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Therapeutics, Targets, and Chemical Biology

LSD1 Inhibitor T-3775440 Inhibits SCLC CellProliferation by Disrupting LSD1 Interactions withSNAG Domain Proteins INSM1 and GFI1BShinji Takagi1, Yoshinori Ishikawa1, Akio Mizutani1, Shinji Iwasaki2, Satoru Matsumoto3,Yusuke Kamada4, Toshiyuki Nomura1, and Kazuhide Nakamura1

Abstract

T-3775440 is an irreversible inhibitor of the chromatindemethylase LSD1, which exerts antiproliferative effects by dis-rupting the interaction between LSD1 and GFI1B, a SNAGdomain transcription factor, inducing leukemia cell transdiffer-entiation. Here, we describe the anticancer effects and mecha-nism of action of T-3775440 in small-cell lung cancer (SCLC). T-3775440 inhibited proliferation of SCLC cells in vitro andretarded SCLC tumor growth in vivo. T-3775440 disrupted theinteraction between LSD1 and the transcriptional repressorINSM1, thereby inhibiting expression of neuroendocrine-asso-ciated genes, such as ASCL1. INSM1 silencing phenocopied the

effects of T-3775440 on gene expression and cell proliferation,consistent with the likelihood T-3775440 mediated its effects inSCLC by inhibiting INSM1. T-3775440 also inhibited prolifer-ation of an SCLC cell line that overexpressed GFI1B, rather thanINSM1, by disrupting the interaction between LSD1 and GFI1B.Taken together, our results argue that LSD1 plays an importantrole in neuroendocrine-associated transcription and cell prolif-eration of SCLC via interactions with the SNAG domain proteinsINSM1 and GFI1B. Targeting these critical interactions withLSD1 inhibitors offers a novel rational strategy to therapeuticallymanage SCLC. Cancer Res; 77(17); 4652–62. �2017 AACR.

IntroductionSmall-cell lung cancer (SCLC) accounts for approximately 15%

of all lung cancers and represents themost aggressive subtype. The5-year survival rate of SCLC is reportedly less than 7%. Althoughan initial response to standard chemotherapy is observed inmostSCLC cases, nearly all patients relapse within 6–12months. Manyclinical trials investigating SCLC treatments have been conducted;however, there has been little improvement in treatment out-comes over the past few decades (1). In contrast to non–small celllung cancer (NSCLC), effective molecularly targeted therapeuticsfor SCLC have not been developed due to an incomplete under-

standing of the genomic aberrations and biology associated withSCLC.

As SCLC is a neuroendocrine (NE) tumor, it exhibits molecularfeatures associated with neuroendocrine cells. Some NE-associ-ated molecules, including synaptophysin (SYP), chromogranin A(CHGA), and neural cell adhesion molecule (NCAM or CD56),are used as diagnostic and prognostic markers in SCLC patients(2–4). The transcriptional machinery that drives SCLC differen-tiation and proliferation has been extensively investigated.Achaete-scute homolog 1 (ASCL1 or ASH1), a basic helix-loop-helix (bHLH) transcription factor, is a master regulator of neu-roendocrine differentiation in lung development (5), and of cellproliferation and survival in SCLC (6, 7). Neurogenic differenti-ation 1 (NeuroD1), a bHLH transcription factor, regulates SCLCsurvival and migration by functioning as a regulatory hub ofsignaling pathways associated with these processes (8). In addi-tion, insulinoma-associated protein 1 (INSM1), a zinc-fingerprotein with high expression in SCLC cells (9), contributes toNE differentiation by activating ASCL1 expression (10).

Lysine-specific demethylase 1A (LSD1) is a flavin-dependentmonoamine oxidase that demethylates monomethylated ordimethylated lysine 4 or lysine 9 of histone H3, thereby epige-netically regulating the activation or repression of gene transcrip-tion in different contexts (11–13). LSD1 knockoutmice exhibitedan arrest in embryonic development prior to embryonic day 7.5(13, 14).Moreover, studies using a pituitary-specific LSD1-knock-out allele revealed that LSD1 is required for late cell lineagedetermination and differentiation during pituitary gland organ-ogenesis (13). LSD1 is also required for normal hematopoiesis inadults (15). A conditional LSD1 knockout allele in adult hemato-poietic stem cells induced the loss of terminally differentiatedcells and pancytopenia (16). LSD1 forms a large, transcriptionalrepressor complex with GFI1B, CoREST, HDAC1, and HDAC2that controls hematopoietic differentiation (17). The interaction

1Oncology Drug Discovery Unit, Pharmaceutical Research Division, TakedaPharmaceutical Company Ltd., Fujisawa, Kanagawa, Japan. 2Drug Metabolismand Pharmacokinetics Research Laboratories, Pharmaceutical Research Divi-sion, Takeda Pharmaceutical Company Ltd., Fujisawa, Kanagawa, Japan. 3Inte-grated Technology Research Laboratories, Pharmaceutical Research Division,Takeda Pharmaceutical Company Ltd., Fujisawa, Kanagawa, Japan. 4Biomolec-ular Research Laboratories, Pharmaceutical Research Division, Takeda Pharma-ceutical Company Ltd., Fujisawa, Kanagawa, Japan.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Current address for S. Takagi: Oncology Science Unit, Merck Sharp & Dohme,Tokyo 102-8667, Japan.

