USP7 Knockdown Research Articles

USP7-stabilised HIPK2 promotes high glucose-induced endothelial cell dysfunctions to accelerate diabetic foot ulcers

Background: This study aimed to explore the molecular mechanism of homeodomain-interacting protein kinase 2 (HIPK2) in diabetic foot ulcers (DFU). Methods: High glucose (HG)-induced human umbilical vein endothelial cells (HUVECs) were used to construct DFU cell models. Cell functions were determined using CCK8 assay, EdU assay, flow cytometry, transwell assay, wound healing assay and tube formation assay. Quantitative real-time PCR and western blot were applied to measure the gene expression. Results: HG treatment suppressed HUVECs proliferation, invasion, migration, and angiogenesis, while enhanced apoptosis. HIPK2 was overexpressed in DFU patients, and its knockdown alleviated HG-induced HUVECs dysfunctions. USP7 stabilised HIPK2 protein by reducing its ubiquitination. USP7 overexpression promoted HG-induced HUVECs dysfunctions, and HIPK2 upregulation also reversed the regulation of USP7 knockdown on HG-induced HUVECs dysfunctions. USP7/HIPK2 axis inhibited the activity of PI3K/AKT pathway. Conclusion: Our study revealed that USP7-stabilised HIPK2 contributed to HG-induced HUVECs dysfunctions, thus accelerating DFU process.

Structural and functional characterization of USP47 reveals a hot spot for inhibitor design

USP47 is widely involved in tumor development, metastasis, and other processes while performing a more regulatory role in inflammatory responses, myocardial infarction, and neuronal development. In this study, we investigate the functional and biochemical properties of USP47, whereby depleting USP47 inhibited cancer cell growth in a p53-dependent manner—a phenomenon that enhances during the simultaneous knockdown of USP7. Full-length USP47 shows higher deubiquitinase activity than the catalytic domain. The crystal structures of the catalytic domain, in its free and ubiquitin-bound states, reveal that the misaligned catalytic triads, ultimately, become aligned upon ubiquitin-binding, similar to USP7, thereby becoming ready for catalysis. Yet, the composition and lengths of BL1, BL2, and BL3 of USP47 differ from those for USP7, and they contribute to the observed selectivity. Our study provides molecular details of USP47 regulation, substrate recognition, and the hotspots for drug discovery by targeting USP47.

Inhibition of USP7 induces p53-independent tumor growth suppression in triple-negative breast cancers by destabilizing FOXM1.

Although numerous studies indicate that inhibition of USP7 suppresses tumor growth by activating p53, the precise mechanism by which USP7 contributes to tumor growth through the p53-independent manner is not well understood. p53 is frequently mutated in most triple-negative breast cancers (TNBC), characterized as the very aggressive form of breast cancers with limited treatment options and poor patient outcomes. Here, we found that the oncoprotein Forkhead Box M1 (FOXM1) acts as a potential driver for tumor growth in TNBC and, surprisingly, through a proteomic screen, we identified USP7 as a major regulator of FOXM1 in TNBC cells. USP7 interacts with FOXM1 both in vitro and in vivo. USP7 stabilizes FOXM1 through deubiquitination. Conversely, RNAi-mediated USP7 knockdown in TNBC cells, dramatically reduced the levels of FOXM1. Moreover, based upon the proteolysis targeting chimera (PROTAC) technology, we generated PU7-1 (protein degrader for USP7-1), as a USP7 specific degrader. PU7-1 induces rapid USP7 degradation at low nanomolar concentrations in cells but shows no obvious effect on other USP family proteins. Strikingly, the treatment of TNBC cells with PU7-1 significantly abrogates FOXM1 functions and effectively suppresses cell growth in vitro. By using xenograft mouse models, we found that PU7-1 markedly represses tumor growth in vivo. Notably, ectopic overexpression of FOXM1 can reverse the tumor growth suppressive effects induced by PU7-1, underscored the specific effect on FOXM1 induced by USP7 inactivation. Together, our findings indicate that FOXM1 is a major target of USP7 in modulating tumor growth in a p53-independent manner and reveals the USP7 degrader as a potential therapeutic tool for the treatment of triple-negative breast cancers.

