Targeting G9a/DNMT1 methyltransferase activity impedes IGF2-mediated survival in hepatoblastoma.

BACKGROUND: As the variable clinical outcome of patients with hepatoblastoma (HB) cannot be explained by genetics alone, the identification of drugs with the potential to effectively reverse epigenetic alterations is a promising approach to overcome poor therapy response. The gene ubiquitin like with PHD and ring finger domains 1 (UHRF1) represents an encouraging epigenetic target due to its regulatory function in both DNA methylation and histone modifications and its clinical relevance in HB. METHODS: Patient-derived xenograft in vitro and in vivo models were used to study drug response. The mechanistic basis of CM-272 treatment was elucidated using RNA sequencing and western blot experiments. RESULTS: We validated in comprehensive data sets that UHRF1 is highly expressed in HB and associated with poor outcomes. The simultaneous pharmacological targeting of UHRF1-dependent DNA methylation and histone H3 methylation by the dual inhibitor CM-272 identified a selective impact on HB patient-derived xenograft cell viability while leaving healthy fibroblasts unaffected. RNA sequencing revealed downregulation of the IGF2-activated survival pathway as the main mode of action of CM-272 treatment, subsequently leading to loss of proliferation, hindered colony formation capability, reduced spheroid growth, decreased migration potential, and ultimately, induction of apoptosis in HB cells. Importantly, drug response depended on the level of IGF2 expression, and combination assays showed a strong synergistic effect of CM-272 with cisplatin. Preclinical testing of CM-272 in a transplanted patient-derived xenograft model proved its efficacy but also uncovered side effects presumably caused by its strong antitumor effect in IGF2-driven tumors. CONCLUSIONS: The inhibition of UHRF1-associated epigenetic traces, such as IGF2-mediated survival, is an attractive approach to treat high-risk HB, especially when combined with the standard-of-care therapeutic cisplatin.

DNMT1 inhibition by pUG-fold quadruplex RNA.

Aberrant DNA methylation is one of the earliest hallmarks of cancer. DNMT1 is responsible for methylating newly replicated DNA, but the precise regulation of DNMT1 to ensure faithful DNA methylation remains poorly understood. A link between RNA and chromatin-associated proteins has recently emerged, and several studies have shown that DNMT1 can be regulated by a variety of RNAs. In this study, we have confirmed that human DNMT1 indeed interacts with multiple RNAs, including its own nuclear mRNA. Unexpectedly, we found that DNMT1 exhibits a strong and specific affinity for GU-rich RNAs that form a pUG-fold, a noncanonical G-quadruplex. We find that pUG-fold-capable RNAs inhibit DNMT1 activity by inhibiting binding of hemimethylated DNA, and we additionally provide evidence for multiple RNA binding modes with DNMT1. Together, our data indicate that a human chromatin-associated protein binds to and is regulated by pUG-fold RNA.

DNMT1-Mediated DNA Methylation Targets CDKN2B to Promote the Repair of Retinal Ganglion Cells in Streptozotocin-Induced Mongolian Gerbils during Diabetic Retinopathy.

Objective: DNA methylation played a vital role in the progression of diabetic retinopathy. In this study, we aimed to explore the role of DNA cytosine-5-methyltransferase 1 (DNMT1) in the development of early diabetic retinopathy and its potential underlying mechanism. Methods: Eight-week-old healthy Mongolian gerbils were used to establish type 1 diabetes using streptozotocin (STZ). Alteration of weight, fasting blood glucose, density of RGCs (Tuj1-labeled), and H&E-stained retinal cross sections were applied to evaluate the diabetic retinopathy mouse model. The global DNA methylation level of the retina at different time points after STZ injection was measured using the global methylation assay. Western blot was used to detect the protein expression of DNMT1, DNA methyltransferase 3A (DNMT3A), and 3B (DNMT3B). Quantitative reverse transcription-polymerase chain reactions (qRT-PCR) and western blot were used to determine the expression of CDKN2B. Cell proliferation and cell cycle were evaluated by the MTS assay and flow cytometry. Results: STZ injection caused the increased global DNA methylation level, which reached a maximum at 6 weeks after injection. Moreover, STZ injection caused the damage of RGCs. At 6 weeks after STZ injection, the expression levels of DNMT1 and DNMT3B were significantly increased in the STZ group. DNMT1-induced DNA hypermethylation inhibited the expression of CDKN2B (a negative regulator of cell cycle). DNMT1-mediated DNA methylation facilitated RGC proliferation via regulating the expression of CDKN2B. Conclusion: DNMT1-mediated DNA methylation played an important role in STZ-induced diabetic retinopathy via modulating CDKN2B expression.

