n­cmyc tag human usp10  (Addgene inc)

Journal: Advanced Science

Article Title: Tuning Immune‐Cold Tumor by Suppressing USP10/B7‐H4 Proteolytic Axis Reinvigorates Therapeutic Efficacy of ADCs

doi: 10.1002/advs.202400757

Figure Lengend Snippet: Identification of USP10 as a binding partner of B7‐H4 that upregulates B7‐H4 protein levels in immune‐cold TNBC breast tumors. A) Coomassie blue staining of B7‐H4 immunoprecipitated from MDA‐MB‐468 cells expressing FLAG‐tagged B7‐H4 reveals USP10 as a binding partner by mass spectrometry analysis. B,C) Confirmation of the B7‐H4/USP10 interaction via co‐immunoprecipitation by coimmunoprecipitation of endogenous USP10 (B) and coimmunoprecipitation of endogenous B7‐H4 (C). D) Immunofluorescence staining of MDA‐MB‐468 cells expressing FLAG‐B7‐H4 shows colocalization with USP10. Scale bar = 30 µm. E) MDA‐MB‐468‐FLAG‐B7‐H4 cells were subjected to Duolink in Situ PLA assay with FLAG mouse antibody and USP10 rabbit antibody. Red dots indicate the interaction of the two proteins. Scale bar = 30 µm. F) Overexpression of USP10 in MDA‐MB‐468 cells enhances B7‐H4 protein levels, evidenced by immunoblotting (left panel) and subsequent quantification (right panel). G–J) USP10 knockdown results in downregulation of B7‐H4 protein but not mRNA in MDA‐MB‐468 cells. Three USP10 knockdown cell lines were established with different sgRNAs with CRISPR/Cas9 system. Empty vector CRISPRv2 used as the control. (G) Representative images of immunoblotting of USP10 and B7‐H4. (H) Quantification of USP10 and (I) B7‐H4 protein normalized with β‐actin amount in biological triplicates. (J) Quantification of B7‐H4 mRNA levels with qPCR. K) Decreased membrane and total B7‐H4 in USP10 knockdown cells. MDA‐MB‐468 cells were stained with PE conjugated anti‐human B7‐H4 antibody without or with permeabilization for surface B7‐H4 and total B7‐H4, respectively and subjected to flow cytometry analysis. Representative images are shown. L) Inhibition of USP10 with spautin‐1 leads to decreased B7‐H4 protein in MDA‐MB‐468 cells, validated by western blot (left panel) and quantified intensity (right panel). M) Spautin‐1 treatment increases B7‐H4 turnover. MDA‐MB‐468 cells were treated with DMSO or 10 µM spautin‐1 for 24 h and then treated with 100 µg mL −1 cycloheximide (CHX) at the indicated time points. Representative images of immunoblotting of B7‐H4 (left panel). Quantification of B7‐H4 (40 kDa) intensity normalized to time 0 (right panel). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Data (mean ± SEM) are representative of at least three independent experiments.

Article Snippet: The N‐cMyc tag human USP10 (#184 034) were purchased from Addgene and then corresponding cMyc‐USP10 coding sequence was cloned into lentiviral vector Phage‐PGK‐Flag‐via Gibson assembly (ClonExpress II One Step Cloning Kit C112 from Vazyme) and validated by sequencing.

Techniques: Binding Assay, Staining, Immunoprecipitation, Expressing, Mass Spectrometry, Immunofluorescence, In Situ, Over Expression, Western Blot, Knockdown, CRISPR, Plasmid Preparation, Control, Membrane, Flow Cytometry, Inhibition

Journal: Advanced Science

Article Title: Tuning Immune‐Cold Tumor by Suppressing USP10/B7‐H4 Proteolytic Axis Reinvigorates Therapeutic Efficacy of ADCs

