hdac5 s a  (Addgene inc)

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Representative macroscopic tumor images and tumor weights from KPC Hdac5-WT and KPC Hdac5-KO mouse models treated with vehicle or MRTX1133 (30 mg/kg, i.p., twice daily [bid]) ( n = 5). ( B ) Kaplan-Meier survival curves with log-rank test ( n = 5). ( C and D ) Representative IHC images of tumors in C . IHC scores were quantified in D . Scale bars = 100 μm. n = 5 biologically independent repeats and 3 independent IHC quantifications. ( E ) C57BL/6 mice were orthotopically injected with KPC-Luc cells expressing shNc or shHdac5. Bioluminescence imaging was performed on day 7, followed by treatment with vehicle or MRTX1133 (30 mg/kg, i.p., bid). Representative bioluminescence images and corresponding quantification were acquired on day 28 ( n = 5). ( F ) Tumor growth curves in PDX models with treatment with vehicle or MRTX1133 (30 mg/kg, i.p., bid) ( n = 5). ( G and H ) Representative IHC images of PDXs and quantified IHC scores ( H ). Scale bars = 100 μm. n = 5 biologically independent repeats and 3 independent IHC quantifications. All data are presented as the mean ± SD. Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple comparisons test ( A , D – F , and H ). * P < 0.05, *** P < 0.001.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Injection, Expressing, Imaging

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Venn diagram showing the overlap of genes upregulated in KPC Hdac5-KO mice ( n = 5 per genotype; log 2 [fold-change] > 1, P < 0.05, 2,410 genes) identified via RNA-Seq and genes upregulated in shHDAC5-treated PANC-1 cells ( n = 3 per condition; log 2 [fold-change] > 1, P < 0.05, 2,683 genes), revealing a shared subset of 378 genes. ( B ) Bar graph showing the top 10 enriched transcription factors from transcription factor analysis of 378 genes in A using Enrichr. ATF2, activating transcription factor 2. ( C ) Dual-luciferase reporter assays were performed to assess the transcriptional activity of c-Myc in HDAC5-depleted PANC-1 and AsPC-1 cells. Data are presented as mean ± SD ( n = 5). Statistical significance was determined by 1-way ANOVA followed by Dunnett’s multiple comparisons test. *** P < 0.001. ( D ) Heatmap of MYC ChIP-Seq signal intensity (±3 kb around MYC binding sites) in control vs. HDAC5-knockdown PANC-1 cells. ( E ) The average ChIP signal of MYC centered at transcription start site (±3 kb) in indicated groups. ( F ) Venn diagram depicting the overlap between genes with enhanced MYC promoter occupancy after shHDAC5 knockdown, as identified by ChIP-Seq, and genes upregulated in PANC-1 cells following shHDAC5 knockdown, as determined by RNA-Seq. P = 6 × 10 –71 . ( G ) KEGG pathway enrichment analysis of 821 intersecting genes in F . ( H and I ) Western blot analysis of canonical MAPK pathway protein expression under indicated conditions.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: RNA Sequencing, Luciferase, Activity Assay, ChIP-sequencing, Binding Assay, Control, Knockdown, Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Representative macroscopic images of pancreatic tumors from KPC Hdac5-WT and KPC Hdac5-KO mice after sacrifice. ( B ) Tumor weight analysis in KPC mice treated with vehicle, MRTX1133 (30 mg/kg, i.p. bid), MYCi975 (50 mg/kg, i.p. bid), or their combination ( n = 5). ( C ) Kaplan-Meier survival curves with log-rank test ( n = 5). *** P < 0.001. ( D and E ) Representative IHC images of tumors from treated mice and quantified IHC scores ( E ). Scale bars = 100 μm. n = 5 biologically independent repeats and 3 independent quantifications. ( F and G ) Representative bioluminescence images and quantification ( n = 5). ( H ) Macroscopic images of PDX tumors after 30 days of treatment. ( I and J ) Tumor growth curves ( I ) and final tumor weights ( J ) in PDX models treated with Vehicle + LV-Control, MRTX1133 (30 mg/kg, i.p. bid) + LV-Control, Vehicle + LV-sgMYC (50 μL lentivirus, s.c., weekly), or MRTX1133 + LV-sgMYC. All data are presented as the mean ± SD. Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple comparisons test ( B , E , G , I , and J ). *** P < 0.001.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Control

