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. 2025 Dec 11;136(3):e195814.
doi: 10.1172/JCI195814. eCollection 2026 Feb 2.

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

Taoyu Chen  1   2 , Haixin Yu  3 , Keshan Wang  4 , Gengdu Qin  1   2 , Yuhan Zhao  1   2 , Xueyi Liang  1   2 , Yuxuan Li  1   2 , Tianhao Zou  1   2 , Jiaying Liu  5 , Jingyuan Zhao  1   2 , Zhiqiang Liu  1   2 , Ruozheng Wei  1   2 , Bo Wang  1   2 , Shanmiao Gou  1   2 , Tao Yin  1   2 , Heshui Wu  1   2 , Xin Jin  3 , Yingke Zhou  1   2
Affiliations

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

Taoyu Chen et al. J Clin Invest. .

Abstract

KRAS mutations serve as key oncogenic drivers in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC). Despite the advancement of KRAS inhibitors, such as MRTX1133, for PDAC treatment, intrinsic and acquired resistance remain major barriers to their clinical efficacy. This study underscored the role of histone deacetylase 5 (HDAC5) loss in mediating intrinsic resistance to KRASG12D inhibitors. Mechanistically, HDAC5 promoted c-Myc degradation by deacetylating K148, thereby facilitating NEDD4-mediated ubiquitination at this site. The loss of HDAC5 resulted in hyperacetylation of c-Myc at K148, impeding the ubiquitination and subsequent degradation process of c-Myc following deacetylation. Consequently, c-Myc stability and transcriptional activity were sustained even under KRAS/MEK/ERK pathway inhibition, reinforcing MAPK signaling and promoting cell survival despite KRAS suppression. Our data further demonstrated that pharmacological or genetic inhibition of c-Myc effectively reversed the resistance phenotype mediated by HDAC5 loss, suggesting a therapeutic strategy centered on KRAS-MYC dual-node blockade. Furthermore, the expression levels of HDAC5 and the acetylation status of c-Myc may serve as biomarkers for predicting the therapeutic response to MRTX1133. These findings provide insights into overcoming resistance to KRASG12D inhibitors and offer potential biomarkers and combinatorial therapeutic strategies for precision treatment of PDAC.

Keywords: Cancer; Cell biology; Drug therapy; Epigenetics; Oncology.

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Figures

Figure 1
Figure 1. Loss of HDAC5 induces intrinsic resistance to KRAS inhibitor in vitro.
(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 IC50 ratio of different KRASG12D inhibitors determined by cell counting kit 8 (CCK8) assay in PANC-1 and AsPC-1 cells (n = 2). The normalized IC50 ratio was calculated as the fold-change in IC50 of shHDAC5 relative to shNC. (C) The IC50 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.
Figure 2
Figure 2. Loss of HDAC5 induces intrinsic resistance to KRAS inhibitor in vivo.
(A) Representative macroscopic tumor images and tumor weights from KPCHdac5-WT and KPCHdac5-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.
Figure 3
Figure 3. Loss of HDAC5 upregulates MAPK signaling via c-Myc.
(A) Venn diagram showing the overlap of genes upregulated in KPCHdac5-KO mice (n = 5 per genotype; log2[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; log2[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.
Figure 4
Figure 4. Loss of HDAC5 promotes the stabilization of c-Myc protein.
(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.
Figure 5
Figure 5. HDAC5 promotes c-Myc ubiquitination and degradation through its deacetylation.
(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.
Figure 6
Figure 6. HDAC5 loss disrupts the acetylation-ubiquitination homeostasis at lysine 148 of c-Myc.
(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 H2O 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.
Figure 7
Figure 7. HDAC5-mediated c-Myc deacetylation facilitates NEDD4-mediated ubiquitination at K148 of c-Myc.
(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.
Figure 8
Figure 8. Pharmacological or genetic inhibition of c-Myc overcomes the resistance to KRAS inhibitor induced by HDAC5 loss in vitro.
(A) IC50 values of MRTX1133 in PANC-1 and AsPC-1 cells expressing indicated plasmids for 48 hours, with or without MYCi975 (10 μM), measured by CCK8 assay. (B) Cell viability of PANC-1 and AsPC-1 cells transfected with the indicated plasmids and treated with DMSO, MRTX1133, MYCi975 (10 μM), or the combination, assessed by CCK8 assay. (C and D) PANC-1 and AsPC-1 cells were infected with the indicated shRNAs. At 48 hours postinfection, the cells were treated with vehicle (DMSO), MRTX1133 (10 μM for PANC-1; 5 nM for AsPC-1), MYCi975 (10 μM), or the combination. Colony formation assays were then performed. The resulting colonies were imaged and quantified using ImageJ (NIH) (D) (n = 3). (E and F) Representative images and size quantification of PDOs treated with DMSO, MRTX1133 (1 μM), MYCi975 (4 μM), or their combination. Scale bar = 200 μm (n = 5). (G) Western blot validation of MYC gene editing efficiency using a CBE in PANC-1 and AsPC-1 cells. (H) IC50 values of MRTX1133 under indicated conditions, assessed by CCK8 assay. (I) Cell viability of PANC-1 and AsPC-1 cells transfected with vector or CBE-MYC and treated with DMSO or MRTX1133 (PANC-1: 10 μM; AsPC-1: 5 nM) (n = 3). All data are presented as the mean ± SD. Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple comparisons test (B, D, F, and I). *P < 0.05, ***P < 0.001.
Figure 9
Figure 9. Pharmacological or genetic inhibition of c-Myc overcomes the resistance to KRAS inhibitor induced by HDAC5 loss in vivo.
(A) Representative macroscopic images of pancreatic tumors from KPCHdac5-WT and KPCHdac5-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.
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