Lysine-specific Demethylase 1 Overexpression Research Articles

The Epigenetic Controller Lysine-Specific Demethylase 1 (LSD1) Regulates the Outcome of Hepatitis C Viral Infection.

Hepatitis C virus (HCV) alters gene expression epigenetically to rearrange the cellular microenvironment in a beneficial way for its life cycle. The host epigenetic changes induced by HCV lead to metabolic dysfunction and malignant transformation. Lysine-specific demethylase 1 (LSD1) is an epigenetic controller of critical cellular functions that are essential for HCV propagation. We investigated the putative role of LSD1 in the establishment of HCV infection using genetic engineering and pharmacological inhibition to alter endogenous LSD1 levels. We demonstrated for the first time that HCV replication was inhibited in LSD1-overexpressing cells, while specific HCV proteins differentially fine-tuned endogenous LSD1 expression levels. Electroporation of the full-length HCV genome and subgenomic replicons in LSD1 overexpression enhanced translation and partially restored HCV replication, suggesting that HCV might be inhibited by LSD1 during the early steps of infection. Conversely, the inhibition of LSD1, followed by HCV infection in vitro, increased viral replication. LSD1 was shown to participate in an intriguing antiviral mechanism, where it activates endolysosomal interferon-induced transmembrane protein 3 (IFITM3) via demethylation, leading endocytosed HCV virions to degradation. Our study proposes that HCV-mediated LSD1 oscillations over countless viral life cycles throughout chronic HCV infection may promote epigenetic changes related to HCV-induced hepatocarcinogenesis.

LSD1 regulates the expressions of core cardiogenic transcription factors and cardiac genes in oxygen and glucose deprivation injured mice fibroblasts in vitro

Cardiac reprogramming has emerged as a novel therapeutic approach to regenerating the damaged heart by directly converting endogenous cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs). Cardiac reprogramming requires the activation of the cardiogenic transcriptional program in concert with the repression of the fibroblastic transcriptional program. Lysine-specific demethylase 1 (LSD1) plays an instrumental role in many physiological processes such as cell growth, differentiation and metabolism. The epigenetic modifications of histones are essential for the accurate expression of genes in cardiomyocytes and the normal functioning of the heart. However, the effect of LSD1 in regulating the cardiogenic transcriptional program under myocardial ischemia/reperfusion (I/R) injury remains unclear. Thus, mice I/R injury was induced by 4 and 24 h reperfusion after 1-h occlusion of the left anterior descending coronary artery. The primary CFs and CMs were exposed under oxygen and glucose deprivation (OGD) to mimic I/R injury. The expression of LSD1 significantly decreased in I/R injured heart tissue and OGD-injured primary CFs and CM, and methylated histone presented a notable increase in OGD-injured primary CFs. Overexpression of LSD1 inhibited the injury of primary CFs induced by OGD, but showed limited inhibition on injured primary CMs. Under the OGD condition, LSD1 overexpression significantly increased cell viability, decreased cell apoptosis and reactive oxygen species (ROS) production of primary CFs. The expression of core cardiogenic transcription factors and cardiac genes were significantly decreased in OGD injured primary CFs, whereas LSD1 overexpression reversed the decrease of transcription factors and cardiac genes under the OGD condition. In conclusion, the overexpression of LSD1 has a protective role in I/R injury by inhibiting the histone methylation of primary CFs and regulates the expressions of core cardiogenic transcription factors and cardiac genes, which can prove to be a potential approach for direct cardiac reprogramming.

LSD1 downregulates p21 expression in vascular smooth muscle cells and promotes neointima formation

Neointima formation is characterized by the proliferation of vascular smooth muscle cells (VSMC). Although lysine-specific demethylase 1 (LSD1) has critical functions in several diseases, its role in neointima formation remains to be clarified. In this study, we aimed to explore the crucial role of LSD1 on neointima formation using a carotid artery injury model in mice. We observed that aberrant LSD1 expression was increased in human and mouse stenotic arteries and platelet-derived growth factor-BB (PDGF-BB)-treated VSMC. Furthermore, LSD1 knockdown significantly mitigated neointima formation in vivo and inhibited PDGF-BB-induced VSMC proliferation in vitro. We further uncovered that LSD1 overexpression exhibited opposite phenotypes in vivo and in vitro. Finally, LSD1 knockdown inhibited VSMC proliferation by increasing p21 expression, which is associated with LSD1 mediated di-methylated histone H3 on lysine 4 (H3K4me2) modification. Taken together, our data suggest that LSD1 may be a potential therapeutic target for the treatment of neointima formation.

Lysine-specific demethylase 1 induced epithelial-mesenchymal transition and promoted renal fibrosis through Jagged-1/Notch signaling pathway.

