Knockdown Of Lysine-specific Demethylase 1 Research Articles

Abstract 2673: Several LSD1 inhibitors have a potential to disturb fatty acid metabolism necessary &It glycolysis-inhibited pancreatic cancers to survive

Abstract Despite increased aerobic glycolysis is generally seen in human cancers, pancreatic ductal adenocarcinoma (PDAC) cells keep alive in glycolysis-suppressed conditions (1). Here, we sought there are potential therapeutic targets in glycolysis-suppressed PDAC cells. By screening anti-cancer metabolism compounds, we identified SP2509, a selective lysine specific demethylase 1 (LSD1) inhibitor (2). We also tested the effect of another three LSD1 inhibitors (Iadademstat, OG-L002 and T-3775440) on PDAC cells viability. The results showed that OG-L002, but not Iadademstat or T-3775440, lowered glycolysis-suppressed PDAC cells viability, like SP2509. Importantly, only knockdown of LSD1 or LSD2 failed to lower the viability of glycolysis suppressed PDAC cells. In our previous study, we proved that glucose-starvation make PDAC cells yield survival to mitochondrial oxidative phosphorylation (1). In this study, we cleared fatty acids metabolism played a crucial role for glycolysis suppressed PDAC cells survival. Then, we confirmed that SP2509 and OG-L002 can lead lipid droplets accumulation in glycolysis-suppressed conditions. In conclusion, our results showed that SP2509 and OG-L002 may involve regulatory effects on lipid droplets homeostasis, which control storage and release of fatty acids, not only by targeting LSD1 or LSD2. Moreover, SP2509 and OG-L002 significantly lowered viability of glycolysis-suppressed PDAC cells, supposing a novel therapeutic potential of approaches to override resistance of glucose-starvation in PDAC cells. 1.Shiratori R, Furuichi K, Yamaguchi M, Miyazaki N, Aoki H, Chibana H, et al. Glycolytic suppression dramatically changes the intracellular metabolic profile of multiple cancer cell lines in a mitochondrial metabolism-dependent manner. Sci Rep. 2019;9(1):18699. 2.Fiskus W, Sharma S, Shah B, Portier BP, Devaraj SGT, Liu K, et al. Highly effective combination of LSD1 (KDM1A) antagonist and pan-histone deacetylase inhibitor against human AML cells. Leukemia. 2017;31(7):1658. Citation Format: Zhiheng Zhang, Haruna Aoki, Keitaro Umezawa, Taichi Oshima, Takuya Hirao, Yuri Miura, Kousei Ito, Shigeki Aoki. Several LSD1 inhibitors have a potential to disturb fatty acid metabolism necessary &It glycolysis-inhibited pancreatic cancers to survive [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2673.

LSD1 in beige adipocytes protects cardiomyocytes against oxygen and glucose deprivation.

Epicardial adipose tissue (EpAT) is known for its role in supporting the cardiomyocytes. Lysine-specific demethylase 1 (LSD1), a typical lysine demethylase, is an essential regulator for the maintenance of beige adipocytes. However, the effect of LSD1 in the adipogenic differentiation of beige adipocytes in EpAT, and its function on oxygen and glucose deprivation (OGD)-injured cardiomyocytes remain unclear. Heart tissues from young mice and elder mice were collected for immunohistochemical staining. LSD1 in 3T3-L1 cells was knocked down by LSD1-shRNA lentivirus infection. The qRT-PCR, western blotting, and Oil Red O staining were employed to detect the adipogenic differentiation of 3T3-L1 cells and formation of beige adipocytes. The cardiomyocytes co-cultured with beige adipocytes were used for OGD treatment. Cell apoptosis was analyzed by flow cytometry. The lactate dehydrogenase (LDH) and superoxide dismutase (SOD) activity were analyzed using commercially available kits. The decrease of LSD1 was related to the age-dependent loss of beige adipocytes in mice EpAT. LSD1 knockdown inhibited the adipogenic differentiation of 3T3-L1 cells and formation of beige adipocytes. The down-regulation of LSD1 in 3T3-L1 cells decreased the protective effect of mature adipocytes on OGD-injured cardiomyocytes. The decreased expression of LSD1 in mice EpAT was associated with age-dependent ablation of beige adipocytes. The protective effect of beige adipocytes on OGD-injured cardiomyocytes is reduced by knockdown of LSD1 in adipocytes. The present study provided exciting insights into establishing novel therapies against age-dependent cardiac diseases.