S. Takagi and K. Nakamura share senior authorship of this article.

Corresponding Authors: Kazuhide Nakamura, Takeda PharmaceuticalCompany Ltd., 26-1 Muraoka Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan. Phone: 81-466-32-2609; Fax: 81-466-29-4410; E-mail:[email protected]; and Shinji Takagi, Merck Sharp & Dohme,Tokyo 102-8667, Japan. Phone: 81-3-6272-1112; E-mail:[email protected] or [email protected]

doi: 10.1158/0008-5472.CAN-16-3502

�2017 American Association for Cancer Research.

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between LSD1 and GFI1B is mediated by the SNAG domain ofGFI1B, and this domain is also required for the transcriptionalrepression of GFI1B. Elevated LSD1 expression levels areassociated with a poor prognosis in many types of solidcancers, including prostate cancer, breast cancer, esophagealcancer, and Ewing sarcoma (18–24). As LSD1 is also stronglyoverexpressed in a number of hematologic malignancies, it hasgained attention as a potential therapeutic target, especially inacute myeloid leukemia (AML; ref. 25). Small-molecule orsiRNA-mediated inhibition of LSD1 induces differentiationand inhibits proliferation of AML cells (25–29). We previouslyreported that T-3775440, a cyclopropylamine-derived irrevers-ible LSD1 inhibitor, inhibited the proliferation of a subset ofAML cell lines by disrupting the interaction between LSD1 andGFI1B, consequently inducing the transdifferentiation of AMLcells (30).

Herein, we report that T-3775440 exhibits anticancer activityin SCLC cells by disrupting the interaction between LSD1 andthe SNAG domain proteins INSM1 and GFI1B, thereby inhibit-ing LSD1-mediated NE transcription. Our findings provide newinsights into SCLC biology that might facilitate the develop-ment of novel molecularly targeted therapies for patients withSCLC.

Materials and MethodsCells and reagents

Cells were purchased from ATCC in 2014 and maintained inRPMI1640 media (Wako) supplemented with 10% (v/v) heat-inactivated FBS (HyClone Laboratories Inc.). All experimentswere performed within 16 passages from the original frozenstocks. NCI-H1417, NCI-H510A, and NCI-H526 cells wereauthenticated using short tandem repeat DNA profiling in2016. Mycoplasma test was performed by Central Institute forExperimental Animals and all cell lines were confirmed to benegative for mycoplasma. T-3775440, N-(4-{(1S,2R)-2-[(Cyc-lopropylmethyl)amino]cyclopropyl}phenyl)-1-methyl-1H-pyra-zole-4-carboxamide hydrochloride was synthesized at TakedaPharmaceutical Company Ltd. GSK-LSD1 was purchased fromSanta Cruz Biotechnology.

Cell growth inhibition assayCellswere seeded in 96-well plates and treatedwith T-3775440.

Cell viability was determined using the CellTiter-Glo assay (Pro-mega) as described previously (31). EC50 values for cell growthinhibition were calculated using GraphPad Prism software(GraphPad Software Inc).

T-3775440 sensitivity screening of a large panel of cell linesThe screen was conducted at Eurofins (http://www.eurofins.

com). Briefly, after a 10-day treatment with the compound, cellproliferation was determined according to the signal intensity ofthe incorporated nuclear dye.

Quantitative PCRTotal RNA was extracted from cells using the RNeasy Mini

Kit (Qiagen), and cDNA was synthesized using the SuperScriptVILO cDNA Synthesis Kit (Thermo Fisher) according to themanufacturer's protocol. Gene expression assays (AppliedBiosystems) were used as follows: INSM1 (Hs00357871_s1),GFI1B (Hs01062469_m1), ASCL1 (Hs04187546_g1), NEU-

ROD1 (Hs01922995_s1), SYP (Hs00300531_m1), CHGA(Hs00900375_m1), GRP (Hs01107047_m1), KRT19(Hs00761767_s1), UMODL1 (Hs00543151_m1), ZEB1(Hs00232783_m1), PTPN14 (Hs00193643_m1), VIM(Hs00185584_m1), and GAPDH (Hs02758991_g1). The Ct

value of each target gene was normalized to the Ct value ofGAPDH as described previously (31).

Immunoprecipitation and immunoblot analysisSCLC cells were lysed in lysis buffer [1% NP-40, 0.25% deoxy-

cholic acid, 50mmol/L Tris-HCl (pH7.5), 150mmol/LNaCl, and1 mmol/L EDTA) supplemented with cOmplete EDTA-free Pro-tease Inhibitor Cocktail and PhosSTOP phosphatase inhibitor(Roche Diagnostics). LSD1 was immunoprecipitated from thelysates with anti-LSD1 antibody and Protein G Sepharose 4 FastFlow (GE Healthcare). Immunoblot assays were conducted asdescribed previously (31). The following antibodies were used inthe immunoprecipitation or immunoblot analysis: anti-LSD1(pAb-067-050) (Diagenode), anti-INSM1 (sc-271408), anti-GFI1B (sc-28356), and normal rabbit IgG (sc-2027; Santa CruzBiotechnology).

