Colby College Colby College

Digital Commons @ Colby Digital Commons @ Colby

Honors Theses Student Research

2016

Development of a Chemical Genetic Screen to Determine Development of a Chemical Genetic Screen to Determine

Synergistic Compounds with Laromustine in Treating Synergistic Compounds with Laromustine in Treating

Glioblastoma Multiforme Cultured Cells Glioblastoma Multiforme Cultured Cells

Ryan Weeks Colby College

Follow this and additional works at: https://digitalcommons.colby.edu/honorstheses

Part of the Biochemistry Commons, Chemical Actions and Uses Commons, and the Pharmaceutical

Preparations Commons

Colby College theses are protected by copyright. They may be viewed or downloaded from this

site for the purposes of research and scholarship. Reproduction or distribution for commercial

purposes is prohibited without written permission of the author.

Recommended Citation Recommended Citation

Weeks, Ryan, "Development of a Chemical Genetic Screen to Determine Synergistic Compounds

with Laromustine in Treating Glioblastoma Multiforme Cultured Cells" (2016). Honors Theses.

Paper 823.

https://digitalcommons.colby.edu/honorstheses/823

This Honors Thesis (Open Access) is brought to you for free and open access by the Student Research at Digital Commons @ Colby. It has been accepted for inclusion in Honors Theses by an authorized administrator of Digital Commons @ Colby.

DevelopmentofaChemicalGeneticScreentoDetermineSynergisticCompoundswithLaromustineinTreating

GlioblastomaMultiformeCulturedCells

ByRyanA.Weeks

PresentedtotheDepartmentofChemistry,

ColbyCollege,Waterville,MEInPartialFulfillmentoftheRequirementsforGraduation

WithHonorsinChemistry

SubmittedMay16,2016

ii

DevelopmentofaChemicalGeneticScreentoDetermineSynergisticCompoundswithLaromustineinTreating

GlioblastomaMultiformeCulturedCells

ByRyanA.Weeks

Approved:

___________________________________________________________ (KevinP.Rice,AssociateProfessorofChemistry)

____________________________________________________Date

___________________________________________________________(JulieT.Millard,TheDr.GeraldandMyraDorrosProfessorofLifeSciences)

____________________________________________________Date

iii

Vitae RyanAdamWeekswasbornonSeptember30,1993andgrewupinFramingham,

Massachusetts.HegraduatedfromFraminghamHighSchoolin2012inthetop1%ofhis

class.HematriculatedatColbyCollegeinWaterville,MEasaWilliamD.AdamsPresidential

Scholar.WhileatColby,Weeksdoublemajoredinchemistrywithaconcentrationin

biochemistryandmathematicalsciences.HejoinedAssociateProfessorKevinRice’s

biochemistrylaboratoryduringhisfirstyearasaColbyAcademicResearchAssistantand

continuedresearchwithProf.Riceuntilgraduation.Additionally,hewasafour-year

student-athleteasamemberofthevarsityswimminganddivingteam,aCo-Presidentof

Hillel,andaCCAKmentorinWinslow,ME.During2015summer,Weekswasavisiting

researchassistantatDana-FarberCancerInstituteinthelaboratoryofAssistantProfessor

TimurYusufzaistudyingchromodomainhelicases.Inthefallof2016,hewilljointhe

Chemistry-BiologyInterfaceProgramatJohnsHopkinsUniversityinhispursuitofaPh.D.

inChemicalBiology.

PermanentAddress:

33SummerLaneFramingham,MA01701

iv

Acknowledgments

Firstandforemost,IwouldliketothankProfessorKevinRiceforhisguidanceon

thisprojectandduringmyentireundergraduatecareer.Hissupportasaresearchmentor

hasbeeninstrumentalinhelpingcompletethisprojectandgrowasascientist.His

instructioninbiochemistryandthemethodsofbiochemistryresearchhasbeen

instrumentalinallowingmetodevelopthespecificskillsneededtoconductresearchinthe

lab.Additionally,IwouldliketoProfessorJulieMillardforbeingthereadertothisthesisas

wellasforherinstructioninbiochemistry.IwouldliketoacknowledgetherestoftheRice

labmembers,pastandpresent,fortheirdevotiontobiochemistryresearchandbringing

passiontothelab.IamespeciallythankfultoKatieCoe’14forherpreviousworkonthe

project,aswell.Finally,IwouldliketothankEdmundKlinkerchforhisworkin

synthesizingthelaromustineusedduringthisprojectaswellasAmyPoulinfor

administrativesupport.ThisprojectwasfundedbygrantsfromtheNationalCenterfor

ResearchResourcesandtheNationalInstituteofGeneralMedicalSciencesoftheNational

InstitutesofHealththroughtheMaineIdeaNetworkofBiomedicalResearchExcellence

andwasmadepossiblebyfundingandfacilitysupportfromColbyCollege.

v

TableofContents

Vitae.........................................................................................................................................................iiiAcknowledgments..............................................................................................................................iv

TableofContents..................................................................................................................................v

Abstract...................................................................................................................................................1Introduction...........................................................................................................................................2

MaterialsandMethods.......................................................................................................................9

ResultsandDiscussion.....................................................................................................................12FutureWork........................................................................................................................................21

References............................................................................................................................................23

1

Abstract

Laromustineisachemotherapeuticsulfonylhydrazineprodrugusedinclinicaltrials

totreatacutemyeloidleukemia(AML)andglioblastomamultiforme(GBM).While

treatmentofAMLwithlaromustinehasmoredemonstrativeclinicalsuccess,thereare

enoughpromisingdataagainstGBMtopursueadditionalpre-clinicalandclinical

experiments.TodeterminethesynergisticeffectscausedbytreatingculturedGBMcells

withlaromustineandalibraryofFDA-approvedcompounds,achemicalgeneticscreenwas

developed.Tooptimizethescreen,optimalculturedGBMcellseeddensity,growthperiod

andmaximumwellcapacityweredetermined.Thetreatmentperiodforalethaldoseof

laromustineinculturedGBMcellswasfoundtobe6hours;causingacutecelldeathinhalf

asmuchtimeasthetreatmentwithalethaldoseofTemozolomide,thecurrentGBM

treatment.TheLD50forlaromustineinculturedGBMcellswasobservedtobe

approximately700µMwhentreatedfor6hours.Usingthesestandardsofoptimizationfor

maximumreproducibility,achemicalgeneticscreenwillbeusedtodeterminethe

synergisticeffectsoflaromustinewithalibraryofcharacterizedsmallmolecules.

