Targeting KDM4A epigenetically activates tumor-cell-intrinsic immunity by inducing DNA replication stress.

Developing strategies to activate tumor-cell-intrinsic immune response is critical for improving tumor immunotherapy by exploiting tumor vulnerability. KDM4A, as a histone H3 lysine 9 trimethylation (H3K9me3) demethylase, has been found to play a critical role in squamous cell carcinoma (SCC) growth and metastasis. Here we report that KDM4A inhibition promoted heterochromatin compaction and induced DNA replication stress, which elicited antitumor immunity in SCC. Mechanistically, KDM4A inhibition promoted the formation of liquid-like HP1γ puncta on heterochromatin and stall DNA replication, which activated tumor-cell-intrinsic cGAS-STING signaling through replication-stress-induced cytosolic DNA accumulation. Moreover, KDM4A inhibition collaborated with PD1 blockade to inhibit SCC growth and metastasis by recruiting and activating CD8+ T cells. In vivo lineage tracing demonstrated that KDM4A inhibition plus PD1 blockade efficiently eliminated cancer stem cells. Altogether, our results demonstrate that targeting KDM4A can activate anti-tumor immunity and enable PD1 blockade immunotherapy by aggravating replication stress in SCC cells.

Spinal nerve transection-induced upregulation of KDM4A in the dorsal root ganglia contributes to the development and maintenance of neuropathic pain via promoting CCL2 expression in rats.

Studies indicate that the lysine-specific demethylase 4A (KDM4A), acts as a key player in neuropathic pain, driving the process through its involvement in promoting neuroinflammation. Emerging evidence reveals that C-C Motif Chemokine Ligand 2 (CCL2) participates in neuroinflammation, which plays an important role in the development and maintenance of neuropathic pain. However, it remains unclear if KDM4A plays a role in regulating CCL2 in neuropathic pain. This study found that following spinal nerve transection (SNT) of the lumbar 5 nerve root in rats, the expression of KDM4A and CCL2 increased in the ipsilateral L4/5 dorsal root ganglia (DRG). Injecting KDM4A siRNA into the DRGs of rats post-SNT resulted in a higher paw withdrawal threshold (PWT) and paw-withdrawal latency (PWL) compared to the KDM4A scRNA group. In addition, prior microinjection of AAV-EGFP-KDM4A shRNA also alleviates the decrease in PWT and PWL caused by SNT. Correspondingly, microinjection of AAV-EGFP-KDM4A shRNA subsequent to SNT reduced the established mechanical and thermal hyperalgesia. Furthermore, AAV-EGFP-KDM4A shRNA injection decreased the expression of CCL2 in DRGs. ChIP-PCR analysis revealed that increased binding of p-STAT1 with the CCL2 promoter induced by SNT was inhibited by AAV-EGFP-KDM4A shRNA treatment. These findings suggest that KDM4A potentially influences neuropathic pain by regulating CCL2 expression in DRGs.

KDM4A promotes malignant progression of breast cancer by down-regulating BMP9 inducing consequent enhancement of glutamine metabolism.