Surface plasmon resonance experimentSurface plasmon resonance (SPR) biosensing experiments

were performed as described previously (30). Briefly, theLSD1/CoREST complex was immobilized on the sensor chipusing a standard amine coupling procedure. INSM1 peptide(PRGFLVKRSKKSTPVSYRVR, purchased from SCRUM Inc.) orGFI1B peptide (PRSFLVKSKKAHTYHQPPRVQ, purchased fromSCRUM Inc.) were injected at concentrations of 50 nmol/L and 1mmol/L, respectively, for 120 seconds at a flow rate of 50 mL/minute. To inactivate the LSD1/CoREST complex prior to pep-tide injection, T-3775440 or GSK-LSD1 was injected at con-centarations of 10 mmol/L for 180 seconds at a flow rate of 50mL/minute. BIAevaluation ver. 4.1.1 (GE Healthcare) was usedfor subtraction data processing.

siRNA transfectionNonsilencing ON-TARGETplus SMARTpool (L-HUMN-XX-

0005) and LSD1 ON-TARGETplus SMARTpool (siLSD1_#1, L-009223-00-0005) were purchased from GE Dharmacon. ThesiLSD1_#2 (108660), siINSM1_#1 (s7474), siINSM1_#2(s7475), siGFI1B_#1 (s15850), and siGFI1B_#2 (s15851) siRNAswere purchased from Ambion. AllStars Hs Cell Death ControlsiRNA (siCellDeath) was purchased from Qiagen. The cells weretransfected with siRNA nanoparticles prepared at Takeda Inte-grated Technology Research Laboratories (30) or siRNA usingRNAiMAX Transfection Reagent (Invitrogen) according to themanufacturer's protocol.

Microarray analysisNCI-H510A, NCI-H526, and NCI-H1417 cells were treated

with 500 nmol/L of T-3775440 for 3 days (n ¼ 3). Total RNAwas extracted using an RNeasy Plus Kit (Qiagen). The cells werelabeled and scanned at Macrogen (http://www.macrogen.com)using a SurePrint G3 Human Gene Expression 8 � 60K v2Microarray Kit (Agilent). Ingenuity Pathway Analysis (IPA, Qia-gen) was used to search for possible biological pathways. Allmicroarray data are deposited in the NCBI GEO repository (acces-sion number: GSE100169).

Interaction between LSD1 and INSM1/GFI1B Drives SCLC Growth

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http://www.eurofins.com

http://www.eurofins.com

http://www.macrogen.com


Tumor xenograft modelsSCLC cells were subcutaneously implanted in female BALB/

cAJcl-nu/nu mice (CLEA Japan Inc.). The tumor volume wasmeasured as described previously (30). The treatment/control(T/C) values (%) were statistically analyzed using Williams' test.All of the animal studies were conducted in accordance with theguidelines of the Takeda Institutional Animal Care and UseCommittee in a facility accredited by the American Associationfor Accreditation of Laboratory Animal Care.

Analysis of pharmacodynamic markers in vivoThe tumors were excised and preserved in RNAlater (Ambion).

Total RNA was extracted using an RNeasy Mini Kit (Qiagen), andcDNA was synthesized using a SuperScript VILO cDNA SynthesisKit (Thermo Fisher) according to the manufacturer's protocol.Quantitative PCR was conducted as described previously (31).

ResultsThe LSD1 inhibitor T-3775440 inhibits SCLC cell proliferation

T-3775440 is an irreversible inhibitor of LSD1 (30). We exam-ined the effect of long-term treatment (10 days) with T-3775440on 240 cancer cell lines derived from awide range of tumor types.T-3775440 inhibited the growth of five leukemia cell lines, oneovarian cancer cell line (ES-2), and one SCLC cell line (DMS53)(Fig. 1A). However, the antiproliferative effects on ES-2 cells werenot reproducible (data not shown). To confirm the inhibitoryeffect observed in the one SCLC cell line, we evaluated severalother SCLC cell lines. As a result, T-3775440 inhibited the pro-liferation of three additional SCLC cell lines (NCI-H510A, NCI-H526, and NCI-H1417) in a concentration-dependent manner(Fig. 1B–D). The inhibitory effect of T-3775440 on cell growthwas only observed after 4 or more days of treatment. The EC50

values of T-3775440 in NCI-H510A, NCI-H526, and NCI-H1417cells were highly similar, ranging from 18 to 61 nmol/L (Supple-mentary Fig. S1). Themaximumgrowth inhibition rate associatedwith T-3775440 (10 mmol/L) was greater inNCI-H1417 andNCI-H510A cells (82% and 86% inhibition, respectively) comparedwith NCI-H526 cells (57% inhibition).

