Lysine Methyltransferase 9 (KMT9) Is an Actionable Target in Muscle-Invasive Bladder Cancer.

Novel treatment modalities are imperative for the challenging management of muscle-invasive and metastatic BC to improve patient survival rates. The recently identified KMT9, an obligate heterodimer composed of KMT9α and KMT9ß, regulates the growth of various types of tumors such as prostate, lung, and colon cancer. While the overexpression of KMT9α was previously observed to be associated with aggressive basal-like MIBC in an analysis of patients' tissue samples, a potential functional role of KMT9 in this type of cancer has not been investigated to date. In this study, we show that KMT9 regulates proliferation, migration, and invasion of various MIBC cell lines with different genetic mutations. KMT9α depletion results in the differential expression of genes regulating the cell cycle, cell adhesion, and migration. Differentially expressed genes include oncogenes such as EGFR and AKT1 as well as mediators of cell adhesion or migration such as DAG1 and ITGA6. Reduced cell proliferation upon KMT9α depletion is also observed in Pten/Trp53 knockout bladder tumor organoids, which cannot be rescued with an enzymatically inactive KMT9α mutant. In accordance with the idea that the catalytic activity of KMT9 is required for the control of cellular processes in MIBC, a recently developed small-molecule inhibitor of KMT9 (KMI169) also impairs cancer cell proliferation. Since KMT9α depletion also restricts the growth of xenografts in mice, our data suggest that KMT9 is an actionable novel therapeutic target for the treatment of MIBC.

Discovery of a Potent, Selective, and Cell-Active SPIN1 Inhibitor.

The methyl-lysine reader protein SPIN1 plays important roles in various human diseases. However, targeting methyl-lysine reader proteins has been challenging. Very few cellularly active SPIN1 inhibitors have been developed. We previously reported that our G9a/GLP inhibitor UNC0638 weakly inhibited SPIN1. Here, we present our comprehensive structure-activity relationship study that led to the discovery of compound 11, a dual SPIN1 and G9a/GLP inhibitor, and compound 18 (MS8535), a SPIN1 selective inhibitor. We solved the cocrystal structure of SPIN1 in complex with 11, confirming that 11 occupied one of the three Tudor domains. Importantly, 18 displayed high selectivity for SPIN1 over 38 epigenetic targets, including G9a/GLP, and concentration dependently disrupted the interactions of SPIN1 and H3 in cells. Furthermore, 18 was bioavailable in mice. We also developed 19 (MS8535N), which was inactive against SPIN1, as a negative control of 18. Collectively, these compounds are useful chemical tools to study biological functions of SPIN1.

Structure-guided design of a selective inhibitor of the methyltransferase KMT9 with cellular activity.

Inhibition of epigenetic regulators by small molecules is an attractive strategy for cancer treatment. Recently, we characterised the role of lysine methyltransferase 9 (KMT9) in prostate, lung, and colon cancer. Our observation that the enzymatic activity was required for tumour cell proliferation identified KMT9 as a potential therapeutic target. Here, we report the development of a potent and selective KMT9 inhibitor (compound 4, KMI169) with cellular activity through structure-based drug design. KMI169 functions as a bi-substrate inhibitor targeting the SAM and substrate binding pockets of KMT9 and exhibits high potency, selectivity, and cellular target engagement. KMT9 inhibition selectively downregulates target genes involved in cell cycle regulation and impairs proliferation of tumours cells including castration- and enzalutamide-resistant prostate cancer cells. KMI169 represents a valuable tool to probe cellular KMT9 functions and paves the way for the development of clinical candidate inhibitors as therapeutic options to treat malignancies such as therapy-resistant prostate cancer.

Quinoline-based thiazolyl-hydrazones target cancer cells through autophagy inhibition.

