Histone Methyltransferase KMT2D Promotes Castration-Resistant Prostate Cancer Progression by Reactivating AR through FOXA1

Histone Methyltransferase KMT2D Promotes Castration-Resistant Prostate Cancer Progression by Reactivating AR through FOXA1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Histone Methyltransferase KMT2D Promotes Castration-Resistant Prostate Cancer Progression by Reactivating AR through FOXA1 Qiang Wei, Mayao Luo, Chenwei Wu, Manli Zhou, Yifan Zhang, Yadong Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7812403/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Prostate cancer (PCa) progression, particularly to castration-resistant prostate cancer (CRPC), is driven by androgen receptor (AR) reactivation and epigenetic alterations. Here, we identify lysine methyltransferase 2D (KMT2D) as a critical epigenetic oncogene in PCa. KMT2D expression is elevated in PCa and correlates with poor prognosis. Mechanistically, KMT2D facilitates AR signaling by recruiting the pioneer factor FOXA1 to AR-specific enhancers, promoting chromatin accessibility and activating AR target genes. FOXA1 mutations impair this regulation, demonstrating their functional interplay. Furthermore, KMT2D-FOXA1-AR axis modulates ketone body metabolism via transcriptional control of HMGCS2, supporting tumor growth. Pharmacological inhibition of UTX, a COMPASS complex demethylase essential for KMT2D function, disrupts H3K4me1 deposition and suppresses AR signaling and tumor proliferation. Altogether, we characterize KMT2D as a key driver of AR-dependent PCa progression and propose UTX inhibition as a promising therapeutic strategy. Biological sciences/Genetics/Epigenetics Biological sciences/Cancer/Urological cancer/Prostate cancer Biological sciences/Cancer/Cancer metabolism prostate cancer androgen receptor KMT2D FOXA1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Background Prostate cancer (PCa) is one of the most prevalent malignancies in men worldwide and the second leading cause of cancer mortality 38 . Although androgen deprivation therapy (ADT) as the standard initial treatment significantly improves survival, most patients ultimately progress to castration-resistant prostate cancer (CRPC), which is characterized by metastatic spread and poor prognosis 8 . Epigenetic alterations, including DNA methylation, histone modification, and chromatin remodeling, critically influence CRPC progression 40 . These modifications modulate gene expression and promote cancer cell survival, proliferation, and resistance to therapy. The androgen receptor (AR) signaling pathway cooperates with these epigenetic changes, amplifying AR activity and accelerating CRPC advancement 12 . Lysine methyltransferase 2D (KMT2D) or mixed-lineage leukemia 4 (MLL4) monomethylates histone H3 at lysine 4 (H3K4me1) in mammalian systems 15 . KMT2D collaborates with transcription factors and co-regulators to maintain an open chromatin architecture across cell types, governing processes such as proliferation, embryonic differentiation, and tumorigenesis through H3K4me1-mediated transcriptional control and pathways such as PI3K/Akt, Notch, and Wnt 4 , 28 , 30 , 44 . Aberrant KMT2D expression or mutations contribute to malignancies, including lymphoma 33 , ovarian 22 , bladder 41 , breast 44 , and lung cancers 3 , 29 . In PCa, KMT2D acts as an oncogene by activating proliferative and metastatic signaling cascades 25 , 26 . Although KMT2D modulates estrogen receptor (ER) transcriptional activity via Forkhead Box A1 (FOXA1) in breast cancer 44 , its interaction with AR in PCa remains poorly understood. We demonstrated that KMT2D critically regulated AR transcriptional activity by recruiting FOXA1, thereby driving PCa progression. FOXA1 mutations in the forkhead DNA-binding domain (FKHD), such as D226G and M253K, disrupt KMT2D-mediated AR regulation and cell proliferation. Furthermore, inhibiting UTX (also called KDM6A), a COMPASS complex component, markedly reduced KMT2D-dependent H3K4me1 catalysis and AR activity. These findings elucidate the mechanistic basis of KMT2D-driven CRPC progression and highlight its potential as a therapeutic target for PCa. 2. Methods Cell culture and reagents The LNCaP, C4-2, and HEK293T were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China). LNCaP and C4-2 cells were maintained in RPMI 1640 medium (Corning), whereas HEK293T cells were grown in DMEM (Corning). All media contained 10% FBS (Haixing Biosciences, China) or charcoal-stripped FBS (CSS, Biological Industries), along with 100 U/mL penicillin, and 100 µg/mL streptomycin (Thermo Fisher Scientific) and were incubated at 37°C with 5% CO₂. PCR confirmed the absence of mycoplasma contamination. Enzalutamide (MedChem Express, HY-70002) and dihydrotestosterone ( M6033) were purchased from commercial sources. The phenol red-free RPMI 1640 medium was purchased from Thermo Fisher Scientific. RNA interference : KMT2D siRNA (siKMT2D) and negative control siRNA (siNC) were obtained from Dharmacon (Catalog ID: L-004828-00-0020 for KMT2D and D-001810-10-20 for the negative control). Cells were transfected with 100 nM siRNA using Lipofectamine 3000 (Thermo Fisher Scientific), according to the manufacturer’s protocol. CRISPR/Cas9-mediated KMT2D gene knockout pLV-CMV-FOXA1(D226G)-3FLAG and pLV-CMV-FOXA1(M253K)-3FLAG plasmids were constructed as previously described (17). The mutations were confirmed by Sanger sequencing (Supplementary Figure S4). FOXA1 mutation plasmids construction Construction of the pLV-CMV-FOXA1(D226G)-3FLAG and pLV-CMV-FOXA1(M253K)-3FLAG plasmids was conducted as previously described 17 . The mutations were confirmed by Sanger sequencing (Supplementary Figure S4). Cell viability assay Cells were directly plated in 96-well plates at a density of 3,000–5,000 cells per well and allowed to adhere for 24 h. Subsequent treatments were performed under designated experimental conditions. Following a 48-h incubation period, the CCK-8 kit (Apexbio, K1018, America) was used to evaluate cell viability, following the manufacturer’s instructions. 5-ethynyl-2’-deoxyuridine (EdU) proliferation assay PCa cells (3 × 10⁴ cells/well) were plated in 96-well plates and allowed to adhere for 24 h. The cells were then cultured in either standard medium with 10 nM Dihydrotestosterone (DHT) or in phenol red-free RPMI-1640 supplemented with CSS. Following 48 h of treatment, 50 µM EdU (Apexbio) was added for 2 h before processing, according to the manufacturer’s protocol. Nuclear staining was performed using Hoechst 33342 (5 µg/mL) for 30 min, followed by fluorescence microscopy. ImageJ software was used to quantify the EdU-positive cell density by comparing nuclear Hoechst 33342 staining with negative controls. Colony formation assays 1,000 PCa cells were seeded into six-well plates per well for two weeks. After washing with PBS, cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Colony counts were quantified using the ImageJ software. RNA extraction, reverse transcription, and quantitative real-time PCR (qRT-PCR) PCa cells were cultured in 12-well plates and subjected to the specified treatments. Total RNA was extracted after 48 h using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. Reverse transcription was performed with 1 µg of RNA using the HiScript® III All-in-one RT SuperMix kit (Vazyme Biotech Co. Ltd.). Quantitative PCR analysis was performed using the Taq Pro Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.) on a QuantStudio™ 6 Flex system (Thermo Fisher Scientific). The relative gene expression levels were calculated using the 2 –ΔΔCt method. The primer details are listed in Supplementary Table S1 . Western blot PCa cells were lysed using RIPA buffer supplemented with an EDTA-free protease inhibitor cocktail (Bimake, China), and protein concentrations were determined using the BCA Protein Assay Kit. samples were mixed with loading buffer and denatured by heating at 100°C for 10 min. Proteins (30 µg per lane) were resolved by SDS-PAGE and transferred to 0.45 µm PVDF membranes (Bio-Rad). The membranes were probed with primary antibodies, followed by incubation with HRP-conjugated Goat Anti-Rabbit IgG or Goat Anti-Mouse IgG. Immunoreactive bands were visualized using an ultra-high sensitivity ECL kit and quantified using the ImageJ software. Uncropped Western blots are provided in the Supplementary Materials. RNA-Seq and data analysis All cell lines were cultured in their respective media under the specified treatment conditions. Total RNA was isolated using TRIzol reagent (Invitrogen), according to the manufacturer’s protocol. Libraries were prepared and sequenced on the NovaSeq 6000 platform. The resulting sequencing reads were aligned to the human genome reference sequence (hg38) using HISAT2 (v2.0.5) with default parameters. Subsequently, gene-level quantification was performed using FeatureCounts (v1.5.0-p3). Differential expression analysis was conducted using EdgeR (version 3.22.5), incorporating the Benjamini-Hochberg procedure to adjust for multiple hypothesis testing and to control the false discovery rate (FDR). Gene ontology (GO) enrichment was analyzed using clusterProfiler (version 3.8.1), and pathway enrichment was evaluated using gene set enrichment analysis (GSEA, version 4.2.2). Chromatin immunoprecipitation-sequencing (ChIP-seq) and data analysis ChIP assays were conducted according to the manufacturer's protocol (9003, Cell Signaling Technology,USA). LNCaP and C4-2 cells were subjected to crosslinking, lysis, and enzymatic digestion to generate chromatin fragments. These fragments were immunoprecipitated overnight with either an anti-AR antibody (Cell Signaling Technology, 5153) or a control IgG. The Novogene Corporation (Beijing, China) prepared ChIP-seq libraries from purified DNA. Sequencing was performed on the Illumina NovaSeq 6000 platform. Bowtie2 (v2.2.5) aligned the reads to the hg38 human genome using default parameters, followed by SAM-to-BAM conversion using Samtools (v1.6). MACS2 (v2.2.6) identified enriched regions, whereas Homer (v4.11) analyzed the DNA motifs. Peak visualization across samples was achieved using Integrative Genomics Viewer (IGV; v2.16.2). Cleavage under targets and tagmentation (CUT&Tag) assay and data analysis CUT&Tag was performed according to manufacturer's protocol (Yeasen, 12597ES) according to the manufacturer’s protocol. Briefly, 1 × 10⁵ cells were collected, washed, and incubated with concanavalin A-coated beads. After sequential incubation with primary and secondary antibodies, DNA fragments were generated using pA-Tn5 transposase. For library preparation, purified DNA was amplified using 2×HiFi Amplification Mix and uniquely barcoded primers from the Hieff NGS® Tagment Index Kit for Illumina® (Yeasen, 12416), followed by purification with DNA Selection Beads. All libraries were sequenced on an Illumina NovaSeq 6000 platform. Fastp (v0.22.0) filtered low-quality reads, and Bowtie2 (v2.2.5) aligned the reads to the hg38 genome using parameters “-X 2000 --very-sensitive-local -no-discordant.” Sambamba (v0.6.6) removed PCR duplicates and Samtools (v1.6) converted SAM to BAM files. MACS2 (v2.2.6) called the peaks at the q-value threshold of 0.05. DeepTools (v3.5.2) generated BigWig files, profile plots, and heatmaps, whereas Homer (v4.11) identified motifs. IGV (v2.16.2) visualized the signal tracks, and EdgeR (v3.22.5) performed differential peak analysis. Assay for transposase-accessible chromatin library preparation, sequencing, and data analysis The assay for transposase-accessible chromatin with sequencing (ATAC-seq) samples was performed in duplicate using the Hyperactive ATAC-seq Library Prep Kit (TD711, Vazyme Biotech Co., Ltd.) following the manufacturer’s instructions. Briefly, 100,000 cells were collected, washed, and tagged with the Tn5 transposase. The digested DNA was then purified using magnetic beads. Libraries were amplified and sequenced using the Illumina platform. The Fastp tool (v.0.22.0) was used to obtain quality control statistics of the samples. The ATAC-seq clean reads were mapped to the hg38 using Bowtie2 (v2.2.5) using parameters “-X 2000 --very-sensitive-local -no-discordant.” PCR-duplicated reads were removed using Sambamba (v0.6.6). SAM files were converted to BAM files using Samtools (v1.6). MACS2 (v2.2.6) was used to identify peaks with parameters “-q 0.05 --extsize 200 –shift 100.’’ DeepTools (v3.5.2) was used to convert the BAM files to BigWig files to generate a profile plot and heatmap. Homer (v4.11) was used for motif analysis. The ATAC-Seq signal tracks were visualized using IGV (v2.16.2). The R package EdgeR (3.22.5) was used to perform differential peak analysis. Ketone body detection A Ketone Body Content Assay Kit (Solarbio, BC5065) was used to measure the cellular ketone body content, following the manufacturer’s instructions. Patient-derived tumor fragments (PDTF) The PDTF collection was approved by the Ethics Committee of Nanfang Hospital (NFEC-2022-299). Ex vivo culture of PDTFs was conducted as previously described 45 . Briefly, prostate tumor tissue was collected in a 50 mL tube, kept on ice, and transferred to a pre-chilled collection medium. The prostate tumor tissue was then cut into approximately 1 mm 3 sections. PDTFs from two patients with prostate cancer were collected and frozen in vials containing freezing medium. For the PDTF culture, cryopreserved PDTFs were thawed, washed, and embedded in the prepared extracellular matrix in 96-well plates. A tumor medium supplemented with either 10 µM GSK-J4 or DMSO was added to the top of the matrix. The PDTFs were cultured for 48 h before RNA and total protein extraction. Xenograft study : All animal procedures were approved by the Ethics Committee of Ganzhou Hospital, Southern Medical University (approval no. TY-DKY2023-020-01). Four-week-old male BALB/c nude mice (strain no. D000521) from GemPharmatech (Nanjing, China) was subcutaneously injected with 2×10 6 C4-2 sgNC or C4-2 sgKMT2D cells suspended in a 1:1 PBS-Matrigel mixture. In separate inhibitor experiments, castrated male mice were implanted with 2×10 6 Enzalutamide resistance (EnzR) cells and randomly assigned to treatment groups when tumors reached 100 mm³. The control group received vehicle (5% DMSO, 40% PEG300, 5% Tween80), while the experimental group received 10 mg/kg/day GSK-J4 via intraperitoneal injection for 15 days. Tumor dimensions were recorded every 72 h using calipers and volumes were calculated as L×W 2 /2. Following euthanasia, the excised tumors were subjected to qRT-PCR, western blotting, and histopathological examination. Histology and Immunohistochemistry (IHC) Subcutaneous tumors excised from nude mice were fixed overnight in 4% paraformaldehyde, dehydrated using a series of ethanol concentrations, and embedded in paraffin. Consecutive 4 µm tissue sections from the formalin-fixed samples were subjected to routine hematoxylin and eosin (H&E) staining and immunohistochemistry. Quantification of digital IHC images was performed using ImageJ (Fiji) to assess the extent of staining (75%=4) and staining intensity (0 = negative; 1 = weak; 2 = moderate; 3 = strong) for each specimen. The immunoreactive score (IRS), ranging from 0 to 12, was calculated by multiplying the two values. Statistical analysis Data analyses were performed using GraphPad Prism 10 software, with statistical significance set at p < 0.05. Each experimental setup included a minimum of two biological replicates to ensure reliable statistical assessment. Results are presented as the mean ± standard error of the mean (SEM), with error bars indicating variability among three or more biological replicates or independent experiments. Comparisons between two groups were conducted using unpaired two-tailed Student’s t-tests (*P < 0.05, **P < 0.01, ***P < 0.001), whereas multi-group comparisons were conducted using one-way ANOVA. Survival analyses were performed using Kaplan-Meier curves, and differences were tested using two-sided log-rank tests. 3. Results 3.1 KMT2D expression and activity are upregulated in prostate cancer and related to poor prognosis To comprehensively evaluate KMT2D expression and activity in prostate cancer, we initially assessed its mRNA levels in the Renji cohort. Tumor tissues exhibited significantly higher KMT2D expression than adjacent normal tissues (P < 0.01, Fig. 1 A). Independent validation in GSE35988 and GSE70770 revealed elevated KMT2D expression in castration-resistant prostate cancer compared to localized PCa (Figs. 1 B and 1 C, P < 0.001). TCGA-PRAD survival analysis (Fig. 1 D) revealed that high KMT2D expression was correlated with shorter disease-free survival (log-rank P = 0.018), suggesting its prognostic value. Immunohistochemical staining from the Human Protein Atlas and Nanfang cohorts confirmed increased KMT2D protein expression in prostate cancer tissues (Fig. 1 E), consistent with transcriptomic findings. Gene effect analysis based on CRISPR knockout screens in DepMap (Fig. 1 F) revealed that KMT2D is critical for cell viability in prostate adenocarcinoma as well as in multiple other cancer types, including acute myeloid leukemia, invasive breast carcinoma, ovarian epithelial tumors, and diffuse glioma (all adjusted P < 0.001). We then employed CUT&Tag and RNA-seq in C4-2 and LNCaP cell lines to map KMT2D-regulated genes and identify 61 direct targets (Fig. 1 G). Analysis of GSE181294 single-cell RNA-seq data distinguished benign from malignant epithelial clusters (Fig. 1 H), with KMT2D activity markedly increased in tumor cells (Figs. 1 I and 1 J, P < 0.001), supporting its association with malignant transformation at single-cell resolution. Together, these findings establish that KMT2D is consistently overexpressed in prostate cancer, particularly in CRPC, where it is correlated with poor prognosis. 3.2 KMT2D Enhances Androgen Receptor Signaling Through Direct Regulation of AR Target Genes Previous research established endogenous AR reporter cell lines (LNCaP_mCherry_PSA) which mCherry expression is directly regulated by the AR transcriptional complex. Functional CRISPR screen was performed and KMT2D was identified as an AR coactivator 31 (Fig. 2 A). To investigate KMT2D's function in AR signaling, we analyzed pathway enrichment related to KMT2D-activated genes. Gene set enrichment revealed significant associations between KMT2D and AR-related pathways, including the androgen response, AR ChIP-seq targets, and prostate cancer signatures (Fig. 2 B). Analysis of TCGA-PRAD data revealed a significant positive correlation between KMT2D expression and AR signature scores (R = 0.39, P < 0.0001) as well as between the KMT2D signature and AR signature (R = 0.71, P < 0.0001) at the transcript level (Supplementary Fig. 2A). Single-cell RNA-seq analysis revealed markedly higher AR activity in tumor cells than in normal prostate epithelial cells (Fig. 2 C, P < 0.05). The AR and KMT2D activities exhibited a robust positive correlation at the single-cell level (Fig. 2 D, R = 0.58, P < 2.2e-16). We then examined KMT2D's functional impact through CRISPR knockout in C4-2 cells and siRNA knockdown in LNCaP cells (Supplementary Fig. 2B and 2C). Both genetic approaches markedly suppressed KLK3, TMPRSS2, and NKX3-1 transcript levels under both regular and androgen-depleted (CSS) conditions (Fig. 2 E and Supplementary Fig. 2C and 2E). Western blot analyses confirmed that KLK3 protein level decreased upon KMT2D silencing in LNCaP and C4-2 cells treated with dihydrotestosterone (DHT) or CSS medium (Fig. 2 F). Consistently, the inhibitory effect on KLK3 expression showed a clear dose dependence in both cell lines (Fig. 2 G and Supplementary Fig. 2D). To investigate the regulatory role of KMT2D in AR, we conducted RNA-seq analysis after knocking down KMT2D in LNCaP and C4-2 cells exposed to DHT or cultured under CSS conditions. Heatmaps revealed that androgen-induced genes were substantially downregulated upon KMT2D depletion in both cell lines under all treatment conditions (Supplementary Fig. 2F). AR activity scores derived from RNA-seq data also decreased significantly after KMT2D knockdown in LNCaP and C4-2 cells, whether stimulated with DHT or deprived of androgens (Fig. 2 H). GSVA confirmed this attenuated AR activity by showing reduced expression across multiple androgen-responsive gene sets (Fig. 2 I). Broader GSEA analysis across treatment groups consistently demonstrated suppressed androgen signaling pathways following KMT2D loss (Fig. 2 J and Supplementary Fig. 2G), underscoring KMT2D's essential function in maintaining AR transcriptional activity. To investigate KMT2D-AR interactions at the chromatin level, we performed genome-wide CUT&Tag profiling of LNCaP and C4-2 cells. Venn analysis revealed extensive overlap between KMT2D and AR binding sites, indicating frequent co-occupancy of the regulatory elements (Fig. 2 K). Combining RNA-seq with AR CUT&Tag through BETA analysis showed that KMT2D predominantly activates AR transcription (Kolmogorov-Smirnov test, P < 0.0001; Fig. 2 L), highlighting its functional role at AR target loci. AR ChIP-seq also showed that KMT2D depletion substantially reduced the AR binding intensity at promoters and enhancers in both cell lines (Fig. 2 M). Genomic annotation of lost AR sites revealed enrichment in distal intergenic and promoter regions aligned with known transcriptional regulatory elements (Fig. 2 N). ChIP-qPCR validated the significantly reduced AR occupancy at canonical target genes (KLK3, NKX3-1, KLK2, and TMPRSS2) upon KMT2D silencing (Fig. 2 O). Genome browser tracks further confirmed the diminished AR peaks at these loci after KMT2D knockdown (Supplementary Fig. 2H). Collectively, these results establish KMT2D as a critical facilitator of AR chromatin binding and transcriptional activation in prostate cancer cells. 