The promotive role of USP1 inhibition in coordinating osteogenic differentiation and fracture healing during nonunion.

BACKGROUND: Nonunion is a failure of fracture healing and a major complication after fractures. Ubiquitin-specific protease 1 (USP1) is a deubiquitinase that involved in cell differentiation and cell response to DNA damage. Herein we investigated the expression, function and mechanism of USP1 in nonunion. METHODS AND RESULTS: Clinical samples were used to detect the USP1 expression in nonunion. ML323 was selected to inhibit USP1 expression throughout the study. Rat models and mouse embryonic osteoblasts cells (MC3T3-E1) were used to investigate the effects of USP1 inhibition on fracture healing and osteogenesis in vivo and in vitro, respectively. Histological changes were examined by micro-computerized tomography (Micro-CT), hematoxylin & eosin (H&E) staining and Masson staining. Alkaline phosphatase (ALP) activity detection and alizarin red staining were used for osteogenic differentiation observation. The expression of related factors was detected by quantitative real-time PCR, western blot or immunohistochemistry (IHC). It was shown that USP1 was highly expressed in nonunion patients and nonunion rats. USP1 inhibition by ML323 promoted fracture healing in nonunion rats and facilitated the expression of osteogenesis-related factors and the signaling of PI3K/Akt pathway. In addition, USP1 inhibition accelerated osteogenic differentiation and promoting PI3K/Akt signaling in MC3T3-E1 cells. CONCLUSIONS: USP1 inhibition plays a promotive role in coordinating osteogenic differentiation and fracture healing during nonunion. PI3K/Akt may be the downstream pathway of USP1.

Ubiquitin specific peptidases and prostate cancer.

Protein ubiquitination is an important post-translational modification mechanism, which regulates protein stability and activity. The ubiquitination of proteins can be reversed by deubiquitinating enzymes (DUBs). Ubiquitin-specific proteases (USPs), the largest DUB subfamily, can regulate cellular functions by removing ubiquitin(s) from the target proteins. Prostate cancer (PCa) is the second leading type of cancer and the most common cause of cancer-related deaths in men worldwide. Numerous studies have demonstrated that the development of PCa is highly correlated with USPs. The expression of USPs is either high or low in PCa cells, thereby regulating the downstream signaling pathways and causing the development or suppression of PCa. This review summarized the functional roles of USPs in the development PCa and explored their potential applications as therapeutic targets for PCa.

Discovery and Characterization of BAY-805, a Potent and Selective Inhibitor of Ubiquitin-Specific Protease USP21.

USP21 belongs to the ubiquitin-specific protease (USP) subfamily of deubiquitinating enzymes (DUBs). Due to its relevance in tumor development and growth, USP21 has been reported as a promising novel therapeutic target for cancer treatment. Herein, we present the discovery of the first highly potent and selective USP21 inhibitor. Following high-throughput screening and subsequent structure-based optimization, we identified BAY-805 to be a non-covalent inhibitor with low nanomolar affinity for USP21 and high selectivity over other DUB targets as well as kinases, proteases, and other common off-targets. Furthermore, surface plasmon resonance (SPR) and cellular thermal shift assays (CETSA) demonstrated high-affinity target engagement of BAY-805, resulting in strong NF-κB activation in a cell-based reporter assay. To the best of our knowledge, BAY-805 is the first potent and selective USP21 inhibitor and represents a valuable high-quality in vitro chemical probe to further explore the complex biology of USP21.

Ubiquitin-Specific Proteases (USPs) and Metabolic Disorders.