Lysine-specific demethylase 1 in primary sensory neurons participates in chronic compression of dorsal root ganglion–induced neuropathic pain

Low back and radicular pain syndromes, usually caused by local inflammation and irritation to the nerve root and dorsal root ganglion (DRG), are common throughout medical practice, but sufficient pain relief is scarce. In this study, we employed a chronic compression of DRG (CCD)–induced radicular pain model in rats to explore whether lysine-specific demethylase 1 (LSD1), a histone demethylase and transcriptional co-repressor, is involved in the pathological process of radicular pain. We found that LSD1 was expressed in various-sized DRG neurons by immunohistochemistry. CCD induced the upregulation of LSD1 in compressed L4–L5 DRGs. Moreover, either LSD1 small interfering RNAs or LSD1 inhibitor attenuated CCD–induced pain hypersensitivities. LSD1 was also upregulated in the injured lumbar 4 (L4) DRG in a spinal nerve ligation (SNL)–induced neuropathic pain mouse model. Nevertheless, LSD1 was not altered in L3–L5 DRGs in complete Freund’s adjuvant–induced inflammatory pain mouse model, paclitaxel– or streptozotocin–induced neuropathic pain models. Furthermore, knockdown of LSD1 in the injured L4 DRG reversed SNL–induced pain hypersensitivities in mice. Therefore, we speculate that nerve injury induced the upregulation of LSD1 in the injured DRGs, which contributes to neuropathic pain hypersensitivities; thus, LSD1 may serve as a potential target for the treatment of radicular pain and neuropathic pain.

Abstract 3329: Efficacy of EGFR/PI3K signaling inhibition is enhanced with LSD1 inhibition in glioblastoma stem cell (GSC) models

Abstract Mutated and dysregulated protein kinases, such as EGFR and PI3K, have become major targets in cancer therapy due to the growth advantage they confer and the frequency of alterations. In glioblastoma (GBM), EGFR and PI3K are two of the most mutated genes and result in hyperactivation of these kinases. However, single agent inhibition has offered minimal success, largely due to the development of resistance from compensatory downstream signaling pathways. One strategy to circumvent resistance and improve efficacy in GBM is to use combination therapy, such as concurrent inhibition of EGFR or PI3K with inhibition of relevant epigenetic modulators. In GBM, lysine-specific demethylase 1 (LSD1) is an important epigenetic regulator that has shown to promote kinase signaling in cancer models. Therefore, the present study sought to define the relationship between the EGFR/PI3K signaling pathway and LSD1 and to evaluate the efficacy of co-inhibition of EGFR/PI3K and LSD1 in GBM.Transcriptional changes were examined following knockdown of LSD1 in isogenic human GBM cells using RNA-seq. These data were analyzed by GSEA to evaluate biological processes associated with the LSD1 expression. We identified several kinase signaling processes that were enriched in GBM cells with wild type LSD1 such as gene sets for regulation of PI3K activity, RTK binding, and transmembrane RTK activity. The effect of kinase inhibition on LSD1 expression was assessed using western blot analysis. We also evaluated the effects of LSD1 inhibition on expression of downstream kinase signaling proteins. Three kinase inhibitors directed against either EGFR or PI3K were evaluated in GSCs as single agents and in combination with LSD1 inhibitors. The three kinase inhibitors, osimertinib, erlotinib, and BKM120, all have evidence of some brain penetrance. We also evaluated a novel dual kinase inhibitor, MTX-241, which targets both EGFR and PI3K simultaneously. Five LSD1 inhibitors were assessed for their ability to enhance efficacy of EGFR/PI3K inhibitors.Our results demonstrate that LSD1 protein expression can be modulated by stimulation of EGFR in patient-derived GSCs. We observed an increase in LSD1 protein expression following the addition of EGF in GSC 17 cells. The concurrent inhibition of EGFR/PI3K and LSD1 enhanced the in vitro efficacy compared to single agent, supporting convergence of kinase signaling and LSD1 dependent pathways. In fact, several treatment combinations of EGFR/PI3K inhibitors and LSD1 inhibitors resulted in synergistic effects in multiple GSC lines.In summary, our results highlight the need for effective therapy combinations that can reduce the population of GSCs and avoid the adaptive resistance that is typical of kinase inhibitors. Future studies will focus on evaluating the efficacy and tolerability of the most promising treatment combinations in vivo using orthotopic xenograft models of the GSCs. Citation Format: Lea Stitzlein, Matthew Luetzen, Caitlin McCabe, Maninder Khosla, Melissa Singh, Xiaoping Su, Yue Lu, Joy Gumin, Frederick Lang, Christopher Whitehead, Judith Sebolt-Leopold, Joya Chandra. Efficacy of EGFR/PI3K signaling inhibition is enhanced with LSD1 inhibition in glioblastoma stem cell (GSC) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3329.

