Otud4 promotes the progression of glioblastoma by deubiquitinating cdk1 and activating mapk signaling pathway

ABSTRACT Glioblastoma, IDH-Wild type (GBM, CNS WHO Grade 4) is a highly heterogeneous and aggressive primary malignant brain tumor with high morbidity, high mortality, and poor patient

prognosis. The global burden of GBM is increasing notably due to limited treatment options, drug delivery problems, and the lack of characteristic molecular targets. OTU deubiquitinase 4

(OTUD4) is a potential predictive factor for several cancers such as breast cancer, liver cancer, and lung cancer. However, its function in GBM remains unknown. In this study, we found that

high expression of OTUD4 is positively associated with poor prognosis in GBM patients. Moreover, we provided in vitro and in vivo evidence that OTUD4 promotes the proliferation and invasion

of GBM cells. Mechanism studies showed that, on the one hand, OTUD4 directly interacts with cyclin-dependent kinase 1 (CDK1) and stabilizes CDK1 by removing its K11, K29, and K33-linked

polyubiquitination. On the other hand, OTUD4 binds to fibroblast growth factor receptor 1 (FGFR1) and reduces FGFR1’s K6 and K27-linked polyubiquitination, thereby indirectly stabilizing

CDK1, ultimately influencing the activation of the downstream MAPK signaling pathway. Collectively, our results revealed that OTUD4 promotes GBM progression via OTUD4-CDK1-MAPK axis, and may

be a prospective therapeutic target for GBM treatment. SIMILAR CONTENT BEING VIEWED BY OTHERS _USP18_ DEUBIQUITINATES AND STABILIZES SOX9 TO PROMOTE THE STEMNESS AND MALIGNANT PROGRESSION

OF GLIOBLASTOMA Article Open access 15 May 2025 ATXN3 DEUBIQUITINATES ZEB1 AND FACILITATES EPITHELIAL–MESENCHYMAL TRANSITION IN GLIOBLASTOMA Article Open access 06 March 2025 FBXO22 PROMOTES

GLIOBLASTOMA MALIGNANT PROGRESSION BY MEDIATING VHL UBIQUITINATION AND DEGRADATION Article Open access 23 March 2024 INTRODUCTION Glioblastoma, IDH-Wild type(GBM, CNS WHO Grade 4 [1,2,3])

is a highly heterogeneous and aggressive primary malignant brain tumor with a median survival of 14 months [4, 5]. Despite improved surgical strategies and vigorous radiation and

chemotherapy [6], the prognosis for GBM patients is poor [7]. It is urgent to find new drug targets for the treatment of GBM. OTU deubiquitinase 4 (OTUD4) is a deubiquitinase that belongs to

the Ovarian tumor-associated proteases domain-containing proteins (OTUDs) family. Research confirmed that OTUD4 is a potential predictor of several human cancers [8] and is also involved in

DNA alkylation damage repair, which is important in cancer radiation and chemotherapy [9, 10]. However, the biological function of OTUD4 in GBM has not been elucidated. Here, we report that

OTUD4 is significantly overexpressed in glioblastoma and is important for cell proliferation, invasion, and clonogenic capacity. Mechanistically, on the one hand, we demonstrated a direct

protein interaction between OTUD4 (181-300aa) and CDK1, and OTUD4 regulates the stability of CDK1 by deubiquitination. OTUD4, on the other hand, indirectly stabilizes CDK1 by binding to and

deubiquitinating FGFR1, which then positively affects the MAPK pathway. In conclusion, our findings demonstrated that the OTUD4-CDK1-MAPK axis is critical for promoting GBM progression. Our

study, therefore, may offer insights for finding novel, feasible glioblastoma targets and new anticancer therapies. MATERIALS AND METHODS CELL CULTURE Human glioblastoma cell lines (A172,

LN229, U87MG, and U118MG), human astroglia cells SVGP12, and human embryonic kidney (HEK) 293FT cells were originally obtained from American Type Culture Collection (ATCC, Beijing, China).