Heterocyclic pharmacophores such as thiazole and quinoline rings have a significant role in medicinal chemistry. They are considered privileged structures since they constitute several Food and Drug Administration (FDA)-approved drugs for cancer treatment. Herein, we report the synthesis, in silico evaluation of the ADMET profiles, and in vitro investigation of the anticancer activity of a series of novel thiazolyl-hydrazones based on the 8-quinoline (1a-c), 2-quinoline (2a-c), and 8-hydroxy-2-quinolyl moiety (3a-c). The panel of several human cancer cell lines and the nontumorigenic human embryonic kidney cell line HEK-293 were used to evaluate the compound-mediated in vitro anticancer activities, leading to [2-(2-(quinolyl-8-ol-2-ylmethylene)hydrazinyl)]-4-(4-methoxyphenyl)-1,3-thiazole (3c) as the most promising compound. The study revealed that 3c blocks the cell-cycle progression of a human colon cancer cell line (HCT-116) in the S phase and induces DNA double-strand breaks. Also, our findings demonstrate that 3c accumulates in lysosomes, ultimately leading to the cell death of the hepatocellular carcinoma cell line (Hep-G2) and HCT-116 cells, by the mechanism of autophagy inhibition.

Four-and-a-Half LIM-Domain Protein 2 (FHL2) Induces Neuropeptide Y (NPY) in Macrophages in Visceral Adipose Tissue and Promotes Diet-Induced Obesity.

Obesity is characterized by the expansion of the adipose tissue, usually accompanied by inflammation, with a prominent role of macrophages infiltrating the visceral adipose tissue (VAT). This chronic inflammation is a major driver of obesity-associated comorbidities. Four-and-a-half LIM-domain protein 2 (FHL2) is a multifunctional adaptor protein that is involved in the regulation of various biological functions and the maintenance of the homeostasis of different tissues. In this study, we aimed to gain new insights into the expression and functional role of FHL2 in VAT in diet-induced obesity. We found enhanced FHL2 expression in the VAT of mice with Western-type diet (WTD)-induced obesity and obese humans and identified macrophages as the cellular source of enhanced FHL2 expression in VAT. In mice with FHL2 deficiency (FHL2KO), WTD feeding resulted in reduced body weight gain paralleled by enhanced energy expenditure and uncoupling protein 1 (UCP1) expression, indicative of activated thermogenesis. In human VAT, FHL2 was inversely correlated with UCP1 expression. Furthermore, macrophage infiltration and the expression of the chemokine MCP-1, a known promotor of macrophage accumulation, was significantly reduced in WTD-fed FHL2KO mice compared with wild-type (wt) littermates. While FHL2 depletion did not affect the differentiation or lipid metabolism of adipocytes in vitro, FHL2 depletion in macrophages resulted in reduced expressions of MCP-1 and the neuropeptide Y (NPY). Furthermore, WTD-fed FHL2KO mice showed reduced NPY expression in VAT compared with wt littermates, and NPY expression was enhanced in VAT resident macrophages of obese individuals. Stimulation with recombinant NPY induced not only UCP1 expression and lipid accumulation but also MCP-1 expression in adipocytes. Collectively, these findings indicate that FHL2 is a positive regulator of NPY and MCP-1 expression in macrophages and herewith closely linked to the mechanism of obesity-associated lipid accumulation and inflammation in VAT. Thus, FHL2 appears as a potential novel target to interfere with the macrophage-adipocyte crosstalk in VAT for treating obesity and related metabolic disorders.

Overexpression of KMT9α Is Associated with Aggressive Basal-like Muscle-Invasive Bladder Cancer.

Muscle-invasive bladder cancer (MIBC) is associated with limited response rates to systemic therapy leading to a significant risk of recurrence and death. A recently discovered histone methyltransferase KMT9, acts as an epigenetic regulator of carcinogenesis in different tumor entities. In this study, we investigated the presence and association of histological and molecular subtypes and their impact on the survival of KMT9α in MIBC. We performed an immunohistochemical (IHC) analysis of KMT9α in 135 MIBC patients undergoing radical cystectomy. KMT9α was significantly overexpressed in the nucleus in MIBC compared to normal urothelium and low-grade urothelial cancer. Using the HTG transcriptome panel, we assessed mRNA expression profiles to determine molecular subtypes and identify differentially expressed genes. Patients with higher nuclear and nucleolar KMT9α expression showed basal/squamous urothelial cancer characteristics confirmed by IHC and differentially upregulated KRT14 expression. We identified a subset of patients with nucleolar expression of KMT9α, which was associated with an increased risk of death in uni- and multivariate analyses (HR 2.28, 95%CI 1.28-4.03, p = 0.005). In conclusion, basal-like MIBC and the squamous histological subtype are associated with high nuclear KMT9α expression. The association with poor survival makes it a potential target for the treatment of bladder cancer.

Nutrient regulation of the islet epigenome controls adaptive insulin secretion.