T-3775440 disrupts the interaction between LSD1 and INSM1In a previous report, we demonstrated that T-3775440 inhib-

ited the proliferation of a subset of AML cell lines by disruptingthe interaction between LSD1 and GFI1B and inducing tran-scriptional reprograming and transdifferentiation (30). There-fore, we investigated whether T-3775440 also affects the inter-action between LSD1 and its binding partners in SCLC cells. Wefocused on the potential interaction between LSD1 and INSM1,as INSM1 is strongly expressed in SCLC cells (Fig. 2A; Supple-mentary Figs. S2 and S3) and similar to GFI1B, INSM1 pos-sesses a SNAG domain (Fig. 2B). Furthermore, INSM1 isreported to interact with LSD1 in pituitary endocrine cells(32). To evaluate the interaction between LSDI and INSM1 inNCI-H510A cells, we conducted immunoprecipitation experi-ments with an antibody against LSD1. INSM1 coimmunopre-cipitated with anti-LSD1 in DMSO-treated control cells, but notin T-3775440–treated cells, suggesting that LSD1 interacts withINSM1 in SCLC cells and that T-3775440 disrupts this inter-action (Fig. 2C). In addition, LSD1 interacted with CoREST,HDAC1, and HDAC2 in NCI-H510A cells. However, theseinteractions were unaffected by T-3775440. To test whether

T-3775440 directly disrupts the LSD1–INSM1 interaction, wedeveloped a SPR biosensing assay using recombinant LSD1proteins and a peptide consisting of the SNAG domain ofINSM1. Binding of the INSM1 or GFI1B peptide to the LSD1protein significantly elevated the SPR signal, whereas T-3775440 pretreatment diminished this elevation (Fig. 2D).The publicly available LSD1 inhibitor GSK-LSD1 showed asimilar effect (Supplementary Fig. S4). These results indicatethat these irreversible LSD1 inhibitors, cyclopropylamine deri-vatives, directly disrupt the interaction between LSD1 andINSM1.

INSM1 plays a role in the proliferation of NCI-H510A and NCI-H1417 cells

Next, we investigated whether INSM1 is involved in prolifer-ation of SCLC cells. NCI-H510A, NCI-526, and NCI-H1417 cellswere transfected with siRNA targeting INSM1 (siINSM1) or non-silencing siRNA (siControl). INSM1 knockdown was confirmedusing quantitative PCR (qPCR) and immunoblot analysis (Fig.2E; Supplementary Fig. S5A). Cell proliferation was significantlyinhibited in NCI-H510A and NCI-H1417 cells transfected withsiINSM1 compared with siControl (Fig. 2F and G), but this effectwas not observed in NCI-H526 cells (Fig. 2H). Compared withT-3775440 treatment, the siINSM1-mediated antiproliferativeeffect was marginal because of poor transfection efficiency, asshown in siCellDeath-transfected cells and difference in thekinetics of INSM inhibition between siRNAs and drug treatment.

GFI1B plays a critical role in the morphology and proliferationof NCI-H526 cells

As INSM1 knockdown did not significantly affect NCI-H526cell proliferation, we hypothesized that the antiproliferativeeffects of T-3775440 on NCI-H526 cells were mediated byINSM1-independent mechanisms. NCI-H526 cells treated withT-3775440 underwent a distinct morphologic change (Fig. 3A).DMSO-treated NCI-H526 cells formed sheet-like cell aggregates,whereas T-3775440–treated cells formed spheroid structures.Unlike NCI-H510A and NCI-H1417 cells, NCI-H526 cells unex-pectedly expressed high levels of GFI1B, and these levels were ashigh as in some AML cell lines (Fig. 3B). Similar to what wepreviously demonstrated inAML cells (30), immunoprecipitationassays revealed that LSD1 interacted with GFI1B, CoREST, andHDAC1/2 in NCI-H526 cells and that T-3775440 disrupted theinteraction between LSD1 and GFI1B (Fig. 3C). To determinewhether GFI1B is involved in T-3775440–associated changes inthe morphology and growth of NCI-H526 cells, we inhibited theexpression of LSD1, INSM1, and GFI1B using siRNA. siRNA-mediated knockdown of LSD1 or GFI1B, but not INSM1, mim-icked the effects of T-3775440 on cell morphology (Fig. 3D–K;Supplementary Fig. S5B) and inhibited proliferation of NCI-H526 cells (Fig. 3L). These results suggest that the LSD1–GFI1Bcomplex plays a critical role in the morphology and proliferationofNCI-H526 cells and that the effects of T-3775440onNCI-H526cells in this context are primarilymediatedby thedisruption of theLSD1–GFI1B complex.

T-3775440 affects the expression of neuroendocrine-relatedgenes in SCLC cells

To identify the genes affected by LSD1 inhibition inT-3775440–sensitive SCLC cell lines, we evaluated NCI-H1417,NCI-H510A, and NCI-H526 cells treated with T-3775440 for

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Cancer Res; 77(17) September 1, 2017 Cancer Research4654




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T-3775440 exhibits antiproliferative activity in a subset of SCLC cell lines. A, Cell proliferation assay of 240 cell lines. The seven most sensitive cell lines arehighlighted. The SCLC cell line DMS53was sensitive to T-3775440.B–D, Time- and concentration-dependent antiproliferative effects of T-3775440onNCI-H1417 (B),NCI-H510A (C), and NCI-H526 (D) cells. The y-axis represents relative light units (RLU). Data represent the mean þ SD.






