LSD1 Inhibition Attenuates Tumor Growth by Disrupting PLK1 Mitotic Pathway Priya S

Published OnlineFirst February 13, 2019; DOI: 10.1158/1541-7786.MCR-18-0971

Chromatin, Epigenetics, and RNA Regulation Molecular Cancer Research LSD1 Inhibition Attenuates Tumor Growth by Disrupting PLK1 Mitotic Pathway Priya S. Dalvi1,2, Iris F. Macheleidt1,2, So-Young Lim1,2, Sonja Meemboor1,2, Marion Muller€ 1, Hannah Eischeid-Scholz1, Stephan C. Schaefer1,3,4, Reinhard Buettner1,2,3,4, Sebastian Klein1,5,6, and Margarete Odenthal1,2

Abstract

Lysine-specific demethylase 1 (LSD1) is a histone modifier Mechanistically, LSD1 directly regulates expression of PLK1 by that is highly overexpressed in lung adenocarcinoma, which binding to its promoter region that subsequently affects results in aggressive tumor biology. Tumor cell proliferation expression of its downstream target genes. Notably, using lung and migration analysis after LSD1 inhibition in the lung adenocarcinoma TCGA datasets a significant correlation adenocarcinoma cell line PC9, using the LSD1 inhibitor between LSD1 and PLK1 along with its downstream targets HCI-2509 and siRNA, demonstrated that LSD1 activity was was observed. Furthermore, the LSD1/PLK1 linkage was con- essential for proliferation and migration capacities of tumor firmed by IHC analysis in a clinical lung adenocarcinoma cells. Moreover, reduced proliferation rates after LSD1 inhibi- cohort (n ¼ 43). Conclusively, this is the first study showing a tion were shown to be associated with a cell-cycle arrest of direct transcriptional link between LSD1 and PLK1. the tumor cells in the G2–M-phase. Expression profiling followed by functional classification and pathway analysis Implications: These findings point to a role of LSD1 indicated prominent repression of the polo-like kinase 1 in regulating PLK1 and thus efficient G2–M-transition– (PLK1) pathway upon LSD1 inhibition. In contrast, transient mediating proliferation of tumor cells and suggest targeting overexpression of exogenous PLK1 plasmid rescued the LSD1 the LSD1/PLK1 axis as a novel therapeutic approach for lung inhibition–mediated downregulation of PLK1 pathway genes. adenocarcinoma treatment.

Introduction that significantly affects transcriptomic changes by demethylating histone tails. LSD1 belongs to the amine oxidase superfamily of Lung cancer is ranked first among cancer-related deaths world- proteins, and catalyzes FAD-dependent oxidative demethyla- wide (1). The most common subtype of non–small cell lung tion (8). Specifically, it demethylates mono/di-methylated his- cancer (NSCLC), lung adenocarcinoma, is associated with a poor tone 3 lysine 4 (K4) and lysine 9 (K9) (H3K4 and H3K9) residues, 5-year survival rate of less than 20% after diagnosis, and is defined thereby altering the epigenetic marks and inducing gene expres- as a disease with genetic and cellular heterogeneity (2, 3). Com- sion changes (8, 9). In addition, LSD1 also demethylates lysine prehensive molecular characterization of lung adenocarcinoma residues of nonhistone proteins like E2F1, TP53, and DNMT1, has revealed a huge complexity of not only genetic alterations but resulting in a change in their cellular properties (10, 11). LSD1 also epigenetic alterations (4, 5). Importantly, tumor progression performs these functions in association with different histone and development in lung adenocarcinoma has been shown to be modifying complexes and transcription coactivating or corepres- linked with epigenetic silencing of crucial tumor suppressor sing complexes (12). LSD1 is also crucial for the development genes (6, 7). and maintenance of normal tissue homeostasis and for stem Posttranslational modification of histone tails is a complex cell differentiation, and has been shown to be involved in mechanism regulating gene expression changes. Lysine-specific embryogenesis (13–15). demethylase 1 (LSD1/KDM1A) is one such epigenetic modifier Furthermore, high expression of LSD1 has been linked to poor prognosis in various cancer types, including breast cancer, hepa- 1Institute of Pathology, University Hospital of Cologne, Cologne, Germany. tocellular carcinoma, esophageal cancer, lung cancer, neuroblas- 2Center for Molecular Medicine Cologne, Cologne, Germany. 3Center for Inte- toma, as well as acute myeloid leukemia (13). As a result, many grated Oncology Cologne Bonn, Cologne, Germany. 4Lung Cancer Group compounds targeting LSD1 are being employed in preclinical 5 Cologne, University Hospital of Cologne, Cologne, Germany. Department of studies (16, 17). Among these inhibitors, HCI-2509 a reversible Translational Genomics, University of Cologne, Cologne, Germany. 6Else Kroner€ LSD1 inhibitor identified by extensive screening approaches has Forschungskolleg Cologne, University Hospital of Cologne, Cologne, Germany. been shown to be highly specific for LSD1 (18). This compound Note: Supplementary data for this article are available at Molecular Cancer has demonstrated to significantly inhibit tumor growth in pro- Research Online (http://mcr.aacrjournals.org/). state and Ewing sarcoma tumor cell lines and is expected to enter Corresponding Author: Margarete Odenthal, Institute of Pathology, University phase I clinical trials within the near future (19–21). Hospital of Cologne, Kerpener Str. 62, Cologne 50937, Germany. Phone: 221- In this study, we used HCI-2509 to disrupt LSD1 activity 478-6367; Fax: 221-478-6360; E-mail: [email protected] in lung adenocarcinoma cells. Inhibition of LSD1 reduced doi: 10.1158/1541-7786.MCR-18-0971 cell proliferation, downregulated major cell growth regulatory 2019 American Association for Cancer Research. pathways, and most predominantly led to downregulation of