Decitabine potentiates efficacy of doxorubicin in a preclinical trastuzumab-resistant HER2-positive breast cancer models.

Acquired drug resistance and metastasis in breast cancer (BC) are coupled with epigenetic deregulation of gene expression. Epigenetic drugs, aiming to reverse these aberrant transcriptional patterns and sensitize cancer cells to other therapies, provide a new treatment strategy for drug-resistant tumors. Here we investigated the ability of DNA methyltransferase (DNMT) inhibitor decitabine (DAC) to increase the sensitivity of BC cells to anthracycline antibiotic doxorubicin (DOX). Three cell lines representing different molecular BC subtypes, JIMT-1, MDA-MB-231 and T-47D, were used to evaluate the synergy of sequential DAC + DOX treatment in vitro. The cytotoxicity, genotoxicity, apoptosis, and migration capacity were tested in 2D and 3D cultures. Moreover, genome-wide DNA methylation and transcriptomic analyses were employed to understand the differences underlying DAC responsiveness. The ability of DAC to sensitize trastuzumab-resistant HER2-positive JIMT-1 cells to DOX was examined in vivo in an orthotopic xenograft mouse model. DAC and DOX synergistic effect was identified in all tested cell lines, with JIMT-1 cells being most sensitive to DAC. Based on the whole-genome data, we assume that the aggressive behavior of JIMT-1 cells can be related to the enrichment of epithelial-to-mesenchymal transition and stemness-associated pathways in this cell line. The four-week DAC + DOX sequential administration significantly reduced the tumor growth, DNMT1 expression, and global DNA methylation in xenograft tissues. The efficacy of combination therapy was comparable to effect of pegylated liposomal DOX, used exclusively for the treatment of metastatic BC. This work demonstrates the potential of epigenetic drugs to modulate cancer cells' sensitivity to other forms of anticancer therapy.

Targeting DNMT1 by demethylating agent OR-2100 increases tyrosine kinase inhibitors-sensitivity and depletes leukemic stem cells in chronic myeloid leukemia.

ABL1 tyrosine kinase inhibitors (TKIs) dramatically improve the prognosis of chronic myeloid leukemia (CML), but 10-20% of patients achieve suboptimal responses with low TKIs sensitivity. Furthermore, residual leukemic stem cells (LSCs) are involved in the molecular relapse after TKIs discontinuation. Aberrant DNA hypermethylation contributes to low TKIs sensitivity and the persistence of LSCs in CML. DNMT1 is a key regulator of hematopoietic stem cells, suggesting that aberrant DNA hypermethylation targeting DNMT1 represents a potential therapeutic target for CML. We investigated the efficacy of OR-2100 (OR21), the first orally available single-compound prodrug of decitabine. OR21 exhibited anti-tumor effects as a monotherapy, and in combination therapy it increased TKI-induced apoptosis and induction of tumor suppressor genes including PTPN6 encoding SHP-1 in CML cells. OR21 in combination with imatinib significantly suppressed tumor growth in a xenotransplant model. OR21 and combination therapy decreased the abundance of LSCs and inhibited engraftment in a BCR-ABL1-transduced mouse model. These results demonstrate that targeting DNMT1 using OR21 exerts anti-tumor effects and impairs LSCs in CML. Therefore, combination treatment of TKIs and OR21 represents a promising treatment strategy in CML.