3 days using DNAmicroarray analysis (Supplementary Table S1).Ninety-two genes (183 probes) and 25 genes (39 probes) weresignificantly upregulated and downregulated (P < 0.05), respec-tively, with a 1.5-fold change or greater in T-3775440–treatedcells compared with control cells (Fig. 4A and B; SupplementaryTable S2). These genes were considered to contain putativepharmacodynamic markers of T-3775440 in SCLC cells. Toidentify the molecular mechanism underlying the antiproli-ferative effect of T-3775440, we further evaluated the differen-tially expressed genes. Gene ontology (GO) analysis revealedthat T-3775440–affected genes were associated with neuronaldevelopment and differentiation (Fig. 4C and D). Furthermore,Ingenuity Pathway Analysis (IPA) revealed that the transcrip-tion factors ASCL1 and NeuroD1, which are commonlyexpressed in SCLC cells (refs. 5, 7, 8, 33; Supplementary Fig.S6A and S6B), are upstream regulators of T-3775440–affectedgenes (Fig. 4E). T-3775440 inhibited the expression of ASCL1

and NeuroD1 at both the mRNA and protein levels in NCI-H1417 and NCI-H510 cells, but not in NCI-H526 cells (Fig.4F–H; Supplementary Fig. S7A and S7B). T-3775440 alsoinhibited the expression of the NE markers SYP and CHGA,and gastrin-releasing peptide (GRP), a well-established serummarker of SCLC, in NCI-H1417 and NCI-H510A cells, but notin NCI-H526 cells (Fig. 4I–K; Supplementary Fig. S7C–S7E). Inaddition, the effects of the siRNA-mediated knockdown ofASCL1 or NeuroD1 on the proliferation of NCI-H1417 andNCI-H510A cells were investigated. All of the three siASCL1ssignificantly inhibited proliferation in NCI-H1417 and NCI-H510A cells (Fig. 4L–N). However, one of the three siNeuroD1s(siNeuroD1_#2) did not significantly inhibit the proliferationof NCI-H1417 cells, and growth-inhibitory effects of siNeur-oD1s were less pronounced than those of siASCL1s in H1417and H510A cells. These results suggest that the antiproliferativeeffects of T-3775440 are mainly mediated by a decrease in

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INSM1 interacts with LSD1 and is involved in the proliferation of a subset of SCLC cells. A, INSM1 mRNA expression levels as determined using Cancer Cell LineEncyclopedia data. B, Schematic representation of human GFI1B and INSM1. The amino acid sequences of the SNAG domains are shown. The prolineresidue highlighted in red is predicted to be critical for the interaction between GFI1B and LSD1 (17). C, Immunoprecipitation of endogenous INSM1with anti-LSD1 in NCI-H510A cells treated with DMSO or T-3775440 (1 mmol/L) for 24 hours. D, SPR sensorgrams of the interaction of LSD1 with INSM1peptide (gray, left) or GFI1B peptide (gray, right). SPR sensorgrams after treatment with T-3775440 are shown in red. E, Confirmation of INSM1knockdown in NCI-H1417 cells transfected with siINSM1 (n¼ 4). F–H, Cell viability in siINSM1-transfected NCI-H510A (F), NCI-H1417 (G), and NCI-H526 (H) cells.SiCellDeath was used as a control siRNA to monitor transfection efficiency. Data represent the mean þ SD. �� , P < 0.001 vs. siControl as calculatedusing Dunnett parametric multiple comparison test. NS, not significant.

Takagi et al.





ASLC1 expression. In contrast, neither ASCL1 nor NeuroD1knockdown affected the proliferation of NCI-H526 cells(Fig. 4O).

INSM1 knockdown mimics the effects of T-3775440 on geneexpression in SCLC cells

Next, we investigated the potential role of INSM1 in T-3775440–induced changes in gene transcription. In both T-3775440–treated and INSM1 knocked down NCI-H1417 cells,expression levels of the NE-associated genes ASCL1, NeuroD1,SYP, CHGA, andGRP decreased (Fig. 4F, 4G, 4I–K, and Fig. 5A–F),and expression levels of keratin 19 (KRT19), uromodulin like 1(UMODL1), and vimentin (VIM) increased (Supplementary Fig.S8A–C; Supplementary Table S2). These results suggest that T-3775440–mediated changes in gene expression were at leastpartially attributable to the perturbation of INSM1 function viathe disruption of its interaction with LSD1.