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Scatterplot depicting the antiproliferative effects of a single dose (10 μM) of 1,737 FDA-approved anticancer compounds on PANC-1 and Mia PaCa-2 cells, treated with either short hairpin RNA negative control (shNC) or shHDAC5. ( B ) Heatmap shows the normalized IC 50 ratio of different KRAS G12D inhibitors determined by cell counting kit 8 (CCK8) assay in PANC-1 and AsPC-1 cells ( n = 2). The normalized IC 50 ratio was calculated as the fold-change in IC 50 of shHDAC5 relative to shNC. ( C ) The IC 50 of MRTX1133 was assessed by CCK8 assay in PANC-1 and AsPC-1 cells with HDAC5 knockdown. ( D and E ) Representative images of 3D-cultured HDAC5-depleted cells treated with DMSO or MRTX1133 (PANC-1: 10 μM; AsPC-1: 5 nM; 48 hours). Scale bars = 100 μm. Relative survival of cells ( E ) ( n = 3). ( F ) Cell viability of PANC-1 and AsPC-1 cells expressing shNC or shHDAC5 and treated with DMSO or MRTX1133 (10 μM for PANC-1; 5 nM for AsPC-1), measured by CCK8 assay ( n = 3). ( G ) Representative images and size quantification of PDOs treated with DMSO or MRTX1133 (1 μM) ( n = 5). All data are presented as the mean ± SD. Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple comparisons test ( E – G ). * P < 0.05, *** P < 0.001.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: shRNA, Negative Control, Cell Counting, CCK-8 Assay, Knockdown, Cell Culture, Expressing

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Representative macroscopic tumor images and tumor weights from KPC Hdac5-WT and KPC Hdac5-KO mouse models treated with vehicle or MRTX1133 (30 mg/kg, i.p., twice daily [bid]) ( n = 5). ( B ) Kaplan-Meier survival curves with log-rank test ( n = 5). ( C and D ) Representative IHC images of tumors in C . IHC scores were quantified in D . Scale bars = 100 μm. n = 5 biologically independent repeats and 3 independent IHC quantifications. ( E ) C57BL/6 mice were orthotopically injected with KPC-Luc cells expressing shNc or shHdac5. Bioluminescence imaging was performed on day 7, followed by treatment with vehicle or MRTX1133 (30 mg/kg, i.p., bid). Representative bioluminescence images and corresponding quantification were acquired on day 28 ( n = 5). ( F ) Tumor growth curves in PDX models with treatment with vehicle or MRTX1133 (30 mg/kg, i.p., bid) ( n = 5). ( G and H ) Representative IHC images of PDXs and quantified IHC scores ( H ). Scale bars = 100 μm. n = 5 biologically independent repeats and 3 independent IHC quantifications. All data are presented as the mean ± SD. Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple comparisons test ( A , D – F , and H ). * P < 0.05, *** P < 0.001.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Injection, Expressing, Imaging