The DNMT1-associated lncRNA UCA1 was upregulated in TK6 cells transformed by long-term exposure to hydroquinone and benzene-exposed workers via DNA hypomethylation.

Exposure to benzene or its metabolite hydroquinone (HQ) is a risk factor for a series of myeloid malignancies, and long noncoding RNAs play an important role in the process of pathogenesis. Urothelial cancer-associated 1 (UCA1) functions as an oncogene in the development of acute myeloid leukemia. However, the association between DNMT1 and UCA1 with benzene or HQ exposure has not been explored. We characterized UCA1 expression in cells briefly exposed to HQ (HQ-ST cells) and HQ-induced malignantly transformed (TK6-HT cells) treated with 5-aza-2'-deoxycytidine (5-AzaC) or trichostatin A (TSA). Compared to that in control cells, UCA1 expression was increased, whereas DNMT1 was decreased in HQ-ST cells and TK6-HT cells treated with 5-AzaC or TSA. Moreover, UCA1 expression was also upregulated and positively correlated with benzene exposure time in benzene-exposed workers. Furthermore, the expression of UCA1 was negatively associated with the DNA methylation level of its promoter in benzene-exposed workers. DNMT1 rather than DNMT3b knockout in TK6-HT cells activated the expression of UCA1 by inducing its promoter hypomethylation. These results suggest that benzene or HQ exposure leads to UCA1 upregulation via DNA hypomethylation in the UCA1 promoter, which is mediated by DNMT1.

5-Aza-4'-thio-2'-deoxycytidine, a New Orally Bioavailable Nontoxic "Best-in-Class": DNA Methyltransferase 1-Depleting Agent in Clinical Development.

DNA methyltransferase (DNMT) 1 is an enzyme that functions as a maintenance methyltransferase during DNA replication, and depletion of this enzyme from cells is considered to be a rational goal in DNA methylation-dependent disorders. Two DNMT1-depleting agents 5-aza-2'-deoxycytidine (aza-dCyd, decitabine) and 5-aza-cytidine (aza-Cyd, azacitidine) are currently used for the treatment of myelodysplastic syndromes and acute myeloid leukemia and have also been investigated for nononcology indications, such as sickle cell disease. However, these agents have several off-target activities leading to significant toxicities that limit dosing and duration of treatment. Development of more selective inhibitors of DNMT1 could therefore afford treatment of long durations at effective doses. We have discovered that 5-aza-4'-thio-2'-deoxycytidine (aza-T-dCyd) is as effective as aza-dCyd in depleting DNMT1 in mouse tumor models but with markedly low toxicity. In this review we describe the preclinical studies that led to the development of aza-T-dCyd as a superior DNMT1-depleting agent with respect to aza-dCyd and will describe its pharmacology, metabolism, and mechanism of action. In an effort to understand why aza-T-dCyd is a more selective DNMT1 depleting agent than aza-dCyd, we will also compare and contrast the activities of these two agents. SIGNIFICANCE STATEMENT: Aza-T-dCyd is a potent DNMT1-depleting agent. Although similar in structure to decitabine (aza-dCyd), its metabolism and mechanism of action is different than that of aza-dCyd, resulting in less off-target activity and less toxicity. The larger therapeutic index of aza-T-dCyd (DNMT1 depletion vs. toxicity) in mice suggests that it would be a better clinical candidate to selectively deplete DNMT1 from target cells and determine whether or not depletion of DNMT1 is an effective target for various diseases.

A novel small-molecule antagonist enhances the sensitivity of osteosarcoma to cabozantinib in vitro and in vivo by targeting DNMT-1 correlated with disease severity in human patients.