Ubiquitination and deubiquitination are reversible processes that modify the characteristics of target proteins, including stability, intracellular localization, and enzymatic activity. Ubiquitin-specific proteases (USPs) constitute the largest deubiquitinating enzyme family. To date, accumulating evidence indicates that several USPs positively and negatively affect metabolic diseases. USP22 in pancreatic ß-cells, USP2 in adipose tissue macrophages, USP9X, 20, and 33 in myocytes, USP4, 7, 10, and 18 in hepatocytes, and USP2 in hypothalamus improve hyperglycemia, whereas USP19 in adipocytes, USP21 in myocytes, and USP2, 14, and 20 in hepatocytes promote hyperglycemia. In contrast, USP1, 5, 9X, 14, 15, 22, 36, and 48 modulate the progression of diabetic nephropathy, neuropathy, and/or retinopathy. USP4, 10, and 18 in hepatocytes ameliorates non-alcoholic fatty liver disease (NAFLD), while hepatic USP2, 11, 14, 19, and 20 exacerbate it. The roles of USP7 and 22 in hepatic disorders are controversial. USP9X, 14, 17, and 20 in vascular cells are postulated to be determinants of atherosclerosis. Moreover, mutations in the Usp8 and Usp48 loci in pituitary tumors cause Cushing syndrome. This review summarizes the current knowledge about the modulatory roles of USPs in energy metabolic disorders.

Post-ovulatory aging is associated with altered patterns for small ubiquitin-like modifier (SUMO) proteins and SUMO-specific proteases.

Mammalian oocytes are ovulated arrested at metaphase of the second meiotic division. If they are not fertilized within a short period, the oocyte undergoes several progressive morphological, structural, and molecular changes during a process called oocyte aging. Herein, we focused on those functional events associated with proper cytoskeleton organization and those that correlate with spindle displacement and chromosome misalignment or scatter. Post-translational modifications by Small Ubiquitin-like Modifier (SUMO) proteins are involved in spindle organization and here we demonstrate that the SUMO pathway is involved in spindle morphology changes and chromosome movements during oocyte aging. SUMO-2/3 as well as the SUMO-specific proteases SENP-2 localization are affected by postovulatory aging in vitro. Consistent with these findings, UBC9 decreases during oocyte aging while differential ubiquitination patterns also correlate with in vitro oocyte aging. These results are consistent with postovulatory aging-related alterations in the posttranslational modifications of the spindle apparatus by SUMO and its SENP proteases. These findings are suggestive that such age-related changes in SUMOylation and the deSUMOylation of key target proteins in the spindle apparatus and kinetochore may be involved with spindle and chromosome alignment defects during mammalian oocyte postovulatory aging. Such findings may have implications for ART-related human oocyte aging in vitro regarding the activities of the SUMO pathway and fertilization success.

Human hematopoietic stem cell vulnerability to ferroptosis.

Hematopoietic stem cells (HSCs) have a number of unique physiologic adaptations that enable lifelong maintenance of blood cell production, including a highly regulated rate of protein synthesis. Yet, the precise vulnerabilities that arise from such adaptations have not been fully characterized. Here, inspired by a bone marrow failure disorder due to the loss of the histone deubiquitinase MYSM1, characterized by selectively disadvantaged HSCs, we show how reduced protein synthesis in HSCs results in increased ferroptosis. HSC maintenance can be fully rescued by blocking ferroptosis, despite no alteration in protein synthesis rates. Importantly, this selective vulnerability to ferroptosis not only underlies HSC loss in MYSM1 deficiency but also characterizes a broader liability of human HSCs. Increasing protein synthesis rates via MYSM1 overexpression makes HSCs less susceptible to ferroptosis, more broadly illustrating the selective vulnerabilities that arise in somatic stem cell populations as a result of physiologic adaptations.

Deubiquitinase catalytic activity of MYSM1 is essential in vivo for hematopoiesis and immune cell development.