Evaluation of in vitro and in vivo efficacy of pharmacological lysine‐specific demethylase 1 (LSD1) inhibitors in glioblastoma stem cell (GSC) models

Epigenetically directed cancer therapeutics, such as targeting the histone demethylase LSD1, are an emerging approach to the treatment of glioblastoma (GBM), with dozens of candidates in preclinical development. To date, knockdown (KD) of LSD1 exerts effects on proliferation, but an evaluation and comparison of pharmacological LSD1 inhibitors in GBM is lacking. Therefore, the present study evaluated the effect of LSD1 KD compared with pharmacological inhibition and studied four LSD1 inhibitors in patient derived GSCs in vitroand in vivo for therapeutic efficacy and genes associated with resistance.Changes in gene expression were observed upon KD of LSD1 and compared with pharmacological inhibition of LSD1 in GBM cells. Four LSD1 inhibitors were assessed in a panel of nine GSC lines for their effects on cell viability and colony formation. The radioresistant GSC line, GSC 20, was used to establish an orthotopic xenograft model to evaluate the in vivo activity of LSD1 inhibition. In parallel, we identified five genes associated with resistance to LSD1 inhibition using RNA‐seq comparing non‐transformed and isogenic resistant GBM lines. Lastly, we probed for the presence of this gene set in our in vitro and in vivo GSC experiments with LSD1 inhibitors and in GBM patient exome data.In GBM cell lines, we identified an upregulation of genes associated with cell death, regulation of cell proliferation, and regulation of intracellular signaling upon both LSD1 KD and LSD1 inhibition using tranylcypromine treatment. Additional, more selective, pharmacological inhibitors of LSD1 (GSK‐LSD1, RN‐1 and SP‐2577) in vitro had differential responses in the GSCs that were more varied, and responses were dependent on the specific LSD1 inhibitor and GSC line. Importantly, the response to LSD1 inhibition in vitro was not dependent on the GSC radiosensitivity or molecular subtype indicating unique determinants of sensitivity. When assessing in vivoefficacy, the GSC tumor‐bearing mice treated with GSK‐LSD1 had a strong reduction in tumor burden that occurred seven weeks into treatment. However, tumor regrowth occurred and overall survival was not significantly better in LSD1 inhibitor treated mice. To understand mechanisms associated with tumor regrowth, we mined our existing data on selectivity and sensitivity to LSD1 blockade and identified five genes potentially associated with resistance to LSD1 inhibition, including HKDC1, RAB3IL1, RAB39B, FTH1, and FAM213A. These genes were found to be upregulated in treatment resistant GSCs and in the brain tissue of GSK‐LSD1 treated mice after they succumbed to tumor burden. Finally, the resistant‐associated genes were present in exome sequencing data from GBM patients where they showed an inverse relationship with LSD1 expression.Future studies will focus on designing combination therapies with LSD1 inhibitors that will enhance their effects and overcome resistance associated with treatment by targeting pathways associated with the genes we have uncovered.