TGF-β1-induced excessive deposition of extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT) process of tubular epithelial cells play critical roles in the progression of renal fibrosis. We are aimed to explore the effects of lysine-specific demethylase 1 (LSD1) in TGF-β1-treated HK-2 cells and in rats with unilateral ureteral obstruction (UUO), and to investigate the underlying molecular mechanism. TGF-β1-treated HK-2 cells and UUO-treated rats were used to establish the model of renal fibrosis in vitro and in vivo, respectively. Protein expression of LSD1, E-cadherin, a-smooth muscle actin (a-SMA), Vimentin, Jagged-1, Notch-1 and Notch-2 were detected by Western blot. The concentrations of type I collagen (Col-I) and Fibronectin (FN) were measured by ELISA. Transwell assay were used to assess cell invasion. LSD1 was dramatically increased in TGF-β1-stimulated HK-2 cells. Knockdown of LSD1 decreased the TGF-β1-induced secretion of Col-I and FN, and suppressed TGF-β1-induced expression of E-cadherin,α-SMA and Vimentin, while suppressed cell invasion. Consistent with the in vitro data, the severe histopathological damage, collagen deposition and reduced E-cadherin, increased α-SMA induced by UUO was abated by the knockdown of LSD1 in vivo. Moreover, knockdown of LSD1 suppressed TGF-β1-induced expression of Jagged-1, Notch-1 and Notch-2. Furthermore, we found that inhibition of Notch signaling by a γ-secretase inhibitor RO4929097 almost recapitulated the effects of LSD1 knockdown in TGF-β1-induced HK-2 cells, and at least in part reversed the effects of LSD1 overexpression on EMT and ECM deposition in HK-2 cells. Taken together, LSD1 significantly impact on the progression of TGF-β1-mediated EMT and ECM deposition in HK-2 cells, and it may represent novel target for the prevention strategies of renal fibrosis.

Abstract 3833: Antitumor activity of novel reversible LSD1 inhibitor in preclinical models of AML and SCLC

Abstract Introduction: Global changes in the epigenetic landscape are known as the hallmark in cancer and the histone demethylase Lysine-specific demethylase 1 (LSD1) is one of the novel targets for the therapy of various malignant diseases. Also LSD1 overexpression is associated with poor prognosis in various cancers. LSD1-mediated epigenetic modification is known to play a key role in the regulation of gene expression by removing the methyl groups from methylated lysine 4 and lysine 9 of histone H3. Alterations in histone methylation lead to aberrant silencing of expression of multiple genes involved in tumor survival and in cell cycle. Moreover, epigenetic dysregulation plays a critical role in pathogenesis of various types of cancers such as acute myeloid leukemia (AML) or small cell lung cancer (SCLC). In this study, we have characterized a novel LSD1 inhibitor and assessed its potential as a novel therapy for AML and SCLC patients. Materials and Methods: Novel LSD1 inhibitor, HM97211 and its derivatives were designed and synthesized as the active biologic inhibitory compound against the AML and SCLC cells. Biochemical enzyme assay was performed to identify enzyme selectivity of HM97211 derivatives. Standard proliferation assay, immunoblotting, and apoptosis analysis were carried out to validate the potency of HM97211 derivatives in AML and SCLC cell lines. In vivo anti-tumor activity was evaluated in AML and SCLC cell xenograft mice models. Tumor sizes were measured and tumor samples were analyzed to define the mechanisms of action. Results: HM97211 derivatives showed higher selectivity toward LSD1 compared to other FAD-utilizing enzyme as well as other epigenetic enzymes. HM97211 derivatives alter expression of various genes and induce differentiation and apoptosis, resulting in growth suppression in a panel of AML and SCLC cells. Moreover, HM97211 derivatives significantly induced programmed cell death in AML and SCLC cells. Oral administration of HM97211 derivatives inhibited tumor growth of human AML and SCLC xenograft models as a single agent at doses exhibiting significant. Efficacy of HM97211 derivatives was further evaluated in combination treatment with standard chemotherapies in human AML and SCLC xenograft model. Conclusion: Collectively, results of this study suggested that novel reversible LSD1 inhibitor, HM97211 derivatives, can be a strong therapeutic agent for AML and SCLC patients. Hanmi presents a new insight to epigenetics application in anticancer therapy. Citation Format: InHwan Bae, WonJeoun Kim, JiSook Kim, JiYoung Song, JungSoo Nam, Moonsub Lee, Jooyun Byun, Hyeongki Kim, TaeHun Song, Seokhyun Hong, KyuHang Lee, YoungGil Ahn, YoungHoon Kim, Kwee Hyun Suh. Antitumor activity of novel reversible LSD1 inhibitor in preclinical models of AML and SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3833.