doi: 10.1002/advs.202400757

Figure Lengend Snippet: USP10 is a pivotal regulator to stabilize B7‐H4 via deubiquitylation and modulate T cell function. A,B) USP10 knockdown leads to increased B7‐H4 turnover in MDA‐MB‐468. A) Pulse‐chase analysis for the control CRISPR v2 and USP10 knockdown MDA‐MB‐468 cells with 100 µg mL −1 cycloheximide. B) Quantification of the amount of B7‐H4 (40 kDa) normalized to the initial time point. The half‐life t 1/2 is estimated based on the plot. C) USP10 overexpression reduces B7‐H4 ubiquitylation in HEK‐293T stable cell line expressing FLAG‐B7‐H4. FLAG‐B7‐H4 was immunoprecipitated followed by immunoblotting with anti‐ubiquitin. D) USP10 knockdown enhances B7‐H4 ubiquitylation in MDA‐MB‐468 cells. USP10 was knocked down in MDA‐MB‐468 cells stably expressing HA‐tagged B7‐H4. Following immunoprecipitation of HA‐B7‐H4, ubiquitylation levels were assessed via immunoblotting with an anti‐ubiquitin antibody. E–G) Flow cytometry analysis of MDA‐MB‐468 cells cocultured with PBMCs shows increased apoptosis in USP10 knockdown cells (E). Quantitative analysis of the percentage of total apoptosis (Annexin V+) (F) and late apoptosis (Annexin V+ and PI+) cancer cells (G). H,I) USP10 knockdown or B7‐H4 knockdown in MDA‐MB‐468 releases the suppression of the function of cytotoxic T cells by cancer cells as determined by the percentage of TNFα positive cytotoxic T cells (H) and the percentage of Ki67+ cytotoxic T cells (I). J,K) Flow cytometry analysis of MDA‐MB‐468 cells cocultured with PBMCs shows decreased apoptosis in USP10 overexpression cells (J). Quantitative analysis of the percentage of late apoptosis (Annexin V+ and PI+) cancer cells (K). L,M) USP10 overexpression in MDA‐MB‐468 further suppressed the function of cytotoxic T cells by cancer cells as determined by the percentage of TNFα+cytotoxic T cells (L) and the percentage of IFN‐γ+ cytotoxic T cells (M). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by the one‐way ANOVA test or two‐way ANOVA test, Data (mean ± SEM) are representative of at least three independent experiments.

Article Snippet: The N‐cMyc tag human USP10 (#184 034) were purchased from Addgene and then corresponding cMyc‐USP10 coding sequence was cloned into lentiviral vector Phage‐PGK‐Flag‐via Gibson assembly (ClonExpress II One Step Cloning Kit C112 from Vazyme) and validated by sequencing.

Techniques: Cell Function Assay, Knockdown, Pulse Chase, Control, CRISPR, Over Expression, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Flow Cytometry

Journal: Advanced Science

Article Title: Tuning Immune‐Cold Tumor by Suppressing USP10/B7‐H4 Proteolytic Axis Reinvigorates Therapeutic Efficacy of ADCs

doi: 10.1002/advs.202400757

Figure Lengend Snippet: Molecular mapping the domains on USP10 and B4‐H4 that facilitates the interaction between USP10 and B7‐H4. A–E) Schematic diagram of human USP10 domains and strategy to engineer a series of USP10 fragments (A) and USP10 deletion mutants (D). Mapping of the molecular domain on USP10 involving in the interaction with B7‐H4. The interactions between B7‐H4 and USP10 fragments were examined by co‐IP experiments in HEK‐293T cells. Amino acid stretches from 516 to 615 amino acid on USP10 (B‐E) were identified as the region that mediates its interaction with B7‐H4. F) Schematic diagram of human B7‐H4 domains and strategy to engineer a series of B7‐H4 deletion fragments. G) The interactions between USP10 and B7‐H4 fragments were examined by co‐IP experiments in HEK‐293T cells. Amino acid stretches from 24 to 146 (including Ig like V‐type domain) on B7‐H4 were identified as the region that mediates the interaction between USP10 and B7‐H4. H) Molecular docking model of the interaction between USP10‐B7‐H4‐AMFR complex. AMFR is the E3 ligase for B7‐H4. The ring motif of AMFR is in green; the Ig‐like V‐type and Ig‐like‐C2 type domain of B7‐H4 is in red and 516–615 residues of USP10 is in blue. The docking model shows that USP10 binds to the Ig‐like V‐type domain of B7‐H4 and the deubiquitinase AMFR binds to the Ig‐like‐C2 type domain of B7‐H4, where the interacting residues are highlighted in red and green line. I) The Structural model for the interaction of USP10 (sky blue) with B7‐H4 (red) investigated through docking studies (left panel). Docking simulations have explored the potential binding sites and energetics of the B7‐H4‐USP10 interaction with lowest energy −654.5 kJ mol −1 , and also revealed amino acid residues (Lue122, Glu144 Lys146 and Thr147) of B7‐H4 (yellow) that forming conventional hydrogen bond interaction with USP10 (green) residue (Lys518, Ser516, Glu522, Tyr527) and further stabilized by other hydrophobic interactions between (Ile531, Pro601, Ile602, Phe606) and (His35, Val39, Ala43, Asn142), respectively. The zoomed view of interface indicating the close interaction between B7‐H4 (stick yellow) with USP10 (stick green) shows in (right panel).

