Chapter16 The Rational Treatment of Cancer Ali substances are poisonous, there isnone that is not a poison; the right dose differentiates a poison from a remedy. Paracelsus(Auroleus Phillip us Theostratus Bombastus von Hohenheim), alchemist and physician,1538 Doctors are men who prescribe medicines of which they know little,tocurediseasesof which theyknow less,inhuman beingsof whom they know nothing. Voltaire(Franc,:ois-Marie Arouet),author and philosopher, 1760 Theresearchdescribedthroughoutthisbook representsarevolutioninour understandingofcancerpathogenesis.In1975,therewerevirtuallyno insightsintothemolecularalterationswithinhumancellsthatleadtothe appearance of malignancies. One generation later, we possess this knowledge in abundance. Indeed,the available information and concepts about cancer's origins can truly be said to constitute a science with a logical and coherent conceptual structure. Inspiteof these extraordinary leapsforward,relatively littleprogress hasbeen made in exploiting these insights into etiology (Le.,the causative mechanisms of disease)topreventthediseaseand,equally important,totreatit.Mostof the anti-cancer treatments in widespread use today were developed prior to1975, at a time when the development of therapeutics was not yet informed by detailed knowledge of the genetic and biochemicalmechanisms of cancer pathogenesis. 725 I Chapter 16: The Rational Treatment of Cancer (A)(8) 10060 5590 Q)c 50 Q)C 80 ~0~0\,....,;:; \.....;::;45 70s::.'"s::.'"+-'-:;40+-'-:; '"e.'"e.60Q) 0Q) 035"De.colon andrectum9"De.lung9 "Do 30 "Do50 ~ o J ~ o "'0 uterus "'025::J ::J ~ o ~ o"Do"Do20' P ~' P ~ Q)~Q)~ O'IQ) 15 O'IQ)",e. '"e.10 5 0 0I-no celllines(groupB;blue line).Thisillustrates ~0.7 '"established graphically whytumor xenografts..... '"0.6c:: producedfromestablishedca ncercell +J0.5 C1!linesusually failto recapitulatethe ro 0. '+- 0.4 .-..... group B (n=168) propertiesofthetumorstypically . -.....0 Iencounteredina cancerclinic(sincethe c:: 0.30 cancercelllinesusuallyderivefrom "13 0.2 tumors at the farendofthe~ '+- celllines establishedgroup A(n= 35)0.1spectrum-themostaggressivesubset). (FromYShimada,M.Maeda, 0.0 020406080100120140160180 G.WatanabeetaI.,Clin.Cancer Res. survival(months)9243-249,2003) 749 Chapter 16: The Rational Treatment of Cancer Figure 16.19 Pharmacokinetics and pharmacodynamics of Gleevec The pharmacokineticsof a drugrepresent thekinetics of itsaccumulationinand disappearancefromtheplasma,whichin turnarepresumedtoprovidea good indicationofthedrugconcentrations that tumor cellsexperienceina laboratory animalora patientunder therapy.Theplasmalevelof thedrug Gleevec,plottedona logarithmic scale (left ordinate), fluctuatesdramatically followinginjectionof thedruginto a mouse (bluecurve).Itsconcentrationis indicatedhereasa multipleof thedrug concentrationknowntoinhibit thefiring of thet yrosinekinaseof theKitreceptor by50%(i.e,theICsoofthisagent). (Thetyrosinekinasedomainof theKit growth factorreceptorisalsoa target of inhibitionbyGleevec. ) Asseenhere, the amount of phosphotyrosineassociated withtheKitreceptor(areflectionofKit tyrosinekinaseactivity)expressedby eng raftedhumanmastcellleukemia cells (red curve),whichwasinitiall y set as100%,isreducedto 1 %of preexistinglevelswithinanhourafter druginjectionbutreboundswithin8 hoursastheconcentrationof thedrug declinesintheplasma.(Courtesy of D.L.Emerson,051PharmaceuticalsInc) clearance rate of paclitaxel 1000 120 A ;::;: til 100 -0 :::r'" 0 100

0

til"' -u;:R-o0.80o:::r00 ...,... ,,'< Olu 60 o::>'-- 10 '-- Q) ::> ...- . -0> ::>,+-0 011> .:::;;" 40

0

::>...020 11> u ::> .-+ 0.10 04812162024 time after dosing (hours) The pharmacokinetics (PK)of a drug represent a key determinant of its efficacy in vivo:Does it accumulate tosignificant levelsinthe plasmaortissues foran extendedperiodof time?Orisitpresentinthebodyonlytransiently,being excretedbythekidneyswithinminutesof enteringintothecirculation?Isit resistant torapid degradation, or do certain drug-metabolizing systems, such as the cytochrome P-450s(Cyps)that weencountered inChapter 12,rapidly convert itinto an innocuous molecular species(Figure16.19)?(Akey pharmacokineticparameter that isoftenmeasuredisthe "areaunder thecurve, " or AUC, calculated by integrating the concentration of a drug in the plasma as a function of time;the AUCisthought toreflect the cumulative drug dose experienced by cellsin a tumor.)Andcan itbe administered oraJly rather than requiring injection? Laboratory animalsgivesomeroughindicationof adrug'spharmacokinetics, but are by no means accurate predictors of how humans metabolize and excrete variousagents.Moreover,aswereadearlier(Sidebar12.5),theratesatwhich various compounds, including drugs, are metabolized or excreted can even vary dramaticaJlyfromonepersontoanother(forexample,seeFigure16.20).