BACKGROUND: Recent studies have found that histone-modified genes play an increasingly important role in tumor progression. Lysine(K) specific demethylase 4A (KDM4A) is a histone lysine-specific demethylase highly expressed in a variety of malignant tumors, data showed that KDM4A was negatively correlated with the Bone Morphogenetic Protein 9 (BMP9) in breast cancer. And previous experiments have demonstrated that exogenous BMP9 significantly inhibits breast cancer development. MATERIALS AND METHODS: We detected the expression of KDM4A in breast cancer and the relationship between KDM4A and BMP9 using real-time quantitative PCR (RT-qPCR) and Western blot, and verified the interaction between KDM4A and BMP9 by ChIP experiments. At the same time, we also detected whether KDM4A had effects on the RNA and protein stability of BMP9 using actinomycin D and cycloheximide. Measurement of alpha-ketoglutarate (α-KG) level by ELISA to observe the effect of BMP9 on glutamine metabolism in breast cancer cells. Nucleoplasmic distribution of KDM4A after exogenous BMP9 treatment in breast cancer cells were observed by immunofluorescence staining and Western blot. A subcutaneous xenograft tumor model in nude mice was used to study the therapeutic effects of exogenous BMP9 and KDM4A inhibitor (JIB-04) in breast cancer. CCK-8, conoly formation, Transwell, wound healing, and immunohistochemistry were used to monitor the growth of tumor and cell function. RESULTS: We found that KDM4A was abnormally highly expressed in breast cancer, and silenced BMP9 expression by removing histone methyl groups from the BMP9 gene region. Meanwhile, KDM4A could also reduce the stability of BMP9 protein. BMP9 inhibit glutamine metabolism in breast cancer, resulting in a decrease in its product α-KG, is confirmed by ELISA. Altered nucleoplasmic distribution of KDM4A due to decreased α-KG was confirmed by immunofluorescence staining and Western blot. Animal experiments confirm that the combination of exogenous BMP9 and JIB-04 shows significantly better results in breast cancer. CONCLUSIONS: KDM4A silences BMP9 expression by removing histone methyl groups from the BMP9 gene region, leading to further enhancement of glutamine metabolism, which contributes to malignant tumor progression. In addition, using JIB-04 in combination with exogenous BMP9 could inhibit the malignant progression of breast cancer cells and the growth of tumors more significantly.

Construction of a prognostic model for disulfidptosis-related long noncoding RNAs in R0 resected hepatocellular carcinoma and analysis of their impact on malignant behavior.

BACKGROUND: Disulfidptosis is an emerging form of cellular death resulting from the binding of intracellular disulfide bonds to actin cytoskeleton proteins. This study aimed to investigate the expression and prognostic significance of hub disulfidptosis-related lncRNAs (DRLRs) in R0 resected hepatocellular carcinoma (HCC) as well as their impact on the malignant behaviour of HCC cells. METHODS: A robust signature for R0 resected HCC was constructed using least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression and was validated in an independent internal validation cohort to predict the prognosis of R0 HCC patients. Comprehensive bioinformatics analysis was performed on the hub DRLRs (KDM4A-AS1, MKLN1-AS, and TMCC1-AS1), followed by experimental validation using quantitative real-time polymerase chain reaction (qRT‒PCR) and cellular functional assays. RESULTS: The signature served as an independent prognostic factor applicable to R0 HCC patients across different age groups, tumour stages, and pathological characteristics. Gene Ontology (GO) and gene set enrichment analysis (GSEA) revealed hub pathways associated with this signature. The high-risk group presented an increased abundance of M0 macrophages and activated memory CD4 T cells as well as elevated macrophage and major histocompatibility complex (MHC) class I expression. High-risk R0 HCC patients also presented increased tumour immune dysfunction and exclusion scores (TIDEs), mutation frequencies, and tumour mutational burdens (TMBs). Drug sensitivity analysis revealed that high-risk patients were more responsive to drugs, including GDC0810 and osimertinib. High expression levels of the three hub DRLRs were detected in R0 HCC tissues and HCC cell lines. Functional assays revealed that the three hub DRLRs enhanced HCC cell proliferation, migration, and invasion. CONCLUSIONS: A signature was constructed on the basis of three DRLRs, providing novel insights for personalized precision therapy in R0 HCC patients.

Functional mechanism of hypoxia-like conditions mediating resistance to ferroptosis in cervical cancer cells by regulating KDM4A SUMOylation and the SLC7A11/GPX4 pathway.