Article Snippet: The N‐cMyc tag human USP10 (#184 034) were purchased from Addgene and then corresponding cMyc‐USP10 coding sequence was cloned into lentiviral vector Phage‐PGK‐Flag‐via Gibson assembly (ClonExpress II One Step Cloning Kit C112 from Vazyme) and validated by sequencing.

Techniques: Co-Immunoprecipitation Assay, Binding Assay, Residue

Journal: Advanced Science

Article Title: Tuning Immune‐Cold Tumor by Suppressing USP10/B7‐H4 Proteolytic Axis Reinvigorates Therapeutic Efficacy of ADCs

doi: 10.1002/advs.202400757

Figure Lengend Snippet: Structural modeling identified the critical role of E144/K146 on B7‐H4 in mediating USP10‐mediated stabilization of B7‐H4. A) The structure model of USP10‐B7‐H4 E144R/K146D . Mutation of B7‐H4 (red) residue Glu144 and Lys146 to Arg144 and Asp146 highlighted as sticks and colored in yellow. The double mutation disrupts the interaction: Glu144 (yellow)‐Tyr527(green) interaction, and Lys146(yellow)‐Glu522(green) in the previous interface with USP10 (sky blue) and shows long distance (63.6 and 64.1 Å) to each other. USP10 binds to a region other than the critical residues of B7‐H4 (left). Zoom in view of the mutations in the B7‐H4 and its hydrophobic interactions with USP10. B,C) Validation of interaction between USP10 with B7‐H4 and B7‐H4 E144R/K146D by coimmunoprecipitation of ectopic Flag‐tagged B7‐H4 WT and Flag‐tagged B7‐H4 E144R/K146D in HEK‐293T cells. The ubiquitylation status of B7‐H4 WT and B7‐H4 E144R/K146D were also determined using an antibody against ubiquitin. D) MDA‐MB‐468‐B7‐H4 WT and MDA‐MB‐468‐B7‐H4 E144R/K146D cells were treated with 100 µg mL −1 CHX. Cell lysates were collected at indicated time points and followed by measuring B7‐H4 protein turnover. Quantification of B7‐H4 turnover were illustrated in the bottom panel. E–H) Annexin V/Propidium Iodide apoptosis assay shows that in comparison with WT, E121R&K123D mutation leads to increased cancer cell apoptosis in the coculture with human PBMC. MDA‐MB‐468 (E.F) and SKBR3 (G,H) cells expressing either B7‐H4 WT and B7‐H4 E144R/K146D mutant were cocultured with activated human PBMCs. Representative images of flow cytometry (E, G). Quantitative data show the percentage of cells in late apoptosis (Annexin V+ and PI+) stage (F, H). * p < 0.05 and **** p < 0.0001. Data (mean ± SEM) are representative of at least three independent experiments.

Article Snippet: The N‐cMyc tag human USP10 (#184 034) were purchased from Addgene and then corresponding cMyc‐USP10 coding sequence was cloned into lentiviral vector Phage‐PGK‐Flag‐via Gibson assembly (ClonExpress II One Step Cloning Kit C112 from Vazyme) and validated by sequencing.

Techniques: Mutagenesis, Residue, Biomarker Discovery, Ubiquitin Proteomics, Modified Annexin V/Propidium Iodide Apoptosis Assay, Comparison, Expressing, Flow Cytometry