(In somepharmaceuticalcompanies,thepharmacokineticsofcandidatecompounds may be measured evenbefore any testsof therapeutic efficacyagainst xenograftedtumors;thoseshowing poorpharmacokineticsinlaboratory animals are often eliminated from further testing. Discarding such drugs may occasionaJly be premature, given the dramatically differing rates of drug metabolism and excretion between rodents and humans.) In fact,Figure16.19reveals a second attribute of a drug-its pharmacodynamics(PD),in this case those of Gleevec. Pharmacodynamics gauge the ability of a drug toaffectatargetedbiochemical functionin atumor under treatment.In the PDpresentedin this figure,asisoften thepractice,a surrogate marker of the targetedBcr-Ablfunctionwasmeasured- thebehavior of theKitreceptor. As we willread in more detail later, Kit isone of several tyrosi ne kinases affected by Gleevec,and itsresponses tothe drug presumably parallel thoseof Bcr-Abl. Figure 16.19 reveaJs that Kit activity in this experiment was inhibited only briefly at the time when the highest concentration of drug waspresent inthe circulation.Suchatransientinhibition-only afractionof acellcycle-isgenerally insufficient to elicit a substantial biologicaJ response, such astumor cell killing. Figure16.20 Inter-individual variability in drug clearancePaclitaxel isa chemotherapeuticdrugusedto treata number of malignancies;it worksbystabilizingmicrotubules,therebyinterferingwiththe progressionof cellsthroughMphase.Asseenhere,inthisstudyof 22ovariancancerpatients,therelativeratesof clearanceof thisdrug fromtheplasmaafter initialinjectionvariedovera factor of3. These ratesmaybeinfluencedbychangesintherateofmetabolismby enzymessuchascytochromec andbyexcretioninthekidneys.(From M . Nakajima,YFujiki,S.KyoetaI.,IClin.Pharm.45:674-682,2005.) 750 Pharmacokineticsand pharmacodynamics During the course of animaltesting,informationmay surfaceabout thetoxic side-effects that the drug elicits in whole organisms. They represent the bane of almostallexistingcancer treatments.Quitefrequently,variousnormalorgan systems, including the liver,kidneys,gastrointestinal tract,and the hematopoieticsystem, show toxiceffectsof adrug whenitisusedat the concentrations required to kill tumor cells. These toxicities are rarely predicted by in.vitro tissue culturetests,andtoxicitiesdetectedinlaboratoryanimals,includingdogs, monkeys,mice,and rats,mayor may not be predictive of human responses. Such observationsdirectour attentions once again tothe therapeutic index of an agent-the efficiency vvith which it affectscancerous tissues compared with itstoxi c effectsinnormaltissues.Clearly, idealcancer treatments shouldhave hightherapeutic indices, wreaking havoc on cancer cells while leavingnormal tissues relatively untouched. The fundamental obstacle to achieving such selectivityissuggestedbythefactthatthevastmajorityof the20,000or so genes expressed in cancer cells are also being expressed by their normal counterparts. The failure of animalmodels to predict the toxic side effects of a drug in humans createsseriousproblems.Theroughly80millionyearsof independent evolution that separate us from our rodent cousins have led to substantial differences inmetabolism;wemayreacttocertaindrugsmuchdifferentlyfrommiceor rats, or even the more closely related Old World monkeys, which may eventually be exposedtoacandidatedruginorder toobtain amarginally more accurate prediction of toxi citiesin humans. Inthe event that a drug passes these various tests without raising too many warning flags,it may be promoted to a candidate fortesting in humans. 16.8Promising candidate drugsmust besubjected to rigorous and extensiveclinical trialsin Phase Itrials in humans The discussions above explain why the first true tests of a drug's tolerability usuallycomeininitialpatientexposures,whicharetermedPhaseItrialsinthe UnitedStates.Here,candidate drugsaretestedat variousdoses,including the presumed therapeutic doses, to gauge toxic side-effects. The usual practice isto beginthese trials at drug dosagesthat are likely tobefarbelow the level of any overt toxicity (e. g., one-tenth the drug concentration that created toxicity in laboratoryanimals)andthen,inaseriesof patients,increasethedosagesincrementally until drug levelsare reached that begin toinduce unacceptable toxicities.This"doseescalation"yieldsavalue-themaximumtolerateddose (MTD)-thatisthenusedtoguidefurthertreatmentprotocols.Certainside effects,such asa ski n rash or transient nausea, may betolerable and not scuttle further drug development, while others, such as massive diarrhea or bone marrow depletion,may be soburdensome or life-threatening that they cause rapid abandonment of all further development of a drug. DuringthesePhaseItrials,pharmacokineticmeasurements,likethosemade previously in animals, will alsobe taken in order toascertain whether the drug is reaching tumor cells at a sufficient concentration and foran extended period of time.Still,these measurements giveno indication whether thecancer cellsare respondinginanyway-thepropertyofpharmacodynamics.Forexample,in Figure16.21,weseethepharmacodynamicresponsestotreatmentswithEGF receptor antagonists(whichhappen inthiscasetoincludeboth amonoclonal antibody and a low-molecular-weight tyrosine kinase inhibitor). The oncologists who undertook this particular clinical trial wished toobtain some measure ofthe effects of therapies on the EGF-R in patients' tumors. Todo so,they chose to use, as a surrogate marker, the EGF-R of patients' skin cells, which were far more easily monitored, simply by taking small skin biopsies frompatients in treatment. 751 Chapter 16: The Rational Treatment of Cancer Asseen in Figure 16.21A, patient exposure to an EGF-R tyrosine kinase inhibitor resulted ina strong suppression of EGF-Rsignaling in the skin. Inaddition, the activity of MAP kinase, which functions as an important downstream transducer ofEGF-Rsignaling(Section6.5),wasalsosuppressed,indicatingsuccessful inhibition of downstream mitogenic signaling. Similarresultswereobservedinbiopsiestakenfromacoloncancerpatient's tumorfollowingtreatmentwithananti-EGF-Rmonoclonalantibody(Figure 16.21B).Pharmacodynamic measurements likethese providereassurance that the administered treatment (in this case a monoclonal antibody)isreaching its intended target at concentrations that suffice to shut down much of the activity of itsintended target. Interestingly,anumberofthesignal-transducingproteinsoperatingdownstream of the EGF-R,including Akt/PKB, were only minimally suppressed in the colonictumor(Figure16.21B),indicatingthatthetumorcellshadacquired alternative means for activating these signaling molecules. Hence, pharmacodynamicsmeasurementsensure that one preconditionof therapeuticsuccessdelivery of the therapeutic agent to the targeted cells and molecules-has been satisfied,but donot, on their own,guarantee that the therapy willsucceed, as other factors may thwart it. Whentakentogether,themeasurementsof maximumtolerateddose(MTD), pharmacokinetics(PK) ,andpharmacodynamics(PD)definethetherapeutic window-the range of concentrations that are higher than that needed to elicit atherapeuticeffectandlowerthanthemaximumtolerateddose(Figure 16.21C).Ideally, a therapeutic window of a drug should be broad soas toallow clinicianssomeflexibilityinadministeringthedrug,adjustingdosagetothe ' patient and the condition being treated. As the therapeutic window narrows, the likelihood that a candidate drug willprove clinically usefuldiminishes. Occasionally,PhaseIclinicaltrials,whichareusuallyundertakenwithvery smallgroupsof patientvolunteerswhohavefailedotheravailabletherapies, may reveal some favorableresponses in terms of tumor regression or halting of furthertumorgrowth,doingsoatacceptably10v\Tlevelsof toxicity.However, even if there are hints of clinical efficacy, the positiveresults observedin Phase Itrialsareneverstatisticallysignificantandthusnotregardedasdefinitive. Instead,thesetrialsarereallyundertakentodiscoverunanticipatedtoxicities and tolerable levels of drug dosage. 16.9Phase II and IIItrialsprovidecredible indicationsof clinical efficacy Acceptably low levels of toxicity in a Phase I trial willencourage testing a candidate drug'sefficacy in aPhase IItrial,in which larger groups of cancer patients areinvolved.Now,forthe firsttime,critical decisions must be made about the indicationsforenlistingspecificpatientsinthetrial-that is,whichtypeof tumor or what stage of tumor progression willjustify enrolling patients insuch atrial? Sometimes the clinical indications areobvious.For example,as wenoted earlier,the effects of an agent targetedagainst the Bcr-Abloncoprotein should be testedinpatientsdiagnosedwithchronicmyelogenousleukemia(CML). AnotherdrugdirectedagainsttheHER2/Neureceptormoleculeshouldbe testedintheapproximately 30%of breastcancer patients whosetumorcells overexpressthisprotein. Yetanother agent-an inhibitor of Raf kinases-can be tried inpatients with advanced melanomas, in which the B-Rafkinase molecule is often (70%of cases) mutant and constitutively activated.