Adaptation of the islet ß cell insulin-secretory response to changing insulin demand is critical for blood glucose homeostasis, yet the mechanisms underlying this adaptation are unknown. Here, we have shown that nutrient-stimulated histone acetylation plays a key role in adapting insulin secretion through regulation of genes involved in ß cell nutrient sensing and metabolism. Nutrient regulation of the epigenome occurred at sites occupied by the chromatin-modifying enzyme lysine-specific demethylase 1 (Lsd1) in islets. ß Cell-specific deletion of Lsd1 led to insulin hypersecretion, aberrant expression of nutrient-response genes, and histone hyperacetylation. Islets from mice adapted to chronically increased insulin demand exhibited shared epigenetic and transcriptional changes. Moreover, we found that genetic variants associated with type 2 diabetes were enriched at LSD1-bound sites in human islets, suggesting that interpretation of nutrient signals is genetically determined and clinically relevant. Overall, these studies revealed that adaptive insulin secretion involves Lsd1-mediated coupling of nutrient state to regulation of the islet epigenome.

Focal structural variants revealed by whole genome sequencing disrupt the histone demethylase KDM4C in B-cell lymphomas.

Histone methylation-modifiers, such as EZH2 and KMT2D, are recurrently altered in B-cell lymphomas. To comprehensively describe the landscape of alterations affecting genes encoding histone methylation-modifiers in lymphomagenesis we investigated whole genome and transcriptome data of 186 mature B-cell lymphomas sequenced in the ICGC MMML-Seq project. Besides confirming common alterations of KMT2D (47% of cases), EZH2 (17%), SETD1B (5%), PRDM9 (4%), KMT2C (4%), and SETD2 (4%), also identified by prior exome or RNA-sequencing studies, we here found recurrent alterations to KDM4C in chromosome 9p24, encoding a histone demethylase. Focal structural variation was the main mechanism of KDM4C alterations, and was independent from 9p24 amplification. We also identified KDM4C alterations in lymphoma cell lines including a focal homozygous deletion in a classical Hodgkin lymphoma cell line. By integrating RNA-sequencing and genome sequencing data we predict that KDM4C structural variants result in loss-offunction. By functional reconstitution studies in cell lines, we provide evidence that KDM4C can act as a tumor suppressor. Thus, we show that identification of structural variants in whole genome sequencing data adds to the comprehensive description of the mutational landscape of lymphomas and, moreover, establish KDM4C as a putative tumor suppressive gene recurrently altered in subsets of B-cell derived lymphomas.

KMT9 Controls Stemness and Growth of Colorectal Cancer.

Colorectal cancer is among the leading causes of cancer-associated deaths worldwide. Treatment failure and tumor recurrence due to survival of therapy-resistant cancer stem/initiating cells represent major clinical issues to overcome. In this study, we identified lysine methyltransferase 9 (KMT9), an obligate heterodimer composed of KMT9α and KMT9ß that monomethylates histone H4 at lysine 12 (H4K12me1), as an important regulator in colorectal tumorigenesis. KMT9α and KMT9ß were overexpressed in colorectal cancer and colocalized with H4K12me1 at promoters of target genes involved in the regulation of proliferation. Ablation of KMT9α drastically reduced colorectal tumorigenesis in mice and prevented the growth of murine as well as human patient-derived tumor organoids. Moreover, loss of KMT9α impaired the maintenance and function of colorectal cancer stem/initiating cells and induced apoptosis specifically in this cellular compartment. Together, these data suggest that KMT9 is an important regulator of colorectal carcinogenesis, identifying KMT9 as a promising therapeutic target for the treatment of colorectal cancer. SIGNIFICANCE: The H4K12 methyltransferase KMT9 regulates tumor cell proliferation and stemness in colorectal cancer, indicating that targeting KMT9 could be a useful approach for preventing and treating this disease.

Analysis of tripartite motif (TRIM) family gene expression in prostate cancer bone metastases.