T-3775440 exhibits antitumor activity in SCLC tumor xenograftmodels

We evaluated the antitumor activity of T-3775440 in vivo usingSCLC tumor xenograft mouse models that received two cycles

(5 days on/2 days off) of once-daily oral doses of T-3775440.T-3775440 significantly inhibited tumor growth at 10 or30 mg/kg, with treatment/control values (T/C) values of 54%and 45%, respectively, in a NCI-H510A model on day 15(Fig. 6A). It also inhibited tumor growth at 15 and 30 mg/kgwith T/C values of 34% and 20%, respectively, in a NCI-H526model (Fig. 6B). Body weight loss was observed in mice treatedwith 30 mg/kg T-3775440 (Supplementary Fig. S9A and S9B)and the treatment had to be discontinued in one of five micebearing NCI-H510A on day 10, when the humane endpoint wasreached. None of the mice that received 10 or 15 mg/kgT-3775440 required treatment discontinuation. Consistent withthe in vitro results, in vivo tumor growth inhibition was notdetectable in the first few days after treatment, but was apparentby the second week of treatment. In addition, expression levels ofthe putative pharmacodynamic (PD) markers KRT19, UMODL1,ZEB1, and PTPN14 increased 8–24 hours after a single dose ofT-3775440 (30 mg/kg) compared with the vehicle control(Fig. 6C–F). Plasma concentrations of T-3775440 reached peaklevels 15 minutes to 2 hours after T-3775440 administration(Tmax ¼ 1.08 hours) and returned to near baseline levels after24 hours (Supplementary Fig. S10; Supplementary Table S3).

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Figure 3.

GFI1B, but not INSM1, plays a role in the morphology and viability of NCI-H526 cells. A, NCI-H526 cells were treated with DMSO (control), or 100 nmol/L T-3775440.Representative images of cell morphology on day 4 are shown. Scale bars, 100 mm. B, GFI1B mRNA expression levels were determined using qPCR. Geneexpression levels were normalized to GAPDH levels. TF-1a and HEL92.1.7: AML cell lines. RKO, colon cancer cell line. C, Immunoblot analyses of proteins thatimmunoprecipitated with anti-LSD1 in NCI-H526 cells treated with DMSO or T-3775440 (1 mmol/L) for 24 hours. D–K, NCI-H526 cells were transfected with siRNAtargeting INSM1, LSD1, orGFI1B. Representative images on day 3 are shown. L, The effects of INSM1, LSD1, orGFI1B knockdown on the viability of NCI-H526 cells. Datarepresent the mean þ SD. �� , P < 0.001 versus siControl as calculated using Dunnett parametric multiple comparison test. N.S., not significant.






DiscussionIn this study, we demonstrated that the irreversible LSD1

inhibitor T-3775440 exerts anticancer effects in SCLC in vitro andin vivo. Previous reports have demonstrated that LSD1 is over-expressed inmany types of human cancers, including lung, breast,prostate, and blood cancers (18, 19, 25, 34, 35). However,preclinical data supporting a therapeutic role for LSD1 inhibitorshave been limited to AML. We recently reported that T-3775440exerts anticancer effects in a subset of AML cell lines (30). Duringthe preparation of this manuscript, Mohammad and colleaguesreported that the LSD1 inhibitor GSK2879552 exerted antitumoreffects in a subset of SCLC cell lines (36). The three T-3775440–sensitive SCLC cell lines identified in this study (NCI-H510, NCI-H1417, and NCI-H526) were also sensitive to GSK2879552.Consistent with the reported effects of GSK2879552, the anti-proliferative effects of T-3775440 were primarily cytostatic.Mohammad and colleagues utilized similar cell growth assays,

and both LSD1 inhibitors contain a cyclopropylamine moiety.Together, these results suggest that a commonmechanism under-lies the antitumor effects of T-3775440 andGSK2879552 in SCLCcells. However, the precise mechanism underlying the antitumoreffects of GSK2879552 remains unclear.

We found that T-3775440 disrupted the interaction betweenLSD1 and the SNAG domain transcription factor INSM1 (Fig. 2Cand D). INSM1 knockdown inhibited proliferation of NCI-H510A and NCI-H1417 cells (Fig. 2E and F), and mimicked theeffects of T-3775440 on gene expression patterns (Figs. 4 and 5).These results suggest that the antiproliferative effects of T-3775440 in SCLC cells are at least partially dependent on theinhibition of INSM1-mediated gene transcription. It has beenreported that INSM1 interacts with LSD1 via its SNAG domainand that the resulting complex controls the differentiation ofendocrine cells in the anterior pituitary gland (32). Our findingssuggest that the LSD1–INSM1 complex plays a similar role inneuroendocrine differentiation in SCLC cells. Indeed, T-3775440

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Figure 4.

T-3775440 affects the neuroendocrine-related transcriptional program in SCLC cells. A and B, Microarray analysis was conducted to identify genes affectedbyT-3775440 inT-3775440–sensitive SCLC cell lines. AVenndiagramof upregulatedgenes (A) and downregulated genes (B) that exhibited a statistically significantchange in expression levels of 1.5-fold or greater (P < 0.05). C and D, GO analysis of genes that were upregulated (C) and downregulated (D) by T-3775440in NCI-H510A cells. E, IPA analysis of transcription factors affected by T-3775440. F, G, and I–K, qPCR analysis of genes associated with the neuroendocrinephenotype:ASCL1 (F),NeuroD1 (G), SYP (I),CHGA (J), andGRP (K).H, Immunoblot analysis of DMSO- and T-3775440-treated cells. L,M, andO, Cell viability assay incells transfected with siASCL or siNeuroD1: NCI-H1417 (L), NCI-H510A (M), and NCI-H526 (O). Data represent the mean þ SD. �� , P < 0.001; �� , P < 0.01;� , P < 0.05 versus siControl as calculated using Dunnett parametric multiple comparison test. N.S., not significant. N, Immunoblot analysis of NCI-H510A cellstransfected with siASCL1 or siNeuroD1.