All cell lines were tested mycoplasma-negative [11]. Cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco: Grand Island, NY, USA) supplemented with 10% (vol/vol)

fetal bovine serum (Vivacell: Chongqing, China) at 37 °C, in 5% CO2 humid atmosphere. REAGENTS AND ANTIBODIES Cycloheximide (CHX) (Merck: Kenilworth, NJ, USA, CAS: 66-81-9), MG132 (MCE:

Chongqing, China, CAS:133407-82-6). The primary antibodies used are described in Table 1. PLASMIDS, TRANSFECTION, AND INFECTION For OTUD4 and FGFR1 reduction, short hairpin RNA (shRNA)

sequences were synthesized by Sangon biotech (Shanghai, China) and were inserted into the pLKO.1-puro vector (Table 2). Flag-tagged OTUD4 were subcloned into the pCDH-CMV-MCS-EF1-GFP-Puro

vector (Youbio, Hunan, China). The recombinant plasmids expressing HA-tagged wild-type ubiquitin were purchased from Addgene (Beijing, China). HA-tagged-ubiquitin mutant plasmids (K6R, K11R,

K27R, K29R, K33R, K48R, K63R) were purchased from Unibio Biotech (Changsha, China). Plasmids encoding MYC-tagged CDK1 mutants were obtained from GeneCreate Biotech (Wuhan, China). The

transfection and infection experiments were performed as described previously [12]. WESTERN BLOT ANALYSIS For western blot analysis, cell lysates were prepared in RIPA lysis buffer

containing protease inhibitors and phosphatase inhibitors. Proteins of different molecular weights were separated by SDS/PAGE and then transferred onto a polyvinylidene difluoride membrane,

which was blocked with BSA or nonfat milk for 2 h. The membrane was then sequentially incubated with specific primary antibodies and secondary antibodies. We finally visualized it using the

ECL Prime western blot detection system (Thermo Fisher, Shanghai, China). PROXIMITY LIGATION ASSAY (PLA) Proximity ligation assay was carried out using Duolink® In Situ Red Starter Kit

(#DUO92101, Sigma-Aldrich) as described previously [13]. QUANTITATIVE REAL-TIME PCR (QRT-PCR) Total cellular RNA was extracted using TriZol and cDNA was prepared by the Reverse Transcription

Kit (Promega). SYBR qPCR SuperMix Plus (Novo Protein: Shanghai, China) was used for qRT-PCR analyses. Expression levels of the target genes were calculated according to the comparative Ct

method (∆∆CT) (Table 3). MTT ASSAY A total of 1 × 103 cells per well were seeded into a 96-well plate and cultured at 37 °C. 2-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide

(MTT, Sigma) was added and incubated for 2 h, then cultured with 200 µL DMSO per well. Absorbance at a wavelength of 560 nm was measured by a microplate reader to detect the cell

proliferation curves. Repeat the steps for 7 days. TRANSWELL ASSAY A total of 5 × 104 cells per well were seeded into the upper chamber (Corning, Beijing, China, pore size 8-µm) with 200 µL

serum-free medium. For invasion experiments, Matrigel (BD Biosciences) was additionally added to coat the membrane. About 500 µL medium containing 10% FBS was added below the chamber. After

approximately 8–10 h, the migration and invasion cells were fixed with paraformaldehyde solution, stained with crystal violet, and examined for numbers by microscopy after wiping. COLONY

FORMATION ASSAY A total of 1 × 103 cells per well were seeded into a 6-well plate and cultured at 37 °C for 7 days. The colonies were fixed with 4% paraformaldehyde, stained with crystal

violet, and scanned under a scanner. CELL CYCLE ANALYSIS Collected and resuspended cells in 75% ethanol at 4 °C overnight, then centrifugated and washed them with 1× PBS. Finally, stained

cells with RNase A (100 µg/ml) and propidium iodide (PI, 50 µg/ml) for 30 min at 37 °C in the dark, then analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA). UBIQUITINATION AND