USP7 imparts partial EMT state in colorectal cancer by stabilizing the RNA helicase DDX3X and augmenting Wnt/β-catenin signaling

Epithelial mesenchymal transition (EMT) is a fundamental and highly regulated process that is normally observed during embryonic development and tissue repair but is deregulated during advanced cancer. Classically, through the process of EMT, cancer cells gradually transition from a predominantly epithelial phenotype to a more invasive mesenchymal phenotype. Increasing studies have, however, brought into light the existence of unique intermediary states in EMT, often referred to as partial EMT states. Through our studies we have found the deubiquitinase USP7 to be strongly associated with the development of such a partial EMT state in colon cancer cells, characterized by the acquisition of mesenchymal characteristics but without the reduction in epithelial markers. We found USP7 to be overexpressed in colon adenocarcinomas and to be closely associated with advancing tumor stage. We found that functional inhibition or knockdown of USP7 is associated with a marked reduction in mesenchymal markers and in overall migration potential of cancer cells. Starting off with a proteomics-based approach we were able to identify and later on verify the DEAD box RNA helicase DDX3X to be an interacting partner of USP7. We then went on to show that USP7, through the stabilization of DDX3X, augments Wnt/β-catenin signaling, which has previously been shown to be greatly associated with colorectal cancer cell invasiveness. Our results indicate USP7 as a novel key player in establishing a partial mesenchymal phenotype in colorectal cancer.

USP7 Promotes deubiquitination and stabilization of MyD88 to enhance immune responses.

Toll-like receptors (TLRs) are involved in the sensing of pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), flagellin, unmethylated double-stranded DNA (CpG), single-stranded RNA (ssRNA) and lipoproteins. Myeloid differentiation primary response protein 88 (MyD88) is a canonical adaptor for the Toll-like receptor family which has crucial roles in host defense against infection by microbial pathogens. The dysregulation of MyD88 may also induce autoimmune diseases. Here, we demonstrate that the deubiquitinase USP7 interacts with MyD88 in chicken, with knockdown or overexpression of USP7 leading to the regulation of MyD88 protein in a positive manner. Consequently, USP7 positively regulates the expression of proinflammatory factors upon LPS challenge. Furthermore, we observed USP7-deficient mice to be more susceptible to infection by Salmonella typhimurium. Collectively, our findings demonstrate MyD88 as a bona fide substrate of USP7 and uncover a mechanism by which USP7 regulates innate immune signaling.

TRIM27-USP7 complex promotes tumour progression via STAT3 activation in human hepatocellular carcinoma.

TRIM27 is stabilized by binding to USP7 and mediates tumour progression in several cancers; however, the roles of TRIM27-USP7 complex on STAT3 activation in HCC are unknown. Regulations and functions of TRIM27 for activating STAT3 in HCC were assessed using 207 HCC samples or HCC cells. TRIM27 expression was increased in some cases of HCC. High TRIM27 expression was an independent predictor for poor prognosis in HCC after surgery. It was correlated with the expression of EpCAM, vimentin, MMP-9, and activation of STAT3 in HCC. TRIM27 expression was correlated with USP7 expression, and HCC with high TRIM27 expression together with high USP7 expression showed enhanced STAT3 activation, resulting in poorer prognosis. p-JAK1 expression was correlated with STAT3 activation in HCC with high TRIM27 expression. In vitro, USP7 knockdown decreased TRIM27 expression, suggesting that USP7 was essential for TRIM27 stabilization. Knocking down of TRIM27 or USP7 suppressed STAT3 activation and overexpression of TRIM27 accelerated STAT3 activation; therefore, the formation of TRIM27-USP7 complex was needed for STAT3 activation, which led to aggressive tumour proliferation and invasion by enhancing EMT and CSC-like property. Binding of JAK1 to TRIM27-USP7 complex was confirmed in vitro. Deletion of TRIM27-USP7 complex by USP7 inhibitor significantly inhibited tumour cell invasion by suppressing STAT3 activation. TRIM27 is stabilized by binding to USP7 and is related to aggressive tumour progression in HCC via STAT3 activation, resulting in poor prognosis after operation. Therefore, TRIM27-USP7 complex is a useful prognostic predictor and a promising therapeutic target for HCC.

DRPPM-EASY: A Web-Based Framework for Integrative Analysis of Multi-Omics Cancer Datasets.