Advanced osteosarcoma (OSA) is highly aggressive and can lead to distant metastasis or recurrence. Here, a novel small-molecule inhibitor/antagonist of DNA methyltransferase 1 (DNMT-1) named DI-1 (inhibitor of DNMT-1) was explored to enhance the antitumor effect of a molecular-targeted agent, cabozantinib, on OSA cell lines. In patients with OSA, expression of DNMT-1 was negatively related with that of microRNA (miR)-34a and associated with a poor prognosis. In OSA cell lines (OSA cell line U2OS and an OSA cell line U2OSR resistance to cabozantinib), DI-1 treatment enhanced miR-34a expression by inhibiting hypermethylation of the promoter region of miR-34a mediated by DNMT-1. DI-1 enhanced the sensitivity of OSA cells (U2OS, 143B and MG63) to cabozantinib and other molecular-targeted agents by enhancing miR-34a expression and repressing activation of the Notch pathway. Mechanistically, DI-1 repressed recruitment of DNMT-1 to the promoter region of miR-34a and, in turn, decreased the methylation rate in the promoter region of miR-34a in OSA cells. These results suggest that repressing DNMT-1 activation by DI-1 enhances miR-34a expression in OSA cells and could be a promising therapeutic strategy for OSA.

Antitumoral activity of liraglutide, a new DNMT inhibitor in breast cancer cells in vitro and in vivo.

Breast cancer (BC) is the most frequently diagnosed female cancer and second leading cause of death. Despite the discovery of many antineoplastic drugs for BC, the current therapy is not totally efficient. In this study, we investigated the potential of repurposing the well-known diabetes type II drug liraglutide to modulate epigenetic modifications in BC cells lines in vitro and in vivo via Ehrlich mice tumors models. The in vitro results revealed a significant reduction on cell viability, migration, DNMT activity and displayed lower levels of global DNA methylation in BC cell lines after liraglutide treatment. The interaction between liraglutide and the DNMT enzymes resulted in a decrease profile of DNA methylation for the CDH1, ESR1 and ADAM33 gene promoter regions and, consequently, increased their gene and protein expression levels. To elucidate the possible interaction between liraglutide and the DNMT1 protein, we performed an in silico study that indicates liraglutide binding in the catalytic cleft via hydrogen bonds and salt bridges with the interdomain contacts and disturbs the overall enzyme conformation. The in vivo study was also able to reveal that liraglutide and the combined treatment of liraglutide and paclitaxel or methotrexate were effective in reducing tumor growth. Moreover, the modulation of CDH1 and ADAM33 mouse gene expression by DNA demethylation suggests a role for liraglutide in DNMT activity in vivo. Altogether, these results indicate that liraglutide may be further analysed as a new adjuvant treatment for BC.

8a, a New Acridine Antiproliferative and Pro-Apoptotic Agent Targeting HDAC1/DNMT1.

Epigenetic therapy using histone deacetylase (HDAC) inhibitors has become an attractive project in new drug development. However, DNA methylation and histone acetylation are important epigenetic ways to regulate the occurrence and development of leukemia. Given previous studies, N-(2-aminophenyl)benzamide acridine (8a), as a histone deacetylase 1 (HDAC1) inhibitor, induces apoptosis and shows significant anti-proliferative activity against histiocytic lymphoma U937 cells. HDAC1 plays a role in the nucleus, which we confirmed by finding that 8a entered the nucleus. Subsequently, we verified that 8a mainly passes through the endogenous (mitochondrial) pathway to induce cell apoptosis. From the protein interaction data, we found that 8a also affected the expression of DNA methyltransferase 1 (DNMT1). Therefore, an experiment was performed to assess the binding of 8a to DNMT1 at the molecular and cellular levels. We found that the binding strength of 8a to DNMT1 enhanced in a dose-dependent manner. Additionally, 8a inhibits the expression of DNMT1 mRNA and its protein. These findings suggested that the anti-proliferative and pro-apoptotic activities of 8a against leukemia cells were achieved by targeting HDAC1 and DNMT1.