PROTEIN HALF-LIFE ASSAY For ubiquitination detection, the required plasmids, such as Flag-OTUD4, MYC-CDK1, and HA-UB were co-transfected into 293FT cells. Cells were treated with the

proteasome inhibitor MG132 (10 µM) for 6–8 h before collection and then used for the evaluation of the ubiquitination levels of CDK1 or FGFR1. For the protein half-life assay, cells were

treated with the protein synthesis inhibitor cycloheximide (CHX), after which cells were collected in a temporal gradient for the indicated durations. Protein levels were analyzed by western

blot analysis and ImageJ. XENOGRAFT STUDIES Animal experiments were approved by the Institutional Animal Care and Use Committee of Southwest University (IACUC-20221028-01) and were

conducted strictly with the “Guidelines for Animal Care and Use” (Ministry of Science and Technology of China, 2006). Four-week-old female nude mice (BALB/c-nu) were purchased from Hunan SJA

Laboratory Animal Co., Ltd (Hunan, China), housed in an SPF chamber with constant temperature and humidity, and were randomly assigned into two groups (12/group). The mice were anesthetized

before injection to reduce pain. Then 1 × 105 human GBM cells stably transfected with shGFP and shOTUD4 were collected, resuspended in 6 µl PBS, and injected into the brain of each mouse.

After that, mice were sterilized with 75% medical alcohol. According to literatures and previous experimental experience, the mice were killed by cervical dislocation after about 4–6 weeks,

and the brains were immersed in 4% paraformaldehyde. Randomization and single blinding were used for following experiments. IMMUNOHISTOCHEMISTRY STAINING (IHC) The samples were dehydrated,

paraffin-embedded, cut into 5–10-μm-thick cross sections, which were sequentially dewaxed, rehydrated, antigen repaired, and inactivated endogenous peroxidase and organisms. After being

sealed with goat serum and incubated with antibodies overnight at 4 °C, a reaction enhancer and an enhanced enzyme-labeled goat anti-rabbit/mouse IgG polymer were added. The sections were

cleaned with PBS between each step. Finally, the DAB system was used to amplify the detection signal, and hematoxylin was used for re-staining, then dehydrated and sealed and observed under

a microscope [14]. IHC assessment was produced by two independent observers (and a third in the case of strong disagreement) in a blinded fashion (i.e., no prior knowledge of clinical

picture and patient outcome) [15]. STATISTICAL ANALYSIS Statistics in the experiments were all performed by GraphPad Prism 7.0. All data in this study were analyzed and presented as mean ± 

standard deviation (SD) of at least three independent experiments. The two-tailed unpaired Student’s t-test was used to compare the results differences between groups. If the _p_-value < 

0.05, the data were confirmed to be significant; *_p_ < 0.05, **_p_ < 0.01, ***_p_ < 0.001. RESULTS OTUD4 IS UPREGULATED IN HUMAN GBM AND POSITIVELY ASSOCIATED WITH POOR PROGNOSIS

To explore the expression of OTUD4 in glioblastoma, we analyzed the TCGA, CGGA, and GlioVis databases and found that compared with matched adjacent normal tissues, the expression level of

OTUD4 in glioblastoma tissues was higher (Fig. 1A), and OTUD4 expression increased with glioma clinical grade deepened (Fig. 1B). Meanwhile, OTUD4 has the highest expression in GBM (the top

level of glioma), and the increase of OTUD4 gene copy number was significantly correlated with its expression level in glioma (Supplementary Fig. 1A, B). The Human Protein Atlas database

also revealed that the positive rate of OTUD4 in glioblastoma was significantly higher than that in normal human tissues (Supplementary Fig. 1C). Consistent with the database results,

immunohistochemical staining on tissue samples from glioma patients showed that the expression level of OTUD4 was significantly increased in glioma, especially in grade 4 tissues (Fig. 1C,

