Lysine-specific Demethylase 1A Research Articles

KDM3A histone demethylase functions as an essential factor for activation of JAK2−STAT3 signaling pathway

Janus tyrosine kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) signaling pathway is essential for modulating cellular development, differentiation, and homeostasis. Thus, dysregulation of JAK2-STAT3 signaling pathway is frequently associated with human malignancies. Here, we provide evidence that lysine-specific demethylase 3A (KDM3A) functions as an essential epigenetic enzyme for the activation of JAK2-STAT3 signaling pathway. KDM3A is tyrosine-phosphorylated by JAK2 in the nucleus and functions as a STAT3-dependent transcriptional coactivator. JAK2-KDM3A signaling cascade induced by IL-6 leads to alteration of histone H3K9 methylation as a predominant epigenetic event, thereby providing the functional and mechanistic link between activation of JAK2-STAT3 signaling pathway and its epigenetic control. Together, our findings demonstrate that inhibition of KDM3A phosphorylation could be a potent therapeutic strategy to control oncogenic effect of JAK2-STAT3 signaling pathway.

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.

1832PD - Phase I study of CC-90011 in patients with advanced solid tumors and relapsed/refractory non-Hodgkin lymphoma (R/R NHL)

Background: CC-90011 is a selective, reversible inhibitor of the epigenetic target, lysine-specific demethylase 1A (LSD1) that has demonstrated anti-tumor activity against neuroendocrine cells in vitro and in xenograft models. Methods: This is a phase I, first-in-human clinical study of CC-90011 in patients (pts) with advanced unresectable solid tumors and R/R NHL. Pts received oral CC-90011 once/week in 28-day cycles. Primary objectives were to determine safety, maximum-tolerated dose (MTD), and/or recommended phase II dose (RP2D). Secondary objectives were to measure preliminary efficacy, pharmacokinetics (PK), and pharmacodynamics (PD). Results: As of 15 Jan 2018, 35 pts were enrolled; 34 with advanced solid tumors, including 17 with neuroendocrine tumors (NETs) and 1 with R/R NHL. They received escalating doses of CC-90011 at 1.25 mg (n = 4), 2.5 mg (n = 5), 5 mg (n = 6), 10 mg (n = 4), 20 mg (n = 4), 40 mg (n = 6), or 80 mg (n = 4). The MTD and RP2D have not yet been established. The median age was 61 y (range, 22-75), 19 (54%) were male, with 2 (range, 1-4) median number of prior systemic anticancer therapies. No dose-limiting toxicities were reported. The only treatment-related grade 3/4 adverse event (AE) was thrombocytopenia (9%). Serious AEs occurred in 24% of pts; none were treatment-related. Peak plasma concentrations were 2-4 hours post-dose and terminal elimination half-life was approximately 64 hours; increase in plasma exposures was dose proportional. Blood biomarker analyses showed increasing suppression of MMD and MYL9 expression with higher CC-90011 doses suggesting target engagement at doses ≥ 40 mg. Overall, 13 (39%) pts had stable disease (SD). Prolonged SD (≥4 mo) was observed in 5 pts with solid tumors; notably, 3 of those pts had bronchial NETs [5 mg (n = 1), 20 mg (n = 1) and 40 mg (n = 1)]. Preliminary PD results from NETs showed CC-90011 decreased chromogranin A levels, correlating with clinical benefit. Conclusions: CC-90011 has dose-proportional PK with target engagement at tolerable doses with preliminary evidence of antitumor activity in advanced solid tumors and R/R NHL, including prolonged SD particularly in pts with NETs. Clinical trial identification: NCT02875223. Editorial acknowledgement: Editorial assistance was provided by BioConnections, LLC. Legal entity responsible for the study: Celgene Corporation. Funding: Celgene Corporation. Disclosure: A. Hollebecque: Honoraria: Merck Serono; Consulting or advisory role: Gritstone Oncology; Travel, accommodations: Amgen. J. de Bono: Consulting or advisory role: AstraZeneca, GlaxoSmithKline, Genentech, Pfizer, Merck, Sharp & Dhome, Bayer, Merck Serone, Janssen, Astellas, Seattle Genetics; Research funding: AstraZeneca, Sanofi, Genentech, GlaxoSmithKline, Daiichi, Taiho, Merck Serone, Merck Sharp & Dhome, Sierra; Speaker's bureau: AstraZeneca. R. Plummer: Honoraria: Novartis, Bristol-Myers Squibb, AstraZeneca; Consulting or advisory role: Clovis Oncology, Bayer, Novartis, Pierre Faber, Karus Therapeutics, Genmab, Octimet; Research funding: Merck, Genmab, AstraZeneca, Menarini; Patents, royalties, other intellectual property: Clovis Oncology; Travel, accommodations: Merck Sharpe & Dhome, Bristol-Myers Squibb. S. Mora, A. Harding, Z. Nikolova: Employment, equity ownership, travel, accommodations: Celgene, Inc. A. Nguyen: Employment, equity ownership, patents, royalties, other intellectual property: Celgene Corporation. E. Filvaroff: Employment: Celgene; Equity ownership: Celgene, Amgen, Gilead, Genentech, Roche; Patents, royalties, other intellectual property: Celgene, Genentech. M. Lamba: Employment, equity ownership, research funding: Pfizer Inc., Celgene Corporation; Patents, royalties, other intellectual property: Pfizer, Inc. K. Liu, M. Zuraek: Employment, equity ownership: Celgene Corporation. J. DiMartino: Employment, leadership, equity ownership: Celgene Corporation. All other authors have declared no conflicts of interest.

