed that it significantly

shortened the half-life of exogenous ICN1 from 9.0 hours to 4.2 hours (Fig. 4D).

Similar to Fbw7, a well-characterized ICN1 E3 ligase (30, 31), wild-type Stub1

significantly inhibited ICN1-induced luciferase activity. In contrast, Stub1 E3

ligase-inactive H260Q mutant or substrate-binding deficient K30A mutant failed to

induce any noticeable effects (Fig. 4E). Co-immunoprecipitation demonstrated a

robust physical interaction of ICN1 and Stub1 when co-expressed in 293T cells (Fig.




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4F). As such, only wild-type Stub1, but not its inactivating H260Q or K30A mutant,

resulted in robust ICN1 polyubiquitination, and Hsp90 inhibition by PU-H71 further

enhanced polyubiquitination densities (Fig. 4G). In sum, these results identify Stub1

as the E3 ligase that is largely responsible for ICN1 degradation via the

ubiquitin-proteasome pathway upon Hsp90 inhibition in T-ALL cells.

Hsp90 inhibition induces T-ALL cell apoptosis in vitro and impedes xenograft

growth in vivo

To determine the biological outcomes of Hsp90 inhibition in T-ALL, we first

assessed the effect of 17-AAG or PU-H71 on survival of multiple T-ALL cell lines

that harbor Notch1 gain-of-function mutations and rely on Notch1 activity for

efficient expansion. In T-ALL cell lines we tested, 17-AAG or PU-H71 induced robust

apoptosis (Supplementary Fig. S1A) and effectively decreased cell viability in a

dose-dependent manner (Fig. 5A). Notch1 activation was shown to sustain aerobic

glycolysis (the Warburg effect) in T-ALL cells (32, 33). Consistently, we found that

Hsp90 pharmacological inhibitors, which caused ICN1 depletion, markedly inhibited

glucose uptake and subsequent lactate secretion in HPB-ALL and CUTLL1 cells

(Supplementary Fig. S1B). To test if ICN1 degradation contributes to the anti-tumor

effects of Hsp90 inhibition in T-ALL cells, we overexpressed ICN1 in HPB-ALL cells,

and found that ectopic ICN1 expression partially but significantly rescued 17-AAG

induced growth inhibition (Fig. 5B), arguing that ICN1 degradation is an important

route that mediates the cytotoxic effects of Hsp90 inhibition.




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To translate the effects of Hsp90 deficiency in vitro into an in vivo setting, we

assessed leukemia burden in T-ALL xenograft bearing mice with or without

PU-H71, which had been shown great efficacies in various preclinical tumor models

(15, 34-36) and is currently under clinical examination (27). For this, CUTLL1 cells

were intravenously injected into immunodeficient NSI mice. Engrafted mice were

randomized and respectively treated with 75 mg/kg PU-H71 or vehicle. This dose is

well tolerated with minimal body weight loss (Supplementary Fig. S2A), consistent

with previous reports that chronic PU-H71 (75 mg/kg) therapy is not associated with

significant toxicities (27, 34). The percentage of human CD45+CD8+ leukemic cells

was significantly reduced in bone marrow and spleen of each PU-H71-treated mouse

(Fig. 5C and Supplementary Fig. S2B), resulting in bones with more reddish color and

spleens with much smaller sizes (Fig. 5C and Supplementary Fig. S2C). H&E staining

showed that, in comparison to vehicle treatment, PU-H71 significantly reduced

lymphoblastic leukemia burdens in bone marrow and subsequent leukemia cell

infiltration into spleens and livers (Supplementary Fig. S3). Consistent with the in

vitro results shown in Fig. 2B and Fig. 5A, splenocytes from PU-H71-treated mice

exhibited reduced ICN1 and PCNA (a marker indicating cell proliferation)

immunochemical staining (Fig. 5D). As such, Hsp90 inactivation significantly

decreased Notch1 target gene expression in sorted human CD45+ splenocytes

(Supplementary Fig. S2D), arguing that PU-H71 functions in part through Notch1

downregulation. Administration of PU-H71 did not substantially improve overall

survival (Supplementary Fig. S2E), suggesting that PU-H71, as a single agent, is




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insufficient for a prolonged survival in our disease model. Most likely, a drug

combination with a standard chemotherapy would achieve a more desirable outcome.