Journal: Advanced Science

Article Title: Tuning Immune‐Cold Tumor by Suppressing USP10/B7‐H4 Proteolytic Axis Reinvigorates Therapeutic Efficacy of ADCs

doi: 10.1002/advs.202400757

Figure Lengend Snippet: Stabilization of B7‐H4 by USP10 impedes the effectiveness of Sacituzumab govitecan through its immunosuppressive role in immune‐cold breast tumors. A) The MSigDB‐based pathway correlation analysis using GSE72362 database shows that tumor cell related adaptive immune response pathways are negatively correlated with SG payload induced DNA damage response. B) Spearman's rank gene correlation analysis using GSE72362 database shows that USP10 expression is highly positively correlated with DNA topoisomerase expression. C) MDA‐MB‐468 cells were treated with 0.5 µg mL −1 SG for 24 h. USP10 and B7‐H4 protein levels were determined by immunoblotting (left panel). Quantification of USP10 and B7‐H4 protein normalized with β‐actin amount in biological triplicates (right panel). D) MDA‐MB‐468 cells either with or without USP10 knockdown were treated with either PBS (control) or 100 ng mL −1 SG for 24 h, followed by treatment with 100 µg mL −1 cycloheximide (CHX) at specified time points (hours). The left panel shows representative immunoblotting images for B7‐H4. The right panel provides the quantification of B7‐H4 (40 kDa) intensity at 2 h normalized to the level at time 0. E) MDA‐MB‐468‐B7‐H4 KD and MDA‐MB‐468‐USP10 KD cells were cocultured with human PBMCs at Effector (E) to Target (T) ratio (3:1) and treated with SG (0.1 µg mL −1 ), cell proliferation rate was measured by the cell confluence normalized to the corresponding vehicle control of each cell line (%). F–J) MDA‐MB‐468‐B7‐H4 KD and MDA‐MB‐468‐USP10 KD cells were treated with SG (0.5 µg mL −1 ), surface expression of damage‐associated molecular patterns (DAMPs) markers: HSP70, HSP90 and Calreticulin (CRT) was measured by flow cytometry. (F, H) representative flow cytometry images. G, I, J) Quantification of the percentage of positive cells. K,L) The control CRISPR v2 and USP10 knockdown (KD) MDA‐MB‐468 cells were pretreated with SG (100 ng mL −1 ) for 24 h and then cocultured with purified human dendritic cells at a 1:1 ratio. The cancer cells and purified human dendritic cells were stained with cell tracker green CMFDA and red CMTPX, respectively, and subjected to fluorescence imaging after a 3‐h coculture. K) Representative microscopic images. Scale bar = 500 µm. L) Quantification of phagocytosis based on fluorescence microscopic images. The phagocytosis percentage is the number of dendritic cells with engulfed cancer cells, indicated by the colocalization of green and red signals, over the total number of dendritic cells per view. M,N) SG (100 ng mL −1 ) pretreated control CRISPR v2 and USP10 knockdown (KD) MDA‐MB‐468 cells were stained with cell tracker green CMFDA, seeded on a plate, and cultured with human dendritic cells for 2 h. The dendritic cells were then collected and subjected to immunostaining with anti‐CD11c‐APC and analyzed by flow cytometry. M) Representative flow cytometry images. N) Quantification of the percentage of phagocytosis, indicated as the percentage of CMFDA+ dendritic cells. O,P) SG (100 ng mL −1 ) pretreated control CRISPR v2 and USP10 knockdown (KD) MDA‐MB‐468 cells were cultured with the human dendritic cells for 2 h. Dendritic cells were collected and subjected to immunostaining with anti‐MHC‐I‐PE‐Dazzle‐594 and analyzed by flow cytometry. O) Representative flow cytometry image showing the distribution of surface MHC‐I expression levels in dendritic cells. P) Quantification of the median membrane staining of MHC‐I in dendritic cells cocultured with the corresponding cancer cells. * p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.0001 by the one‐way or two‐way ANOVA test. Data (mean ± SEM) are representative of at least three independent experiments.

Article Snippet: The N‐cMyc tag human USP10 (#184 034) were purchased from Addgene and then corresponding cMyc‐USP10 coding sequence was cloned into lentiviral vector Phage‐PGK‐Flag‐via Gibson assembly (ClonExpress II One Step Cloning Kit C112 from Vazyme) and validated by sequencing.

