WP1130 reveals USP24 as a novel target in T-cell acute lymphoblastic leukemia
T-cell acute lymphoblastic leukemia (T-ALL) is a lymphoid malignancy caused by the oncogenic transformation of immature T-cell progenitors with poor outcomes. WP1130 has shown potent activity against a variety of cancer but whether WP1130 has anti-T-ALL activity is not clear. USP24, one target of WP1130, is one of the largest deubiquitinases and its detailed mechanism is poorly understood. The aim of this study was to explore whether WP1130 could suppress T-ALL and the role of USP24 in T-ALL.
Molecular docking and cellular thermal shift assay were performed to determine whether and how WP1130 directly interact with USP24. Mitochondrial transmembrane potential assay was measured via Rhodamine 123 staining. USP24 was reactivated using the deactivated CRISPR-associated protein 9 (dCas9)-synergistic activation mediator (SAM) system. The in vivo results were examined by tumor xenografts in NOD-SCID mice. All statistical analyses were performed with the SPSS software package.
WP1130 treatment decreased the viability and induces apoptosis of T-ALL cells both in vitro and in vivo. Furthermore, we demonstrated that knockdown of USP24 but not USP9X could significantly induce growth inhibition and apoptosis of T-ALL cells. Oncomine database showed that USP24 expression was upregulated in T-ALL samples and Kaplan–Meier results indicated that the USP24 was negatively but USP9X was positively associated with survival in T-ALL patients. Additionally, we proposed that WP1130 directly interacts with the activity site pocket of USP24 in T-ALL cells, which leads to the decrease of its substrates Mcl-1. Mechanistically, WP1130 induces apoptosis by accelerating the collapse of mitochondrial transmembrane potential via USP24-Mcl-1 axis.
Altogether, using WP1130 as a chemical probe, we demonstrate that USP24 but not USP9X is a novel target in T-ALL cells. Moreover, we uncovered that WP1130 induces apoptosis by accelerating the collapse of mitochondrial transmembrane potential via USP24-Mcl-1 axis. These results provide that USP24-Mcl-1 axis may represent a novel strategy in the treatment of T-ALL and WP1130 is a promising lead compound for developing anti-T-ALL drugs.
KeywordsWP1130 USP24 dCas9-SAM T-cell acute lymphoblastic leukemia Mcl-1
T-cell acute lymphoblastic leukemia
T cell lymphoma
deactivated CRISP associated protein 9
synergistic activation mediator
cellular thermal shift assay
ubiquitin C-term hydrolase
T-cell acute lymphoblastic leukemia (T-ALL) is a lymphoid malignancy caused by the oncogenic transformation of immature T-cell progenitors with poor outcomes. It accounts for about 15% of pediatric and 25% of adult ALL cases, and males has higher incidence than females . T cell lymphoma (TCL) and T-ALL were classified together by the World Health Organization (WHO), though the clinical presentation of them is different . Despite some progress have been made, the molecular mechanisms of T-ALL development and the prognostic factors of T-ALL are still not fully understood [3, 4]. Most of the T-ALL patients will die of relapse or drug resistance. Therefore, a more effective treatment is urgently required. To this end, understanding the molecular pathogenesis of this disease may not only provide novel insights into the biology of T-ALL, but also facilitate targeted therapy for the treatment of T-ALL.
WP1130, known as Degrasyn previously , is a reported partially selective deubiquitinase inhibitor for USP9X, USP24 and UCH37. WP1130 has shown potent activity against a variety of cancer , including B-cell lymphoma (BCL) , glioblastoma , non-small cell lung cancer , breast cancer  and other malignant tumors [11, 12, 13]. However, whether WP1130 has anti-T-ALL activity is not clear.
The ubiquitin-specific protease USP24 is one of the largest deubiquitinases belonging to the ubiquitin-specific protease family. It has 2620 amino acids that contain one ubiquitin C-term hydrolase (UCH) domain as the catalytic domain and the ubiquitin-associated domain (UBA) which can bind to the ubiquitin signal on substrate proteins. A few reports show that USP24 is related with Parkinson disease [14, 15]. Through removing the ubiquitin moieties from the protein, USP24 can regulate the stability, localization and interaction of its substrates such as Mcl-1 , p53 , DDB2 , p300  and NCOA4 . Accordingly, USP24 is involved in cell death control, iron metabolism, DNA damage repair, transcription regulation and tumorigenesis [20, 21, 22]. Nevertheless, the role of USP24 in cancer is far from clear.
