USP44 accelerates the growth of T-cell acute lymphoblastic leukemia through interacting with WDR5 and repressing its ubiquitination

T-cell acute lymphoblastic leukemia (T-ALL) is a common hematologic malignancy. Based on the data from GSE66638 and GSE141140, T-ALL patients depicted a higher USP44 level. However, its role in T-ALL is still unclear. In the present study, we investigated the role of USP44 in T-ALL growth. USP44 overexpression elevated the proliferation of CCRF-CEM cells, while USP44 knockdown suppressed the proliferation of Jurkat and MOLT-4 cells. In addition, USP44 accelerated the cell cycle progression, with boosted cyclinD and PCNA levels. However, USP44 knockdown induced apoptosis in Jurkat and MOLT-4 cells, with an upheaval among cleaved caspase-3 and PARP levels. Mechanistically, USP44 co-localized and interacted with WDR5, leading to the repression of its ubiquitination and degradation. Interestingly, WDR5 overexpression abolished the apoptosis induced by USP44 knockdown. Consistently, the in vivo study revealed that USP44 knockdown restricted the leukemic engraftments in the bone marrow and spleens and reduced the infiltration of T-ALL cells in the livers and lungs. In conclusion, this study indicated that USP44 enhanced the growth of T-ALL through interacting with WDR5 and repressing its ubiquitination. This study highlights the potential use of USP44 as a therapeutic target of T-ALL.


Introduction
T-cell acute lymphoblastic leukemia (T-ALL), a common malignancy in the hematologic system, is arisen from genetic lesion accumulation during T-cell development, leading to the differentiation arrest and abnormal proliferation of immature progenitors.
During T-ALL, leukocytosis initiates and infiltrates within the lymph nodes and other organs [1]. T-ALL accounts for 15% of pediatric and 25% of adult ALL patients [1]. So far, chemotherapy is the primary treatment for T-ALL. However, high-intensity chemotherapy will result in short-term and long-term side effects, lacking effective targeted therapy. Therefore, it is urgent to understand the molecular pathogenesis of T-ALL.
Ubiquitination, a form of post-translation modification, leads to protein degradation. The ubiquitination/deubiquitination system is critical in various physiological events by participating in intracellular signaling pathways [2,3]. Ubiquitinspecific protease 44 (USP44), a kind of deubiquitinase, is implicated in modulating innate immune responses [4], spindle checkpoints and centrosome positioning [5], stem cell differentiation [6] and DNA damage response [7]. Recently, several studies revealed the characteristics of USP44 in cancers. USP44 is highly expressed in gastric cancer and glioma [8,9]. In contrast, it is poorly expressed in lung, colon, breast, renal and pancreatic cancers [10][11][12][13][14], and is implicated in their growth and metastasis. Interestingly, Ying et al. demonstrated a high USP44 level in T-ALL [15], with little knowledge about its role.
The WD repeat protein family members regulate various cellular events and are considered as potential targets for disease treatment [16]. WD repeat domain 5 (WDR5) is a core component of the histone H3 lysine 4 (H3K4) methyltransferase complex, which catalyzes Ivyspring International Publisher histone H3K4 tri-methylation (H3K4me3) [17]. H3K4me3 is associated with the transcription activation of target genes [18,19] and regulates the expression of genes associated with protein synthesis [20]. On the other hand, WDR5 is also a crucial co-activator for recruiting transcription factors, including c-Myc, to chromatin and transcription activation [21,22]. It is implicated in multiple biological processes, such as tumorigenesis, vertebrate development, and self-renewal. WDR5 is highly expressed in various cancer cells and is associated with clinicopathological parameters and poor prognoses. The WDR5 expression in T-ALL patients is also higher than normal controls and is related to the increased risk of leukemia [23]. Knockdown of WDR5 suppresses the proliferation of leukemia cells [23]. Based on data from the HitPredict Database (http://www.hitpredict.org/), WDR5 may bind with USP44. However, whether WDR5 mediates the functions of USP44 is unknown.
In this study, the role of USP44 in T-ALL was explored. It was revealed that USP44 accelerated the growth of T-ALL cells by interacting with WDR5 and suppressing its ubiquitination and degradation. This study depicted a potential role of USP44 as a candidate target for treating T-ALL.