(Interestingly, 752 Early clinical trialsof toxicity and efficacy in the last case, a B-Raf inhibitor failed to effectively stop further proliferation of metastaticmelanomas,whileitsuseincombinationwithaconventional chemotherapeutic drug has yielded dramatic, albeit only anecdotal responses.) Butmoreoftenthan not,thechoiceof indications isneither rationalnor optimal. Which classof cancer patients should be treated,forexample, with a drug (A) pre-treatmentpost-treatment activated EGF-R inskin activated MAPK in skin (B) overall EGF-R pEGF-R pAktJPKB day 0 Figure16.21Measurementsof pharmacodynamics and determinationof the therapeutic window Theextentofinhibitionof the EGF-Rina tumor can,inprinciple,be gaugedbymeasuringeffectsof drug treatment ontheEGF-Rintheskin;the latter isreadilyassessedthroughsmall skinbiopsies.Inthecasesillustrated here,patientsundertreatment were sufferingfroma varietyoftumors, includingcarcinomasof the ovary,lung, colon,prostate,andhead-and-neck. (A)Shownherearetheeffectsof treatinga cancerpatient withIressa,a low-molecular-weight EGF-Rtyrosine kinaseinhibitor (seeFigure1631)The upperpanels show immunohistochemistryusinganantibodyagainst phospho-EGF-R(brown), i.e.,the acti vatedformofthereceptor.Thelower panelsusedanantibodyagainst phospho-MAPK,theactivatedformof thiskinase.Bothmeasurements dependedonthenormallyintense signalingoccurringinkeratinocytes presentinthehairfollicles.(B) The effectsof ananti-EGFreceptor(EGF-R) monoclonalantibody (termedEMD7200) weregaugedbyimmunohistochemical stainingof a coloncarcinomabiopsy.In thiscase,long-termtreatmentresulted ina minimalreductionintheoveralllevel oftheEGF-R(brown) anda st rong reductioninthelevelofphosphorylated (andthereforeactivated)receptor (brown;pEGF-R). Thereductioninthe levelofphosphorylated,activated AktlPKB(brown;pAktlPKB) w asslight andthepatientshowedonlya partial responsetothis antibodytherapy,w hich mayhavereflectedthisminimal reductionof AktlPKBacti vityintumor cells.(C)Measurementsof pharmacodynamicssuchasthese,takentogether withstudiesof pharmacokinetics and toxicity,definethetherapeuticwindow inw hicha drugshouldbegi ven-the rangeof concentrationsthatare efficaciouswithout creatingan unacceptablelevelof toxicsideeffect s. (AandB,courtesyof J.Baselga.) (C)therapeutic window ,-----, >_tolerated dose ,...u o ,'f;+".:;ro Qj30 'f;30 rot 0Qj0.+" Q) o E20~20 >~ '.j:; 0. ~ e:: 10 0 10 o ShhNp++ ~I (C)60 >, +".:; 'f;40 ro Qj t o 0. e:: Q) .i;20 ro ~ Lo 310cyclopamine- +- +- +- + LI____________~ levelof Smo 505050.5 ~ LI-----.Jcontrol+ cyclopaminecontrolcyclopamine expressionwild(flM)oncogenic typeSmoothened Smoothened Figure16.41Actionsof cyclopamineonthePatchedSmoothenedpathway (A)Intheexperimentshownhere,the activityofSmoothenedwasgaugedindirectlybymeasuringthe activityof areportergenewhosetranscriptionisdrivenbyGliin mouseNIH3T3cells.Intheabsenceof addedSonichedgehog ligand(ShhNp),aHedgehogvariantthatisalsoa ligandof the Patchedreceptor,therewasnoactivity ofGli(light greenbar)In thepresenceofShhNp,Gliactivity wasstrongly stimulated(dark greenbar),andthisinductionwasreversedbycyclopamine treatment(pink,red bars).Thisdemonstratedthatcyclopamine counteractstheeffects ofHedgehogligandandisthereforelikely to liedownstreamof thePatchedreceptorinthesignalingpathway. (B)Thetargetof cyclopamineactionwasfurtherlocalizedbythis experiment,inwhichtheactivity ofGli(measuredasinpanelA) wasmeasuredinPTCnl- cells.Gliactivitywas,asbefore, suppressedbycyclopamine,confirmingthat thisdrugislikelyto interferewitha stepdownstreamof andindependent ofPatched. (C)Whenwild-typeSmoothened(Smo)wasexpressedathighlevels inNIH3T3cells,itsactivity was,onceagain,suppressedbyaddition of cyclopamine,asindicatedbytheactivityof theGli-regulated reportergene(blue,orangebars,left).However,whena mutant, dominantly acting,oncogenicSmoothenedwasexpressedatthe sameorlowerlevels(right bars)signalingwasquiteresistantt o cyclopamineinhibition.ThisindicatedthatSmoothenedwaseither downstreamof oradirecttarget of cyclopamineaction. Subsequentstudiesgenerateda seriesof mutant,const itutively activeSmoothenedproteinsthat wereallresistanttocycl opami ne inhibition,reinforcingthenotionthat cyclopamineinteractsdirectly withSmoothened(seeFigure1640A)Biochemicalanalysesthen demonstratedthedirectbindingofthecyclopaminemoleculeto Smoothened(not shown)(FromJTaipale,JK.Chen,M.K.Cooper et al.,Nature 406:1005-1009,2000.) 