SP2509, a Selective Inhibitor of LSD1, Suppresses Retinoblastoma Growth by Downregulating β-catenin Signaling.

PurposeTo study the role of lysine-specific demethylase 1 (LSD1) in retinoblastoma (RB) growth and to determine whether the LSD1 inhibitor SP2509 can inhibit RB progression.MethodsWe detected the levels of LSD1 in 12 RB tissue samples, two RB cell lines (Y79 and Weri-RB1), and a retinal pigment epithelium cell line (ARPE-19). Overexpression or knockdown of LSD1 was performed to examine the role of LSD1 in RB cancer cell survival. In vitro and in vivo experiments were conducted to detect the antitumor effect of SP2509, and the antitumor mechanism of SP2509 was examined by RNA sequencing and Western blot.ResultsLSD1 is overexpressed in RB tissues and cells and increases RB cancer cell viability and colony formation ability. The LSD1 inhibitor SP2509 inhibits RB cell proliferation in vitro and in vivo. Treatment with SP2509 increases the levels of dimethylated histone 3 lysine 4 (H3K4me2) and inhibits the expression of β-catenin signaling pathway–related proteins in RB cells.ConclusionsWe demonstrated that LSD1 is overexpressed in RB cells and promotes RB cell survival. The LSD1 inhibitor SP2509 exerted strong growth inhibition in vitro and in vivo, which was at least partially mediated by suppression of the β-catenin pathway.

Histone lysine-specific demethylase 1 induced renal fibrosis via decreasing sirtuin 3 expression and activating TGF-\u03b21/Smad3 pathway in diabetic nephropathy

ObjectiveDiabetic nephropathy (DN) is the leading cause of end-stage renal disease. Histone lysine-specific demethylase 1 (LSD1) is a flavin-containing amino oxidase that can repress or activate transcription. The aim of this study is to explore the mechanism of LSD1 aggravating DN-induced renal fibrosis.MethodsThe STZ-induced DN rat model was established for in vivo study. The rats were divided into four groups: Sham, STZ, STZ + Ad-shNC and Ad-shLSD1. The Hematoxylin–eosin (HE) staining was used to evaluate the renal injury. The Immunofluorescence assay was used to determine the LSD1, Fibronectin and α-SMA expression. The related protein expression was detected by western blot.ResultsKnockdown of LSD1 alleviated STZ-induced renal injury. Moreover, knockdown of LSD1 decreased the expression of serum biochemical markers, containing urine output (24 h), urinary protein (24 h), serum creatinine, BUN and UACR. Furthermore, we proved that knockdown of LSD1 alleviated renal fibrosis in STZ-induced DN rats. In vitro, knockdown of LSD1 suppressed NRK-49F cells activation and overexpression of LSD1 induced renal fibrosis. In addition, knockdown of LSD1 could deactivate TGF-β1/Smad3 pathway and promote sirtuin 3 (SIRT3) expression in vivo and in vitro. The rescue experiments confirmed that LSD1 induced renal fibrosis via decreasing SIRT3 expression and activating TGF-β1/Smad3 pathway.ConclusionLSD1 deficiency leads to alleviate STZ-induced renal injury and overexpression of LSD1 induces renal fibrosis via decreasing SIRT3 expression and activating TGF-β1/Smad3 pathway, which provides a reasonable strategy for developing novel drugs targeting LDS1 to block renal fibrosis.

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.