Myb-like SWIRM and MPN domains 1 (MYSM1) is a chromatin binding protein with deubiquitinase (DUB) catalytic activity. Rare MYSM1 mutations in human patients result in an inherited bone marrow failure syndrome, highlighting the biomedical significance of MYSM1 in the hematopoietic system. We and others characterized Mysm1-knockout mice as a model of this disorder and established that MYSM1 regulates hematopoietic function and leukocyte development in such models through different mechanisms. It is, however, unknown whether the DUB catalytic activity of MYSM1 is universally required for its many functions and for the maintenance of hematopoiesis in vivo. To test this, here we generated a new mouse strain carrying a Mysm1D660N point mutation (Mysm1DN) and demonstrated that the mutation renders MYSM1 protein catalytically inactive. We characterized Mysm1DN/DN and Mysm1fl/DN CreERT2 mice, against appropriate controls, for constitutive and inducible loss of MYSM1 catalytic function. We report a profound similarity in the developmental, hematopoietic, and immune phenotypes resulting from the loss of MYSM1 catalytic function and the full loss of MYSM1 protein. Overall, our work for the first time establishes the critical role of MYSM1 DUB catalytic activity in vivo in hematopoiesis, leukocyte development, and other aspects of mammalian physiology.

USP51/ZEB1/ACTA2 axis promotes mesenchymal phenotype in gastric cancer and is associated with low cohesion characteristics.

poorly cohesive (PC) gastric cancer (GC) (PC-GC) is a distinct histological subtype of GC and is defined as a tumor consisting of isolated or small clusters of tumor cells with poorly differentiated and metastatic characteristics. According to multiple studies, PC-GC is intrinsically heterogeneous, with mesenchymal variants being the most aggressive. However, to date, the molecular mechanisms associated with PC-GC are still not fully understood. This study investigated the role of the USP51/ZEB1/ACTA2 axis in promoting GC metastasis. Single-cell sequencing revealed that E-box binding homeobox 1 (ZEB1) expression was significantly increased in a subpopulation of low-adherent cells and was an independent prognostic factor in GC patients. Furthermore, the bulk transcriptome analysis revealed a significant positive correlation between Ubiquitin Specific Peptidase 51 (USP51), ZEB1, and Actin Alpha 2 (ACTA2), and our data further confirmed that all three were highly co-localized in PC-GC tissues. According to the findings of in vitro and in vivo experiments, USP51 was able to maintain ZEB1 expression to promote ACTA2 transcription, thereby activating the mesenchymal phenotype of GC cells and promoting tumor metastasis. Moreover, USP51 could recruit and activate stromal cells, including M2-like macrophages and fibroblasts, through cancer cells. Clinical data suggested that overexpression of USP51 predicts that patients have difficulty benefiting from immunotherapy and is associated with immune-exclusion tumor characteristics. Collectively, the findings of this study shed light on a key mechanism by which elevated USP51 expression induces Epithelial-mesenchymal transition (EMT) in GC cells, hence facilitating GC cell proliferation, survival, and dissemination. In this view, USP51/ZEB1/ACTA2 may serve as a candidate therapeutic target against GC metastasis.

USP15 promotes pulmonary vascular remodeling in pulmonary hypertension in a YAP1/TAZ-dependent manner.

Pulmonary hypertension (PH) is a life-threatening cardiopulmonary disease characterized by pulmonary vascular remodeling. Excessive growth and migration of pulmonary artery smooth muscle cells (PASMCs) are believed to be major contributors to pulmonary vascular remodeling. Ubiquitin-specific protease 15 (USP15) is a vital deubiquitinase that has been shown to be critically involved in many pathologies. However, the effect of USP15 on PH has not yet been explored. In this study, the upregulation of USP15 was identified in the lungs of PH patients, mice with SU5416/hypoxia (SuHx)-induced PH and rats with monocrotaline (MCT)-induced PH. Moreover, adeno-associated virus-mediated functional loss of USP15 markedly alleviated PH exacerbation in SuHx-induced mice and MCT-induced rats. In addition, the abnormal upregulation and nuclear translocation of YAP1/TAZ was validated after PH modeling. Human pulmonary artery smooth muscle cells (hPASMCs) were exposed to hypoxia to mimic PH in vitro, and USP15 knockdown significantly inhibited cell proliferation, migration, and YAP1/TAZ signaling in hypoxic hPASMCs. Rescue assays further suggested that USP15 promoted hPASMC proliferation and migration in a YAP1/TAZ-dependent manner. Coimmunoprecipitation assays indicated that USP15 could interact with YAP1, while TAZ bound to USP15 after hypoxia treatment. We further determined that USP15 stabilized YAP1 by inhibiting the K48-linked ubiquitination of YAP1. In summary, our findings reveal the regulatory role of USP15 in PH progression and provide novel insights into the pathogenesis of PH.