Takagi et al.





treatment disturbed neuroendocrine lineage-associated transcrip-tion in SCLC cells, as evidenced by a decrease in the expression ofneuroendocrine markers such as CHGA and GRP (Fig. 4J and K).These findings were further supported by Gene Ontology analysis(Fig. 4C and D).

Furthermore, our results suggest that the downstream effects ofT-3775440–mediated INSM1 inhibition are mainly dependenton ASCL1, a master regulator of neuroendocrine differentiation(Fig. 4E), because ASCL1 expression was downregulated in T-3775440–treated cells as well as in INSM1 knockdown cells (Figs.4F and H and 5A and B). Notably, INSM1 has been reported tobind to the promoter region of ASCL1 and positively regulateASCL1mRNA expression (10). Therefore,ASCL1 downregulationmay be a direct downstream effect of T-3775440–mediatedINSM1 inhibition. Moreover, ASCL1 knockdown inhibited SCLCgrowth (Fig. 4L andM), a finding consistent with previous reportsshowing that ASCL1 is essential for the growth and survival ofneuroendocrine lung cancers (6, 7) Although NeuroD1 isreported to promote SCLC cell survival and metastasis (8), Neu-roD1 knockdown had a less pronounced effect on SCLC cell

proliferation than ASCL1 knockdown in our experiment. Theseresults suggest that T-3775440 targets the LSD1/INSM1 complex–ASCL1 axis and suppresses SCLC cell growth. Lenhart and col-leagues recently reported that the BET inhibitor JQ1 inhibits SCLCproliferation via downregulation of ASCL1 (37). This supportsour mechanistic insight that T-3775440–mediated INSM1 inhi-bition leads to growth inhibition via ASCL1 downregulation.

It should be noted that the anticancer effects of T-3775440would be mediated by a similar mechanism in SCLC and AMLcells. T-3775440 disrupted the interaction between LSD1 andINSM1 and altered INSM1-dependent neuroendocrine-relatedtranscription in SCLC cells. Similarly, T-3775440 disrupted theinteraction between LSD1 and GFI1B, and altered GFI1B-depen-dent megakaryocyte/erythroid–related transcription in megakar-yoblastic leukemia/erythroleukemia cells (30). Unexpectedly, wefound thatGFI1B is highly expressed inNCI-H526 cells. Similar towhat was observed in AML cells, T-3775440 disrupted the inter-action between LSD1 and GFI1B in NCI-H526 cells and alteredthe corresponding downstream transcriptional program, therebyinhibiting cell growth. These results suggest that INSM1 and

Figure 5.

INSM1 knockdown inhibits the expressionof neuroendocrine-related genes.NCI-H1417 cellswere transfectedwith siControl or siINSM1, andmRNAexpression levels ofthe indicated genes were measured using qPCR. Human GAPDH was used as an internal control. Expression levels of INSM1 (A), ASCL1 (B), NeuroD1 (C),SYP (D),CHGA (E), andGRP (F). Data is presented as themeanþ SD. �� , P <0.001 versus siControl as calculated using Dunnett parametric multiple comparison test.






GFI1B play similar roles via their interaction with LSD1 in themaintenance of the neuroendocrine lineage in SCLC cells,although the morphologic change caused by T-3775440 wereonly observed in GFI1B-expressing NCI-H526 cells. Moreover,among23pulmonary tissue samples resected fromSCLCpatients,many of which overexpressed INSM1, one sample overexpressedGFI1B rather than INSM1, suggesting that GFI1B plays a clinicallyrelevant role in patients with SCLC (Supplementary Fig. S11A andS11B; ref. 38).

Our findings indicate that the anticancer effects of T-3775440are primarily mediated by the inhibition of the interactionbetween LSD1 and INSM1 or GFI1B, and by subsequent changesin downstream transcription associated with neuroendocrinephenotype. Stricker and colleagues reported that the redifferen-tiation of glioblastoma-derived iPS cells to non-neural lineagecells suppressed the malignant behavior (39). These results sug-

gest that concordance between genetic mutation and differenti-ation status is important for the maintenance of the malignantphenotype. Thus, disturbance in the neuroendocrine lineageinduced by T-3775440 may override the activity of geneticallyaltered pathways in driving unconstrained proliferation. Howev-er,we cannot eliminate thepossibility that the anticancer effects ofT-3775440 are also mediated by catalytic inhibition of LSD1.