3.3 KMT2D Depletion Inhibits Prostate Cancer Cell Proliferation and Tumor Growth To investigate the functional role of KMT2D in prostate cancer cell proliferation, we conducted loss-of-function studies in LNCaP and C4-2 cells. KMT2D knockdown using siRNA or sgRNA substantially inhibited cell proliferation after 72 h, as quantified by CCK-8 assays (Fig. 3 A, P < 0.001). Colony formation assays also confirmed this effect, showing a significantly diminished clonogenic potential in both cell lines following KMT2D depletion (Fig. 3 B). EdU incorporation assays showed that KMT2D silencing reduced proliferating cell populations in LNCaP and C4-2 cells under standard culture conditions, regardless of DHT stimulation and in CSS medium (Fig. 3 C). KMT2D depletion also attenuated cell viability with increasing DHT levels, suggesting its involvement in androgen-mediated proliferation (Fig. 3 D and 3 E). In C4-2 xenograft models, KMT2D knockout delayed tumor progression and reduced tumor volume (Figs. 3 F and 3 G). Excised tumors from the KMT2D-deficient groups weighed significantly less than those from the controls (Figs. 3 H and 3 I). qRT-PCR analyses showed decreased expression of AR target genes KLK3, TMPRSS2, and NKX3.1 in KMT2D-deficient tumors (Fig. 3 J). Western blotting confirmed reduced KLK3 protein and H3K4me1 levels in KMT2D knockout group (Fig. 3 K). Immunohistochemistry revealed lower Ki-67 positivity and KLK3 expression in the KMT2D-deficient group xenografts, indicating impaired proliferation and AR signaling in vivo (Figs. 3 L and 3 M). These results confirmed that KMT2D is a regulator of prostate cancer proliferation and tumorigenesis, mainly through the maintenance of AR-dependent transcription. 3.4 KMT2D Modulates Chromatin Accessibility and Facilitates FOXA1-Mediated AR Target Gene Regulation To investigate how KMT2D modulates AR signaling, we analyzed AR ChIP-seq data and found that KMT2D depletion significantly reduced the number of AR-binding sites (Fig. 4 A). Motif analysis of KMT2D-dependent AR binding sites revealed strong enrichment of FOXA1 and androgen response elements (AREs; Fig. 4 B), suggesting that KMT2D and FOXA1 may cooperatively regulate AR target genes. We further explored the relationship between KMT2D and FOXA1 expression in prostate cancer. TCGA-PRAD transcriptomic analysis demonstrated a robust positive correlation between KMT2D and FOXA1 signature expression (Supplementary Fig. 4A). Both CRISPR-Cas9 knockout and RNA interference screens identified FOXA1 as the top transcription factor with the highest relative importance score in DepMap (Supplementary Fig. 4B). We also performed CUT&Tag of H3K4me1 and H3K27ac in LNCaP and C4-2 cells, with or without KMT2D knockdown. As shown in Fig. 4 C, AR-binding sites exhibited strong co-enrichment of FOXA1, KMT2D, and active enhancer marks H3K4me1 and H3K27ac, supporting a coordinated regulation of androgen receptor target regions by these factors and epigenetic modifications. KMT2D knockdown globally reduced H3K4me1 and H3K27ac binding, particularly at AR-dependent and AR-FOXA1 co-binding sites (Fig. 4 D, 4 E left panel; Supplementary Fig. 4C-4H). FOXA1 motifs were significantly enriched in KMT2D-dependent H3K4me1/H3K27ac sites (Fig. 4 D, 4 E, right panel), further implicating FOXA1 in KMT2D-mediated regulation. FOXA1 CUT&Tag demonstrated decreased global binding upon KMT2D silencing, particularly at AR-dependent loci (Fig. 4 F, 4 G left panel; Supplementary Fig. 4I-4J). Quantitative PCR confirmed reduced FOXA1 occupancy at canonical AR targets (KLK2, KLK3, TMPRSS2, and NKX3-1) in both cell lines (Fig. 4 F, 4 G right panel), establishing KMT2D's necessity for FOXA1 recruitment. To evaluate changes in global chromatin accessibility, we conducted ATAC-seq in LNCaP and C4-2 cells with or without KMT2D knockdown. ATAC-seq analysis showed that KMT2D depletion broadly diminished chromatin accessibility, particularly in the AR-dependent and FOXA1-AR co-dependent regions (Fig. 4 J, 4 K; Supplementary Fig. 4K-4L). FOXA1 and forkhead family motifs dominated these KMT2D-dependent accessible regions (Fig. 4 L), suggesting that KMT2D cooperated with forkhead family to maintain chromatin openness. Collectively, these findings support a model in which KMT2D promotes prostate cancer progression by sustaining active enhancer histone modifications, recruiting FOXA1, and maintaining chromatin accessibility at the AR regulatory elements. 3.5 FOXA1 Missense Mutations Impair KMT2D-Mediated Regulation of AR Signaling and Prostate Cancer Cell Proliferation To determine whether KMT2D-driven proliferation requires FOXA1, we performed combinatorial knockdown in C4-2 cells. Silencing FOXA1 substantially reduced both cell proliferation and colony formation, while dual knockdown of KMT2D and FOXA1 produced no additional suppression compared to FOXA1 depletion alone (Figs. 5 A–C), demonstrating FOXA1's downstream position relative to KMT2D. Similarly, FOXA1 knockdown strongly attenuated DHT-induced KLK3 expression, while concurrent KMT2D knockdown failed to further diminish this effect (Fig. 5 D). Further qRT-PCR analysis revealed that FOXA1 knockdown alone substantially decreased KLK3, TMPRSS2, and NKX3-1 mRNA levels, whereas simultaneous depletion of FOXA1 and KMT2D failed to induce further suppression (Fig. 5 E). Corresponding protein level reductions were verified by western blot analysis (Fig. 5 F). Next, we examined two clinically relevant FOXA1 missense mutations, D226G and M253K, within the forkhead DNA-binding (FKHD) domain (Fig. 5 G and Supplementary Fig. 5A). Previous work has demonstrated that FKHD domain missense mutations (FKHD-MSs) disrupt FOXA1 chromatin binding at AR-dependent enhancers, suppressing AR transcriptional activity. The stable expression of these mutants in C4-2 cells reduced the mRNA levels of KLK3, TMPRSS2, and NKX3-1, along with KLK3 protein expression (Supplementary Fig. 5B and 5C). KMT2D depletion resulted in no growth suppression in cells with FOXA1-D226G or FOXA1-M253K mutations (Figs. 5 H–J; Supplementary Fig. 5D and 5E), indicating that these mutations confer resistance to KMT2D loss. Next, we evaluated how KMT2D knockdown affected AR target gene expression in cells expressing FOXA1 mutants. qRT-PCR demonstrated that KMT2D depletion failed to substantially decrease KLK3, TMPRSS2, or NKX3-1 mRNA levels in cells with either D226G or M253K mutations (Fig. 5 K). Correspondingly, Western blot analysis indicated that KLK3 protein expression showed minimal changes following KMT2D knockdown in both the mutant cell lines (Fig. 5 L). CUT & Tag profiling showed that FOXA1 chromatin occupancy persisted in D226G and M253K mutant cells despite KMT2D knockdown (Fig. 5 M; Supplementary Fig. 5H). Integration with AR CUT&Tag data identified extensive FOXA1-AR co-binding sites that remained stable following KMT2D depletion in mutant cells (Figs. 5 N; Supplementary Fig. 5F and 5G). Genome browser visualization of key AR target loci confirmed sustained FOXA1 binding in mutant cells upon KMT2D knockdown (Fig. 5 O). These findings imply that D226G and M253K mutations in FOXA1 confer resistance to the inhibitory effects of KMT2D loss on AR signaling in prostate cancer cells. (B, C) Representative images and quantification of colony formation assays under indicated knockdowns. (D) Dose-response of KLK3 mRNA expression to DHT stimulation after knockdowns measured by qRT-PCR. (E) Expression of AR target genes KLK3, TMPRSS2, and NKX3-1 following knockdowns quantified by qRT-PCR. (F) Immunoblot of FOXA1 and KLK3 proteins in C4-2 cells with indicated knockdowns; β-Actin as loading control. (G) Schematic of FOXA1 protein domains highlighting D226G and M253K mutations. (H) Cell viability assays in C4-2 cells stably expressing FOXA1 mutants with or without KMT2D knockout. (I, J) EdU incorporation assay images and quantification in FOXA1 mutant cells post-KMT2D knockout. (K) AR target gene expression assessed by qRT-PCR in FOXA1 mutant cells with KMT2D depletion. (L) Immunoblot of KLK3 in FOXA1 mutant cells after KMT2D knockout. β-Actin as loading control. (M) CUT&Tag heatmaps of FOXA1 binding in mutant cells ± KMT2D knockout. (N) Venn diagrams of FOXA1 and AR co-binding sites in mutant cells. (O) Genome browser tracks showing FOXA1 occupancy at AR target loci in FOXA1 mutants with or without KMT2D knockout. 3.6 KMT2D-FOXA1-AR axis regulates ketogenesis via HMGCS2 in PCa To investigate the biological functions mediated by the KMT2D-FOXA1-AR axis, we re-analyzed the GSEA results after KMT2D knockdown under both DHT and CSS conditions. The enrichment of metabolic pathways, including fatty acid metabolism and cholesterol homeostasis, suggests that KMT2D is involved in metabolic regulation (Fig. 6 A). We identified downstream genes by selecting those with reduced expression and diminished chromatin accessibility by ATAC-seq following KMT2D inhibition in C4-2 cells (Fig. 6 B). Among these, 394 genes were consistently downregulated in C4-2 cells after KMT2D knockdown (Fig. 6 C). Pathway enrichment demonstrated significant associations with fatty acid, triacylglycerol, and ketone body metabolism in Planet, along with PPAR signaling pathway enrichment in KEGG (Fig. 6 D). Similar patterns were also observed in LNCaP cells, corroborating the C4-2 cell findings (Supplementary Fig. 6A–E). Focusing on the genes within these pathways, we identified six candidates for further study (Fig. 6 E). HMGCS2 mRNA levels decreased in both LNCaP and C4-2 cells upon KMT2D knockdown (Fig. 6 F and 6 G). Subsequent analysis revealed that KMT2D inhibition reduced AR, FOXA1, H3K27ac, and H3K4me1 binding, along with chromatin accessibility, to the enhancer of HMGCS2 (Fig. 6 H). HMGCS2 catalyzes HMG-CoA formation from acetyl-CoA, which is the rate-limiting enzyme in ketogenesis. Ketone bodies, primarily β-hydroxybutyrate (β-OHB), acetoacetate, and acetone, serve as critical energy sources for extrahepatic tissues, with β-OHB constituting ~ 70% of the circulating ketone bodies (Fig. 6 I). KMT2D knockdown significantly decreased ketone body levels in both the cell lines (Fig. 6 J). siRNA-mediated HMGCS2 suppression inhibited LNCaP and C4-2 cell proliferation, while reducing ketone body production (Fig. 6 K and 6 L; Supplementary Figure S6F–I). Supplementation with 10 mM β-OHB restored proliferation of KMT2D-deficient cells (Fig. 6 M and 6 O; Supplementary Fig. 6J), indicating that KMT2D promotes prostate cancer growth partly through ketone body metabolism. These results established that the KMT2D-FOXA1-AR axis modulates ketogenesis by controlling HMGCS2 expression. 3.7 Targeting KMT2D-Associated H3K4me1 Methylation Suppresses AR Signaling and Prostate Cancer Growth KMT2D operates within the COMPASS complex to establish H3K4me1 marks at enhancers. The complex contains core WRAD subunits (WDR5, RBBP5, ASH2L, and DPY30) as well as KMT2C/D-specific partners, including PTIP, PA1, and H3K27me3 demethylase UTX (KDM6A), which are required for KMT2D's catalytic function and chromatin localization (Fig. 7 A). We systematically evaluated the functional interactions between these components in prostate cancer using multi-dataset correlation analyses. Notably, UTX showed the most robust co-expression and co-dependency with KMT2D in TCGA PRAD samples as well as in the DepMap CRISPR and RNAi screening datasets (Fig. 7 B). TCGA cohort analysis further revealed that UTX expression was positively associated with AR signaling activity (Supplementary Fig. 7A), suggesting coordinated roles for UTX and KMT2D in modulating the AR pathway. Given UTX's prominent association with KMT2D, we investigated whether the pharmacological inhibition of UTX demethylase activity with GSK-J4, a selective UTX/JMJD3 inhibitor, affects prostate cancer cell viability through KMT2D-dependent mechanisms. GSK-J4 treatment reduced the viability of both LNCaP and C4-2 cell lines in a dose-dependent manner (Fig. 7 C; Supplementary Fig. 7B). Correspondingly, mRNA expression levels of KLK3, TMPRSS2, and NKX3.1 were markedly downregulated upon GSK-J4 treatment in both cell lines (Fig. 7 D). Western blot analyses also demonstrated that GSK-J4 reduced KLK3 protein expression and decreased H3K4me1 levels (Fig. 7 E). PCa is known to have a high level of heterogeneity. PDTFs are increasingly being utilized as effective models for studying tumor biology and evaluating therapeutic strategies. Therefore, to validate these findings in a clinically relevant model, we exposed PDTF cells to GSK-J4 under ex vivo conditions (Fig. 7 F). The compound markedly reduced KLK3 and TMPRSS2 mRNA expression (Fig. 7 G), mirroring the results from cell line experiments, while simultaneously decreasing KLK3 and H3K4me1 protein levels (Fig. 7 H). These observations demonstrate that the pharmacological inhibition of UTX/KMT2D-mediated epigenetic regulation effectively disrupts AR signaling in prostate cancer. 3.8 KMT2D Promotes Enzalutamide Resistance by Sustaining AR Signaling and Epigenetic Remodeling in Prostate Cancer Enzalutamide (Enz) resistance has emerged as a major challenge in prostate cancer treatment. Previous studies have established enzalutamide-resistant cell models and have demonstrated AR reactivation as a hallmark of resistance. To investigate KMT2D’s role in the development of enzalutamide resistance, we established an enzalutamide-resistant (EnzR) LNCaP cell model using prolonged drug exposure (Fig. 8 A). Dose-response assays confirmed that EnzR cells exhibited a markedly higher IC50 for enzalutamide compared to parental LNCaP cells (25.66 µM vs. 3.36 µM), validating the resistant phenotype (Supplementary Fig. 7C). qRT-PCR revealed significantly increased levels of KMT2D and AR mRNA in the EnzR sublines relative to parental cells (Supplementary Fig. 7D). Compared to parental LNCaP cells, EnzR cells displayed elevated levels of AR and H3K4me1, indicating epigenetic reprogramming associated with resistance (Fig. 8 B). CCK-8 assays revealed that KMT2D knockout markedly reduced EnzR cell proliferation (Fig. 8 C, left panel) and increased sensitivity to enzalutamide (Fig. 8 C, right panel). We also calculated AR signature scores using RNA-seq data, revealing significantly elevated AR signaling in EnzR cells relative to that in parental LNCaP cells (Fig. 8 D). KMT2D knockdown in EnzR cells markedly reduced AR pathway activity, establishing its essential function in maintaining AR axis activation in enzalutamide-resistant prostate cancer cells. ATAC-seq was performed in EnzR cells following KMT2D knockdown. As shown in Figs. 8 E and 8 F, ATAC-seq profiling revealed widespread loss of chromatin accessibility upon KMT2D knockdown in EnzR cells. Motif analysis revealed that FOXA1 motifs were significantly enriched in KMT2D-dependent chromatin sites (Fig. 8 G and 8 H). We also treated EnzR cells with GSK-J4 to investigate its effects on cell proliferation and AR signaling pathways. GSK-J4 dose-dependently reduced EnzR cell viability relative to DMSO controls, as shown by the proliferation assays (Fig. 8 I). This growth inhibition was further supported by the colony formation assays (Supplementary Fig. 7E). qRT-PCR analysis revealed that GSK-J4 markedly decreased KLK3, TMPRSS2, and NKX3.1 mRNA expression (Fig. 8 J). Western blotting confirmed reduced KLK3 protein levels and H3K4me1 expression following GSK-J4 treatment (Fig. 8 K). The effects of GSK-J4 were investigated in vivo . Mice with tumors were treated with vehicle or GSK-J4 for 15 d (Fig. 8 L). GSK-J4 treatment significantly suppressed tumor growth in vivo (Fig. 8 M-O). Additionally, decreased levels of Ki-67 and H3K4me1 were observed in tumors treated with GSK-J4, further supporting the effect of GSK-J4 on KMT2D function and PCa proliferation (Fig. 8 P). 4. Discussion Our findings established KMT2D as an epigenetic oncogene that drives prostate cancer proliferation and progression in both cellular and animal models. KMT2D exerts its oncogenic effects by modulating androgen receptor transcriptional activity through downstream signaling pathways. Integrated multiomics analyses combining RNA-seq, ChIP-seq, and ATAC-seq revealed that KMT2D enhances chromatin accessibility, facilitating AR and FOXA1 binding to the regulatory elements of androgen-responsive genes. We further identified the KMT2D-FOXA1-AR axis as a novel regulator of ketogenesis via HMGCS2 expression control. While no direct KMT2D inhibitors exist, pharmacological targeting of the COMPASS complex with GSK-J4 inhibits prostate cancer growth by disrupting KMT2D-mediated H3K4me1 modification. Epigenetic dysregulation plays a critical role in tumorigenesis, particularly cancer progression, invasion, metastasis, and drug resistance 13 . Among the various epigenetic alterations, aberrant histone modifications characterize multiple malignancies, with histone methylation emerging as a key modulator of chromatin dynamics and transcriptional regulation 9 . Lysine methyltransferases (KMTs) and demethylases (KDMs) predominantly govern this methylation process 5 . As a member of this family, KMT2D (MLL4) mediates H3K4 monomethylation to epigenetically control gene expression across diverse signaling cascades. In AR-negative prostate cancer (PCa), KMT2D drives tumor progression and metastasis through upregulation of LIFR and KLF4, subsequently activating the PI3K/Akt pathway 25 . Additionally, KMT2D deficiency in AR-negative PCa triggers ROS-dependent DNA damage by disrupting FOXO3-mediated antioxidant responses, ultimately inducing apoptosis and suppressing tumor growth 26 . However, the functional consequences of KMT2D activity are highly context-dependent across cancer types 14 . Genetic studies in mouse models reveal KMT2D's tumor-suppressive role in lymphoma 33 , medulloblastoma 34 , melanoma 27 , and lung adenocarcinoma (LUAD) 3 , while clinical evidence supports its oncogenic function in breast 44 , esophageal 1 , and gastric cancers 24 . This functional dichotomy underscores the tissue-specific nature of KMT2D activity in carcinogenesis, warranting further investigations in other malignancies. Our current findings establish that KMT2D facilitates AR-positive PCa progression through the FOXA1-dependent epigenetic activation of AR signaling. In CRPC, tumor progression is driven by AR reactivation via multiple mechanisms. Current therapies targeting the AR-ligand axis, including potent AR antagonists (enzalutamide, apalutamide, and darolutamide) and intratumoral androgen synthesis inhibitors (abiraterone), often fail because of acquired resistance mediated by AR alterations 7 . However, these approaches are limited by the resistance mechanisms that emerge as a result of AR alterations. Aberrant alterations in the AR have been reported in up to 58.78% of patients with CRPC 16 . Therefore, targeting molecular pathways beyond the AR to disrupt AR signaling has emerged as a promising strategy for mitigating AR reactivation in CRPC. Emerging evidence implicates epigenetic dysregulation in PCa progression, with several epigenetic-targeting therapies demonstrating clinical potential 11 . In this study, we established KMT2D as a regulator of AR recruitment and transcriptional activity in CRPC, contributing to AR reactivation. LSD1 (KDM1A), another epigenetic modulator, demethylates H3K4me1/2 while functioning as an AR coactivator through its interaction with the CoREST complex 18 . Furthermore, LSD1 converts H3K4me2 to H3K4me1 at enhancers of AR-stimulated genes 6 , suggesting that KMT2D and LSD1 cooperatively regulate AR transcriptional activity through this active enhancer mark, underscoring its broad function as an epigenetic regulator of nuclear receptor signaling 44 . The transcription factor forkhead box A1 (FOXA1) induces an open chromatin conformation, enabling lineage-specific transcription factors such as AR to bind and drive prostate cancer (PCa) growth, survival, and drug resistance 43 . FOXA1 chromatin binding is enhanced by reduced DNA methylation or increased levels of histone methylation markers, notably H3K4me1 and H3K4me2. We found that FOXA1 recruitment to AR-specific enhancer sites depends on KMT2D-mediated H3K4me1, establishing an open chromatin state that facilitates AR binding and activation and ultimately accelerates PCa progression. Consistent with our findings, recent studies have shown that FOXA1 is one of the most frequently mutated genes in PCa 18 . Our experiments revealed that FOXA1 forkhead-domain mutants (D226G and M253K) exhibited diminished binding to AR enhancer sites. KMT2D knockout did not compromise FOXA1 binding, suggesting that KMT2D primarily regulates AR activity via FOXA1. However, the precise mechanisms governing the regulation of FOXA1 activity remain unclear. Although FOXA1 inhibition attenuates AR transcriptional activity and curbs PCa cell proliferation, it paradoxically enhances tumor invasiveness, complicating its therapeutic targeting 39 . LSD1 inhibition markedly impairs PCa growth and progression 23 , implying that KMT2D suppression effectively disrupts the FOXA1-AR axis in castration-resistant PCa. Metabolic reprogramming is a hallmark of cancer and contributes to the initiation, progression, metastasis, and drug resistance 2 . Ketogenesis in cancer cells has been reported to suppress or enhance cell growth and proliferation 19 . Studies show that KMT2D regulates tumorigenesis and progression through metabolic reprogramming in pancreatic cancer 20 , lung cancer 3 , and melanoma 27 by regulating glycolysis. However, the role of KMT2D in the ketone body metabolism remains unclear. This study showed that the expression of 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 2 (HMGCS2), the key rate-limiting enzyme involved in ketogenesis, is regulated by the KMT2D-FOXA1-AR axis, which facilitates PCa proliferation and progression. This indicates that metabolic reprogramming of ketone bodies plays an oncogenic role in PCa. In contrast, another study reported that HMGCS2 acts as a tumor suppressor in PCa and that a ketogenic diet or calorie-restricted diet can inhibit PCa growth 46 , 48 . However, consistent with our results, Labanca et al. demonstrated that relapsed PCa after ADT shows higher ketone body levels, along with elevated levels of key ketone catabolic enzymes 21 . In addition, Punit et al. revealed that HMGCS2 is highly expressed in high-grade PCa and an androgen-independent PCa cell line, suggesting that it could serve as a diagnostic or prognostic marker 35 , further supporting our results. Collectively, KMT2D was identified as an epigenetic modulator involved in the upregulation of HMGCS2 expression, thereby driving ketone metabolic reprogramming to promote PCa progression. KMT2D functions as a catalytic subunit within the mammalian complex of proteins associated with SET1 (COMPASS) and mediates H3K4me1 and H3K4me3. UTX, also known as KDM6A, is a H3K27-specific histone demethylase and a component of COMPASS. Studies have shown that the function of UTX depends on its association with COMPASS and that the interaction between KMT2D and other components of the COMPASS complex does not rely on its enzymatic domain 10 , 32 , 42 . However, the effect of UTX deficiency on KMT2D function remains unclear. This study showed that GSK-J4, a UTX inhibitor, significantly suppressed the proliferation of PCa cells. Mechanistically, GSK-J4 reduced the expression of H3K4me1 and AR signaling pathway genes including KLK3, TMPRSS2, and NKX3.1. These findings indicate that inhibition of UTX by GSK-J4 impairs the catalytic activity of KMT2D. Consistent with our findings, the loss of KMT2D induces brain metastasis in triple-negative breast cancer (TNBC). However, this effect can be mitigated by blocking UTX, suggesting that the function of KMT2D may depend on UTX 36 . Wang et al. reported that UTX inhibition significantly impaired KMT2D function by disrupting its cooperative relationship with p300. Mechanistically, UTX facilitated the recruitment of p300, enhancing H3K27 acetylation, a process necessary for MLL4-mediated H3K4me1 deposition. In the absence of UTX, this synergy is disrupted, resulting in reduced enhancer activity and impaired transcriptional activation 47 . Additionally, Shi et al. reported that UTX facilitates the localization of histone methyltransferase KMT2D to the same condensates, enhancing KMT2D H3K4 methylation activity 37 . These studies further support our findings that UTX inhibition impairs KMT2D function in PCa cells. 5. Conclusion Our study established KMT2D as a pivotal epigenetic regulator that promotes prostate cancer progression through FOXA1-mediated enhancement of AR signaling and metabolic reprogramming. The KMT2D-FOXA1-AR axis facilitates chromatin remodeling and ketogenesis, thereby driving tumor growth. Targeting UTX effectively impaired KMT2D enzymatic activity and suppressed AR signaling and PCa cell proliferation. These insights highlight the therapeutic potential of disrupting the KMT2D-associated epigenetic mechanisms in advanced prostate cancer. Abbreviations ADT Androgen Deprivation Therapy AR Androgen Receptor ARE Androgen Response Element ATAC-seq Assay for Transposase-Accessible Chromatin with sequencing ChIP-seq Chromatin Immunoprecipitation sequencing COMPASS Complex of Proteins Associated with Set1 CRPC Castration-Resistant Prostate Cancer CSS Charcoal-Stripped Serum CUT&Tag Cleavage Under Targets and Tagmentation DHT Dihydrotestosterone EdU 5-Ethynyl-2′-deoxyuridine EnzR Enzalutamide-Resistant ER Estrogen Receptor FKHD Forkhead DNA-binding domain FOXA1 Forkhead Box A1 GSEA Gene Set Enrichment Analysis GSVA Gene Set Variation Analysis H3K4me1 Histone H3 lysine 4 monomethylation HMGCS2 3-Hydroxy-3-Methylglutaryl-CoA Synthase 2 IHC Immunohistochemistry KMT2D Lysine Methyltransferase 2D PCa Prostate Cancer PDTF Patient-Derived Tumor Fragment qRT-PCR Quantitative Real-Time Polymerase Chain Reaction RNA-seq RNA sequencing siRNA Small Interfering RNA TCGA-PRAD The Cancer Genome Atlas-Prostate Adenocarcinoma UTX Ubiquitously Transcribed Tetratricopeptide Repeat, X Chromosome Declarations Conflict of Interest The authors declare no potential conflicts of interest. Competing interests The authors declare no competing interests Author Contribution Statement M.Y. Luo, C.W. Wu, M.L. Zhou, and Y.D. Li designed and developed the methodology. Q. Wei, S.D. Lv, and C.C. Du conducted the investigations. M.Y. Luo, Y.F. Zhang, and Y.P. Liao performed data visualization. Q. Wei and S.D. Lv supervised the project. M.Y. Luo drafted the manuscript. Q. Wei and S.D. Lv acquired funding, provided resources, and contributed to supervision. All authors reviewed and approved the final manuscript. Consent for publication Not applicable. Ethic approval and consent to participate The mouse experiments were approved by the Ethics Committee of Ganzhou Hospital, Southern Medical University, Southern Hospital (Approval No.TY-DKY2023-020-01). The PDTF collection was approved by the Ethics Committee of Nanfang Hospital (NFEC-2022-299). Funding This work was funded in part by the Bethune Oncology Basic Research Program (J202101E005, Qiang Wei), Ganpo Talent Support Program - Major Discipline Academic and Technological Leader Training Project (20232BCJ22018, Qiang Wei), Ganzhou Municipal Science and Technology Project (2022–RC1341, Qiang Wei), Natural Science Foundation of Jiangxi Province (20224ACB206007, Qiang Wei), National Natural Science Foundation of China (82472756, 82103276, to Shidong Lv), and Natural Science Foundation of Guangdong Province (2023A1515010321, to Qiang Wei; 2024A1515010331, to Shidong Lv). Acknowledgments Not applicable. Data availability The ChIP-seq, ATAC-seq, CUT&Tag, and RNA-seq datasets generated in this study are available in the GEO database under accession numbers GSE285691, GSE285692, and GSE285790, respectively, with public release upon publication. We also analyzed external datasets from the DepMap database, single-cell RNA-seq data (GSE181294), and TCGA-PRAD cohorts, all of which were accessible through their original repositories. References Abudureheman A, Ainiwaer J, Hou Z, Niyaz M, Turghun A, Hasim A et al . High MLL2 expression predicts poor prognosis and promotes tumor progression by inducing EMT in esophageal squamous cell carcinoma. J Cancer Res Clin Oncol 2018; 144: 1025–1035. Ahmad F, Cherukuri MK, Choyke PL. Metabolic reprogramming in prostate cancer. Br J Cancer 2021; 125: 1185–1196. Alam H, Tang M, Maitituoheti M, Dhar SS, Kumar M, Han CY et al . KMT2D Deficiency Impairs Super-Enhancers to Confer a Glycolytic Vulnerability in Lung Cancer. Cancer Cell 2020; 37: 599–617 e597. Ang SY, Uebersohn A, Spencer CI, Huang Y, Lee JE, Ge K et al . KMT2D regulates specific programs in heart development via histone H3 lysine 4 di-methylation. Development 2016; 143: 810–821. Black JC, Van Rechem C, Whetstine JR. Histone lysine methylation dynamics: establishment, regulation, and biological impact. Mol Cell 2012; 48: 491–507. Cai C, He HH, Gao S, Chen S, Yu Z, Gao Y et al . Lysine-specific demethylase 1 has dual functions as a major regulator of androgen receptor transcriptional activity. Cell Rep 2014; 9: 1618–1627. Cai M, Song XL, Li XA, Chen M, Guo J, Yang DH et al . Current therapy and drug resistance in metastatic castration-resistant prostate cancer. Drug Resist Updat 2023; 68: 100962. Chandrasekar T, Yang JC, Gao AC, Evans CP. Mechanisms of resistance in castration-resistant prostate cancer (CRPC). Transl Androl Urol 2015; 4: 365–380. Cheng Y, He C, Wang M, Ma X, Mo F, Yang S et al . Targeting epigenetic regulators for cancer therapy: mechanisms and advances in clinical trials. Signal Transduct Target Ther 2019; 4: 62. Cocciadiferro D, Augello B, De Nittis P, Zhang J, Mandriani B, Malerba N et al . Dissecting KMT2D missense mutations in Kabuki syndrome patients. Hum Mol Genet 2018; 27: 3651–3668. Conteduca V, Hess J, Yamada Y, Ku SY, Beltran H. Epigenetics in prostate cancer: clinical implications. Transl Androl Urol 2021; 10: 3104–3116. Cucchiara V, Yang JC, Mirone V, Gao AC, Rosenfeld MG, Evans CP. Epigenomic Regulation of Androgen Receptor Signaling: Potential Role in Prostate Cancer Therapy. Cancers (Basel) 2017; 9. Dai W, Qiao X, Fang Y, Guo R, Bai P, Liu S et al . Epigenetics-targeted drugs: current paradigms and future challenges. Signal Transduct Target Ther 2024; 9: 332. Dhar SS, Lee MG. Cancer-epigenetic function of the histone methyltransferase KMT2D and therapeutic opportunities for the treatment of KMT2D-deficient tumors. Oncotarget 2021; 12: 1296–1308. Froimchuk E, Jang Y, Ge K. Histone H3 lysine 4 methyltransferase KMT2D. Gene 2017; 627: 337–342. Fujita K, Nonomura N. Role of Androgen Receptor in Prostate Cancer: A Review. World J Mens Health 2019; 37: 288–295. Gao S, Chen S, Han D, Barrett D, Han W, Ahmed M et al . Forkhead domain mutations in FOXA1 drive prostate cancer progression. Cell Res 2019; 29: 770–772. Gao S, Chen S, Han D, Wang Z, Li M, Han W et al . Chromatin binding of FOXA1 is promoted by LSD1-mediated demethylation in prostate cancer. Nat Genet 2020; 52: 1011–1017. Hwang CY, Choe W, Yoon KS, Ha J, Kim SS, Yeo EJ et al . Molecular Mechanisms for Ketone Body Metabolism, Signaling Functions, and Therapeutic Potential in Cancer. Nutrients 2022; 14. Koutsioumpa M, Hatziapostolou M, Polytarchou C, Tolosa EJ, Almada LL, Mahurkar-Joshi S et al . Lysine methyltransferase 2D regulates pancreatic carcinogenesis through metabolic reprogramming. Gut 2019; 68: 1271–1286. Labanca E, Bizzotto J, Sanchis P, Anselmino N, Yang J, Shepherd PDA et al . Prostate cancer castrate resistant progression usage of non-canonical androgen receptor signaling and ketone body fuel. Oncogene 2021; 40: 6284–6298. Li M, Shi M, Xu Y, Qiu J, Lv Q. Histone Methyltransferase KMT2D Regulates H3K4 Methylation and is Involved in the Pathogenesis of Ovarian Cancer. Cell Transplant 2021; 30: 9636897211027521. Li M, Liu M, Han W, Wang Z, Han D, Patalano S et al . LSD1 Inhibition Disrupts Super-Enhancer-Driven Oncogenic Transcriptional Programs in Castration-Resistant Prostate Cancer. Cancer Res 2023; 83: 1684–1698. Li Q, Wu R, Wu F, Chen Q. KMT2D promotes proliferation of gastric cancer cells: evidence from ctDNA sequencing. J Clin Lab Anal 2021; 35: e23721. Lv S, Ji L, Chen B, Liu S, Lei C, Liu X et al . Histone methyltransferase KMT2D sustains prostate carcinogenesis and metastasis via epigenetically activating LIFR and KLF4. Oncogene 2018; 37: 1354–1368. Lv S, Wen H, Shan X, Li J, Wu Y, Yu X et al . Loss of KMT2D induces prostate cancer ROS-mediated DNA damage by suppressing the enhancer activity and DNA binding of antioxidant transcription factor FOXO3. Epigenetics 2019; 14: 1194–1208. Maitituoheti M, Keung EZ, Tang M, Yan L, Alam H, Han G et al . Enhancer Reprogramming Confers Dependence on Glycolysis and IGF Signaling in KMT2D Mutant Melanoma. Cell Rep 2020; 33: 108293. Oswald F, Rodriguez P, Giaimo BD, Antonello ZA, Mira L, Mittler G et al . A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes. Nucleic Acids Res 2016; 44: 4703–4720. Pan Y, Han H, Hu H, Wang H, Song Y, Hao Y et al . KMT2D deficiency drives lung squamous cell carcinoma and hypersensitivity to RTK-RAS inhibition. Cancer Cell 2023; 41: 88–105 e108. Pang L, Tian H, Gao X, Wang W, Wang X, Zhang Z. KMT2D deficiency disturbs the proliferation and cell cycle activity of dental epithelial cell line (LS8) partially via Wnt signaling. Biosci Rep 2021; 41. Parolia A, Eyunni S, Verma BK, Young E, Liu Y, Liu L et al . NSD2 is a requisite subunit of the AR/FOXA1 neo-enhanceosome in promoting prostate tumorigenesis. Nat Genet 2024; 56: 2132–2143. Rickels R, Wang L, Iwanaszko M, Ozark PA, Morgan MA, Piunti A et al . A small UTX stabilization domain of Trr is conserved within mammalian MLL3-4/COMPASS and is sufficient to rescue loss of viability in null animals. Genes Dev 2020; 34: 1493–1502. Saffie R, Zhou N, Rolland D, Onder O, Basrur V, Campbell S et al . FBXW7 Triggers Degradation of KMT2D to Favor Growth of Diffuse Large B-cell Lymphoma Cells. Cancer Res 2020; 80: 2498–2511. Sanghrajka RM, Koche R, Medrano H, El Nagar S, Stephen DN, Lao Z et al . KMT2D suppresses Sonic hedgehog-driven medulloblastoma progression and metastasis. iScience 2023; 26: 107831. Saraon P, Cretu D, Musrap N, Karagiannis GS, Batruch I, Drabovich AP et al . Quantitative proteomics reveals that enzymes of the ketogenic pathway are associated with prostate cancer progression. Mol Cell Proteomics 2013; 12: 1589–1601. Seehawer M, Li Z, Nishida J, Foidart P, Reiter AH, Rojas-Jimenez E et al . Loss of Kmt2c or Kmt2d drives brain metastasis via KDM6A-dependent upregulation of MMP3. Nat Cell Biol 2024; 26: 1165–1175. Shi B, Li W, Song Y, Wang Z, Ju R, Ulman A et al . UTX condensation underlies its tumour-suppressive activity. Nature 2021; 597: 726–731. Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin 2024; 74: 12–49. Song B, Park SH, Zhao JC, Fong KW, Li S, Lee Y et al . Targeting FOXA1-mediated repression of TGF-beta signaling suppresses castration-resistant prostate cancer progression. J Clin Invest 2019; 129: 569–582. Sugiura M, Sato H, Kanesaka M, Imamura Y, Sakamoto S, Ichikawa T et al . Epigenetic modifications in prostate cancer. Int J Urol 2021; 28: 140–149. Sun P, Wu T, Sun X, Cui Z, Zhang H, Xia Q et al . KMT2D inhibits the growth and metastasis of bladder Cancer cells by maintaining the tumor suppressor genes. Biomed Pharmacother 2019; 115: 108924. Sze CC, Shilatifard A. MLL3/MLL4/COMPASS Family on Epigenetic Regulation of Enhancer Function and Cancer. Cold Spring Harb Perspect Med 2016; 6. Teng M, Zhou S, Cai C, Lupien M, He HH. Pioneer of prostate cancer: past, present and the future of FOXA1. Protein Cell 2021; 12: 29–38. Toska E, Osmanbeyoglu HU, Castel P, Chan C, Hendrickson RC, Elkabets M et al . PI3K pathway regulates ER-dependent transcription in breast cancer through the epigenetic regulator KMT2D. Science 2017; 355: 1324–1330. Voabil P, de Bruijn M, Roelofsen LM, Hendriks SH, Brokamp S, van den Braber M et al . An ex vivo tumor fragment platform to dissect response to PD-1 blockade in cancer. Nat Med 2021; 27: 1250–1261. Wan S, Xi M, Zhao HB, Hua W, Liu YL, Zhou YL et al . HMGCS2 functions as a tumor suppressor and has a prognostic impact in prostate cancer. Pathol Res Pract 2019; 215: 152464. Wang SP, Tang Z, Chen CW, Shimada M, Koche RP, Wang LH et al . A UTX-MLL4-p300 Transcriptional Regulatory Network Coordinately Shapes Active Enhancer Landscapes for Eliciting Transcription. Mol Cell 2017; 67: 308–321 e306. Weber DD, Aminzadeh-Gohari S, Tulipan J, Catalano L, Feichtinger RG, Kofler B. Ketogenic diet in the treatment of cancer - Where do we stand? Mol Metab 2020; 33: 102–121. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files Supplementalinformation.pdf Supplemental information Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 20 Nov, 2025 Review # 2 received at journal 10 Nov, 2025 Review # 1 received at journal 07 Nov, 2025 Reviewer # 2 agreed at journal 27 Oct, 2025 Reviewer # 1 agreed at journal 27 Oct, 2025 Reviewers invited by journal 27 Oct, 2025 Submission checks completed at journal 09 Oct, 2025 Editor assigned by journal 08 Oct, 2025 First submitted to journal 08 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7812403","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":527123941,"identity":"684baa78-5bfe-4308-ab2f-edcd5900f55d","order_by":0,"name":"Qiang Wei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArUlEQVRIiWNgGAWjYJCCAx8gtAHxWg7OIFkLMw9JWvhuJB88bPNnW2IDe/M2CYaaO4S1SN5ISzic23Y7sYHnWJkEw7FnhLUY3M4xOJzbANQikWMmwdhwmBgt+R8OW/wBapF/Q7SWHIbDDGwgW3iI1CJ5/5nBwd6228ZtPGnFFgnHiNDCd+bw4w8//tyW7Wc/vPHGhxoitDAcgNJsICKBCA0ILaNgFIyCUTAKcAIA3iY/jj4zqAQAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-6875-7697","institution":"Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Southern Medical University","correspondingAuthor":true,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Wei","suffix":""},{"id":527123942,"identity":"0ce3057d-a0ba-44e7-af5a-addd5a7ca735","order_by":1,"name":"Mayao Luo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Mayao","middleName":"","lastName":"Luo","suffix":""},{"id":527123943,"identity":"cff7d738-56a0-406d-b597-bd0f5a08880f","order_by":2,"name":"Chenwei Wu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Chenwei","middleName":"","lastName":"Wu","suffix":""},{"id":527123944,"identity":"a6d5162c-1465-4487-9084-2165c1450ca2","order_by":3,"name":"Manli Zhou","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Manli","middleName":"","lastName":"Zhou","suffix":""},{"id":527123945,"identity":"5374258f-1f2b-45ce-bc9e-358f2be17aa9","order_by":4,"name":"Yifan Zhang","email":"","orcid":"","institution":"Nanfang Hospital, Southern Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yifan","middleName":"","lastName":"Zhang","suffix":""},{"id":527123946,"identity":"ae105069-0075-4d9d-8886-cf7827134314","order_by":5,"name":"Yadong Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yadong","middleName":"","lastName":"Li","suffix":""},{"id":527123947,"identity":"b544508a-48f3-44e2-8559-7c5ffa4e69f1","order_by":6,"name":"Yuanpeng Liao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yuanpeng","middleName":"","lastName":"Liao","suffix":""},{"id":527123948,"identity":"e7b0ce7a-118f-4e61-8c27-de2f208486f0","order_by":7,"name":"Xin Huang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Huang","suffix":""},{"id":527123949,"identity":"b815458c-1025-4c53-a46b-eb034b4a1227","order_by":8,"name":"Chuance Du","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Chuance","middleName":"","lastName":"Du","suffix":""},{"id":527123950,"identity":"89f1e0b0-d79e-4093-a4dd-65c3051be1bb","order_by":9,"name":"Shidong Lv","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shidong","middleName":"","lastName":"Lv","suffix":""}],"badges":[],"createdAt":"2025-10-09 03:10:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7812403/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7812403/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":95355871,"identity":"0ea226e7-6f9c-4b92-873e-eea271c0c82f","added_by":"auto","created_at":"2025-11-07 06:33:41","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4041944,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/60f19e6edd55474a726a5e26.tif"},{"id":95355875,"identity":"00e43f1c-5a8a-40fe-9075-ad4cff87acb8","added_by":"auto","created_at":"2025-11-07 06:33:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9550250,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/52625e394bc5f4eb2e6ca949.docx"},{"id":95355872,"identity":"ae6e596e-62c1-4c0a-b0c9-61741c80f4e1","added_by":"auto","created_at":"2025-11-07 06:33:41","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5445928,"visible":true,"origin":"","legend":"","description":"","filename":"Figure.2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/0f796c20cce2dc30443e4d72.tif"},{"id":95525145,"identity":"38b44dae-4dee-483d-9421-d94704d6bbba","added_by":"auto","created_at":"2025-11-10 10:04:20","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5659200,"visible":true,"origin":"","legend":"","description":"","filename":"Figure.3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/40d53c723028b0fe28cb2567.tif"},{"id":95524347,"identity":"f4536cd2-d48d-4b97-a315-ae84a8d01467","added_by":"auto","created_at":"2025-11-10 10:02:38","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4299988,"visible":true,"origin":"","legend":"","description":"","filename":"Figure.4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/e45ade6c82c49f4993930043.tif"},{"id":95526062,"identity":"0de2d983-a671-4a8a-9948-1767d6734066","added_by":"auto","created_at":"2025-11-10 