Eradication of Central Nervous System Leukemia of T-Cell Origin with a Brain-Permeable LSD1 Inhibitor.

Lysine-specific demethylase 1 (LSD1) regulates several biological processes via the bifunctional modulation of enhancer functions. Recently, we reported that LSD1 overexpression is a founder abnormality of T-cell leukemogenesis and is maintained in fully transformed T-cell acute lymphoblastic leukemia (T-ALL) cells. On the basis of this finding, we attempted to develop novel LSD1 inhibitors effective for T-ALL with central nervous system (CNS) involvement. We chemically modified the prototype LSD inhibitor tranylcypromine (TCP) and screened for cytotoxicity against TCP-resistant T-ALL cell lines. In vivo efficacy of novel LSD1 inhibitors was examined in immunodeficient mice transplanted with luciferase-expressing T-ALL cell lines, which faithfully reproduce human T-ALL with CNS involvement. We found robust cytotoxicity against T-ALL cells, but not normal bone marrow progenitors, for two N-alkylated TCP derivatives, S2116 and S2157. The two compounds induced apoptosis in TCP-resistant T-ALL cells in vitro and in vivo by repressing transcription of the NOTCH3 and TAL1 genes through increased H3K9 methylation and reciprocal H3K27 deacetylation at superenhancer regions. Both S2116 and S2157 significantly retarded the growth of T-ALL cells in xenotransplanted mice and prolonged the survival of recipients as monotherapy and in combination with dexamethasone. Notably, S2157 could almost completely eradicate CNS leukemia because of its ability to efficiently pass through the blood-brain barrier. These findings provide a molecular basis and rationale for the inclusion of a brain-permeable LSD1 inhibitor, S2157, in treatment strategies for T-ALL with CNS involvement.

LSD1 inhibition suppresses the growth of clear cell renal cell carcinoma via upregulating P21 signaling

Histone lysine-specific demethylase 1 (LSD1) has been implicated in the disease progression of several types of solid tumors. This study provides the first evidence showing that LSD1 overexpression occurred in 62.6% (224/358) of clear cell renal cell carcinomas (ccRCC). LSD1 expression was associated with the progression of ccRCC, as indicated by TNM stage (P=0.006), especially tumor stage (P=0.017) and lymph node metastasis (P=0.030). High LSD1 expression proved to be an independent prognostic factor for poor overall survival (P<0.001) and recurrence-free survival (P<0.001) of ccRCC patients. We further show that LSD1 inhibition by siRNA knockdown or using the small molecule inhibitor SP2509 suppressed the growth of ccRCC in vitro and in vivo. Mechanistically, inhibition of LSD1 decreased the H3K4 demethylation at the CDKN1A gene promoter, which was associated with P21 upregulation and cell cycle arrest at G1/S in ccRCC cells. Our findings provide new mechanistic insights into the role of LSD1 in ccRCC and suggest the therapeutic potential of LSD1 inhibitors in ccRCC treatment.

Abstract 4871: Antitumor activity of novel reversible LSD1 inhibitor, HM97211 in preclinical models of SCLC

Abstract Introduction: Global changes in the epigenetic landscape are known as the hallmark in cancer and the histone demethylase Lysine-specific demethylase 1 (LSD1) is one of the novel target for the therapy of various malignant diseases and LSD1 overexpression is associated with poor prognosis in various cancers. LSD1-mediated epigenetic modification is known to play a key role in the regulation of gene expression by removing the methyl groups from methylated lysine 4 and lysine 9 of histone H3. Alterations in histone methylation lead to aberrant silencing of expression of multiple genes involved in tumor survival and in cell cycle. Moreover, epigenetic dysregulation plays a critical role in pathogenesis of various types of cancers such as acute myeloid leukemia (AML) or small cell lung cancer (SCLC). Herein, Hanmi introduces, a safe drug profile of the novel LSD1 inhibitor, H97211on hematopoiesis differentiation by reversible binding on LSD1. Materials and Methods: Novel reversible LSD1 inhibitor, HM97211 was designed and synthesized as the active biologic inhibitory compound against the SCLC cells. The binding mode on reversibility for HM97211 and biochemical activity on LSD1 were determined by LSD1 Fluorescent assay kit (BPS Bioscience; Venkataswamy Sorna et al, 2013). In vitro proliferation assay was performed by using the CellTiter-Glo (Promega) In vivo therapeutic efficacy of HM97211 was evaluated in NCI-H1417 xenograft mice models. 5 x 106 NCI-H1417 cells/mouse were implanted subcutaneously and mice were treated by daily oral administration of (30mg/kg) HM97211. Tumor sizes were measured and tumor samples were analyzed of the mechanisms of action. Results: HM97211 enhanced not only global methylation of H3K4 but also apoptotic signaling in NCI-H1417 cell line. HM97211 altered expression of neuroendocrine factor ASCL1, inducing programmed cell death in SCLC cells. In NCI-H1417 xenograft models, daily oral administration of HM97211 induced tumor regression without remarkable toxicity. Analyses of tumor samples revealed the important role of potent small molecule LSD1 inhibitor in neuroendocrine-associated transcription and cell proliferation of SCLC. Conclusion: Collectively, results of this study suggested that novel reversible LSD1 inhibitor, HM97211, can be a strong therapeutic agent for SCLC patients. Hanmi presents a new insight to epigenetics application in anticancer therapy. Citation Format: InHwan Bae, JiSook Kim, ChangHee Park, Hyeongki Kim, Namgoong Gwangmog, Joo-Yun Byun, Seokhyun Hong, Teahun Song, JaeHo Lee, Kyuhang Lee, Myoungsil Ko, Cheolkyung Kim, YoungMi Lee, Ho Jeong Lee, YoungHoon Kim, YoungGil Ahn, KweeHyun Suh, Sun-Jin Kim. Antitumor activity of novel reversible LSD1 inhibitor, HM97211 in preclinical models of SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4871.