HPV16 E7-induced upregulation of KDM2A promotes cervical cancer progression by regulating miR-132-radixin pathway.

Human papillomavirus (HPV) infection and viral proteins expression cause a number of epigenetic alterations leading to cervical carcinogenesis. The recent discovery of a large amount of histone methylation modifiers reveals important roles of these enzymes in regulating tumor progression. The changes in expression of 48 histone methylation modifiers were assessed following knockdown of HPV16 E7 in CaSki cells. Lysine-specific demethylase 2A (KDM2A)-regulated microRNAs (miRNAs) in cervical cancer pathogenesis were disclosed using quantitative real-time polymerase chain reaction. The function of KDM2A-miRNAs on cervical cancer was investigated in vitro and in vivo. Upregulation of KDM2A induced by HPV16 E7 promotes cervical cancer cell proliferation and invasion and is correlated with poor prognosis in patients with cervical cancer. KDM2A physically interacts with the promoter of miR-132 and suppresses its expression by removing the mono or dimethyl group from H3K36 at the miR-132 locus. Functionally, miR-132 represses cancer cell proliferation and invasion by inhibiting radixin (RDX). Upregulated KDM2A promotes cervical cancer progression by repressing miR-132, which results in a derepression of RDX. Therefore, KDM2A functions as a tumor activator in cervical cancer pathogenesis by binding miR-132 promoter and abrogating its tumor suppressive function. Our results suggest a function for KDM2A in cervical cancer progression and suggest its candidacy as a new prognostic biomarker and target for clinical management of cervical cancer.

Lysine-Specific Demethylase 1A as a Promising Target in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematopoietic malignancy characterized by the accumulation of incompletely differentiated progenitor cells (blasts) in the bone marrow and blood, and by suppression of normal hematopoiesis. It has recently become apparent that the AML genome is characterized by recurrent mutations and dysregulations in epigenetic regulators. These mutations frequently occur before the onset of full blown leukemia, at the pre-leukemic phase, and persist in residual disease that remains after therapeutic intervention, thus suggesting that targeting the AML epigenome may help to eradicate minimal residual disease and prevent relapse. Within the AML epigenome, lysine-specific demethylase 1 A (LSD1) is a histone demethylase that is found frequently overexpressed, albeit not mutated, in AML. LSD1 is a required constituent of critical transcription repressor complexes like CoREST and nucleosome remodeling and deacetylase (NuRD), and abrogation of LSD1 expression results in impaired self-renewal and proliferation, and increased differentiation and apoptosis in AML models and primary cells, particularly in AMLs with MLL- and AML1-rearrangements, or erythroid and megakaryoblastic differentiation block. On this basis, a number of LSD1 inhibitors have been developed in the past decade, and few of them are currently being tested in clinical trials for patients with AML, along with other malignancies. To date, the most promising application of this therapeutic strategy appears to be combination therapy of LSD1 inhibitors with all-trans retinoic acid (ATRA) to reactivate myeloid differentiation in cells that are not spontaneously susceptible to ATRA treatment. In this review, we provide an overview of LSD1 function in normal hematopoiesis and leukemia, and of the current clinical application of LSD1 inhibitors for the treatment of patients with AML.