781 ---Chapter 16: The Rational Treatment of Cancer (A) 800 QJ CJ) c '" u :oR ~400 o ~ to .2 a - 100 cyclopamine treatment duration Figure16.42 Effect of cydopamine andanalogous drugs on tumor growth (A)Humancholangiosarcoma (bileducttumor)cellsformedtumor xenograftsinmiceof180mm 3 volume andeitherwereleft untreated(red line) orwerethentreatedfor22dayswith cyclopamine(blueline). Inthelatter case,thetumorshrank anddidnot reappearinthe76daysthatfollowedin theabsenceof furthercyclopamine treatment.(B)Micewitha Ptc+l-p53-igenotypedevelopmedulloblastomas throughout theircerebellaearlyinlife. By5 weeksof age,thecerebellumina wi ld-typemouse(topleft)isfarsmaller thaninthetumor-pronemutant (top right) . ASmoothenedantagonist, termedHhAntag, wasidentifiedthrough screeningof a druglibrary.Ifthemutant miceweretreatedwiththedrugtw ice dailybetweenthethirdandthefifth weekoflife,witheither20mgor100 mgperkgofbodyweight,thetumors regressedpartiallyorcompletely(bottom left and Tight).Subsequenttreatmentsof 8- and10-week-oldmutantmicewith farlargertumors haveyielded comparabletherapeuticresponses(not shown).(A,fromD.M.Berman, 5.5.Karhadkar, A.Maitraetai, Nature 425:846-851 ,2003; B,fromJ.T.Romer, H.Kimura,S.Magdalenoetal.,Cancer Cell6229-240,2004) (B)cerebella Ptc+l- p53-1- untreated wild-type Ptc+l- p53-1- treated with 20mg/kgHhAntag 9498days Ptc+l- p53-1- treated with100mg/kgHhAntag In order to test these new compounds, a mouse model of human medulloblastoma hasbeen createdthat dependson inactivation(seeSidebar 7.10)of one copy of the Pte geneandbothcopiesof thep53 geneinthemousegermline, yieldingaPte+ l -p5:r1- genotype;virtuallyallsuchmicedevelopmedulloblastomasby3months of age.An inhibitor of Smoothened, termedHhAntag,was synthesizedthathas10timesthepotency of cyclopamineandisabletopass easilythroughtheblood-brainbarrier,thespecializedbiologicalbarrierthat protects the brain tissue fromthe contents of the circulation. Asseen in Figure 16.42B, treatment of3-week-old mutant mice that developed medulloblastomas with HhAntag causes a regression of the tumor within twoweeks; this occurred \ovithlittle if any systemic toxicity. Inthe caseof pancreatic cancer,theprospect of developing aclinicallyuseful inhibitor of the Hedgehog signaling pathway isan exciting one. Atpresent, this carcinoma,in which Hedgehog signaling often playsaprominentrole, has an almostinevitablefataloutcome:oncethiscancerhasbeendiagnosedina patient,theprobability of surviving foranother fiveyearsislessthan 4%.This contrastswiththefive-yearsurvivalin1998of Americanpatientsdiagnosed withbreast cancer (86%)and prostate cancer (97%). Medulloblastomas,largely pediatric tumors,occur about one-tenth asoften as pancreaticcarcinomas;atpresent,almosttwo-thirdsof patientsarecuredof thistumorthroughacombinationof surgery,radiation,andchemotherapy; thesetreatmentscan,however,leavesurvivors\ovithsignificantneurological impairment,includingcompromisedcognitivefunctions.Ironically,however, the major economic incentive fordeveloping cyclopamine mimetics islikely to derivefromtheneedtotreatthemostbenignbutalsothemostcommon human cancer type-basal cell carcinomas of the skin. 16.15 mTOR, a master regulator of cell physiology, representsanattractivetarget foranti-cancer therapy Thefinalanecdoteisthe shortest of all,if only because it describes aregulatorycircuitthat isstillincompletely understoodandhas yieldedfewclinical successes to date.Nonetheless, this circuit has all the attributes of generating 782 (A)(B) Me mTOR/0 binding Me o regionMe OMe ~ # Me rapamycin Me Figure16.43 Rapamycin.FKBP12andmTOR(A)Rapamycinis describedchemicallyasa macrocycliclactoneandbiologicallyasa macrolideantibiotic,oneof manythataremadebybacteriabelongingto theStreptomycesgenus.Rapamycinanditschemicalderivativesactas potentimmunosuppressants withoutinducingseveresideeffectsinother organsystemsinthebody.Someof itseffectsareduetoitsabilityto inhibitmTORsignaling.(B)Thebindingof rapamycin(green,red stick figure)to FKBP12(blueribbonand space-filling model,right)occursw ith highaffinity,thedissociationconstant (Kd)beingintherangeof 0.2to OAnM. Thisbimolecular complexformsamolecular surfacethatcan thenassociatewithmTOR(redribbonand space-fillingmodel,left)and preventthelatter fromfunctioningasa serine/threoninekinase.Inthis image,onlytheFRB(FKBP12-rapamycin-binding)domainof mTORis shown.