The discovery of ferroptosis has unveiled new perspectives for cervical cancer (CC) management. We elucidated the functional mechanism of hypoxia-like conditions in CC cell ferroptosis resistance. CC cells were subjected to normoxia or hypoxia-like conditions, followed by erastin treatment to induce ferroptosis. The assessment of cell viability/ferroptosis resistance was performed by MTT assay/Fe2+, MDA, and glutathione measurement by colorimetry. KDM4A/SUMO1/Ubc9/SENP1 protein levels were determined by Western blot. Interaction and binding sites between KDM4A and SUMO1 were analyzed and predicted by immunofluorescence/co-immunoprecipitation and GPS-SUMO 1.0 software, with the target relationship verified by mutation experiment. SLC7A11/GPX4/H3K9me3 protein levels, and H3K9me3 level in the SLC7A11 gene promoter region were determined by RT-qPCR and Western blot/chromatin immunoprecipitation. H3H9me3/SLC7A11/GPX4 level alterations, and ferroptosis resistance after KDM4A silencing or KDM4A K471 mutation were assessed. Hypoxia-like conditions increased CC cell ferroptosis resistance and KDM4A, SUMO1, and Ubc9 protein levels, while it decreased SENP1 protein level. KDM4A and SUMO1 were co-localized in the nucleus, and hypoxia-like conditions promoted their interaction. Specifically, the K471 locus of KDM4A was the main locus for SUMO1ylation. Hypoxia-like conditions up-regulated SLC7A11 and GPX4 expression levels and decreased H3K9me3 protein level and H3K9me3 abundance in the SLC7A11 promoter region. KDM4A silencing or K471 locus mutation resulted in weakened interaction between KDM4A and SUMO1, elevated H3K9me3 levels, decreased SLC7A11 expression, ultimately, a reduced CC cell ferroptosis resistance. CoCl2-stimulated hypoxia-like conditions enhanced SUMO1 modification of KDM4A at the K471 locus specifically, repressed H3K9me3 levels, and up-regulated SLC7A11/GPX4 to enhance CC cell ferroptosis resistance.

SUMO Modification of Histone Demethylase KDM4A in Kaposi's Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma.

Primary effusion lymphoma (PEL) is a fatal B-cell lymphoma caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Inducing KSHV lytic replication that causes the death of host cells is an attractive treatment approach for PE; however, combination therapy inhibiting viral production is frequently needed to improve its outcomes. We have previously shown that the KSHV lytic protein K-bZIP can SUMOylate histone lysine demethylase 4A (KDM4A) at lysine 471 (K471) and this SUMOylation is required for virus production upon KSHV reactivation. Here, we demonstrate that SUMOylation of KDM4A orchestrates PEL cell survival, a major challenge for the success of PEL treatment; and cell movement and angiogenesis, the cell functions contributing to PEL cell extravasation and dissemination. Furthermore, integrated ChIP-seq and RNA-seq analyses identified interleukin-10 (IL-10), an immunosuppressive cytokine, as a novel downstream target of KDM4A. We demonstrate that PEL-induced angiogenesis is dependent on IL-10. More importantly, single-cell RNA sequencing (scRNA-seq) analysis demonstrated that, at the late stage of KSHV reactivation, KDM4A determines the fates of PEL cells, as evidenced by two distinct cell populations; one with less apoptotic signaling expresses high levels of viral genes and the other is exactly opposite, while KDM4A-K417R-expressing cells contain only the apoptotic population with less viral gene expression. Consistently, KDM4A knockout significantly reduced cell viability and virus production in KSHV-reactivated PEL cells. Since inhibiting PEL extravasation and eradicating KSHV-infected PEL cells without increasing viral load provide a strong rationale for treating PEL, this study indicates targeting KDM4A as a promising therapeutic option for treating PEL. IMPORTANCE PEL is an aggressive and untreatable B-cell lymphoma caused by KSHV infection. Therefore, new therapeutic approaches for PEL need to be investigated. Since simultaneous induction of KSHV reactivation and apoptosis can directly kill PEL cells, they have been applied in the treatment of this hematologic malignancy and have made progress. Epigenetic therapy with histone deacetylase (HDAC) inhibitors has been proved to treat PEL. However, the antitumor efficacies of HDAC inhibitors are modest and new approaches are needed. Following our previous report showing that the histone lysine demethylase KDM4A and its SUMOylation are required for lytic reactivation of KSHV in PEL cells, we further investigated its cellular function. Here, we found that SUMOylation of KDM4A is required for the survival, movement, and angiogenesis of lytic KSHV-infected PEL cells. Together with our previous finding showing the importance of KDM4A SUMOylation in viral production, KDM4A can be a potential therapeutic target for PEL.