Lysine-specific demethylase 2A expression is associated with cell growth and cyclin D1 expression in colorectal adenocarcinoma.

Lysine-specific demethylase 2A (KDM2A), a specific H3K36me1/2 demethylase, has been reported to be closely associated with several types of cancer. In this study, we aimed to investigate the expression and function of KDM2A in colorectal adenocarcinoma. A total of 215 colorectal adenocarcinoma specimens were collected, and then subjected to immunohistochemistry assay to evaluate the expression levels of KDM2A, cyclin D1 and other proteins in colorectal adenocarcinoma tissues. Real-time polymerase chain reaction, Western blot, and other molecular biology methods were used to explore the role of KDM2A in colorectal adenocarcinoma cells. In this study, we report that the expression level of KDM2A is high in colorectal adenocarcinoma tissues, and this high expression promotes the proliferation and colony formation of colorectal adenocarcinoma cells, as demonstrated by KDM2A knockdown experiments. In addition, the expression of KDM2A is closely associated with cyclin D1 expression in colorectal adenocarcinoma tissues and cell lines. Our study reveals a novel role for high-expressed KDM2A in colorectal adenocarcinoma cell growth, and that the expression of KDM2A is associated with that of cyclin D1 in colorectal adenocarcinoma.

Abstract A33: Histone demethylase KDM3A in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children, adolescents, and young adults, presenting a myogenic phenotype. RMS can be divided into two major histopathologic subtypes: driver oncofusion (PAX3/7-FOXO1) positive, alveolar (ARMS), and oncofusion-negative embryonal (ERMS). Histone methylation is a key element of chromatin-based modifications that regulates a number of cellular processes, including DNA replication, DNA repair, and gene transcription. Histone demethylase gene family members (HDMs) regulate gene expression by removing methyl marks on histone tails to activate or repress transcription. KDM3A (lysine-specific demethylase 3A or JMJD1A) is a histone demethylase belonging to the Jumonji C-terminal domain (JmjC)-containing histone demethylases that selectively removes mono- and dimethyl groups from H3K9. It has been demonstrated that KDM3A is overexpressed in many kinds of cancer, such as bladder, lung, and breast cancer, and, previously shown by our laboratory, in Ewing sarcoma, contributing to carcinogenesis. We and others have identified KDM3A as overexpressed in RMS, but the functional consequences of this overexpression have not been examined. The aim of this study was to analyze the influence of KDM3A on RMS biology. Two different RMS cells lines were used, RH30 (ARMS) and RD (ERMS), to perform stable shRNA-mediated KDM3A knockdown. The protein and RNA levels were analyzed by Western blotting and RT-qPCR, respectively. The phenotypic effects of KDM3A knockdown were analyzed by proliferation assays (MTT and clonogenic assay) and cell migration (transwell migration assay). KDM3A knockdown was able to inhibit the proliferation, colony formation, and migration of RMS cell lines, demonstrating an important role in RMS biology. Citation Format: Lays M. Sobral, Paul Jedlicka. Histone demethylase KDM3A in rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A33.

Sumoylation of the histone demethylase KDM4A is required for binding to tumor suppressor p53 in HCT116 colon cancer cell lines

ABSTRACTThe histone demethylase lysine-specific demethylase 4A (KDM4A/Jmjd2A) has diverse functions, including involvement in gene regulation and cell cycle, and plays an oncogenic role in cancer cells. The modulation of KDM4A through post-translational modifications remains unclear. Here, we show that small ubiquitin-like modifier (SUMO) 1-mediated modification of KDM4A was required for interaction with tumor suppressor p53. Our data revealed that KDM4A is mainly sumoylated at lysine residue 471. However, the SUMO modification resulted in little change in subcellular localization, demethylase activity, or protein stability of KMD4A. Intriguingly, co-immunoprecipitation data revealed that sumoylation-defective mutants of KDM4A had a lower binding ability with p53 compared to that of wild-type KDM4A, suggesting a positive role for sumoylation in the interaction between KDM4A and p53. Together, these data suggest that KDM4A is post-translationally modified by SUMO, and this sumoylation may be a novel regulatory switch for controlling the interplay between KDM4A and p53.

Expanding the Oro-Dental and Mutational Spectra of Kabuki Syndrome and Expression of KMT2D and KDM6A in Human Tooth Germs.