2

Introduction Despitenumerousadvancesmadeinthetreatmentofcancer,itstillremainsasone

oftheleadingcausesofdeathintheUnitedStates.Canceriscategorizedbyailmentscaused

byuncontrolledandunregulatedgrowthofabnormalcellsinthebody.Someofthemain

treatmentplansforcancerintheUnitedStatesincluderadiationtherapy,surgical

resection,andchemotherapy.Chemotherapyisthetreatmentofcancerthroughthe

introductionofchemicalsthatdamageordestroycancercells,ofteninsultingnormalcells

intheprocess.Whileexistingchemotherapyissuccessfulinmanycases,therearestill

numeroustreatmentslefttobediscovered,withover4775activeclinicaltrialsranging

fromphaseItophaseIVacrosstheUnitedStates(2)

Glioblastomasarehighlymalignanttumorsgenerallyfoundinthecerebral

hemispheresofthebrain.Theyarisefromastrocytes,star-shapedadhesivecellsfoundin

thesupportivetissueofthebrain.(3).Glioblastomasmultiforme,orgrade4glioblastoma,

isthemostcommon,yetmostaggressiveformofglioblastomainhumans.Becauseofthe

tumors’aggressiveformandlowsurvivalrateoflessthanayear,successfultreatments

havebeenevadingresearchersforthelast50years(4).Currenttreatmentsinvolve

immediatesurgicalresectionofthetumor,followedbytreatmentwithin3-4weekswith

radiationtherapyandconcurrentoradjuvantchemotherapy(5).However,treatment

remainsdifficultbecauseofthecomplexnatureofglioblastomamultiformeinaspects

rangingfromvaryingformsmicroscopically,grossly,andgenetically(4).Researchhas

estimatedthatglioblastomamultiformecellsmayhavemutationsinanygeneatarateof1

in1,000cells,makingitextremelydifficulttotargetduetoitsmutatingnature(6).Current

researchfortreatmenthasspannedmanyapproaches,includingimmunologicavenues,

3

Figure1:Decompositionoflaromustineto90CEandmethylisocyante(1)

genetherapy,aswellasadditionalchemotherapeuticpossibilities,thoughtherehasbeen

littlesuccessinextendingthemeansurvival(7,8).

Laromustine(Cloretazine,Onrigin,1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-

[(methylamino)carbonyl]hydrazine),isanexperimentalchemotherapeuticagentusedin

clinicaltrialstocombatacutemyeloidleukemia(AML)andglioblastomamultiforme

(GBM).Laromustine,asulfonylhydrazineprodrug,undergoesbase-catalyzedactivationto

produce90CE(1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-hydrazine),achloroethylating

species,andmethylisocyanate,whichcancarbamoylatethiolsandprimaryamines(Figure

1).Researchdemonstratesthat90CEchloroethylatesDNAattheO6positionofguanine,

whichleadstoaninterstrandcrosslinkwithcytosineontheoppositestrand;these

linkagesareconsideredthelethallesionsthatdisruptDNAreplicationandcausecelldeath

(9).DatasuggestthatlaromustineproducesmorethantwicethemolaryieldofDNAcross-

4

linkscomparedtocommonnitrosoureas,lipophilicDNAalkylatingagentscontaining

nitrosoandureagroupsthatareoftenusedintreatinggliomas(10,11).

OneofthemainprocessesofDNArepairisbaseexcisionrepairinwhichenzymes

removeerroneousnitrogenousbasesandreplacethemwiththecorrectbase.

Laromustine’scarbomoylatingactivitieshasbeenshowntoinhibittheactivityoftherepair

enzymeDNApolymeraseβ(Polβ),whichisinvolvedinbaseexcisionrepair(12).Cells

deficientinPolβhaveshownhypersensitivitytosomecrosslinkingagents,creatinga

possiblesynergismbetweenlaromustine’s2-chloroethylatingspeciesandcarbomoylating

species(12,13).Atthesametime,methylisocyanateishypothesizedtointerferewith

tumorangiogenesisbyinducingdissociationofASK1fromthioredoxin(1,14).Additionally,

itisthoughtthatmethylisocyantemaypromotecross-linkingby90CE,perhapsthrough

theinhibitionoftheDNArepairproteinO6-alkylguanine-DNA-alkyltransferase(AGT)or

otherDNArepairprocesses(12,15).

Laromustinehasyieldedpromisingpreclinicaldatainculturedneoplasticcellsand

invivoactivityagainstAML.Itinducesadose-dependentinhibitionofproliferation,

reductionincellviabilityandanincreaseinapoptosisinallsamples,effectsonlyenhanced

whencombinedwiththeotherAMLdrugscytarabineordaunorubicin(16,17).Intreating

patientswithAML,laromustinehasbeenusedinphaseIItrials,whichsuggestpossible

benefitsoverotherchemotherapydrugs.Forexample,inatrialwith85poor-riskelderly

patientswithpreviouslyuntreatedAML,theoverallresponseratewas32%;however,

therewasnorandomizedsettingduringthestudywhichmadeitimpossibletodetermineif

laromustinewasmoresuccessfulthancurrenttreatmentstrategies(10,18,19).Ina

separatestudy,laromustinecombinedwithcytarabineshoweda37%responseratewhile

5

thecontrolofjustcytarabineshowedonlya19%responserate.Thestudywasstoppeddue

tohighlevelofdeathfrommyelosuppressionwiththeintentofcontinuingthetrialwith

lowerdosagesduetoitsresponsesuccess(16).Clinicaltrialssuggestthatlaromustine

shouldbeinvestigatedfurtherwithothernon-traditionalcytotoxicagentswithalackof

toxicitythatmaysynergisticallyextendthebenefitsoflaromustine(16).