Targeting the T-Cell Lymphoma Epigenome Induces Cell Death, Cancer Testes Antigens, Immune-Modulatory Signaling Pathways.

The peripheral T-cell lymphomas (PTCL) could be considered the prototypical epigenetic disease. As a disease, they are uniquely sensitive to histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors, both alone and in combination, are characterized by a host of mutations in epigenetic genes, and can develop spontaneously in genetically engineered murine models predicated on established recurring mutations in (RHOAG17V) and TET2, an epigenetic gene governing DNA methylation. Given the clinical benefit of HDAC inhibitors (HDACi) and hypomethlyation agents alone and in combination in PTCL, we sought to explore a mechanistic basis for these agents in PTCL. Herein, we reveal profound class synergy between HDAC and DNMT inhibitors in PTCL, and that the combination induces degrees of gene expression that are substantially different and more extensive than that observed for the single agents. A prominent signature of the combination relates to the transcriptional induction of cancer testis antigens and genes involved in the immune response. Interestingly, TBX21 and STAT4, master regulators of TH1 differentiation, were among the genes upregulated by the combination, suggesting the induction of a TH1-like phenotype. Moreover, suppression of genes involved in cholesterol metabolism and the matrisome were also identified. We believe that these data provide a strong rationale for clinical studies, and future combinations leveraging an immunoepigenetic platform.

DNMT1 Inhibitor Restores RUNX2 Expression and Mineralization in Periodontal Ligament Cells.

Periodontal ligament cells (PDLCs) have well documented osteogenic potential; however, this commitment can be highly heterogenous, limiting their applications in tissue regeneration. In this study, we use PDLC populations characterized by high and low osteogenic potential (h-PDLCs and l-PDLCs, respectively) to identify possible sources of such heterogeneity and to investigate whether the osteogenic differentiation can be enhanced by epigenetic modulation. In h-PDLCs, low basal expression levels of pluripotency markers (NANOG, OCT4), DNA methyltransferases (DNMT1, DNMT3B), and enzymes involved in active DNA demethylation (TET1, TET3) were prerequisite to high osteogenic potential. Furthermore, these genes were downregulated upon early osteogenesis, possibly allowing for the increase in expression of the key osteogenic transcription factors, Runt-related transcription factor 2 (RUNX2) and SP7, and ultimately, mineral nodule formation. l-PDLCs appeared locked in the multipotent state and this was further enhanced upon early osteogenic stimulation, correlating with low RUNX2 expression and impaired mineralization. Further upregulation of DNMTs was also evident, while pretreatment with RG108, the DNMTs' inhibitor, enhanced the osteogenic program in l-PDLCs through downregulation of DNMTs, increased RUNX2 expression and nuclear localization, accelerated expression of osteogenic markers, and increased mineralization. These findings point toward the role of DNMTs and Ten Eleven Translocations (TETs) in osteogenic commitment and support application of epigenetic approaches to modulate biomineralization in PDLCs.

Design and Synthesis of Novel Epigenetic Inhibitors Targeting Histone Deacetylases, DNA Methyltransferase 1, and Lysine Methyltransferase G9a with In Vivo Efficacy in Multiple Myeloma.

Concomitant inhibition of key epigenetic pathways involved in silencing tumor suppressor genes has been recognized as a promising strategy for cancer therapy. Herein, we report a first-in-class series of quinoline-based analogues that simultaneously inhibit histone deacetylases (from a low nanomolar range) and DNA methyltransferase-1 (from a mid-nanomolar range, IC50 < 200 nM). Additionally, lysine methyltransferase G9a inhibitory activity is achieved (from a low nanomolar range) by introduction of a key lysine mimic group at the 7-position of the quinoline ring. The corresponding epigenetic functional cellular responses are observed: histone-3 acetylation, DNA hypomethylation, and decreased histone-3 methylation at lysine-9. These chemical probes, multitarget epigenetic inhibitors, were validated against the multiple myeloma cell line MM1.S, demonstrating promising in vitro activity of 12a (CM-444) with GI50 of 32 nM, an adequate therapeutic window (>1 log unit), and a suitable pharmacokinetic profile. In vivo, 12a achieved significant antitumor efficacy in a xenograft mouse model of human multiple myeloma.