Techniques: Expressing, Western Blot, Knockdown, Control, Flow Cytometry, CRISPR, Purification, Staining, Fluorescence, Imaging, Cell Culture, Immunostaining, Membrane

Journal: Advanced Science

Article Title: Tuning Immune‐Cold Tumor by Suppressing USP10/B7‐H4 Proteolytic Axis Reinvigorates Therapeutic Efficacy of ADCs

doi: 10.1002/advs.202400757

Figure Lengend Snippet: Suppression of USP10/B7‐H4 proteolytic axis in vivo reinvigorates therapeutic efficacy of ADCs through regulating tumor immune response. A–D) 4T1 control and 4T1‐USP10 overexpression breast cancer cells were orthotopically injected into the right fourth mammary gland of the BALB/c wild‐type (WT) mice (A). Phosphate‐buffered saline (PBS) was used in the control group. Tumor growth curve was plotted (B) and tumor was harvested at 21 days after tumor challenge, weight was imaged and measured (C,D). E) Representative images of IHC staining of mouse USP10, mouse B7‐H4 and mouse CD8+ cells level of control and USP10 OE mouse tumors. F,G) 4T1 control and 4T1‐USP10 KD tumors were harvested 21 days after tumor challenge and analyzed. Tumor growth curve was plotted (F) and tumor weight (G) was measured at the end point. H) Schematic diagram of 4T1‐hTROP2, where the basal mouse TROP2 was replaced with a human counterpart, and 4T1‐hTROP2 cells were orthotopically injected into the left fourth mammary fat pad and allow to grow ≈100 mm 3 , followed by injection of spautin‐1 (20 mg kg −1 , i.p.) for two times per week, and SG (10 mg kg −1 , i.p.) for three times. PBS and IgG control were used in control groups. I–K) 4T1‐hTROP2 and 4T1‐hTROP2 mB7‐H4 OE breast cancer cells were orthotopically injected into the left fourth mammary fat pad followed by injection of SG (10 mg kg −1 , i.p.) for three times. Tumor growth curve was plotted (I) and tumor was harvested after tumor challenge, weight was imaged (J) and measured (K). L,M) 4T1‐hTROP2 cells were orthotopically injected into the left fourth mammary fat pad and allow to grow ≈100 mm 3 , followed by injection of spautin‐1 (20 mg kg −1 , i.p.) for two times per week, and SG (10 mg kg −1 , i.p.) for three times. PBS and IgG control were used in control groups. The tumor growth was monitored twice per week. Tumor growth (L) and survival curve (M) of the mice were plotted. N) Representative images of IHC staining of mouse CD8, granzyme B and TOX level of 4T1‐hTrop2 mouse tumors after the treatment of SG treatment or SG + Spautin‐1 combination. Scale bar = 20 µm. O,P). USP10 expression level and B7‐H4 expression level were analyzed and quantified by IHC using TMA consisting of TNBC samples. Representative IHC images (K). Quantification of USP10 and B7‐H4 expression (L). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by the two‐way ANOVA test. Data (means ± SEM) are representative of at least two independent experiments with five to ten independently analyzed mice per group.

Article Snippet: The N‐cMyc tag human USP10 (#184 034) were purchased from Addgene and then corresponding cMyc‐USP10 coding sequence was cloned into lentiviral vector Phage‐PGK‐Flag‐via Gibson assembly (ClonExpress II One Step Cloning Kit C112 from Vazyme) and validated by sequencing.

Techniques: In Vivo, Drug discovery, Control, Over Expression, Injection, Saline, Immunohistochemistry, Expressing

Journal: Advanced Science

Article Title: Tuning Immune‐Cold Tumor by Suppressing USP10/B7‐H4 Proteolytic Axis Reinvigorates Therapeutic Efficacy of ADCs

doi: 10.1002/advs.202400757

Figure Lengend Snippet: Proposed working model. In immune‐cold triple‐negative breast cancers, elevated B7‐H4 expression is a barrier that attenuates tumor immune responses and impedes the efficacy of targeted therapies such as sacituzumab govitecan. B7‐H4 levels is governed by a “yin” and “yang” proteolytic regulation through E3 ligase AMFR and deubiquitinase USP10, creating a delicate balance between degradation and stabilization. The stabilization of B7‐H4 by USP10 leads to diminished immune activity and lessened response to ADCs. Pharmacological inhibition of USP10 restore B7‐H4 turnover, reviving tumor immunogenicity and enhancing the therapeutic impact of Sacituzumab govitecan.

Article Snippet: The N‐cMyc tag human USP10 (#184 034) were purchased from Addgene and then corresponding cMyc‐USP10 coding sequence was cloned into lentiviral vector Phage‐PGK‐Flag‐via Gibson assembly (ClonExpress II One Step Cloning Kit C112 from Vazyme) and validated by sequencing.

Techniques: Expressing, Activity Assay, Inhibition, Immunopeptidomics