Currently,themediansurvivalforanadultwithglioblastomais14.6monthswith

concurrentradiationtherapyandtreatmentwithtemozolomide,analkylating

chemotherapeuticagentthatpassesthroughtheblood-brainbarrier(20).Previous

researchhasbeendoneregardingthetreatmentofglioblastomapatientswithlaromustine;

however,theresultshavenotproventobeconclusive.Patientswhodidnotrespondto

radiationandtemozolomidetreatmentweregivenlaromustineandshoweda6-month

survivalrateof6%withamedianprogressionfreesurvivalrateof6.3weeks(21,22).

Despitethemodestsuccess,laromustineisstillbeinginvestigatedfortreatmentofGBM

duetoitsability,liketemozolomide,topasstheblood-brainbarrier.Additionally,although

evidencesuggeststhatlaromustineandtemozolomideareeffectiveintreatingpatients

withrefractoryAML,furtherstudieshavenotbeendonetoexaminetheeffectsofpartner

drugswithlaromustineinGBMtreatment(23).

High-throughputscreeninghasbecomeanimportanttoolforresearchers,asit

createsanefficientwayforthemtodevelopnewtherapeuticcompoundsandstudytheir

effectswithotherknowndrugsaswellasacrossnumerousbiochemicalpathways.A

chemicalgeneticscreenincorporatesthecanonicalgeneticsrationaletosolvea

biochemicalproblem.Inatypicalforwardgeneticexperiment,randommutationsare

introducedintoapopulationthatisthenscreenedforthedesiredphenotype.Inchemical

6

genetics,smallmoleculesthatinhibittheactivityofgeneproductsareused,instead,to

interferewithbiochemicalphenomena,andthepopulationisscreenedforadesired

phenotype.Aswithtraditionalgeneticsexperiments,therearetwoformsofachemical

geneticscreen,forwardandreverse.Aforwardscreencanbeusedtodeterminewhich

molecules,andthereforewhichgeneproductsacteduponbythecompounds,producea

desiredphenotype;alibraryofmoleculesisexaminedforacertainphenotypeand

individualmoleculesarethenstudiedtounderstandthedeterminedphenotype(24).

Reversescreens,alternatively,canbeusedtovalidatedrugtargets;proteinsarescreened

fortheiraffinitytolibrarycompoundsthenusedtoobservebiologicalphenomena(25,26).

Theresultsofachemicalgeneticscreencanprovideresearcherswithinformation

astohowcertainpathways,treatments,andprocessesreacttotheintroductionofforeign

specieswithknowninteractions.Chemicalgeneticscreenshaveprovensuccessfulinmany

modelsystemsfromculturedtumorcellstozebrafish.Todetermineinhibitorsofthe

PI3K/PTEN/Aktsignaltransductionpathway,achemicalgeneticscreenwasperformedon

PTENlipidphosphatase,atumorsuppressorandnegativeregulatorofPI3K/Aktpathway,

nullcellsusingalibraryofcompounds.Successfulinhibitorsfromthefirstroundof

treatmentwerestudiedindepthtodetermineasetofmoleculesthatsuccessfulinhibitsthe

PI3K/AktpathwayinPTENnullcells,exemplifyingtheuseofthechemicalgeneticscreen

toidentifyknowncompoundsfornoveluses(27).Inzebrafish,achemicalgeneticscreen

wasusedtoidentifyknowncompoundsthathadnotbeenpreviouslyknowntohavecell

cycleactivity.Thelibraryof320compoundswasusedtofindalterationsinthemitotic

markerphosphor-histoneH3:revealing14compoundsthatmaybeusefulinstudyingcell

cyclebiologyandindevelopingchemotherapeuticagents(28).Mostsimilartothedesired

7

resultswithlaromustine,achemicalgeneticscreenwasdevelopedtoshowsynergistic

cytotoxicityinninedifferentmelanomacelllines.Amongthe300drugcombinations

tested,synergybetweentwoknowndrugs,sorafenib,amultikinaseinhibitor,and

diclofenac,anonsteroidalanti-inflammatorydrug,exhibitedthemostcytotoxicity(29).The

studyshowsthatitispossibletoidentifypreviouslyunknownusesandtargetsforalready

knowndrugsaswellasshowsthatthediscoveryofsynergisticmoleculesispossibleusing

achemicalgeneticscreen.

Toaidresearchersincreatingscreenswithalargenumberofcompoundsthe

NationalInstitutesofHealthdevelopedtheNIHClinicalCollectionfromtheNIHSmall

MolecularRepository.Thecollectionconsistsofapproximately450clinicallytested,US

FoodandDrugAdministrationapproved,compounds.Thelibraryhasbeentestedtoallow

researchersaccesstoalargenumberofclinicallyapprovedandunderstoodmoleculesfor

high-throughoutscreening.Ithasbeenusedinscreenstoidentifypreviouslyunknown

usesfortheknowndrugs,suchasobservingunknowninhibitoryeffectsonadenylyl

cyclaseisoforms;expertshaveexpressedmediumtohighlevelsofconfidenceinthe

performanceofthecollection(30,31).

Toexaminethepossiblyundiscoveredsynergisticeffectsintreatingglioblastoma

multiformebetweenlaromustinewhenpairedwithcompoundsfromtheNIHClinical

Collection,aforwardchemicalgeneticscreenwillbeperformedinwhichviabilityof

culturedcellstreatedwithlaromustineandthelibraryofcompoundsisassessed(Figure

2).Whilelaromustinelikelyhasmultiplemechanismsofaction,asdiscussed,nobiaswill

beplacedonaparticularofthecytotoxiceffects;butinsteadontheeffectsasawhole.If

moleculesareidentified,theywillbestudiedmoreindepthtodeterminethemechanisms

8

behindtheirsynergismwithlaromustine.Resultsfromthisscreencouldleadtoabetter

understandingofglioblastomamultiformeandtoanimprovedtreatmentforthedeadly

cancer.