Simple SummaryWith the influx of multi-omics profiling, effective integration of these data remains the bottleneck for omics-driven discovery. Thus, we developed DRPPM-EASY, an R Shiny framework for integrative multi-omics analysis of cancer datasets. Our tool enables the exploration of multi-omics data by providing a simple user interface that minimizes the need for computational experience. Furthermore, the interface can be deployed locally or on a webserver to facilitate scientific collaboration and discovery.High-throughput transcriptomic and proteomic analyses are now routinely applied to study cancer biology. However, complex omics integration remains challenging and often time-consuming. Here, we developed DRPPM-EASY, an R Shiny framework for integrative multi-omics analysis. We applied our application to analyze RNA-seq data generated from a USP7 knockdown in T-cell acute lymphoblastic leukemia (T-ALL) cell line, which identified upregulated expression of a TAL1-associated proliferative signature in T-cell acute lymphoblastic leukemia cell lines. Next, we performed proteomic profiling of the USP7 knockdown samples. Through DRPPM-EASY-Integration, we performed a concurrent analysis of the transcriptome and proteome and identified consistent disruption of the protein degradation machinery and spliceosome in samples with USP7 silencing. To further illustrate the utility of the R Shiny framework, we developed DRPPM-EASY-CCLE, a Shiny extension preloaded with the Cancer Cell Line Encyclopedia (CCLE) data. The DRPPM-EASY-CCLE app facilitates the sample querying and phenotype assignment by incorporating meta information, such as genetic mutation, metastasis status, sex, and collection site. As proof of concept, we verified the expression of TP53 associated DNA damage signature in TP53 mutated ovary cancer cells. Altogether, our open-source application provides an easy-to-use framework for omics exploration and discovery.

RNA interference screens discover proteases as synthetic lethal partners of PI3K inhibition in breast cancer cells.

Rationale: PI3K/mTOR signaling is frequently upregulated in breast cancer making inhibitors of this pathway highly promising anticancer drugs. However, PI3K-inhibitors have a low therapeutic index. Therefore, finding novel combinatory treatment options represents an important step towards clinical implementation of PI3K pathway inhibition in breast cancer therapy. Here, we propose proteases as potential synergistic partners with simultaneous PI3K inhibition in breast cancer cells.Methods: We performed mRNA expression studies and unbiased functional genetic synthetic lethality screens by a miR-E based knockdown system targeting all genome-encoded proteases, i.e. the degradome of breast cancer cells. Importantly theses RNA interference screens were done in combination with two PI3K pathway inhibitors. Protease hits were validated in human and murine breast cancer cell lines as well as in non-cancerous cells by viability and growth assays.Results: The degradome-wide genetic screens identified 181 proteases that influenced susceptibility of murine breast cancer cells to low dose PI3K inhibition. Employing independently generated inducible knockdown cell lines we validated 12 protease hits in breast cancer cells. In line with the known tumor promoting function of these proteases we demonstrated Usp7 and Metap2 to be important for murine and human breast cancer cell growth and discovered a role for Metap1 in this context. Most importantly, we demonstrated that Usp7, Metap1 or Metap2 knockdown combined with simultaneous PI3K inhibition resulted in synergistic impairment of murine and human breast cancer cell growthConclusion: We successfully established proteases as combinatory targets with PI3K inhibition in human and murine breast cancer cells. Usp7, Metap1 and Metap2 are synthetic lethal partners of simultaneous protease/PI3K inhibition, which may refine future breast cancer therapy.