(C)Thedetailsof theinterfacebetweenrapamycin(yellow,red stickfigure)andthe surfacesof thetwoproteinsareshownhere.Areas of highstereochemicalcomplementaritybetweenrapamycinandthe proteinsarehighlightedinpurple.Someof thehighaffinity association depends ontheinsertionof chemicalgroups of rapamycininto deep cavitiesw ithinFKBP12(right)andtheFRBdomainof mTOR(left). (BandC,courtesyof YMaoandJ.Clardy,andfromJ.Choi,J.Chen, S.L.SchreiberandJ.Clardy,Science273239-242,1996 ) therapies that will rival and even eclipse some of those that have been described earlier in this chapter. This story alsostarts withanaturalproduct-rapamycin-that wasisolated in the1960s fromStreptomyces hygroscopicus bacteria growing in the soil of Rapa Nui, known tous as Easter Island, in the middle of the Pacific. In the early 1970s, it was re-isolated by a drug company, which developed it as an antifungal agent. In the decades that followed,it became clear that rapamycin (Figure 16.43A)can acttohaltthegrmvthof anextraordinarilywidespectrumof eukaryoticcells, ranging fromthose of yeast tomammals. Rapamycin was also found to have powerful immunosuppressive powers, even when used at low concentrations. In 1999, it was approved by the U.S.Food and DrugAdministration(FDA)topreventimmunerejectionoftransplanted organs,largelykidneys.Thisdrug,alsocalledsirolimus,functionssynergisticallywithother immunosuppressants,specificallycyclosporineand steroids, toensure long-termengraftment without causingmajor sideeffectsintransplant recipients. The reasonsforitsselective actions in preferentially affecting theimmune systemarenotfullyunderstood.[Intriguingly,immunosuppressionbycyclosporine inorgantransplantrecipientsleadstoincreasedriskof mTOR inhibition by rapamycin FRBdomain of mTOR rapamycin (C)deep burialof rapamycin methyl group inmTOR deep burial of rapamycin pipecolinylgroup inFKBP12 78. Chapter 16: The Rational Treatment of Cancer malignancies (see Section 15.9), while rapamycin-induced immunosuppression in these patients actually decreases the risk of post-transplantation lymphoproliferative disorders.Hence the notion that immunosuppression always leads to increased cancer risk needs to be refined, since some types of immunosuppression yield increased tumor incidence while other types do not.] Biochemicalanalyses show that rapamycin binds directly toalow-molecularweight protein,called FKBP12(FK506-binding protein of 12kD),originally discovered because it is also bound by FK506, a similarly acting drug. Once formed, therapamycin-FKBPl2complex (Figure16.43B)associates withaproteinthat wasidentifiedin1994,termedmTOR(mammaliantargetof rapamycin),and shuts it down.mTORisa large (289kD)protein that functions as a serine/threoninekinase;itskinasedomainresemblesthatofPI3kinaseandrelated enzymes. mTORisof special interest, because it operates as a criticalnode in the control circuitry of mammalian cells (Figure 16.44A). Thus, mTOR integrates a variety of afferent(Le.,incoming)signals,includingnutrientavailabilityandmitogens, and, having done so,acts tocontrol glucose import and protein synthesis. More specifically,mTORphosphorylatestwokeygovernorsoftranslation:p70S6 kinase(S6Kl)and4E-BPl.ThisphosphorylationactivatesS6Kl,whichthen proceeds to phosphorylate the S6protein of the small (40-S)ribosomal subunit, Figure16.44 ThemTORcircuitandtumor responsestomTOR inhibitors (A)mTORsitsinthemiddleof a complexregulatorycircuit that integrates incomingsignalsaboutnutrient availability,oxygen tension,ATPlevels,andmitogenicsignalsand,inresponse,releases signalsthatgovernribosomebiogenesis,proteinsynthesis,cell proliferation,protectionfromapoptosis,angiogenesis,andevencell motility.mTORexistsintwo alternativecomplexeswithitsRictor(left) andRaptor(right)partners;the twocomplexesintercommunicateinstillobscureways.ThemTOR- Rictor complexgovernsthe activityof AktJPKB by addinga criticalsecondphosphate tothelatter andthereby gains controloverAktJPKB 'smultipledownstreameffectors.Exposureto rapamycin(lower right)rapidlyinhibitsthemTOR-Raptor complexand, afterextendedperiods,causesa progressiveshutdownof the mTOR-Rictor complex.(B)BALB/cmicebearinginjectedcellsof a syngeneic colonadenocarcinomacellline developlarge,well-vascularized tumors (left)by 35daysafterinjection.However,ifthetumors are allowedtogrow for a weekafter whichthemicereceivecontinuous treatment withdosesofrapamycincomparabletothoseusedinhumans forimmunosuppression,thetumorsaremuchsmaller(right)andthe density ofmicrovesselsinthesetumorsislessthanhalf of thatseenin thecontroltumors (not shown).