10:06:10","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4452960,"visible":true,"origin":"","legend":"","description":"","filename":"Figure.5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/7ecfa1d75d8a2b5f45cb3cd1.tif"},{"id":95525739,"identity":"e40bc105-cb01-4ec9-90ec-8a784526cfd7","added_by":"auto","created_at":"2025-11-10 10:05:38","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4012596,"visible":true,"origin":"","legend":"","description":"","filename":"Figure.6.tif","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/e343857f2f8f7532b13ecb45.tif"},{"id":95524475,"identity":"dd0c3945-43ba-4814-b879-fe4485a65121","added_by":"auto","created_at":"2025-11-10 10:02:47","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2458688,"visible":true,"origin":"","legend":"","description":"","filename":"Figure.7.tif","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/261db540fa414d137f8dc1e4.tif"},{"id":95355916,"identity":"71d8047a-7c9d-400a-9f63-d27b9a06ecaa","added_by":"auto","created_at":"2025-11-07 06:33:43","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5286016,"visible":true,"origin":"","legend":"","description":"","filename":"Figure.8.tif","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/8bb6971ec28cf4c9f5413ed3.tif"},{"id":95355878,"identity":"5cdfd4e0-117a-4aec-8227-21c6c9703934","added_by":"auto","created_at":"2025-11-07 06:33:41","extension":"json","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10238,"visible":true,"origin":"","legend":"","description":"","filename":"ONC202503102.json","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/af1e932c4f69b8796fc8fec9.json"},{"id":95355881,"identity":"a330c869-84bb-439c-ab1d-36a464db392c","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6465500,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/2027e3e7f7c30274115d8090.pdf"},{"id":95355874,"identity":"b4332c02-dad5-474f-b740-591bb3c7d4da","added_by":"auto","created_at":"2025-11-07 06:33:41","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":162238,"visible":true,"origin":"","legend":"","description":"","filename":"ONC2025031020enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/b1063968cc9d275303e6d4de.xml"},{"id":95525737,"identity":"03db1999-9006-4831-bb2c-d0bb056d469e","added_by":"auto","created_at":"2025-11-10 10:05:38","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4041944,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/31e6ad70f583289efc0bf597.tif"},{"id":95524478,"identity":"0aec2ebc-e12d-4f49-952d-5269ebb58838","added_by":"auto","created_at":"2025-11-10 10:02:47","extension":"tif","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5445928,"visible":true,"origin":"","legend":"","description":"","filename":"Figure.2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/a42e82d238ea889dce6e7f5d.tif"},{"id":95355908,"identity":"a3b5ae68-109c-434c-b79d-561a5ead85a7","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5659200,"visible":true,"origin":"","legend":"","description":"","filename":"Figure.3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/33a2332590c3d4a9c0e65ce2.tif"},{"id":95525711,"identity":"08c706cb-2a18-45ad-807b-653f4fa9ed31","added_by":"auto","created_at":"2025-11-10 10:05:36","extension":"tif","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4299988,"visible":true,"origin":"","legend":"","description":"","filename":"Figure.4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/0420d887160153714f58d44e.tif"},{"id":95355890,"identity":"4da634ca-1998-4bcf-b3b5-7e21dc73f361","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"tif","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4452960,"visible":true,"origin":"","legend":"","description":"","filename":"Figure.5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/4ae9fbe7ecfc54cef7a80c54.tif"},{"id":95525780,"identity":"fe680632-3f41-44f8-943b-fd87aaf974a4","added_by":"auto","created_at":"2025-11-10 10:05:41","extension":"tif","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4012596,"visible":true,"origin":"","legend":"","description":"","filename":"Figure.6.tif","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/02797618fe1bf24880bf6239.tif"},{"id":95355913,"identity":"dd97ddb8-d65e-42af-829f-c7ab4c878ff4","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"tif","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2458688,"visible":true,"origin":"","legend":"","description":"","filename":"Figure.7.tif","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/2ab136c30c2d3cbbc206d84c.tif"},{"id":95355883,"identity":"57127a08-a026-4b85-9b03-3c78c72fe88b","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"tif","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5286016,"visible":true,"origin":"","legend":"","description":"","filename":"Figure.8.tif","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/e763958923d1e4dc1c072aba.tif"},{"id":95355910,"identity":"07c99556-3ee3-409b-a353-0b4d49afa873","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"jpeg","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1384768,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/48eb09b7355a15707d097b2f.jpeg"},{"id":95355887,"identity":"eb59e0c1-4448-4024-82f1-8d42628e862c","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"jpeg","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1092890,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/7067e3a439402ae43d880a7d.jpeg"},{"id":95524644,"identity":"d0c8ca73-cb0e-460f-9ee4-8b98de2be28a","added_by":"auto","created_at":"2025-11-10 10:03:08","extension":"jpeg","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1482726,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/6e04affee17466cddd007aad.jpeg"},{"id":95355891,"identity":"0814b7d5-436d-438a-8d85-18bf1596d2e0","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"jpeg","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1253256,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/98ade47756a45c012ce33f91.jpeg"},{"id":95355893,"identity":"c201f88a-e4aa-4d1a-921d-df4c0083a67f","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"jpeg","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1211852,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/a21c6650c4c6886eb53959f8.jpeg"},{"id":95524364,"identity":"124b8422-4863-4497-88c2-53f0d7fee379","added_by":"auto","created_at":"2025-11-10 10:02:40","extension":"jpeg","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1033134,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/0c87c8f0b5a78755b7763341.jpeg"},{"id":95355904,"identity":"9441d783-5491-4905-868b-3a3f29f9d691","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"jpeg","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":569296,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/292e7e7edab62ccf1de92ebd.jpeg"},{"id":95525777,"identity":"9938dc3b-4cf8-40d0-9cc0-b341cc6ce720","added_by":"auto","created_at":"2025-11-10 10:05:41","extension":"jpeg","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1345962,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/5433f80b61e7b1c06fb7d4ca.jpeg"},{"id":95355897,"identity":"94136f85-dca8-4e9b-8f3c-eda56f7db533","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":481239,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/9c32a5a7269acd7c19122a25.png"},{"id":95525510,"identity":"557150c2-b320-4cb1-8956-27fadd3f69c9","added_by":"auto","created_at":"2025-11-10 10:05:10","extension":"png","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":483689,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/770c40a3b1508a25b45566e2.png"},{"id":95355902,"identity":"13ad1967-537d-4bb7-b1e5-6ea33f97d5eb","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"png","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":519169,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/004fc3a6b1eeeed9ee144fe2.png"},{"id":95355895,"identity":"1f5e4163-8f5c-4cb2-a9db-93c2f185701f","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"png","order_by":31,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":351008,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/7cb46951debd020e2fd13f27.png"},{"id":95525117,"identity":"9562066e-d58f-4ee5-9f97-10064ab5d397","added_by":"auto","created_at":"2025-11-10 10:04:16","extension":"png","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":381594,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/0970e7f1420bac116c622421.png"},{"id":95525984,"identity":"6a2b7361-d8fc-4fb6-aea5-42532996267d","added_by":"auto","created_at":"2025-11-10 10:05:59","extension":"png","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":391219,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/2020509b8fb72eaade641ded.png"},{"id":95524948,"identity":"74737304-647e-4df5-a3b4-364905723631","added_by":"auto","created_at":"2025-11-10 10:03:54","extension":"png","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":200480,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure.7.png","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/beaa6f7b73a79e40dc995561.png"},{"id":95524970,"identity":"84c17c6f-79a1-4ee5-83e4-c769dd16be95","added_by":"auto","created_at":"2025-11-10 10:03:56","extension":"png","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":488065,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure.8.png","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/5bc6f0c1654a27ef4640ff64.png"},{"id":95525812,"identity":"716a38a3-8674-484c-a894-a1e99923a7f1","added_by":"auto","created_at":"2025-11-10 10:05:42","extension":"png","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":229824,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/e0f49a1950e3220953da60e3.png"},{"id":95355896,"identity":"b7b75845-6971-42ef-a78a-09f7e4ffedc3","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"png","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":167829,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/59b468ce965806b3c3de9cd4.png"},{"id":95355898,"identity":"8373a9fd-fc80-4197-a0b0-2f7b8efd89cb","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"png","order_by":38,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":237264,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/8de9806a0471e85db425d188.png"},{"id":95525779,"identity":"9d812ef7-b7af-4db3-bedc-094a388ac1dc","added_by":"auto","created_at":"2025-11-10 10:05:41","extension":"png","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":156899,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/c0c1b2e64083bd71acfd54fe.png"},{"id":95524248,"identity":"737ae3ab-4285-4716-a25d-0c7d70192c80","added_by":"auto","created_at":"2025-11-10 10:02:33","extension":"png","order_by":40,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":176730,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/66ec772617621886021a4ee4.png"},{"id":95525782,"identity":"ee9ed1dc-afed-4124-91d8-9b398de640bc","added_by":"auto","created_at":"2025-11-10 10:05:41","extension":"png","order_by":41,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":172357,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/5900321ab0fa306ca51b84ae.png"},{"id":95524796,"identity":"32430fa5-3f2f-4786-82b9-3ec2156ea98d","added_by":"auto","created_at":"2025-11-10 10:03:33","extension":"png","order_by":42,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":81330,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/e9aa4f59ea5123139c9587b7.png"},{"id":95525661,"identity":"d3f8d943-0486-4dd0-95ee-6af674399ac0","added_by":"auto","created_at":"2025-11-10 10:05:31","extension":"png","order_by":43,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":189955,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/865aebb86063ff1aad3aff10.png"},{"id":95355906,"identity":"0e56d69f-4b58-482b-aa8a-5a9a9a501fa4","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"xml","order_by":44,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":160882,"visible":true,"origin":"","legend":"","description":"","filename":"ONC2025031020structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/2418d586a97337c1120997c5.xml"},{"id":95525763,"identity":"87a16857-b647-4e98-a9de-7b12380a5fe2","added_by":"auto","created_at":"2025-11-10 10:05:39","extension":"html","order_by":45,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":173056,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/6611dfdcf3878c96df65d57e.html"},{"id":95355862,"identity":"d651e258-ad80-4d1c-a00a-6a1cac32fd4f","added_by":"auto","created_at":"2025-11-07 06:33:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2129679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKMT2D expression and activity is upregulated in prostate cancer and related to the poor prognosis. (A) \u003c/strong\u003eComparison of normalized KMT2D mRNA expression between tumor tissues and adjacent normal tissues in the Renji cohort. Data are presented as mean ± SD; \u003cstrong\u003e(B, C)\u003c/strong\u003eValidation of elevated KMT2D mRNA expression in castration-resistant prostate cancer (CRPC) compared to localized prostate cancer in independent cohorts GSE35988 (B) and GSE70770 (C); \u003cstrong\u003e(D) \u003c/strong\u003eKaplan–Meier analysis of disease-free survival in TCGA-PRAD patients stratified by KMT2D expression levels. High KMT2D expression is associated with shorter survival (log-rank P = 0.018); \u003cstrong\u003e(E)\u003c/strong\u003e Representative immunohistochemical staining of KMT2D protein in normal prostate and prostate cancer tissues from the Human Protein Atlas (left) and Nanfang cohorts (right). Scale bars: 250 µm (overview), 25 µm (insets) in Human Protein Atlas; 200 µm (overview), 50 µm (insets) in Nanfang cohorts; \u003cstrong\u003e(F) \u003c/strong\u003eGene effect scores of KMT2D knockout across multiple cancer types from DepMap CRISPR screens; \u003cstrong\u003e(G) \u003c/strong\u003eVenn diagram integrating CUT\u0026amp;Tag and RNA-seq datasets from C4-2 and LNCaP cells identifies 61 direct KMT2D target genes activated by KMT2D; \u003cstrong\u003e(H)\u003c/strong\u003e UMAP visualization of single-cell RNA-seq data from GSE181294, distinguishing benign (pink shades) and tumor (blue shades) epithelial cell clusters; \u003cstrong\u003e(I)\u003c/strong\u003e UMAP plots overlaying inferred KMT2D activity scores onto benign and tumor epithelial clusters, showing elevated activity in tumor cells; \u003cstrong\u003e(J)\u003c/strong\u003e Violin plot quantifying KMT2D activity scores in benign versus tumor epithelial cells from GSE181294.\u003c/p\u003e","description":"","filename":"Figure.1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/d33e948ca4568136ee11d716.jpg"},{"id":95525624,"identity":"4503d540-0c46-4a17-833b-0db3e108af1b","added_by":"auto","created_at":"2025-11-10 10:05:27","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3009058,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKMT2D enhances androgen receptor (AR) signaling through direct regulation of AR target genes. \u003c/strong\u003e(A) Enrichment rank plot of sgRNAs calculated from the guide RNA ratio in mCherry-LOW versus mCherry-HIGH cells. (B) Pathway enrichment analysis of KMT2D-activated genes. (C) Violin plot comparing AR activity scores between normal and tumor prostate epithelial cells. (D) Correlation plot showing KMT2D activity and AR activity at the single-cell level (Pearson R = 0.58, P \u0026lt; 2.2e-16). (E) Quantitative RT-PCR analysis of AR target genes (KLK3, TMPRSS2, NKX3-1) in C4-2 cells following CRISPR-mediated KMT2D knockout under regular, dihydrotestosterone (DHT)-stimulated, and charcoal-stripped serum (CSS) conditions. Data represent mean ± SD; ***P \u0026lt; 0.001 by Student’s t-test. (F) Immunoblot of KLK3 protein in LNCaP cells following siRNA-mediated KMT2D knockdown and in C4-2 cells following sgRNA-mediated KMT2D knockout under regular, DHT, or CSS conditions. β-Actin served as loading control. (G) qRT-PCR analysis of KLK3 mRNA levels in C4-2 cells with sgNC or sgKMT2D, treated with the indicated concentrations of DHT. (H) AR activity analysis based on the sum of Z-score values for AR signature genes in LNCaP and C4-2 cells following KMT2D inhibition treated with or without DHT (10 nM, 24 h). (I) GSVA heatmap depicting downregulation of multiple androgen-responsive gene sets upon KMT2D silencing in LNCaP and C4-2 cells under different culture conditions. (J) GSEA dot plot illustrating consistent suppression of androgen signaling pathways in LNCaP and C4-2 cells after KMT2D knockdown. Normalized enrichment scores (NES) and adjusted P values are shown. (K) Venn diagrams showing extensive overlap of AR and KMT2D CUT\u0026amp;Tag binding sites in LNCaP and C4-2 cells. (L) BETA analysis combining RNA-seq and AR CUT\u0026amp;Tag data reveals that KMT2D predominantly activates transcription at AR binding sites (Kolmogorov–Smirnov test, P \u0026lt; 0.0001). (M) Heatmap and average intensity curves of ChIP-seq reads at AR-binding sites in LNCaP and C4-2 cells following KMT2D inhibition, treated with DHT (10 nM, 24 h). (N) Genomic annotation of KMT2D-dependent AR binding sites. (O) ChIP-qPCR of AR binding at the KLK2, KLK3, TMPRSS2, and NKX3.1 enhancer sites in LNCaP and C4-2 cells, treated with DHT (10 nM, 24 h).\u003c/p\u003e","description":"","filename":"Figure.2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/c2f8aafc35a96d000ea0969b.jpg"},{"id":95355863,"identity":"ee6cf5a7-6f44-4e07-a612-dea1a1d681a8","added_by":"auto","created_at":"2025-11-07 06:33:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2826111,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKMT2D Depletion Inhibits Prostate Cancer Cell Proliferation and Tumor Growth.\u003c/strong\u003e (A) Cell viability assays in LNCaP cells with negative control (siNC) or KMT2D siRNAs (siKMT2D) and C4-2 cells with vector (sgNC) or KMT2D sgRNA (sgKMT2D). (B) Representative colony formation assay and quantification in LNCaP cells with siNC or siKMT2D and C4-2 cells with sgNC or sgKMT2D. (C) Representative image and quantification of EdU assay in LNCaP cells with siNC or siKMT2D and C4-2 cells with sgNC or sgKMT2D under regular, DHT-stimulated, and CSS conditions. (D) Quantification of EdU assay in LNCaP cells with siNC or siKMT2D and C4-2 cells with sgNC or sgKMT2D, treated with indicated concentrations of DHT. (E) Cell viability assays in LNCaP cells with siNC or siKMT2D and C4-2 cells with sgNC or sgKMT2D, treated with indicated concentrations of DHT. (F) Tumor volume changes in C4-2 cells with sgNC (n = 7) or sgKMT2D (n = 3). (G) Reverse Kaplan-Meier plot of tumor grafting of C4-2 sgNC, sgKMT2D cells. (H) Representative images of excised tumors from sgKMT2D and control groups. (I) Tumor weight of sgNC and sgKMT2D C4-2 xenografts. (J) qRT-PCR analysis of KLK3, TMPRSS2, and NKX3.1 in xenograft tumors with KMT2D knockout versus controls. Data are mean ± SD. (K) Western blot analysis of KLK3 and H3K4me1 protein levels in C4-2 xenografts. (L, M) Representative immunohistochemical staining and quantification of Ki-67 (L) and KLK3 (M) in xenograft tumors from sgKMT2D and control groups. Scale bars, 50 µm.\u003c/p\u003e","description":"","filename":"Figure.3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/105b855f95831951e9129df8.jpg"},{"id":95524639,"identity":"fb1afe02-1840-445b-bdac-c4a7968ad13b","added_by":"auto","created_at":"2025-11-10 10:03:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2477816,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKMT2D Modulates Chromatin Accessibility and Facilitates FOXA1-Mediated AR Target Gene Regulation.\u003c/strong\u003e (A) Venn diagrams showing AR-binding sites in LNCaP and C4-2 cells after KMT2D inhibition, treated with DHT (10 nM, 24 h). (B) Enriched motifs for AR-binding sites lost after KMT2D inhibition. (C) Heatmap of AR, FOXA1, KMT2D, H3K4me1, and H3K27ac CUT\u0026amp;Tag signal intensities at AR-binding sites in C4-2 cells. (D and E) Average intensity curves of CUT\u0026amp;Tag reads at H3K4me1- or H3K27ac-binding sites in C4-2 cells following KMT2D inhibition (left). Enriched motifs ranking at H3K4me1 or H3K27ac binding sites that are lost after KMT2D inhibition in C4-2 cells. (F and H) Average intensity curves of CUT\u0026amp;Tag reads at FOXA1 binding sites in LNCaP and C4-2 cells following KMT2D inhibition. (G and I) CUT\u0026amp;Tag-qPCR for FOXA1 binding at KLK2, KLK3, TMPRSS2, and NKX3.1 enhancer sites in LNCaP and C4-2 cells. (J) Heatmaps and intensity curves of ATAC-Seq in LNCaP and C4-2 cells. (K) Venn diagrams showing differentially accessible sites in LNCaP and C4-2 cells following KMT2D inhibition. (L) Enriched motifs for less accessible sites after KMT2D inhibition in LNCaP and C4-2 cells.\u003c/p\u003e","description":"","filename":"Figure.4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/fbb9b9e375be1c6c8c5ecbf0.jpg"},{"id":95355864,"identity":"eb5572be-c5e5-4340-80cb-ae93c8f9c49f","added_by":"auto","created_at":"2025-11-07 06:33:41","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2693858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFOXA1 Missense Mutations Impair KMT2D-Mediated Regulation of AR Signaling and Prostate Cancer Cell Proliferation.