Kabuki syndrome is a rare genetic disorder characterized by distinct dysmorphic facial features, intellectual disability, and multiple developmental abnormalities. Despite more than 350 documented cases, the oro-dental spectrum associated with kabuki syndrome and expression of KMT2D (histone-lysine N-methyltransferase 2D) or KDM6A (lysine-specific demethylase 6A) genes in tooth development have not been well defined. Here, we report seven unrelated Thai patients with Kabuki syndrome having congenital absence of teeth, malocclusion, high-arched palate, micrognathia, and deviated tooth shape and size. Exome sequencing successfully identified that six patients were heterozygous for mutations in KMT2D, and one in KDM6A. Six were novel mutations, of which five were in KMT2D and one in KDM6A. They were truncating mutations including four frameshift deletions and two nonsense mutations. The predicted non-functional KMT2D and KDM6A proteins are expected to cause disease by haploinsufficiency. Our study expands oro-dental, medical, and mutational spectra associated with Kabuki syndrome. We also demonstrate for the first time that KMT2D and KDM6A are expressed in the dental epithelium of human tooth germs.

Abstract A169: Combination of TPC-144, a reversible LSD1 inhibitor, and a hypomethylating agent resulted in synergistic antitumor efficacy in preclinical models of AML

Abstract Background: Lysine-specific demethylase 1A (LSD1/KDM1A) is a flavin adenine dinucleotide (FAD)-dependent histone demethylase that specifically modifies histone H3 lysine 4 and lysine 9. LSD1 activity is implicated in the pathogenesis of several human cancers, and recent studies indicated that the inhibition of LSD1 is a promising therapeutic strategy for acute myeloid leukemia (AML). Decitabine and azacitidine, FDA-approved agents for the treatment of AML and myelodysplastic syndrome, alter global DNA methylation by inhibiting DNA methyltransferases (DNMTs), resulting in apoptosis and differentiation. Since it is reported that LSD1 directly demethylates and stabilizes DNMT1 by protection from degradation, we hypothesized that an LSD1 inhibitor could augment anti-AML activity of DNMT inhibitors. Previously, we demonstrated that TPC-144, a novel reversible LSD1 inhibitor, was efficacious in preclinical models of AML. Here, we report the activity of TPC-144 in combination with decitabine in cell-based and in vivo studies. Material and Methods: In vitro studies on growth inhibition and apoptosis were conducted using AML cell lines by measuring cellular ATP content (Cell-Titer Glo, Promega) and sub-G1 population, respectively. For expression changes of surface markers, cells were stained with specific antibodies (BD Pharmingen) and analyzed by flow cytometry. mRNA levels of LSD1 target genes were quantitated by real-time PCR. LINE-1 methylation levels were evaluated in Global DNA Methylation-LINE-1 Kit (Active Motif). In vivo efficacy was evaluated in human AML xenograft models in SCID mice. TPC-144 and decitabine was administered orally or intraperitoneally, respectively. Results: The combination of TPC-144 and decitabine showed synergistic growth inhibition of human AML cells in vitro. TPC-144 enhanced apoptosis induction in combination with low-dose decitabine at the concentrations at which TPC-144 decreased DNMT1 protein levels and LINE-1 methylation levels. Combination treatment strongly increased expression of surface markers of differentiation such as CD86 and CD11b, compared with single-agent treatment. In AML xenograft models, the combination of TPC-144 and decitabine was tolerated without signs of hematopoietic toxicities, and demonstrated curative antitumor efficacy compared with each inhibitor alone in a decitabine-sensitive xenograft model. Importantly, the combination treatment of TPC-144 and decitabine exhibited strong antitumor efficacy even in a decitabine-insensitive AML model. Conclusions: TPC-144, a novel potent and selective reversible LSD1 inhibitor, demonstrated synergistic antitumor efficacy in combination with decitabine in human AML cell lines and xenograft models. This combination was well tolerated and showed strong efficacy not only in decitabine-sensitive but also in decitabine-insensitive AML models. Therefore, combination of TPC-144 and decitabine could enhance their therapeutic value for the treatment of AML patients. Citation Format: Akiko Osada, Aki Kawagishi, Ryo Hatanaka, Takumitsu Machida, Kenjiro Ito, Satoshi Yamashita, Takahiro Ogawa, Tadashi Imaoka, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. Combination of TPC-144, a reversible LSD1 inhibitor, and a hypomethylating agent resulted in synergistic antitumor efficacy in preclinical models of AML [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A169.