Deubiquitinase USP7 Regulates Erythroid Development Via Deubiquinating GATA1

Erythropoiesis is the process which produces mature red blood cells from haematopoietic stem cell. Ubiquitination/deubiquititation plays important roles in many cellular processes. Previous studies have documented the roles of ubiquitination and several ubiquitinases in erythropoiesis. In contrast, the roles of deubiquitiation/deubiquitinases in erythropoiesis remain unexplored. In this study, we found that the expression of deubiquitinase USP7 was significantly upregulated during erythropoiesis. We then employed shRNA mediated knockdown approach to explore the role USP7, we found that USP7 knockdown significantly delayed terminal erythroid differentiation, inhibited the hemoglobin expression and cell proliferation, and induced apoptosis. To explore the underlying mechanisms for the observed phenotypic changes, we compared the expression of several key erythroid transcription factors including GATA1, KLF1, LSD1, SOX1 and IKAROS between control and USP7-knockdown cells by western blot analysis. We found that only the protein level of GATA1 but not other transcription factors was significantly decreased following USP7 knockdown. Moreover, GATA1 mRNA level was not affected by USP7 knockdown. The effect of USP7 on the stability of GATA1 protein was further demonstrated by the findings that inhibition of USP7 by USP7 inhibitors also resulted in decreased GATA1 protein levels and that knockdown/inhibition of USP7 shorten the GATA1 half-life and increased the ubiquitination of GATA1. Furthermore, we performed Co-IP, pull down and in vitro ubiquitization experiment which documented that USP7 directly binds to and deubiquinates GATA1. Our findings demonstrate that GATA1 is the substrate of USP7 and that USP7 stabilizes GATA1 by deubiquinating GATA1. To examine whether the impaired erythropoiesis following USP7 knockdown is due to decreased GATA1, we ectopically expressed GATA1 in USP7 knockdown cells and showed that the delayed differentiation was rescued by GATA1 overexpression. To demonstrate the role of USP7 in vivo, we established a stress erythropoiesis mouse model by phlebotomy and then treated the mice by intraperitoneal injection of USP7 inhibitor. We found USP7 inhibitor delayed the recovery of anemia. Collectively, our findings not only identified previously unknown roles of USP7 in erythropoiesis but also identified a novel regulatory mechanism for GATA1 expression.AcknowledgmentsThis work was supported in part by grants 81270576, 81470362 and 81530005 from Natural Science Foundation of China, by NIH grants DK100810 and DK26263, and by postgraduate innovation project of Central South University of China 2016zzts165.The authors declare no conflict of interest. DisclosuresNo relevant conflicts of interest to declare.

USP7 promotes hepatoblastoma progression through activation of PI3K/AKT signaling pathway.

Hepatoblastoma (HB) is an embryonic solid tumor and the most common primary malignant liver tumor in children. HB usually occurs in infants and children. Although treatment diversity is increasing, some patients still have very poor prognosis. Many studies have investigated USP7 inhibitors for tumors. Using database information, we found that USP7 is highly expressed in HB. Lentivirus-mediated USP7 knockdown and overexpression was performed in HB cell lines HepG2 and Huh6. CCK8 and transwell assays were used to determine cell viability and metastasis. Flow cytometry was used to study cell cycle and apoptosis. Levels of proteins were detected using western blots. Downregulation of USP7 resulted in significant decrease in cell proliferation, clonal formation, and cell migration and invasion. With overexpression of USP7, cellular malignant behavior increased. Cell cycle assays showed that USP7 knockdown inhibited G1 to S phase transition in the cell cycle. Upregulation of USP7 promoted the transition. Animal experiments showed USP7 facilitated tumor growth in vivo. Western blots indicated that USP7 may affect HB tumorigenesis through the PI3K/AKT signaling pathway. Furthermore, USP7 inhibitor P5091 inhibited HB development and PI3K/AKT pathway. USP7 upregulation contributed to HB genesis and development through the PI3K/AKT signaling pathway. USP7 could be a potential target for future HB treatment.

Deubiquitinase USP7 contributes to the pathogenicity of spinal and bulbar muscular atrophy.

Polyglutamine (polyQ) diseases are devastating, slowly progressing neurodegenerative conditions caused by expansion of polyQ-encoding CAG repeats within the coding regions of distinct, unrelated genes. In spinal and bulbar muscular atrophy (SBMA), polyQ expansion within the androgen receptor (AR) causes progressive neuromuscular toxicity, the molecular basis of which is unclear. Using quantitative proteomics, we identified changes in the AR interactome caused by polyQ expansion. We found that the deubiquitinase USP7 preferentially interacts with polyQ-expanded AR and that lowering USP7 levels reduced mutant AR aggregation and cytotoxicity in cell models of SBMA. Moreover, USP7 knockdown suppressed disease phenotypes in SBMA and spinocerebellar ataxia type 3 (SCA3) fly models, and monoallelic knockout of Usp7 ameliorated several motor deficiencies in transgenic SBMA mice. USP7 overexpression resulted in reduced AR ubiquitination, indicating the direct action of USP7 on AR. Using quantitative proteomics, we identified the ubiquitinated lysine residues on mutant AR that are regulated by USP7. Finally, we found that USP7 also differentially interacts with mutant Huntingtin (HTT) protein in striatum and frontal cortex of a knockin mouse model of Huntington's disease. Taken together, our findings reveal a critical role for USP7 in the pathophysiology of SBMA and suggest a similar role in SCA3 and Huntington's disease.