(C)Metastatic osteosarcomasareoften difficult to treat.However, inthecaseof a23-year-oldosteosarcoma patient,treatmentwithAP23573,ananalogof rapamycin,yieldeda morethan50%decreaseinthemaximumstandarduptakevalue (SUVmax)ofradiolabeledglucosebya metastasiswithin5daysof treatment,anda morethan85%decreaseby54daysof treatment (white arrows).Whilesuchresponsesarenot typical,theyindicate the potentialof thistypeof treatment andthepossibilitythat,inthefuture, conditions w illbefoundto enablesimilarresponsesina significant proportionof suchpatients.Eachoftheseimagesisa fusionoftwo imagesinitially obtainedbyCT(computerizedX-raytomography) and PET(positron-emissiontomography);thelattermeasurestheextent ofuptakeofradiolabeledglucose,whichisgenerallyelevatedin neoplastictissue . (A,fromDA GuertinandD.M.Sabatini,Trends Mol.Med.11 :353-361,2005;B,fromM.Guba,vonBreitenbuch, M.SteinbaueretaI.,Nat.Med.8: 128-135,2002;C,courtesyof S.P ChawlaandK.K.Sankhala,CenturyCityDoctors'HospitalandJohn WayneCancerInstitute,andof C.L.Bedrosian,AriadPharmaceuticals, Inc.) 784 Rapamycinasananti-cancer agent enabling this subunit toparticipate in ribosome formation(by associating with the large ribosomal subunit) and thus in protein synthesis. In addition, by phosphorylating 4E-BP1, mTOR causes 4E-BPlto release its grip on the keytranslational initiation factoreIF4E (eukaryotic initiation factor 4E); once liberated, eIF4E formscomplexes with several other initiation factors,and thereSUltingcomplexesenableribosomestoinitiatetranslationofcertain (A) s AktlPKB I cellgrowth 1 protein synthesis - I T transcription (s;;

G- G J'(L I\ 8 eFKBP12GTP T rapamycin - hypoxia/stress -AMPK ....f--r nutrients (amino acids,glucose) /11/1\\\\ ! ;oli feration I'cell survival] (B)(C)control+ rapamycin beginning of5 days oftreatment54daysof treatment AP23573treatment 785 Chapter 16: The Rational Treatment of Cancer mRNAs,specificallythose witholigopyrimidinetractsintheir 5',untranslated regions. Together, these various actions allow mTOR to be a key governor of cell growth(rather than cell proliferation; see Figure 8.2). Untilrecently,mTORwasthoughttobeoneof themultipledownstream substrates of AktlPKB, specifically the one allOwing AktlPKB to regulate cell growth by controlling protein synthesis. But the tables have been turned: mTORisnow reali zedtobeakeyupstreamactivator of AktlPKB(Sidebar 540 ).Thisshift puts mTORina farmore powerful position inthe cell.Bycontrolling AktlPKB, mTORcan regulate apoptosisand proliferation inaddition toitsknown ability to regulate cell growth. Infact,mTORappears intwoplacesin the circuitry depictedinFigure16.44A, sinceitisabletoassociatewithtwoalternativepartners,calledRaptorand Rictor.The mTOR-Rictor complex (together with athird protein,isregulatedinunknownwaysbygrmvthfactorsandisresponsibleforactivating Akt/PKB. ThemTOR-Raptor complex(+about whichmoreisknown,is responsible foractivatingprotein synthesis(byphosphorylating S6K1and 4EBPI) .ActingtogetherwithFKBP12,rapamycindirectlyinteractswiththe mTOR-Raptor complex,which israpidly inhibited after thisdrug isappliedto cells.If, however,rapamycin treatment iscontinued for many hours, eventually themTOR-Rictorcomplexisalsoshutdown,resultingintheinhibitionof AktlPKB.Themechanismbywhichrapamycinsucceedsininhibitingthe mTOR-Rictor complex is poorly understood. This inhibitory effect on AktlPKB signaling seems to be responsible formuch of rapamycin'seffecton cancer cellsthat exhibit ahyperactivatedPI3Kor lossof PTENexpression.It isplausiblethatsuchcells,muchlikethesmall-celllung carcinomacellswithmutantEGFreceptors(Sidebar16.3),havebecome "addicted"toAktlPKBsignalsandlurchintoapoptosisthemomenttheyare deprivedofthesesignalsbytheactionsofrapamycinandrelateddrugs. However,thepreciserulesthatdeterminesensitivitytorapamycintreatment are yet tobe worked out. The regulatory circuit shown in Figure 16.44A intersects in additional ways with cancerpathogenesis.Forexample,TSCland TSC2(alsocalledhamartinand tuberin) have already appeared in this book in the context of their role as tumor suppressor proteins. Lossof either of these proteins leads totuberous sclerosis (Table7.1) ; and,as seeninFigure 8.2, lossofTSClresultsin the formationof giant cells in both flies and humans. TSC2 acts as a GAP(GTPase-activating protein; see, for example, Sidebar 5.11) for Rheb, a small Ras-like protein. Aslong as itremains in its GTP-bound state,Rhebcontributes in unknown ways to stimulating the complex; however,once TSC2has induced Rheb to hydrolyze its GTp,Rheb loses this stimulatory activity. Yet other signaling connections between the mTOR circuit and critical growth-inducing and mitogenic proteins are being forgedby ongoing research. Variousderivativesof rapamycinhavebeenproduced,and three areinearlyphaseclinicaltrial.Theirdevelopmenthas beenencouraged,inpart,bythe observation that drugs likerapamycin can be tolerated forextended