KDM4A, involved in the inflammatory and oxidative stress caused by traumatic brain injury-hemorrhagic shock, partly through the regulation of the microglia M1 polarization.

BACKGROUND: Microglial polarization and the subsequent neuroinflammatory response and oxidative stress are contributing factors for traumatic brain injury (TBI) plus hemorrhagic shock (HS) induced brain injury. In the present work, we have explored whether Lysine (K)-specific demethylase 4 A (KDM4A) modulates microglia M1 polarization in the TBI and HS mice. RESULTS: Male C57BL/6J mice were used to investigate the microglia polarization in the TBI + HS model in vivo. Lipopolysaccharide (LPS)-induced BV2 cells were used to examine the mechanism of KDM4A in regulating microglia polarization in vitro. We found that TBI + HS resulted in neuronal loss and microglia M1 polarization in vivo, reflected by the increased level of Iba1, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, malondialdehyde (MDA) and the decreased level of reduced glutathione (GSH). Additionally, KDM4A was upregulated in response to TBI + HS and microglia were among the cell types showing the increased level of KDM4A. Similar to the results in vivo, KDM4A also highly expressed in LPS-induced BV2 cells. LPS-induced BV2 cells exhibited enhanced microglia M1 polarization, and enhanced level of pro-inflammatory cytokines, oxidative stress and reactive oxygen species (ROS), while this enhancement was abolished by the suppression of KDM4A. CONCLUSION: Accordingly, our findings indicated that KDM4A was upregulated in response to TBI + HS and microglia were among the cell types showing the increased level of KDM4A. The important role of KDM4A in TBI + HS-induced inflammatory response and oxidative stress was at least partially realized through regulating microglia M1 polarization.

Hypoxia drives transient site-specific copy gain and drug-resistant gene expression.

Copy number heterogeneity is a prominent feature within tumors. The molecular basis for this heterogeneity remains poorly characterized. Here, we demonstrate that hypoxia induces transient site-specific copy gains (TSSGs) in primary, nontransformed, and transformed human cells. Hypoxia-driven copy gains are not dependent on HIF1α or HIF2α; however, they are dependent on the KDM4A histone demethylase and are blocked by inhibition of KDM4A with a small molecule or the natural metabolite succinate. Furthermore, this response is conserved at a syntenic region in zebrafish cells. Regions with site-specific copy gain are also enriched for amplifications in hypoxic primary tumors. These tumors exhibited amplification and overexpression of the drug resistance gene CKS1B, which we recapitulated in hypoxic breast cancer cells. Our results demonstrate that hypoxia provides a biological stimulus to create transient site-specific copy alterations that could result in heterogeneity within tumors and cell populations. These findings have major implications in our understanding of copy number heterogeneity and the emergence of drug resistance genes in cancer.

Effects of overexpression of histone H3K9me3 demethylase on development of porcine cloned embryos.