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the polo-like kinase (PLK) mitotic pathway. We demonstrate (Applied Biosystems). GoTaq QPCR Master Mix (Promega) was that PLK1 is a direct transcriptional target of LSD1, and that used to perform real-time PCR. Primers used are listed in Sup- depletion of LSD1 activates G2–M arrest. Moreover, we show plementary File S1; Supplementary Table S2. The real-time PCR that LSD1 binds to the PLK1 promoter region and thereby was run on CFX96 Thermo Cycler (Bio-Rad) or Roche Lightcycler regulates its expression and tumor cell proliferation. In sum- 480 (Roche). mary, we propose that therapeutic targeting of LSD1 offers a mechanistic rationale for the treatment of lung adenocarcino- Microarray ma and other cancers that exhibit PLK1 addiction. Gene expression–profiling analysis was performed on untreated PC9 cells and PC9 cells treated with the LSD1 inhibitor HCI- Materials and Methods 2509. RNA isolation from cells was performed using the Ambion- PureLink RNA Mini Kit (Life Technologies) following the man- Cell culture ufacturer's instructions. The Human Gene 2.0 ST Array Platform Information about all cell lines used in this study is listed in (Affymetrix) was used for performing the microarray analysis. Supplementary File S1; Supplementary Table S1. Cell lines were Significance parameters used: P < 0.05 and fold change > 1.5. All either cultured in DMEM or RPMI media, supplemented with details regarding the Gene Ontology and pathway analysis 10% FCS, in presence or absence of L-Glutamine. All cell cultures can be found in the Supplementary File S1; Supplementary were maintained in 5% CO2 at 37 C. Cells were passaged when Methods section. The microarray data generated are available in 80% confluency was reached. the Gene Expression Omnibus repository under accession num- Cell lines used in this study are not listed in the International ber GSE117702. Cell Line Authentication Committee's misidentified cell lines database. All cell lines used in the study were tested for Protein isolation and Western blot analysis Mycoplasma using Venor GeM OneStep Kit (Minerva Biolabs; Protein lysates were extracted from cells using the Pierce RIPA last test, November 2017). On an average all the cell lines used Buffer (Thermo Fisher Scientific) and blotted as described previ- were in between 15–40 passage number and were used for ously (23). For studying phosphorylated proteins by Western blot cellular assays after 5–8 passages postthawing. analysis, protein lysates were extracted from cells using Cell Lysis Buffer 10X (New England Biolabs) as per the manufacturer's Cell treatment with the LSD1 inhibitor instructions. The membranes were incubated overnight with 2 One day prior to treatment, cells were plated on 20 cm plates primary antibodies in blocking solution (5% milk powder in and then supplemented with 2 mmol/L HCI-2509 (Xcess Bio- PBST) at 4C using the antibodies listed in Supplementary File S1; sciences). For microarray analysis and the corresponding valida- Supplementary Table S3. Developed blots were imaged using tion experiments, and for the expression analysis in different ChemiDoc XRsþ System (Bio-Rad) and processed using the cancer lines, HCI-2509 treatments were performed for 48 hours, associated Image Lab software v 4.0. while for all other experiments it was for 72 hours. Cells were harvested after 48 or 72 hours for either protein or RNA isolation. Cell-cycle analysis CycletestPlusDNAKit(BDBiosciences) was used to deter- Transfection with silencing RNA and plasmids mine percentage of cells in different phases of the cell cycle Transfections were performed using control siRNA (SIC001; following the manufacturer's instructions. Cell-cycle measure- Sigma) and three different siRNA sequences against LSD1 at a final ment analysis was performed on FACSVerse Flow Cytometer concentration of 50 nmol/L for 72 hours using Lipofectamine (BD Biosciences). All experiments were performed in tripli- 2000 as described earlier (Thermo Fisher Scientific; ref. 22). The cates. Percentage of cells in different phases of cell cycle was following siRNA oligonucleotides were used in the knockdown calculated by plotting propidium iodide area histogram. experiments: siLSD1-S1: sense 50-CUGCAGUUGUGGUUG- GAUAtt-30 and antisense 50-UAUCCAACCACAACUGCAGtg-30; Immunofluorescence siLSD1-S2: sense 50-CACAAGGAAAGCUAGAAGAtt-30 and anti- Cells were grown in 12-well plates on coverslips and cell sense 50-UCUUCUAGCUUUCCUUGUGtt-30; and siLSD1-S3: treatments were performed as mentioned above. Seventy-two sense 50-AGGCCUAGACAUUAAACUGaa-30 and antisense 50- hours later cells were fixed using 100% ice cold methanol for CAGUUUAAUGUCUAGGCCUaa-30. Plasmid transfections were 10 minutes and blocked using 0.1% fish gelatin (G7041; Sigma) performed using a PLK1 overexpression construct [mCherry- in PBS for 1 hour at room temperature. Cells were then sequen- PLK1-N-16 was a gift from Michael Davidson, Florida State tially stained with primary (overnight at 4C) and fluorescent University, Tallahassee, FL (Addgene plasmid catalog no. secondary antibodies (2 hours at room temperature) with inter- 55119; http://n2t.net/addgene:55119; RRID: Addgene_55119)] mediate washing steps using the blocking buffer. Antibodies used and a mock control, [pSicoR-Ef1a-mCh-Puro was a gift from Bruce have been listed in Supplementary File S1; Supplementary Table Conklin, Gladstone Institute of Cardiovascular Disease, San S3. Confocal images were collected on Olympus Fluoview FV Francisco, CA (Addgene plasmid catalog no 31845; http://n2t. 1000 scanning confocal microscope using a 60 oil objective and net/addgene:31845; RRID: Addgene_31845)] using Lipofecta- 1 magnification. Olympus Fluoview FV 1000 software was used mine 2000 according to the manufacturer's instructions. for data acquisition. Image files were processed using Fiji v 2.0.0 (ImageJ) and Adobe Photoshop. RNA isolation and quantitative PCR Isolation of RNA from cells was performed using the TCGA lung adenocarcinoma RNA-seq analysis Maxwell LEV simplyRNA Tissue Kit (Promega) according to the A processed dataset (z-transformation) of RNA-seq clinical manufacturer's instructions. For real-time PCR, RNA was reverse data from lung adenocarcinoma (TCGA, Provisional) was down- transcribed into cDNA using TaqMan Reverse Transcription Kit loaded from the cancer genomics repository cBioPortal (24).