A ubiquitin-specific protease functions in regulating cell death and immune responses in rice.

Ubiquitin-specific proteases (UBPs) process deubiquitination in eukaryotic organisms and are widely involved in plant development and responses to environmental stress. However, their role in cell death and plant immunity remains largely unknown. Here, we identified a rice lesion mimic mutant (LMM) and cloned its causative gene, LMM22. Both dysfunction and overexpression of LMM22 gave rise to the hypersensitive response-like cell death, reactive oxygen species bursts, and activated defence responses. LMM22 encodes an active UBP that is localised to the endoplasmic reticulum (ER) and displays a constitutive expression pattern in rice. LMM22 interacts with SPOTTED LEAF 35 (SPL35), a coupling of ubiquitin conjugation to ER degradation domain-containing protein that is known to participate in ubiquitination and the regulation of cell death and disease response in rice. Additional analyses suggest that LMM22 can positively regulate and stabilise the abundance of SPL35 protein likely through its deubiquitination activity. These data therefore improve our understanding of the function of UBP in rice innate immune responses by demonstrating that LMM22 functions as a critical regulator of SPL35 in cell death and disease resistance.

Over-expression of USP15/MMP3 predict poor prognosis and promote growth, migration in non-small cell lung cancer cells.

Aberrant ubiquitin modifications caused by an imbalance in the activities of ubiquitinases and de-ubiquitinases are emerging as important mechanisms underlying non-small cell lung cancer (NSCLC) progression. The deubiquitinating enzyme ubiquitin-specific peptidase 15 (USP15) has been identified as an important factor in oncogenesis and a potential therapeutic target. However, the expression profile and function of USP15 in NSCLC remain elusive. In the present study, we investigated the expression pattern and the potential biological functions of USP15 in NSCLC both in cells and animal models. Our data revealed that USP15 was highly expressed in NSCLC tissues and cells compared with normal counterpart. We subsequently knocked down USP15 expression in two NSCLC cell lines, which significantly suppressed cell proliferation. In addition, knocking down USP15 expression reduced NSCLC cell migration and invasion according to the results from Matrigel-Transwell analysis. NSCLC animal model results showed that USP15 knockdown also reduced NSCLC size. Biochemical analysis revealed that USP15 knockdown inhibited matrix metalloproteinase (MMP)3 and MMP9 expression. Furthermore, high levels of USP15 and MMP3 expression were associated with poor prognosis in NSCLC. In conclusion, the results from the present study suggest that the high expression of USP15 promotes NSCLC tumorigenesis. Therefore, it is proposed that USP15 and MMPs may represent novel biomarkers for NSCLC progression and prognosis.

EOAI, a ubiquitin-specific peptidase 5 inhibitor, prevents non-small cell lung cancer progression by inducing DNA damage.

OBJECTIVE: Targeting deubiquitinases (DUBs) has emerged as a promising avenue for anticancer drug development. However, the effect and mechanism of pan-DUB inhibitor EOAI on non-small cell lung cancer (NSCLC) remains to be studied. MATERIALS AND METHODS: The expression of ubiquitin-specific peptidase 5 (USP5) in NSCLC was evaluated by immunohistochemistry. The effect of the USP5 inhibitor, EOAI, on NSCLC cell growth and cell cycle was evaluated by CCK-8 and PI staining. Apoptosis was detected by Annexin V-FITC/PI double staining. Autophagy was examined by LC3 immunofluorescence. Comet assay and γ-H2AX immunofluorescence staining were used to detect DNA damage, and Western blotting was used to detect the expression of apoptosis, cycle, autophagy and DNA damage-related proteins. In vivo experiments demonstrated the effect of EOAI on NSCLC. RESULTS: We also found that USP5 was significantly upregulated in NSCLC tissues in this study. In addition, we show that EOAI can cause DNA damage in NSCLC cells while modulating the transcriptional activity of P53, thereby inducing cell cycle arrest in NSCLC cells, autophagy and apoptosis. In vivo experiments have shown that EOAI can inhibit tumors and synergistically enhance the anti-tumor effect of cisplatin. CONCLUSION: USP5-mediated epigenetic regulation of oncogenes promotes the occurrence of NSCLC, which provides ideas for developing potential targeted therapy.