\u003c/strong\u003e (A) Cell proliferation in C4-2 cells following siRNA knockdown of KMT2D and/or FOXA1 measured by CCK-8 assay.\u003cbr\u003e\n(B, C) Representative images and quantification of colony formation assays under indicated knockdowns. (D) Dose-response of KLK3 mRNA expression to DHT stimulation after knockdowns measured by qRT-PCR. (E) Expression of AR target genes KLK3, TMPRSS2, and NKX3-1 following knockdowns quantified by qRT-PCR. (F) Immunoblot of FOXA1 and KLK3 proteins in C4-2 cells with indicated knockdowns; β-Actin as loading control. (G) Schematic of FOXA1 protein domains highlighting D226G and M253K mutations. (H) Cell viability assays in C4-2 cells stably expressing FOXA1 mutants with or without KMT2D knockout. (I, J) EdU incorporation assay images and quantification in FOXA1 mutant cells post-KMT2D knockout. (K) AR target gene expression assessed by qRT-PCR in FOXA1 mutant cells with KMT2D depletion. (L) Immunoblot of KLK3 in FOXA1 mutant cells after KMT2D knockout. β-Actin as loading control. (M) CUT\u0026amp;Tag heatmaps of FOXA1 binding in mutant cells ± KMT2D knockout. (N) Venn diagrams of FOXA1 and AR co-binding sites in mutant cells. (O) Genome browser tracks showing FOXA1 occupancy at AR target loci in FOXA1 mutants with or without KMT2D knockout.\u003c/p\u003e","description":"","filename":"Figure.5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/878f0bf0654e6e5e75ef69b2.jpg"},{"id":95526082,"identity":"2126b6e8-3138-465b-8658-857df6b06c9e","added_by":"auto","created_at":"2025-11-10 10:06:12","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2752389,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKMT2D-FOXA1-AR axis regulates ketogenesis via HMGCS2 in PCa. \u003c/strong\u003e(A) Bar plots showing enriched pathways in GSEA comparing KMT2D knockdown (siKMT2D) vs. negative control (siNC) in LNCaP and C4-2 cells treated with DHT (10 nM, 24 h). (B) Venn diagrams showing the overlap between downregulated genes and less accessible ATAC sites in C4-2 cells with KMT2D inhibition. (C) RNA-Seq heatmap of overlapping genes (n=394) in LNCaP and C4-2 cells following KMT2D inhibition treated with or without DHT (10 nM, 24 h). (D) Enrichr analysis of overlapping genes in molecular signature and biological pathway databases. (E) Venn diagrams representing overlap genes (n=6) between lipid and lipoprotein metabolism pathway, fatty acid, triacylglycerol, and ketone body metabolism pathways, and PPAR signaling pathway. (F) RNA-Seq analysis of HMGCS2 gene expression in LNCaP and C4-2 cells under DHT and CSS conditions. (G) qRT-PCR analysis of HMGCS2 mRNA level in LNCaP and C4-2 cells with KMT2D inhibition. (H) IGV tracks showing KMT2D, AR, FOXA1, H3K27ac, and H3K4me1 occupancy and ATAC accessible sites at the HMCGS2 loci in LNCaP and C4-2 cells. (I) Schematic showing ketogenesis and ketone body metabolism. (J) Ketobody content detection in LNCaP and C4-2 cells with KMT2D inhibition. (K) Cell viability assays of sgNC C4-2 and sgKMT2D C4-2 cells treated with DMSO or 10 μM β-OHB. (L) Cell viability assays of LNCaP and C4-2 cells transfected with HMGCS2 siRNA under the indicated condition.\u003c/p\u003e","description":"","filename":"Figure.6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/4446b8e2e55f83f76f739a70.jpg"},{"id":95355868,"identity":"da10503b-96b2-4f89-923e-b03e8b9d7019","added_by":"auto","created_at":"2025-11-07 06:33:41","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1335519,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeting KMT2D-associated H3K4me1 methylation suppresses AR signaling and prostate cancer growth.\u003c/strong\u003e (A) Schematic of the KMT2D COMPASS complex components responsible for H3K4me1 deposition, including core WRAD subunits and KMT2D-specific partners such as UTX. (B) Heatmap showing correlation and co-dependency between KMT2D and COMPASS complex members, highlighting UTX across TCGA-PRAD, DepMap CRISPR, and RNAi datasets. (C) Dose-dependent reduction in cell viability of LNCaP (left) and C4-2 (right) cells treated with the UTX inhibitor GSK-J4 over 3 days. (D) qRT-PCR analysis showing decreased mRNA levels of AR target genes KLK3, TMPRSS2, and NKX3.1 upon GSK-J4 treatment in LNCaP and C4-2 cells. (E) Western blot demonstrating reduced KLK3 protein and global H3K4me1 levels in LNCaP and C4-2 cells treated with GSK-J4. β-Tubulin serves as a loading control. (F) Workflow schematic of ex vivo treatment of patient-derived tumor fragments (PDTFs) with GSK-J4. (G) qRT-PCR analysis of KLK3 and TMPRSS2 expression in two independent PDTF samples treated with GSK-J4 or DMSO control. (H) Immunoblot showing decreased AR, KLK3, and H3K4me1 protein levels in PDTFs following GSK-J4 exposure. β-Tubulin served as loading control.\u003c/p\u003e","description":"","filename":"Figure.7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/6f5563949286cdacaac9fa3a.jpg"},{"id":95355879,"identity":"15b10a9a-5af3-4f83-b7de-ef010d9e9748","added_by":"auto","created_at":"2025-11-07 06:33:42","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2602850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKMT2D promotes enzalutamide resistance by sustaining AR signaling and epigenetic remodeling in prostate cancer.\u003c/strong\u003e (A) Schematic illustrating the development of enzalutamide resistance in LNCaP cells through prolonged drug exposure leading to EnzR cells. (B) Western blot analysis showing elevated AR and H3K4me1 protein levels in EnzR cells compared to parental LNCaP and C4-2 cells. β-tubulin served as loading control. (C) Left: CCK-8 assay showing reduced proliferation of EnzR cells upon sgRNA-mediated KMT2D knockout. Right: Dose-response curves indicating increased enzalutamide sensitivity following KMT2D depletion. (D) Heatmap of AR signature scores derived from RNA-seq data demonstrating enhanced AR activity in EnzR cells and its suppression by KMT2D knockdown. (E) ATAC-seq heatmap displaying global loss of chromatin accessibility in EnzR cells after KMT2D depletion. (F) Aggregation plot showing decreased chromatin accessibility at accessible sites upon KMT2D knockdown. (G) Venn diagram of accessible chromatin regions in EnzR cells with or without KMT2D knockdown. (H) Motif enrichment analysis of chromatin sites losing accessibility after KMT2D knockdown, highlighting FOXA1 and related forkhead motifs. (I) CCK-8 assay showing dose-dependent reduction in EnzR cell viability following treatment with UTX inhibitor GSK-J4. (J) qRT-PCR analysis of AR target genes (KLK3, TMPRSS2, NKX3.1) demonstrating decreased expression after GSK-J4 treatment in EnzR cells. (K) Western blot showing reduced KLK3 protein and global H3K4me1 levels in EnzR cells treated with GSK-J4. β-Tubulin is loading control. (L) Experimental timeline for in vivo treatment of EnzR xenograft-bearing mice with vehicle or GSK-J4. (M) Tumor growth curves indicating significant suppression of EnzR xenograft progression by GSK-J4 treatment. (N) Representative images of excised tumors from vehicle- and GSK-J4-treated mice. (O) Quantification of tumor weights confirming reduced tumor burden following GSK-J4 administration. (P) Immunohistochemical staining and quantification of Ki-67 proliferation marker and H3K4me1 modification in xenografts, showing decreased proliferation and epigenetic mark levels after GSK-J4 treatment. (Q) Proposed model illustrating how KMT2D and associated epigenetic regulators sustain AR signaling and ketone metabolism to promote tumor growth, and how GSK-J4-mediated inhibition disrupts this axis to suppress prostate cancer progression.\u003c/p\u003e","description":"","filename":"Figure.8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/011334eaf2587e8529f96072.jpg"},{"id":95654266,"identity":"db2f5275-9b57-48a4-a97c-713c9e77894b","added_by":"auto","created_at":"2025-11-11 16:10:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21413494,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/8905e36e-5e08-4159-8817-1b32248742d8.pdf"},{"id":95355869,"identity":"7777d292-13b3-4388-a2f7-424f77fc870a","added_by":"auto","created_at":"2025-11-07 06:33:41","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6465500,"visible":true,"origin":"","legend":"Supplemental information","description":"","filename":"Supplementalinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7812403/v1/a0292e8c8e6665c517258b17.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Histone Methyltransferase KMT2D Promotes Castration-Resistant Prostate Cancer Progression by Reactivating AR through FOXA1","fulltext":[{"header":"1. Background","content":"\u003cp\u003eProstate cancer (PCa) is one of the most prevalent malignancies in men worldwide and the second leading cause of cancer mortality \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Although androgen deprivation therapy (ADT) as the standard initial treatment significantly improves survival, most patients ultimately progress to castration-resistant prostate cancer (CRPC), which is characterized by metastatic spread and poor prognosis \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEpigenetic alterations, including DNA methylation, histone modification, and chromatin remodeling, critically influence CRPC progression \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. These modifications modulate gene expression and promote cancer cell survival, proliferation, and resistance to therapy. The androgen receptor (AR) signaling pathway cooperates with these epigenetic changes, amplifying AR activity and accelerating CRPC advancement \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eLysine methyltransferase 2D (KMT2D) or mixed-lineage leukemia 4 (MLL4) monomethylates histone H3 at lysine 4 (H3K4me1) in mammalian systems \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. KMT2D collaborates with transcription factors and co-regulators to maintain an open chromatin architecture across cell types, governing processes such as proliferation, embryonic differentiation, and tumorigenesis through H3K4me1-mediated transcriptional control and pathways such as PI3K/Akt, Notch, and Wnt \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Aberrant KMT2D expression or mutations contribute to malignancies, including lymphoma \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, ovarian \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, bladder \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, breast \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, and lung cancers \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In PCa, KMT2D acts as an oncogene by activating proliferative and metastatic signaling cascades \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Although KMT2D modulates estrogen receptor (ER) transcriptional activity via Forkhead Box A1 (FOXA1) in breast cancer \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, its interaction with AR in PCa remains poorly understood.\u003c/p\u003e\u003cp\u003eWe demonstrated that KMT2D critically regulated AR transcriptional activity by recruiting FOXA1, thereby driving PCa progression. FOXA1 mutations in the forkhead DNA-binding domain (FKHD), such as D226G and M253K, disrupt KMT2D-mediated AR regulation and cell proliferation. Furthermore, inhibiting UTX (also called KDM6A), a COMPASS complex component, markedly reduced KMT2D-dependent H3K4me1 catalysis and AR activity. These findings elucidate the mechanistic basis of KMT2D-driven CRPC progression and highlight its potential as a therapeutic target for PCa.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture and reagents\u003c/strong\u003e\u003cp\u003eThe LNCaP, C4-2, and HEK293T were obtained from Procell Life Science \u0026amp; Technology Co., Ltd. (Wuhan, China). LNCaP and C4-2 cells were maintained in RPMI 1640 medium (Corning), whereas HEK293T cells were grown in DMEM (Corning). All media contained 10% FBS (Haixing Biosciences, China) or charcoal-stripped FBS (CSS, Biological Industries), along with 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin (Thermo Fisher Scientific) and were incubated at 37\u0026deg;C with 5% CO₂. PCR confirmed the absence of mycoplasma contamination. Enzalutamide (MedChem Express, HY-70002) and dihydrotestosterone ( M6033) were purchased from commercial sources. The phenol red-free RPMI 1640 medium was purchased from Thermo Fisher Scientific.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eRNA interference\u003c/em\u003e: KMT2D siRNA (siKMT2D) and negative control siRNA (siNC) were obtained from Dharmacon (Catalog ID: L-004828-00-0020 for KMT2D and D-001810-10-20 for the negative control). Cells were transfected with 100 nM siRNA using Lipofectamine 3000 (Thermo Fisher Scientific), according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCRISPR/Cas9-mediated KMT2D gene knockout\u003c/strong\u003e\u003cp\u003epLV-CMV-FOXA1(D226G)-3FLAG and pLV-CMV-FOXA1(M253K)-3FLAG plasmids were constructed as previously described (17). The mutations were confirmed by Sanger sequencing (Supplementary Figure S4).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFOXA1 mutation plasmids construction\u003c/strong\u003e\u003cp\u003eConstruction of the pLV-CMV-FOXA1(D226G)-3FLAG and pLV-CMV-FOXA1(M253K)-3FLAG plasmids was conducted as previously described \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The mutations were confirmed by Sanger sequencing (Supplementary Figure S4).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCell viability assay\u003c/strong\u003e\u003cp\u003eCells were directly plated in 96-well plates at a density of 3,000\u0026ndash;5,000 cells per well and allowed to adhere for 24 h. Subsequent treatments were performed under designated experimental conditions. Following a 48-h incubation period, the CCK-8 kit (Apexbio, K1018, America) was used to evaluate cell viability, following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e5-ethynyl-2\u0026rsquo;-deoxyuridine (EdU) proliferation assay\u003c/strong\u003e\u003cp\u003ePCa cells (3 \u0026times; 10⁴ cells/well) were plated in 96-well plates and allowed to adhere for 24 h. The cells were then cultured in either standard medium with 10 nM Dihydrotestosterone (DHT) or in phenol red-free RPMI-1640 supplemented with CSS. Following 48 h of treatment, 50 \u0026micro;M EdU (Apexbio) was added for 2 h before processing, according to the manufacturer\u0026rsquo;s protocol. Nuclear staining was performed using Hoechst 33342 (5 \u0026micro;g/mL) for 30 min, followed by fluorescence microscopy. ImageJ software was used to quantify the EdU-positive cell density by comparing nuclear Hoechst 33342 staining with negative controls.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eColony formation assays\u003c/strong\u003e\u003cp\u003e1,000 PCa cells were seeded into six-well plates per well for two weeks. After washing with PBS, cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Colony counts were quantified using the ImageJ software.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eRNA extraction, reverse transcription, and quantitative real-time PCR (qRT-PCR)\u003c/strong\u003e\u003cp\u003ePCa cells were cultured in 12-well plates and subjected to the specified treatments. Total RNA was extracted after 48 h using TRIzol reagent (Invitrogen) following the manufacturer\u0026rsquo;s instructions. Reverse transcription was performed with 1 \u0026micro;g of RNA using the HiScript\u0026reg; III All-in-one RT SuperMix kit (Vazyme Biotech Co. Ltd.). Quantitative PCR analysis was performed using the Taq Pro Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.) on a QuantStudio\u0026trade; 6 Flex system (Thermo Fisher Scientific). The relative gene expression levels were calculated using the 2\u003csup\u003e\u0026ndash;ΔΔCt\u003c/sup\u003e method. The primer details are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003cp\u003ePCa cells were lysed using RIPA buffer supplemented with an EDTA-free protease inhibitor cocktail (Bimake, China), and protein concentrations were determined using the BCA Protein Assay Kit. samples were mixed with loading buffer and denatured by heating at 100\u0026deg;C for 10 min. Proteins (30 \u0026micro;g per lane) were resolved by SDS-PAGE and transferred to 0.45 \u0026micro;m PVDF membranes (Bio-Rad). The membranes were probed with primary antibodies, followed by incubation with HRP-conjugated Goat Anti-Rabbit IgG or Goat Anti-Mouse IgG. Immunoreactive bands were visualized using an ultra-high sensitivity ECL kit and quantified using the ImageJ software. Uncropped Western blots are provided in the Supplementary Materials.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eRNA-Seq and data analysis\u003c/strong\u003e\u003cp\u003eAll cell lines were cultured in their respective media under the specified treatment conditions. Total RNA was isolated using TRIzol reagent (Invitrogen), according to the manufacturer\u0026rsquo;s protocol. Libraries were prepared and sequenced on the NovaSeq 6000 platform. The resulting sequencing reads were aligned to the human genome reference sequence (hg38) using HISAT2 (v2.0.5) with default parameters. Subsequently, gene-level quantification was performed using FeatureCounts (v1.5.0-p3). Differential expression analysis was conducted using EdgeR (version 3.22.5), incorporating the Benjamini-Hochberg procedure to adjust for multiple hypothesis testing and to control the false discovery rate (FDR). Gene ontology (GO) enrichment was analyzed using clusterProfiler (version 3.8.1), and pathway enrichment was evaluated using gene set enrichment analysis (GSEA, version 4.2.2).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eChromatin immunoprecipitation-sequencing (ChIP-seq) and data analysis\u003c/strong\u003e\u003cp\u003eChIP assays were conducted according to the manufacturer's protocol (9003, Cell Signaling Technology,USA). LNCaP and C4-2 cells were subjected to crosslinking, lysis, and enzymatic digestion to generate chromatin fragments. These fragments were immunoprecipitated overnight with either an anti-AR antibody (Cell Signaling Technology, 5153) or a control IgG. The Novogene Corporation (Beijing, China) prepared ChIP-seq libraries from purified DNA. Sequencing was performed on the Illumina NovaSeq 6000 platform. Bowtie2 (v2.2.5) aligned the reads to the hg38 human genome using default parameters, followed by SAM-to-BAM conversion using Samtools (v1.6). MACS2 (v2.2.6) identified enriched regions, whereas Homer (v4.11) analyzed the DNA motifs. Peak visualization across samples was achieved using Integrative Genomics Viewer (IGV; v2.16.2).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCleavage under targets and tagmentation (CUT\u0026amp;Tag) assay and data analysis\u003c/strong\u003e\u003cp\u003eCUT\u0026amp;Tag was performed according to manufacturer's protocol (Yeasen, 12597ES) according to the manufacturer\u0026rsquo;s protocol. Briefly, 1 \u0026times; 10⁵ cells were collected, washed, and incubated with concanavalin A-coated beads. After sequential incubation with primary and secondary antibodies, DNA fragments were generated using pA-Tn5 transposase. For library preparation, purified DNA was amplified using 2\u0026times;HiFi Amplification Mix and uniquely barcoded primers from the Hieff NGS\u0026reg; Tagment Index Kit for Illumina\u0026reg; (Yeasen, 12416), followed by purification with DNA Selection Beads.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eAll libraries were sequenced on an Illumina NovaSeq 6000 platform. Fastp (v0.22.0) filtered low-quality reads, and Bowtie2 (v2.2.5) aligned the reads to the hg38 genome using parameters \u0026ldquo;-X 2000 --very-sensitive-local -no-discordant.\u0026rdquo; Sambamba (v0.6.6) removed PCR duplicates and Samtools (v1.6) converted SAM to BAM files. MACS2 (v2.2.6) called the peaks at the q-value threshold of 0.05. DeepTools (v3.5.2) generated BigWig files, profile plots, and heatmaps, whereas Homer (v4.11) identified motifs. IGV (v2.16.2) visualized the signal tracks, and EdgeR (v3.22.5) performed differential peak analysis.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAssay for transposase-accessible chromatin library preparation, sequencing, and data analysis\u003c/strong\u003e\u003cp\u003eThe assay for transposase-accessible chromatin with sequencing (ATAC-seq) samples was performed in duplicate using the Hyperactive ATAC-seq Library Prep Kit (TD711, Vazyme Biotech Co., Ltd.) following the manufacturer\u0026rsquo;s instructions. Briefly, 100,000 cells were collected, washed, and tagged with the Tn5 transposase. The digested DNA was then purified using magnetic beads. Libraries were amplified and sequenced using the Illumina platform.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eThe Fastp tool (v.0.22.0) was used to obtain quality control statistics of the samples. The ATAC-seq clean reads were mapped to the hg38 using Bowtie2 (v2.2.5) using parameters \u0026ldquo;-X 2000 --very-sensitive-local -no-discordant.