PU-H71 impedes T-ALL leukemia progression in a murine allograft model

Notch1 gain-of-function mutations at the heterodimerization domain are weak

alleles to elicit leukemogenesis but capable of accelerating T-ALL development in

K-rasG12D transgenic background (4). We obtained these primary T-ALL cells driven

by K-rasG12D and Notch1L1601P mutants (Notch1L1601P is expressed in MigR1 virus

with GFP as a surrogate marker) and also found that Hsp90 antagonism decreased

ICN1 levels (Fig. 6A) and its transcriptional activity (Supplementary Fig. S4A).

These murine T-ALL cells were then intravenously injected into half-lethally

irradiated C57BL/6 mice for a secondary transplant. After three weeks of 50mg/kg of

either vehicle or PU-H71 treatment, mice were sacrificed and assessed for in vivo

leukemia cell expansion (Fig. 6B). PU-H71 treatment significantly reduced the GFP+

leukemic cell percentages in the bone marrows and spleens (Fig. 6C and 6D). Murine

T-ALL cell population characterized by CD4+CD8+ positive staining were markedly

reduced in PU-H71 treated mice compared with the vehicle treated group (Fig. 6E and

6F). Moreover, PU-H71 treatment ameliorated splenomegaly (Fig. 6G) and leukemia

cell infiltration into spleens (Fig. 6H). Again, the Notch1 targets were significantly

downregulated in GFP+ splenocytes from PU-H71-injected mice (Supplementary Fig.

S4B). Altogether, these results show that pharmacological inactivation of Hsp90

reduces leukemia burden in vivo and provide proof-of-concept in administration of




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Hsp90 inhibitors as a potential therapeutic regimen for Notch1-addicted T-ALLs.

Discussion

Much progress has been made in identifying transcriptional oncogenic programs

activated by Notch1 during T-ALL pathogenesis. Yet not much is known about the

molecular mechanisms sustaining the aberrant Notch1 activities, especially those

critical for Notch1 stabilization. In the current study, we show that Hsp90 is

responsible for aberrant ICN1 accumulation in T-ALL cells. Upon Hsp90 inhibition,

the ubiquitin E3 ligase Stub1 mediates ICN1 polyubiquitination and subsequent

proteasome-dependent degradation. These data describe a previously unsuspected

pathway, amenable to pharmacological manipulation, which mediates ICN1 stability.

Conventional chemotherapy is still the mainstay treatment guideline for patients

with T-ALL and overall prognosis of T-ALL remains unsatisfied (2, 37, 38). Effective

targeted therapies are currently lacking. Inhibition of Notch by γ-secretase inhibitors

have not achieved impressive efficacy and yielded considerable side-effects (19). In

this report, we show that Hsp90 inhibition depletes ICN1 expression and two distinct

Hsp90 inhibitors (17-AAG and PU-H71) demonstrate efficacy in Notch1-dependent

T-ALL cells and murine models. These effects were associated with dose-dependent,

potent in vitro and in vivo inhibition of Notch1 activation (via ICN1 degradation) and

downstream target expression. In addition to T-ALL, aberrant Notch activation has

been identified in ovarian cancer, breast cancer, lung carcinoma and cancers of the

pancreas and prostate (39). Although not yet FDA approved, the clinical development




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of Hsp90 inhibitors is making steady progress. There are currently more than twenty

active clinical trials involving Hsp90 inhibitors. Conceivably, these Hsp90 inhibitors

would act as potential, alternative therapeutic regimens to benefit patients with

Notch-dependent malignancies. More interestingly, we have identified the particular

ICN1 sequence (peptide H) which mediates the interaction between ICN1 and Hsp90.

In principle, cellular delivery of this peptide would block ICN1-Hsp90 interaction and

lead to selective ICN1 degradation, which holds great promise for therapeutic

purposes in Notch-addicted tumors.