In this study, using WP1130 as a chemical probe, we found that USP24 but not USP9X plays important role in the survival of T-ALL cells. We further demonstrated that targeting USP24-Mcl-1 axis may represent a novel strategy in the treatment of T-ALL. Developing USP24 specific inhibitor warrants further investigation.
Materials and methods
Antibodies and reagents
Antibodies against USP9X (sc-365353) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-USP24 (ab72241) was purchased from Abcam. Antibodies against Mcl-1 (#94296), PARP-1 (#9532), caspase 3 (#9662), cleaved caspase-3 (#9661), K48-Ub (#8081), K63-Ub (#5621) were purchased from Cell Signaling Technology (CST). Lipofectamine 2000 (11668-019) was purchased from Invitrogen. WP1130 were purchased from Targetmol Company (Manassas, VA, USA).
The human T-cell lymphoma cell lines Jurkat, Molt-4, HPB and CCRF-CEM were purchased from the ATCC (Manassas, VA, USA) and cultured in RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen). All cells were cultured and maintained at 37 °C in 5% CO2 incubator. PBMCs were isolated from the blood of T-ALL patients or healthy volunteers by Ficoll-Hypaque (Pharmacia, Piscataway, NJ, USA) density sedimentation.
Cell viability assay
The cell viability was detected by Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Tumor cells (3 × 104/well) were seeded onto 96-well plates at a total volume (containing the WP1130 at the indicated doses) of 200 µL per well for 24 h. Then, 10 µL/well CCK-8 solutions were added and incubated with the plates for another 4 h, and the absorbance was determined at 450 nm using an MRX II microplate reader (Dynex, Chantilly, VA, USA).
Hoechst 33342 and PI double staining
The cell death induction of WP1130 was measured using Hoechst 33342 and PI double staining. The treated cells were washed with PBS and stained with PI (10 μg/mL) and Hoechst 33342 (5 μg/mL) at room temperature for 10 min. Then the indicated cells were observed under fluorescence microscope.
Real-time quantitative PCR
The primers sequences for genes used in the study
Cell apoptosis assay
After cancer cells were exposed to WP1130, cells (5 × 105) were centrifuged and washed twice with PBS. Cells were resuspended in 0.5 mL of cold Annexin V binding buffer, and Annexin V-FITC and Propidium iodide were added, samples were incubated at room temperature for 15 min in the dark. Annexin V positive cells were analyzed by flow cytometry using a FACScan analyzer (Becton–Dickinson, USA).
Homology modeling and molecular docking
The homology modeling of USP24 (1689–2039) 3D structure model used SWISS-MODEL  (https://www.swissmodel.expasy.org/), based on the structure of USP9X (protein data bank, PDB code: 5WCH). Covalent binding between USP24 and compound WP1130 were analyzed using software AUTODOCK4. WP1130 was attached to residue CYS1698, then, during the docking, flexible side chain method was used . A water molecule was docked to the binding pocket, which was centered on the residue CYS1698. The connected complex (CYS1698-WP1130) is treated as a fully flexible side chain. The binding site was covered by preparing a 60 × 60 × 60 size of grid box with grid spacing of 0.375 Å of spacing between grid points.
Cellular thermal shift assay (CETSA)
CETSA was performed according to the method described . PBS diluted Jurkat or Molt-4 cells suspensions were freeze-thawed three times with liquid nitrogen. The soluble fraction (lysate) was separated from the cell debris by centrifugation at 20,000g for 20 min at 4 °C. The cell lysates were diluted with PBS and divided into two aliquots, with one aliquot treated with DMSO and the other aliquot with WP1130. After 30 min incubation at room temperature the respective lysates were divided into smaller aliquots (20 μL) and heated individually at different temperatures for 3 min (Veriti thermal cycler, Applied Biosystems/Life Technologies) followed by cooling for 3 min at room temperature. The appropriate temperatures were determined in preliminary CETSA experiments (data not shown). The heated lysates were centrifuged at 20,000g for 20 min at 4 °C in order to separate the soluble fractions from precipitates. The supernatants were transferred to new micro tubes and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blot analysis. Dose effect of WP1130 on the stability of USP24 was evaluated similarly.