The abnormal modification of histone is an important factor restricting development of porcine cloned embryos. Overexpression of histone H3K9me3 demethylase KDM4 family can effectively improve the developmental efficiency of cloned embryos. In order to explore the effects of overexpression of H3K9me3 demethylase on the development of porcine cloned embryos, KDM4A mRNA and KDM4D mRNA were injected respectively into porcine cloned embryos at the 1-cell stage and 2-cell stage to detect the blastocyst rate; 2-cell stage cloned embryos injected with KDM4A mRNA and embryo injection water (the control group) at the 1-cell stage were collected to detect the expression level of H3K9me3, and 4-cell stage cloned embryos were collected for single cell transcriptome sequencing, then the sequencing data was analyzed with KEGG and GO. The results showed that the blastocyst rate of porcine cloned embryos injected with KDM4A mRNA at 1-cell stage was significantly higher than that of the control group (25.32 ± 0.74% vs 14.78 ± 0.87%), while cloned embryos injected with KDM4D mRNA had a similar blastocyst rate with cloned embryos in control group (16.27 ± 0.77% vs 14.78 ± 0.87%). Porcine cloned embryos injected with KDM4A mRNA and KDM4D mRNA at 2-cell stage had a similar blastocyst rate with cloned embryos in control group (32.18 ± 1.67%, 30.04 ± 0.91% vs 31.22 ± 1.40%). The expression level of H3K9me3 in cloned embryos injected with KDM4A mRNA at 1-cell stage was lower than that in control group. There were 133 differentially expressed genes detected by transcriptome sequencing, including 52 up-regulated genes and 81 down-regulated genes. Pathways enriched by GO analyses were mainly related to protein localization. Pathways enriched by KEGG analyses were related to cellular senescence and acute myeloid leukemia. These results suggest that overexpression of histone H3K9me3 demethylase KDM4A can significantly improve the developmental efficiency of porcine cloned embryos.

KDM4A-mediated histone demethylation of SLC7A11 inhibits cell ferroptosis in osteosarcoma.

Osteosarcoma (OS) is the most common type of bone tumor that seriously affects limb function and induces great pain in patients. Lung metastasis and chemotherapy resistance are two key issues leading to the poor prognosis of OS patients, therefore new treatment targets and strategies are urgently needed. In our study, we uncovered the role of histone demethylase KDM4A in regulating OS cell ferroptosis and tumor progression. KDM4A was significantly upregulated in OS specimens and high KDM4A expression was associated with poorer prognosis in OS patients. Our data indicated that targeting KDM4A significantly increased OS cell death, enhanced cisplatin response, and attenuated migration ability in vitro. KDM4A depletion dramatically inhibited tumor progression and lung metastasis of OS in vivo Further experiments confirmed that KDM4A knockdown promoted OS cell ferroptosis, a special non-apoptotic form of cell death. KDM4A regulates SLC7A11 transcription and OS cell ferroptosis by controlling H3K9me3 demethylation in the promoter region of SLC7A11. Our findings deepened the recognition of epigenetic regulatory mechanism in OS tumorigenesis, chemoresistance, and metastasis, suggesting that KDM4A activity may be a potential therapeutic target for future OS treatment.

Histone demethylase KDM4A overexpression improved the efficiency of corrected human tripronuclear zygote development.

Human zygotes are difficult to obtain for research because of limited resources and ethical debates. Corrected human tripronuclear (ch3PN) zygotes obtained by removal of the extra pronucleus from abnormally fertilized tripronuclear (3PN) zygotes are considered an alternative resource for basic scientific research. In the present study, eight-cell and blastocyst formation efficiency were significantly lower in both 3PN and ch3PN embryos than in normal fertilized (2PN) embryos, while histone H3 lysine 9 trimethylation (H3K9me3) levels were much higher. It was speculated that the aberrant H3K9me3 level detected in ch3PN embryos may be related to low developmental competence. Microinjection of 1000 ng/µl lysine-specific demethylase 4A (KDM4A) mRNA effectively reduced the H3K9me3 level and significantly increased the developmental competence of ch3PN embryos. The quality of ch3PN zygotes improved as the grading criteria, cell number and pluripotent expression significantly increased in response to KDM4A mRNA injection. Developmental genes related to zygotic genome activation (ZGA) were also upregulated. These results indicate that KDM4A activates the transcription of the ZGA program by enhancing the expression of related genes, promoting epigenetic modifications and regulating the developmental potential of ch3PN embryos. The present study will facilitate future studies of ch3PN embryos and could provide additional options for infertile couples.