Figure2:Aschematicdepictingtheoverallprocedureofthechemicalgeneticscreentodeterminesynergisticcompoundswithlaromustine.CellswillbetreatedwithlaromustineandtheNIHClinicalCollectionseparately,andacutecelldeathwillbedetermined.Thelibraryandlaromustinewillthenbecombinedfortreatmentandcombinationsthatshowgreatercelldeathwillbestudiedindepth.(Note:Allgraphsarepredictions.Ingraphpredictingcelldeathwithlibrary+laromustine,diamondpointsrepresentcelldeathwithjustlibrarycompound,squarerepresentsdeathwithlibrarycompoundandlaromustine,circledsquaresrepresentpositivehitsforincreasedcelldeath.

9

MaterialsandMethodsU138CellCultureBetween25-75%Confluence:

U138humanglioblastomacellsweregrowninEagleMinimumEssentialMedium

with0.1%gentamycin,1%L-glutamine,and10%fetalbovineserum.Cellsweregrownat

37˚Cwith5%CO2and100%relativehumidity.

U138CellSeeding

Beforeeachexperiment,80–90%confluentU138cellswerewashedtwicewith

0.12mL/cm2ofHEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid)buffered

salinesolution(150mMNaCl,20mMHEPES,pH7.4)andsubjectedtotrypsin/EDTA

(Ethylenediaminetetraaceticacid)for5mintodetachthecellsfromtheflask.Thetrypsin

wasneutralizedusingtwicethevolumeofmedia,anddetachedcellswerecentrifugedat

1100rpmfor5min.Thesupernatantwasremoved,andthecellswerere-suspendedin3

mLfreshmedia.ThecellswerecountedwithaCellometerAutoT4andviabilitywas

determinedusingtheTrypanblueexclusionassay.DetachedU138cellswereincubatedin

a384-wellwhiteµClearplatewith25µL/wellattheindicatedseeddensitiesfortwodays

undertheaforementionedconditions.

DeterminationofOptimumSeedingConcentration:

Todeterminetheoptimumcellcountfortheassay,culturedU138cellswereseeded

inquadruplicateatconcentrationsrangingfrom250cells/wellto25,000cells/well(3,000

cells/cm2–300,000cells/cm2)andanegativecontrolwithoutcells,andincubatedfortwo

days.Att=48hr,25µLofCellTiter-Gloreagent(CellTiter-GloLuminescentCellViability

Assay,Promega,Madison,WI)wasaddedtoeachwellandrelativeluminescencewas

obtainedusingaMolecularDevicesSpectraMaxM5Microplatereader.Datawereanalyzed

10

foraverageluminescencebyquadruplicateanderrormeasuredbystandarddeviationof

quadruplicates.

DeterminationofU138DoublingTimeandMaximumCellsperWell

ToassesstheincubationperiodforU138cellstodoubleinwellsandthemaximum

capacityofcellsineachwell,U138cellswereseededintriplicateatconcentrationsranging

from250cells/wellto25,000cells/well(3,000cells/cm2–300,000cells/cm2)and

incubatedfortwodayswithanegativecontrolwithoutcells.Attimet=48hr,60hr,74hr,

84hr,96hr,120hr,144hr,and168hr,25µLofCellTiter-Gloreagentwasaddedtoeach

wellandrelativeluminescencewasobtained.Datawereanalyzedforaverageluminescence

bytriplicatesanderrormeasuredbystandarddeviationoftriplicates.

DeterminationofIncubationPeriodoflaromustinewithlethaldosetreatment:

Todeterminetheamountoftimeneededforlethaldoselaromustinetocauseacute

celldeathU138cellswereseededintriplicateat5,000cells/well(60,000cells/cm2)as

describedpreviouslyfortwodayswithanegativecontrolwithoutcells.Att=48hr,culture

mediawasaspiratedandcellsweretreatedwith25µLofmediacontaining1mM

laromustine,1mMtemozolomideorthecorrespondingvolumeof1%DMSObyvolume.At

eachtimet=10min,20min,30min,1hr,2hr,3hr,6hr,9hr,12hr,18hr,and24hr,25

µLofCellTiter-Gloreagentwasaddedtothewellsandrelativeluminescencewasobtained.

Datawereanalyzedbyaverageluminescencebytriplicatesanderrormeasuredasa

standarddeviationoftriplicates.Averageluminescenceacrossthetimescalewere

comparedtodeterminetimeneededforlaromustinetocauseacutecelldeathincultured

U138cells.

11

Determinationof50%LethalDose:

Todeterminethemediandoseoflaromustineisneededtocause50%acutecell

death,U138cellswereseededintriplicateat2500cells/well(30,000cells/cm2)as

describedpreviouslyandincubatedfortwodayswithanegativecontrolwithoutcells.Att

=48hr,mediawasaspiratedandcellsweretreatedwith25µLmediacontaininga

concentrationoflaromustinerangingfrom2000µMto1µMinatwo-foldserialdilution

withthecomparativevolumeof1%DMSObyvolumeasacontrol.Attimet=6hrafter

seeding,25µLofCellTiter-Gloreagentwasaddedtowellsandrelativeluminescencewas

obtained.Datawereanalyzedbyaverageluminescenceoftriplicatesoverthreetrialsand

errormeasuredbystandarddeviationoftriplicates.Curvefitanalysiswasusedto

determinetheLD50forlaromustinewhenusingculturedU138cells.

VerificationofassayoptimizationusingTemozolomide:

Toverifytheassayconditionsbeforebeginningthescreeningprocess,

Temozolomidewasusedtoassesssynergisticeffectswithlaromustine.CulturedU138cells

wereseededintriplicateat2500cells/well(30,000cells/cm2)asdescribedpreviouslyand

incubatedfortwodayswithanegativecontrolwithoutcells.Att=48hr,mediawas

aspiratedandcellsweretreatedwith25µLmediacontainingaconcentrationthefollowing

conditions:1000µMTemozolomidewith500µMlaromustineandwithoutlaromustine,

100µMTemozolomidewith500µMlaromustineandwithoutlaromustine,100µM

Temozolomidewith500µMlaromustineandwithoutlaromustine,500µMlaromustine

andacontrolof3%DMSObyvolume.Attimet=6hrandt=12hrafterseeding,25µLof

CellTiter-Gloreagentwasaddedtowellsandrelativeluminescencewasobtained.Data

wereanalyzedbyaverageluminescenceoftriplicatesanderrormeasuredbystandard

12

deviationoftriplicates.Averageluminescencewerecomparedbetweendatafor

Temozolomide/laromustinetreatmentsandTemozolomideandlaromustinetreatments

separately.