Lysine-specific demethylase 1 (LSD1) promotes ovarian cancer cell progression by Forkhead box O 3a (FOXO3a) inhibition

Recently, LSD1 is considered as a possible therapeutic mark for ovarian epithelial cancer (OEC). Though, the underlying molecular mechanism by which LSD1 endorses the oncogenesis of OEC has not been fully understood. Here, we revealed that overexpression of LSD1 downregulated Forkhead box O 3a (FOXO3a), while knockdown or pharmacological inhibition of LSD1 upregulated FOXO3a expression. Specifically, LSD1 interacted with demethylated FOXO3a. The LSD1-demethylated FOXO3a degraded via an ubiquitin-proteasome pathway. Biologically, LSD1 destabilized FOXO3a to abrogate its functions in the suppression of soft agar colony and cell proliferation formation in HO8910 ovarian cancer cells. Knockdown of FOXO3a rescued the restricted cell proliferation by LSD1 downregulation. As a whole, our study clarifies a way in ovarian cancer cell growth through the negative regulation of FOXO3a by LSD1.

Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer.

Activation of the epithelial-to-mesenchymal transition (EMT) program is a critical mechanism for initiating cancer progression and migration. Colorectal cancers contain many genetic and epigenetic alterations that can contribute to EMT. Mutations activating the PI3K/AKT signaling pathway are observed in >40% of patients with colorectal cancer contributing to increased invasion and metastasis. Little is known about how oncogenic signaling pathways such as PI3K/AKT synergize with chromatin modifiers to activate the EMT program. Lysine-specific demethylase 1 (LSD1) is a chromatin-modifying enzyme that is overexpressed in colorectal cancer and enhances cell migration. In this study, we determine that LSD1 expression is significantly elevated in patients with colorectal cancer with mutation of the catalytic subunit of PI3K, PIK3CA, compared with patients with colorectal cancer with WT PIK3CA. LSD1 enhances activation of the AKT kinase in colorectal cancer cells through a noncatalytic mechanism, acting as a scaffolding protein for the transcription-repressing CoREST complex. In addition, growth of PIK3CA-mutant colorectal cancer cells is uniquely dependent on LSD1. Knockdown or CRISPR knockout of LSD1 blocks AKT-mediated stabilization of the EMT-promoting transcription factor Snail and effectively blocks AKT-mediated EMT and migration. Overall, we uniquely demonstrate that LSD1 mediates AKT activation in response to growth factors and oxidative stress, and LSD1-regulated AKT activity promotes EMT-like characteristics in a subset of PIK3CA-mutant cells. IMPLICATIONS: Our data support the hypothesis that inhibitors targeting the CoREST complex may be clinically effective in patients with colorectal cancer harboring PIK3CA mutations.

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

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

Lysine-specific demethylase-1 regulates fibroblast activation in pulmonary fibrosis via TGF-β1/Smad3 pathway

Pulmonary fibrosis is a progressive and fatal fibrotic lung disease with mysterious pathogenesis and limited effective therapies. The aberrantly activated lung myofibroblasts with resultant excessive accumulation of extracellular matrix is a central event in the progression of pulmonary fibrosis. Lysine-specific demethylase 1 (LSD1) has been suggested to epigenetically regulate cell differentiation, migration and invasion in tumor microenvironment. However, its function in pulmonary fibrosis remains unclear. The present study aimed to investigate the potential effect and underlying mechanisms of LSD1 in pulmonary fibrosis. Here, we found that LSD1 expression was elevated in lung tissues of mice with bleomycin-induced pulmonary fibrosis and lung fibroblasts treated with transforming growth factor-β1 (TGF-β1). In vivo knockdown of LSD1 by lentiviral shRNA transfection attenuated pulmonary fibrosis in mice, as evidenced by improved lung morphology, decreased lung coefficient and collagen secretion, and down-regulated α-SMA, collagen type I alpha and fibronectin expression in lungs. Additionally, in vitro knockdown of LSD1 inhibited the differentiation of fibroblasts to myofibroblasts, and decreased myofibroblast migration. By further mechanistic analysis, we demonstrated that knockdown of LSD1 prevented fibroblast--to-myofibroblast differentiation and subsequent pulmonary fibrosis by suppressing TGF-β1/Smad3 signaling pathway through modulation of a balance between histone H3 lysine 9 methylation and histone H3 lysine 4 methylation. Together, our data indicate that LSD1 activation contributes to pulmonary myofibroblast differentiation and fibrosis by targeting TGF-β1/Smad3 signaling, and suggest LSD1 as a therapeutic target for the treatment of pulmonary fibrosis.