Stub1 is a co-chaperone that interacts with Hsp70/90 and substrates. Stub1

remodels Hsp90 machinery and induces proteasome-mediated substrate degradation

when Hsp90 is inactivated (17). Consistently, we demonstrate that 17-AAG-induced

ICN1 turnover is largely blocked when Stub1 is silenced. It is well supported that

Stub1 modulates protein triage decisions that regulate the balance between protein

folding and degradation for charperone substrates. When overexpressed, Stub1

negatively regulates Hsp90 chaperone function evidenced by inhibiting the

glucocorticoid receptor, a well-characterized Hsp90 client (29). Similarly, we show

here enhanced expression of Stub1 promotes ICN1 polyubiquitination and subsequent

protein degradation, suggesting a critical role of Stub1 in the triage decision of ICN1

protein folding or degradation. This observation raises a possibility that manipulation

of Stub1 levels may affect T-ALL cell growth. Indeed, depletion of Stub1 significantly

enhanced whereas its overexpression inhibited T-ALL cell growth in vitro

(Supplementary Fig. S5). Considering the negative roles of Stub1 in regulation of




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oncogenic substrates, it is unsurprising that Stub1 could play a tumor suppressive role

(40-42). Whether manipulation of Stub1 offers a therapeutic opportunity in cancer

treatment awaits more vigorous in vivo investigation using complementary disease

models.

As inhibition of Hsp90 simultaneously downregulates multiple oncogenic client

proteins crucial for cell viability and tumor development, it is likely that the effects of

17-AAG and PU-H71 result from inhibition of multiple target proteins in addition to

ICN1. Several oncogenic Hsp90 substrates, including AKT (43), JAK (36) and Tyk2

(44, 45), also play important roles in T-ALL pathogenesis. By adding an important

new member in this list, our data provide a rationale for immediate clinical

development of Hsp90 inhibitors in treating T-ALL and other Notch1-addicted

malignancies.

Disclosure of Potential Conflicts of Interest

The authors declare no competing financial interests.

Authors' Contributions

Conception and design: G. Qing

Development of methodology: Z. Wang, Y. Hu

Acquisition of data (provided animals, acquired and managed patients, provided

facilities, etc.): Z. Wang, Y. Hu, D. Xiao, P. Li

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,

computational analysis): Z. Wang, Y. Hu, D. Xiao, H. Liu, G. Qing

Writing, review, and/or revision of the manuscript: Z. Wang, Y. Hu, H. Liu, G.




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Qing

Administrative, technical, or material support (i.e., reporting or organizing data,

constructing databases): J. Wang, C. Liu, Y. Xu, X. Shi, P. Jiang, L. Huang

Study supervision: G. Qing

Acknowledgements

We thank Drs. Warren Pear (University of Pennsylvania, Philadelphia, USA) and

Bin Li (Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China)

for generous provision of reagents.

Grant Support

This work was supported by grants from the National Natural Science Foundation

of China (81470332 to HL and 81372205 to GQ).

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Figure legends

Figure 1. ICN1 interacts with the Hsp90 chaperone.

(A) Total cell extracts prepared from 293T cells expressing Flag-tagged ICN1 or

vector alone were subjected to immunoprecipitation using anti-Flag beads. Proteins

were resolved by SDS-PAGE and visualized by silver staining. (B) LC-MS/MS

spectrometry of the purified Flag-ICN1 associated peptides corresponding to Hsp90.

(C) Flag-tagged ICN1 and/or HA-tagged Hsp90 were overexpressed in 293T cells and

subjected to reciprocal co-immunoprecipitation (co-IP) to detect protein interaction.

(D) Co-IP with anti-ICN1 antibody to detect interaction between endogenous Hsp90

and ICN1. (E) Schematic presentation of various ICN1 truncations and potential

Hsp90 binding region (termed as Peptide H) used in Hsp90 binding assays. 293T cells

expressing HA-tagged Hsp90 and/or various Flag-tagged ICN1 truncations were

subjected to co-IP using anti-Flag beads (Lanes 1-8). A Flag epitope followed by a

tentative Hsp90 binding peptide (Peptide H) or a control peptide (Peptide Ctrl) was

synthesized (ABclonal) and subjected to co-IP using anti-Flag beads (Lane 9 and 10).

Figure 2. Hsp90 inactivation downregulates ICN1 expression and its

transcriptional activity.