Synergistic anti-AML effects of the LSD1 inhibitor T-3775440 and the NEDD8-activating enzyme inhibitor pevonedistat via transdifferentiation and DNA rereplication

Lysine-specific demethylase 1A (LSD1, KDM1A) specifically demethylates di- and monomethylated histones H3K4 and K9, resulting in context-dependent transcriptional repression or activation. We previously identified an irreversible LSD1 inhibitor T-3775440, which exerts antileukemic activities in a subset of acute myeloid leukemia (AML) cell lines by inducing cell transdifferentiation. The NEDD8-activating enzyme inhibitor pevonedistat (MLN4924, TAK-924) is an investigational drug with antiproliferative activities in AML, and is also reported to induce cell differentiation. We therefore tested the combination of these two agents in AML models. The combination treatment resulted in synergistic growth inhibition of AML cells, accompanied by enhanced transdifferentiation of an erythroid leukemia lineage into granulomonocytic-like lineage cells. In addition, pevonedistat-induced rereplication stress during the S phase was greatly augmented by concomitant treatment with T-3775440, as reflected by the increased induction of apoptosis. We further demonstrated that the combination treatment was markedly effective in subcutaneous tumor xenograft models as well as in a disseminated model of AML, leading to tumor eradication or prolonged survival in T-3775440/pevonedistat cotreated mice. Our findings indicate the therapeutic potential of the combination of LSD1 inhibitors and pevonedistat for the treatment of AML.

Abstract 4052: Enhancing estrogen receptor beta mediated tumor suppression by lysine specific demethylase 1A inhibitor for the treatment of ovarian cancer

Abstract Ovarian cancer (OCa) is the deadliest of all gynecologic cancers in the United States. Although patients with OCa respond initially to standard combinations of surgical and cytotoxic therapy, nearly 90% will develop recurrence and inevitably succumb to chemotherapy-resistant disease. Estrogen mediates its effects through the estrogen receptor α (ESR1) and estrogen receptor β (ESR2). Recent studies suggest that OCa cells and OCa stem cells express ESR2, which functions as a tumor suppressor and identified synthetic compound LY500307 as a potent ESR2 specific agonist. However, ESR2 expression decreases during tumor progression and under the selection pressure of chemotherapy; this decrease occurs via epigenetic mechanisms. The lysine-specific histone demethylase 1A (KDM1A/LSD1) is overexpressed in OCa and plays a vital role in metastasis and cancer stemness. Our group recently developed a novel KDM1A-specific inhibitor (NCD-38) based on an innovative concept of direct delivery of phenylcyclopropylamine to the KDM1A active site. We reason that agents that restore the expression and functions of ESR2 by increasing active histone marks may provide a novel therapeutic opportunity to reactivate ESR2 for suppression of OCa. The objective of this study is to test whether KDM1A inhibitor could enhance the expression and tumor suppressive functions of ESR2 and to test the combination of KDM1A inhibitor along with ESR2 agonist using in vitro and in vivo preclinical models. Our results demonstrated that combination of ESR2 agonist and KDM1A inhibitor reduces the cell viability of OCa cells synergistically. Further caspase-3/7 activity and Annexin V assays showed synergistic induction of apoptosis upon combination therapy. Matrigel invasion assays revealed reduction in cell invasion of OCa cell lines following ESR2 agonist and KDM1A inhibitor treatment. RT-qPCR analysis demonstrated that KDM1A inhibitor treatment significantly increases the expression of ESR2 and its target genes leading to increased tumor suppression. Further, co-immunoprecipitation analysis revealed that KDM1A interacts with ESR2 and Chromatin immunoprecipitation assays demonstrated that KDM1A inhibition increases the recruitment of active histone methylation mark H3K4-me2 at ESR2 0N promoter. RNA-sequencing analysis demonstrated that ESR2 agonist treatment modulates the several pathways related to cell cycle, apoptosis and DNA damage check point regulation. Finally, ESR2 agonist and KDM1A inhibitor treatment significantly reduced the in vivo tumor growth in orthotopic models. Since ESR2 agonists and KDM1A inhibitors are currently in clinical trials for other clinical indications and are well tolerated, identification of KDM1A inhibitor and ESR2 agonist combination therapy as a novel therapeutic can be readily transferred to clinical use for enhancing survival in OCa patients. Citation Format: Gangadhara Reddy R. Sareddy, Jinyou Liu, Lauren Garcia, Suryavathi Viswanadhapalli, Mei Zhou, Takayoshi Suzuki, Edward Kost, Ratna K. Vadlamudi. Enhancing estrogen receptor beta mediated tumor suppression by lysine specific demethylase 1A inhibitor for the treatment of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4052. doi:10.1158/1538-7445.AM2017-4052

Genome-Wide Association Study Identifies Loci Associated with Resistance to Viral Nervous Necrosis Disease in Asian Seabass.