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The dataset were grouped in two by filtering for LSD1 expres- capacities in vitro, as well as in vivo (28–30). Here, we could sion; LSD1 levels above the 90th percentile was defined as a high confirm these results using the LSD1 inhibitor HCI-2509 or with LSD1–expressing group, while levels below the 10th percentile siRNA, that also led to reduced cell proliferation and migration were defined as a low LSD1–expressing group. Differentially capacities in the lung adenocarcinoma cell line PC9 (Supple- expressed genes after LSD1 inhibition were plotted against the mentary File S1; Supplementary Fig. S1). To identify the under- grouped dataset in an unsupervised way following Spearman lying molecular mechanisms of LSD1 inhibition in lung correlation with the help of R (25). The association of LSD1 and adenocarcinoma, we first examined gene expression patterns of PLK1 mRNA expression in the TCGA lung adenocarcinoma data the EGFR-mutated (DE746-A750) lung adenocarcinoma cell line were calculated by Spearman correlation coefficient. PC9, in presence of the LSD1 inhibitor HCI-2509 using hybrid- ization arrays. Comparison of the transcriptome of untreated PC9 IHC staining of tissue microarray cells and LSD1-inhibited PC9 cells identified 2,570 (>1.5-fold Staining was performed as described previously (26) on par- change, P < 0.05) genes to be differentially regulated (Fig. 1A). affin-embedded tumor sections of prepared tissue microarrays Gene ontology analysis using PANTHER classification system, (TMA) consisting of 43 lung adenocarcinoma patients, using revealed enrichment of genes involved in multiple biological a-LSD1 and PLK1 antibodies as per the manufacturer's instruc- processes, including cellular and metabolic processes, as well as tions. Staining intensities for LSD1 and PLK1 were calculated immune response–related processes (Fig. 1B). Next, we subject- using QuPath (27), where the DAB channel intensity was ed the dataset for gene set enrichment analysis (GSEA) to analyze extracted for each represented core of the TMA. In addition, the differential expression of biologically relevant sets of genes immunostaining results for LSD1 and PLK1 were scored on a that share common biological functions after LSD1 inhibition. scale of 1–3(1 ¼ low, 2 ¼ medium, and 3 ¼ high), by The top 25 up- and downregulated genes, exhibited several genes experienced pathologists (S.C. Schaefer and S. Klein). The asso- associated with cell cycle to be significantly depleted after LSD1 ciation between LSD1 and PLK1 protein levels was calculated by inhibition (Fig. 1C). Among them the most prominent genes were Spearman correlation coefficient. PLK1, BORA, KIF18A, HISTH2AC, and HIST1H3I. The study was approved by the local ethics committee (KEK No. 200/2014) and informed consent was obtained from all patients. Gene expression profiling suggests a role of LSD1 in cell-cycle All methods were performed in accordance with the relevant regulation guidelines and regulations of the institution. An unbiased transcriptome profiling using GSEA showed that the untreated group was negatively enriched in cell-cycle progres- Chromatin immunoprecipitation sion genes (Fig. 1D). In contrast, the HCI-2509–treated group was Chromatin immunoprecipitation (ChIP) was performed using positively enriched with genes associated with cell-cycle arrest. SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Tech- GSEA revealed that the G2–M cell cycle and PLK1 pathways were nology) as per the manufacturer's instructions. Two steps of the among the most enriched gene sets in the untreated control protocol were optimized; sonication and amount of nuclease, by group as compared with the HCI-2509–treated group. To further using different setups for sonication and different concentrations validate these results, we used ingenuity pathway analysis (IPA) of nuclease to obtain appropriate fragmentation of chromatin. to examine the relationship between the highly significant genes For sonication, eight cycles of 30 seconds ON and 30 seconds OFF and to determine the key canonical pathways which are dysregu- each was determined to be optimal and 0.5 mg nuclease per 10 lated by LSD1. In line with the results of the GSEA analysis, IPA million cells. ChIP was performed using 20 mg lysate with 5 mg analysis revealed two growth regulatory pathways playing a each of LSD1 antibody and nonspecific rabbit IgG antibody. central role in cell cycle to be significantly altered. Both pathways, Eluted DNA was tested for successful working of protocol by namely, the G2–M DNA damage checkpoint regulation pathway analyzing binding of LSD1 to known gene targets by qPCR. and the mitotic role of PLKs (Fig. 1E) pathway had an activation Primers specific to different regions of PLK1 promoter were z-score of >2. Together, a consistent finding using both GSEA designed; for CDKN1A, promoter region sequences were and IPA was that multiple genes related to cell-cycle regulation obtained from previous reports (22). Each promoter region was were significantly altered after LSD1 inhibition using HCI-2509. analyzed in triplicate qPCR reactions per experiment and three These results indicated a reduced cell growth through down- independent experiments were performed. Data are presented regulation of cell-cycle regulatory genes. relative to percent of input. List of primers used for ChIP qPCR is provided in Supplementary File S1; Supplementary Table S2. LSD1 inhibition represses the PLK1 mitotic pathway With an in depth analysis of transcriptional changes using Statistical analysis GSEA and IPA, we recognized that the PLK1 mitotic pathway was Student t test (two-tailed) and two-way ANOVA tests were used most significantly downregulated upon LSD1 inhibition. By to analyze the in vitro and the lung adenocarcinoma clinical data pharmacologically disrupting LSD1 function due to HCI-2509 by GraphPad Prism v5 and "R" (25). Significance parameters treatment, several of the PLK1 target genes, including ECT2, used: P < 0.05. CDC20, CDC25C, KIF11, TERF11, TOP2A, BUB1, AURKA, ORC2, CCNB1, KIF20A, BORA, and PLK1 itself were downregulated (Fig. 2A). To further verify these results, the expression level of Results PLK1 target genes was quantified by qPCR. Indeed, the qPCR LSD1 inhibition results in a differential gene expression results were in accordance with the microarray analysis (Fig. 2B). signature Furthermore, we examined the effect of LSD1 inhibition on PLK1 Previous studies have shown that inhibition or silencing of expression on a panel of 23 different cancer cell lines from breast, LSD1 leads to reduced tumor cell proliferation and migration liver, esophageal, neuroblastoma, and lung cancer by treating