D). Then, we detected that abnormal activation of OTUD4 expression was positively correlated with poor prognosis in GBM patients (Fig. 1E). Additionally, western blot analysis showed that

OTUD4 protein expression in four GBM cell lines was higher than that in astrocytes SVGP12 (Fig. 1F). Taken together, these results indicated that OTUD4 is overexpressed in GBM, and OTUD4

might be a potential predictor of poor prognosis in GBM. OTUD4 PROMOTES GBM CELL PROLIFERATION AND INVASION To further confirm whether OTUD4 affects GBM progression, stable OTUD4

downregulated GBM cell lines were constructed (Fig. 2A). OTUD4-knockdown GBM cells showed a dramatic decrease in cell number, accompanied by significant morphological changes (Supplementary

Fig. 2A). Next, MTT assay showed that OTUD4 knockdown restrained the growth rate of GBM cells (Fig. 2B). Meanwhile, the results of plate cloning assay also revealed that the colony forming

ability of GBM cells was decreased after OTUD4 knockdown (Fig. 2C, Supplementary Fig. 2B). Then, flow cytometry experiment observed that OTUD4 knockdown induced cell cycle arrest mainly in

the G2/M phase (Fig. 2D, Supplementary Fig. 2C). Western blot consistently confirmed that the expression of G2/M-phase signature proteins CDK1 and cyclinB1 reduced, p21 and p27 upregulated

(Fig. 2E). Next, transwell assay and wound-healing assay results indicated that the invasion capacity of GBM cells were markedly decreased after OTUD4 knockdown (Fig. 2F, G, Supplementary

Fig. 3A). Subsequently, we examined marker proteins of invasion, the protein expression levels of N-cadherin and MMP9 were notably reduced, while E-cadherin expression was increased (Fig.

2H). To further avoid the mistarget effect, recovery assays were performed and indicated that OTUD4 overexpression could partially rescue the proliferation and invasion capacity of GBM cells

with OTUD4 knockdown (Supplementary Fig. 2D–F, Supplementary Fig. 3B–D). In addition, overexpression of OTUD4 notably promoted the proliferation and invasion of LN229 cells (Supplementary

Fig. 2G–I, Supplementary Fig. 3E–G). Overall, our results concluded that OTUD4 is a key regulator of the proliferation and invasion of GBM cells. THE 181-300AA OF OTUD4 INTERACTS WITH CDK1

We next explored the mechanism of OTUD4 in glioblastoma. We used the CGGA database for GSEA analysis, and the results showed that the high expression of OTUD4 was significantly positively

correlated with the mitotic and cell cycle G2/M checkpoint in GBM (Fig. 3A, Supplementary Fig. 4A). Western blot analysis in the previous period also verified that, after OTUD4 was knocked

down or overexpressed, the expression levels of CDK1, cyclinB1, p21 and p27 changed correspondingly (Fig. 2E, Supplementary Fig. 2F, I). However, further co-IP experiments showed that only

CDK1 has an obvious and strong interaction with OTUD4 (Fig. 3B). And considering that CDK1 is an important regulator of cell proliferation and invasion [16], it is highly upregulated in GBM

and is positively associated with poor patient prognosis (Supplementary Fig. 4B–D), we ultimately excluded other genes and selected CDK1 as a potential target gene of OTUD4 for follow-up

experiments. Previous experiments discovered that the cell cycle was arrested in the G2/M phase after OTUD4 depletion, and the expression of CDK1 was markedly downregulated, but there was no

significant difference in CDK1 mRNA levels (Supplementary Fig. 4E), indicating that the downregulation of CDK1 may occur at the post-transcriptional level. Ubiquitin-proteasome protein

degradation pathway plays a key role in regulating protein levels. Interestingly, OTUD4 is a deubiquitylase. As expected, ectopic expression of OTUD4 resulted in CDK1 elevation in a