Viral nervous necrosis disease (VNN), caused by nervous necrosis virus (NNV), is one major threat to mariculture. Identifying loci and understanding the mechanisms associated with resistance to VNN are important in selective breeding programs. We performed a genome-wide association study (GWAS) using genotyping-by-sequencing (GBS) to study the genomic architecture of resistance to NNV infection in Asian seabass. We genotyped 986 individuals from 43 families produced by 15 founders with 44498 bi-allelic genetic variants using GBS. The GWAS identified three genome-wide significant loci on chromosomes 16, 19, and 20, respectively, and six suggestive loci on chromosomes 1, 8, 14, 15, 21, and 24, respectively, associated with resistance to NNV infection measured as binary and quantitative traits. Using the 500 most significant markers in combination with a training population of 800 samples could reach a genomic prediction accuracy of 0.7. Candidate genes significantly associated with resistance to NNV, including lysine-specific demethylase 2A, beta-defensin 1, and cystatin-B, which play important roles in immune responses against virus infection, were identified. Almost all the candidate genes were differentially expressed in different tissues against NNV infection. The significant genetic variants can be used in genomic selection and help understand the mechanism of resistance to VNN. Future studies should use populations of large effective size and whole genome resequencing to identify more useful genetic variants.

Androgen and AR contribute to breast cancer development and metastasis: an insight of mechanisms.

The role of androgen and androgen receptor (AR) in breast carcinogenesis has long been a disputed issue. This report provides a mechanistic insight into how androgen and AR contributes to invasion and metastasis of breast cancer. We find that dihydrotestosterone (DHT) is able to induce the epithelial-to-mesenchymal transition in breast cancer cells in an AR-dependent/estrogen receptor-independent manner. This process is dependent on the demethylation activity of lysine-specific demethylase 1A (LSD1) by epigenetically regulating the target genes E-cadherin and vimentin. In vivo, DHT promotes metastasis in a nude mouse model, and AR and LSD1 are indispensable in this process. We establish that higher expression of nucleus AR to cytoplasm AR associated with worse prognostic outcomes in breast cancer patient samples. This study maps an 'androgen-AR/LSD1-target genes' pathway in breast carcinogenesis, implicating the importance of hormonal balance in women, and the potential clinical significance of serum androgen and AR in prediction of breast cancer and selection of breast cancer therapy.

Downregulation of KDM4A Suppresses the Survival of Glioma Cells by Promoting Autophagy.

Glioma is the most common type of primary intracranial tumor and has a poor prognosis. It has been reported that lysine-specific demethylase 4A (KDM4A) can promote tumor progression; however, its role in human glioma remains unclear. Western blot and qRT-PCR analyses showed that KDM4A was highly expressed in U87MG and T98G cells. 48h after transfection with siKDM4A, the protein level of KDM4A was significantly downregulated. The silenced expression of KDM4A in T98G or U87MG cells inhibited cell viability and invasion, and aggravated cell apoptosis. We found that the siKDM4A led to a significant increase in acidic vesicular organelles (AVOs) and upregulated the expression of autophagy-related proteins, including LC3B-phosphatidylethanolamine conjugate, a cytosolic form of LC3B (LC3B-II/LC3B-I) and Beclin 1 in T98G and U87MG cells. Further studies demonstrated that after pretreatment with 3-MA (3mmol/L) for 48h, siKDM4A-transfected cells showed a prominent decrease in LC3B-II/LC3B-I and Beclin 1, accompanied by increased viability and invasion and decreased apoptosis. Our results suggest that the inhibition of KDM4A expression might efficiently suppress glioma cell survival by promoting autophagy, providing a promising agent for treating malignant gliomas.