Combination of Inhibitors of USP7 and PLK1 has a Strong Synergism against Paclitaxel Resistance.

USP7 is a promising target for the development of cancer treatments because of its high expression and the critical functions of its substrates in carcinogenesis of several different carcinomas. Here, we demonstrated the effectiveness of targeting USP7 in advanced malignant cells showing high levels of USP7, especially in taxane-resistant cancer. USP7 knockdown effectively induced cell death in several cancer cells of lung, prostate, and cervix. Depletion of USP7 induced multiple spindle pole formation in mitosis, and, consequently, resulted in mitotic catastrophe. When USP7 was blocked in the paclitaxel-resistant lung cancer NCI-H460TXR cells, which has resistance to mitotic catastrophe, NCI-H460TXR cells underwent apoptosis effectively. Furthermore, combination treatment with the mitotic kinase PLK1 inhibitor volasertib and the USP7 inhibitor P22077 showed a strong synergism through down-regulation of MDR1/ABCB1 in paclitaxel-resistant lung cancer. Therefore, we suggest USP7 is a promising target for cancer therapy, and combination therapy with inhibitors of PLK1 and USP7 may be valuable for treating paclitaxel-resistant cancers, because of their strong synergism.

Ubiquitin-Specific-Processing Protease 7 Regulates Female Germline Stem Cell Self-Renewal Through DNA Methylation

Ubiquitin-specific-processing protease 7 (Usp7) is a key deubiquitinase controlling epigenetic modification and regulating the self-renewal, proliferation, and differentiation of stem cells. However, the functions and mechanisms of action of Usp7 on female germline stem cells (FGSCs) are unknown. Here, we demonstrated that Usp7 regulated FGSC self-renewal via DNA methylation. The results of Cell Counting Kit-8 and 5-ethynyl-20-deoxyuridine assays showed that the viability and proliferation of FGSCs were negatively regulated by Usp7. Moreover, Usp7 downregulated the expression of self-renewal genes, such as Oct4, Etv5, Foxo1, and Akt, but upregulated the expression of differentiation-related genes including Stra8 and Sycp3. Mechanistically, RNA-seq results showed that Usp7 negatively regulated FGSC self-renewal but positively modulated differentiation in FGSCs. Meanwhile, both overexpression and knockdown of Usp7 resulted in significant changes in DNA methylation and histone modification in FGSCs. Additionally, RNA-seq and MeDIP-seq analyses showed that Usp7 regulates the self-renewal and differentiation of FGSCs mainly through DNA methylation rather than histone modification, which was also confirmed by a rescue assay. Our study not only offers a novel method to research FGSC self-renewal and differentiation in view of epigenetic modifications, but also provides a deep understanding of FGSC development.Graphical

Abstract 6057: Discovery of a novel function for USP7 inhibitors: Reprogramming the tumor microenvironment