dose-dependent manner (Fig. 3C). Therefore, we speculated that OTUD4 may interact with CDK1 to deubiquitinate and stabilize it. Next, binding assays revealed that endogenous expression OTUD4

and CDK1 have physical interaction (Fig. 3D, E). Moreover, the proximity ligation assay (PLA) assay also corroborated a direct in situ interaction of OTUD4 with CDK1(Fig. 3F). To further

validate their interaction regions, we constructed a set of Flag-tagged plasmids expressing different OTUD4 truncated mutants (full-length 1–1114aa, 1–180aa, 1–550aa, Δ181–300aa, Δ301–550aa,

and 301–1114aa), and MYC-tagged CDK1 deletion mutant plasmids (N-terminal deletion of 1–99aa, intermediate deletion of 100–198aa, C-terminal deletion of 199–297aa) for transfection

experiments. Results demonstrated that the 181–300aa domain of OTUD4 (Fig. 3G) and the full-length of CDK1 (1–297aa) (Supplementary Fig. 4F) mediated their physical interaction. This result

is also consistent with our simulated molecular docking results of HADDOCK database [17,18,19,20,21] (Supplementary Fig. 4G). OTUD4 MAINTAINS CDK1 STABILITY THROUGH DEUBIQUITINATION Next, we

aimed to verify the possibility of the deubiquitination effect by OTUD4 on CDK1. MG132 (an inhibitor of the ubiquitin-proteasome pathway) dramatically blocks the downregulation of CDK1

caused by OTUD4 knockdown (Fig. 4A), suggesting that OTUD4 protects CDK1 from proteasome-dependent degradation. Then, cycloheximide (CHX, an inhibitor of protein synthesis) assay showed that

the half-life of CDK1 was largely prolonged after OTUD4 overexpression, suggesting that OTUD4 specifically stabilizes CDK1(Fig. 4B, Supplementary Fig. 5A). Moreover, ubiquitination assays

showed that the ubiquitination level of CDK1 could dramatically decrease after OTUD4 ectopic expression, and the decrease was concentration gradient dependent (Fig. 4C, D). In contrast, the

downregulation of OTUD4 increased CDK1 polyubiquitylation level (Fig. 4E, Supplementary Fig. 6I). In conclusion, these results confirmed our speculation that OTUD4 directly stabilizes CDK1

by deubiquitination. Meanwhile, further assays showed that only when the full-length or 1–550aa of OTUD4 was overexpressed, the ubiquitination level of CDK1 was significantly reduced. And

when 181–300aa was deleted or 301–1114aa was overexpressed, the ubiquitination level of CDK1 was partially reduced (Fig. 4F). Based on these, we speculated that 301–550aa of OTUD4 is the

catalytic domain for deubiquitination of CDK1, and 181–300aa of OTUD4 might promote the DUB activity of OTUD4 on CDK1 by enhancing their interaction ability. On the one hand, in order to

identify which type of polyubiquitin modifications on CDK1 was affected by OTUD4, we performed a thorough deubiquitylation assay. The results suggested that downregulation of OTUD4

specifically enhanced wild-type and other ubiquitin mutants-linked polyubiquitylation of CDK1, except for K11R, K29R, and K33R (Fig. 4G). It was consistent with the results of ubiquitination

experiments when OTUD4 is overexpressed (Supplementary Fig. 5B). On the other hand, to identify the possible key amino acid residues of CDK1 in this progression, we constructed two

MYC-tagged CDK1 mutants with a single-point mutation (K9R, K56R) according to the PLMD (Protein Lysine Modifications Database) and UbiNet databases (Supplementary Fig. 5C). Co-IP results

indicated that K56 residue of CDK1 is essential for OTUD4 deubiquitination (Fig. 4H). OTUD4 INTERACTS WITH FGFR1 AND STABILIZES FGFR1 BY DEUBIQUITINATION GSEA analysis using the CGGA

database showed that the first two most significantly enriched signaling pathways among the high OTUD4 gene expression phenotype are those associated with FGFR1 and oncogenic MAPK signaling

pathways (Fig. 5A, Fig. 6A, Supplementary Fig. 6A). FGF/FGFR signaling is essential for growth and invasion of human GBM cells [22]. Databases also displays that FGFR1 is highly expressed in