Gene expression of USP44
GEO DataSets (GSE66638 and GSE141140) were utilized to analyze the expression of USP44 within T-ALL samples and normal cells.

CCK-8
After infection or transfection for 48 h, the cells were seeded into 96-well plates (4×10 3 /well). According to the instruction, the cell viability was assessed using CCK-8 reagent (KeyGen Biotechnology Co., Ltd, Nanjing, China) at indicated time points. Absorbance at 450 nm was measured with a microplate reader (BIOTEK, Winooski, USA).
Based on the instruction, the cell lysates were incubated with bead-USP44 or bead-WDR5 for immunoprecipitation after measurement of protein concentration. The immunoprecipitates were washed with PBS, eluted with 50 μl elution buffer and subjected to western blot as described above. The antibodies used for the western blot were USP44 (1: 300) and WDR5 (1: 300).

Ubiquitination assay
HA-Ubiquitin, Flag-WDR5, and His-USP44 were co-transfected into HEK-293T cells. After 48 h, the cells were treated using 5 μM MG132 (Aladdin Biotechnology Co., Ltd) for 6 h to repress the proteasomal degradation. After that, the cells were harvested, and the ubiquitination status of WDR5 was determined through immunoprecipitation, as description above. Briefly, the whole cell lysates were immunoprecipitated with bead-flag and subjected to western blot. The antibodies utilized in western blot were flag (1: 3000, Abcam Technology Co., Ltd, Cambridge, UK), his (1: 1000; Beyotime Biotechnology Co., Ltd, Shanghai, China) and HA (1: 1000; Beyotime Biotechnology Co., Ltd).

Flow cytometry analysis
For cell cycle determination, the cells were incubated with 10 μM BrdU (Aladdin Biotechnology Co., Ltd) for 40 min after indicated treatment and then harvested, fixed, permeabilized and labeled using a FITC-BrdU determination kit (KeyGen Biotechnology Co., Ltd) and a 7-ADD cell cycle determination kit (Bestbio, Changsha, China) based on the protocol. After that, the cells were analyzed through a flow cytometer (NovoCyte, Agilent Technologies, Inc., Santa Clara, USA). For determinating cell apoptosis, the cells were harvested, washed using PBS and stained with a cell apoptosis determination kit (KeyGen Biotechnology Co., Ltd) following the protocol. Thereafter, the cell apoptosis was determined with a flow cytometer.
The femurs and tibias of mice were harvested to obtain the bone marrow. The spleen tissues of mice were also harvested. Cells in the bone marrow and spleens were incubated with CD45 primary antibody (MultiSciences Biotechnology Co., Ltd, Hangzhou, China) at 4 °C for 30 min. Later, the cells were analyzed with a flow cytometry.

Protein half-life assay
In order to determine the half-time of WDR5 in Jurkat cells with USP44 knockdown, the cells were treated with 10 μg/ml CHX (Aladdin Biotechnology Co., Ltd) to block protein translation. After treatment with CHX for 0, 2, 4, and 8 h, the cells were collected, and western blot was conducted to determine the WDR5 protein level.

Animal experiment protocol
Six-week-old NOD-SCID mice were procured from Beijing Huafukang Biotechnology Co., Ltd (Beijing, China) and fed within a standard condition. USP44 knockdown cells (LV-shUSP44-1#) were injected into mice through the caudal vein (2×10 5 ). At the end of the study, mice were sacrificed. The bone marrow and spleens were obtained to determine CD45 + cells. The images and weight of spleens were recorded. The lung and liver tissues were harvested for hematoxylin and eosin (HE) and immunohistochemistry (IHC) staining using CD3. All the experiments were conducted following the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Ethics Committee of Shengjing Hospital of China Medical University.