Mitochondrial transmembrane potential assay
After being exposed to WP1130, cells (5 × 105) were centrifuged and washed twice with PBS. Cells were suspended in 0.5 mL of cold PBS, and incubated with 10 mg/L Rhodamine 123 (Rh123) at 37 °C for 30 min. Rh123 is a cationic lipophilic fluorochrome that is taken up by mitochondria in proportion to the ΔΨm. Then, 50 mg/L PI, a membrane-impermeable DNA-binding dye, was added to the cells. The fluorescent intensities were determined with flow cytometry (Becton–Dickinson). Ten thousand cells were analyzed in every sample. All data were collected, stored, and analyzed using LYSIS II software (Becton–Dickinson).
Deactivated CRISPR-associated protein 9 (dCas9)-synergistic activation mediator (SAM) system
Sequences of dCas-sgNC or dCas-sgUSP24
RNA interference and transfection
The sequences of shRNA targeting USP9X and USP24
Western blot analysis
Cells were centrifuged and washed twice with PBS and then suspended with cold PBS, followed by addition of an equal volume of 2× cell lysis buffer. The protein concentration was quantified using the Bradford Protein Assay Kit (Thermo, Rockford, IL, USA). Cell lysates were separated by SDS-PAGE and proteins were transferred to nitrocellulose filter membranes (NC) (Millipore, Billerica, MA, USA). The membranes were then incubated with corresponding antibodies (Santa Cruz, USA) at 4 °C overnight. The membranes were washed three times with TBS/T and then incubated with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Protein expression was detected by chemiluminescence (GE Healthcare, Piscataway, NJ, USA).
Xenograft mouse model
NOD-SCID mice bearing Jurkat (2 × 107 per mouse) xenograft tumors were treated with vehicle or WP1130 (25 mg/kg) every 2 days after tumor volumes reached 100 mm3. Tumor volume was calculated with formula mm3 = 0.5 a × b2, where a is the length and b is the width. All animals were handled according to the protocols approved by the Committee for the Humane Treatment of Animals at Shanghai Jiao Tong University School of Medicine.
A Student’s unpaired two-tailed t test was used to assess the statistical significance. Values with P < 0.05 were considered statistically significant. All statistical analyses were performed with the SPSS software package (version 17.0, SPSS, Inc., Chicago, IL).
WP1130 inhibits proliferation and reduces viability of T-ALL cells
WP1130 induces apoptosis of T-ALL cells
Down-regulation of USP24 but not USP9X induces growth inhibition and apoptosis of T-ALL cells
USP24 is overexpressed in T-ALL and negatively associated with survival in patients with T-ALL
WP1130 interacts with USP24 in cells
In order to investigate whether the WP1130 directly interacts with USP24, a cellular thermal shift assay (CETSA) was conducted [25, 29]. As shown in Fig. 5b, with the presence of increasing concentrations of WP1130, the thermal stability of USP24 markedly decreased. Consistent with this, WP1130 markedly decreased the thermal stability of USP24 at different temperatures in Jurkat (Fig. 5c, e) and Molt-4 (Fig. 5d, f) cells. As a negative control, WP1130 did not change the thermal stability of vinculin in cells. These data suggest that WP1130 directly interacts with USP24 in T-ALL cells.
WP1130 induces apoptosis by accelerating the collapse of mitochondrial transmembrane potential via USP24-Mcl-1 axis
WP1130 inhibits T-ALL cells growth in vivo
Notably, WP1130 is not only inhibiting USP9X, but also other DUBs such as USP24, an USP9X closely related DUB . Role of USP9X in cancer is tissue specific and USP9X displays both oncogenic and tumor suppressor activities [32, 33, 34]. Interestingly, different from Peterson’s finding that USP9X is an oncogene in B-cell malignancies , we found that knockdown of USP9X did not induce cell death of T-ALL cells, indicating that the role of USP9X is cell type dependent. Consistent with our findings, Spinella et al. reported that USP9X may play a tumor suppressor role in T-ALL . It is interesting to observe that knockdown of USP9X resulted in a feedback upregulation of USP24, which may keep cell alive through maintaining the stability of Mcl-1. Interestingly, we found that knock down of USP9X shows no effect but knock down of USP24 could recapitulates the effects of WP1130 in T-ALL cells. Currently, the mechanism of knockdown of USP9X-induced upregulation USP24 is not known. However, we showed that the upregulation of USP24 does not occur at transcription level, as knockdown of USP9X does not change the mRNA level of USP24. Our preliminary data showed that USP9X overexpression resulted in the degradation of USP24, suggesting that USP9X may regulate the stability of USP24.