Ogasawara and colleagues reported that the cyclopropylamine-derived LSD1 inhibitors form covalent bond with flavin adeninedinucleotide (FAD) in the active site cavity of LSD1, where severalresidues of the histone tail substrate can be accommodated (40).Lin and colleagues have additionally reported that the sequence ofthe SNAG domain is similar to that of the histone H3 tail and thatthe interaction of LSD1 and Snail1, another SNAG domainprotein, can be blocked by a histone H3 peptide (41). Thesedata suggest that cyclopropylamine-derived LSD1 inhibitors

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T-3775440 pharmacodynamics andantitumor efficacy in SCLC xenograftmodels. A and B, The efficacy of T-3775440 was analyzed in NCI-H510A(A) and NCI-H526 (B) tumor xenograftmodels. Tumor-bearing mice weretreated with the vehicle control orT-3775440 at the indicated oral doseonce daily (QD) from day 1 to day 12.Data represent mean tumor volume �SEM. �� , P < 0.01; �� , P < 0.001 versusvehicle as calculated using Williams'test.C–F,Gene expression levels inNCI-H526 tumors isolated from micetreated with a single oral dose ofT-3775440 (30 mg/kg) weredetermined using qPCR. HumanGAPDHwas used as an internal control.

Takagi et al.





specifically disrupt the interaction between LSD1 and SNAGdomain proteins through covalent bond formation with FAD ofLSD1. Indeed, interaction of CoREST or HDAC1/2 proteins withLSD1 is not affected by T-3775440 (Fig. 2C), suggesting itsselectivity for SNAG domain proteins. Other SNAG domainproteins interact with LSD1 and are selectively recruited to specificloci to ensure the fine-tuning of transcription and downstreameffects. For example, the SNAG domain protein Snai1 recruitsLSD1 to epithelial gene promoters, mediating transcriptionalrepression of epithelial markers during epithelial–mesenchymaltransition (41, 42). Thus, it would be worth investigating whetherT-3775440 affects the interaction with other SNAG domainproteins and their functions in different cell types.

We demonstrated the antitumor effects of T-3775440 in vivo intumor xenograft models generated from INSM1-overexpressingNCI-H510 cells and GFI1B-overexpressing NCI-H526 cells (Fig.6A and B). T-3775440 upregulated the same subset of genes inboth cell types in vitro and in vivo, suggesting that T-3775440functions via the same mechanism in both settings. In addition,prolonged pharmacodynamic responses were observed even 24hours after a single dose of T-3775440, despite the rapid clearanceof the compound (Tmax of approximately 1 hour). This effectmight be attributable to the irreversible mode of action of T-3775440 and/or a delayed response to its downstream transcrip-tional effects. At efficacious doses, T-3775440 was generallytolerated in mice; however, thrombocytopenia was observed inT-3775440–treated mice (data not shown). This observation isconsistent with that of previous reports in AML models and isconsidered as amechanism-driven, reversible effect of T-3775440(15, 30). Additional in vivo studies are needed to determine theoptimal T-3775440 dose and dosing schedule in SCLC.

In conclusion, we demonstrated that the LSD1 inhibitor T-3775440 exerts anticancer effects in SCLC. Mechanistically, T-3775440 disrupted the interaction between LSD1 and the SNAGdomain proteins INSM1 and GFI1B, thereby distorting the neu-roendocrine-related gene expression program of SCLC. Our find-ings provide a novel insight into the functional interaction

between LSD1 and SNAG domain proteins in SCLC and suggestthat targeting these interactions using LSD1 inhibitors is a poten-tial strategy to treat patients with SCLC. Moreover, it is generallydifficult to directly target transcription factors using small-mol-ecule inhibitors. However, emerging evidences suggest that theinhibition of factors involved in super-enhancer regulation mayrepresent a strategy to target transcription factor–driven cancers,such as BRD4 inhibition (43, 44). Our results provide an exampleof the regulation of intractable oncogenic programs driven bysequence-specific transcription factors by targeting obligate epi-genetic coregulators.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S. Takagi, K. NakamuraDevelopment of methodology: S. Matsumoto, Y. KamadaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Takagi, Y. Ishikawa, A. Mizutani, S. Iwasaki,Y. KamadaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Takagi, A. Mizutani, S. IwasakiWriting, review, and/or revision of the manuscript: S. Takagi, S. Iwasaki,K. NakamuraStudy supervision: S. Takagi, T. Nomura, K. Nakamura

AcknowledgmentsWewould like to thank all the LSD1projectmembers for helpful discussions;

SyuMorita for technical assistance; andKoji Yamamoto for processing ofAgilentraw data. We would also like to express cordial gratitude to Daisuke Tomita andShinichi Imamura for providing us with T-3775440 and Hiroshi Miyake andChristopher Claiborne for their guidance and support during the course of thiswork.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 22, 2016; revised May 13, 2017; accepted June 26, 2017;published OnlineFirst June 30, 2017.

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Takagi et al.




2017;77:4652-4662. Published OnlineFirst June 30, 2017.Cancer Res Shinji Takagi, Yoshinori Ishikawa, Akio Mizutani, et al. and GFI1BDisrupting LSD1 Interactions with SNAG Domain Proteins INSM1 LSD1 Inhibitor T-3775440 Inhibits SCLC Cell Proliferation by

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