ResultsandDiscussionCellsshouldbeseededat2500cells/wellandincubatedfor48hourspriortodrugtreatment

ToensurethatculturedU138cellsaregiventhesufficientspaceandtimetogrow

beforedrugtreatment,theoptimalcellseedingconcentrationwasdetermined.Thiswould

ensurethatthetotalnumberofcellsperwellwasnotimpedingthecells’abilitytodivide

comfortably,butalsotoensurethattherewereenoughcellsperwelltoachieveastable

luminescencesignal.Ideally,thetransparentbottomofeachwellwouldbemaximally

coveredsuchthatthelargestnumberofcellsperareacouldbetreated.Concentrations

rangingfrom250cells/wellto25,000cells/well(3,000cells/cm2–300,000cells/cm2)

wereseededandviabilityafter48hoursofincubationwascomparedbyluminescence

(Figure3).Wellscontainingmorethan7500cells/wellatseedingproducedluminescence

ofapproximately3.5x104RLU.Astherelativeluminescencepeakedatthisvalue,despite

theincreasingcellconcentration,itislikelythatthewellseitherbecametoodensefor

survivalortheCellTiter-Glowasalimitingagent.Asitispreferredfortherelative

luminescencetobeonthethresholdsuchthatthenumberofcellstreatedisashighas

possiblewithoutimpairinggrowth,therelativeluminescenceofwellsseededat250-1250

cells/wellwastoolow.Wellsseededat2500cells/welland5000cells/wellresultedin

luminescencejustbeforetheluminescencethresholdatapproximately3.0x104RLUand

3.3x104RLU,respectively.

13

Figure3:SeedconcentrationofU138culturedcells.Cellswerelysed48hafterseedingandluminescencerecorded.Errorisstandarddeviationofquadruplicates.

Becausetheluminescencesignalplateauedat3.5x104RLU,itwasnecessaryto

ensurethattheCellTiter-Glowasnotthelimitingagentineachreactionmixture.Volumes

of25–50µLofCellTiter-Gloreagentwereaddedtowellscontaining1.0x105cells/wellat

thetimeofseeding.Luminescenceresultsshowaninverselyproportionalrelationship

betweenthevolumeofCellTiter-Gloandrelativeluminescence(Figure4).Increasingthe

volumeofCellTiter-Glomaycauseunnecessarycompetitionortheaddedluciferasemay

interferewithluminescenceinsomeway.Theresultsconfirmthatthe25µLofCellTiter-

Gloistheoptimalvolumetoproducepeakluminescence.

0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

40,000

45,000

0 5,000 10,000 15,000 20,000 25,000

RelativeLum

inescence(RLU)

ConcentrationatSeeding(cells/well)

14

Figure4:DeterminationofoptimalvolumeofCellTiter-Glousedtolysecellsandshowluminescencerelativetoviabilityofcellsineachwell.Errorisstandarddeviationoftriplicates.

Basedonpreviouswork,cellswereincubatedfor48hours,asittakes

approximately24hoursforcellstoadheretothewellbottom,providinganadditional24

hoursforcellsnumbertodoubleatleastonce.Toconfirmthat48hoursprovidesample

timeforcellstoadhereanddivide,wellswereseededwith1250cells/wellto6250cells/

well,astotrytoavoidthethresholdluminescenceat3.5x104RLU.Forupto48hours,

cellswerelysedandluminescencerecordedevery12hours,thenevery24hoursfollowing

until144hoursafterseeding(Figure5).Thedataappeartoshowthatforlesser

concentrations,at48hours,luminescenceisapproximatelydoubletheluminescenceat0

hours,whileforhigherconcentrationstheluminescenceisapproximately1.5timesthe

luminescenceat0hours.Thismayhaveresultedbecauseittooklongerforcellsinwells

withhigherconcentrationstoadheretothewellbottomduetocompetitionforspaceand

nutrientstodivide.Atperiodsbeyond48hours,erroramongsttriplicatesbecamelargeras

20,000

22,000

24,000

26,000

28,000

30,000

32,000

34,000

25 30 35 40 45 50RelativeLum

inescence(RLU)

VolumeofCellTiterGlo(µL)

15

luminescencevariedbygreateramounts,suggestingthatcellsweredividingatdifferent

ratesandtheconditionsineachwellcouldnotbecontrolledtomaintainconsistency.

Figure5:DeterminationofdoublingtimeofU138culturedcells.Wellswereseededwithvariousconcentrationsofcellsandluminescencemeasuredatmultipletimepoints.Errorisstandarddeviationoftriplicates.

However,despiteerror,thedatasuggestthatcelldoublingstilloccursroughlyevery48

hoursuntilwellsbecometoodensewithcells.Thewellsseededat2500cells/wellshow

approximatedoublingafter48hours,withconsiderablylowerror.After48hours,the

relativeluminescenceofthewellsseededat2500cells/wellwas2.5x104RLU,similarto

therelativeluminescenceemittedwhendeterminingtheoptimalseedingconcentration.

Thecellsseededinwellsat2500cells/wellduplicateinapproximately48hoursanderror

increasesmarkedlyafter48hoursofincubation;seedingcellsat2500cells/wellwith48

0

20,000

40,000

60,000

80,000

100,000

0 24 48 72 96 120 144

RelativeLum

inescence(RLU)

IncubationTime(hr)

NOCells1250cells/well2500cells/well3750cells/well5000cells/well6250cells/well

16

hoursofincubationpriortotreatmentproducesthemostpreciseconditionsforthe

chemicalgeneticscreen.