Abstract During the transition from healthy tissue to cancer and metastasis, the tumor microenvironment (TME) initiates crucial changes that licenses the progression from primary tumors to aggressive and metastatic disease. Our understanding of the specific factors and molecular mechanisms underpinning these transitions in the TME is critical for influencing the potency and scope of existing cancer therapy strategies but is still evolving and subject to substantial and widespread investigations. Here we uncover a novel molecular mechanism actioned by fibroblasts in the TME to modulate the secretion of the vascular endothelial growth factor (VEFG) via the hypoxia-inducible transcription factor (HIF1α). Utilizing primary human cells co-culture systems in combination with highly potent and selective USP7 inhibitors, we demonstrate that the deubiquitinating enzyme USP7 modulates the stability of HIF1α in primary human fibroblasts and secreted VEGF levels in primary fibroblasts as well as cancer-associated fibroblasts. This USP7-mediated modulation of VEGF secretion in the TME does not occur in cancer cells, immune cells or endothelial cells, rather it is restricted to the fibroblastic compartment. Importantly, the modulation of VEGF secretion by USP7 in fibroblasts is not mediated via the Mdm2 axis: Mdm2 antagonists are unable to recapitulate the VEGF modulation in fibroblasts. Knock-down of USP7 in primary fibroblasts validates the phenotype obtained with potent and selective USP7 inhibitors. In line with their effects on the TME in primary cell cultures, potent, selective USP7 inhibitors demonstrated tumour growth inhibition in vivo in a syngeneic cancer xenograft model that does not respond to USP7 inhibitors in vitro. Collectively, our studies reveal a molecular mechanism whereby USP7 inhibition leads to TME reprogramming mediated by an increased destabilization of HIF1α leading to reduced levels of VEGF in the TME and improved anti-tumor activity. Our results support further investigations of combination of a USP7 inhibitor with immune checkpoint inhibitors, as well as other TME modulating agents. Citation Format: Xavier Jacq, Anamarija Jurisic, Julien Daubriac, Ian T. Lobb, Mark Wappett, Aaron Cranston, Peggy Sung, Gerald Gavory, Colin O'Dowd, Tim Harisson. Discovery of a novel function for USP7 inhibitors: Reprogramming the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6057.

The deubiquitinase USP7 stabilizes Maf proteins to promote myeloma cell survival

The Maf proteins, including c-Maf, MafA, and MafB, are critical transcription factors in myelomagenesis. Previous studies demonstrated that Maf proteins are processed by the ubiquitin-proteasome pathway, but the mechanisms remain elusive. This study applied MS to identify MafB ubiquitination-associated proteins and found that the ubiquitin-specific protease USP7 was present in the MafB interactome. Moreover, USP7 also interacted with c-Maf and MafA and blocked their polyubiquitination and degradation. Consistently, knockdown of USP7 resulted in Maf protein degradation along with increased polyubiquitination levels. The action of USP7 thus promoted Maf transcriptional activity as evidenced by luciferase assays and by the up-regulation of the expression of Maf-modulated genes. Furthermore, USP7 was up-regulated in myeloma cells, and it was negatively associated with the survival of myeloma patients. USP7 promoted myeloma cell survival, and when it was inhibited by its specific inhibitor P5091, myeloma cell lines underwent apoptosis. These results therefore demonstrated that USP7 is a deubiquitinase of Maf proteins and promotes MM cell survival in association with Maf stability. Given the significance of USP7 and Maf proteins in myeloma genesis, targeting the USP7/Maf axle is a potential strategy to the precision therapy of MM.

USP7 is a novel Deubiquitinase sustaining PLK1 protein stability and regulating chromosome alignment in mitosis

BackgroundThe deubiquitinase USP7 has been identified as an oncogene with key roles in tumorigenesis and therapeutic resistance for a series of cancer types. Recently small molecular inhibitors have been developed to target USP7. However, the anticancer mechanism of USP7 inhibitors is still elusive.MethodsCell viability or clonogenicity was tested by violet crystal assay. Cell apoptosis or cell cycle was analyzed by flow cytometry, and chromosome misalignment was observed by a fluorescent microscopy. The protein interaction of PLK1 and USP7 was detected by tandem affinity purification and high throughput proteomics, and further confirmed by co-immunoprecipitation, GST pull-down and protein co-localization. The correlation between USP7 level of tumor tissues and taxane-resistance was evaluated.ResultsPharmacological USP7 inhibition by P5091 retarded cell proliferation and induced cell apoptosis. Further studies showed that P5091 induced cell cycle arrest at G2/M phase, and particularly induced chromosome misalignment, indicating the key roles of USP7 in mitosis. USP7 protein was detected in the PLK1-interacted protein complex. USP7 interacts with PLK1 protein through its PBD domain by catalytic activity. USP7 as a deubiquitinase sustained PLK1 protein stability via the C223 site, and inversely, USP7 inhibition by P5091 promoted the protein degradation of PLK1 through the ubiquitination-proteasome pathway. By overexpressing PLK1, USP7 that had been depleted by RNAi ceased to induce chromosome misalignment in mitosis and again supported cell proliferation and cell survival. Both USP7 and PLK1 were overexpressed in taxane-resistant cancer cells, and negatively correlated with the MP scores in tumor tissues. Either USP7 or PLK1 knockdown by RNAi significantly sensitized taxane-resistant cells to taxane cell killing.ConclusionThis is the first report that PLK1 is a novel substrate of USP7 deubiquitinase, and that USP7 sustained the protein stability of PLK1. USP7 inhibition induces cell apoptosis and cell cycle G2/M arrest, and overcomes taxane resistance by inducing the protein degradation of PLK1, resulting in chromosome misalignment in mitosis.