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Venn diagram showing the overlap of genes upregulated in KPC Hdac5-KO mice ( n = 5 per genotype; log 2 [fold-change] > 1, P < 0.05, 2,410 genes) identified via RNA-Seq and genes upregulated in shHDAC5-treated PANC-1 cells ( n = 3 per condition; log 2 [fold-change] > 1, P < 0.05, 2,683 genes), revealing a shared subset of 378 genes. ( B ) Bar graph showing the top 10 enriched transcription factors from transcription factor analysis of 378 genes in A using Enrichr. ATF2, activating transcription factor 2. ( C ) Dual-luciferase reporter assays were performed to assess the transcriptional activity of c-Myc in HDAC5-depleted PANC-1 and AsPC-1 cells. Data are presented as mean ± SD ( n = 5). Statistical significance was determined by 1-way ANOVA followed by Dunnett’s multiple comparisons test. *** P < 0.001. ( D ) Heatmap of MYC ChIP-Seq signal intensity (±3 kb around MYC binding sites) in control vs. HDAC5-knockdown PANC-1 cells. ( E ) The average ChIP signal of MYC centered at transcription start site (±3 kb) in indicated groups. ( F ) Venn diagram depicting the overlap between genes with enhanced MYC promoter occupancy after shHDAC5 knockdown, as identified by ChIP-Seq, and genes upregulated in PANC-1 cells following shHDAC5 knockdown, as determined by RNA-Seq. P = 6 × 10 –71 . ( G ) KEGG pathway enrichment analysis of 821 intersecting genes in F . ( H and I ) Western blot analysis of canonical MAPK pathway protein expression under indicated conditions.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: RNA Sequencing, Luciferase, Activity Assay, ChIP-sequencing, Binding Assay, Control, Knockdown, Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A and B ) Western blot ( A ) and RT-qPCR ( B ) analyses of c-Myc expression in PANC-1 and AsPC-1 cells infected with indicated shRNAs for 48 hours ( n = 3). ( C ) Western blot analysis of c-Myc protein levels in PANC-1 and AsPC-1 cells transfected with indicated plasmids and treated with DMSO or MG132 (10 μM, 8 hours). EV, empty vector. ( D ) RT-qPCR analysis of c-Myc mRNA in PANC-1 and AsPC-1 cells transfected with indicated plasmids for 48 hours ( n = 3). ( E ) Immunofluorescence analysis of c-Myc in PANC-1 cells transfected with indicated plasmids for 48 hours. Representative images and fluorescence intensity quantification are shown ( n = 5). ( F ) Western blot analysis and quantification of c-Myc protein stability in control or HDAC5-knockdown PANC-1 and AsPC-1 cells treated with cycloheximide (CHX, 50 μg/mL) for indicated times ( n = 3). ( G ) Colocalization analysis of the merged images in E , showing pixel intensity profiles along the white line from left to right in each panel. Colors correspond to the merged images: green for c-Myc and blue for DAPI. All data are presented as the mean ± SD. Statistical significance was determined by 1-way ANOVA followed by Dunnett’s multiple comparisons test ( B , D , and E ) or 2-way ANOVA followed by Tukey multiple comparisons test ( F ). ** P < 0.01, *** P < 0.001.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Infection, Transfection, Plasmid Preparation, Immunofluorescence, Fluorescence, Control, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Co-IP assay showing interaction between c-Myc and HDAC5 in PANC-1 and AsPC-1 cells. ( B ) Schematic diagrams of the truncations of GST-HDAC5. ( C ) Western blot analysis of full-length c-Myc protein in PANC-1 whole-cell lysate pulled down by GST or GST-HDAC5 recombinant proteins. Arrows indicate expected bands. ( D ) Co-IP detection of acetylated lysine on c-Myc in HDAC5-deficient PANC-1 and AsPC-1 cells. ( E ) Co-IP detection of acetylated lysine on c-Myc in PANC-1 and AsPC-1 cells overexpressing plasmids as indicated. ( F and G ) Co-IP analysis of c-Myc ubiquitination levels in HDAC5-deficient ( F ) or HDAC5-overexpressing ( G ) PANC-1 cells. ( H ) A hypothetical model depicting that HDAC5 deacetylates c-Myc, promoting its ubiquitination and degradation.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Recombinant, Ubiquitin Proteomics