Atlethaldose,laromustineresultsinsignificantlossofviabilityinU138culturedcellsafter6

hours

Theexacttimeaftertreatmentforlaromustinetoentercellsandcausemarkedloss

incellviabilityinU138culturedcellsisimportanttoassessbeforecompletingthechemical

geneticscreen.Thedrugrequirestimetoenterthecellandbegintocauseitsdeleterious

effects.Thecellshouldrespondtothetreatmentbytryingtoassuagethedrug’seffects.The

seriesofeventswillnothappenimmediately,thustheamountoftimeforthedrugto

significantlyaffectcellsmustbedetermined.Thisisdoneusingalethaldoseofdrugand

measuringtheviabilityatvarioustimesaftertreatment.TheviabilityofU138culturedcells

wasmeasuredatdifferenttimesfrom10minto24hoursaftertreatmentwithlethaldoses

oflaromustineandtemozolomide(Figure6,7).Within6hoursoftreatment,therewas

significantlossofviabilityinthecellstreatedwithlaromustinecomparedtotheDMSO

control,from1.43timeswithin3hoursoftreatmentto0.20timesthecontrolat6hours

aftertreatment.Theviabilityofcellstreatedwithlaromustinecontinuedtodecreasein

subsequenthours,untilthecellsshowedapproximately1%viabilityrelativetheDMSO

control.Similarresultswereobservedinthecellstreatedwithtemozolomide,though

decreasesinviabilityoccurred12hoursaftertreatment.Viabilityofcellstreatedwith

temozolomidedecreasedto10%relativetheDMSOcontrolafter12hoursbutremainedat

approximately10%duringsubsequenthours.

17

Figure6:Determinationoflethaldosetimeresponseoflaromustinecomparedtotemozolomide.U138cellsweretreatedwith1000µMlaromustineand1000µMtemozolomideandluminescencedeterminedatvaryingtimepointsovercourseof24hours.Errorisstandarddeviationoftriplicates.

Figure7:Determinationoflethaldosetimeresponseoflaromustinecomparedtotemozolomide.ShowsdatafromFigure5aingreaterdetailfrom0to6hoursaftertreatment.Errorisstandarddeviationoftriplicates.

0

0.5

1

1.5

2

2.5

0 6 12 18 24

RelativeLum

inesencetoDMSO

Control

TimeafterTreatment(hr)

1mMLaromustine1mMTemozolomide

0

0.5

1

1.5

2

2.5

0 1 2 3 4 5 6

RelativeLum

inesencetoDMSO

Control

TimeafterTreatment(hr)

1mMLaromustine1mMTemozolomide

18

Thecellstreatedwithlaromustineshowedsignificantlossofviabilitywithin6hours

oftreatment.Thisprovidesevidencethatlaromustinerequires6hoursofincubation

followingtreatmenttodrasticallyaffectcellsandimpedetheirabilitysurviveand

duplicate.LaromustinemaybecausinglossofviabilityinU138cellsinbothdirectand

indirectways.Thehighdoseofthedrugiscytotoxictothecell,thoughthenatureofthe

drug’sacutetoxicityisunknown.Thedrugmayaffectcellsviaitsknownmethodof

crosslinkingDNA,impedingcellsfromreplicatingandforcingthemintoapoptosis.

Similarly,temozolomidecausessignificantlylossofviabilitywithin12hoursoftreatment.

Atlethaldoses,laromustineaffectsviabilityinhalftheamountoftime,indicatingthat

laromustineisabletoentercellsandcauseharmfuleffectsquickerthantemozolomide.

Additionally,laromustinecausesanincreasedlossofviabilitycomparedtotemozolomide.

At24hoursaftertreatment,laromustineshowed1.5%viabilityrelativetotheDMSO

controlwhiletemozolomideremainedat11.5%viabilityrelativetotheDMSOcontrol.This

preliminary10-folddifferenceinviability,withinerror,impliesthatlaromustinemaybe

moreeffectiveintreatingU138culturedcells,thoughmoreresultsarenecessaryto

confirm.

ThemedianlethaldoseoflaromustineintreatingU138culturedcellsfor6hoursis400µM

Alethaldoseoflaromustineisshowntocauselossofviabilitysuchthatcellsremain

aslittleas1%viablecomparedtoDMSOcontrol.Inordertoassesstheeffectivesynergistic

effectsbetweenlaromustineandothercompounds,aconcentrationoflaromustinemustbe

foundsuchthatmorethan1%viabilityremains.Ideally,amedianlethaldoseisfoundsuch

thatcellsare50%viableafter6hoursoftreatmentrelativetotheDMSOcontrol.Usinga

medianlethaldoseallowsforcomparisonoflossofviabilityamongcellstreatedwithsolely

19

laromustineandcellstreatedwithbothlaromustineandaseparatecompound.Themedian

lethaldosewasdeterminedbytreatingU138culturedcellswithatwofolddilutionof

laromustinefrom2000µMto1µMandallowing6hoursofincubationbeforelysingand

recordingluminescence(Figure8).Significantdosesover1000µMareeffectivein

Figure8:DeterminationofLD50oflaromustineintreatingculturedU138cells.CurvefitanalysisshowsLD50of708µM.Datashownareofthreetrials.

drasticallyaffectingcellviability,whiledosesbelow250µMwerelesseffective.Data

analysisshowsasigmoidalcurvewithamedianlethaldoseof708µM.Previousstudies

demonstratethatlaromustineiseffectiveatconcentrationslowerthan50µMintreatingan

AMLcellline,HL60.Theabilityforthecelltoprotectitselfagainstlowlevelsoflaromustine

providesmotivationforthechemicalgeneticscreen.Itisplausible,thatwithanadditional

0

0.2

0.4

0.6

0.8

1

10 100 1000 10000

FractionControlViability

[laromustine](µM)

20

infectingagent,laromustinemaybemoreeffectiveindecreasingtheviabilityofcultured

braincancercellsatalowerdose.