A feasibility study of combined epigenetic and vaccine therapy in advanced colorectal cancer with pharmacodynamic endpoint.

Epigenetic therapies may modulate the tumor microenvironment. We evaluated the safety and optimal sequence of combination DNA methyltransferase inhibitor guadecitabine with a granulocyte macrophage-colony-stimulating-factor (GM-CSF) secreting colon cancer (CRC) vaccine (GVAX) using a primary endpoint of change in CD45RO + T cells. 18 patients with advanced CRC enrolled, 11 underwent paired biopsies and were evaluable for the primary endpoint. No significant increase in CD45RO + cells was noted. Grade 3-4 toxicities were expected and manageable. Guadecitabine + GVAX was tolerable but demonstrated no significant immunologic activity in CRC. We report a novel trial design to efficiently evaluate investigational therapies with a primary pharmacodynamic endpoint.Trial registry Clinicaltrials.gov: NCT01966289. Registered 21 October, 2013.

Newly Synthesized DNA Methyltransferase Inhibitors as Radiosensitizers for Human Lung Cancer and Glioblastoma Cells.

BACKGROUND/AIM: Improvement of the efficacy of radiotherapy for lung cancer and glioblastoma is urgently needed. MATERIALS AND METHODS: We synthesized several novel DNA methyltransferase inhibitors and evaluated their potentials as possible radiosensitizers. Eleven non-nucleoside compounds were synthesized and evaluated along with one known compound using human lung cancer (A549) and glioblastoma (U373MG) cells. Cytotoxicity and radiosensitizing effects were evaluated using clonogenic assay. Sensitizer enhancement ratios at a survival fraction of 0.5 were calculated, and statistical analysis was performed using the ratio paired t-test. The inhibitory effects of three selected compounds on the activity of DNA methyltransferase 1 (DNMT1) and the pharmacokinetic profiles were analyzed. RESULTS: All twelve compounds demonstrated various levels of cytotoxicity. Of the twelve compounds, eleven and eight compounds radiosensitized A549 and U373MG cells, respectively, with at least marginal significance (p<0.10). The sensitizer enhancement ratios in A549 and U373MG ranged 1.166-2.537 and 1.083-1.743 among compounds with radiosensitizing effects, respectively. The three selected compounds inhibited DNMT1 activity by 26.5-78.5%. Elimination half-lives ranged from 0.3 to 1.3 h. CONCLUSION: Novel DNA methyltransferase inhibitors with significant radiosensitizing capacity and improved biostability were synthesized. These materials will serve as a basis for the development of novel radiosensitizers.

Identification of DNA Methyltransferase-1 Inhibitor for Breast Cancer Therapy through Computational Fragment-Based Drug Design.

Epimutation by DNA Methyltransferase 1 (DNMT1), an epigenetic regulator enzyme, may lead to the proliferation of breast cancer. In this report, 168,686 natural products from the PubChem database were screened and modified by in silico method to acquire the potential inhibitor of DNMT1. The initial screening of PubChem natural products using Lipinski's and Veber's rules of three and toxic properties have resulted in 2601 fragment candidates. Four fragments from pharmacophore-based molecular docking simulation were modified by utilizing FragFP and the Lipinski's and Veber's rules of five, and resulted in 51,200 ligands. The toxicological screening collected 13,563 ligands for a series of pharmacophore-based molecular docking simulations to sort out the modified ligands, which had the better binding activity and interactions to DNMT1 compared to the standards, SAH, SAM, and SFG. This step resulted in five ligand candidates, namely C-7756, C-5769, C-1723, C-2129, and C-2140. The ADME-Tox properties prediction showed that the selected ligands are generally better than standards in terms of druglikeness, GI absorption, and oral bioavailability. C-7756 exhibited a stronger affinity to DNMT1 as well as better ADME-Tox properties compared to the other ligands.