Transposon mutagenesis identifies genes and cellular processes driving epithelial-mesenchymal transition in hepatocellular carcinoma

Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), leading to their poor prognosis. The genes driving EMT in HCC are not yet fully understood, however. Here, we show that mobilization of Sleeping Beauty (SB) transposons in immortalized mouse hepatoblasts induces mesenchymal liver tumors on transplantation to nude mice. These tumors show significant down-regulation of epithelial markers, along with up-regulation of mesenchymal markers and EMT-related transcription factors (EMT-TFs). Sequencing of transposon insertion sites from tumors identified 233 candidate cancer genes (CCGs) that were enriched for genes and cellular processes driving EMT. Subsequent trunk driver analysis identified 23 CCGs that are predicted to function early in tumorigenesis and whose mutation or alteration in patients with HCC is correlated with poor patient survival. Validation of the top trunk drivers identified in the screen, including MET (MET proto-oncogene, receptor tyrosine kinase), GRB2-associated binding protein 1 (GAB1), HECT, UBA, and WWE domain containing 1 (HUWE1), lysine-specific demethylase 6A (KDM6A), and protein-tyrosine phosphatase, nonreceptor-type 12 (PTPN12), showed that deregulation of these genes activates an EMT program in human HCC cells that enhances tumor cell migration. Finally, deregulation of these genes in human HCC was found to confer sorafenib resistance through apoptotic tolerance and reduced proliferation, consistent with recent studies showing that EMT contributes to the chemoresistance of tumor cells. Our unique cell-based transposon mutagenesis screen appears to be an excellent resource for discovering genes involved in EMT in human HCC and potentially for identifying new drug targets.

594 Histone Lysine-Specific Demethylase 1A inhibitor sensitizes keratinocytes to ultraviolet radiation

594 Histone Lysine-Specific Demethylase 1A inhibitor sensitizes keratinocytes to ultraviolet radiation

Lysine-Specific Demethylase 1A (KDM1A/LSD1): Product Recognition and Kinetic Analysis of Full-Length Histones.

Lysine-specific demethylase 1A (KDM1A/LSD1) is a FAD-dependent enzyme that catalyzes the oxidative demethylation of histone H3K4me1/2 and H3K9me1/2 repressing and activating transcription, respectively. Although the active site is expanded compared to that of members of the greater amine oxidase superfamily, it is too sterically restricted to encompass the minimal 21-mer peptide substrate footprint. The remainder of the substrate/product is therefore expected to extend along the surface of KDM1A. We show that full-length histone H3, which lacks any posttranslational modifications, is a tight-binding, competitive inhibitor of KDM1A demethylation activity with a Ki of 18.9 ± 1.2 nM, a value that is approximately 100-fold higher than that of the 21-mer peptide product. The relative H3 affinity is independent of preincubation time, suggesting that H3 rapidly reaches equilibrium with KDM1A. Jump dilution experiments confirmed the increased binding affinity of full-length H3 was at least partially due to a slow off rate (koff) of 1.2 × 10(-3) s(-1), corresponding to a half-life (t1/2) of 9.63 min, and a residence time (τ) of 13.9 min. Independent affinity capture surface plasmon resonance experiments confirmed the tight-binding nature of the H3/KDM1A interaction, revealing a Kd of 9.02 ± 2.3 nM, a kon of (9.3 ± 1.5) × 10(4) M(-1) s(-1), and a koff of (8.4 ± 0.3) × 10(-4) s(-1). Additionally, no other core histones exhibited inhibition of KDM1A demethylation activity, which is consistent with H3 being the preferred histone substrate of KDM1A versus H2A, H2B, and H4. Together, these data suggest that KDM1A likely contains a histone H3 secondary specificity element on the enzyme surface that contributes significantly to its recognition of substrates and products.

Abstract A52: Gene alterations associated with clinical characteristics of bladder cancer