Mechanistic insights into KDM4A driven genomic instability.

Alterations in global epigenetic signatures on chromatin are well established to contribute to tumor initiation and progression. Chromatin methylation status modulates several key cellular processes that maintain the integrity of the genome. KDM4A, a demethylase that belongs to the Fe-II dependent dioxygenase family that uses α-ketoglutarate and molecular oxygen as cofactors, is overexpressed in several cancers and is associated with an overall poor prognosis. KDM4A demethylates lysine 9 (H3K9me2/3) and lysine 36 (H3K36me3) methyl marks on histone H3. Given the complexity that exists with these marks on chromatin and their effects on transcription and proliferation, it naturally follows that demethylation serves an equally important role in these cellular processes. In this review, we highlight the role of KDM4A in transcriptional modulation, either dependent or independent of its enzymatic activity, arising from the amplification of this demethylase in cancer. KDM4A modulates re-replication of distinct genomic loci, activates cell cycle inducers, and represses proteins involved in checkpoint control giving rise to proliferative damage, mitotic disturbances and chromosomal breaks, ultimately resulting in genomic instability. In parallel, emerging evidence of non-nuclear substrates of epigenetic modulators emphasize the need to investigate the role of KDM4A in regulating non-nuclear substrates and evaluate their contribution to genomic instability in this context. The existence of promising KDM-specific inhibitors makes these demethylases an attractive target for therapeutic intervention in cancers.

Targeting USP1-dependent KDM4A protein stability as a potential prostate cancer therapy.

The histone demethylase lysine-specific demethylase 4A (KDM4A) is reported to be overexpressed and plays a vital in multiple cancers through controlling gene expression by epigenetic regulation of H3K9 or H3K36 methylation marks. However, the biological role and mechanism of KDM4A in prostate cancer (PC) remain unclear. Herein, we reported KDM4A expression was upregulation in phosphatase and tensin homolog knockout mouse prostate tissue. Depletion of KDM4A in PC cells inhibited their proliferation and survival in vivo and vitro. Further studies reveal that USP1 is a deubiquitinase that regulates KDM4A K48-linked deubiquitin and stability. Interestingly, we found c-Myc was a key downstream effector of the USP1-KDM4A/androgen receptor axis in driving PC cell proliferation. Notably, upregulation of KDM4A expression with high USP1 expression was observed in most prostate tumors and inhibition of USP1 promotes PC cells response to therapeutic agent enzalutamide. Our studies propose USP1 could be an anticancer therapeutic target in PC.

MiR-10a functions as a tumor suppressor in prostate cancer via targeting KDM4A.

Deregulation of microRNAs contributes to the abnormal cell growth which is frequently observed in cancer. In the current study, we detected the expression and regulatory relationship between miR-10a and Lysine-specific demethylase 4A (KDM4A) to reveal their function in prostate cancer (PCa) progression. We found that miR-10a levels were significantly decreased in PCa cell lines in comparison with the normal epithelial cell line RWPE-1. Downregulation of miR-10a levels was also observed in tumor tissues from PCa patients compared with the adjacent normal tissues. Enhanced expression of miR-10a inhibited cell proliferation and colony forming capability of PCa cells. In addition, quantitative real-time polymerase chain reaction and Western blot analysis showed a significant decrease of KDM4A in response to miR-10a elevation in PCa cells. Using dual luciferase assay, we confirmed that KDM4A was a target gene for miR-10a. Furthermore, Western blot analysis indicated that miR-10a overexpression inactivated YAP signaling and suppressed transcription of YAP target genes. Additionally, cell growth arrest and colony forming capacity inhibition induced by miR-10a overexpression could be reversed by YAP overexpression in PCa cells. More importantly, miR-10a mimics inhibited PC-3 tumor growth in nude mice accompanied with a remarkable reduction of KDM4A and YAP expression. In conclusion, our results uncovered a tumor suppressor role of miR-10a in PCa via negative regulation of KDM4A and its downstream Hippo-YAP pathway.