LSD1 modulates the non-canonical integrin \u03b23 signaling pathway in non-small cell lung carcinoma cells

The epigenetic writer lysine-specific demethylase 1 (LSD1) is aberrantly upregulated in many cancer types and its overexpression correlates with poor survival and tumor progression. In this study, we analysed LSD1 function in non-small cell lung cancer adenocarcinomas. Expression profiling of 182 cases of lung adenocarcinoma proved a significant correlation of LSD1 overexpression with lung adenocarcinoma progression and metastasis. KRAS-mutated lung cancer cell clones were stably silenced for LSD1 expression. RNA-seq and comprehensive pathway analysis revealed, that genes related to a recently described non-canonical integrin β3 pathway, were significantly downregulated by LSD1 silencing. Hence, invasion and self-renewal capabilities were strongly decreased. Notably, this novel defined LSD1/integrin β3 axis, was also detected in human lung adenocarcinoma specimens. Furthermore, the linkage of LSD1 to an altered expression pattern of lung-lineage specific transcription factors and genes, which are involved in alveolar epithelial differentiation, was demonstrated. Thus, our findings point to a LSD1-integrin β3 axis, conferring attributes of invasiveness and tumor progression to lung adenocarcinoma.

Inhibition of LSD1 epigenetically attenuates oral cancer growth and metastasis.

Lysine-specific demethylase 1 (LSD1) is a nuclear histone demethylase and a member of the amine oxidase (AO) family. LSD1 is a flavin-containing AO that specifically catalyzes the demethylation of mono- and di-methylated histone H3 lysine 4 through an FAD-dependent oxidative reaction. LSD1 is inappropriately upregulated in lung, liver, brain and esophageal cancers, where it promotes cancer initiation, progression, and metastasis. However, unlike other lysine-specific demethylases, the role and specific targets of LSD1 in oral squamous cell carcinoma (OSCC) pathogenesis remain unknown. We show that LSD1 protein expression was increased in malignant OSCC tissues in a clinical tissue microarray, and its expression correlated with progressive tumor stages. In an orthotopic oral cancer mouse model, LSD1 overexpression in aggressive HSC-3 cells promoted metastasis whereas knockdown of LSD1 inhibited tumor spread, suggesting that LSD1 is a key regulator of OSCC metastasis. Pharmacological inhibition of LSD1 using a specific small molecule inhibitor, GSK-LSD1, down-regulated EGF signaling pathway. Further, GSK-LSD1 attenuates CTGF/CCN2, MMP13, LOXL4 and vimentin expression but increased E-cadherin expression in pre-existing, patient-derived tonsillar OSCC xenografts. Similarly, GSK-LSD1 inhibited proliferation and CTGF expression in mesenchymal cells, including myoepithelial cells and osteosarcoma cells. In addition, gene set enrichment analysis revealed that GSK-LSD1 increased p53 expression and apoptosis while inhibiting c-myc, β-catenin and YAP-induced oncogenic transcriptional networks. These data reveal that aberrant LSD1 activation regulates key OSCC microenvironment and EMT promoting factors, including CTGF, LOXL4 and MMP13.

3-(Piperidin-4-ylmethoxy)pyridine Containing Compounds Are Potent Inhibitors of Lysine Specific Demethylase 1.