Thoughnotconfirmedbylaboratorywork,laromustineandTemozolomideshouldact

synergisticallyininducingcelldeathofU138cells

Aspreviouslymentioned,Temozolomideshouldshowsynergisticeffectswith

laromustinedueitsabilitytointerferencewithAGTenzymefunction.Decreasingthe

effectivenessofAGTshouldallowforalargermolarquantityofcrosslinkingby

laromustine.Preliminarytestshaveshownthatthereisnotanincreasedcelldeathwhen

usingbothlaromustineandtemozolomidetogetherinrelationtowhenusingthem

separately.Whilethereisdrasticcelldeath,resultsshowthatitismostlikelyrelatedsolely

totheactionoflaromustine,astheTemozolomidecontrolof1000µM,alethaldose,isnot

provingtoinducemarkedcelldeath.Theseresultsarestillforthcomingandworkwill

continuetobedoneinthefuture.DespitealackofconfirmationwithTemozolomide,the

chemicalgeneticscreenhasbeenfullyoptimizedtocomparecelldeathbetween

laromustineandlaromustineinconjunctionwithalibraryofsmallmolecules.Usingthe

dataobtainedfromthescreen,compoundsshowingsynergisticeffectswithlaromustine

canbeverified,laromustine’smechanismsofactionscanbebetterunderstood,andthe

possibilityofmoresuccessfultreatmentsofglioblastomamultiformewithlaromustinecan

beinvestigated.

21

FutureWork

Littleworkremainstocompletethisproject.First,thesmall-scalecombinationwith

Temozolomidemustbecompleted.ItisexpectedthatTemozolomideandlaromustinewill

havesynergisticeffectsduetotheirdeterminedlysimilarmechanismsofactionand

Temozolomide’sabilitytoinhibitAGT.Oncethetestcombinationiscompleted,the

standarddeathforeachcompoundintheNIHClinicalCollectionwillbedetermined,as

writtenbelow.Then,withtheassistanceofProfessorRobertWheelerattheUniversityof

MaineOrono,thechemicalgeneticscreenwillberuntodeterminethecelldeathwhen

laromustineandcompoundsfromthelibraryareusedinconjunction.Thechemicalgenetic

screenwillbeconductedasdescribedbelow.Basedontheresultsofthescreen,further

researchcanbedoneintothemechanismsoflaromustineandtheeffectivenessofthe

compoundsthatshowedsynergisticeffectsfromthescreen.

DeterminationofStandardDeathforNIHClinicalCollection

Cellswillbeseededat2500cells/well(30,000cells/cm2)with25µL/wellas

previouslydescribedinmethodssectionandincubatedfortwodays.Attimet=48hr,

mediawillbeaspiratedandmediacontainingoneofeachofthecompoundsfromtheNIH

ClinicalCollectionwillbeaddedtoeachwell.Attimet=6hrafterseeding,25µLof

CellTiter-Gloreagentwillbeaddedtoeachwellandrelativeluminescenceobtained.

ChemicalGeneticScreenusingLaromustineandNIHClinicalCollection

Cellswillbeseededat2500cells/well(30,000cells/cm2)with25µL/wellas

previouslydescribedinthemethodssectionandincubatedfortwodays.Attimet=48hr,

mediawillbeaspiratedandmediacontainingLD50concentrationoflaromustineaswellas

22

oneofeachofthecompoundsfromtheNIHClinicalCollectionistobeaddedtoeachwell.

Attimet=6hrafterseeding,25µLofCellTiter-Gloreagentwillbeaddedtoeachwelland

relativeluminescenceobtained.Combinationsofdrugsthatshowadecreased

luminescencethanwhenlaromustineandtheNIHClinicalCollectionseparatelywillbe

testedagainasdescribed,thoughintriplicate,toensurevalidityofdata.

23

References1. Rice,K.P.,Klinkerch,E.J.,Gerber,S.A.,Schleicher,T.R.,Kraus,T.J.,andBuros,C.M.

(2012)Thioredoxinreductaseisinhibitedbythecarbamoylatingactivityoftheanticancersulfonylhydrazinedruglaromustine.Molecularandcellularbiochemistry370,199-207

2. Trials,N.C.I.C.(2016).3. Jordan,J.T.,Gerstner,E.R.,Batchelor,T.T.,Cahill,D.P.,andPlotkin,S.R.(2016)

Glioblastomacareintheelderly.Cancer122,189-1974. Holland,E.C.(2000)Glioblastomamultiforme:Theterminator.Proceedingsofthe

NationalAcademyofSciencesoftheUnitedStatesofAmerica97,6242-62445. Henson,J.W.(1999)Glioblastomamultiformeandanaplasticgliomas:

Apatientguide.MassachusettsGeneralHospitalBrainTumorCenter6. Livingstone,L.R.,White,A.,Sprouse,J.,Livanos,E.,Jacks,T.,andTlsty,T.D.(1992)

Alteredcellcyclearrestandgeneamplificationpotentialaccompanylossofwild-typep53.Cell70,923-935

7. Carson,W.E.,3rd,Jakowatz,J.G.,Yamamoto,R.,Fitzgerald,T.,Gupta,S.,Vayuvegula,B.,Lucci,J.A.,3rd,Beckman,M.T.,Dulkanchainun,S.,Granger,G.A.,andetal.(1991)Ratmitogen-stimulatedlymphokine-activatedTkillercells:productionandeffectsonC6gliomacellsinvitroandinvivointhebrainofWistarrats.Journalofimmunotherapy:officialjournaloftheSocietyforBiologicalTherapy10,131-140

8. Culver,K.W.,Ram,Z.,Wallbridge,S.,Ishii,H.,Oldfield,E.H.,andBlaese,R.M.(1992)Invivogenetransferwithretroviralvector-producercellsfortreatmentofexperimentalbraintumors.Science(NewYork,N.Y.)256,1550-1552

9. Rockwell,S.,Liu,Y.,Seoq,H.,Ishiguro,K.,Baumann,R.,Penketh,P.,Shyam,K.,Akintujoye,O.,Glazer,P.,andSartorelli,A.(2012).InternationalJournalofRadiationBiology88,277-285

10. Schiller,G.J.,O'Brien,S.M.,Pigneux,A.,DeAngelo,D.J.,Vey,N.,Kell,J.,Solomon,S.,Stuart,R.K.,Karsten,V.,Cahill,A.L.,Albitar,M.X.,andGiles,F.J.(2010)Single-agentlaromustine,anovelalkylatingagent,hassignificantactivityinolderpatientswithpreviouslyuntreatedpoor-riskacutemyeloidleukemia.JournalofClinicalOncology28,815-821