Abstract 1888: The FAD-directed LSD1 specific inhibitor, INCB059872, is a promising epigenetic agent for AML therapy by inducing differentiation of leukemic stem/progenitor cells

Abstract Numerous studies have elucidated that the most pivotal functions of lysine specific demethylase-1 (LSD1) are associated with regulating normal or malignant hematopoiesis by maintaining stem cell self-renewal and regulating myeloid differentiation. In preclinical models, studies with either pharmacological inhibition or genetic knockdown of LSD1 demonstrated that LSD1 is essential for differentiation of progenitor cells during normal hematopoiesis. In the clinic, AML manifests itself via clonal expansion of abnormal differentiation and proliferation of myeloid cells and, therefore, the inhibition of LSD1 activity with small molecule inhibitors could be a promising therapeutic approach for AML. Previously, we reported upon the identification of a flavin adenine dinucleotide (FAD) directed LSD1 specific inhibitor, INCB059872, which is efficacious in preclinical mouse models utilizing human AML cell lines and primary AML cells by inducing cell differentiation as indicated by the induction of CD11b and CD86 markers. Using a larger panel of myeloid and HSC flow cytometry markers, our currents efforts expanded upon these observations to ascertain whether INCB059872 enhanced lineage commitment at hematopoietic stem cell (HSC) and/ or promoted monocytic/granulocytic differentiation of human primary AML cells ex vivo and in human systemic AML PDX models. In both human AML PDX models and human primary AML samples, INCB059872 increased myeloid differentiation with increasing populations of monocytes (CD14+) and granulocytes (CD15+). Furthermore, INCB059872 induced the differentiation of early hematopoietic progenitors, CD34+/CD38- to more committed CD34+/CD38+ multipotent/oligopotent progenitors, which in turn gave rise to lineage specific progenitors in the human AML PDX models. These studies support further exploration of INCB059872 as a promising novel epigenetic agent for AML therapy whose mechanism of action lies in part through the induction of differentiation of leukemic stem/progenitor cells to more committed hematopoietic lineages. Citation Format: Antony Chadderton, Min Ye, Melody Diamond, Valerie Roman, Michael Weber, Chunhong He, Liangxing Wu, Swamy Yeleswaram, Alan Roberts, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Bruce Ruggeri, Sang Hyun Lee. The FAD-directed LSD1 specific inhibitor, INCB059872, is a promising epigenetic agent for AML therapy by inducing differentiation of leukemic stem/progenitor cells [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 1888.

LSD1 negatively regulates autophagy through the mTOR signaling pathway in ovarian cancer cells.

Lysine-specific demethylase1 (LSD1) plays a key role in cell proliferation, differentiation and carcinogenesis. In the present study we revealed that LSD1 functioned as an autophagy suppressor in ovarian cancer HO8910 cells. Pharmacological inhibition or genetic knockdown of LSD1 resulted in the elevation of the LC3‑II protein, enhancement of autophagosomal formation and stimulation of the autophagic flux. In addition, knockdown of LSD1 further promoted the serum starvation- and rapamycin-induced autophagy. Furthermore, we demonstrated that LSD1 regulated autophagy via the mTOR signaling pathway. Collectively, our findings identified LSD1 as a novel negative regulator of autophagy through the mTOR signaling pathway in ovarian cancer HO8910 cells and indicated that LSD1 may function as a driving factor of ovarian cancer progression via deregulating autophagy.

Inhibition of lysine-specific demethylase 1 prevents proliferation and mediates cisplatin sensitivity in ovarian cancer cells.