METTL5 stabilizes c-Myc by facilitating USP5 translation to reprogram glucose metabolism and promote hepatocellular carcinoma progression.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in the world, with a high likelihood of metastasis and a dismal prognosis. The reprogramming of glucose metabolism is critical in the development of HCC. The Warburg effect has recently been confirmed to occur in a variety of cancers, including HCC. However, little is known about the molecular biological mechanisms underlying the Warburg effect in HCC cells. In this study, we sought to better understand how methyltransferase 5, N6-adenosine (METTL5) controls the development of HCC and the Warburg effect. METHODS: In the current study, quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression of METTL5 in HCC tissues and cell lines. Several different cell models and animal models were established to determine the role of METTL5 in glucose metabolism reprogramming and the underlying molecular mechanism of HCC. Glutathione-S-transferase pulldown, coimmunoprecipitation, RNA sequencing, non-targeted metabolomics, polysome profiling, and luciferase reporter assays were performed to investigate the molecular mechanisms of METTL5 in HCC cells. RESULTS: We discovered that METTL5 drove glucose metabolic reprogramming to promote the proliferation and metastasis of HCC. Mechanistically, upregulation of METTL5 promoted c-Myc stability and thus activated its downstream glycolytic genes lactate dehydrogenase A (LDHA), enolase 1 (ENO1), triosephosphate isomerase 1 (TPI1), solute carrier family 2 member 1 (SLC2A1), and pyruvate kinase M2 (PKM2). The c-Box and ubiquitin binding domain (UBA) regions of ubiquitin specific peptidase 5 (USP5) binded to c-Myc protein and inhibited K48-linked polyubiquitination of c-Myc. Further study revealed that METTL5 controled the USP5 translation process, which in turn regulated the ubiquitination of c-Myc. Furthermore, we identified cAMP responsive element binding protein 1 (CREB1)/P300 as a critical transcriptional regulator of METTL5 that promoted the transcription of METTL5 in HCC. In patient-derived tumor xenograft (PDX) models, adenovirus-mediated knockout of METTL5 had a good antitumor effect and prolonged the survival of PDX-bearing mice. CONCLUSIONS: These findings point to a novel mechanism by which CREB1/P300-METTL5-USP5-c-Myc controls abnormal glucose metabolism and promotes tumor growth, suggesting that METTL5 is a potential therapeutic target and prognostic biomarker for HCC.

USP39 stabilizes ß-catenin by deubiquitination and suppressing E3 ligase TRIM26 pre-mRNA maturation to promote HCC progression.

Ubiquitin-specific protease 39(USP39) plays an important role in modulating pre-mRNA splicing and ubiquitin-proteasome dependent proteolysis as a member of conserved deubiquitylation family. Accumulating evidences prove that USP39 participates in the development of hepatocellular carcinoma (HCC). However, little is known about the mechanism especially deubiquitinating target of USP39 in regulating hepatocellular carcinoma (HCC) growth. Here, we prove that USP39 promotes HCC cell proliferation and migration by directly deubiquitin ß-catenin, a key molecular of Wnt/ß-catenin signaling pathway whose abnormal expression or activation results in several tumors, following its co-localization with USP39. In this process, the expression of E3 ligase TRIM26, which is proved to restrain HCC in our previous research, shows a decreasing trend. We further demonstrate that TRIM26 pre-mRNA splicing and maturation is inhibited by USP39, accompanied by its reduction of ubiquitinating ß-catenin, facilitating HCC progression indirectly. In summary, our data reveal a novel mechanism in the progress of HCC that USP39 promotes the proliferation and migration of HCC through increasing ß-catenin level via both direct deubiquitination and reducing TRIM26 pre-mRNA maturation and splicing, which may provide a new idea and target for clinical treatment of HCC.