Methylation of histone lysine residues plays important roles in gene expression regulation as well as cancer initiation. Lysine specific demethylase 1 (LSD1) is responsible for maintaining balanced methylation levels at histone H3 lysine 4 (H3K4). LSD1 is a drug target for certain cancers, due to important functions of methylated H3K4 or LSD1 overexpression. We report the design, synthesis, and structure-activity relationships of 3-(piperidin-4-ylmethoxy)pyridine containing compounds as potent LSD1 inhibitors with Ki values as low as 29 nM. These compounds exhibited high selectivity (>160×) against related monoamine oxidase A and B. Enzyme kinetics and docking studies suggested they are competitive inhibitors against a dimethylated H3K4 substrate and provided a possible binding mode. The potent LSD1 inhibitors can increase cellular H3K4 methylation and strongly inhibit proliferation of several leukemia and solid tumor cells with EC50 values as low as 280 nM, while they had negligible effects on normal cells.

Lysine-specific demethylase 1 promotes tumorigenesis and predicts prognosis in gallbladder cancer.

Gallbladder Cancer (GBC), characterized by invasive growth and infiltrative dissemination, is difficult to diagnose and has poor prognosis. Emerging evidence demonstrates that Lysine-Specific Demethylase 1 (LSD1) has important roles in carcinogenesis, proliferation and metastasis. We studied the roles and molecular mechanisms of LSD1 in GBC. We examined LSD1 expression in 109 paired samples of GBC and normal gallbladder tissues. We found GBC tissues had upregulated LSD1 compared with normal gallbladder tissues (P = 0.003), and its high expression was associated with tumor-node-metastasis stage (P < 0.0001), Nevin's stage (P = 0.0093) and distant metastases (P = 0.0070). We found positive correlations between LSD1 expression and other proteins: epithelial-mesenchymal transition markers, C-myc and cyclin-related proteins. Inhibiting LSD1 expression in vitro impaired the proliferation and invasiveness of GBC cells and also downregulated c-myc expression and consequently inhibited GBC cell proliferation. LSD1 overexpression promotes GBC development and may be a predictor for a worsened prognosis. LSD1 may be a novel therapeutic target and prognostic tool for gallbladder cancer.

Prognostic role of LSD1 in various cancers: evidence from a meta-analysis.

The prognostic value of lysine-specific demethylase 1 (LSD1) overexpression in various cancers has been investigated by many studies with inconsistent results. A meta-analysis was performed to assess the association between LSD1 and overall survival (OS) in cancer patients. Eligible studies were identified by searching the online databases PubMed and China National Knowledge Infrastructure up to February 2015. Hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated to clarify the correlation between LSD1 expression and prognosis of different cancers. In total, nine studies with 1,149 cancer patients were included for final analysis. The meta-analysis suggested that LSD1 overexpression was associated with poor OS in cancer patients (HR =1.80, 95% CI: 1.39–2.34, P=0.000). Subgroup analysis by ethnicity, cancer type and HR estimate also showed that high levels of LSD1 were significantly correlated with OS. The meta-analysis showed that LSD1 overexpression may be associated with a worse prognosis in cancer patients.

Overexpression of the shortest isoform of histone demethylase LSD1 primes hematopoietic stem cells for malignant transformation

AbstractRecent investigations indicate that epigenetic regulators act at the initial step of myeloid leukemogenesis by forming preleukemic hematopoietic stem cells (HSCs), which possess the increased self-renewal potential but retain multidifferentiation ability, and synergize with genetic abnormalities in later stages to develop full-blown acute myeloid leukemias. However, it is still unknown whether this theory is applicable to other malignancies. In this study, we demonstrate that lysine-specific demethylase 1 (LSD1) overexpression is a founder abnormality for the development of T-cell lymphoblastic leukemia/lymphoma (T-LBL) using LSD1 transgenic mice. LSD1 expression is tightly regulated via alternative splicing and transcriptional repression in HSCs and is altered in most leukemias, especially T-LBL. Overexpression of the shortest isoform of LSD1, which is specifically repressed in quiescent HSCs and demethylates histone H3K9 more efficiently than other isoforms, increases self-renewal potential via upregulation of the HoxA family but retains multidifferentiation ability with a skewed differentiation to T-cell lineages at transcriptome levels in HSCs. Transgenic mice overexpressing LSD1 in HSCs did not show obvious abnormalities but developed T-LBL at very high frequency after γ-irradiation. LSD1 overexpression appears to be the first hit in T-cell leukemogenesis and provides an insight into novel strategies for early diagnosis and effective treatment of the disease.