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A B C Response to stimulus HCI-2509 Untreated

Metabolic process 1092 1478 HEPHL1 Immune sytem process CITED2 Cellular process SCNN1G SCNN1B Biological regulation FN1 STC1 Upregulated 0204060 PARM1 Figure 1. LSD1 inhibition in PC9 cells results Downregulated % Of total LOC70755 (involved genes/total genes) MERTK into differential gene expression. A, CXCL16 Pie chart indicating the total KAT2B number of differentially expressed D CTSS genes upon LSD1 inhibition in PC9 MYO5B PID_PLK1_PATHWAY REACTOME_DNA_REPLICATION SPRR1A cells using HCI-2509. Microarray NES: -2.07 NES: -2.04 VTCN1 data from n ¼ 3 biological FDR qval: 0, P val: 0 FDR qval: 0, P val: 0 ADAMTS9 replicates. B, Bar chart depicting DKK1 the enriched biological processes CD68 on y axis and the total number of TNFAIP3 genes annotated to each biological FBXO32 process on x axis. C, Heatmap KLK5 VGLL3 representing the top 50 genes PI3 identified by GSEA to be MIR324 deregulated in the PC9 cells after DENND2C HCI-2509 treatment (red, relatively HIST1H2BI high gene expression and blue, HIST1H2AM relatively low gene expression). D, PSAT1 GSEA enrichment plots showing AKR1C2 CCDC113 negative enrichment of different PTGIS cell-cycle–related gene sets, as REACTOME_CELL_CYCLE REACTOME_G2_M_CHECKPOINTS CEACAM5 represented by negative NES NES: -1.90 NES: -1.85 HIST2H2AC (normalized enrichment score). FDR qval: 0.001, P val: 0 FDR qval: 0.0027, P val: 0 BORA NES reflects the degree to which ODZ1 the gene set was changed after HIST1H3I treatment as compared with the NFIB fi FTV1 untreated control. Signi cant gene ABCC2 sets were defined as those with a MIR4256 nominal P < 0.05. E, Bar chart GALNT12 depicting predicted canonical MOCOS pathways on the y axis and the PRTFDC1 corresponding significance of IL1RAPL2 expression indicated as log (P) DEPDC1 10 x HIST2H2AB on the axis. Indicated in black ACP6 boxes on the right are activation FLJ45974 z-scores. A positive z-score PLK1 indicates an increased activity of KIF18A the canonical pathway whereas a E negative z-score indicates the decreased activity of the indicated G2–M DNA damage checkpoint regulation +2.5 canonical pathway.

PPAR Signaling -1.8

IL6 Signaling +1.9

Mitotic role of PLK -2.6 02468 - P Z Log10( ) -score

these cell lines with the LSD1 inhibitor HCI-2509. qPCR analyses showed substantial downregulation of PLK1 at the protein level. revealed that the majority of the cell lines showed reduced PLK1 Similarly, the protein expression of PLK1's positively regulat- expression (Fig. 2C). Notably, 13 of the 23 cell lines showed at ed target aurora kinase A (AURKA) was significantly reduced, least 50% downregulation of PLK1 mRNA expression in response whereas the expression of its negatively regulated target cyclin- to LSD1 inhibition by HCI-2509. dependent kinase inhibitor 1A (CDKN1A) was significantly The methylation levels of LSD1 substrate H3K4me2 were increased, suggesting that LSD1 inhibition hampered PLK1 activ- altered after LSD1 inhibition (Fig. 2D). An increase in H3K4me2 ity. Similar effects on the expression of PLK1 and its target genes levels was detected using HCI-2509, confirming its on-target at transcript and protein levels was observed upon LSD1 knock- activity. Compared with the control cells, LSD1 inhibition down using three different siRNAs (Fig. 2E and F).