DNMT1: A key drug target in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Altered epigenetics regulation including DNA hypermethylation by DNA methyltransferase 1 (DNMT1) has been implicated as one of the causes of TNBC tumorigenesis. In this review, the oncogenic functions rendered by DNMT1 in TNBCs, and DNMT1 inhibitors targeting TNBC cells are presented and discussed. In summary, DNMT1 expression is associated with poor breast cancer survival, and it is overexpressed in TNBC subtype. The oncogenic roles of DNMT1 in TNBCs include: (1) Repression of estrogen receptor (ER) expression; (2) Promotion of epithelial-mesenchymal transition (EMT) required for metastasis; (3) Induces cellular autophagy and; (4) Promotes the growth of cancer stem cells in TNBCs. DNMT1 confers these phenotypes by hypermethylating the promoter regions of ER, multiple tumor suppressor genes, microRNAs and epithelial markers involved in suppressing EMT. DNMT1 inhibitors exert anti-tumorigenic effects against TNBC cells. This includes the hypomethylating agents azacitidine, decitabine and guadecitabine that might sensitize TNBC patients to immune checkpoint blockade therapy. DNMT1 represents an epigenetic target for TNBC cells destruction as well as to derail their metastatic and aggressive phenotypes.

Causal roles of stress kinase JNK2 in DNA methylation and binge alcohol withdrawal-evoked behavioral deficits.

Excessive binge alcohol intake is a common drinking pattern in humans, especially during holidays. Cessation of the binge drinking often leads to aberrant withdrawal behaviors, as well as serious heart rhythm abnormalities (clinically diagnosed as Holiday Heart Syndrome (HHS)). In our HHS mouse model with well-characterized binge alcohol withdrawal (BAW)-induced heart phenotypes, BAW leads to anxiety-like behaviors and cognitive impairment. We have previously reported that stress-activated c-Jun NH(2)-terminal kinase (JNK) plays a causal role in BAW-induced heart phenotypes. In the HHS brain, we found that activation of JNK2 (but not JNK1 and JNK3) in the prefrontal cortex (PFC), but not hippocampus and amygdala, led to anxiety-like behaviors and impaired cognition. DNA methylation mediated by a crucial DNA methylation enzyme, DNA methyltransferase1 (DNMT1), is known to be critical in alcohol-associated behavioral deficits. In HHS mice, JNK2 in the PFC (but not hippocampus and amygdala) causally enhanced total genomic DNA methylation via increased DNMT1 expression, which was regulated by enhanced binding of JNK downstream transcriptional factor c-JUN to the DNMT1 promoter. JNK2-specific inhibition either by an inhibitor JNK2I or JNK2 knockout completely offset c-JUN-regulated DNMT1 upregulation and restored the level of DNA methylation in HHS PFC to the baseline levels seen in sham controls. Strikingly, either JNK2-specific inhibition or genetic JNK2 depletion or DNMT1 inhibition (by an inhibitor 5-Azacytidine) completely abolished BAW-evoked behavioral deficits. In conclusion, our studies revealed a novel mechanism by which JNK2 drives BAW-evoked behavioral deficits through a DNMT1-regulated DNA hypermethylation. JNK2 could be a novel therapeutic target for alcohol withdrawal treatment and/or prevention.

Dual Targeting of G9a and DNA Methyltransferase-1 for the Treatment of Experimental Cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors. APPROACH AND RESULTS: Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl4 treatment (JnkΔhepa + DEN + CCl4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in JnkΔhepa + DEN + CCl4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status. CONCLUSIONS: Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.