LSD1 Impairs the Epithelial-Mesenchymal Transition (EMT) and Osteoclastogenesis Potency in Multiple Myeloma and Synergistically Induces Cytotoxicity with HDAC Inhibitors

Lysine-specific demethylase 1 (LSD1) is a FAD-dependent histone demethylase, which selectively removes mono- and di-methyl groups from histone 3 lysine 4 or 9 residues (H3K4, H3K9) leading to either repression or activation of transcriptome. Previous studies have shown that lenalidomide and pomalidomide cause cell cycle arrest in Multiple Myeloma (MM) by modifying the chromatin structure of the p21WAF-1 promoter through LSD1 demethylation. LSD1 forms a co-repression complex with HDAC1 and HDAC2, mSin3a, and MMSET. However, the functional role of LSD1 in MM and its contribution in aggressive traits of the disease is largely unknown.First, we evaluated the expression of LSD1 in different datasets of MM patients (GSE2113, GSE16122) and observed significant overexpression in patients with symptomatic MM and Plasma Cell Leukemia (PCL) (p<.001). The expression of LSD1 in a panel 45 HMCLs was also pronounced. We confirmed the expression and both its nuclear and cytoplasmic localization by immunoblotting analysis in 4 different HMCLs and primary bone marrow plasma cells from newly diagnosed, relapsed MM and PCL patients (N=8).We further evaluated the LSD1-mediated effect on proliferation and survival by performing loss- and gain of function studies. LSD1 knockdown in LP1 and MM1S cells resulted in modest cytotoxicity. After a combination silencing of JARID1 members and LSD1 we were able to observe a further significant decrease in survival of MM cells lacking JARID1C and LSD1, indicating that the overlapping demethylation of H3K4 is of high importance for the cell survival.We examined the post-translational histone modifications by immunobloting after LSD1 knockdown and as expected, we observed significant increase of K4me2/3 and K9me2 marks, but more interestingly, alteration of acetylation status of K9. Therefore, we performed cytotoxicity and proliferation experiments in MM after knockdown of LSD1 in combination with HDAC inhibitors (SAHA, LBH589) and we observed that LSD1 depletion enhances the cytotoxicity effect of HDACs inhibitors. LSD1 depletion resulted in significant reduction of mRNA levels by using real-time PCR and protein expression by immunoblotting of HDAC1 and HDAC2.Furthermore, based on findings of higher expression of LSD1 in more aggressive types of MM, we sought to investigate the impact of LSD1 in epithelial-mesenchymal transition (EMT). LSD1 depletion in MM1S and LP1 cells inhibited significantly the migratory ability estimated by transwell migration assay, invasion and wound healing assays. More importantly, MM cells lacking LSD1 expressed significant lower levels of E-cadherin, N-cadherin and Vimentin evaluated by immunoblotting and immunocytochemistry. We confirmed the suppression of EMT-involved gene expression by performing a PCR-microarray assay.Finally, given the presence of osteolytic lesions as a hallmark of disease, and consequent impact on outcome, we evaluated the impact of LSD1 on osteoblast differentiation and osteoclastogenesis. LSD1 depletion/ and pharmacological inhibition (S2101) resulted in significant inhibition of osteoclastogenesis and RANKL-induced resorption evaluated by double TRAP/ALP staining, survival of OCs, and mRNA expression level of osteoblast markers (APL, BSP, OC). In contrast, LSD1 overexpression confirmed the upregulation of Wnt/b-catenin pathway suggesting a possible underlying mechanism for the osteoclastogenesis potency in MM patients with high expression of LSD1.Taken together, our findings demonstrate a promising epigenetic approach in myeloma therapeutics by targeting the deregulated LSD1-methylome in MM patients earlier than aggressive disease phase. DisclosuresNo relevant conflicts of interest to declare.

Abstract 3679: Inhibition of LSD1 disrupts global EWS/ETS transcriptional function in Ewing sarcoma