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AB 2 CDKN1A 6 TGFB1 CDKN2AIP 4 * ECT2 CDC20 2 Fold change -4 CDC25C *** KIF11 (fold change) 0

TERF1 2 Figure 2. TOP2A -2 * ** ** Log * * ** ** LSD1 inhibition downregulates BUB1 * ** ** ** expression of PLK1 and its target AURKA -4 genes. A, Heatmap showing the ORC2 Downregulated Upregulated PLK1 expression proﬁle of PLK1 target ECT2 KIF11 BUB1 ORC2 CCNB1 Downregulated Upregulated BORA TERF1 TGFB1 CDC20 TOP2A KIF20A CCNB1 genes after LSD1 inhibition in PC9 AURKA

KIF20A CDC25C cells using HCI-2509. B, Validation CDKN1A

PLK1 CDKN2AIP of microarray results of the BORA differentially expressed PLK1 target genes in PC9 cells after LSD1 inhibition using HCI-2509 by qPCR C D in an independent experiment. 2 C, PLK1 gene expression in different human cancer cell lines after LSD1 0 inhibition using HCI-2509 measured by qPCR. Data in B and C are * - * PLK1 60 kDa plotted as mean SD for n ¼ 3 2 * * ** biological replicates; , P < 0.05; ** ** * AURKA 46 kDa , P < 0.01; and , P < 0.001 as (fold change) - ** ** 2 4 * compared with untreated group. * **

D, Immunoblot showing protein Log ** - CDKN1A 21 kDa levels of PLK1, AURKA, CDKN1A, 6

and H3K4me2 in untreated PC9 PC9 H460 OE19 OE21 Calu6 HUH7 H1975 cells and cells treated with H3122 H3K4me2 17 kDa HCT15 POP10 HepG2 SW480 HCC95 HCT116 CLB-Ga HEK293 SW1271 HCC827 GI-ME-N

HCI-2509. E, Bar graph showing IMR-5/75 HCC1833 mRNA expression levels of LSD1 b-Actin 46 kDa ﬁ MD-MBA-231

and PLK1 target genes quanti ed SK-N-BE(2)-C after knockdown of LSD1 in PC9

cells using three different siRNA SW48 KRAS G12V sequences. Data are presented as mean SD for n ¼ 3 biological replicates; , P < 0.05 as compared E 4 F with the scrambled siRNA control. siLSD1-S1 siLSD1-S2 siLSD1-S3 F, Immunoblot showing protein levels of LSD1, PLK1, H3K4me2, and 2 * CDKN1A in scrambled transfected * PC9 cells and cells transfected LSD1 93 kDa with siLSD1. 0 PLK1 60 kDa (fold change) 2 *** -2 * *

Log * H3K4me2 * 17 kDa * -4 CDKN1A 21 kDa PLK1 LSD1 BUB1 b-Actin

CCBN1 46 kDa AURKA CDKN1A

To further control for an antiproliferative effect of LSD1 not overlap with the effect observed after LSD1 inhibition using inhibition in lung adenocarcinoma, we analyzed a publicly HCI-2509, suggesting that related downregulation of PLK1 available dataset containing gene expression proﬁle of the lung target genes is LSD1 speciﬁc and not a general antiproliferative adenocarcinoma cell line A549 after treatment with the anti- effect of the inhibitor. cancer drug cisplatin (GSE6410; ref. 31) for the top hits of the differentially expressed genes. Particularly, we noticed no dif- Transient overexpression of PLK1 overcomes LSD1 ference in the expression of the corresponding genes using a inhibition–mediated alteration of its target genes multivariate analysis (Supplementary File S1; Supplementary To demonstrate that the altered expression of PLK1 and its Fig. S2). The gene expression patterns of the genes evaluated did target genes in response to LSD1 inhibition using HCI-2509

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A B PLK1 15 Untreated HCI-2509 10 Mock Mock + PLK1HCI-2509 + HCI-2509 5 PLK1 Mock + HCI-2509

PLK1 60 kDa (fold change) 2 0 PLK1 + HCI-2509

b-Actin 46 kDa Log -5

C CDKN1A BUB1 3 0.5 Untreated 2 HCI-2509 0.0 1 Mock PLK1 0 (fold change) -0.5 Mock + HCI-2509 2 (fold change) 2 -1 PLK1 + HCI-2509 Log -2 -1.0

AURKA CCBN1 1.5 1.0

1.0 0.5 0.5 0.0 0.0 -0.5 -0.5 (fold change) Log (fold change) 2 2 - -1.0 1.0 Log -1.5 Log -1.5

Figure 3. Ectopic overexpression of PLK1 rescues LSD1 inhibition–mediated effect. Immunoblot showing PLK1 protein levels (A)andbargraphshowingPLK1 mRNA expression levels of PC9 cells transfected with mock and PLK1 overexpression plasmid (B), in absence and presence of HCI-2509. C, Bar graph showing transcript levels of PLK1 target genes in untreated PC9 cells, cells treated with HCI-2509 and PC9 cells transfected with mock and PLK1 overexpression plasmid, and overexpression constructs in combination with HCI-2509. Data in B and C are plotted as mean SD for n ¼ 3 biological replicates.

is a LSD1-specific effect, we transiently overexpressed PLK1 Inhibition of LSD1 results into mitotic arrest plasmid in the lung adenocarcinoma cell line PC9 in combi- PLK1 is highly expressed in cancer cells and its major nation with HCI-2509. As shown in Fig. 3A and B, ectopic role is implemented in cell proliferation by regulating the expression of PLK1 construct in the cells followed with LSD1 G2–M-phase transition of cell cycle. To investigate whether inhibition using HCI-2509, did no longer affect PLK1 protein the repressed PLK1 levels caused by LSD1 inhibition lead to or mRNA levels. Ectopic expression of PLK1 by transient abnormalities in cell-cycle progression, potentially due to the transfection not only blocked LSD1 inhibition–induced alter- inability of cells to transit from G2–M-phase, we performed ation of PLK1, but also its target genes. The positively regulated cell-cycle analysis after treatment of PC9 cells with HCI-2509 targets of PLK1, BUB1, AURKA, and CCBN1, showed a trend of using flow cytometry (Fig. 4A). LSD1 inhibition in PC9 cells increasing mRNA levels after PLK1 overexpression (Fig. 3C). led to accumulation of cells in the S- and G2–M-phase of cell Whereas, HCI-2509–mediated LSD1 inhibition had relatively cycle, while it led to decrease of cells in the G1-phase as less or no effect on the expression of BUB1, AURKA, and compared with the control cells (Fig. 4B). In addition, LSD1 CCBN1 in presence of transgenic PLK1 overexpression. Like- inhibition using HCI-2509 and knockdown using siRNA wise, the negatively regulated target gene of PLK1, CDKN1A, induced depletion of the mitotic marker H3S28ph in PC9 cells showed reduced mRNA levels after PLK1 overexpression, (Fig.4C).Furthermore,weperformedimmunofluorescence whereas, this effect was minimized in combination with (IF) staining to observe the effect of LSD1 inhibition on HCI-2509. Overall, transient overexpression of exogenous mitosis. The most visible effect was reduced LSD1 and PLK1 PLK1 in PC9 cells rescued the LSD1 inhibition–mediated levels in the cells after HCI-2509 treatment and perinuclear downregulation of PLK1 target genes, indicating the positive localization of LSD1 (Fig. 4D). Noticeably, with nuclear regulation of PLK1 by LSD1. counterstaining no mitotic cells were detected after LSD1