Tripartite motif (TRIM) family proteins are post-translational protein modifiers with E3-ubiquitin ligase activity, thereby involved in various biological processes. The molecular mechanisms driving prostate cancer (PCa) bone metastasis (BM) are incompletely understood, and targetable genetic alterations are lacking in the majority of cases. Therefore, we aimed to explore the expression and potential functional relevance of 71 TRIM members in bone metastatic PCa. We performed transcriptome analysis of all human TRIM family members and 770 cancer-related genes in 29 localized PCa and 30 PCa BM using Nanostring. KEGG, STRING and Ubibrowser were used for further bioinformatic gene correlation and pathway enrichment analyses. Compared to localized tumors, six TRIMs are under-expressed while nine TRIMs are over-expressed in BM. The differentially expressed TRIM proteins are linked to TNF-, TGFß-, PI3K/AKT- and HIF-1-signaling, and to features such as proteoglycans, platelet activation, adhesion and ECM-interaction based on correlation to cancer-related genes. The identification of TRIM-specific E3-ligase-substrates revealed insight into functional connections to oncogenes, tumor suppressors and cancer-related pathways including androgen receptor- and TGFß signaling, cell cycle regulation and splicing. In summary, this is the first study that comprehensively and systematically characterizes the expression of all TRIM members in PCa BM. Our results describe post-translational protein modification as an important regulatory mechanism of oncogenes, tumor suppressors, and pathway molecules in PCa progression. Therefore, this study may provide evidence for novel therapeutic targets, in particular for the treatment or prevention of BM.

Structure-Based Design, Docking and Binding Free Energy Calculations of A366 Derivatives as Spindlin1 Inhibitors.

The chromatin reader protein Spindlin1 plays an important role in epigenetic regulation, through which it has been linked to several types of malignant tumors. In the current work, we report on the development of novel analogs of the previously published lead inhibitor A366. In an effort to improve the activity and explore the structure-activity relationship (SAR), a series of 21 derivatives was synthesized, tested in vitro, and investigated by means of molecular modeling tools. Docking studies and molecular dynamics (MD) simulations were performed to analyze and rationalize the structural differences responsible for the Spindlin1 activity. The analysis of MD simulations shed light on the important interactions. Our study highlighted the main structural features that are required for Spindlin1 inhibitory activity, which include a positively charged pyrrolidine moiety embedded into the aromatic cage connected via a propyloxy linker to the 2-aminoindole core. Of the latter, the amidine group anchor the compounds into the pocket through salt bridge interactions with Asp184. Different protocols were tested to identify a fast in silico method that could help to discriminate between active and inactive compounds within the A366 series. Rescoring the docking poses with MM-GBSA calculations was successful in this regard. Because A366 is known to be a G9a inhibitor, the most active developed Spindlin1 inhibitors were also tested over G9a and GLP to verify the selectivity profile of the A366 analogs. This resulted in the discovery of diverse selective compounds, among which 1s and 1t showed Spindlin1 activity in the nanomolar range and selectivity over G9a and GLP. Finally, future design hypotheses were suggested based on our findings.

Epigenetics meets GPCR: inhibition of histone H3 methyltransferase (G9a) and histamine H3 receptor for Prader-Willi Syndrome.

The role of epigenetic regulation is in large parts connected to cancer, but additionally, its therapeutic claim in neurological disorders has emerged. Inhibition of histone H3 lysine N-methyltransferase, especially G9a, has been recently shown to restore candidate genes from silenced parental chromosomes in the imprinting disorder Prader-Willi syndrome (PWS). In addition to this epigenetic approach, pitolisant as G-protein coupled histamine H3 receptor (H3R) antagonist has demonstrated promising therapeutic effects for Prader-Willi syndrome. To combine these pioneering principles of drug action, we aimed to identify compounds that combine both activities, guided by the pharmacophore blueprint for both targets. However, pitolisant as selective H3R inverse agonist with FDA and EMA-approval did not show the required inhibition at G9a. Pharmacological characterization of the prominent G9a inhibitor A-366, that is as well an inhibitor of the epigenetic reader protein Spindlin1, revealed its high affinity at H3R while showing subtype selectivity among subsets of the histaminergic and dopaminergic receptor families. This work moves prominent G9a ligands forward as pharmacological tools to prove for a potentially combined, symptomatic and causal, therapy in PWS by bridging the gap between drug development for G-protein coupled receptors and G9a as an epigenetic effector in a multi-targeting approach.

Nitroreductase-Mediated Release of Inhibitors of Lysine-Specific Demethylase 1 (LSD1) from Prodrugs in Transfected Acute Myeloid Leukaemia Cells.