GBM and is positively associated with poor prognosis (Supplementary Fig. 6B, C). Interestingly, studies have shown that FGFR1 signaling pathway can stimulate CDK activity [23] and inhibit

cyclin kinase inhibitors (CDKNs) [24]. As a result, we conducted further experiments to detect their relationship. Co-IP and PLA assays found that OTUD4 interacts with FGFR1(Fig. 5B, C,

Supplementary Fig. 6D). And western blot showed that overexpression of OTUD4 stabilized whereas depletion of OTUD4 destabilized FGFR1, qRT-PCR assays confirmed that OTUD4 positively

regulated FGFR1 at the protein level but not at the mRNA level (Supplementary Fig. 6E–G). Furthermore, MG132 significantly reduces the downregulation of FGFR1 caused by OTUD4 knockdown (Fig.

5D), suggesting that OTUD4 regulates FGFR1 in a manner related to the proteasome degradation pathway. CHX assay found that OTUD4 overexpression prolongs the half-life of FGFR1 (Fig. 5E,

Supplementary Fig. 6H). We next sought to determine the linkage specificity of OTUD4-mediated deubiquitination of FGFR1. To this end, a specific deubiquitylation assay was adopted and

exhibited that OTUD4 effectively identified and removed the K6 and K27-linked polyubiquitination of FGFR1 (Fig. 5F–I, Supplementary Fig. 6I). Next, our results corroborated that FGFR1

interference exacerbated the CDK1 downregulation caused by OTUD4 knockdown, and FGFR1 may maintain CDK1 protein stability by stabilizing the CDK1-cyclinB1 complex (Fig. 5J, K). THE

OTUD4-CDK1-MAPK AXIS IS REQUIRED IN GBM PROGRESSION MAPK pathway is involved in the regulation of tumor cell proliferation and cell cycle, and also plays a mediating and signal amplification

role in tumor invasion, including GBM [25]. Interestingly, both CDK1 and FGFR1 can activate the downstream MAPK signaling pathway [26,27,28,29,30,31,32,33]. Therefore, we speculated that

OTUD4 might regulate CDK1 and the MAPK signaling pathway, ultimately affecting the progression of GBM. Next, we conducted western blot assays in GBM cells and found that OTUD4 knockdown or

FGFR1 knockdown significantly downregulated the phosphorylation levels of proteins in the MAPK signaling pathway without affecting their total protein levels (Fig. 6B, C). While the

downregulation was partially eliminated after CDK1 expression was restored (Fig. 6D). Furthermore, MTT assay, colony formation assay, transwell assay and western blot assay results exhibited

that overexpression of CDK1 could partially restore the phenotypes of GBM cell proliferation and invasion inhibition caused by OTUD4 knockdown, and the expressions of cell cycle and

EMT-related proteins also changed correspondingly (Fig. 6E–H). Hence, we conclude that OTUD4 does affect the activation of the MAPK signaling pathway through the regulation of CDK1, and

promote the proliferation and invasion ability of GBM cells. OTUD4 CONTRIBUTES TO THE GBM PROGRESSION IN MICE Next, we aimed to explore the correlation between OTUD4 and CDK1 in clinical

specimens. IHC assay found that samples with high OTUD4 expression were more likely to have high CDK1 staining intensity. In contrast, CDK1 staining was weak among OTUD4-low staining samples

(Fig. 7A). Spearman correlation test further demonstrated the positive correlation between OTUD4 and CDK1 expression (Fig. 7B). Moreover, prognostic analysis using the CGGA database also

revealed that patients had the worst prognosis when OTUD4 was highly expressed in combination with high CDK1 or FGFR1 expression (Fig. 7C, D). Together, there is an apparent clinical

association of OTUD4-CDK1/FGFR1 in the human glioma development. To further evaluate the effect of OTUD4 on GBM cells proliferation in vivo, orthotopic implantation assay was performed in