\u0026rdquo; PCR-duplicated reads were removed using Sambamba (v0.6.6). SAM files were converted to BAM files using Samtools (v1.6). MACS2 (v2.2.6) was used to identify peaks with parameters \u0026ldquo;-q 0.05 --extsize 200 \u0026ndash;shift 100.\u0026rsquo;\u0026rsquo; DeepTools (v3.5.2) was used to convert the BAM files to BigWig files to generate a profile plot and heatmap. Homer (v4.11) was used for motif analysis. The ATAC-Seq signal tracks were visualized using IGV (v2.16.2). The R package EdgeR (3.22.5) was used to perform differential peak analysis.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eKetone body detection\u003c/strong\u003e\u003cp\u003eA Ketone Body Content Assay Kit (Solarbio, BC5065) was used to measure the cellular ketone body content, following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003ePatient-derived tumor fragments (PDTF)\u003c/strong\u003e\u003cp\u003eThe PDTF collection was approved by the Ethics Committee of Nanfang Hospital (NFEC-2022-299). \u003cem\u003eEx vivo\u003c/em\u003e culture of PDTFs was conducted as previously described \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Briefly, prostate tumor tissue was collected in a 50 mL tube, kept on ice, and transferred to a pre-chilled collection medium. The prostate tumor tissue was then cut into approximately 1 mm\u003csup\u003e3\u003c/sup\u003e sections. PDTFs from two patients with prostate cancer were collected and frozen in vials containing freezing medium. For the PDTF culture, cryopreserved PDTFs were thawed, washed, and embedded in the prepared extracellular matrix in 96-well plates. A tumor medium supplemented with either 10 \u0026micro;M GSK-J4 or DMSO was added to the top of the matrix. The PDTFs were cultured for 48 h before RNA and total protein extraction.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eXenograft study\u003c/em\u003e: All animal procedures were approved by the Ethics Committee of Ganzhou Hospital, Southern Medical University (approval no. TY-DKY2023-020-01). Four-week-old male BALB/c nude mice (strain no. D000521) from GemPharmatech (Nanjing, China) was subcutaneously injected with 2\u0026times;10\u003csup\u003e6\u003c/sup\u003e C4-2 sgNC or C4-2 sgKMT2D cells suspended in a 1:1 PBS-Matrigel mixture. In separate inhibitor experiments, castrated male mice were implanted with 2\u0026times;10\u003csup\u003e6\u003c/sup\u003e Enzalutamide resistance (EnzR) cells and randomly assigned to treatment groups when tumors reached 100 mm\u0026sup3;. The control group received vehicle (5% DMSO, 40% PEG300, 5% Tween80), while the experimental group received 10 mg/kg/day GSK-J4 via intraperitoneal injection for 15 days. Tumor dimensions were recorded every 72 h using calipers and volumes were calculated as L\u0026times;W\u003csup\u003e2\u003c/sup\u003e/2. Following euthanasia, the excised tumors were subjected to qRT-PCR, western blotting, and histopathological examination.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eHistology and Immunohistochemistry (IHC)\u003c/strong\u003e\u003cp\u003eSubcutaneous tumors excised from nude mice were fixed overnight in 4% paraformaldehyde, dehydrated using a series of ethanol concentrations, and embedded in paraffin. Consecutive 4 \u0026micro;m tissue sections from the formalin-fixed samples were subjected to routine hematoxylin and eosin (H\u0026amp;E) staining and immunohistochemistry. Quantification of digital IHC images was performed using ImageJ (Fiji) to assess the extent of staining (\u0026lt;\u0026thinsp;5%=0; 5\u0026ndash;25%=1; 26\u0026ndash;50%=2; 50\u0026ndash;75%=3; \u0026gt;75%=4) and staining intensity (0\u0026thinsp;=\u0026thinsp;negative; 1\u0026thinsp;=\u0026thinsp;weak; 2\u0026thinsp;=\u0026thinsp;moderate; 3\u0026thinsp;=\u0026thinsp;strong) for each specimen. The immunoreactive score (IRS), ranging from 0 to 12, was calculated by multiplying the two values.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003cp\u003eData analyses were performed using GraphPad Prism 10 software, with statistical significance set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Each experimental setup included a minimum of two biological replicates to ensure reliable statistical assessment. Results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM), with error bars indicating variability among three or more biological replicates or independent experiments. Comparisons between two groups were conducted using unpaired two-tailed Student\u0026rsquo;s t-tests (*P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas multi-group comparisons were conducted using one-way ANOVA. Survival analyses were performed using Kaplan-Meier curves, and differences were tested using two-sided log-rank tests.\u003c/p\u003e\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e3.1 KMT2D expression and activity are upregulated in prostate cancer and related to poor prognosis\u003c/h2\u003e\u003cp\u003eTo comprehensively evaluate KMT2D expression and activity in prostate cancer, we initially assessed its mRNA levels in the Renji cohort. Tumor tissues exhibited significantly higher KMT2D expression than adjacent normal tissues (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Independent validation in GSE35988 and GSE70770 revealed elevated KMT2D expression in castration-resistant prostate cancer compared to localized PCa (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). TCGA-PRAD survival analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) revealed that high KMT2D expression was correlated with shorter disease-free survival (log-rank P\u0026thinsp;=\u0026thinsp;0.018), suggesting its prognostic value. Immunohistochemical staining from the Human Protein Atlas and Nanfang cohorts confirmed increased KMT2D protein expression in prostate cancer tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), consistent with transcriptomic findings. Gene effect analysis based on CRISPR knockout screens in DepMap (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) revealed that KMT2D is critical for cell viability in prostate adenocarcinoma as well as in multiple other cancer types, including acute myeloid leukemia, invasive breast carcinoma, ovarian epithelial tumors, and diffuse glioma (all adjusted P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eWe then employed CUT\u0026amp;Tag and RNA-seq in C4-2 and LNCaP cell lines to map KMT2D-regulated genes and identify 61 direct targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Analysis of GSE181294 single-cell RNA-seq data distinguished benign from malignant epithelial clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), with KMT2D activity markedly increased in tumor cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), supporting its association with malignant transformation at single-cell resolution. Together, these findings establish that KMT2D is consistently overexpressed in prostate cancer, particularly in CRPC, where it is correlated with poor prognosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e3.2 KMT2D Enhances Androgen Receptor Signaling Through Direct Regulation of AR Target Genes\u003c/h2\u003e\u003cp\u003ePrevious research established endogenous AR reporter cell lines (LNCaP_mCherry_PSA) which mCherry expression is directly regulated by the AR transcriptional complex. Functional CRISPR screen was performed and KMT2D was identified as an AR coactivator\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To investigate KMT2D's function in AR signaling, we analyzed pathway enrichment related to KMT2D-activated genes. Gene set enrichment revealed significant associations between KMT2D and AR-related pathways, including the androgen response, AR ChIP-seq targets, and prostate cancer signatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Analysis of TCGA-PRAD data revealed a significant positive correlation between KMT2D expression and AR signature scores (R\u0026thinsp;=\u0026thinsp;0.39, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) as well as between the KMT2D signature and AR signature (R\u0026thinsp;=\u0026thinsp;0.71, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) at the transcript level (Supplementary Fig.\u0026nbsp;2A). Single-cell RNA-seq analysis revealed markedly higher AR activity in tumor cells than in normal prostate epithelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The AR and KMT2D activities exhibited a robust positive correlation at the single-cell level (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, R\u0026thinsp;=\u0026thinsp;0.58, P\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16).\u003c/p\u003e\u003cp\u003eWe then examined KMT2D's functional impact through CRISPR knockout in C4-2 cells and siRNA knockdown in LNCaP cells (Supplementary Fig.\u0026nbsp;2B and 2C). Both genetic approaches markedly suppressed KLK3, TMPRSS2, and NKX3-1 transcript levels under both regular and androgen-depleted (CSS) conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and Supplementary Fig.\u0026nbsp;2C and 2E). Western blot analyses confirmed that KLK3 protein level decreased upon KMT2D silencing in LNCaP and C4-2 cells treated with dihydrotestosterone (DHT) or CSS medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Consistently, the inhibitory effect on KLK3 expression showed a clear dose dependence in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and Supplementary Fig.\u0026nbsp;2D).\u003c/p\u003e\u003cp\u003eTo investigate the regulatory role of KMT2D in AR, we conducted RNA-seq analysis after knocking down KMT2D in LNCaP and C4-2 cells exposed to DHT or cultured under CSS conditions. Heatmaps revealed that androgen-induced genes were substantially downregulated upon KMT2D depletion in both cell lines under all treatment conditions (Supplementary Fig.\u0026nbsp;2F). AR activity scores derived from RNA-seq data also decreased significantly after KMT2D knockdown in LNCaP and C4-2 cells, whether stimulated with DHT or deprived of androgens (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). GSVA confirmed this attenuated AR activity by showing reduced expression across multiple androgen-responsive gene sets (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Broader GSEA analysis across treatment groups consistently demonstrated suppressed androgen signaling pathways following KMT2D loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ and Supplementary Fig.\u0026nbsp;2G), underscoring KMT2D's essential function in maintaining AR transcriptional activity.\u003c/p\u003e\u003cp\u003eTo investigate KMT2D-AR interactions at the chromatin level, we performed genome-wide CUT\u0026amp;Tag profiling of LNCaP and C4-2 cells. Venn analysis revealed extensive overlap between KMT2D and AR binding sites, indicating frequent co-occupancy of the regulatory elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). Combining RNA-seq with AR CUT\u0026amp;Tag through BETA analysis showed that KMT2D predominantly activates AR transcription (Kolmogorov-Smirnov test, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL), highlighting its functional role at AR target loci. AR ChIP-seq also showed that KMT2D depletion substantially reduced the AR binding intensity at promoters and enhancers in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eM). Genomic annotation of lost AR sites revealed enrichment in distal intergenic and promoter regions aligned with known transcriptional regulatory elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN). ChIP-qPCR validated the significantly reduced AR occupancy at canonical target genes (KLK3, NKX3-1, KLK2, and TMPRSS2) upon KMT2D silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eO). Genome browser tracks further confirmed the diminished AR peaks at these loci after KMT2D knockdown (Supplementary Fig.\u0026nbsp;2H). Collectively, these results establish KMT2D as a critical facilitator of AR chromatin binding and transcriptional activation in prostate cancer cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e3.3 KMT2D Depletion Inhibits Prostate Cancer Cell Proliferation and Tumor Growth\u003c/h2\u003e\u003cp\u003eTo investigate the functional role of KMT2D in prostate cancer cell proliferation, we conducted loss-of-function studies in LNCaP and C4-2 cells. KMT2D knockdown using siRNA or sgRNA substantially inhibited cell proliferation after 72 h, as quantified by CCK-8 assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Colony formation assays also confirmed this effect, showing a significantly diminished clonogenic potential in both cell lines following KMT2D depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). EdU incorporation assays showed that KMT2D silencing reduced proliferating cell populations in LNCaP and C4-2 cells under standard culture conditions, regardless of DHT stimulation and in CSS medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). KMT2D depletion also attenuated cell viability with increasing DHT levels, suggesting its involvement in androgen-mediated proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eIn C4-2 xenograft models, KMT2D knockout delayed tumor progression and reduced tumor volume (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Excised tumors from the KMT2D-deficient groups weighed significantly less than those from the controls (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). qRT-PCR analyses showed decreased expression of AR target genes KLK3, TMPRSS2, and NKX3.1 in KMT2D-deficient tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). Western blotting confirmed reduced KLK3 protein and H3K4me1 levels in KMT2D knockout group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). Immunohistochemistry revealed lower Ki-67 positivity and KLK3 expression in the KMT2D-deficient group xenografts, indicating impaired proliferation and AR signaling in vivo (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). These results confirmed that KMT2D is a regulator of prostate cancer proliferation and tumorigenesis, mainly through the maintenance of AR-dependent transcription.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.4 KMT2D Modulates Chromatin Accessibility and Facilitates FOXA1-Mediated AR Target Gene Regulation\u003c/h2\u003e\u003cp\u003eTo investigate how KMT2D modulates AR signaling, we analyzed AR ChIP-seq data and found that KMT2D depletion significantly reduced the number of AR-binding sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Motif analysis of KMT2D-dependent AR binding sites revealed strong enrichment of FOXA1 and androgen response elements (AREs; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), suggesting that KMT2D and FOXA1 may cooperatively regulate AR target genes. We further explored the relationship between KMT2D and FOXA1 expression in prostate cancer. TCGA-PRAD transcriptomic analysis demonstrated a robust positive correlation between KMT2D and FOXA1 signature expression (Supplementary Fig.\u0026nbsp;4A). Both CRISPR-Cas9 knockout and RNA interference screens identified FOXA1 as the top transcription factor with the highest relative importance score in DepMap (Supplementary Fig.\u0026nbsp;4B). We also performed CUT\u0026amp;Tag of H3K4me1 and H3K27ac in LNCaP and C4-2 cells, with or without KMT2D knockdown. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, AR-binding sites exhibited strong co-enrichment of FOXA1, KMT2D, and active enhancer marks H3K4me1 and H3K27ac, supporting a coordinated regulation of androgen receptor target regions by these factors and epigenetic modifications. KMT2D knockdown globally reduced H3K4me1 and H3K27ac binding, particularly at AR-dependent and AR-FOXA1 co-binding sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE left panel; Supplementary Fig.\u0026nbsp;4C-4H). FOXA1 motifs were significantly enriched in KMT2D-dependent H3K4me1/H3K27ac sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, right panel), further implicating FOXA1 in KMT2D-mediated regulation. FOXA1 CUT\u0026amp;Tag demonstrated decreased global binding upon KMT2D silencing, particularly at AR-dependent loci (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG left panel; Supplementary Fig.\u0026nbsp;4I-4J). Quantitative PCR confirmed reduced FOXA1 occupancy at canonical AR targets (KLK2, KLK3, TMPRSS2, and NKX3-1) in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG right panel), establishing KMT2D's necessity for FOXA1 recruitment.\u003c/p\u003e\u003cp\u003eTo evaluate changes in global chromatin accessibility, we conducted ATAC-seq in LNCaP and C4-2 cells with or without KMT2D knockdown. ATAC-seq analysis showed that KMT2D depletion broadly diminished chromatin accessibility, particularly in the AR-dependent and FOXA1-AR co-dependent regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK; Supplementary Fig.\u0026nbsp;4K-4L). FOXA1 and forkhead family motifs dominated these KMT2D-dependent accessible regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL), suggesting that KMT2D cooperated with forkhead family to maintain chromatin openness. Collectively, these findings support a model in which KMT2D promotes prostate cancer progression by sustaining active enhancer histone modifications, recruiting FOXA1, and maintaining chromatin accessibility at the AR regulatory elements.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.5 FOXA1 Missense Mutations Impair KMT2D-Mediated Regulation of AR Signaling and Prostate Cancer Cell Proliferation\u003c/h2\u003e\u003cp\u003eTo determine whether KMT2D-driven proliferation requires FOXA1, we performed combinatorial knockdown in C4-2 cells. Silencing FOXA1 substantially reduced both cell proliferation and colony formation, while dual knockdown of KMT2D and FOXA1 produced no additional suppression compared to FOXA1 depletion alone (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;C), demonstrating FOXA1's downstream position relative to KMT2D. Similarly, FOXA1 knockdown strongly attenuated DHT-induced KLK3 expression, while concurrent KMT2D knockdown failed to further diminish this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Further qRT-PCR analysis revealed that FOXA1 knockdown alone substantially decreased KLK3, TMPRSS2, and NKX3-1 mRNA levels, whereas simultaneous depletion of FOXA1 and KMT2D failed to induce further suppression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Corresponding protein level reductions were verified by western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eNext, we examined two clinically relevant FOXA1 missense mutations, D226G and M253K, within the forkhead DNA-binding (FKHD) domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and Supplementary Fig.\u0026nbsp;5A). Previous work has demonstrated that FKHD domain missense mutations (FKHD-MSs) disrupt FOXA1 chromatin binding at AR-dependent enhancers, suppressing AR transcriptional activity. The stable expression of these mutants in C4-2 cells reduced the mRNA levels of KLK3, TMPRSS2, and NKX3-1, along with KLK3 protein expression (Supplementary Fig.\u0026nbsp;5B and 5C). KMT2D depletion resulted in no growth suppression in cells with FOXA1-D226G or FOXA1-M253K mutations (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH\u0026ndash;J; Supplementary Fig.\u0026nbsp;5D and 5E), indicating that these mutations confer resistance to KMT2D loss. Next, we evaluated how KMT2D knockdown affected AR target gene expression in cells expressing FOXA1 mutants. qRT-PCR demonstrated that KMT2D depletion failed to substantially decrease KLK3, TMPRSS2, or NKX3-1 mRNA levels in cells with either D226G or M253K mutations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK). Correspondingly, Western blot analysis indicated that KLK3 protein expression showed minimal changes following KMT2D knockdown in both the mutant cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL). CUT \u0026amp; Tag profiling showed that FOXA1 chromatin occupancy persisted in D226G and M253K mutant cells despite KMT2D knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM; Supplementary Fig.\u0026nbsp;5H). Integration with AR CUT\u0026amp;Tag data identified extensive FOXA1-AR co-binding sites that remained stable following KMT2D depletion in mutant cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN; Supplementary Fig.\u0026nbsp;5F and 5G). Genome browser visualization of key AR target loci confirmed sustained FOXA1 binding in mutant cells upon KMT2D knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eO). These findings imply that D226G and M253K mutations in FOXA1 confer resistance to the inhibitory effects of KMT2D loss on AR signaling in prostate cancer cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e(B, C) Representative images and quantification of colony formation assays under indicated knockdowns. (D) Dose-response of KLK3 mRNA expression to DHT stimulation after knockdowns measured by qRT-PCR. (E) Expression of AR target genes KLK3, TMPRSS2, and NKX3-1 following knockdowns quantified by qRT-PCR. (F) Immunoblot of FOXA1 and KLK3 proteins in C4-2 cells with indicated knockdowns; β-Actin as loading control. (G) Schematic of FOXA1 protein domains highlighting D226G and M253K mutations. (H) Cell viability assays in C4-2 cells stably expressing FOXA1 mutants with or without KMT2D knockout. (I, J) EdU incorporation assay images and quantification in FOXA1 mutant cells post-KMT2D knockout. (K) AR target gene expression assessed by qRT-PCR in FOXA1 mutant cells with KMT2D depletion. (L) Immunoblot of KLK3 in FOXA1 mutant cells after KMT2D knockout. β-Actin as loading control. (M) CUT\u0026amp;Tag heatmaps of FOXA1 binding in mutant cells\u0026thinsp;\u0026plusmn;\u0026thinsp;KMT2D knockout. (N) Venn diagrams of FOXA1 and AR co-binding sites in mutant cells. (O) Genome browser tracks showing FOXA1 occupancy at AR target loci in FOXA1 mutants with or without KMT2D knockout.