Abstract 3652: USP7 heterozygous loss-of-function affects T-cell differentiation in pediatric T-ALL

Abstract Ubiquitin-specific-processing protease 7 (USP7), a protein deubiquitinase, is one of the most frequently mutated genes (33%) in the TAL1 subtype of pediatric T-lineage acute lymphoblastic leukemia (T-ALL). However, the functional effect of USP7 haploinsufficiency on T-ALL pathogenesis remains elusive. To understand USP7 haploinsufficiency’s impact on T-ALL, we performed gene expression analysis on 42 non-early T-cell precursor T-ALL RNAseq samples downloaded from phs000218. The gene expression analysis suggested that USP7 haploinsufficiency (USP7-mut N = 12; USP7-wt N = 30) down-regulated the expression of T-cell maturation markers (e.g. RAG1, RAG2, and CD1B), which were negatively regulated by TAL1 [1]. RNAseq on a T-ALL cell line with USP7 knocked down by shRNA also found the same TAL1 negatively regulated gene set, further supporting an increase of TAL1 activity in the USP7 mutated T-ALLs. To examine whether the T-cell maturation was affected by USP7 heterozygous knockout, we generated a conditional knockout (cKO) mouse model by cross-breeding the transgenic vav1-cre mice with the USP7fl/fl mice to obtain heterozygous USP7fl/wt-vav1-cre. Thymocytes isolated from cKO mice were co-cultured with the OP9-Δ1 cells in the medium supplied with cytokines. The cell surface differentiation markers, CD4 and CD8 were stained for flow cytometry detection, and we observed an increase of double-negative cells and a decrease of double-positive cells in USP7-het-KO mice versus control (N = 4 in each group; p-value < 0.05), consolidating the disrupted T-cell development ex vivo. To further understand USP7’s mechanism to regulate TAL1, we analyzed proteins that interacted with USP7 by affinity purification-mass spectrometry (AP-MS) and immune-precipitated followed by western-blotting (IP/WB). AP-MS with the anti-USP7 antibody revealed that USP7 directly interacts with TAL1 in Jurkat cells; AP-MS with the anti-TAL1 antibody also reciprocated the USP7-TAL1 interactions. Whole proteome analysis, through tandem-mass-tag and two-dimensional liquid chromatography-tandem mass spectrometry, revealed that USP7 knockdown down-regulated TRIM27, a deubiquitin target of USP7. IP/WB further confirmed the interaction between USP7, TRIM27, and TAL1, suggesting a possible synergistic relationship between USP7 and TRIM27 to regulate TAL1. In conclusion, our finding demonstrates heterozygous loss of function of USP7 dysregulates T-cell maturation by enhancing TAL1 activity. Future work on TRIM27 could further shed light on the mechanism underlying USP7’s ability to regulate TAL1. Reference: [1] Sanda et al. Cancer Cell 22, 209 (2012). Citation Format: Timothy I. Shaw, Li Dong, Anthony High, Yu Liu, Bensheng Ju, Kanisha Kavdia, Vishwajeeth Pagala, Bridget Shaner, John Easton, Chenxi Qian, Jiyang Yu, Janke Janke, John Kim Choi, Junmin Peng, Wei Gu, James R. Downing, Jinghui Zhang. USP7 heterozygous loss-of-function affects T-cell differentiation in pediatric T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3652.