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Illustration of c-Myc acetylation at K148 identified by mass spectrometry. ( B ) Mass spectrometry quantification of c-Myc-K148ac intensity in siControl vs. siHDAC5 groups. si, siRNA. ( C ) Different peptides dissolved in double-distilled H 2 O were applied to the nitrocellulose membrane, followed by immunoblotting with anti–c-Myc-K148ac antibody. ( D ) Co-IP detection of c-Myc-K148ac in PANC-1 cells expressing c-Myc (WT/K148R/K148Q) and treated with shNC or shHDAC5. ( E – G ) PANC-1 and AsPC-1 cells were transfected with equal amounts of c-Myc (WT/K148R/K148Q) plasmids for 48 hours. Afterward, cells were harvested for RT-qPCR analysis ( E ) and Western blot analysis ( F ), followed by protein quantification analysis ( G ) ( n = 3). ( H ) Western blot analysis was conducted to evaluate c-Myc protein levels and normalized protein intensity in PANC-1 cells transfected with equal amounts of c-Myc (WT/K148R/K148Q) plasmids after treatment with 50 μg/mL CHX for the indicated durations ( n = 3). ( I ) Co-IP detection of c-Myc ubiquitination in PANC-1 cells expressing c-Myc (WT/K148R/K148Q) and treated with shNC or shHDAC5. ( J ) Illustration of c-Myc ubiquitination at K148 identified by mass spectrometry. ( K ) A bar graph showing the intensities of c-Myc ubiquitination at K148 identified by mass spectrometry in shNC and shHDAC5 groups. ( L ) Luciferase reporter activities of c-Myc were assessed in PANC-1 cells infected with shHDAC5 and knockin c-Myc (WT/K148R/K148Q) ( n = 5). ( M ) A hypothetical model illustrating how HDAC5 regulates c-Myc degradation through the competition between K148 acetylation and ubiquitination. All data are presented as the mean ± SD. Statistical significance was determined by 1-way ANOVA ( E and G ) or 2-way ANOVA ( H and L ), followed by Tukey’s multiple comparisons test. ** P < 0.01, *** P < 0.001.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Mass Spectrometry, Membrane, Western Blot, Co-Immunoprecipitation Assay, Expressing, Transfection, Quantitative RT-PCR, Ubiquitin Proteomics, Luciferase, Infection, Knock-In

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Mass spectrometry identified NEDD4 peptide fragments coprecipitated with c-Myc antibody, showing reduced signals upon HDAC5 knockdown. ( B and C ) Western blot analysis of PANC-1 cells infected with indicated siRNAs for 48 hours. ( D ) Western blot analysis and quantification of c-Myc protein stability in control or NEDD4-knockdown PANC-1 cells after CHX (50 μg/mL) treatment. All data are presented as the mean ± SD ( n = 3). Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple comparisons test. * P < 0.05. ( E ) Endogenous co-IP analysis of the NEDD4/c-Myc interaction in PANC-1 cells transfected with indicated siRNAs. ( F and G ) Co-IP detection of c-Myc ubiquitination ( F ) and c-Myc-K148ac ( G ) in PANC-1 cells expressing c-Myc-WT and treated with siHDAC5, siNEDD4, or both. ( H ) Co-IP detection of c-Myc ubiquitination in PANC-1 cells expressing c-Myc (WT/K148R/K148Q) and treated with siControl or siNEDD4. ( I ) In vitro ubiquitination assay evaluating NEDD4 regulation of c-Myc (WT/K148R/K148Q) ubiquitination at K148. ( J ) A hypothetical model illustrating how HDAC5 and NEDD4 competitively modify the acetylation and ubiquitination of c-Myc at the K148 site, collaboratively promoting c-Myc protein degradation.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Mass Spectrometry, Knockdown, Western Blot, Infection, Control, Co-Immunoprecipitation Assay, Transfection, Ubiquitin Proteomics, Expressing, In Vitro

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Representative macroscopic images of pancreatic tumors from KPC Hdac5-WT and KPC Hdac5-KO mice after sacrifice. ( B ) Tumor weight analysis in KPC mice treated with vehicle, MRTX1133 (30 mg/kg, i.p. bid), MYCi975 (50 mg/kg, i.p. bid), or their combination ( n = 5). ( C ) Kaplan-Meier survival curves with log-rank test ( n = 5). *** P < 0.001. ( D and E ) Representative IHC images of tumors from treated mice and quantified IHC scores ( E ). Scale bars = 100 μm. n = 5 biologically independent repeats and 3 independent quantifications. ( F and G ) Representative bioluminescence images and quantification ( n = 5). ( H ) Macroscopic images of PDX tumors after 30 days of treatment. ( I and J ) Tumor growth curves ( I ) and final tumor weights ( J ) in PDX models treated with Vehicle + LV-Control, MRTX1133 (30 mg/kg, i.p. bid) + LV-Control, Vehicle + LV-sgMYC (50 μL lentivirus, s.c., weekly), or MRTX1133 + LV-sgMYC. All data are presented as the mean ± SD. Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple comparisons test ( B , E , G , I , and J ). *** P < 0.001.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Control