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.6 KMT2D-FOXA1-AR axis regulates ketogenesis via HMGCS2 in PCa\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eTo investigate the biological functions mediated by the KMT2D-FOXA1-AR axis, we re-analyzed the GSEA results after KMT2D knockdown under both DHT and CSS conditions. The enrichment of metabolic pathways, including fatty acid metabolism and cholesterol homeostasis, suggests that KMT2D is involved in metabolic regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). We identified downstream genes by selecting those with reduced expression and diminished chromatin accessibility by ATAC-seq following KMT2D inhibition in C4-2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Among these, 394 genes were consistently downregulated in C4-2 cells after KMT2D knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Pathway enrichment demonstrated significant associations with fatty acid, triacylglycerol, and ketone body metabolism in Planet, along with PPAR signaling pathway enrichment in KEGG (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Similar patterns were also observed in LNCaP cells, corroborating the C4-2 cell findings (Supplementary Fig.\u0026nbsp;6A\u0026ndash;E).\u003c/p\u003e\u003cp\u003eFocusing on the genes within these pathways, we identified six candidates for further study (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). HMGCS2 mRNA levels decreased in both LNCaP and C4-2 cells upon KMT2D knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Subsequent analysis revealed that KMT2D inhibition reduced AR, FOXA1, H3K27ac, and H3K4me1 binding, along with chromatin accessibility, to the enhancer of HMGCS2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). HMGCS2 catalyzes HMG-CoA formation from acetyl-CoA, which is the rate-limiting enzyme in ketogenesis. Ketone bodies, primarily β-hydroxybutyrate (β-OHB), acetoacetate, and acetone, serve as critical energy sources for extrahepatic tissues, with β-OHB constituting\u0026thinsp;~\u0026thinsp;70% of the circulating ketone bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). KMT2D knockdown significantly decreased ketone body levels in both the cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). siRNA-mediated HMGCS2 suppression inhibited LNCaP and C4-2 cell proliferation, while reducing ketone body production (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL; Supplementary Figure S6F\u0026ndash;I). Supplementation with 10 mM β-OHB restored proliferation of KMT2D-deficient cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eO; Supplementary Fig.\u0026nbsp;6J), indicating that KMT2D promotes prostate cancer growth partly through ketone body metabolism. These results established that the KMT2D-FOXA1-AR axis modulates ketogenesis by controlling HMGCS2 expression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Targeting KMT2D-Associated H3K4me1 Methylation Suppresses AR Signaling and Prostate Cancer Growth\u003c/h2\u003e\u003cp\u003eKMT2D operates within the COMPASS complex to establish H3K4me1 marks at enhancers. The complex contains core WRAD subunits (WDR5, RBBP5, ASH2L, and DPY30) as well as KMT2C/D-specific partners, including PTIP, PA1, and H3K27me3 demethylase UTX (KDM6A), which are required for KMT2D's catalytic function and chromatin localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). We systematically evaluated the functional interactions between these components in prostate cancer using multi-dataset correlation analyses. Notably, UTX showed the most robust co-expression and co-dependency with KMT2D in TCGA PRAD samples as well as in the DepMap CRISPR and RNAi screening datasets (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). TCGA cohort analysis further revealed that UTX expression was positively associated with AR signaling activity (Supplementary Fig.\u0026nbsp;7A), suggesting coordinated roles for UTX and KMT2D in modulating the AR pathway.\u003c/p\u003e\u003cp\u003eGiven UTX's prominent association with KMT2D, we investigated whether the pharmacological inhibition of UTX demethylase activity with GSK-J4, a selective UTX/JMJD3 inhibitor, affects prostate cancer cell viability through KMT2D-dependent mechanisms. GSK-J4 treatment reduced the viability of both LNCaP and C4-2 cell lines in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC; Supplementary Fig.\u0026nbsp;7B). Correspondingly, mRNA expression levels of KLK3, TMPRSS2, and NKX3.1 were markedly downregulated upon GSK-J4 treatment in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Western blot analyses also demonstrated that GSK-J4 reduced KLK3 protein expression and decreased H3K4me1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003ePCa is known to have a high level of heterogeneity. PDTFs are increasingly being utilized as effective models for studying tumor biology and evaluating therapeutic strategies. Therefore, to validate these findings in a clinically relevant model, we exposed PDTF cells to GSK-J4 under ex vivo conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). The compound markedly reduced KLK3 and TMPRSS2 mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG), mirroring the results from cell line experiments, while simultaneously decreasing KLK3 and H3K4me1 protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). These observations demonstrate that the pharmacological inhibition of UTX/KMT2D-mediated epigenetic regulation effectively disrupts AR signaling in prostate cancer.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.8 KMT2D Promotes Enzalutamide Resistance by Sustaining AR Signaling and Epigenetic Remodeling in Prostate Cancer\u003c/h2\u003e\u003cp\u003eEnzalutamide (Enz) resistance has emerged as a major challenge in prostate cancer treatment. Previous studies have established enzalutamide-resistant cell models and have demonstrated AR reactivation as a hallmark of resistance. To investigate KMT2D\u0026rsquo;s role in the development of enzalutamide resistance, we established an enzalutamide-resistant (EnzR) LNCaP cell model using prolonged drug exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Dose-response assays confirmed that EnzR cells exhibited a markedly higher IC50 for enzalutamide compared to parental LNCaP cells (25.66 \u0026micro;M vs. 3.36 \u0026micro;M), validating the resistant phenotype (Supplementary Fig.\u0026nbsp;7C). qRT-PCR revealed significantly increased levels of KMT2D and AR mRNA in the EnzR sublines relative to parental cells (Supplementary Fig.\u0026nbsp;7D). Compared to parental LNCaP cells, EnzR cells displayed elevated levels of AR and H3K4me1, indicating epigenetic reprogramming associated with resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eCCK-8 assays revealed that KMT2D knockout markedly reduced EnzR cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, left panel) and increased sensitivity to enzalutamide (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, right panel).\u003c/p\u003e\u003cp\u003eWe also calculated AR signature scores using RNA-seq data, revealing significantly elevated AR signaling in EnzR cells relative to that in parental LNCaP cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). KMT2D knockdown in EnzR cells markedly reduced AR pathway activity, establishing its essential function in maintaining AR axis activation in enzalutamide-resistant prostate cancer cells. ATAC-seq was performed in EnzR cells following KMT2D knockdown. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF, ATAC-seq profiling revealed widespread loss of chromatin accessibility upon KMT2D knockdown in EnzR cells. Motif analysis revealed that FOXA1 motifs were significantly enriched in KMT2D-dependent chromatin sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003eWe also treated EnzR cells with GSK-J4 to investigate its effects on cell proliferation and AR signaling pathways. GSK-J4 dose-dependently reduced EnzR cell viability relative to DMSO controls, as shown by the proliferation assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI). This growth inhibition was further supported by the colony formation assays (Supplementary Fig.\u0026nbsp;7E). qRT-PCR analysis revealed that GSK-J4 markedly decreased KLK3, TMPRSS2, and NKX3.1 mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ). Western blotting confirmed reduced KLK3 protein levels and H3K4me1 expression following GSK-J4 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eK). The effects of GSK-J4 were investigated \u003cem\u003ein vivo\u003c/em\u003e. Mice with tumors were treated with vehicle or GSK-J4 for 15 d (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eL). GSK-J4 treatment significantly suppressed tumor growth \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eM-O). Additionally, decreased levels of Ki-67 and H3K4me1 were observed in tumors treated with GSK-J4, further supporting the effect of GSK-J4 on KMT2D function and PCa proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eP).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur findings established KMT2D as an epigenetic oncogene that drives prostate cancer proliferation and progression in both cellular and animal models. KMT2D exerts its oncogenic effects by modulating androgen receptor transcriptional activity through downstream signaling pathways. Integrated multiomics analyses combining RNA-seq, ChIP-seq, and ATAC-seq revealed that KMT2D enhances chromatin accessibility, facilitating AR and FOXA1 binding to the regulatory elements of androgen-responsive genes. We further identified the KMT2D-FOXA1-AR axis as a novel regulator of ketogenesis via HMGCS2 expression control. While no direct KMT2D inhibitors exist, pharmacological targeting of the COMPASS complex with GSK-J4 inhibits prostate cancer growth by disrupting KMT2D-mediated H3K4me1 modification.\u003c/p\u003e\u003cp\u003eEpigenetic dysregulation plays a critical role in tumorigenesis, particularly cancer progression, invasion, metastasis, and drug resistance \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Among the various epigenetic alterations, aberrant histone modifications characterize multiple malignancies, with histone methylation emerging as a key modulator of chromatin dynamics and transcriptional regulation \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Lysine methyltransferases (KMTs) and demethylases (KDMs) predominantly govern this methylation process \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. As a member of this family, KMT2D (MLL4) mediates H3K4 monomethylation to epigenetically control gene expression across diverse signaling cascades. In AR-negative prostate cancer (PCa), KMT2D drives tumor progression and metastasis through upregulation of LIFR and KLF4, subsequently activating the PI3K/Akt pathway \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Additionally, KMT2D deficiency in AR-negative PCa triggers ROS-dependent DNA damage by disrupting FOXO3-mediated antioxidant responses, ultimately inducing apoptosis and suppressing tumor growth \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. However, the functional consequences of KMT2D activity are highly context-dependent across cancer types \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Genetic studies in mouse models reveal KMT2D's tumor-suppressive role in lymphoma \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, medulloblastoma \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, melanoma \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, and lung adenocarcinoma (LUAD) \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, while clinical evidence supports its oncogenic function in breast \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, esophageal \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, and gastric cancers \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. This functional dichotomy underscores the tissue-specific nature of KMT2D activity in carcinogenesis, warranting further investigations in other malignancies. Our current findings establish that KMT2D facilitates AR-positive PCa progression through the FOXA1-dependent epigenetic activation of AR signaling.\u003c/p\u003e\u003cp\u003eIn CRPC, tumor progression is driven by AR reactivation via multiple mechanisms. Current therapies targeting the AR-ligand axis, including potent AR antagonists (enzalutamide, apalutamide, and darolutamide) and intratumoral androgen synthesis inhibitors (abiraterone), often fail because of acquired resistance mediated by AR alterations \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, these approaches are limited by the resistance mechanisms that emerge as a result of AR alterations. Aberrant alterations in the AR have been reported in up to 58.78% of patients with CRPC \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Therefore, targeting molecular pathways beyond the AR to disrupt AR signaling has emerged as a promising strategy for mitigating AR reactivation in CRPC. Emerging evidence implicates epigenetic dysregulation in PCa progression, with several epigenetic-targeting therapies demonstrating clinical potential \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In this study, we established KMT2D as a regulator of AR recruitment and transcriptional activity in CRPC, contributing to AR reactivation. LSD1 (KDM1A), another epigenetic modulator, demethylates H3K4me1/2 while functioning as an AR coactivator through its interaction with the CoREST complex \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Furthermore, LSD1 converts H3K4me2 to H3K4me1 at enhancers of AR-stimulated genes \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, suggesting that KMT2D and LSD1 cooperatively regulate AR transcriptional activity through this active enhancer mark, underscoring its broad function as an epigenetic regulator of nuclear receptor signaling \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe transcription factor forkhead box A1 (FOXA1) induces an open chromatin conformation, enabling lineage-specific transcription factors such as AR to bind and drive prostate cancer (PCa) growth, survival, and drug resistance \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. FOXA1 chromatin binding is enhanced by reduced DNA methylation or increased levels of histone methylation markers, notably H3K4me1 and H3K4me2. We found that FOXA1 recruitment to AR-specific enhancer sites depends on KMT2D-mediated H3K4me1, establishing an open chromatin state that facilitates AR binding and activation and ultimately accelerates PCa progression. Consistent with our findings, recent studies have shown that FOXA1 is one of the most frequently mutated genes in PCa \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Our experiments revealed that FOXA1 forkhead-domain mutants (D226G and M253K) exhibited diminished binding to AR enhancer sites. KMT2D knockout did not compromise FOXA1 binding, suggesting that KMT2D primarily regulates AR activity via FOXA1. However, the precise mechanisms governing the regulation of FOXA1 activity remain unclear. Although FOXA1 inhibition attenuates AR transcriptional activity and curbs PCa cell proliferation, it paradoxically enhances tumor invasiveness, complicating its therapeutic targeting \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. LSD1 inhibition markedly impairs PCa growth and progression \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, implying that KMT2D suppression effectively disrupts the FOXA1-AR axis in castration-resistant PCa.\u003c/p\u003e\u003cp\u003eMetabolic reprogramming is a hallmark of cancer and contributes to the initiation, progression, metastasis, and drug resistance \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Ketogenesis in cancer cells has been reported to suppress or enhance cell growth and proliferation \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Studies show that KMT2D regulates tumorigenesis and progression through metabolic reprogramming in pancreatic cancer \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, lung cancer \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, and melanoma\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e by regulating glycolysis. However, the role of KMT2D in the ketone body metabolism remains unclear. This study showed that the expression of 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 2 (HMGCS2), the key rate-limiting enzyme involved in ketogenesis, is regulated by the KMT2D-FOXA1-AR axis, which facilitates PCa proliferation and progression. This indicates that metabolic reprogramming of ketone bodies plays an oncogenic role in PCa. In contrast, another study reported that HMGCS2 acts as a tumor suppressor in PCa and that a ketogenic diet or calorie-restricted diet can inhibit PCa growth \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. However, consistent with our results, Labanca et al. demonstrated that relapsed PCa after ADT shows higher ketone body levels, along with elevated levels of key ketone catabolic enzymes \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In addition, Punit et al. revealed that HMGCS2 is highly expressed in high-grade PCa and an androgen-independent PCa cell line, suggesting that it could serve as a diagnostic or prognostic marker \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, further supporting our results. Collectively, KMT2D was identified as an epigenetic modulator involved in the upregulation of HMGCS2 expression, thereby driving ketone metabolic reprogramming to promote PCa progression.\u003c/p\u003e\u003cp\u003eKMT2D functions as a catalytic subunit within the mammalian complex of proteins associated with SET1 (COMPASS) and mediates H3K4me1 and H3K4me3. UTX, also known as KDM6A, is a H3K27-specific histone demethylase and a component of COMPASS. Studies have shown that the function of UTX depends on its association with COMPASS and that the interaction between KMT2D and other components of the COMPASS complex does not rely on its enzymatic domain \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. However, the effect of UTX deficiency on KMT2D function remains unclear. This study showed that GSK-J4, a UTX inhibitor, significantly suppressed the proliferation of PCa cells. Mechanistically, GSK-J4 reduced the expression of H3K4me1 and AR signaling pathway genes including KLK3, TMPRSS2, and NKX3.1. These findings indicate that inhibition of UTX by GSK-J4 impairs the catalytic activity of KMT2D. Consistent with our findings, the loss of KMT2D induces brain metastasis in triple-negative breast cancer (TNBC). However, this effect can be mitigated by blocking UTX, suggesting that the function of KMT2D may depend on UTX \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Wang et al. reported that UTX inhibition significantly impaired KMT2D function by disrupting its cooperative relationship with p300. Mechanistically, UTX facilitated the recruitment of p300, enhancing H3K27 acetylation, a process necessary for MLL4-mediated H3K4me1 deposition. In the absence of UTX, this synergy is disrupted, resulting in reduced enhancer activity and impaired transcriptional activation \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Additionally, Shi et al. reported that UTX facilitates the localization of histone methyltransferase KMT2D to the same condensates, enhancing KMT2D H3K4 methylation activity \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. These studies further support our findings that UTX inhibition impairs KMT2D function in PCa cells.