Abstract Ewing sarcoma is an aggressive pediatric bone tumor characterized by an absolute reliance on the transcriptional activity of the EWS/ETS family of transcription factor fusion oncoproteins. The most common fusion is EWS/FLI, present in 85-95% of cases, though less common fusions include EWS/ERG, EWS/ETV1, EWS/ETV4 and EWS/FEV. EWS/FLI utilizes lysine-specific demethylase 1 (LSD1) as member of the NuRD complex to mediate transcriptional repression of critical tumor suppressors in Ewing sarcoma. LSD1 overexpression has been observed in clinical samples from Ewing sarcoma patients and small molecule blockade of LSD1 using tranylcypromine has been suggested as a therapeutic approach for Ewing sarcoma. We therefore evaluated the effects of LSD1 inhibition with the small molecule inhibitor, HCI-2509, in both in vitro and in vivo models of Ewing sarcoma. HCI-2509 is an LSD1 inhibitor with a Ki of ∼30 nM and multiple Ewing sarcoma cell lines show IC50s in cell viability assays from 500 nM-1 μM. Using RNA-seq, we show that HCI-2509 dramatically reverses both the up- and downregulated transcriptional profiles of both EWS/FLI and EWS/ERG accompanied by the induction of apoptosis and disruption of oncogenic phenotypes modulated by EWS/FLI. We further developed a 9-gene panel based on the RNA-seq data to assess the transcriptional phenotype of HCI-2509 in additional cell lines and showed that it HCI-2509 disrupted both EWS/FLI-activated and -repressed genes similarly across the tested Ewing sarcoma cell lines. HCI-2509 impaired transformation of Ewing sarcoma cell lines in colony forming assays with IC50s from from 25 nM-1 μM. Notably, HCI-2509 displayed single-agent efficacy in multiple xenograft models. We investigated the PK/PD relationship using tumor histone H3K4 and H3K9 methylation. Taken together, these data suggest that epigenetic modulation through LSD1 inhibition may be a therapeutic strategy for Ewing sarcoma, and highlight a critical dual role for LSD1 in the oncogenic transcriptional activity of EWS/ETS proteins. Citation Format: Emily Rose Theisen, Savita Sankar, Jared Bearss, Timothy Mulvihill, Venkataswamy Sorna, Sunil Sharma, Stephen L. Lessnick. Inhibition of LSD1 disrupts global EWS/ETS transcriptional function in Ewing sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3679. doi:10.1158/1538-7445.AM2014-3679

LSD1 promotes oxidative metabolism of white adipose tissue.

Exposure to environmental cues such as cold or nutritional imbalance requires white adipose tissue (WAT) to adapt its metabolism to ensure survival. Metabolic plasticity is prominently exemplified by the enhancement of mitochondrial biogenesis in WAT in response to cold exposure or β3-adrenergic stimulation. Here we show that these stimuli increase the levels of lysine-specific demethylase 1 (LSD1) in WAT of mice and that elevated LSD1 levels induce mitochondrial activity. Genome-wide binding and transcriptome analyses demonstrate that LSD1 directly stimulates the expression of genes involved in oxidative phosphorylation (OXPHOS) in cooperation with nuclear respiratory factor 1 (Nrf1). In transgenic (Tg) mice, increased levels of LSD1 promote in a cell-autonomous manner the formation of islets of metabolically active brown-like adipocytes in WAT. Notably, Tg mice show limited weight gain when fed a high-fat diet. Taken together, our data establish LSD1 as a key regulator of OXPHOS and metabolic adaptation in WAT.

Overexpression of lysine specific demethylase 1 predicts worse prognosis in primary hepatocellular carcinoma patients

To investigate the clinicopathological features and prognostic value of lysine specific demethylase 1 (LSD1) in hepatocellular carcinoma (HCC). We examined LSD1 expression in 60 paired liver cancer tissues and adjacent noncancerous tissues by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting. In addition, we analyzed LSD1 expression in 198 HCC samples by immunohistochemistry. The relationship between LSD1 expression, clinicopathological features and patient survival was investigated. Immunohistochemistry, Western blotting, and qRT-PCR consistently confirmed LSD1 overexpression in HCC tissues compared to adjacent non-neoplastic tissues (P < 0.01). Additionally, immunostaining showed more LSD1-positive cells in the higher tumor stage (T3-4) and tumor grade (G3) than in the lower tumor stage (T1-2, P < 0.001) and tumor grade (G1-2, P < 0.001), respectively. Moreover, HCC patients with high LSD1 expression had significantly lower 5-year overall survival rates (P < 0.001) and lower 5-year disease-free survival rates (P < 0.001), respectively. A Cox proportional hazards model further demonstrated that LSD1 over-expression was an independent predictor of poor prognosis for both 5-year disease-free survival [hazards ratio (HR) = 1.426, 95%CI: 0.672-2.146, P < 0.001] and 5-year overall survival (HR = 2.456, 95%CI: 1.234-3.932, P < 0.001) in HCC. Our data suggest for the first time that the overexpression of LSD1 protein in HCC tissues indicates tumor progression and predicts poor prognosis.