(A-C) ICN1 proteins were analyzed by immunoblot upon Hsp90 inhibition. ICN1

band intensities were measured by ImageJ and relative intensities are shown. (A)

Multiple T-ALL cells were treated with increasing concentrations of 17-AAG for 24

hours. (B) T-ALL cells were treated with PU-H71 (1 μmol/L) for the indicated time




26

points. (C) Hsp90 was depleted using lentiviruses expressing shRNAs targeting

Hsp90β or GFP (Ctrl). Transduced cells were purified upon puromycin (2μg/mL)

treatment for 48 hours. Before selection, initial transduction efficiencies of shGFP and

shHsp90 were about 60% and 50% respectively. (D) CUTLL1 cells were treated with

PU-H71 (1 μmol/L) or DMSO for 16 hours. Gene expression was quantified by qPCR.

Data shown represent the means (± SD) of triplicate (**P<0.01). (E) ChIP was

performed in SIL-ALL cells treated with DMSO or 1 µmol/L PU-H71 for 16 hours.

Eluted DNAs were analyzed by qPCR with primers flanking the Hes1 CSL binding

site. Data shown represent the means (± SD) of triplicates (**P<0.01, n.s. means not

significant). All the experiments were repeated at least three times.

Figure 3. Hsp90 inhibition accelerates ICN1 proteasomal degradation.

(A-C) Time course analysis of ICN1 levels by immunoblot in CUTLL1 (A) or

SIL-ALL cells (B). Cells were pretreated with 5 μmol/L DAPT for 2 hours, followed

by DMSO or 1 μmol/L 17-AAG treatment for indicated time points. (C) Exogenous

ICN1 was analyzed in 293T cells. 293T cells expressing ectopic Flag-tagged ICN1

were pretreated with cycloheximide (CHX, 100 μg/mL) for 2 hours. Cells were

harvested at the indicated time points upon DMSO or 17-AAG (1 μmol/L) treatment.

ICN1 band intensities were quantified, normalized to β-actin and then normalized to

t=0 controls. Data shown are averages of three independent experiments. (D)

Indicated T-ALL cells were treated with DMSO or 17-AAG (1 μmol/L) for 24 hours.

Before harvest, cells were treated with MG132 (10 µM) for 5 hours as indicated.




27

ICN1 levels were analyzed by immunoblot with β-actin as a loading control. (E) In

vivo ICN1 polyubiquitination assay. 293T cells were transfected with plasmids

expressing HA-tagged ubiquitin and Flag-tagged ICN1, followed by DMSO or

17-AAG (1 μmol/L) treatment for 16 hours. MG132 (10 µmol/L) was added 5 hours

prior to harvest; Cells were subsequently lysed in RIPA buffer (24).

Ubiquitin-conjugated ICN1 proteins were immunoprecipitated with Flag-tag antibody

and subjected to immunoblot with ubiquitin antibody (Lane 1-3). ICN1 and

polyubiquitination from input extract (Lane 4-6) were shown on the right.

Figure 4. Stub1 promotes ICN1 ubiquitination and degradation.

(A) HPB-ALL and MOLT-4 cells were transduced with lentivirus carrying shRNAs

against the control GFP or Stub1. Transduced cells were purified upon puromycin

(2μg/mL) treatment for 48 hours. Before selection, initial transduction efficiency of

shGFP or shStub1 was about 50%. Cells were subsequently treated with 17-AAG (1

μmol/L) for 24 hours. ICN1 and Stub1 protein levels were assessed by immunoblot

with β-actin as a loading control. (B) 293T, HPB-ALL and CUTLL1 cells were

infected with lentiviruses expressing pCDH-Stub1 or empty vector for 48 hours.

Initial transduction efficiencies of pCDH and pCDH-Stub1 were about 40% and 30%

respectively. After 2μg/mL puromycin selection for 48 hours, ICN1 and Stub1

protein levels were assessed by immunoblot with β-actin as a loading control. (C)

293T cells were transfected with Myc-tagged Stub1 and/or Flag-tagged ICN1 for 48

hours. Cells were pre-treated with CHX (100 μg/mL) for 2 hours, followed by




28

17-AAG treatment (1 μmol/L) for indicated time points. ICN1 and Stub1 protein

levels were assessed by immunoblot with β-actin as a loading control. (D) ICN1 band

intensities were quantified, normalized to β-actin and then normalized to t=0 controls.