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eOur study established KMT2D as a pivotal epigenetic regulator that promotes prostate cancer progression through FOXA1-mediated enhancement of AR signaling and metabolic reprogramming. The KMT2D-FOXA1-AR axis facilitates chromatin remodeling and ketogenesis, thereby driving tumor growth. Targeting UTX effectively impaired KMT2D enzymatic activity and suppressed AR signaling and PCa cell proliferation. These insights highlight the therapeutic potential of disrupting the KMT2D-associated epigenetic mechanisms in advanced prostate cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eADT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAndrogen Deprivation Therapy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAndrogen Receptor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eARE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAndrogen Response Element\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eATAC-seq\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAssay for Transposase-Accessible Chromatin with sequencing\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eChIP-seq\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eChromatin Immunoprecipitation sequencing\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCOMPASS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eComplex of Proteins Associated with Set1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCRPC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCastration-Resistant Prostate Cancer\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCSS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCharcoal-Stripped Serum\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCUT\u0026amp;Tag\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCleavage Under Targets and Tagmentation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDHT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDihydrotestosterone\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEdU\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e5-Ethynyl-2\u0026prime;-deoxyuridine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEnzR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEnzalutamide-Resistant\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eER\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEstrogen Receptor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFKHD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eForkhead DNA-binding domain\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFOXA1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eForkhead Box A1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGSEA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGene Set Enrichment Analysis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGSVA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGene Set Variation Analysis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eH3K4me1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHistone H3 lysine 4 monomethylation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHMGCS2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e3-Hydroxy-3-Methylglutaryl-CoA Synthase 2\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eImmunohistochemistry\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKMT2D\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eLysine Methyltransferase 2D\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePCa\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eProstate Cancer\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePDTF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePatient-Derived Tumor Fragment\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eqRT-PCR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eQuantitative Real-Time Polymerase Chain Reaction\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRNA-seq\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRNA sequencing\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003esiRNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSmall Interfering RNA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTCGA-PRAD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eThe Cancer Genome Atlas-Prostate Adenocarcinoma\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eUTX\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eUbiquitously Transcribed Tetratricopeptide Repeat, X Chromosome\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of Interest\u003c/h2\u003e\u003cp\u003eThe authors declare no potential conflicts of interest.\u003c/p\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eAuthor Contribution Statement\u003c/h2\u003e\u003cp\u003eM.Y. Luo, C.W. Wu, M.L. Zhou, and Y.D. Li designed and developed the methodology. Q. Wei, S.D. Lv, and C.C. Du conducted the investigations. M.Y. Luo, Y.F. Zhang, and Y.P. Liao performed data visualization. Q. Wei and S.D. Lv supervised the project. M.Y. Luo drafted the manuscript. Q. Wei and S.D. Lv acquired funding, provided resources, and contributed to supervision. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConsent for publication\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthic approval and consent to participate\u003c/h2\u003e\u003cp\u003e The mouse experiments were approved by the Ethics Committee of Ganzhou Hospital, Southern Medical University, Southern Hospital (Approval No.TY-DKY2023-020-01). The PDTF collection was approved by the Ethics Committee of Nanfang Hospital (NFEC-2022-299).\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was funded in part by the Bethune Oncology Basic Research Program (J202101E005, Qiang Wei), Ganpo Talent Support Program - Major Discipline Academic and Technological Leader Training Project (20232BCJ22018, Qiang Wei), Ganzhou Municipal Science and Technology Project (2022\u0026ndash;RC1341, Qiang Wei), Natural Science Foundation of Jiangxi Province (20224ACB206007, Qiang Wei), National Natural Science Foundation of China (82472756, 82103276, to Shidong Lv), and Natural Science Foundation of Guangdong Province (2023A1515010321, to Qiang Wei; 2024A1515010331, to Shidong Lv).\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe ChIP-seq, ATAC-seq, CUT\u0026amp;Tag, and RNA-seq datasets generated in this study are available in the GEO database under accession numbers GSE285691, GSE285692, and GSE285790, respectively, with public release upon publication. We also analyzed external datasets from the DepMap database, single-cell RNA-seq data (GSE181294), and TCGA-PRAD cohorts, all of which were accessible through their original repositories.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbudureheman A, Ainiwaer J, Hou Z, Niyaz M, Turghun A, Hasim A \u003cem\u003eet al\u003c/em\u003e. High MLL2 expression predicts poor prognosis and promotes tumor progression by inducing EMT in esophageal squamous cell carcinoma. J Cancer Res Clin Oncol 2018; 144: 1025\u0026ndash;1035.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhmad F, Cherukuri MK, Choyke PL. Metabolic reprogramming in prostate cancer. Br J Cancer 2021; 125: 1185\u0026ndash;1196.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlam H, Tang M, Maitituoheti M, Dhar SS, Kumar M, Han CY \u003cem\u003eet al\u003c/em\u003e. KMT2D Deficiency Impairs Super-Enhancers to Confer a Glycolytic Vulnerability in Lung Cancer. Cancer Cell 2020; 37: 599\u0026ndash;617 e597.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAng SY, Uebersohn A, Spencer CI, Huang Y, Lee JE, Ge K \u003cem\u003eet al\u003c/em\u003e. KMT2D regulates specific programs in heart development via histone H3 lysine 4 di-methylation. Development 2016; 143: 810\u0026ndash;821.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBlack JC, Van Rechem C, Whetstine JR. Histone lysine methylation dynamics: establishment, regulation, and biological impact. Mol Cell 2012; 48: 491\u0026ndash;507.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCai C, He HH, Gao S, Chen S, Yu Z, Gao Y \u003cem\u003eet al\u003c/em\u003e. Lysine-specific demethylase 1 has dual functions as a major regulator of androgen receptor transcriptional activity. Cell Rep 2014; 9: 1618\u0026ndash;1627.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCai M, Song XL, Li XA, Chen M, Guo J, Yang DH \u003cem\u003eet al\u003c/em\u003e. Current therapy and drug resistance in metastatic castration-resistant prostate cancer. Drug Resist Updat 2023; 68: 100962.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChandrasekar T, Yang JC, Gao AC, Evans CP. Mechanisms of resistance in castration-resistant prostate cancer (CRPC). Transl Androl Urol 2015; 4: 365\u0026ndash;380.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCheng Y, He C, Wang M, Ma X, Mo F, Yang S \u003cem\u003eet al\u003c/em\u003e. Targeting epigenetic regulators for cancer therapy: mechanisms and advances in clinical trials. Signal Transduct Target Ther 2019; 4: 62.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCocciadiferro D, Augello B, De Nittis P, Zhang J, Mandriani B, Malerba N \u003cem\u003eet al\u003c/em\u003e. Dissecting KMT2D missense mutations in Kabuki syndrome patients. Hum Mol Genet 2018; 27: 3651\u0026ndash;3668.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eConteduca V, Hess J, Yamada Y, Ku SY, Beltran H. Epigenetics in prostate cancer: clinical implications. Transl Androl Urol 2021; 10: 3104\u0026ndash;3116.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCucchiara V, Yang JC, Mirone V, Gao AC, Rosenfeld MG, Evans CP. Epigenomic Regulation of Androgen Receptor Signaling: Potential Role in Prostate Cancer Therapy. Cancers (Basel) 2017; 9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDai W, Qiao X, Fang Y, Guo R, Bai P, Liu S \u003cem\u003eet al\u003c/em\u003e. Epigenetics-targeted drugs: current paradigms and future challenges. Signal Transduct Target Ther 2024; 9: 332.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDhar SS, Lee MG. Cancer-epigenetic function of the histone methyltransferase KMT2D and therapeutic opportunities for the treatment of KMT2D-deficient tumors. Oncotarget 2021; 12: 1296\u0026ndash;1308.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFroimchuk E, Jang Y, Ge K. Histone H3 lysine 4 methyltransferase KMT2D. Gene 2017; 627: 337\u0026ndash;342.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFujita K, Nonomura N. Role of Androgen Receptor in Prostate Cancer: A Review. World J Mens Health 2019; 37: 288\u0026ndash;295.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGao S, Chen S, Han D, Barrett D, Han W, Ahmed M \u003cem\u003eet al\u003c/em\u003e. Forkhead domain mutations in FOXA1 drive prostate cancer progression. Cell Res 2019; 29: 770\u0026ndash;772.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGao S, Chen S, Han D, Wang Z, Li M, Han W \u003cem\u003eet al\u003c/em\u003e. Chromatin binding of FOXA1 is promoted by LSD1-mediated demethylation in prostate cancer. Nat Genet 2020; 52: 1011\u0026ndash;1017.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHwang CY, Choe W, Yoon KS, Ha J, Kim SS, Yeo EJ \u003cem\u003eet al\u003c/em\u003e. Molecular Mechanisms for Ketone Body Metabolism, Signaling Functions, and Therapeutic Potential in Cancer. Nutrients 2022; 14.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKoutsioumpa M, Hatziapostolou M, Polytarchou C, Tolosa EJ, Almada LL, Mahurkar-Joshi S \u003cem\u003eet al\u003c/em\u003e. Lysine methyltransferase 2D regulates pancreatic carcinogenesis through metabolic reprogramming. Gut 2019; 68: 1271\u0026ndash;1286.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLabanca E, Bizzotto J, Sanchis P, Anselmino N, Yang J, Shepherd PDA \u003cem\u003eet al\u003c/em\u003e. Prostate cancer castrate resistant progression usage of non-canonical androgen receptor signaling and ketone body fuel. Oncogene 2021; 40: 6284\u0026ndash;6298.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi M, Shi M, Xu Y, Qiu J, Lv Q. Histone Methyltransferase KMT2D Regulates H3K4 Methylation and is Involved in the Pathogenesis of Ovarian Cancer. Cell Transplant 2021; 30: 9636897211027521.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi M, Liu M, Han W, Wang Z, Han D, Patalano S \u003cem\u003eet al\u003c/em\u003e. LSD1 Inhibition Disrupts Super-Enhancer-Driven Oncogenic Transcriptional Programs in Castration-Resistant Prostate Cancer. Cancer Res 2023; 83: 1684\u0026ndash;1698.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Q, Wu R, Wu F, Chen Q. KMT2D promotes proliferation of gastric cancer cells: evidence from ctDNA sequencing. J Clin Lab Anal 2021; 35: e23721.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLv S, Ji L, Chen B, Liu S, Lei C, Liu X \u003cem\u003eet al\u003c/em\u003e. Histone methyltransferase KMT2D sustains prostate carcinogenesis and metastasis via epigenetically activating LIFR and KLF4. Oncogene 2018; 37: 1354\u0026ndash;1368.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLv S, Wen H, Shan X, Li J, Wu Y, Yu X \u003cem\u003eet al\u003c/em\u003e. Loss of KMT2D induces prostate cancer ROS-mediated DNA damage by suppressing the enhancer activity and DNA binding of antioxidant transcription factor FOXO3. Epigenetics 2019; 14: 1194\u0026ndash;1208.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaitituoheti M, Keung EZ, Tang M, Yan L, Alam H, Han G \u003cem\u003eet al\u003c/em\u003e. Enhancer Reprogramming Confers Dependence on Glycolysis and IGF Signaling in KMT2D Mutant Melanoma. Cell Rep 2020; 33: 108293.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOswald F, Rodriguez P, Giaimo BD, Antonello ZA, Mira L, Mittler G \u003cem\u003eet al\u003c/em\u003e. A phospho-dependent mechanism involving NCoR and KMT2D controls a permissive chromatin state at Notch target genes. Nucleic Acids Res 2016; 44: 4703\u0026ndash;4720.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePan Y, Han H, Hu H, Wang H, Song Y, Hao Y \u003cem\u003eet al\u003c/em\u003e. KMT2D deficiency drives lung squamous cell carcinoma and hypersensitivity to RTK-RAS inhibition. Cancer Cell 2023; 41: 88\u0026ndash;105 e108.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePang L, Tian H, Gao X, Wang W, Wang X, Zhang Z. KMT2D deficiency disturbs the proliferation and cell cycle activity of dental epithelial cell line (LS8) partially via Wnt signaling. Biosci Rep 2021; 41.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eParolia A, Eyunni S, Verma BK, Young E, Liu Y, Liu L \u003cem\u003eet al\u003c/em\u003e. NSD2 is a requisite subunit of the AR/FOXA1 neo-enhanceosome in promoting prostate tumorigenesis. Nat Genet 2024; 56: 2132\u0026ndash;2143.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRickels R, Wang L, Iwanaszko M, Ozark PA, Morgan MA, Piunti A \u003cem\u003eet al\u003c/em\u003e. A small UTX stabilization domain of Trr is conserved within mammalian MLL3-4/COMPASS and is sufficient to rescue loss of viability in null animals. Genes Dev 2020; 34: 1493\u0026ndash;1502.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaffie R, Zhou N, Rolland D, Onder O, Basrur V, Campbell S \u003cem\u003eet al\u003c/em\u003e. FBXW7 Triggers Degradation of KMT2D to Favor Growth of Diffuse Large B-cell Lymphoma Cells. Cancer Res 2020; 80: 2498\u0026ndash;2511.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSanghrajka RM, Koche R, Medrano H, El Nagar S, Stephen DN, Lao Z \u003cem\u003eet al\u003c/em\u003e. KMT2D suppresses Sonic hedgehog-driven medulloblastoma progression and metastasis. iScience 2023; 26: 107831.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaraon P, Cretu D, Musrap N, Karagiannis GS, Batruch I, Drabovich AP \u003cem\u003eet al\u003c/em\u003e. Quantitative proteomics reveals that enzymes of the ketogenic pathway are associated with prostate cancer progression. Mol Cell Proteomics 2013; 12: 1589\u0026ndash;1601.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSeehawer M, Li Z, Nishida J, Foidart P, Reiter AH, Rojas-Jimenez E \u003cem\u003eet al\u003c/em\u003e. Loss of Kmt2c or Kmt2d drives brain metastasis via KDM6A-dependent upregulation of MMP3. Nat Cell Biol 2024; 26: 1165\u0026ndash;1175.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShi B, Li W, Song Y, Wang Z, Ju R, Ulman A \u003cem\u003eet al\u003c/em\u003e. UTX condensation underlies its tumour-suppressive activity. Nature 2021; 597: 726\u0026ndash;731.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin 2024; 74: 12\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSong B, Park SH, Zhao JC, Fong KW, Li S, Lee Y \u003cem\u003eet al\u003c/em\u003e. Targeting FOXA1-mediated repression of TGF-beta signaling suppresses castration-resistant prostate cancer progression. J Clin Invest 2019; 129: 569\u0026ndash;582.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSugiura M, Sato H, Kanesaka M, Imamura Y, Sakamoto S, Ichikawa T \u003cem\u003eet al\u003c/em\u003e. Epigenetic modifications in prostate cancer. Int J Urol 2021; 28: 140\u0026ndash;149.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSun P, Wu T, Sun X, Cui Z, Zhang H, Xia Q \u003cem\u003eet al\u003c/em\u003e. KMT2D inhibits the growth and metastasis of bladder Cancer cells by maintaining the tumor suppressor genes. Biomed Pharmacother 2019; 115: 108924.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSze CC, Shilatifard A. MLL3/MLL4/COMPASS Family on Epigenetic Regulation of Enhancer Function and Cancer. Cold Spring Harb Perspect Med 2016; 6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTeng M, Zhou S, Cai C, Lupien M, He HH. Pioneer of prostate cancer: past, present and the future of FOXA1. Protein Cell 2021; 12: 29\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eToska E, Osmanbeyoglu HU, Castel P, Chan C, Hendrickson RC, Elkabets M \u003cem\u003eet al\u003c/em\u003e. PI3K pathway regulates ER-dependent transcription in breast cancer through the epigenetic regulator KMT2D. Science 2017; 355: 1324\u0026ndash;1330.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVoabil P, de Bruijn M, Roelofsen LM, Hendriks SH, Brokamp S, van den Braber M \u003cem\u003eet al\u003c/em\u003e. An ex vivo tumor fragment platform to dissect response to PD-1 blockade in cancer. Nat Med 2021; 27: 1250\u0026ndash;1261.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWan S, Xi M, Zhao HB, Hua W, Liu YL, Zhou YL \u003cem\u003eet al\u003c/em\u003e. HMGCS2 functions as a tumor suppressor and has a prognostic impact in prostate cancer. Pathol Res Pract 2019; 215: 152464.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang SP, Tang Z, Chen CW, Shimada M, Koche RP, Wang LH \u003cem\u003eet al\u003c/em\u003e. A UTX-MLL4-p300 Transcriptional Regulatory Network Coordinately Shapes Active Enhancer Landscapes for Eliciting Transcription. Mol Cell 2017; 67: 308\u0026ndash;321 e306.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWeber DD, Aminzadeh-Gohari S, Tulipan J, Catalano L, Feichtinger RG, Kofler B. Ketogenic diet in the treatment of cancer - Where do we stand? Mol Metab 2020; 33: 102\u0026ndash;121.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"prostate cancer, androgen receptor, KMT2D, FOXA1","lastPublishedDoi":"10.21203/rs.3.rs-7812403/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7812403/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProstate cancer (PCa) progression, particularly to castration-resistant prostate cancer (CRPC), is driven by androgen receptor (AR) reactivation and epigenetic alterations. Here, we identify lysine methyltransferase 2D (KMT2D) as a critical epigenetic oncogene in PCa. KMT2D expression is elevated in PCa and correlates with poor prognosis. Mechanistically, KMT2D facilitates AR signaling by recruiting the pioneer factor FOXA1 to AR-specific enhancers, promoting chromatin accessibility and activating AR target genes. FOXA1 mutations impair this regulation, demonstrating their functional interplay. Furthermore, KMT2D-FOXA1-AR axis modulates ketone body metabolism via transcriptional control of HMGCS2, supporting tumor growth. Pharmacological inhibition of UTX, a COMPASS complex demethylase essential for KMT2D function, disrupts H3K4me1 deposition and suppresses AR signaling and tumor proliferation. Altogether, we characterize KMT2D as a key driver of AR-dependent PCa progression and propose UTX inhibition as a promising therapeutic strategy.\u003c/p\u003e","manuscriptTitle":"Histone Methyltransferase KMT2D Promotes Castration-Resistant Prostate Cancer Progression by Reactivating AR through FOXA1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-07 06:33:36","doi":"10.21203/rs.3.rs-7812403/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-11-20T16:25:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-11-10T21:26:30+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-11-07T15:52:40+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-27T18:58:43+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-27T18:19:49+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-10-27T17:54:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-09T13:01:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-09T03:09:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oncogene","date":"2025-10-09T03:09:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"af5954bf-9a42-4bc8-94ff-7dcd9bda7ec0","owner":[],"postedDate":"November 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56030532,"name":"Biological sciences/Genetics/Epigenetics"},{"id":56030533,"name":"Biological sciences/Cancer/Urological cancer/Prostate cancer"},{"id":56030534,"name":"Biological sciences/Cancer/Cancer metabolism"}],"tags":[],"updatedAt":"2026-04-16T12:21:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-07 06:33:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7812403","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7812403","identity":"rs-7812403","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}