Abstract Next-generation sequencing (NGS) of urologic tumor genomes has identified frequently altered cancer genes. We recently examined the exomes of bladder tumors (BCa) from 54 U.S. patients and 99 Chinese patients to identify novel, altered cancer genes associated with clinical characteristics of disease. Exome capture of DNA from tumor and normal urothelial tissue using probes for ~180,000 protein-coding exons and microRNA loci; DNA fragment libraries were sequenced on a HiSeq 2000 platform (Illumina); and base calls on paired-end, 100 bp reads were generated using the Genome Analyzer Pipeline, v. 1.3 and standard parameters. Gene expression was analyzed using quantitative PCR, telomere length by a standard assay, and KDM6A function using cell-based assays in T24T BCa cells. Copy number variation (CNV), aneuploidy, and CpG methylation were analyzed as recently described. Telomerase (TERT) was the most frequently altered by somatic sequence changes in 37/54 (69%) of U.S. patient tumors. The TERT promoter was altered by frequent germline nucleotide changes in 30/54 (56%) tumors. Most of the TERT germline variants were novel (19/20) and three variants were confirmed in distinct tumors as either somatic or germline, including the most frequent variant, c. –245 T>C (rs2853669). The somatic TERT promoter alterations did not correlate with other somatic BCa gene alterations, but were associated with increased TERT expression and with significantly shorter telomeres in tumors as compared to matched normal tissue. The stromal antigen 2 (STAG2) gene was the most frequently altered novel BCa gene in Chinese patients, by somatic, deleterious sequence changes in 11% of tumors; CNV deletions (5%); and promoter CpG hypermethylation leading to presumed expression silencing (7/30, 23%). STAG2 encodes a component of the cohesion complex involved in sister chromatid cohesion and segregation. STAG2 mutations were associated with an increased number of chromosomal arm copy number changes corresponding to higher tumor cell aneuploidy, and mutations were associated with a significantly reduced patient survival by Kaplan-Meier survival analysis. In total, 32/99 (32%) of tumors displayed SCCS pathway gene alterations. The most frequently altered epigenetic gene was the histone lysine-specific demethylase 6A (KDM6A/UTX) in 24% of U.S. and 30% of Chinese patient tumors. Experimentally, we show KDM6A loss in human BCa T24T cells enhanced in vitro proliferation and cell migration, and in vivo tumor growth in mice. Re-expression reversed these effects, confirming KDM6A loss drives the BCa phenotype. Somatic KDM6A alterations were associated with somatic BRCA1-associated protein-1 (BAP1) alterations observed in 15% of U.S. tumors. Somatic, deleterious sequence changes occurred preferentially in U.S. Caucasian as compared to Chinese BCa patient tumors; are associated with papillary histologic features in a subset of tumors; and contribute to a significant number of tumors (10/14, 71%) that displayed alteration of genes encoding BRCA DNA repair pathway proteins. Genes encoding BRCA pathway proteins, including BAP1, BRCA1, BRCA2, PALB2, and ATM were altered by a combination of somatic and rare germline variants. We identified altered genes associated with clinical characteristics of BCa, including ‘immortalization' (TERT), aneuploidy (STAG2), proliferation (KDM6A), and papillary features in tumors (BAP1). STAG2 alterations are associated with a reduced patient survival, indicating a lethal subtype of disease; and altered X chromosome genes (STAG2 and KDM6A) are single copy in males and likely contribute to the cancer gender bias. Finally, we identified BRCA pathway alterations in BCa that are similar to those in breast, ovarian, prostate and kidney tumors, that have a common DNA repair deficiency that can be exploited by therapy targeting poly (ADP ribose) polymerase. Citation Format: Mike Nickerson, Kate M. Im, Guangwu Guo, Yaoting Gui, Sevilay Turan, James C. Costello, Quan Zhou, Zhiming Cai, Song Wu, M. Scott Lucia, Lee E. Moore, Michael Dean, Dan Theodorescu. Gene alterations associated with clinical characteristics of bladder cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A52.

Discovery of a Novel Inhibitor of Histone Lysine-Specific Demethylase 1A (KDM1A/LSD1) as Orally Active Antitumor Agent

We report the stereoselective synthesis and biological activity of a novel series of tranylcypromine (TCPA) derivatives (14a-k, 15, 16), potent inhibitors of KDM1A. The new compounds strongly inhibit the clonogenic potential of acute leukemia cell lines. In particular three molecules (14d, 14e, and 14g) showing selectivity versus MAO A and remarkably inhibiting colony formation in THP-1 human leukemia cells, were assessed in mouse for their preliminary pharmacokinetic. 14d and 14e were further tested in vivo in a murine acute promyelocytic leukemia model, resulting 14d the most effective. Its two enantiomers were synthesized: the (1S,2R) enantiomer 15 showed higher activity than its (1R,2S) analogue 16, in both biochemical and cellular assays. Compound 15 exhibited in vivo efficacy after oral administration, determining a 62% increased survival in mouse leukemia model with evidence of KDM1A inhibition. The biological profile of compound 15 supports its further investigation as a cancer therapeutic.