BALB/c-nu mice. The H&E results found that the tumor growth capability of GBM cells was significantly decreased after OTUD4 knockdown (Fig. 7E). IHC experiments displayed that the

positive expressions of Ki67(a well-known marker of cell proliferation), OTUD4, CDK1, FGFR1 and p-BRAF were obviously decreased in tumor xenografts with OTUD4 knockdown (Fig. 7F). These

results suggested that OTUD4 is required for GBM progression in vivo. DISCUSSION Glioblastoma is a common primary glioma known for its resistance to chemotherapy and radiation, its tendency

to relapse frequently, and its poor prognosis [1]. It is urgent to find new drug targets for the treatment of GBM. More and more evidence has proved that OTUs is closely related to the

development of glioma. For example, OTUD3 promotes the progression of PTEN-associated glioma [34]. OTUB1 promotes the invasion of glioma cells, and inhibits CNS autoimmune [35, 36]. OTULIN

maintains GSC self-renewal and is associated with poor prognosis in GBM [37]. As a member of the OTU family, OTUD4 has also been shown to affect cancer progression, for example, on the one

hand, OTUD4 acts as an oncogene to facilitate the metastasis of triple-negative breast cancer [38] and melanoma [39]; on the other hand, OTUD4 acts as a tumor suppressor gene to inhibit the

proliferation of liver cancer and non-small cell lung cancer, and regulate the radiosensitivity of nasopharyngeal carcinoma [40]. However, the effects of OTUD4 on GBM have not been reported.

In this study, we report for the first time that OTUD4 may play an oncogenic role in GBM and may be considered as a potential prognostic biomarker for GBM. In addition, we found for the

first time that OTUD4 directly or indirectly regulates the stability of CDK1 through deubiquitination, thus affecting the activation of the downstream MAPK signaling pathway, ultimately

facilitating the GBM process (Fig. 7G). It is generally accepted that OTU domain(1–180aa) is the main catalytic domain of OTUD4. However, studies reported that the deubiquitase recruitment

domain (DRD, 181–550aa) of OTUD4 interacts with and deubiquitinates ALKBH3 [41, 42], intrinsically disordered regions (IDRs, such as 885–1114aa) of OTUD4 interacts with RNA [43], the

ubiquitin interaction motif (UIM, 271–300aa) of OTUD4 promotes its DUB activity against MyD88 [10]. Similarly, in our study, 301–550aa of OTUD4 is the catalytic domain for deubiquitination

of CDK1, and 181–300aa of OTUD4 might promote the DUB activity of OTUD4 on CDK1 by enhancing their interaction ability. These suggested that the domains that mediate the binding of OTUD4 to

different substrates and the domains responsible for its DUB catalytic activity may be different. Moreover, most human OTU enzymes are linkage specific, preferentially cleaving one, two, or

a defined subset of linkage types. For example, OTUD4 has been reported to preferentially cleave K48-linked polyubiquitin chains [41, 44]. Phosphorylation of OTUD4 activates its K63-specific

DUB activity [10]. Consistent with previous findings, our research also confirmed that K11-linked polyubiquitination is closely related to the cell cycle [45], and K11, K29-linked

ubiquitination is essential for proteasomal degradation [46, 47]. In addition, we further speculated that K6-, K27-, and K33-linked ubiquitin chains may also be related to protein

degradation. Currently, CDK1 inhibitors for GBM have achieved certain effects [48, 49]. But deficiencies such as drug resistance have gradually emerged. Our findings established that