11. Schabel,F.M.,Jr.(1976)Nitrosoureas:areviewofexperimentalantitumoractivity.Cancertreatmentreports60,665-698

12. Frederick,A.M.,Davis,M.L.,andRice,K.P.(2009)InhibitionofhumanDNApolymeraseβactivitybytheanticancerprodrugCloretazine.Biochemicalandbiophysicalresearchcommunications378,419-423

13. Ochs,K.,Sobol,R.W.,Wilson,S.H.,andKaina,B.(1999)CellsdeficientinDNApolymerasebetaarehypersensitivetoalkylatingagent-inducedapoptosisandchromosomalbreakage.Cancerresearch59,1544-1551

14. Ji,W.,Yang,M.,Praggastis,A.,Li,Y.,Zhou,H.J.,He,Y.,Ghazvinian,R.,Cincotta,D.J.,Rice,K.P.,andMin,W.(2014)CarbamoylatingactivityassociatedwiththeactivationoftheantitumoragentlaromustineinhibitsangiogenesisbyinducingASK1-dependentendothelialcelldeath.PLoSONE9

24

15. Baumann,R.P.,Seow,H.A.,Shyam,K.,Penketh,P.G.,andSartorelli,A.C.(2005)Theantineoplasticefficacyoftheprodrugcloretazine™isproducedbythesynergisticinteractionofcarbamoylatingandalkylatingproductsofitsactivation.OncologyResearch15,313-325

16. Giles,F.J.(2009)Laromustine:Thereturnofalkylatorstonon-myeloablativetherapyofAML.LeukemiaResearch33,1022-1023

17. Morris,K.L.,Adams,J.A.,andLiuYin,J.A.(2009)Effectofcloretazine(VNP40101M)onacutemyeloidleukaemiablastcellsinvitroasasingleagentandcombinedwithcytarabineanddaunorubicin.LeukemiaResearch33,1096-1099

18. Nazha,A.F.(2014)Acutemyeloidleukemiaintheelderly:doweknowwhoshouldbetreatedandhow?Leukemia&Lymphoma55,979-987

19. Lancet,J.E.(2013)Newagents:Greatexpectationsnotrealized.BestPractice&ResearchClinicalHaematology26,269-274

20. Stupp,R.,Mason,W.,andBent,M.v.d.(2005).NewEnglandJournalofMedicine352,987-996

21. Badruddoja,M.A.,Penne,K.,Desjardins,A.,Reardon,D.A.,Rich,J.N.,Quinn,J.A.,Sathornsumetee,S.,Friedman,A.H.,Bigner,D.D.,Herndon,J.E.,Cahill,A.,Friedman,H.S.,andVredenburgh,J.J.(2007)PhaseIIstudyofCloretazineforthetreatmentofadultswithrecurrentglioblastomamultiforme.JNeuro-Oncology13,428-436

22. Olson,J.J.,Nayak,L.,Ormond,D.R.,Wen,P.Y.,andKalkanis,S.N.(2014)Theroleofcytotoxicchemotherapyinthemanagementofprogressiveglioblastoma:Asystematicreviewandevidence-basedclinicalpracticeguideline.JournalofNeuro-Oncology118,501-555

23. Rizzieri,D.,LoRusso,S.,Tse,W.,Khan,K.,Advani,A.,Moore,J.,Karsten,V.,Cahill,A.,andLGerson,S.(2010).ClinicalLymphoma,MyelomaandLeukemia10,211-216

24. Choi,H.,Kim,J.Y.,Chang,Y.T.,andNam,H.G.(2014)Forwardchemicalgeneticscreening.Methodsinmolecularbiology(Clifton,N.J.)1062,393-404

25. Kawasumi,M.,andNghiem,P.(2007)ChemicalGenetics:ElucidatingBiologicalSystemswithSmall-MoleculeCompounds.JournalofInvestigativeDermatology127,1577-1584

26. Thorpe,D.S.(2003)Forward&reversechemicalgeneticsusingSPOS-basedcombinatorialchemistry.Combinatorialchemistry&highthroughputscreening6,623-647

27. Kau,T.R.,Schroeder,F.,Ramaswamy,S.,Wojciechowski,C.L.,Zhao,J.J.,Roberts,T.M.,Clardy,J.,Sellers,W.R.,andSilver,P.A.(2003)AchemicalgeneticscreenidentifiesinhibitorsofregulatednuclearexportofaForkheadtranscriptionfactorinPTEN-deficienttumorcells.CancerCell4,463-476

28. Murphey,R.D.,Stern,H.M.,Straub,C.T.,andZon,L.I.(2006)AChemicalGeneticScreenforCellCycleInhibitorsinZebrafishEmbryos.ChemicalBiology&DrugDesign68,213-219

29. Roller,D.G.,Axelrod,M.,Capaldo,B.J.,Jensen,K.,Mackey,A.,Weber,M.J.,andGioeli,D.(2012)Syntheticlethalscreeningwithsmall-moleculeinhibitorsprovidesapathwaytorationalcombinationtherapiesformelanoma.MolecularCancerTherapeutics11,2505-2515

30. Conley,J.M.,Brand,C.S.,Bogard,A.S.,Pratt,E.P.,Xu,R.,Hockerman,G.H.,Ostrom,R.S.,Dessauer,C.W.,andWatts,V.J.(2013)Developmentofahigh-throughput

25

screeningparadigmforthediscoveryofsmall-moleculemodulatorsofadenylylcyclase:identificationofanadenylylcyclase2inhibitor.TheJournalofpharmacologyandexperimentaltherapeutics347,276-287

31. Oprea,T.I.,Bologa,C.G.,Boyer,S.,Curpan,R.F.,Glen,R.C.,Hopkins,A.L.,Lipinski,C.A.,Marshall,G.R.,Martin,Y.C.,Ostopovici-Halip,L.,Rishton,G.,Ursu,O.,Vaz,R.J.,Waller,C.,Waldmann,H.,andSklar,L.A.(2009)AcrowdsourcingevaluationoftheNIHchemicalprobes.NatChemBiol5,441-447