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A 1,500 1,500 1,500 G0–G1 Untreated HCI-2509 1 mmol/L HCI-2509 2 mmol/L

1,000 1,000 1,000

G0–G1 G0–G1 500 500 500 Count G –M G –M G2–M 2 S 2 S Sub G1 S 4N Sub G1 4N Sub G1 4N 0 0 0 0 50 100 150 200 250 0 50 100 150 200 250 0 50 100 150 200 250 ×1000 ×1000 ×1000 Propidium iodide area

B C

80 Untreated HCI-2509 1 mmol/L 60 HCI-2509 2 mmol/L # # H3S28ph 40 # # # # (15 kDa) 20 b-Actin (46 kDa) 0 Cell-cycle distribution (%) 1 1 S –M 4N –G 2 0 G Sub G G

D DAPI LSD1 PLK1

Untreated

HCI-2509

Figure 4. LSD1 inhibition leads to reduction in mitotic activity. A, Representative histograms showing cell-cycle distribution of untreated and PC9 cells treated with HCI-2509, measured by propidium iodide staining followed by ﬂow cytometry (n ¼ 3). B, Bar graph representing the percent cell-cycle distribution in each condition. Data presented are mean SD for n ¼ 3 biological replicates; #, P < 0.001 as compared with untreated group by two-way ANOVA. C, Immunoblot showing protein levels of H3S28ph in untreated PC9 cells, cells treated with HCI-2509 and cells transfected with scrambled control and siRNA against LSD1. D, Representative microscopic images of untreated PC9 cells (top) and HCI-2509 treated PC9 cells (bottom), stained with DAPI (blue), LSD1 (green), and PLK1 (magenta). White arrows indicate mitotic cells. Scale bar, 25 mm; n ¼ 3 biological replicates.

inhibition. These results are in accordance with our experi- LSD1 and PLK1 expression positively correlates at mRNA mental observations showing that LSD1 inhibition using and protein levels in lung adenocarcinoma HCI-2509 or siLSD1 results into less cell growth and prolifer- While the results of LSD1 inhibition showed a picture of ation in PC9 cells. differentially expressed genes, we next asked whether this

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couldbetranslatedintopatient-relateddata.Toidentify, (r ¼ 0.53; P ¼ 1.1e06) between the two proteins immuno- whether there is a correlation between LSD1 and PLK1, we histochemically on the TMA (Fig. 5B and C). In a previous ﬁrst analyzed RNA-seq dataset of TCGA from patients with study, we showed that LSD1 levels correlate with tumor grade lung adenocarcinoma (517 lung adenocarcinoma cases). in lung adenocarcinoma (26). As evident from Fig. 5C, on Impressively, we observed a statistically signiﬁcant correlation similar lines, we observed that PLK1 expression was higher in between LSD1 and PLK1 mRNA expression (r ¼ 0.42; P < high LSD1–expressing tumors (higher tumor grade, g3) and 0.05; Fig. 5A). To further determine, whether the correlation lower in low LSD1–expressing tumors (lower tumor grade, that we found at transcript level also exists at protein level, we g1). Nuclear LSD1 expression was observed all over the tumor inspected 43 lung adenocarcinoma clinical samples for LSD1 area, however a scattered PLK1 expression was observed in the and PLK1 protein expression. Strikingly, in the studied lung tumor cells, with mitotic cells expressing the most (Supple- adenocarcinoma specimens we detected a strong correlation mentary File S1; Supplementary Fig. S3).

A B r = 0.53, P = 1.1e–06 0.30 1 LSD2 0.8

LSD1 0.6 0.25 0.4 PLK1

0.2 PLK1 0.20 PLK4 0 - PLK5 0.2 Figure 5. Correlation -0.4 0.15 LSD1 and PLK1 expression correlates at mRNA and protein levels in clinical lung PLK2 -0.6 adenocarcinoma samples. A, Figure -0.8 PLK3 0.04 0.06 0.08 0.10 0.12 shows Spearman correlation matrix between LSD1 and PLK family members in -1 LSD1 a TCGA lung adenocarcinoma mRNA expression dataset. Correlation between C LSD1 and PLK1 is indicated with a red box. Values that reach statistical significance (P < 0.05) have been represented with a circle (bigger the circle, higher is the significance). Blank spaces indicate a nonsignificant correlation defined by a P > 0.01. The color of the circles in the matrix represent the level of correlation, LSD1 with 1 indicating positive correlation (dark blue) and 1 indicating negative correlation (dark red) between the two protein families. B, Correlation plot between LSD1 and PLK1 protein expression analyzed by IHC on the lung adenocarcinoma TMA. C, Exemplary images of IHC staining performed on lung adenocarcinoma patient samples; top, tumor grade 3 (poorly differentiated) lung adenocarcinoma sample, showing corresponding high LSD1 and PLK1 levels, bottom, tumor grade 1 (well differentiated) showing corresponding low LSD1 and PLK1 levels. Scale bar, 250 mm. PLK1