HDAC and LSD1 Inhibitors Synergize to Induce Cell Death in Acute Leukemia Cells

Abstract 1427Histone deacetylase inhibitors (HDACi) are a class of emerging epigenetic therapies which are being used to treat cancer. Two HDACi (vorinostat and romidepsin) are FDA approved for cutaneous T-cell lymphoma. HDACi have been employed in clinical trials for acute leukemia, but single agent activity has been limited. Improved efficacy is observed when combined with other anticancer agents. In the current study we addressed acute leukemia models using vorinostat, a pan-HDACi that inhibits HDAC class I, II, and IV and entinostat, a newer HDACi that inhibits HDAC class I more specifically. These HDACi were combined with inhibition of another histone modifying enzyme: lysine specific demethylase 1 (LSD1). The LSD1 gene encodes a favin-dependent monoamine oxidase, which demethylates mono- and di-methylated lysines, specifically lysines 4 and 9 on histone 3 (H3K4 and H3K9), thus it is also involved in gene regulation through post-translational histone modification. LSD1 overexpression has been linked to human carcinogenesis in bladder carcinomas, lung cancer, and poorly differentiated neuroblastoma. However, it has not been studied in hematologic malignancies. Because LSD1 is structurally similar to monoamine oxidase (MAO), it has been shown that nonselective MAO inhibitors also inhibit LSD1. Here we employed tranylcypromine, a monoamine oxidase inhibitor (MAOi), as an irreversible LSD1 inhibitor. Recently published work from our laboratory has shown synergistic effects of combined HDAC and LSD1 inhibition in brain tumors (glioblastoma multiforme). Similar results have been published in breast cancer cells, but no work has been done in hematological malignancies. The objective of this study was to investigate the possible synergy of HDAC and LSD1 inhibitors in acute leukemia cells. LSD1 protein expression in several leukemia cells lines was analyzed by Western blot analysis. LSD1 was expressed in all leukemia cell lines tested, which included T-cell ALL (Jurkat, Sub-T1, MOLT4), B-cell ALL (JM-1,697), and Philadelphia chromosome positive ALL (Z33, Z119, Z181). To determine whether synergy exists between HDACi and LSD1 inhibitors, Jurkat cells were exposed to different concentrations of tranylcypromine and vorinostat or entinostat. After 24 hr, DNA fragmentation was assessed by propidium iodide (PI) staining followed by flow cytometric analysis. A combination index (CI) less than 1.0 is representative of synergism as measured by Calcusyn software. Results showed a synergistic effect on DNA fragmentation when combining the 2.5 μM dose of vorinostat with a range of tranylcypromine doses (1 mM CI= 0.78, 1.5 mM CI= 0.49, and 2 mM CI= 0.39). The same effect was observed with the combination of 2.5 μM entinostat with 2 mM tranylcypromine (CI=0.52). Viability studies performed with the same drug concentrations in conbination also showed statistically significant cell death. Additional acute leukemia cell lines, 697 and MOLT-4, also demonstrated significantly increased cell death with the combination relative to treatment with either agent alone. Since these agents inhibit histone deacetylation and lysine demethylation, we tested whether these histone modifications were promoted by combination treatment. Jurkat cell lysates were generated by acid extraction of histones and Western blot analysis was conducted. We demonstrated that in fact histone acetylation was increased with combination treatment, indicating that these modifications coordinately regulate each other in acute leukemia cells. A molecular target for LSD1 is p53, a tumor suppressor protein whose activity is regulated by lysine methylation and demethylation. Western blot analysis showed that p53 is downregulated in leukemia cells after exposure to the combination of HDAC and LSD1 inhibitors. Future studies will address if p53 downregulation is a trigger for the synergistic cell death. Taken together, our data shows the efficacy of combining LSD1 inhibitors with HDAC inhibitors in multiple acute leukemia models. Since tranylcypromine is also a FDA-approved agent, these results urge the design of a feasible and effective clinical trial combining LSD1 and HDAC inhibitors for acute leukemia. Disclosures:No relevant conflicts of interest to declare.

(Bis)urea and (Bis)thiourea Inhibitors of Lysine-Specific Demethylase 1 as Epigenetic Modulators

The recently discovered enzyme lysine-specific demethylase 1 (LSD1) plays an important role in the epigenetic control of gene expression, and aberrant gene silencing secondary to LSD1 overexpression is thought to contribute to the development of cancer. We recently reported a series of (bis)guanidines and (bis)biguanides that are potent inhibitors of LSD1 and induce the re-expression of aberrantly silenced tumor suppressor genes in tumor cells in vitro. We now report a series of isosteric ureas and thioureas that are also potent inhibitors of LSD1. These compounds induce increases in methylation at the histone 3 lysine 4 (H3K4) chromatin mark, a specific target of LSD1, in Calu-6 lung carcinoma cells. In addition, these analogues increase cellular levels of secreted frizzle-related protein (SFRP) 2 and transcription factor GATA4. These compounds represent an important new series of epigenetic modulators with the potential for use as antitumor agents.