USP1-regulated reciprocal differentiation of Th17 cells and Treg cells by deubiquitinating and stabilizing TAZ.

The balance between inflammatory T helper type 17 (Th17) and immunosuppressive regulatory T (Treg) cells is critical for maintaining immune homeostasis in the human body and is tightly regulated under healthy conditions. An increasing number of studies have reported that deubiquitinases (DUBs) play a vital role in regulating Th17- and Treg-cell differentiation. However, the biological functions of only a small fraction of DUBs in Th17- and Treg-cell differentiation are well defined. In this study, we identified ubiquitin-specific peptidase 1 (USP1) as a vital regulator of CD4+ T-cell differentiation. USP1 promoted Th17-cell differentiation but attenuated Treg-cell differentiation, thereby promoting the development of inflammatory diseases. Mechanistically, USP1 in CD4+ T cells enhanced the activity of RORγt but promoted the proteasomal degradation of Foxp3 through deubiquitination and stabilization of TAZ in vitro and in vivo. Notably, ML323, a specific inhibitor of the USP1/UAF1 deubiquitinase complex, inhibited Th17-cell differentiation and promoted Treg-cell differentiation in vitro and in vivo, indicating that ML323 might be a promising candidate for the treatment of diseases associated with an imbalance between Th17 and Treg cells. Our study highlights the critical role of USP1 in regulating adaptive immune responses and suggests that USP1 might be a drug target for the treatment of diseases associated with an imbalance between Th17 and Treg cells.

Long non-coding RNA MFSD4A-AS1 promotes lymphangiogenesis and lymphatic metastasis of papillary thyroid cancer.

Lymphatic metastasis is the leading cause responsible for recurrence and progression in papillary thyroid cancer (PTC), where dysregulation of long non-coding RNAs (lncRNAs) has been extensively demonstrated to be implicated. However, the specific lymphatic node metastatsis-related lncRNAs remain not identified in PTC yet. Lymphatic node metastatsis-related lncRNA, MFSD4A-AS1, was explored in the PTC dataset from The Cancer Genome Atlas and our clinical samples. The roles of MFSD4A-AS1 in lymphatic metastasis were investigated in vitro and in vivo. Bioinformatic analysis, luciferase assay and RNA immunoprecipitation assay were performed to identify the potential targets and the underlying pathway of MFSD4A-AS1 in lymphatic metastasis of PTC. MFSD4A-AS1 was specifically upregulated in PTC tissues with lymphatic metastasis. Upregulating MFSD4A-AS1 promoted mesh formation and migration of human umbilical vein endothelial cells and invasion and migration of PTC cells. Importantly and consistently, MFSD4A-AS1 promoted lymphatic metastasis of PTC cells in vivo by inducing the lymphangiogenic formation and enhancing the invasive capability of PTC cells. Mechanistic dissection further revealed that MFSD4A-AS1 functioned as competing endogenous RNA to sequester miR-30c-2-3p, miR-145-3p and miR-139-5p to disrupt the miRNA-mediated inhibition of vascular endothelial growth factors A and C, and further activated transforming growth factor (TGF)-ß signaling by sponging miR-30c-2-3p that targeted TGFBR2 and USP15, both of which synergistically promoted lymphangiogenesis and lymphatic metastasis of PTC. Our results unravel novel dual mechanisms by which MFSD4A-AS1 promotes lymphatic metastasis of PTC, which will facilitate the development of anti-lymphatic metastatic therapeutic strategy in PTC.

The deubiquitinase OTUD1 noncanonically suppresses Akt activation through its N-terminal intrinsically disordered region.