High Low

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LSD1 Inhibition Downregulates PLK1 Mitotic Pathway

Dysregulation of LSD1 in lung adenocarcinoma affects tumor- clustering approach, we identiﬁed a cluster of genes that was associated activation of the PLK1 pathway differentially expressed in LSD1-high versus LSD1-low– Next, we evaluated a TCGA lung adenocarcinoma dataset expressing tumors. Interestingly, among the clustered genes to examine whether the LSD1/PLK1 axis in lung adenocar- many targets of LSD1 and PLK1 were detected (Fig. 6), cinoma governs the expression of PLK1 target genes involved whose expression positively correlated with that of LSD1 in cell-cycle transition. Using an unsupervised hierarchical and PLK1.

Figure 6. PLK1 target genes are highly expressed in high-LSD1–expressing lung adenocarcinoma tumors. Expression levels for differentially expressed genes after LSD1 inhibition plotted in an unsupervised way following Spearman correlation against a grouped (high- and low-LSD1–expressing group) TCGA lung adenocarcinoma dataset. Columns represent samples and rows represent genes ordered by hierarchical clustering analysis. (Red, relatively high gene expression and blue, relatively low gene expression). The cluster of PLK1 target genes that were found to be differentially expressed between the two groups is highlighted.

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A PLK1 B CDKN1A 1.5 IgG 1.5 IgG 1.2 LSD1 1.2 LSD1

0.9 0.9

0.6 0.6 Figure 7. % Of input

% Of input LSD1 binds to PLK1 promoter region. Shown 0.3 0.3 are representative results of IgG and LSD1 ChIP performed on DNA samples from 0 0 untreated and HCI-2509–treated PC9 cells Untreated HCI-2509 Untreated HCI-2509 (top), and siControl and siLSD1-transfected PC9 cells (bottom). ChIP qPCR analysis of PLK1 CDKN1A IgG and LSD1 at PLK1 (A and C) and CDKN1A C D (B and D) gene promoter regions. Data are 0.6 0.4 IgG IgG presented as mean SD of n ¼ 2 technical replicates from one representative 0.5 LSD1 0.3 LSD1 experiment are shown [entire ChIP 0.4 0.2 experiment performed three times (one 0.3 experiment ¼ two technical replicates)]. 0.2

% Of input 0.2 % Of input 0.1 0.1 0 0.0 siControl siLSD1 siControl siLSD1

LSD1 occupies the promoter region of PLK1 associated with cell-cycle progression and therefore leading to To assess the direct involvement of LSD1 in the regulation of a cell-cycle arrest. The gene expression profiles are consistent PLK1, we examined the occupancy of LSD1 on PLK1 genomic with the work of Liang and colleagues, who also showed region by ChIP followed by qPCR. The ChIP assay showed that deregulation of cell-cycle–related genes upon LSD1 inhibi- LSD1 binds to the PLK1 promoter region and that inhibition of tion (35). Interestingly, classification of the differentially LSD1 using HCI-2509 significantly reduced this effect in PC9 cells expressed genes identified the G2–M DNA damage checkpoint (Fig. 7A). As reported earlier, binding of LSD1 to CDKN1A regulation pathway to be significantly activated, and the mitotic promoter region could also be shown, which significantly roles of PLK pathway to be significantly downregulated after decreased after LSD1 inhibition (Fig. 7B). We also validated the LSD1 inhibition. These results are in agreement with the known effect of change in LSD1 occupancy on PLK1 and CDKN1A role of LSD1 in DNA damage response. Predicted activation of promoter regions in PC9 cells after inhibition of LSD1 using the DNA damage checkpoint regulatory pathway implied the siRNA (Fig. 7C and D). These data demonstrate that LSD1 binds at repression of cell-cycle progression, a mechanism known to be the PLK1 promoter site and thereby potentially regulates PLK1 regulated by PLKs (36). expression. PLKs are vital cell-cycle modulators that are overexpressed in various tumors and are regarded as important prognostic indi- cators for antitumor therapy (37–39). PLK1 is the major player Discussion of the PLK family, which regulates diverse cell-cycle activities, LSD1 was the first histone lysine demethylase discovered, including centrosome maturation, mitotic entry, spindle and since then it has been attributed to play various biological assembly, chromosomal alignment, and segregation and cyto- roles in different cellular processes (8, 32). LSD1 is remarkably kinesis (40). To execute the different mitotic events it interacts overexpressed in a variety of tumor types and its expression with and regulates the function of numerous cell-cycle pro- correlates with aggressive tumor biology (33, 34). Therefore, teins (41–44). In our study, PLK1 and its downstream cell-cycle because of its widespread functions in various cancers, disrupt- regulatory genes were differentially expressed after LSD1 inhi- ing LSD1 signal effect in human malignancies is a potential bition with HCI-2509, as well as after siLSD1-mediated knock- novel therapeutic option. In this study, our findings support a down. Furthermore, PLK1 overexpression was sufficient to previously undescribed role of LSD1 in cell-cycle progression, rescue LSD1 inhibition–mediated alteration of PLK1 target by regulating the key cell-cycle kinase PLK1. genes. Mitotic entry and exit is coordinated by PLK1, therefore Previous work from our laboratory showed that LSD1 is repression of downstream targets genes of PLK1 upon LSD1 overexpressed in lung adenocarcinomas and its expression inhibition should lead to a decrease in mitotic activity of the correlates with tumor grade and invasive phenotypes (26). To tumor cells (45). Significantly, cell cycle, H3S28ph mitotic identify the underlying molecular and cellular mechanisms assay and IF analysis confirmed reduced mitotic activity and associated with higher tumor cell proliferation in presence of arrest of cells in S-G2–M-phase. LSD1, we investigated the impact of LSD1 inhibition in lung The diminished levels of LSD1 observed by IF can be very adenocarcinoma cells using the LSD1 inhibitor HCI-2509. well reasoned with the experimental work of Sehrawat and Microarray profiling revealed that LSD1 inhibition led to dif- colleagues, who could clearly show that HCI-2509 reduces ferential expression of genes, and further comprehensive anal- LSD1 stability and targets it for proteasomal degradation (21). ysis showed a significant downregulation of several genes Whereas the altered localization of LSD1 could be due to lower