Lysine-specific demethylase 1 (LSD1) functions as a transcriptional coregulator by modulating histone methylation and has been associated with numerous high-risk cancers. Previously, our group and others identified LSD1 as an upregulated gene in ovarian cancer, and reported that the upregulation of LSD1 was associated with poor prognosis of patients with ovarian cancer. However, the role of LSD1 in ovarian cancer requires further investigation. The present study revealed that the overexpression of LSD1 significantly promoted the proliferation of SKOV3 ovarian cancer cells, while knockdown of LSD1 markedly inhibited cell proliferation and potentiated cisplatin-induced cell apoptosis, supporting LSD1 as an oncogenic protein in ovarian cancer. Mechanistic studies have indicated that LSD1 modulates the expression of cyclin dependent kinase inhibitor 1, Survivin, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X genes, which are known regulators of cell proliferation. Furthermore, LSD1 knockdown plus cisplatin synergistically impaired cell migration via the induction of the epithelial marker E-cadherin and inhibition of the mesenchymal markers, snail family transcriptional repressor 1 and Vimentin. These data of the present study indicated LSD1 as a potential regulator of ovarian cancer cell progression and suggested an unfavorable role of LSD1 in cisplatin-based regimens.

P2.02-012 The Epigenetic Role of LSD1+8a in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is a neuroendocrine tumor which account for 15% of all lung cancers. SCLC is characterized by rapid progression which led metastasis to distant organs and represented poor prognosis. Although there were various molecular targeted therapies in SCLC that were already under investigation, results have been disappointing. The focus on biological pathways has recently shifted from genetic to epigenetic regulations. Histone methylation is one of the epigenetic mechanisms which has pivotal role in gene expression, cell cycle and differentiation. Lysine-specific demethylase 1 (LSD1)/KDM1 is a histone modifier that is expressed in certain human cancers including SCLC. LSD1+8a is one of the isoform of LSD1 that was reported to be restricted in neural tissues and it plays critical role in neuronal differentiation. The aim of this study is to elucidate the epigenetic role of LSD1+8a in SCLC. The expression of LSD1 and LSD1+8a mRNAs were analyzed by qPCR in several cancer cell lines. Neuroendocrine markers were detected by qPCR in several SCLC cell lines. The Pearson correlation test was performed to investigate the correlation between LSD1+8a and neuroendocrine markers. siRNA against specific to exon 8a was designed and knockdown LSD1+8a isoform specifically. Cell proliferation assays following knockdown of LSD1+8a were performed in LSD1+8a expressed SCLC cell lines. LSD1 mRNA was expressed in majority of SCLC cell lines, interestingly LSD1+8a mRNA was detected in small subset of SCLC cell lines. There were positive correlations of LSD1+8a and neuroendocrine marker genes such as chromogranin A (CHGA), enolase2 (ENO2), neural cell adhesion molecule (NCAM) and synaptophysin (SYP) in human cancer cell lines. The suppression of LSD1+8a by siRNA drove to decrease expression of neuroendocrine marker genes in SCLC and inhibited cell proliferation in LSD1+8a expressed cell. LSD1+8a could play an important role in SCLC.

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.

The zinc-finger transcriptional factor Slug transcriptionally downregulates ER\u03b1 by recruiting lysine-specific demethylase 1 in human breast cancer