Circ_SPECC1 enhances the inhibition of miR-526b on downstream KDM4A/YAP1 pathway to regulate the growth and invasion of gastric cancer cells.

Gastric cancer (GC) is a common malignant tumor, and many studies have shown that circular RNAs (circRNAs) play important roles in the progress of GC. This study showed that circ_SPECC1 was down-regulated in various GC cell lines, significantly inhibited GC cell proliferation and invasion, and promote apoptosis, which might play an anti-oncogene role. Circ_SPECC1 was mainly located in the cytoplasm, and its sequence contained multiple potential binding sites of miR-526b. Pull-down experiments with biotinylated miR-526b mimics and circ_SPECC1 probe showed that they could enrich each other. RIP experiments found hat anti-AGO2 antibody could significantly enrich circ_SPECC1. Further dual luciferase reporter gene assay also confirmed that miR-526b could bind directly to circ_SPECC1. miR-526b was also down-regulated in GC cells, and one of its important target genes was KDM4A. Both circ_SPECC1 and miR-526b inhibited the expression of KDM4A and its downstream effector YAP1, but miR-526b inhibitors terminated the above-mentioned inhibition of circ_SPECC1, and KDM4A overexpression reversed the inhibition of circ_SPECC1 and miR-526b on YAP1 expression. Both miR-526b and KDM4A siRNA inhibited GC cell proliferation and invasion, and promote apoptosis; KDM4A overexpression had the opposite effects, and significantly blocked the regulation of miR-526b on cell growth and invasion. Therefore, circ_SPECC1 can enhance miR-526b inhibitory effect on downstream KDM4A/YAP1 pathway by adsorbing it, thus inhibiting GC cell growth and invasion. These findings enrich the mechanism of circRNAs in GC and will provide more new targets for the prevention and treatment of GC.

Targeting lysine specific demethylase 4A (KDM4A) tandem TUDOR domain - A fragment based approach.

The tandem TUDOR domains present in the non-catalytic C-terminal half of the KDM4A, 4B and 4C enzymes play important roles in regulating their chromatin localizations and substrate specificities. They achieve this regulatory role by binding to different tri-methylated lysine residues on histone H3 (H3-K4me3, H3-K23me3) and histone H4 (H4-K20me3) depending upon the specific chromatin environment. In this work, we have used a 2D-NMR based fragment screening approach to identify a novel fragment (1a), which binds to the KDM4A-TUDOR domain and shows modest competition with H3-K4me3 binding in biochemical as well as in vitro cell based assays. A co-crystal structure of KDM4A TUDOR domain in complex with 1a shows that the fragment binds stereo-specifically to the methyl lysine binding pocket forming a network of strong hydrogen bonds and hydrophobic interactions. We anticipate that the fragment 1a can be further developed into a novel allosteric inhibitor of the KDM4 family of enzymes through targeting their C-terminal tandem TUDOR domain.

Regulation of Transient Site-specific Copy Gain by MicroRNA.

Intra-tumor copy number heterogeneity is commonly observed in cancer; however, the molecular mechanisms that contribute to heterogeneity remain poorly understood. Up-regulation of the histone demethylase KDM4A promotes transient site-specific copy gain (TSSG) in cells; therefore, uncovering how KDM4A levels are controlled is important for understanding the regulation of copy number heterogeneity. Here, we demonstrate that KDM4A is regulated by hsa-mir-23a-3p, hsa-mir-23b-3p, and hsa-mir-137. Altering expression of these microRNAs (miRNAs) regulates KDM4A-dependent TSSG. miRNA inhibition promoted copy gains and increased expression of the drug-resistant oncogene CKS1B, which was further substantiated in primary breast tumors. Consistent with increased CKS1B expression, miRNA inhibition reduced breast cancer cell sensitivity to cisplatin. Our data identify these miRNAs as regulators of TSSG and copy gains of a drug resistance gene.