OTUD4/CDK1 is clinically correlated and the OTUD4-CDK1-MAPK axis plays an important role in GBM progression. Considering that, our results help to identify new GBM-targeted inhibitor

markers. In conclusion, this study demonstrated that OTUD4 is an oncogene with prognostic significance in GBM, revealed a new mechanism by which OTUD4 directly deubiquitinates or indirectly

regulates CDK1, and identified specific interaction regions and key amino acid residues of ubiquitin and CDK1(Fig. 7G). Accordingly, these results suggest that OTUD4 may serve as a biomarker

for glioblastoma and provide a novel therapeutic target for GBM patients. DATA AVAILABILITY All data analyzed or generated in this study are included in this article as well as in the

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Inhibition of CDK1 as a potential therapy for tumors over-expressing MYC. Nat Med. 2007;13:820–7. Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS This research was

supported by the Natural Science Foundation of Chongqing (cstc2022ycjh-bgzxm0145), the pilot program of Southwest University (SWU-XDZD22006) and the Chongqing postgraduate Research

Innovation Project (CYS22257). We thank all the participants for their contributions to the study. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * State Key Laboratory of Resource Insects,

Medical Research Institute, Southwest University, Chongqing, 400715, China Mingxin Ci, Gaichao Zhao, Chongyang Li, Ruochen Liu, Xiaosong Hu, Jun Pan, Yang Shen, Guanghui Zhang, Yongsen Li 

& Hongjuan Cui * Jinfeng Laboratory, Chongqing, 401329, China Mingxin Ci, Gaichao Zhao, Chongyang Li, Ruochen Liu, Xiaosong Hu, Jun Pan, Yang Shen, Guanghui Zhang, Yongsen Li & 

Hongjuan Cui * Department of Radiology and Nuclear Medicine, The First Hospital of HeBei Medical University, Hebei, 050000, China Li Zhang * Department of Neurosurgery, Children’s Hospital

of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing, 400014,

China Ping Liang Authors * Mingxin Ci View author publications You can also search for this author inPubMed Google Scholar * Gaichao Zhao View author publications You can also search for

this author inPubMed Google Scholar * Chongyang Li View author publications You can also search for this author inPubMed Google Scholar * Ruochen Liu View author publications You can also

search for this author inPubMed Google Scholar * Xiaosong Hu View author publications You can also search for this author inPubMed Google Scholar * Jun Pan View author publications You can

also search for this author inPubMed Google Scholar * Yang Shen View author publications You can also search for this author inPubMed Google Scholar * Guanghui Zhang View author publications

You can also search for this author inPubMed Google Scholar * Yongsen Li View author publications You can also search for this author inPubMed Google Scholar * Li Zhang View author

publications You can also search for this author inPubMed Google Scholar * Ping Liang View author publications You can also search for this author inPubMed Google Scholar * Hongjuan Cui View

author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS HC, PL, LZ, and MC conceived and designed the experiments. MC, GaZ, JP, YS, GuZ, and YL

performed the experiments, collected the data. MC, GaZ, and XH analyzed the data and prepared the figures. MC, GaZ, CL, RL, and HC wrote and revised the manuscript. All the authors

contributed to the article and approved the final manuscript. CORRESPONDING AUTHORS Correspondence to Li Zhang, Ping Liang or Hongjuan Cui. ETHICS DECLARATIONS COMPETING INTERESTS The

authors declare no competing interests. ETHICS APPROVAL AND CONSENT TO PARTICIPATE Animal experiments were approved by the Institutional Animal Care and Use Committee of Southwest University

(IACUC-20221028-01) and were conducted strictly with the “Guidelines for Animal Care and Use” (Ministry of Science and Technology of China, 2006). ADDITIONAL INFORMATION PUBLISHER’S NOTE

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and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Ci, M., Zhao, G., Li, C. _et al._ OTUD4 promotes the progression of glioblastoma by deubiquitinating CDK1 and activating MAPK signaling

pathway. _Cell Death Dis_ 15, 179 (2024). https://doi.org/10.1038/s41419-024-06569-x Download citation * Received: 15 July 2023 * Revised: 15 February 2024 * Accepted: 21 February 2024 *

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