IHC
The lung and liver tissues were harvested, fixed in 4 % paraformaldehyde, embedded in paraffin, and cut into 5-μm slices. After deparaffinage, rehydration and antigen retrieval, the slices were treated with 3% H 2 O 2 to inactivate endogenous peroxidase, and then blocked with 1% BSA. Then, the slices were incubated with CD3 primary antibody (1: 100; Abclonal Biotechnology Co., Ltd, Wuhan, China) at 4°C overnight. It was followed by incubation with peroxidase-conjugated secondary antibodies (1: 500; ThermoFisher Scientific Technology Co., Ltd, Waltham, USA) for 60 min at 37 °C. The slices were visualized using DAB (MXB Biotechnology, Fuzhou, China) and counterstained with hematoxylin (Solarbio Biotechnology Co., Ltd). The images were captured under a microscope.

Statistical analysis
All the results were represented as mean and SD. Comparison between the groups was analyzed using Student's t test or one-way analysis of variance followed by Tukey's post hoc test. A P-value < 0.05 was considered statistically significant.

USP44 was highly expressed in T-ALL patients
Based on the data from GSE66638 and GSE141140, the USP44 level in T-ALL patients was higher than in normal cells ( Figure 1A). The USP44 level in Jurkat, MOLT-4, and CCRF-CEM cells were determined using quantitative real-time PCR and western blot to further explore the role of USP44 in T-ALL. As shown in Figure 1B-C, Jurkat and MOLT-4 cells revealed a higher USP44 level than the CCRF-CEM cells. Thus USP44 in Jurkat and MOLT-4 cells were knockdown, and USP44 was overexpressed in CCRF-CEM cells through the lentivirus infection system. Thereafter, the infection efficiency was detected by quantitative real-time PCR and western blot. In CCRF-CEM cells, the overexpression of USP44 elevated the USP44 level, while in Jurkat and MOLT-4 cells, knockdown of USP44 decreased the level of USP44 significantly, at both mRNA and protein levels ( Figure 1D-E).

USP44 accelerated the proliferation and cell cycle progression of T-ALL cells
First, we explored the role of USP44 in the proliferation of T-ALL cells. Overexpression of USP44 promoted the proliferation of CCRF-CEM cells. However, the knockdown of USP44 impeded the proliferation of Jurkat and MOLT-4 cells (Figure 2A). To further explore the role of USP44 in cell cycle progression, the cell cycle of T-ALL cells with USP44 overexpression or knockdown was detected with flow cytometry. In cells with USP44 overexpression, the percentages of cells in G2/M phases decreased, while the percentage of cells in S phase increased significantly ( Figure 2B). In cells with USP44 knockdown, the percentage of cells in G2/M phase increased, while those in G0/G1 phase and S phase decreased significantly ( Figure 2B). On the other hand, the protein levels cyclinD and PCNA among cells with USP44 overexpression or knockdown were also determined through western blot. In cells with USP44 overexpression, the levels of PCNA and cyclinD were elevated. In contrast, in cells with USP44 knockdown, the levels of PCNA and cyclinD were reduced ( Figure 2C). These results indicated that USP44 could promote the proliferation and accelerate the cell cycle progression of T-ALL cells.

Knockdown of USP44 induced the apoptosis of T-ALL cells
We explored the role of USP44 in the apoptosis of T-ALL cells. In cells having USP44 knockdown, the total percentage of apoptotic cells was elevated significantly, with both early apoptosis and late apoptosis ( Figure 3A). Meanwhile, the cleaved caspase-3 and cleaved PARP levels were also increased ( Figure 3B). These results depicted that the knockdown of USP44 induced the apoptosis of T-ALL cells. The protein level of USP44 in cells infected with USP44 shRNA or overexpression was determined using western blot. All the results were represented as mean and SD. * p< 0.05, *** p< 0.001.