periods of time by transplant recipients, indicating a tolerably low levelof side-effect toxicity.Inpre-clinical experiments, rapamycin given to mice at levels that are used forchronic immunosuppression has strong effectsin suppressing tumor-associatedneoangiogenesisandthustumor growth(Figure16.44B),an effectthat may beexplainedbythefactthat one of thethree AktlPKBisozymes,Aktl,is critical to the ability of endothelial cells and their precursors to respond to stimulation by vascular endothelial growth factor(VEGF). Insomeclinicaltrials,notablythosefocusedontreatingsarcomas,clinical responses have occasionally been observed that are nothing short of remarkable (Figure16.44C).And in2006,rapamycinwasreportedtoinduce regressionof786 Synopsisand prospects astrocytomas associatedwithtuberous sclerosis(see Figure 8.2B).Indeed, it is clinical responses like these that have motivated discussion of the mTOR circuit in this chapter. They provide tantalizing hints of how this circuit may one day be manipulated to induce cancer celideath, yielding substantial improvements in thetherapy of solidtumors.Theseadvancesarelikelytocomeasoncologists learn which types of cancer cells are particularly sensitive to rapamycin analogs, often in the presence of other coli aborating therapeutic drugs. 16.16 Synopsisand prospects:Challenges and opportunities on theroadahead "When iscancer going to be cured?" This isthe simple and reasonable question posed most often to cancer researchers by those who are not directly involved in this area of biomedical research. In their minds are the histories of other public healthmeasures. Infectiousdiseases,suchaspolioand smallpox,can beprevented, and bacterial infections are, almost invariably, cured. Heart disease is,in the eyes of many, well on its way to being prevented (Sidebar 550 ). Why should cancer be am different? The informationinthisbook provides some insightsintotheanswerstothese questions. As much as we have invoked unifying concepts to portray cancer as a single di sease, the reality-at least in the eyesof clinical oncologists-is fardifferent.Cancer isreallyacollectionof morethan 100diseases,each affectinga distinct cellor tissue type in the body. Pathologi calanalyseshave ledustoembrace thisnumber,or oneabitlarger. (Forexample,thereareatleasteightdistincthistopathologicalcategoriesof breast canceL)However,even the expanded number, largeasitmay be,representsanillusion:thecurrentuseofmoleculardiagnostics,specificallygene expression arrays, is leading to an explosion of subcategories, so that by the second decade of the new millennium, several hundred distinct neoplastic disease entitiesarelikelytoberecognized,eachfollowingitsown,reasonablypredictable clinical course and exhibiting its own responsiveness tospecific forms of therapy. \ \-iththe passage of time, cancer diagnoses will increasingly be made using bioinformatics rather than the trained eyes of apathologist. Sothe initialresponse toquestions about "the cure" isthat there won't be a singlemajorbrea1. 'throughthatwillcureallcancers-adecisivebattlefieldvictory-simply because cancer is not a single disease. Instead, there will be many smallskirmishesthat willsteadilyreducetheoveralldeathratesfrom various typesofcancer.Andbecausecertainmoleculardefectsandpathological processes(e. g .. angiogenesis)are shared bymultiple human cancers,there will beoccasions\\-hen therapeuticadvancesonanumber of frontswillbemade concomitanth. Before we speculate on the future of cancer therapy, it is worthwhile to step back andassessthescopeof thechallenge:(1)Howlargeistheproblem of cancer and,in the future, how desperate will the need be tocure various types of neoplastic disease? (2)How well are we doing now in curing the major solid tumors? Epidemiologyanddemographicsprovidesomeanswerstothefirstquestion. They yield sobering assessments of the road ahead. The statistics in Figure 16.45 demonstratethat cancer islargelyadiseaseof theelderly,whose numbersare growing rapidly and willcontinue todoso, generating progressive increases in the numbers of cancer-related deaths (mortality)over the coming decades. Equallyimportant,westillhaveonlyveryimperfectwaysof measuringincidence-how often thediseasestrikes.Thisgreatlycomplicatesassessmentsof the effectiveness of current therapies and future needs fortherapy. Asindicated787 78.9c280 .2::::70 60 ::::l50 0.(1) 0-0 400.(5 vi-o 30 c::Jro 20 Lf)10 co 0 1990200020102020203020402050 Chapter 16: The Rational Treatment of Cancer (A)(B)>.t:c Vi90roO t.;::; E-340males

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