The function of USP24 in cancer is poorly understood. Accordingly, USP24 could function as a tumor suppressor or oncogene in different kind of cancers or at different stage of cancer progression [18, 20]. A previous study reported that USP24 can stabilize MDM2 then decrease the Suv39h1 level resulting in lung cancer metastasis . We showed that USP24 play an important role in the survival of T-ALL cells. Using Oncomine database to assess the expression of USP24 in T-ALL, we found its expression was upregulated compare to normal. T cell lymphoma (TCL) and T-ALL are considered the same disease, differing by the extent of bone marrow infiltration . We performed an analysis using the R2 microarray database and found that the expression of USP24 was negatively but USP9X was positively associated with survival in patients with TCL. Taken together, these findings suggest that the functional role of USP9X/USP24 is cell type dependent and USP24 play a critical role in the T-ALL cell survival. Considering the possible tumor suppressor role of USP9X and oncogene role of USP24 in T-ALL and the feedback regulation between USP9X and USP24, developing a USP24 specific inhibitor is needed.
In our study, using CETSA we have shown that WP1130 can direct target USP24 in T-ALL cells. Performing molecular docking assessments, we predicted that WP1130 covalently bound to residue Cys1698, and formed polar interactions with residues Asn1693 and Gln1771. Of note, a better understanding of binding mode may be achieved through the crystal structure in future pursuits. Consistent with the inhibition of USP24 activity, treatment of T-ALL cells with WP1130 decreased the levels of its substrates Mcl-1 .Therefore, development of a new selective USP24 inhibitor may be a promising therapeutic strategy for T-ALL.
A robust and rapid reactivation system, which used catalytically-deficient Cas9 (dCas9)-synergistic activation mediator (SAM), was been widely used recently [36, 37, 38]. Using this deactivated CRISP-Cas9-SAM system we successfully reactivated USP24 and demonstrate that endogenously induced USP24 could partially rescue the apoptosis induced by WP1130 in Jurkat cells. Furthermore, we show that the mitochondrial apoptotic pathway is involved in WP1130 induced cell death in T-ALL cells. This is most likely due to the downregulation of Mcl-1 protein. Mcl-1 belongs to the anti-apoptotic proteins of the Bcl-2 family . It helps to maintain mitochondrial integrity, prevent the release of pro-apoptotic factors from the intermembrane space into the cytosol and the subsequent caspase activation . Therefore, our data suggests that WP1130 induces apoptosis by accelerating the collapse of mitochondrial transmembrane potential via USP24-Mcl-1 axis.
Taken together, using WP1130 as a chemical probe, we demonstrate that USP24 but not USP9X is a novel target in T-ALL cells. Moreover, we uncovered that WP1130 induces apoptosis by accelerating the collapse of mitochondrial transmembrane potential via USP24-Mcl-1 axis. These results provide evidence that targeting USP24-Mcl-1 axis may represent a novel strategy in the treatment of T-ALL and WP1130 is a promising lead compound for developing anti-T-ALL drugs.
LH, JB designed the experiments, performed the research, analyzed the data, and wrote the paper. TY, ZL, YL and YJM devoted to the collection of clinical samples. XY, ZYG and HM conducted the cell culture and western blot. CHY, LH and XHZ did literature research and data analysis. WYL, LCX wrote and revised the manuscript. WYL offered professional advices about the whole research. All authors read and approved the final manuscript.
We would like to give our sincere gratitude to the reviewers for their constructive comments.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
The experiments were approved by the Clinical Investigational Review Board of Shanghai Jiao Tong University School of Medicine and performed in accordance with the relevant guidelines and regulations, and informed consent was obtained from all the subjects.
This work was supported in part by grants from the National Key Research and Development Program of China (No. 2017YFA0505200), the National Basic Research Program of China (973 Program) (No. 2015CB910403), the National Natural Science Foundation of China (81570118, 81570112, 81670139,81873436), the Science and Technology Committee of Shanghai (15401901800), and Natural Science Foundation of Shanghai (16ZR1427800).
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