USP44 interacted with WDR5 and reduced its degradation mediated by ubiquitination
Immunofluorescence staining demonstrated that USP44 was co-located with WDR5 (Overlap coefficient=0.76; Figure 4A). USP44 also interacted with WDR5, evidenced by co-immunoprecipitation ( Figure 4B). As USP44 is a deubiquitinase, we also described the influence of USP44 on the ubiquitination of WDR5. Compared with cells transfected without His-USP44, the ubiquitination level of WDR5 among cells transfected with His-USP44 was reduced ( Figure 4C). It indicated that USP44 interacted with WDR5 and decreased its ubiquitination. As ubiquitination is an important event during protein degradation, we also detected the degradation of WDR5. WDR5 degraded more rapidly in cells with USP44 knockdown when compared with negative control cells ( Figure 4D). We wondered whether USP44 performed its function by targeting WDR5. Thus a WDR5 overexpression was transfected into cells with USP44 knockdown. The efficiency of WDR5 overexpression was evidenced through quantitative real-time PCR and western blot ( Figure 4E-F). Interestingly, the cell viability, inhibited by USP44 knockdown, was elevated after WDR5 overexpression ( Figure 4G). Moreover, the cell apoptosis, induced by USP44 knockdown, was suppressed by WDR5 overexpression ( Figure 4H). These data demonstrated that USP44 might promote the growth of T-ALL cells through interacting with WDR5 and reducing its degradation mediated by ubiquitination.

USP44 knockdown repressed the T-ALL progression in vivo
Jurkat cells with/without USP44 knockdown were injected into NOD-SCID mice to verify the function of USP44 on the progression of T-ALL in vivo. CD45 indicated leukemic cells in the bone marrow and spleens. Compared with the negative control (LV-shNC), the percentage of CD45 + cells in the bone marrow decreased when USP44 was knockdown ( Figure 5A). Also, in mice injected with USP44 knockdown cells, the percentage of CD45 + cells in the spleens was reduced ( Figure 5B). These results demonstrated that USP44 knockdown declined leukemic engraftments in the bone marrow and spleens. In addition, the spleens of mice injected with USP44 knockdown cells were much smaller, with a lower spleen weight than those of negative control mice ( Figure 5C). On the other hand, T-lymphocytes were indicated by CD3. The results of HE and IHC staining with anti-CD3 showed that fewer T-ALL cells were infiltrated into the livers and lungs of mice injected with USP44 knockdown cells than the negative control mice ( Figure 5D). These results supported our hypothesis that knockdown of USP44 repressed the T-ALL progression in vivo.