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LSD1 Inhibition Downregulates PLK1 Mitotic Pathway

nuclear import rates or protein sequestration in the cytoplasm. tified. In conclusion, this study sheds new light on to a molecular A number of studies have indicated similar effects on cell-cycle mechanism, why disruption of LSD1 function inhibits cell pro- arrest after inhibition or knockdown of LSD1 (46–48). Taken liferation in tumor cells. Importantly, our data also define down- together, the current findings suggest that LSD1 inhibition regulation of PLK1 as a new biological tumor marker, which could blocked cell-cycle advancement by deregulating the key mitotic be used to monitor tumors in vivo with respect to their responses to mediators. LSD1 inhibitors. To rule out the possibility of a cell line–specific effect of LSD1 fl inhibition on PLK1 expression using HCI-2509, we examined the Disclosure of Potential Con icts of Interest fi fi effect of LSD1 inhibition on different cancer cell lines. The R. Buettner is co-founder and chief scienti cof cer at Targos Molecular Pathology, Inc and has received speakers bureau honoraria from BMS, AZ, majority of the cell lines revealed distinct downregulation of Roche, MSD, Illumina, and Boehringer. No potential conflicts of interest were PLK1 mRNA expression in response to LSD1 inhibition. The disclosed by the other authors. varying effects between cancer cell lines could potentially be explained by their genetic diversity, as well as their distinct Authors' Contributions epigenetic landscape. In addition, decreased protein levels of Conception and design: P.S. Dalvi, S.-Y. Lim, R. Buettner, S. Klein, M. Odenthal € PLK1 and AURKA, and increased levels of CDKN1A following Development of methodology: P.S. Dalvi, M. Muller, R. Buettner, M. Odenthal inhibition and knockdown of LSD1 were observed. Downregu- Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P.S. Dalvi, I.F. Macheleidt, H. Eischeid-Scholz lation of PLK1 and AURKA disrupts the PLK1/AURKA axis Analysis and interpretation of data (e.g., statistical analysis, biostatistics, – necessary for G2 M transition (49). Overall, these data indicate computational analysis): P.S. Dalvi, I.F. Macheleidt, M. Muller,€ S.C. Schaefer, a prominent role of LSD1 in controlling cell proliferation via R. Buettner, S. Klein PLK1. Writing, review, and/or revision of the manuscript: P.S. Dalvi, I.F. Macheleidt, Analyzing a lung adenocarcinoma TCGA dataset revealed S. Meemboor, S. Klein, M. Odenthal Administrative, technical, or material support (i.e., reporting or organizing that PLK1 target genes which were downregulated after LSD1 € – data, constructing databases): P.S. Dalvi, S. Meemboor, M. Muller, H. Eischeid- inhibition had elevated levels in high-LSD1 expressing tumors Scholz, S.C. Schaefer, R. Buettner in contrast to the low-LSD1–expressing tumors. These results Study supervision: S. Klein, M. Odenthal suggest that LSD1 and PLK1 coexpress simultaneously in high Other (conducted enlisted experimental procedures): P.S. Dalvi levels in patients with lung adenocarcinoma. Strikingly, we found a statistically significant correlation between LSD1 and Acknowledgments PLK1 at mRNA, as well as at protein level in two separate The authors are thankful for the excellent technical support of Olivia patient cohorts. Interestingly, two independent previous stud- Kaesgen and Ursula Rommerscheidt-Fuss. This work was supported by the funding received from the Center for Molecular Medicine Cologne (2635 8000 ies showed individual correlation of PLK1 and LSD1 with poor 01) and Federal German Ministry of Science and Education (3632 1407 21 tumor differentiation and advanced clinical stage in all to R. Buettner and M. Odenthal) and the North Rhine-Westfalia Ministry NSCLC (26, 50). This is a first study reporting evidence for of Innovation and Sciences as part of e-Med SMOOSE and PerMed-initiatives. correlation of LSD1 and PLK1 in NSCLC. Together, these results In addition, the project was supported by the Else Kroner-Fresenius€ Stiftung point out to a direct transcriptional regulation of PLK1 expres- (2016-Kolleg-19 received to S. Klein). sion by LSD1. On the basis of these findings, we can confirm that PLK1 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked upregulation by LSD1 is a potential mechanism through which advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate LSD1 drives tumorigenesis and invasiveness in cancer. To date, this fact. several studies have shown that inhibition or silencing of LSD1 – leads to reduced cell proliferation, G2 M-arrest, mitotic defects, Received September 12, 2018; revised January 16, 2019; accepted February 8, nonetheless the underlying rationale behind remained uniden- 2019; published first February 13, 2019.

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LSD1 Inhibition Attenuates Tumor Growth by Disrupting PLK1 Mitotic Pathway

Priya S. Dalvi, Iris F. Macheleidt, So-Young Lim, et al.

Mol Cancer Res Published OnlineFirst February 13, 2019.

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