Lysine-specific demethylase 1 (LSD1) has evolved as a promising therapeutic target for cancer treatment, especially in acute myeloid leukaemia (AML). To approach the challenge of site-specific LSD1 inhibition, we developed an enzyme-prodrug system with the bacterial nitroreductase NfsB (NTR) that was expressed in the virally transfected AML cell line THP1-NTR+ . The cellular activity of the NTR was proven with a new luminescent NTR probe. We synthesised a diverse set of nitroaromatic prodrugs that by design do not affect LSD1 and are reduced by the NTR to release an active LSD1 inhibitor. The emerging side products were differentially analysed using negative controls, thereby revealing cytotoxic effects. The 2-nitroimidazolyl prodrug of a potent LSD1 inhibitor emerged as one of the best prodrug candidates with a pronounced selectivity window between wild-type and transfected THP1 cells. Our prodrugs are selectively activated and release the LSD1 inhibitor locally, proving their suitability for future targeting approaches.

Depletion of histone methyltransferase KMT9 inhibits lung cancer cell proliferation by inducing non-apoptotic cell death.

BACKGROUND: Lung cancer is the leading cause of cancer related death worldwide. Over the past 15 years no major improvement of survival rates could be accomplished. The recently discovered histone methyltransferase KMT9 that acts as epigenetic regulator of prostate tumor growth has now raised hopes of enabling new cancer therapies. In this study, we aimed to identify the function of KMT9 in lung cancer. METHODS: We unraveled the KMT9 transcriptome and proteome in A549 lung adenocarcinoma cells using RNA-Seq and mass spectrometry and linked them with functional cell culture, real-time proliferation and flow cytometry assays. RESULTS: We show that KMT9α and -ß subunits of KMT9 are expressed in lung cancer tissue and cell lines. Importantly, high levels of KMT9α correlate with poor patient survival. We identified 460 genes that are deregulated at the RNA and protein level upon knock-down of KMT9α in A549 cells. These genes cluster with proliferation, cell cycle and cell death gene sets as well as with subcellular organelles in gene ontology analysis. Knock-down of KMT9α inhibits lung cancer cell proliferation and induces non-apoptotic cell death in A549 cells. CONCLUSIONS: The novel histone methyltransferase KMT9 is crucial for proliferation and survival of lung cancer cells harboring various mutations. Small molecule inhibitors targeting KMT9 therefore should be further examined as potential milestones in modern epigenetic lung cancer therapy.

Next-generation sequencing reveals a novel role of lysine-specific demethylase 1 in adhesion of rhabdomyosarcoma cells.

Lysine-specific demethylase 1 (LSD1), a histone lysine demethylase with the main specificity for H3K4me2, has been shown to be overexpressed in rhabdomyosarcoma (RMS) tumor samples. However, its role in RMS biology is not yet well understood. Here, we identified a new role of LSD1 in regulating adhesion of RMS cells. Genetic knockdown of LSD1 profoundly suppressed clonogenic growth in a panel of RMS cell lines, whereas LSD1 proved to be largely dispensable for regulating cell death and short-term survival. Combined RNA and ChIP-sequencing performed to analyze RNA expression and histone methylation at promoter regions revealed a gene set enrichment for adhesion-associated terms upon LSD1 knockdown. Consistently, LSD1 knockdown significantly reduced adhesion to untreated surfaces. Importantly, precoating of the plates with the adhesives collagen I or fibronectin rescued this reduced adhesion of LSD1 knockdown cells back to levels of control cells. Using KEGG pathway analysis, we identified 17 differentially expressed genes (DEGs) in LSD1 knockdown cells related to adhesion processes, which were validated by qRT-PCR. Combining RNA and ChIP-sequencing results revealed that, within this set of genes, SPP1, C3AR1, ITGA10 and SERPINE1 also exhibited increased H3K4me2 levels at their promoter regions in LSD1 knockdown compared to control cells. Indeed, LSD1 ChIP experiments confirmed enrichment of LSD1 at their promoter regions, suggesting a direct transcriptional regulation by LSD1. By identifying a new role of LSD1 in the modulation of cell adhesion and clonogenic growth of RMS cells, these findings highlight the importance of LSD1 in RMS.

A Chemical Probe for Tudor Domain Protein Spindlin1 to Investigate Chromatin Function.