The m6A demethylase FTO promotes the growth of lung cancer cells by regulating the m6A level of USP7 mRNA

Lung cancer is one of the leading causes of cancer-related death in developed countries. Despite decades of intensive efforts to comate this malignant disease, the prognosis of lung cancer remains unfavorable and is especially poor in advanced non-small cell lung cancer (NSCLC). However, whether and how the m6A demethylase FTO functions in lung cancer cells remain unknown. Here in the present study, we show that FTO plays an oncogenic role in human NSCLC. FTO mRNA and protein levels were overexpressed in human NSCLC tissues and cell lines, which was associated with the reduced m6A content. We next knocked down FTO expression in human lung cancer cell lines with lentivirus-mediated shRNAs and the cellular proliferation assay demonstrated that FTO loss-of-function reduced the proliferation rate of cancer cells. FTO knockdown also inhibited the colony formation ability of lung cancer cells. Importantly, our xenograft experiment showed that FTO knockdown reduced lung cancer cells growth in vivo. Mechanism analysis demonstrated that FTO decreased the m6A level and increased mRNA stability of ubiquitin-specific protease (USP7), which was relied on the demethylase activity of FTO. USP7 mRNA level was overexpressed in human lung cancer tissues and USP7 expression was positively correlated with FTO mRNA level. Genetic knockdown or pharmacological inhibition (P5091 or P22027) of USP7 reduced the proliferation rate of lung cancer cells and decreased the capacity of colony formation of lung cancer cells in vitro, whereas lung cancer cells growth inhibition by FTO knockdown is restored by overexertion of USP7. Collectively, our findings demonstrate that the m6A demethylase FTO promotes the growth of NSCLC cells by increasing the expression of USP7.

WDR79 promotes the proliferation of non-small cell lung cancer cells via USP7-mediated regulation of the Mdm2-p53 pathway

WD repeat protein 79 (WDR79) is a member of the WD-repeat protein family and functions as a scaffold protein during telomerase assembly, Cajal body formation and DNA double strand break repair. We have previously shown that WDR79 is frequently overexpressed in cell lines and tissues derived from non-small cell lung cancer (NSCLC) and it accelerates cell proliferation in NSCLC. However, the detailed mechanism underlying the role of WDR79 in the proliferation of NSCLC cells remains unclear. Here, we report the discovery of a molecular interaction between WDR79 and USP7 and show its functional significance in linking the Mdm2-p53 pathway to the proliferation of NSCLC cells. We found that WDR79 colocalized and interacted with USP7 in the nucleus of NSCLC cells. This event, in turn, reduced the ubiquitination of Mdm2 and p53, thereby increasing the stability and extending the half-life of the two proteins. We further found that the functional effects of WDR79 depended upon USP7, because the knockdown of USP7 resulted in their attenuation. Finally, we demonstrated that WDR79 promoted the proliferation of NSCLC cells via USP7. Taken together, our findings reveal a novel molecular function of WDR79 and may lead to broadly applicable and innovative therapeutic avenues for NSCLC.

UbE2E1/UBCH6 Is a Critical in Vivo E2 for the PRC1-catalyzed Ubiquitination of H2A at Lys-119

UbE2E1/UbcH6 is an E2 ubiquitin-conjugating enzyme that is regulated by USP7. We identified UbE2E1 as a novel component of Polycomb repressive complex 1 (PRC1), the E3 ligase complex responsible for histone H2A ubiquitination and gene silencing. We demonstrate that UbE2E1 is critical for the monoubiquitination of H2A at residue Lys-119 (uH2AK119) through its association with the PRC1 complex. UbE2E1 interacts with PRC1 subunits including Ring1A and Ring1B. Overexpression of UbE2E1 results in increased levels of uH2AK119, whereas overexpression of catalytically inactive UbE2E1_C131A or UbE2E1 knockdown results in decreased levels of uH2AK119. The down-regulation of H2A ubiquitination by loss of function of UbE2E1 is correlated with alleviated p16INK4a promoter repression and induced growth inhibition in HCT116 cells. These results are specific to UbE2E1 as knockdown of UbE2D E2s does not show any effect on uH2AK119. We extended the UbE2E1 regulation of uH2AK119 to USP7 and showed that USP7 is also a key regulator for monoubiquitination at H2A Lys-119 as both knockdown and deletion of USP7 results in decreased levels of uH2AK119. This study reveals that UbE2E1 is an in vivo E2 for the PRC1 ligase complex and thus plays an important role in the regulation of H2A Lys-119 monoubiquitination.