Estrogen receptor α (ERα) is related with epithelial–mesenchymal transition, invasion and metastasis, and serves as an important therapeutic predictor and prognostic factor in breast cancer patients. The triple negative breast cancer (TNBC) is characterized by loss of hormone receptors and human epidermal growth factor receptor 2 (Her2), and lacks effective targeted therapy with poor prognosis. Unfortunately, the molecular mechanisms of ERα deficiency, which becomes hormone independent and results in resistance to endocrine therapy, remain to be elucidated in breast cancer. In this study, we observed an inverse correlation between Slug, a zinc-finger transcriptional repressor, and ERα expression in both human breast cancer tissues and cell lines. In ERα-negative breast cancer patients, high Slug messenger RNA expression showed obviously shorter relapse-free survival. We found that Slug binds to the E-box located in the promoter of estrogen receptor 1 gene (ESR1) to suppress its expression. More specifically, Slug recruits lysine-specific demethylase 1 (LSD1) to the E-box and thereby inhibits ERα expression by demethylating H3K4me2, which is evidenced by the interaction between Slug and LSD1. Moreover, the amount of H3K4me2 binding to the E-box was significantly increased after LSD1 knockdown in MDA-MB-231 cells. Functionally, the ability to proliferate, invade and metastasize was significantly suppressed after knockdown of either Slug or LSD1 alone, or both simultaneously. Taken together, these results suggest that Slug transcriptionally inhibits ERα expression by recruiting LSD1 to the ESR1 promoter in breast cancers. Thus, targeted inhibition of Slug and LSD1 may restore ERα and lead to resensitization to hormone therapy, providing a novel therapeutic strategy for ERα-negative breast cancer patients, especially for TNBC.

Effects of cisplatin on the LSD1-mediated invasion and metastasis of prostate cancer cells.

Prostate cancer poses a major public health problem in men. Metastatic prostate cancer is incurable, and ultimately threatens the life of patients. Lysine-specific demethylase 1 (LSD1) is an androgen receptor-interacting protein that exerts a key role in regulating gene expression and is involved in numerous biological processes associated with prostate cancer. Cisplatin, also known as cis-diamminedichloroplatinum or DDP, is a standard chemotherapeutic agent used to treat prostate cancer; however, it has the disadvantage of various serious side effects. The present study aimed to investigate the effects of LSD1 knockdown, and the interplay between LSD1 and DDP, on prostate cancer cell proliferation, apoptosis and invasion, and, therefore, the potential of LSD1 as a target for prostate cancer therapy. Flow cytometric analysis, Cell Counting kit 8 assay, Transwell assay and western blotting results revealed that LSD1 knockdown, in combination with DDP treatment, exerted antiproliferative, proapoptotic and anti–invasive effects on PC3 prostate cancer cells. In addition, knockdown of LSD1 acted synergistically with DDP, thereby enhancing the induction of apoptosis, and the inhibition of proliferation and invasion in prostate cancer cells. These results indicated that LSD1 may serve as a potential therapeutic target, and may enhance the sensitivity of PC3 cells to DDP.

The chromatin modifying complex CoREST/LSD1 negatively regulates notch pathway during cerebral cortex development.

The development of the cerebral cortex is a dynamic and coordinated process in which cell division, cell death, migration, and differentiation must be highly regulated to acquire the final architecture and functional competence of the mature organ. Notch pathway is an important regulator of differentiation and it is essential to maintain neural stem cell (NSC) pool. Here, we studied the role of epigenetic modulators such as lysine-specific demethylase 1 (LSD1) and its interactor CoREST in the regulation of the Notch pathway activity during the development of the cerebral cortex. We found that CoREST and LSD1 interact in vitro with RBPJ-κ in the repressor complex and these proteins are released upon overexpression of Notch intracellular domain (NICD). We corroborated LSD1 and RBPJ-κ interaction in developing cerebral cortex and also found that LSD1 binds to the hes1 promoter. Knock-down of CoREST and LSD1 by in utero electroporation increases Hes1 expression in vivo and decreases Ngn2. Interestingly, we found a functional interaction between CoREST and LSD1 with Notch pathway. This conclusion is based on the observation that both the defects in neuronal migration and the increase in the number of cells expressing Sox2 and Tbr2 were associated to the knock-down of either CoREST or LSD1 and were reversed by the loss of Notch. These results demonstrate that CoREST and LSD1 downregulate the Notch pathway in the developing cerebral cortex, thus suggesting a role of epigenetic regulation in the fine tuning of cell differentiation. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1360-1373, 2016.