Discussion
Several USPs are involved in leukemia. For instance, USP15 deletion injures leukemic progenitor function and derepresses antioxidant responses through the Keap1-Nrf2 pathway [24]. Targeting USP47 can overcome the resistance to tyrosine kinase inhibitors and eradicate leukemia progenitor cells [25]. USP15 safeguards the hematopoiesis and genome integrity within hematopoietic stem cells [26]. In our study, USP44 was highly expressed in T-ALL patients and accelerated the proliferation of T-ALL cells, indicating that USP44 could become a candidate target for treating T-ALL. Consistently, USP44 also aggravates the malignancy of glioma and tumorigenesis of prostatic cancer cells [8,27]. Interestingly, the function of USP44 in tumorigenesis could be tumor-dependent. In several cancers, including colon, breast, pancreatic, renal, and non-small cell lung cancer, USP44 depicted a low expression. USP44 suppressed the growth and metastasis, and induced apoptosis [10][11][12][13][14]. The level of USP44 is also associated with tumor grades, survival and poor prognosis [10,13]. Unfortunately, due to a lack of clinical sample information, the association between USP44 and T-ALL stages, survival, and prognosis remains unclear and requires further exploration.
In our study, USP44 contributed to the growth of T-ALL cells. As the cell cycle is a critical regulator of cell growth, we also investigated the role of USP44 in the cell cycle modulation and depicted that USP44 accelerated cell cycle progression. Noticeably, overexpression of USP44 increased the percentage of cells in S phase, in which phase DNA synthesis is processed, indicating that USP44 might promote the synthesis of DNA. In addition, we showed that knockdown of USP44 increased the percentage of cells in G2/M phase. USP44 has a role in mitosis regulation [28], which is an integral part of the cell cycle. Interestingly, USP44 altered mitotic arrest kinetics, leading to a reinforced mitotic checkpoint [15]. Thus, we hypothesized that USP44 knockdown may suppress the mitosis of T-ALL cells, contributing to its role in the growth of T-ALL cells. Consistently, USP44 increases the level of cyclinB [15], which participates in the transition checkpoint from G2 to M phase. Moreover, we revealed that USP44 also up-regulated the level of cyclinD, which participates in the transition checkpoint from G1 to S phase. This is consistent with our speculation that USP44 could accelerate DNA synthesis.   USP44 also regulated apoptosis of cancer cells in a cell-dependent way. Interestingly, USP44 induced the apoptosis of colon cancer cells [11], while suppressed the apoptosis of glioma [8]. In our study, we demonstrated that USP44 knockdown elevated the apoptosis of T-ALL cells, revealing a suppression role of USP44 during the apoptosis of T-ALL cells. Meanwhile, the enhanced apoptosis induced by USP44 knockdown was also accompanied by increased caspase-3 and PARP activation, which are essential effectors in apoptotic pathways.
WDR5, an oncogene often found in cancers, is associated with the proliferation, cell cycle, and apoptosis of cancer cells [29][30][31][32]. WDR5 could regulate the expression of cyclinD and DNA damage by H3K4me3 [33,34]. The suppression of WDR5 signal can repress the growth of cancer cells [34]. WDR5 also has a critical role in leukemogenesis [17]. Our study observed that USP44 bound with WDR5, while silencing USP44 accelerated WDR5 degradation, indicating that USP44 bound with WDR5 and regulated its stability. Additionally, USP44 decreased the ubiquitination of WDR5, which could contribute to its function during WDR5 degradation. Furthermore, WDR5 overexpression abolished the effects of USP44 on the proliferation and apoptosis of T-ALL cells, revealing that USP44 functioned in T-ALL cells by interacting with WDR5, thereby suppressing the ubiquitination and degradation of WDR5. Interestingly, the WDR5 degrader inhibits the proliferation of acute myeloid leukemia and reduces the level of chromatin-bound c-myc, which is critical in T-ALL pathogenesis [35]. Hence, we wonder whether USP44 affects the interaction between WDR5 and c-myc, and this needs further exploration in our further study. Other signaling pathways, such as the AKT and β-catenin signals [10,11,30,32], were also modulated by WDR5. These signal pathways may also participate in the function of USP44 in T-ALL. However, more explorations are needed to reveal the underlying mechanisms behind USP44 in T-ALL.
Besides, USP44 and WDR5 are also associated with the metastasis of cancer, such as the migration and invasion capability of cancer cells and vasculogenic mimicry [12,13,27,[36][37][38]. WDR5 is also related to the liver infiltration of leukemia cells [23]. In our study, when USP44 was knockdown, the level of leukemia cells in the bone marrow and spleens was reduced, and the level of leukemia cells infiltrated into the livers and lungs also declined. It indicated that USP44 promotes both the growth and infiltration of leukemia cells. Recently, programmed death-ligand 1 (PD-L1), which is associated with immune escape of cancer cells [39], has caught the attention of researchers. WDR5 was reported to promote the expression of PD-L1 in prostate cancer and associated with its radio-and chemo-resistance [34,40]. We speculated that USP44 could also be associated with the immune escapes of T-ALL cells, which needs further investigation.
Collectively, in the present study, USP44 contributed to the growth of leukemia cells and suppressed their apoptosis. It also regulated the progression of T-ALL in vivo and their infiltration in the livers and lungs. Significant research demonstrated that USP44 interacted with WDR5 and repressed its ubiquitination and degradation, contributing to the role of USP44 in T-ALL. Our results depicted that USP44 could become a potential therapeutic target for treating T-ALL.