Data shown are averages of three independent experiments. (E) Hes1 promoter

luciferase activity was determined in 293T cells expressing Flag-ICN1,

Flag-ICN1/Myc-Stub1 (WT or H260Q, K30A mutant) or Flag-ICN1/Myc-Fbw7. Data

shown represent the means (± SD) of triplicates (**P < 0.01, ***P < 0.001, n.s.

means not significant). These experiments were repeated at least three times. (F)

ICN1 and Stub1 protein interaction. Lysates from 293T cells overexpressing

Flag-tagged ICN1 and Myc-tagged Stub1 were subjected to immunoprecipitation

using anti-Flag antibody, and the co-precipitated Stub1 was detected by anti-Myc

antibody. (G) In vivo ICN1 polyubiquitination assay. 293T cells were transfected with

plasmids expressing HA-tagged ubiquitin, Flag-tagged ICN1 and Myc-tagged Stub1

(wild-type or mutants) for 12 hours, followed by PU-H71 (1 µmol/L) or DMSO

treatment for 16 hours. MG132 (10 µmol/L) was included 5 hours prior to harvest;

Cells were subsequently lysed in RIPA buffer (24). Ubiquitin-conjugated ICN1

proteins were immunoprecipitated with Flag-tag antibody and subjected to

immunoblot with ubiquitin antibody.

Figure 5. Hsp90 inhibition exhibits anti-leukemia effects in T-ALL cells and

human T-ALL xenografts.

(A) Assessment of T-ALL cell viability. Notch1-dependent T-ALL cell lines were

treated with 17-AAG or PU-H71 at the various concentrations for 24 hours. Cell




29

viability was determined using CCK8 assay kit. Data shown represent the means

(±SEM) of triplicates (**P<0.01). These experiments were repeated at least three

times. (B) HPB-ALL cells transduced with pCDH or pCDH-ICN1 were subjected to

DMSO or 17-AAG (1 μmol/L) treatment as indicated. Initial transduction efficiencies

of pCDH and pCDH-ICN1 were about 40% and 25%. After puromycin (2μg/mL, 48

hours) selection, viable cells were counted and plotted as shown. Data shown

represent the means (±SEM) of triplicates (**P<0.01). ICN1 overexpression was

confirmed by immunoblot. (C) In vivo CUTLL1 cell expansions in vehicle or PU-H71

treated mice were presented as human CD45+CD8+ percentages in the bone marrow

(BM) or spleen. Representative bone and spleen images from treated or untreated

cohorts are shown at the bottom. (D) Immunohistochemical staining of ICN1 and

PCNA in the spleens from vehicle or PU-H71-treated mice. Representative images are

shown (top: 200×, bottom: 1000×). Relative ICN1 or PCNA staining was quantified

and presented as mean ± SD, five mice per group (**P < 0.01).

Figure 6. PU-H71 reduces tumor burden in a murine T-ALL allograft.

(A) Murine T-ALL cells derived from T-ALL mice driven by K-rasG12D and

Notch1L1601P mutants were treated with PU-H71 (1 μmol/L) for the indicated time

points. ICN1 levels were analyzed by immunoblot and relative band intensities are

shown. (B) Schematic presentation of secondary transplant of T-ALL cells derived

from T-ALL mouse model driven by K-rasG12D and Notch1L1601P mutants. Five days

post-transplantation, PBS (n=5) or PU-H71 (n=5) was administered thrice weekly.




30

After three weeks of treatment, mice were sacrificed and analyzed for T-ALL

progression. (C-D) Leukemia penetrations in the spleen and bone marrow were

assessed by flow cytometry analysis of GFP fluorescence. Representative plots (C)

and GFP+ percentages from all mice (D) are shown. (E-F) Leukemia penetrations in

the spleen and bone marrow were analyzed by flow cytometry analysis of CD4+CD8+

populations. Representative plots (E) and CD4+CD8+ percentages from all mice (F)

are shown. (G) Spleen weights (top) and representative spleen images (bottom) are

presented. (H) Representative spleen H&E stains are shown (top: 200×, bottom:

1000×). *P < 0.05, **P < 0.01.






















Published OnlineFirst January 31, 2017.Clin Cancer Res Zhaojing Wang, Yufeng Hu, Daibiao Xiao, et al. Cell LeukemogenesisStabilization of Notch1 by the Hsp90 Chaperon is Crucial for T

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