Modifications of histone tails, including lysine/arginine methylation, provide the basis of a "chromatin or histone code". Proteins that contain "reader" domains can bind to these modifications and form specific effector complexes, which ultimately mediate chromatin function. The spindlin1 (SPIN1) protein contains three Tudor methyllysine/arginine reader domains and was identified as a putative oncogene and transcriptional coactivator. Here we report a SPIN1 chemical probe inhibitor with low nanomolar in vitro activity, exquisite selectivity on a panel of methyl reader and writer proteins, and with submicromolar cellular activity. X-ray crystallography showed that this Tudor domain chemical probe simultaneously engages Tudor domains 1 and 2 via a bidentate binding mode. Small molecule inhibition and siRNA knockdown of SPIN1, as well as chemoproteomic studies, identified genes which are transcriptionally regulated by SPIN1 in squamous cell carcinoma and suggest that SPIN1 may have a role in cancer related inflammation and/or cancer metastasis.

Discovery of a Potent and Selective Fragment-like Inhibitor of Methyllysine Reader Protein Spindlin 1 (SPIN1).

By screening an epigenetic compound library, we identified that UNC0638, a highly potent inhibitor of the histone methyltransferases G9a and GLP, was a weak inhibitor of SPIN1 (spindlin 1), a methyllysine reader protein. Our optimization of this weak hit resulted in the discovery of a potent, selective, and cell-active SPIN1 inhibitor, compound 3 (MS31). Compound 3 potently inhibited binding of trimethyllysine-containing peptides to SPIN1, displayed high binding affinity, was highly selective for SPIN1 over other epigenetic readers and writers, directly engaged SPIN1 in cells, and was not toxic to nontumorigenic cells. The crystal structure of the SPIN1-compound 3 complex indicated that it selectively binds tudor domain II of SPIN1. We also designed a structurally similar but inactive compound 4 (MS31N) as a negative control. Our results have demonstrated for the first time that potent, selective, and cell-active fragment-like inhibitors can be generated by targeting a single tudor domain.

The Clinically Used Iron Chelator Deferasirox Is an Inhibitor of Epigenetic JumonjiC Domain-Containing Histone Demethylases.

Fe(II)- and 2-oxoglutarate (2OG)-dependent JumonjiC domain-containing histone demethylases (JmjC KDMs) are "epigenetic eraser" enzymes involved in the regulation of gene expression and are emerging drug targets in oncology. We screened a set of clinically used iron chelators and report that they potently inhibit JMJD2A (KDM4A) in vitro. Mode of action investigations revealed that one compound, deferasirox, is a bona fide active site-binding inhibitor as shown by kinetic and spectroscopic studies. Synthesis of derivatives with improved cell permeability resulted in significant upregulation of histone trimethylation and potent cancer cell growth inhibition. Deferasirox was also found to inhibit human 2OG-dependent hypoxia inducible factor prolyl hydroxylase activity. Therapeutic effects of clinically used deferasirox may thus involve transcriptional regulation through 2OG oxygenase inhibition. Deferasirox might provide a useful starting point for the development of novel anticancer drugs targeting 2OG oxygenases and a valuable tool compound for investigations of KDM function.

KMT9 monomethylates histone H4 lysine 12 and controls proliferation of prostate cancer cells.

Histone lysine methylation is generally performed by SET domain methyltransferases and regulates chromatin structure and gene expression. Here, we identify human C21orf127 (HEMK2, N6AMT1, PrmC), a member of the seven-ß-strand family of putative methyltransferases, as a novel histone lysine methyltransferase. C21orf127 functions as an obligate heterodimer with TRMT112, writing the methylation mark on lysine 12 of histone H4 (H4K12) in vitro and in vivo. We characterized H4K12 recognition by solving the crystal structure of human C21orf127-TRMT112, hereafter termed 'lysine methyltransferase 9' (KMT9), in complex with S-adenosyl-homocysteine and H4K12me1 peptide. Additional analyses revealed enrichment for KMT9 and H4K12me1 at the promoters of numerous genes encoding cell cycle regulators and control of cell cycle progression by KMT9. Importantly, KMT9 depletion severely affects the proliferation of androgen receptor-dependent, as well as that of castration- and enzalutamide-resistant prostate cancer cells and xenograft tumors. Our data link H4K12 methylation with KMT9-dependent regulation of androgen-independent prostate tumor cell proliferation, thereby providing a promising paradigm for the treatment of castration-resistant prostate cancer.