Novel inhibitors of lysine (K)-specific Demethylase 4A with anticancer activity.

Lysine (K)-specific demethylase 4A (KDM4A) is a histone demethylase that removes methyl residues from trimethylated or dimethylated histone 3 at lysines 9 and 36. Overexpression of KDM4A is found in various cancer types. To identify KDM4A inhibitors with anti-tumor functions, screening with an in vitro KDM4A enzyme activity assay was carried out. The benzylidenehydrazine analogue LDD2269 was selected, with an IC50 of 6.56 µM of KDM4A enzyme inhibition, and the binding mode was investigated using in silico molecular docking. Demethylation inhibition by LDD2269 was confirmed with a cell-based assay using antibodies against methylated histone at lysines 9 and 36. HCT-116 colon cancer cell line proliferation was suppressed by LDD2269, which also interfered with soft-agar growth and migration of HCT-116 cells. AnnexinV staining and PARP cleavage experiments showed apoptosis induction by LDD2269. Derivatives of LDD2269 were synthesized and the structure-activity relationship was explored. LDD2269 is reported here as a strong inhibitor of KDM4A in in vitro and cell-based systems, with anti-tumor functions.

The requirement of histone modification by PRDM12 and Kdm4a for the development of pre-placodal ectoderm and neural crest in Xenopus.

In vertebrates, pre-placodal ectoderm and neural crest development requires morphogen gradients and several transcriptional factors, while the involvement of histone modification remains unclear. Here, we report that histone-modifying factors play crucial roles in the development of pre-placodal ectoderm and neural crest in Xenopus. During the early neurula stage, PRDM12 was expressed in the lateral pre-placodal ectoderm and repressed the expression of neural crest specifier genes via methylation of histone H3K9. ChIP-qPCR analyses indicated that PRDM12 promoted the occupancy of the trimethylated histone H3K9 (H3K9me3) on the Foxd3, Slug, and Sox8 promoters. Injection of the PRDM12 MO inhibited the expression of presumptive trigeminal placode markers and decreased the occupancy of H3K9me3 on the Foxd3 promoter. Histone demethylase Kdm4a also inhibited the expression of presumptive trigeminal placode markers in a similar manner to PRDM12 MO and could compensate for the effects of PRDM12. ChIP-qPCR analyses revealed that promotion of the occupancy of H3K9me3 on the Foxd3, Slug, and Sox8 promoters was inhibited by Kdm4a overexpression. Taken together, these data indicate that histone modification was essential for pre-placodal ectoderm and neural crest development.

A scintillation proximity assay for histone demethylases.

Covalent modifications, such as methylation and demethylation of lysine residues in histones, play important roles in chromatin dynamics and the regulation of gene expression. The lysine demethylases (KDMs) catalyze the demethylation of lysine residues on histone tails and are associated with diverse human diseases, including cancer, and are therefore proposed as targets for the therapeutic modulation of gene transcription. High-throughput assays have been developed to find inhibitors of KDMs, most of which are fluorescence-based assays. Here we report the development of a coupled scintillation proximity assay (SPA) for 3 KDMs: KDM1A (LSD1), KDM3A (JMJD1A), and KDM4A (JMJD2A). In this assay methylated peptides are first demethylated by a KDM, and a protein methyltransferase (PMT) is added to methylate the resulting peptide with tritiated S-(5'-adenosyl)-l-methionine. The enzyme activities were optimized and kinetic parameters were determined. These robust coupled assays are suitable for screening KDMs in 384-well format (Z' factors of 0.70-0.80), facilitating discovery of inhibitors in the quest for cancer therapeutics.