Akt is commonly activated and serves as a valuable target in human cancer. In this study, OTUD1 is identified as an Akt-associated protein and is downregulated upon Akt activation. Ectopic OTUD1 inhibits Akt phosphorylation; however, its deubiquitinase activity contributes only slightly to this effect. A short peptide (OUN-36) located in the OTUD1 N-terminal intrinsically disordered region strongly binds to the Akt PH domain. The residues in the PH domain, which are required for PtdIns(3,4,5)P3 recognition, are also essential for OUN-36 binding. OUN-36 preferentially inhibits Akt-hyperactive tumor cells' proliferation and interferes with Akt cell membrane localization, presumably by disrupting PH domain-PIP3 interaction. Importantly, OUN-36-based therapy efficiently abrogates Akt feedback reactivation in response to MK-2206 treatment and sensitizes cancer cells to chemotherapy and immunotherapy. We therefore show a mechanism by which OTUD1 modulates Akt activity and suggest a potential peptide-based cancer therapeutic strategy implemented by targeting the Akt PH domain.

Targeting ubiquitin-specific protease 8 sensitizes anti-programmed death-ligand 1 immunotherapy of pancreatic cancer.

Programmed death-1 (PD-1) and its ligand programmed death-ligand 1 (PD-L1) help tumor cells evade immune surveillance, and are regarded as important targets of anti-tumor immunotherapy. Post-translational modification of PD-L1 has potential value in immunosuppression. Here, we identified that ubiquitin-specific protease 8 (USP8) deubiquitinates PD-L1. Pancreatic cancer tissues exhibited significantly increased USP8 levels compared with those in normal tissues. Clinically, the expression of USP8 showed a significant association with the tumor-node-metastasis stage in multiple patient-derived cohorts of pancreatic cancer. Meanwhile, USP8 deficiency could reduce tumor invasion and migration and tumor size in an immunity-dependent manner, and improve anti-tumor immunogenicity. USP8 inhibitor pretreatment led to reduced tumorigenesis and immunocompetent mice with Usp8 knockdown tumors exhibited extended survival. Moreover, USP8 interacted positively with PD-L1 and upregulated its expression by inhibiting the ubiquitination-regulated proteasome degradation pathway in pancreatic cancer. Combination therapy with a USP8 inhibitor and anti-PD-L1 effectively suppressed pancreatic tumor growth by activation of cytotoxic T-cells and the anti-tumor immunity was mainly dependent on the PD-L1 pathway and CD8 + T cells. Our findings highlight the importance of targeting USP8, which can sensitize PD-L1-targeted pancreatic cancer to immunotherapy and might represent a novel therapeutic strategy to treat patients with pancreatic tumors in the future.

N6-methyladenosine-modified USP13 induces pro-survival autophagy and imatinib resistance via regulating the stabilization of autophagy-related protein 5 in gastrointestinal stromal tumors.

Secondary resistance to imatinib (IM) represents a major challenge for therapy of gastrointestinal stromal tumors (GISTs). Aberrations in oncogenic pathways, including autophagy, correlate with IM resistance. Regulation of autophagy-related protein 5 (ATG5) by the ubiquitin-proteasome system is critical for autophagic activity, although the molecular mechanisms that underpin reversible deubiquitination of ATG5 have not been deciphered fully. Here, we identified USP13 as an essential deubiquitinase that stabilizes ATG5 in a process that depends on the PAK1 serine/threonine-protein kinase and which enhances autophagy and promotes IM resistance in GIST cells. USP13 preferentially is induced in GIST cells by IM and interacts with ATG5, which leads to stabilization of ATG5 through deubiquitination. Activation of PAK1 promoted phosphorylation of ATG5 thereby enhancing the interaction of ATG5 with USP13. Furthermore, N6-methyladenosine methyltransferase-like 3 (METTL3) mediated stabilization of USP13 mRNA that required the m6A reader IGF2BP2. Moreover, an inhibitor of USP13 caused ATG5 decay and co-administration of this inhibitor with 3-methyladenine boosted treatment efficacy of IM in murine xenograft models derived from GIST cells. Our findings highlight USP13 as an essential regulator of autophagy and IM resistance in GIST cells and reveal USP13 as a novel potential therapeutic target for GIST treatment.